Metabolic aspects of obesity

Molec, Aspects Med. Vol. 5, pp. 293 to 400, 1982 Printed in Great Britain. All rights reserved.

0098-2997182/050293-10~$54.00/0 Copyright Q 1982 Pergamon Press Ltd.

METABOLIC ASPECTS OF OBESITY M. A. Cawthorne Beecham Pharmaceuticals Research Division, Great Burgh, Yew Tree Bottom Road, Epsom, Surrey, U.K.

Contents Int roduct ion Chapter

i.

296 CDNTROL

OF FOOD

INTAKE

297

Neural factors in the control of food intake Neuroche~ical factors Mechanism of action of centrally-acting anorexic drugs HLrnoral factors in the control of feeding The role of nutrients in the control of food intake Chapter

2.

GONEROL

OF ENERGY

EXP~kTDITURE

Energy cost of ~ight maintenance Experimental overfeeding in r~an Modulations in energy expenditure experimental animals ~nents of the metabolic rate: Basal metabolic rate Physical activity Cold-induced t hermogenesis Dietary induced thermogenesis Psychological t hermogene sis Chapter

3.

THERMOREGULATORY

AND DIETARY-I~

318 318 320 induced

by diet

in 324 326 327 327 327 329 331

THERMOGENESIS

Body size and non-shivering thermogenesis Noradernaline and non-shivering thermogenesis Location of tissues responsible for non-shivering t her~ogene sis Non-shivering thermogenesis in genetically obese Tissue effectors of dietary-induced thermogenesis Thermogenesis in hypothal~mic obesity Non-shivering tbe~nogenesis in man

293

297 298 3O4 310 313

332 333 334

rodents

335 341 345 347 351

294 Chapter

M.A. Cawthorne 4.

CELLULAR

~{ECHANIS%{5

OF ALTERED

ENERGY

EFFICIENCY

Brown adipose tiss~]e Provision of substrates for heat production in brown adipose tissue Defects in brown adipose tissue in obese rodents Brown adipose tissue in Cafeteria-fed rats Brown adipose tissue in man Sodium-Potassium ATP-ase Subst rate cycles Chapter

5.

THE

SY~,{PATHETIC

Chapter

6.

(X~CI/]DINGRFA~LRKS

NERVOUS

SYSTEH

AND

~NERGY

BALANCE

355 ,356 363 367 369 372 372 375 380 389

Acknowledgements

392

References

393

Introduction

In 1976, a Department of Health and Social Security/Medical Research Council working party on research into obesity commented as follows: 'We are unanimous in our belief that obesity is a hazard to health and a detriment to well-being. It is con~Dn enough to constitute one of the most important medical and public health problems of our time, whether we judge importance by a shorter expectation of life, increased morbidity or cost to the c o ~ m m i t y in terms of both money and anxiety' . Obesity is generally defined as a condition in which there is excessive storage of fat. However, what is excessive? There is no precise definition and most workers rely on tables published by life insurance companies that have identified the risk factors of pr~nature mortality. Obesity is a problem of energy balance. Excess energy intake, relative to energy expenditure, results in the storage of extra fat. To illustrate the magnitude of the problem of maintaining energy balance it is useful to examine the calorific requirements of an average man. The U.S. National Research Council's (1974) recc~aended daily allowance for a 23-50 year old male is 2,700 kcals (11.29 MJ). Thus in a year, the total caloric intake will be 985,500 kcals. The calorific value of adipose tissue is approximately 7,700 kcal (32.19 MJ) per kg. Thus, an error of only I% in energy balance over the year will result in a change in the fat stores of I. 25 kg. There are three possible explanations for an increase in the fat stores. are: I. 2. 3.

These

Energy intake has increased but energy expenditure has remained unchanged. Energy intake has remained unchanged but expenditure has reduced. A combination of increased energy intake and reduced energy expenditure.

It therefore follows that an understanding of the nature of the problem of fat storage requires a knowledge of the factors that control food intake and energy expenditure. Are body weight or body fat content or both regulated? Several studies have demonstrated that body weight changes only showly over many years and this has been taken as evidence to support the hypothesis that body weight is regulated. The Ten-State Nutritional survey (1972) showed that body weight remained almost

295

296

M . A . Cawthorne

constant from age 30-60 in white males. In females there was a small increase in weight up to age 40 but it then remained constant. However, stability of body weight should not be regarded as stability of body composition. The fraction of body weight that is fat increases with increasing age, whilst the percentage that is lean tissue declines (Forbes and Reina, 1970). T~o other studies lend support to the concept that body weight and body stores of energy are regulated. Camstock and Stone (1972) found that over a 5 year period weight gain in adult man was only I .I to 1.5 pounds and Chinn, Garrow and Miall (1974) found that most of their subjects did not change in weight over several years. Perhaps the best evidence that body weight is regulated comes from the numerous studies that show that, when animals and man are allowed to eat ad libitum after a period of food restriction, their body weight returns to its prerestriction level. Thus, the general view is that body energy stores are regulated but that there are longterm changes that are relatively small and occur slowly. There are some dissenters from this view (notably Garrow, 1974; Booth, 1976). A major problem in regulating energy balance is that energy expenditure is continual whereas all mammals including man eat discontinuously. Thus, it is perhaps not surprising that studies that have measured body weight daily have revealed short-term variations in weight of the order of 0.5 kg (Adams, Best and Edholm, 1961). It has been argued (see Hirsch, 1978) that for millenia, man's metabolic system has been under pressure to operate well when food was scarce and acquired in unpredictable amounts at considerable energy cost. The ability to be highly efficient in metabolic energy conservation must under these circumstances have had survival value. For such a genetic pool, the relatively new experience of having an abundant supply of food coupled with such factors as the advent of central heating, labour saving devices and transport, all of which have an effect on energy expenditure, places new pressures on the metabolic system. However, this argument is almost certainly an over-simplification since at times in man's past he must have had abundant supplies of fruit and vagetables. If this were not the case, one would not have expected man to evolve an exogenous requirement for vitamins. During these times of abundant food supply pressures to waste or use excess calories might have been expected to evolve. This review will be primarily concerned with the factors regulating energy balance in normal and obese individuals. In such individuals changes in the capacity of the energy store tend to occur slowly. However, there are other situations such as the cachexia that occurs in many cancer patients in which the energy store decreases rapidly. Whilst anorexia is often associated with this disease state, a number of studies have reported weight loss in patients consuming normal amounts of food. Wilson (priv. conm. ) has recently studied this phenomenon in turnout bearing mice and shown that the resting energy expenditure is increased by up to 40% (Brooks, Neville, Rothwell, Stock and Wilson, 1981). Furthermore, these mice had a greater overall capacity for thermogenesis than the control mice. Such findings raise the possibility that cachexia in man results from defective regulation of energy balance through a failure to control energy expenditure.

Chapter 1

Control of Food Intake

~ l s including man eat discrete meals. Feeding can be divided into a sequence of events and behaviours, and in attempting to assess the controlling factors in feeding, the following questions can be posed: I. 2. 3. 4.

What What What What

factors factors factors factors

initiate feeding? sustain the consumption of a meal? cause the termination of a meal? determine the inter meal interval?

Neural Factors in the Control of Food Intake The first description of hypothalamic injury associated with obesity was published by Mohr in 1840. This isolated report was substantiated by Babinski (I900) and Frohlich (1901) who showed that the presence of tumours in the region of the hypothalamus were associated with obesity, atrophy of the gonads and short stature. The documentation that the hypothalamus rather than the pituitary was responsible for the syndrome was primarily obtained from experimental work in animals (Hetherington and Ransome, 1939; 1940). It has recently been shown that the manifestations of- the syndrome in man (Bray and Gallagher, 1975) and experimental animals (Bernardis and Goldman, 1976) are similar. Further studies by Hetherington and Ransome (1942) showed that hyperphagia and obesity were associated with lesions in the medial tubular and posterolateral hypothalamus, whilst lesions in the lateral hypothalamus resulted in aphagia (Anand and Brobeck, 1951). These experiments constituted the beginnings of the dual-centre hypothesis for the control of feeding. This hypothesis proposed that the lateral hypothalamus is the feeding centre and the ventromedial hypothalamus is a satiety centre. Support for this hypothesis came from experiments in which the two centres were stin~lated electrically (Delgado and Anand, 1953; Wyrwicka and Dobrzecka, 1960). In addition, neurophysiological recordings showed that during periods of hunger in dogs, electrical activity was lower in the ventromedial hypothalamus than in the lateral hypothalamus (Anand, Chhina, Sharma, Dua and Singh, 1964; Anand and Pillai, 1967). The administration of glucose intravenously produced an increase in the spikes from the ventromedial hypothalamus and a decrease in those from the lateral hypothalamus. A number of anatomical studies suggested that there were direct connections be297

298

M.A. Cawthorne

tween the ventromedial and lateral hypothalami. Knife-cuts between the two nuclei produce a variety of syndromes depending on the location of the cuts and these studies led to the view that there was reciprocal innervation between the two centres. The role of the ventromedial hypothalamus as a satiety centre has been supported by a number of chemical studies. Gold thioglucose is known to induce hyperphagia and obesity in experimental animals and the substance was shown to be concentrated in the ventromedial hypothalamus (Debons, Krimsby, Likuski, From and Cloutier, 1968). Compounds other than glucose that contained gold do not damage the ventromedial hypothalamus suggesting that glucose transport by glucose sensitive cells was serving as a route of entry for the toxic gold. Further support for a glucoreceptor in the ventromedial hypothalamus was provided by the finding that damage to the ventromedial hypothalamus by gold thioglucose was blocked by glucose and glucose analogues, was blocked by diabetes (Debons et al, 1968) and facilitated by insulin (Debons, Krimsby, From and Cloutier, 1969) and exercise (Baile, McLaughlin, Zinm and Mayer, 1971). Although the dual-centre hypothesis is a useful construct for relating the effects of injury to the hypothalamus with changes in food intake, there are now several lines of evidence to show that it is inadequate. These inadequacies particularly concern the role of the ventromedial hypothalamus as the satiety centre. For example, Reynolds (1965) noted that while electrolytic lesions cause hyperphagia, radio-frequency lesions did not. Other work shows that lesions restricted entirely to this nucleus failed to induce either hyperphagia or obesity (Gold, 1973) and it was suggested that damage ascribed to the ventromedial hypothalamus and causing hyperphagia was in fact due to the destruction of fibre tracts to the ventral noradrenergic bundle.

Neurcchemic al Factors The development of knowledge of central monoaminergic pathways provided by the Swedish school led by Fuxe during the 1960's led to questions on the involvement of monoamines in feeding behaviour. There are relatively few adrenergic neurons or terminals in the tuberal region of the hypothalamus except for the dopaminergic neurons in the median eminence. Two prominent ascending monoaminergic paths have been identified by histofluorescence techniques; a noradrenergic bundle of fibres in the medial forebrain bundle (Fig I. I) and a dopaminergic bundle of fibres (Fig I .2) passing through the most lateral aspect of the hypothalamus. Ungerstedt (1970, 1971a) proposed that the lateral hypothalamic syndrome which results in aphagia is due to the interruption of ascending dopamine containing fibres, particularly the nigro-striatal bundle. In studies using the neurotoxin 6-hydroxydopamine, which causes a degeneration of noradrenaline and dopamine containing neurons (Ungerstedt, 1971b), he found that rats given bilateral injections of 6-hydroxydopamine into the nigrostriatal bundle became aphagic. Destruction of ascending noradrenergic containing fibres or the mesolimbic dopaminergic syste[n did not produce aphagia. Since that time, many workers have noted the similarities between the syndrome induced by 6-hydroxydopamine injections and the lateral hypothalamic syndrome. The general consensus is that the lateral hypothalamic syndrome is due to the destruction of dopamine containing neurons that project into the forebrain. Other similar studies indicate that noradrenergic neurons may be involved in the hyperphagia seen after ventrc~edial hypothalamic damage (Ahlskof and Hoebel, 1973) However, ventral noradrenergic bundle destruction by 6-hydroxydopamine does not exactly replicate the ventromedial hypothalamic lesion and thus it is concluded

Metabolic Aspects of Obesity

299

that other neural systems must also be involved.

Noradrenaline

ventral bundle

Fig. i. 1

Saggital view of the ascending NE pathways. The stripes indicate the major nerve terminal areas. Reproduced from U. Ungerstedt, Acta Physiol. Scand. 367, 1-48, 1971, with permission.

Dopamine

Fig. I. 2

Saggital view of the DA pathways. The stripes indicate the major nerve terminal areas. Reproduced from U. Ungerstedt, Acta Physiol. Scand. 367, 1-48 (1971), with permission.

An understanding of the role of noradrenaline in feeding must include a consideration of those experiments that have examined the effects of centrally administered noradrenaline. Grossman (1960) found that rats given an injection of noradrenaline into the lateral hypothalamus overate in the hour following the injection. These experiments were performed in the daytime, when in normal circumstances rats eat

300

M.A. Cawthorne

little. Experiments carried out at night produced the opposite results, namely anorexia. Since rats eat mainly at night, it follows that destruction of noradrenergic fibres should, overall, result in overeating. It is possible to dissect these actions of noradrenaline using pharmacological techniques. Leibowitz (1970, 1971) has provided evidence for a e-adrenergic 'hunger' system and a 6adrenergic 'satiety' system. Thus, ~-adrenergic agonists injected into the ventromedial hypothalamus produce an increase in food intake whilst ~-adrenergic antagonists suppress eating (Fig. 1.3). 8-adrenergic agonists injected directly into the lateral hypothalamus suppress eating whilst B-antagonists increase feeding (Fig. 1.4).

I

E

I

~6._c .~

o c O

~'4-~"0

Q-

-~.~ 0 Saline

NE (25 n moles)

n

Alpha Beta

Receptor Blockers + NE

Fig. 1.3

Alpha Beta Receptor Blockers alone

Potentiation of feeding effect produced by unilateral norepinephrine (NE) injection into the paraventricular nucleus of 8 hour fooddeprived rats. Local pretreatment with the alphaadrenergic blocker phentolamine (PHT) abolished the NE-induced potentiation of feeding, while the beta-adrenergic blocker l-propranolol (1-PROP) caused a further increase in feeding. When injected alone, neither blocker had any effect on the rats' baseline food intake scores. Reproduced from S.F. Leibowitz in Hunger, basic mechanisms and clinical implications, Raven Press, New York, 1976, with permission.

During the last few years, a number of studies have suggested a role for brain 5-hydroxytryptamine systems in feeding and weight gain. Breisch and Hoebel (1975) showed that intraventricular injection of p-chlorophenylalanine, a reversible depletor of 5-hydroxytryptamine caused rats to overeat. In addition, intraventricular administration of the neurotoxins 5,6-dihydroxy and 5,7-dihydroxytryptamine produced overfeeding and obesity (Diaz, Ellison and Musuoka, 1974; Saller and Stricker, 1976). Various studies in which 5-hydroxytryptamine has been injected intraventricularly, have produced equivocal results. The nutritional state of the animals seems to have a crucial influence on the result obtained. When satiated rats are used only minor changes in food intake are observed, whereas

Metabolic Aspects of Obesity

Food ( g m ) c o n s u m e d 0

1 I

2 I

301

in 9 0 min by h u n g r y rats

3 4 I I

5 I

6 I

7 I

8 I

9 10 11 I I I

Saline

Epinephrine (EPI: 50 n moles)

Beta blockers + EPI

MJ 1999 (300 n moles) ÷ EPI 1- Propranolol (120 n moles) ÷ EPI

d-Propranolol (120 n moles) + EPI

Alpha blocker ÷

Fig. i. 4

EPI

" ' ~

Phentolamine (60 n moles) + EPI

Suppression of feeding effect produced by unilateral epinephrine (EPI) injection into the lateral (perifornical) hypothalamus of 18 hour food-deprived rats. This suppressive effect was abolished by the beta-adrenergic blockers MJ 1999 and l-propranolol, while potentiated by the alphaadrenergic blocker phentolamine. A significant reversal, towards feeding potentiation, was obtained with l-propranolol, while no change was observed with the d-isomer of propranolol. Reproduced from S.F. Leibowitz in Hunger, basic mechanisms and clinical implications, Raven Press, New York, 1976, with permission.

the typical response in food deprived rats is a depression in food consumption (see Blundell, 1977 for review). In addition to the technique of direct administration of the neurotransmitter itself, 5-hydroxytryptamine levels in the brain can he raised by the administration of the precursors tryptophan or 5-hydroxytryptophan. Exogenously administered 5-hydroxytryptophan is rapidly decarboxylated to 5-hydroxytryptamine (Udenfriend, Titus, Weissbach and Peterson, 1956) both centrally and peripherally. Numerous studies have indicated that loading with either of these t~o substances increases the brain levels of 5-hydroxytryptamine (Ashcroft, Eceleston and Crawford, 1965; Harvey and Lints, 1971) and produces a reduction in food intake. The studies with 5-hydroxytryptophan are subject to the possibility that 5-hydroxytryptamine is formed in non-serotonergic systems (Coscina, Warsh, Godse and Stancer, 1974), since the enzyme which decarboxylates 5-hydroxytryptophan is capable of decarboxylating other aromatic amino acids. Because tryptophan hydroxylase is specific for L-tryptophan and occurs only in serotoninergic neurons (Aghajanian and Asher, 1971), studies with tryptophan have the advantage that the enhancement of 5-hydr-

302

M.A. Cawthorne

r

•

CHs--CH-NH2 tryptophan~ ~L~L~N ~J COOH hydroxylase

HO .

./~'..,.

T/ ~

~ " ~ C H T CH-NH~ y COOH N

1

5-hydroxytryptophan I aminoacid decarboxylase

-tryptophan

H O V ~ ~ N ~j] CH2COOH moooam,ne

5-hydroxyindoleaceticacid Fig. 1.5

H O ~ ~ - - - ~ CH2CHgNH2 I II II

oxidaseandaldehyde I ~ ~ I/I dehydrogenase ~ ~Nf 5-hydroxytryptamine

Metabolism of tryptophan

oxytrytamine levels occurs exclusively in serotoninergic neurons. Thus Fernstrom and Wurtman (1972) showed a dose-dependent decrease in food consumption after peripheral injections of tryptophan, and decreases in food intake were pronounced when tryptophan was given to rats that have been pretreated with a monoamine oxidase inhibitor (Barrett and McSharry, 1975). Tryptophan is an essential amino acid in that it cannot be synthesized in the body and thus the diet is the sole source. If 5-hydroxytryptamine does have a real role in the control of feeding it would seem necessary that the concentration of 5hydroxytryptamine in the brain should be modulated by the nutritional state. Although tryptophan hydroxylase is the rate limiting enzyme in the formation of 5-hydroxytryptamine from tryptophan, the km for tryptophan is relatively large (60 ~M) (Carlsson, Kehr, Lindquist, Magnusson and Atack, 1972), whereas the brain concentrations of tryptophan are of the order 20-50 ~M. Thus, the overall rate of formation of 5-hydroxytryptamine is regulated by the concentration of tryptophan in brain and plasma. It has been shown that increases (Green, Greenberg, Erickson, Sawyer and Ellison, 1962) or decreases (Biggio, Fadda, Fanni, Tagliamoute and Gessa, 1974) in dietary intake of tryptophan produce concomitant changes in brain tryptophan. However, the relationship between the amount of dietary tryptophan and brain 5-hydroxytryptamine is not simple for the brain levels of tryptophan and 5-hydroxytryptamine are influenced by insulin (Fernstrom and Wurtman, 1972) , the ratio of tryptophan to plasma neutral amino acids (Fermstrom and Wurtman, 1972) and on the presence of non-esterified fatty acids in the blood (Curzon, Friedel and Knott, 1973). The reason for these inter-relationships is that tryptophan is unique among amino acids in binding to circulating albumin. The ability of albumin to bind tryptophan bears an inverse relationship with the concentration of circulating non-esterified fatty acids (Lipsett, Madras, Wurtman and Munro, 1973). Virtually all circulating non-esterified fatty acids are hound to albumin and the concentration of non-esterified fatty acids in fasting rats or man is of the order I-2 mM whereas the tryptophan concentration is rarely greater than 20 ~M. Insulin decreases the circulating concentration of non-esterified fatty acids by its inhibitory action on lipolysis and it also lowers the concentration of neutral amino acids other than tryptophan by facilitating their uptake by skeletal n~iscle. Since

Metabolic Aspectsof Obesity

303

Oiar I I Dietary

Protein

Carbohydrate (Insulin Secretion)

(Amino Acids)

++1 1 + ~,asma ~

(T+P+L+I+V)

"x~

J ~,asma I

/1 Tryptophan

I PlasmaRatio Try

(T+P+L+I+V)

I Brain

TryptophanI

I Serotonin ra'n I i Fig. 1.6

5-HIAA

I

Proposed model to describe diet-induced changes in brain serotonin concentration in the rat. The ratio of total tryptophan to the combined levels of tyrosine, phenylalanine, leucine, isoleucine, and valine in serum is thought to predict the tryptophan level in brain. The three branched-chain amino acids may be more important suppressors of tryptophan uptake into the brain than the aromatic amino acids. Produced from Fernstrom, J.D. and Wurtman, R.J., Science 178, 414-416. Copy right 1972 by the American Association for the Advancement of Science, with permission.

neutral amino acids are translocated into the brain by the same carrier process as tryptophan (Blasberg and Lajtha, 1965), a decrease in the circulating concentration of the competing amino acids will lead to an increase in the rate of uptake of tryptophan into the brain (Fig. I .6). If a normal rat that has been fasted for 10-12 hours is allowed to consume a protein free meal, the level of tryptophan and 5-hydroxytryptamine in the brain increases significantly in one hour and reaches a maximum by 2 hours (Fernstrom and Wurtman

304

M . A . Cawthorne

1971). These changes occur because of the effects of insulin on lipolysis and utilization of amino acids. The increased binding of tryptophan to albumin allows the total concentration of tryptophan in blood to rise whilst the concentration of free tryptophan falls. Fortunately the binding of tryptophan to albumin does not appear to preclude its uptake into the brain (Madras, Cohen, Messing, Munro and Wurtman, 1974). Paradoxically, if the meal supplied to the rat contains protein, brain tryptophan and 5-hydroxytryptamine levels do not rise as much as when a protein-free meal is administered even though plasma tryptophan levels show a greater increase. Indeed if a high protein meal is given, brain tryptophan and 5hydroxytryptamine levels may actually decrease postprandially. The explanation for this arises from the competitive nature of the brain tryptophan uptake system. Since virtually all natural proteins contain less than I .5% tryptophan, whereas the five competing neutral amino acids (leucine, isoleucine, valine, tryposine, phenylalanine) make up more than 25%, it follows that the circulating concentration of these amino acids increases proportionately more than tryptophan. Furthermore, hepatic tryptophan pyrrolase catabolizes a considerable fraction of the tryptophan entering the liver whereas hepatic metabolism of branch-chain amino acids (valine, leucine and isoleucine) is exceedingly limited. The precise effect of any meal on the brain tryptophan and 5-hydroxytryptamine levels can be expected to depend on the composition of that meal, on the effectiveness of insulin in inhibiting lipolysis and promoting utilization of neutral amino acids, on the duration of fasting from the previous meal (and thereby the level of circulating non-esterified fatty acids), and on the tryptophan content of previous meals. Because of the complex inter-relation involving both amount and type of food (carbohydrate, protein and fat) it is possible that recognition of 5-hydroxytryptamine concentration in the brain could be one means of exerting central control over nutritional strategies. This possibility will be discussed later.

Mechanism of Action of Centrall~-~mting Anorexic Drugs The involvment of eatecholamines and 5-hydroxytryptamine in the control of food intake has also been investigated by extensive experimental animal studies on the mechanism of action of anorexic drugs. The fact that the same drugs produce anorexic effects in both laboratory animals and man suggest that similar control mechanisms could exist in man. Most of the experimental animal work has focussed upon the drug pair amphetamine and fenfluramine and it has attempted to define the locus of interaction of these drugs with the neuroch~mical system. Garratini and Samanin (1976) pointed out that most anorexic drugs used clinically produce profound changes in brain monoamines (Table I .I) and, in turn, procedures that modify the metabolism of monoamines also modify the inhibition of food intake produced by anorexic drugs. On the basis of a number of biochemical studies, it has been suggested that amphetamine acts on brain noradrenaline by releasing it from presynaptic terminals and inhibiting its reuptake into the neurons (Glowinski, Axeirod and Iversen, 1966). As a result of the increase
Metabolic Aspectsof Obesity

305

TABLE 1.1 Effect of Anorectics on Brain Monoamines and some Metabolites

STRIATAL LEVELS

BRAIN LEVELS

DRUG (~g/kg, i.p.) Saline

DA

Ach

HVA(+)

5-HT

5-HIAA

NA

0.43±0.01

0.34±0.01

0.40±0.01

8.2±0.5 3.9±0.1

0.34±0.03

0.24±0.02*

7.5±0.3 5.1±0.5" 416± 9*

212± 6

d,amphetamine sulphate

15

0.40±0.01

d,l,fenfluramine HCl

15

0.16±0.01" 0.20±0.03* 0.38±0.01

Mazindol

15

0.50±0.08

0.34±0.02

0.32±0.03*

-

-

414±45"

Phentermine HCI

15

0.47±0.01

0.37±0.03

0.40±0.01

-

-

338±14"

Diethylpropion HCI

15

0.40±0.01

0.28±0.01

0.30±0.01"

-

-

329±30*

7.2±0.2 2.6±0.1" 477±19"

Each figure represents the mean (ug/g±S.E.) of 6 animals (+) HVA values are expressed in ng/g * p< 0.01 versus saline group (Dunnett's test) 5-HT serotonin 5-HIAA 5-hydroxyindoleacetic acid NA noradrenaline DA dopamine Ach acetylcholine HVA homovanillic acid Reproduced from Garattini and Samanin in Appetite and Food intake, Dahlem Conferenzen, with permission.

acid, which is the major metabolite of dopamine (Garratini, Buczko, Jori and Samanin, 1975). This suggests that both drugs increase the release and turnover of striatal dopamine. The mechanism of the effect is however different for the two compounds. Amphetamine increases the release of dopamine from nerve terminals (Glowinski, 1970) whereas fenfluramine blocks uptake at the receptor in a similar manner to neuroleptic drugs (Fig. I .8). As a result fenfluramine does not produce the stereotyped behaviour commonly seen in animal studies with centrally acting anorexic drugs. In fact, fenfluramine antagonizes the stereotypes behaviour induced by amphetamine (Jori, Cecchetti, Ghezzi and Samanin, 1974). Fenfluramine produces marked and long-lasting depletion of brain 5-hydroxytryptamine levels (Duhault and Verdavaivine, 1967) and a reduction in the level of 5-hydroxyindoleacetic acid, which is a 5-hydroxytryptamine metabolite. Fenfluramine has no effect on 5-hydroxytryptamine synthesis and it appears that these effects arise from an increase in 5-hydroxytryptamine turnover (Costa, Groppetti and Revuelta, 1971). The current view is that fenfluramine increases the release of 5-hydroxytryptamine from storage granules and also blocks its reuptake so that the concentration of 5-hydroxytryptamine at the receptor is increased (Fig. I .9) , (Fuxe, Farnebo, Hamberger and Ogren, 1975). Amphetamine, at minimal anorexic doses, does not significantly alter brain 5-hydroxytryptamine levels or turnover. The studies on the effects of amphetamine and fenfluramine on the level and turnover of brain monoamines suggest that amphetamine has its major effect on the

306

M . A . Cawthorne

/

Receptor Fig. 1.7

Proposed main sites of action of amphetamine on the noradrenergic system

Amphetamine

Fig. 1.8

Proposed main sites of action of amphetamine and fenfluramine on the dopaminergic (DA) system

Metabolic Aspects of Obesity

307

Receptor Fig. 1.9

Proposed main sites of action of fenfluramine on the serotonergic (5HT) system

catecholamine system whereas fenfluramine has its predominant effect on the serotoninergic system. Studies on the effect of various procedures affecting brain catecholamines and 5-hydroxytryptamine on the anorexic effect of amphetamine and fenfluramine endorse this view (see table 1.2 and 1.3). However, these studies do not clarify the relative role of dopamine and noradrenaline in the action of amphetamine. In particular, studies using agents to block dopamine or noradrenaline receptors have produced confusing results. Both pimozide and haloperidol, which are considered to be fairly specific blockers of dopamine receptors in the brain reduced the anorexic activity of amphetamine (Clineschmidt, McGuffin and Werner, 1974), whereas penfluridol (Garratini and Samanin, 1976) a potent central dopamine antagonist, had no effect. Phentolamine, a noradrenaline receptor antagonist, has been shown to block the anorexic action of amphetamine (Frey and Schulz, 1973) although Samanin found no effect of this antagonist. Recent studies have suggested that the antagonistic effects of haloperidol and p~nozide on amphetamine-induced anorexia occur because relatively large doses of amphetamine were used. Such doses induced behavioural changes (through stimulation of dopamine receptors) that were incompatible with feeding. The observed anorexia is a combination of these effects plus the specific effect of amphetamine. By preventing the behavloural changes with dopamine antagonists, there was apparently an inhibition of the anorexic effect of amphetamine. However, at dose levels of amphetamine at which there are no behavioural changes, pimozide had no blocking effect (Burridge and Blundell, 1979). Similar results have been obtained in man. Silverstone has shown that the anorexic effect of amphetamine in man is not blocked by pimozide but it is blocked by the noradrenergic antagonist thymoxamine. Thus, the present concensus is that amphetamine exerts its anorexic effect in man and animals by increasing the availability of noradrenaline at noradrenaline receptors. If amphetamine and fenfluramine interact with different neuroch6~ical systems, it might be anticipated that these two drugs would produce different effects on feeding behaviour. Studies by Blundell, Latham and Leshem (I976) using free-feeding

308

M.A. Cawthorne

rats have shown that amphetamine reduces food intake by delaying the onset of eating whereas when fenfluramine is administered, eating begins normally but is brought to an early halt. The two compounds also affect the local rate of eating; fenfluramine reducing it and amphetamine increasing it. The effects of fenfluramine were also replicated by injections of l-tryptophan (Latham and Blundell, 1979). There findings provide some evidence that catecholamines are involved in the recognition of hunger whereas 5-hydroxytryptamine may he involved in the recognition of satiety. TABLE 1.2 Effect of various procedures affecting Brain Catecholamines on the reduction of Food Intake induced by Amphetamine and Fenfluramine in the Rat

ANOREXIA BY EXPERIMENTAL CONDITION

EFFECT ON CATECHOLAMINES (CA)

AMPHETAMINE

FENFLURAMINE

e-methyl-p-tyrosine

inhibition of CA synthesis

reduced

unchanged

6-hydroxydopamine

destruction of CA-containing neurons

reduced

unchanged, or enhanced

lesion of the ventral noradrenergic bundle

destruction of noradrenergic neurons

reduced

enhanced

lesion of the locus coeruleus

destruction of noradrenergic neurons

unchanged

unchanged

haloperidol

blockade of dopamine receptors

reduced

unchanged

pimozide

blockade of dopamine receptors

reduced

unchanged

penfluridol

blockade of dopamine receptors

unchanged

unchanged

l-propanolol

blockade of B-noradrenergic receptors

reduced, unchanged enhanced or unchanged (+)

phentolamine

blockade of ~-noradrenergic receptors

unchanged or reduced

unchanged

lesion at the level of the lateral hypothalamus

destruction of CA-containing neurons*

reduced

enhanced

* this lesion is likely to affect serotonergic neurons passing through the medial forebrain bundle Reproduced from Garattini, S. and Samanin, R., Anorectic drugs and brain neurotransmitters in Proceedings of the Dahlem, workshop on Appetite and Food Intake, Dahlem Conferenzen, Berlin, 19, with permission.

The drug pair amphetamine and fenfluramine are also yielding interesting information on how free living animals including man might select their nutrients from the range available. In a study in which fasted rats were offered simultaneous access to two isocaloric diets (Wurtman and Wurtman, 1977, 1979) containing either 5% or 45% protein, it was found that following the administration of fenfluramine,

Metabolic Aspects of Obesity

309

the rats preferentially decreased their intake of the 5% protein diet. In this experiment the fat content of the two diets was identical and the net result was that the rats reduced their caloric intake by restricting their carbohydrate intake without a significant reduction in protein intake.

TABLE 1.3 Effect of various procedures affecting Brain 5-HT on the reduction of Food Intake induced by Amphetamine and Fenfluramine in the Rat

EXPERIMENTAL CONDITION

ANOREXIA BY EFFECT ON SEROIONIN AMPHETAMINE

FENFLURAMINE

lesion of the midbrain destruction of 5-HT neurons raphe

unchanged

reduced or unchanged

5,6-dihydroxytryptamine

destruction of 5-HT terminals

unchanged

reduced or unchanged

p-chlorophenylalanine

inhibition of 5-HT synthesis

reduced

unchanged

methergoline

blockade of 5-~h~freceptors

unchanged

reduced

cyproheptadine

blockade of 5-HT receptors

unchanged

reduced

chlorimipradine

inhibition of 5-HT uptake

unchanged or enhanced

reduced

Reproduced from Garattini, S and Samanin, R., Anorectic drugs and brain neurotransmitters in proceedings of the Dahlem workshop on Appetite and Food Intake, Dahlem Conferenzen, Berlin, 19, with permission.

In a similar experiment, Blundell, Latham, Moniz, McArthur and Rogers (1979) examined the effect of fenfluramine and amphetamine on diet selection in rats that were habituated to the selection circumstances. The effect of fenfluramine (table I .4) was to depress equally the intake of both diets so as to preserve the overall percentage intake of protein energy. In contrast, amphetamine severely reduced the consumption of the high protein diet and consequently reduced the percentage intake of protein energy (table I .4). In a further experiment in which rats could individually select carbohydrate, fat or protein (Kanarek, Ho and Meade, 1981) , amphetamine produced a more sustained decrease in fat consumption than it did on either carbohydrate or protein consumption. Experiments in man on the interaction of drugs with the feeding pattern are notoriously difficult to carry out. Even the fact that subjects are taking part in an experiment can produce large changes into their attitude to feeding. In man, both amphetamine and fenfluramine produce significant reductions in food intake over 24 hours. (Blundell, Latham, Moniz, McArthur, and Rogers, 1979). These authors also investigated drug-induced alterations in the selection of basic nutrients (carbohydrate or protein) and changes in the organization of feeding behaviour (latency to begin eating, duration of eating, number of mouthfuls, average size of mouthful, inter-mouthful interval, rate of chewing and rate of ingestion). Table I .5 shows that the higher dose of fenfluramine and amphetamine reduced overall calorie intake by more than 20%. However, amphetamine also gave rise to a significant reduction in the absolute and proportional protein intake whereas fenfluramine did not alter the proportion of protein taken over the whole

310

M . A . Cawthorne

meal. This selection bias was also reflected in a food preference questionaire, which revealed that amphetamine caused subjects to restrict the number of high protein foods that they felt inclined to eat. Analysis of the video-taped recording made whilst the subjects were eating showed that amphetamine produced a small but significant delay in the onset of eating. Since the meal-time was set by the experimenter and there was no opportunity for subjects to delay the beginning of the test-meal session, this observed delay in the initiation of eating within the meal may well be very significant. Amphetamine caused the subjects to load food more quickly into the mouth but the rate of chewing was unaffected. Fenfluramine slowed the rate of eating; that is, once food was placed in the mouth, the subjects chewed the food for a longer period. These results in human volunteers have considerable similarities with the results of animal experiments and thus it is likely that the control mechanisms surrounding feeding behaviour and nutritional choices have similar components. TABLE 1.4 Effects of Amphetamine and Fenflurandne on Total Energy, Protein Energy, and Protein Energy Percentage in Self-selecting, Freely-feeding Rats

TREATMENT

TOTAL ENERGY (Kcal)

PROTEIN E~ERGY (Kcal)

PROTEIN ENERGY %

Amphetamine (0 to lhr) Control

8.17±0.94

2.82±0.49

33.54±3.95

0.5 mg/kg

5.84±1.10

1.36±0.22"

28.54±4.92

1.0 mg/kg

1.83±0.46"**

0.49±0.16"*

22.62±6.06

2.0 irg/kg

2.30±0.39***

0.31±0.06"**

16.05±4.31"

Fenfluramine (0 to 8hr) Control

41.29±1.80

13.08±1.23

31.74±2.54

1.0 mg/kg

38.54±2.03

12.96±0.78

34.04±1.63

2.0 ng/kg

29.06±2.05**

8.70±0.61"

31.74±1.64

4.0 mg/kg

22.15±2.83"**

7.64±0.91"*

35.02±1.45

Mean (±S.D.) values for different doses given intraperitoneally *p< 0.05, **p< 0.01, ***p< 0.001, t-test Reproduced from Blundell e t a l, 1979, Current Med. Res. Opinion ~, suppl, i, 34-54, with permission.

Humoral Factors in the Control of Feeding In a preceeding section, arguments were presented that indicated that elevations in brain 5-hydroxytryptamine arising from increased transport of tryptophan across the blood brain barrier may be used as a satiety signal. Much other research has been carried out to seek mechanisms by which the gut 'talks' to the brain tO indicate that it is sated. The traditional view is that satiety is elicited by gastric distention operating through the vagal nerves (Janowitz, 1967). There is

Metabolic Aspects of Obesity

3t 1

TABLE I .5 Effect of Amphetamine and Fenfluramine on Total Food Intake, Protein Consumption and Protein Energy Percentage in Man

FOOD INTAKE TREATMEWr Kcal

g

Placebo

715± 82

237±27

Amphetamine (I0 ng)

565± 68*

184±10"

Fenfluramine: 30 mg

667±102

219±31

60 mg

526± 96*

173±21"*

PROTEIN INTAKE Kcal

PROTEIN ENERGY %

119±17

16.4±1.3

81±10"

14.8±1.1

106±18

16.4±1.0

92±24

17.0±1.7

Mean (± S.D.) values for 12 subjects. Note: values significantly different from placebo at *p< 0.05 and **p< 0.01 (t-test) Reproduced from Blundell et al, 1979, Current Med. Res. Opinion ~, Suppl i, 34-52, with permission.

considerable evidence that experimental gastric distention can decrease or inhibit feeding but gastric distention does not elicit the behaviour responses (grooming, sleeping) that are associated with satiety in animals including man. Recent studies have concerned the intestine as a site of production of satiety hormones Smith, Gibbs and Young (1974) , equipped rats with a chronic gastric cannula and duodenal catheter. When the cannula is open, ingested food drains out of the stomach and the sham-fed rat feeds continuously. Infusion of liquid food into the duodenum, during sham feeding, stops the sham-feeding process (Fig. 1.10). This probably was not due to aversion or sickness since infusion of saline into the duodenum had no effects. Smith and his colleagues tested a number of hormones produced by the intestine and found that cholecystokinin, which is known for its contractile activity on the gall bladder, inhibited sham feeding in rats and also gave the complete behavioural sequence of satiety (Antin, Gibbs, Holt, Young and Smith, 1975). The synthetic octapeptide of cholecystokinin and purified cholecystokinin have now been tested in a number of species including man (Smith and Gibbs, 1979; Kissileff, Pi-Sunyer, Thornton and Smith, 1981) and been shown to induce satiety of short duration. The doses used have usually been of the order of I to I0 ~g/kg. Unfortunately assays for circulating cholecystokinin are neither reliable nor sufficiently sensitive to determine changes in endogenous release of hormone during a meal. It is therefore not possible to state whether endogenous cholecystokinin is a satiety hormone. However, studies in rats (Anika, Houpt and Houpt, 1979) and monkeys (Gibbs, Faksco and McHugh, 1976) on the effect of substances that are known to he potent releasers of endogenous cholecystokinin such as l-phenylalanine and egg yolk have suggested that cholecystokinin may be a natural satiety hormone. As present there is nO evidence that cholecystokinin can cross the blood brain barrier and thus the mechanism by which the signal is transferred to the brain re~ains a matter of speculation. However, recent studies have shown that infusion of cholecystokinin into the peritoneal cavity alters the pattern of synaptic release of hypothalamic noradrenaline (Myers and McCaleb, 1980; McCaleb and Myers, 1980).

312

M.A. Cawthorne

i

Fig. 1.10

A rat equipped with a chronic gastric cannula and duodenal catheter. When the cannula is open, ingested food drains out of the stomach and the rat sham feeds continuously. During sham feeding, the intestine can be perfused with liquid food through the duodenal catheter and then sham feeding stops. Reproduced from Smith, G.P. and Gibbs, J. Cholecystokinin and Satiety: theoretical and therapeutic implications in Hunger: Basic mechanisms and clinical implications. Raven Press, New York, 1976, with permission.

Another candidate humoral transmitter from the gut to the brain is bombesin, which has recently been shown to reduce meal size (Gibbe, Fauser, Rowe, Rolls, ROlls and Madditon, 1979). This substance was originally identified in frog skin but bombesin-like peptides have now been found in ~ l i a n gut and CNS (Moody and Pert, 1979; Walsh and Holmquist, 1976). Synthetic bombesin was only able to suppress food intake by a maximum of 50%, which is similar to the response produced by cholecystokinin. However, the two substances produce an additive effect on suppression of food intake (Woods, West, Stein, McKay, Lotter, Porte, Kenney and Porte, 1981), suggesting they act by different mechanisms. In another experiment designed to assess the role of intestinal factors in satiety, Koopmans, Selafini, Fichtner and Aravich (1981) surgically transplanted a 10 cm section of lower ileum to the duodenum in rats made obese by hypothalamic lesions and in normal weight controls. Following ileal transposition, the hypothalamic obese rats underate and lost weight to the level of the normal-weight control rats

Metabolic Aspects of Obesity

313

not subject to ileal transposition. Ileal transposition in normal weight rats had no significant effect on body weight. The authors suggest that the lower ileum normally produces a neural or hormonal signal that inhibits feeding. This signal may well be impaired in hypothalamic obese rats. Transposition of the ileum, so that it is in continuity with the duodenum, exposes it to nutrient-rich chyme which initiates the signal to stop feeding. The lower ileum has been shown by Bloom and Polak (1978) to contain large amounts of enteroglucagon and neurotensin. Other studies have shown that food ingestion produces an increase in the level of circulating neurotensin-like activity in dogs and man (Mashford, Nilsson, Rokaeus and Rosell, 1978). Interestingly, in view of the study by KoOpmans in rats, the increase in neurotensin-like inmunoreactivity following fat administration is smaller in very obese subjects than in lean subjects and occurs later (Wiklund, Rokaeus, Hallberg and Rosell, 1980) . If some peptides serve to reduce meal size, it is possible that other peptides might increase meal size and thereby promote obesity. A number of workers have shown that the opiate antagonist naloxone will reduce meal size (Holtzman, 1979; Brands, Thornhill, Hirst and Gowdey, 1979; King, Castellanos, Kastin, Berzas, Mank, Olson and Olson, 1979). However, interest in this subject was really sparked when Margules, Moisset, Lewis, Shihuya and Pert (1978) reported that naloxone reduced food intake more effectively in genetically obese mice and rats than in lean littermates. These authors also reported that the obese rodents had increased levels of B-endorphin within the pituitary. These findings have led Margules (1979) to develop a generalized theory based on excessive production of endorphins as being a cause of some forms of obesity. So far no firm evidence has been provided for such a theory but if there were excessive production of endorphins in the obese, it might be expected that obese subjects would have a greater tolerance to pain. Pradalier, Willer, Boureau and Dry (1980) have indeed shown that obese female subjects have a reduced nociceptive reflex, which correlated with pain threshold.

The Role of Nutrients in the Control of Food Intake As stated previously, glucose has an indirect effect on brain 5-hydroxytryptamine levels through its insulin secretagogue activity. The increased levels of insulin act to increase transport of the 5-hydroxytryptamine precursor, tryptophan, into the brain. This effect is accomplisht~d by the inhibitory action of insulin on lipolysis, which decreases the concentration in the blood of non-esterified fatty acids and so allows more tryptophan to bind to albumin. Secondly it promotes the uptake of the neutral amino acids, which compete with tryptophan for the brain translocation site, into muscle. The importance of insulin in the determination of post-prandial brain serotonin concentration can be judged from studies in diabetic rats (Fernstrom, 1981) (Table 1.6). In diabetic rats, serum and brain tryptophan concentrations are lower than in controls and unlike normal rats are not significantly altered by glucose administration. The concentration of 5-hydroxytryptamine in the brain was also not significantly raised by glucose. Furthermore, the concentration of 5-hydroxyindoleacetic acid, which gives a measure of the flux through the serotoninergic system was significantly raised by glucose treatment to nor~nal rats but not to diabetic rats. The central role of insulin was strengthened by the finding that insulin administration to diabetic rats ingesting glucose resulted in an increase in the serum tryptophan-neutral amino acid ratio, and increases in brain tryptophan and 5-hydroxyindoles. The effect of glucose (or carbohydrate) administration on changes in tryptophan metabolism in obese animals has not been studied. HOwever, most obese subjects have some element of resistance to the action of insulin. It therefore seems possible that carbohydrate-induced changes in tryptophan metabolism could well be

314

M.A. Cawthorne

at least attenuated in obese subjects. Since, it has been suggested earlier that increases in brain 5-hydroxytryptamine may he a signal for satiety for carbohydrate, such attenuation could account for the carbohydrate craving by some obese subjects. TABLE 1.6 Effects of Glucose Intubation of Fastin@Normal and Diabetic Rats on Brain Indole Levels

INDOLE SERUM TRYPTOPHAN RATIO Normal rats Diabetic rats

FASTING

GLUCOSE

0.16±0.01 0.09±0.01t

0.30±0.02* 0.13±0.04t

26.50±2.00 13.70±1.50t

36.30±0.50* 15.70±2.00%

BRAIN SEROTONIN (nmol/g) Normal rats Diabetic rats

4.11±0.07 4.16±0.18

4.48±0.05* 4.30±0.70

BRAIN 5-HIAA (nmol/g) Normal rats Diabetic rats

3.47±0.14 2.95±0.13t

4.38±0.18" 3.25±0.15t

BRAIN TRYPIOPHAN (nmol/g) Normal rats Diabetic rats

Normal and diabetic rats were fasted overnight, and the following morning were intubated with a 2M glucose solution (iml/100g). They were killed 2h later. Control animals continued to fast during the 2h period. Data are presented as the means ± standard errors. *p< 0.05 that value differs significantly from corresponding fasting value; tp< 0.05 that value differs significantly from corresponding value in normal rats. Reproduced from J.D. Fernstrom, Diabetalogia 20, 281-289 (1981), with permission.

Glucose has an important role in the regulation of food intake. Earlier in this chapter it was noted that there may be 'glucoreceptors' in the ventrc~nedial hypothalamus. These cells respond to systemic injections of 2-deoxyglucose, (an analogue of glucose that can enter the cell and be phosphorylated, but cannot be metabolised further and hence blocks glucose utilization) by decreasing the firing rate of ventromedial hypothalamic neurons (Desiralu, Bannerjee and Anand, 1968). On the other hand systemic application of glucose or insulin increases the firing rate (Anand, China, Sharma, Dua and Singh, 1964; BrOwn and Melzack, 1969). Local application of glucose to the ventromedial hypothalamus using an electro-osmotic technique increased the firing rate of neurons in this region suggesting that this area of the hypothalamus is sensitive to glucose. The onset of feeding produced by syst6~nic injections of 2-deoxyglucose has a lag period. Novin, Vander-Weele and Rezek (1973) found that injection of 2-deoxyglucose into the hepatic portal vein of rabbits produced the onset of feeding more rapidly than if the 2-deoxyglucose was injected into a peripheral vein. The effect

Metabolic Aspectsof Obesity

315

of portally administered 2-deoxyglucose was delayed if the vagus nerve was cut. These findings suggested that the liver may have a sensing system for glucose and relays messages to the brain relating to acute changes in nutritional status via the vagus nerve. This possibility is supported by studies by Niijima (1969) who found that the firing rate of nerves from the liver was reduced as the concentration of glucose perfusing the liver was increased. TABLE 1.7 Effect of the Acute Administration of (-)Hydroxycitrate on Appetite and in vivo Lipogenesis a

FOOD INTAKE b

FATTY ACID SYNTHESIS c

DOse

Treatment

Saline

(nmoles/ kg body weight) -

G

12.1±0.9

~moles 3Hconverted G/30min d

nmoles 14Cconverted G/30 m i n d

42.3±6.7

631± 52

(-)-Hydroxycitrate

5.26

8.6i0.8 e

12.0±I.0 f

104i

(-)-Hydroxycitrate

2.63

9.6±0.5 e

14.8±1.2 f

145± 16 f

9f

(-)-Hydroxycitrate

1.32

9.9±0.9

17.5±5.0 e

226± 91 f

(-)-Hydroxycitrate

0.66

9.8±0.8

27.2±4.5

415±105

Rats were fasted 48 hr, then meal-fed a 3-hr meal for 5 to 8 days. On the last day, (-)-hydroxycitrate was administered by stomach gavage immediately before feeding, and livers were assayed immediately after the 3-hr feeding period. Ten rats per group Five rats per group Data are expressed as moles 3H20 converted into fatty acids/g liver/30 min. and nmoles ~4C-alanine converted into fatty acids/g liver/30 min. e

f

p< 0.05 p < 0.01

Reproduced from A.C. Sullivan and J. Triscari, Metabolic flux and appetite, in Hunger: Basic mechanisms and clinical implications, pi15-125, Raven Press, New York (1976), with permission.

What is the nature of the sensing mechanism in the liver? Current evidence suggests that one possibility is liver glycogen stores. Teleologically this would make sense since this store is the primary energy store. If this store is sensed in some way then one would expect glucose and insulin to have effects on feeding behaviour. Further indirect evidence for liver glycogen content being a metabolic sensor for nutritional status comes from studies by Sullivan and her colleagues on the experimental drug (-)-hydroxycitrate (Sullivan and Triscari, 1976). This compound is a potent competitive inhibitor of ATP citrate lyase, the enzyme catalyzing the extra-mitochondrial cleavage of citrate to acetyl coenzyme A (Watson, Fang and Lowenstein, 1969). In conditions of high carbohydrate feeding, most of the acetyl

316

M . A . Cawthorne 14-

["] Control [] (--)-Hydroxycitrate

12108c-

6O 0

.,,I

4-M 2-

0

0

2

4

6

8

10 12

15

18

21

24

Hours after feeding

Fig. i. Ii

Effect of the oral administration of (-)-hydroxycitrate on hepatic glycogen content determined over a 24 hour period. Hepatic glycogen was isolated at the indicated times (i0 rats per group). The amount of glycogen in (-)-hydroxycitratetreated rats from 6-10 hours was significantly greater than controls (p< 0.05). Reproduced from A.C. Sullivan and J. Triscari: Metabolic flux and appetite, in Hunger: Basic mechanisms and clinical implications, pi15-125, Raven Press, New York (1976), with permission.

COA for lipid biosynthesis arises by this mechanism. When administered orally inmediately before feeding, (-)-hydroxycitrate produced a dose-related reduction in both food intake and rates of hepatic liIx)genesis (Table I .7). Further studies, in which any effect on metabolite flux due to differences in caloric intake were normalized by ensuring that both treated and control rats ate an identical amount of food, examined the effect of hydroxycitrate on carbon flux from dietary carbohydrate. In parallel with an inhibition of hepatic lipcgenesis, (-)-hydroxycitrate produced an increase in the apparent in vivo rate of glycogenesis resulting in a statistically significant increase in the hepatic glycogen content (Fig. I. 11). This change in the rate of glycogen synthesis was obtained without any significant change in the concentrations of plasma glucose, insulin or free fatty acids. These data by themselves do not provide evidence as to whether the connection between the increase in glycogenesis and reduction in food intake is causal or merely correlative. However, when added to the previous data they provide support for the idea of a hepatic glucoreceptor monitoring system that feeds information to central regions via the vagus nerve. If the glycogen store is being 'sensed' as a means of providing imput to central areas regarding nutritional requirements, this is one possible defective mechanism in the obese. Wahren and his colleagues (Felig, Wahren and Handler, 1978) using arterio-venous difference methods have shown that in man, 50% of an oral 100g glucose load is taken up by the splanchnic area and presumably much of this is stored as liver glycogen. However, similar studies in

Metabolic Aspectsof Obesity

317

obese maturity on-set diabetics showed that the majority of an oral glucose load escaped the splanchnic bed and appeared in the peripheral circulation (and thereby contributed to the abnormal glucose tolerance). With currently available methodology, it is not possible to measure glycogen synthesis in man. However, in unpublished studies, Simson, Smith and Cawthorne have shown that the genetically obese (ob/ob) mice have a reduced rate of hepatic glycogen synthesis compared with their lean littermates following a glucose load.

Sumaary The control of food intake is probably regulated centrally and this could well involve a monoaminergic syst~n in the ventro-medial and lateral areas of the hypothalamus. Into this system are integrated metabolic, neural and hormonal signals from peripheral monitoring stations including the liver and the gut. Although it has not been discussed in this article, it is clear that in man, at least, there is an additional flow of information regarding the appearance, taste and texture of food. Indeed, Schacter, Goldman and Gordon (1968) have suggested that the eating behaviour of obese subjects was to a large part under external control; that is eating behaviour was initiated and terminated by perceptual factors such as smell, sight, taste and what other people are doing. Schacter considers these things to affect eating behaviour in the obese to a much greater extent than in lean.

Chapter 2

Control of Energy Expenditure

Physicians are accustomed to genetic and enviromental-induced variation in parameters such as height, weight and blood pressure. HOwever, except in those cases of Obesity in which there is an established endocrinological basis such as Cushing's syndrome or hypothyroidism, many are reluctant to accept that there may be variations in metabolic rate and tend to assume that obesity is solely due to either excess food intake or lack of physical activity. In contrast, many patients insist that their obesity is due to a metabolic problem since in spite of limiting their food intake they remain fat. They note that they eat much less than their friend who is 'as thin as a rake', and generally consider that life is very unfair. The physician r ~ r s the first law of thermodynamics and because he does not consider the possibility of a below average metabolic rate, makes the deduction that his patient is cheating with respect to food intake. He may well remind his patient that 'no fat person came out of Belsen' and send him/her away with instructions to keep to the diet and the excess weight will disappear. In this chapter, the evidence for variation in metabolic rate will be reviewed, as will current views on the regulation of the individual energy components that constitute the metabolic rate. In later chapters, mechanisms for disposing of excess energy intake will be discussed.

Energy Cost of Weight Maintenance Garrow (1978) notes that it is far from clear why some individuals can remain lean and in energy balance for many years with no difficulty, while others do so only by conscious effort and still others fail to do so and become obese. Formal studies show that in apparently similar individuals, body weight (and hence constancy of energy balance) can be maintained on widely different food intakes. Widdowson (1962) showed that for any 20 people of the sa~ae age, sex and occupation, one could be found who was eating twice as much food as another. Rose and Williams (1961) carried out measurements on large and small eaters, whose food intakes varied from 1600 to 7400 kcals/day, but whose body weight varied little over a period of scme weeks. Within this group were pairs of individuals of the same weight and similar levels of activity, one of whom was eating twice as much as the other. Other workers have compared the food intakes of lean and obese subjects and in general have come to the conclusion that obese subjects do not eat more than their lean counter-parts. Thus, Maxfield and Konishi (1966) , using 25 age-matched pairs

318

Metabolic Aspects of Obesity

319

of female subjects found a food intake of 1871 ± 873 kcals/day in the obese women and 1986 ± 704 kcals/day in the lean women. McCarthy (1966) found a food intake of 1884 ± 534 kcals/day in 63 obese women and 1978 ± 479 kcals/day in 26 normal weight women. Since these obese subjects were at a stable weight, we must conclude that in spite of their greater size, they had similar energy expenditure to the lean individuals. Thus on a surface area basis, they had a lower energy expenditure. However, in these studies, it is possible that the reduced metabolic rate is a consequence of the obesity rather than a cause. The most compelling evidence that a low metabolic rate might be a cause of obesity comes from work on children from obese parents (Griffiths and Payne, 1976). It is known that obesity is a familial condition with children of obese parents having a greater risk than children with lean parents. In part, this is due to genetic influences since Shields (1962) showed that monozygotic twins raised apart had a closer adult body weight than dizygotic twins raised together. Griffiths and Payne (1976) found that normal weight children of obese parents had a lower energy intake than those from lean families. Thus, it was assumed that these children were only able to maintain their normal weight by eating less than normal. TABLE 2.1

Mean Daily Energy Intake and Expenditure

DIFFERENCES BEqZ~EN GROUPS% GROUP N s.d. GROUP 0 s.d. DIFFERENCE BODY SIZE Height (cm) Weight (kg) Body fat (%) LBM (kg) Standard weight for height (%)

111.3 19.08 14.3 16.2 99.9

7.4 110.7 3.53 19.49 5.2 16.8 2.24 17.0 9.5 100.8

7.5 3.46 3.7 1.71 6.0

ENERGY MEASUREMENTS (kcalorie) Daily intake Intake per kg hody weight Daily expenditure Expenditure per kg body weight Daily expenditure at rest Expenditure at rest per kg body weight Daily expenditure available for activity

1,433 75 1,508 79 1,183 62 371

188 13 352 18 184 9 306

300 8 297 9 146 6 173

GROUPS COMBINED Daily intake Daily expenditure Daily expenditure at resting rate Daily expenditure available for activity

1,325 1,375 1,110 265

263 364 190 266

1,115 57 1,174 60 999 52 190

* * * * *

p<0.01** p<0.01** p<0.05** p<0.02** ~0.05"* p<0.02** p<0.2*

The children were divided into two groups, those with one parent whose adult weight had at some time been more than 20% above desirable weight for height (group 0), and the remainder, neither of whose parents had ever been overweight (group N). * Not significant ** Significant Number in group N=I2; number in group 0=8 (except for intake study when it was 5) Reproduced from Griffiths and Payne, Nature 260, 698-700, copyright 1976 MacMillan Journals Limited, with permission.

320

M . A . Cawthorne

,-, 3.025-

_= 2.0.=_ "

1.5-

¢8

1.0"~

E O

0.5-

1

I

5000

I

I

10000

15000

20000' 25000

Excess c a l o r i e s ( k c a l ]

Fig. 2.1

Gain in weight of two obese women and two thin men plotted against the excess calorie intake during a period of overfeeding. O, O, Women; A , A, men. (Taken from Passmore, R., Strong, J.A., Swindells, Y.E., and el Din, N., Br.J.Nutr. i=~7, 373-383, 1963, with permission.)

Experimental Overfeeding in Man In the weight Gulick intake weight intake

classic luxuskonsumption study, Neumann (1902) found that he could maintain for long periods on energy intakes ranging from 1766 to 2403 kcals/day. (1922) carried out similar experiments on himself. He increased his energy from 1872 to 4113 kcals/day but after a small initial weight gain, his body remained constant. The explanation for these results was that as energy increased, the efficiency of energy utilisation must have decreased.

Additional studies have not always replicated these findings. Thus Passmore, Meiklejohn, Dewar and Thow (1955) found that in a short-term (10-14 days) study, in 3 overfed young men, energy expenditure increased by only 6 to 16% of the excess food intake. In another study, Passmore, Strong, Swindells and Din (1963) found that two obese women overfed for nine days gained w~ight much more quickly than two young men. However, even obese subjects appear to attain a stable upper weight, (Fig. 2. I). A number of studies have shown a remarkable stability of the upper limit of body weight even in obese subjects and there is the classic case of the French gastronome who in spite of massive Overeating could not bridge the gap from 98 to 100kg (Nadal, Nel and Ravina, 1954). The major study on overfeeding was that carried out by Sims, Danforth, Horton, Bray, Glennon and Salans (1973) in lean prisoner volunteers. They persuaded their subjects to consume 7,000-10,000 kcals/day for periods of 200 days or more - an energy excess over their normal requirements of the order of 1 million kcals. There was great individual variation in the ability of lean volunteers to gain weight by overeating. The subjects were expected to increase their body weight by 20-25%. Some reached this goal easily whilst others failed even though they were eating more than those who were successful in reaching this goal.

Metabolic Aspects of Obesity

321

All of these studies suggest there are large variations in people's ability to respond in terms of weight gain to excessive food intake. Consequently, since reduced digestability and absorption of food can be ruled out, it must be presumedthat there is a variable increase in energy expenditure in over-fed subjects. Danforth (1981) has speculated that if lean subjects react to overfeeding by using calories inefficiently, spontaneously obese subjects might lack such a protective mechanism against obesity. Clearly, it would be unethical to overfeed children of obese parents to test this suggestion and we must rely on experiments in animals to test this possibility (see later).

Ex~imental

Underfeeding in Man

The classic studies of starvation conducted by Benedict, Miles, Roth and Smith (1919) and Keys, Brozek, Hanschel, Mickelson and Taylor (1950) both suggest that an adaptive mechanism during starvation reduces the metabolic rate. Keys et al (1950) estimated that approximately 65% of the initial decline in metabolic rate resulted from a loss of the respiring mass, whilst 35% resulted from a decline in the rate of metabolism of the remaining respiring mass. Both of these studies indicated that the adaptive mechanisms led to a sparing effect on the lean body mass. Cahill, Herrera, Morgan, Soeldner, Steinke, Levy, Reichard and Kipnis (1966) extended these studies. They showed that during starvation there was a reducing dependance on glucose derived from gluconeogenesis for energy, thus sparing the breakdown of body protein. Of critical interest to the obese patient is whether they also show a decline in metabolic rate upon reduction of food intake. Even more so, do obese subjects show an exaggerated response? Bray (1969) measured metabolic rate in six obese subjects undergoing dietary restriction. There was a progressive fall in metabolic rate which was much greater than that expected as a result of the decline in body weight (Fig. 2.2). Likewise, Garrow and Warwick (1978) showed a similar response in the majority of 27 obese women following 3 weeks of dietary restriction (Fig. 2.3). Thus, it appears that Obese subjects behave in a similar fashion to lean subjects by reducing their metabolic rate in response to dietary restriction. For many obese subjects this will mean almost inevitably that unless dietary restriction is severe, they will re-achieve energy balance before they have achieved their ideal body weight. Miller and Parsonage (1975) described such an occurrence. They incarcerated obese individuals, who claimed they could not lose weight on recommended diets, in an isolated country house and fed them 1500 kcals/day. The subjects' luggage was searched and their car keys r~aoved so that all subjects were denied access to any food other than that provided. Although 19 of the subjects did lose weight, 9 maintained body weight (± Ikg) and 2 actually gained weight. The subjects showed a good correlation between basal metabolic rate and changes in body weight. These data provide support for the view that within the human population there are individuals maintaining weight on very low energy intakes and they do this by a severe reduction in energy expenditure.

Hypothalamic and Genetic Obesity in Experimental Animals Perhaps the mostpowerful evidence that reduced energy expenditure might be a major factor in the development of obesity comes from the vast volume of work that has been carried out in genetically obese rodents especially those types that are recessively inherited - such as the obese (ob/ob) and diabetic (db/db) mice and fatty (fa/fa) rats. The obese mouse arose spontaneously in 1949 in a strain of mice at the Jackson Laboratories, Maine, U.S.A. Breeding experiments demonstrated that a single autosomal recessive gene was responsible (Ingalls, Dickie and Snell, 1950) and this

322

M . A . Cawthorne

Body weight kg

170-]]_

1oo 140J

co

~

I

_L

20

24

28

i

on 20 L/hr

234

22 _ 4000~ 2000~ 21

Caloric intake kcal/d

4

8

12

16

32

days

Fig. 2.2

Effect of caloric intake on body weight and energy expenditure of six obese patients (Taken from Bray, G.A., Lancet, ii, 397, 1969. With permission.)

was located on chromosome 6 (Coleman and Hunmel, 1971). Obesity in these mice is apparent by visual inspection at about 4 weeks of age and develops progressively up to 6 months. At this time the obese mice are approximately 2.5 times heavier than their lean littermates and 90% of the excess weight is fat (Bates, Nauss, Hegman and Mayer, 1955). The fact that the obesity is not due solely to hyperphagia in these mice is readily demonstrated by long-term food restriction experiments. Even if the food restriction was such that the body weight was kept at or below that of lean controls, mice carrying the ob/ob gene had carcasses with an abnormally high fat content (Alonzo and Maren, 1955; Chloverakis, 1972; Dubuc, 1976). Furthermore, although food intake measurements in ob/ob mice have generally shown that there is excessive intake relative to that in lean animals, it has now been demonstrated that hyperphagia does not develop to any significant extent until after the first month of life. As stated earlier, obesity is visually apparent at this age and carcass analysis of litters from ob/+ v o b / + matings show the existence of obese mice as early as 10 days iThurlby and Trayhurn, 1978). Rath and Thenen (1979) were able to show that ob/ob mice consume no more milk than their lean littermates during the preweaning period and Lin, Rc~sos and Leveille (1977) found no evidence for hyperphagia postweaning until the mice were 28 days of age. Thus, in order to explain the visually apparent obesity of these young mice it is necessary to look for factors other then excessive food intake. A sub-normal metabolic rate in ob/ob mice was reported shortly after their original identification (Mayer, Russell, Bates and Dickie, 1952). Since that time, various authors have measured oxygen consumption in very young mice. Thus, Kaplan and Leveille (1974) found difference in metabolic rate in 18 day old mice such that they could detect preobese ob/ob mice with 90% reliability. Van der Kroon, Van Vroonhover and Douglas,

MetabolicAspectsof Obesity

323

~, 360-~ "~ 340-" 320,~ 300 ] 280260240i 220] == 200t~

18oi

I

I

I

I

I

I

I

i

50 60 70 80 90 100 110120 130 140 Weight (kg) Fig. 2.3

Resting metabolic rate and body weight in 27 obese women before ( O ) and after ( • ) 3 weeks on a diet supplying 3.4 MJ daily. (Taken from Garrow, J.S. and Warwick, P.M., The Diet of Man: Needs and Wants, Yudkin, J., Ed., Applied Science Publishers, Darking, Essex, 1978, 127. With permission.)

(1977) and Biossonneault, Horshuh, Simons, Romsos and Leveille, (1978) found a reduced metabolic rate in those mice destined to become obese at 8 and 6 days of age respectively. Further suggestion for the existence of a lower metabolic rate in ob/ob mice comes from measur6~nent of their sensitivity to the cold. Within a few hours of exposure at 3°C, ob/ob mice die of hypothermia whereas lean mice are able to survive a 7 day exposure to this temperature (Mayer and Barrett, 1953). It was suggested that the inability of the ob/ob mouse to survive cold exposure was due to a failure of chemical heat production, rather than an inability to conserve heat (piloerection) or produce heat by shivering (Davis and Mayer, 1954). Recent work has shown that abnormal thermoregulation to relative cold can be detected as early as 12 days of age. The diabetic (db/db) mouse also arose spontaneously in the Jackson Laboratories, Maine, U.S.A., and is characterized by a severe diabetes in conjunction with obesity (Hurmael, Dickie and Coleman, 1966). The condition is inherited as a single autosomal recessive gene on chromosome 4 and the exact nature of the diabetic condition (but not the obesity) seems to be dependent on the interaction of this gene with the overall genetic background of the mice. Because the db gene is closely linked on chromosome 4 with the coat colour gene misty (m), it is possible by crossbreeding and maintaining the m and db coupling to identify future obese mice as soon as coat-colouring becomes apparent (4-5 days of age). At 10-12 days of age, mdb mice have extra-fat deposits although they are not hyperphagic during the preweaning period (Jeanrenaud, 1978; Le Marchand-Boustel and Jeanrenaud, 1978).

324

M.A. Cawthorne

Furthermore, in a 6 week study in which the food intake of db/db was precisely matched in quantity and frequency of feeding to that of lean mice, the db/db mice gained 42% more weight and accumulated 5 times as much lipid as their lean counterpart (Cox and Powley, 1977). In this study, the lean mice lever-pressed to receive food and this also triggered the delivery of a food pellet to its paired db/db mouse in an adjacent cage. These studies clearly indicate that hyperphagia in db/db is not the primary cause of obesity and indicate that the fundamental cause is an increased efficiency of accretion of stored lipid. This increased metabolic efficiency, when coupled with hyperphagia in the post-weaning period results in massive obesity. As in the ob/ob mouse, it has been found that db/db mice maintain a low~_ body temperature than normal mice at all environmental temperatures below 34 C (Trayhurn, 1979a) and develop hypothermia during short term exposure to an environmental temperature of 4°C (Yen, Fuller and Pearson, 1974) which is rapidly fatal (Trayhurn, 1979a). In genetically ob/ob and db/db mice it appears that a 'thrifty' gene that allows enhanced metabolic efficiency of fuel utilisation has been allowed to develop. This appears to be associated with a lower than normal body temperature and a reduced ability to cope with a cold environment. Such a type of metabolic adaptation is also seen in the wild. Wise (1977) described anomalous thermoregulation in the spiny mouse, which is adapted to living under conditions of severely restricted food supplies. When brought into the laboratory or a zoo and provided with unlimited amounts of food, the mice develop obesity and diabetes. One of the features of this animal is that, at normal ambient temperatures, it's core temperature is maintained at a reduced level and it exhibits profound hypothermia when exposed to cold environments. Other examples of hyper-efficient ~ i s , that have adapted to spartan conditions and become obese when adequately fed, are the Nile rat and the tree shrew. The development of obesity in rats following electrolytic destruction of the ventromedial hypothalamus was noted in the chapter on food intake. Whilst food intake is very often increased in these rats, it is now known that hyperphagia is not essential for the development of obesity. The first suggestion that this was the case came from experiments by Kennedy (1957). He found that destruction of the ventromedial hypothalamus in weanling female rats led to increased adiposity hut neither an increase in body weight or hyperphagia. Subsequent studies demonstrated that the degree to which hyperphagia developed was both age and sex related (Bernardis and Skelton, 1965, 1967). It was suggested that the weanling rat is already eating at a maximal rate due to its rapid growth and it cannot further increase its food intake. However, the finding that food-restricted ventromedial hypothalamic-lesioned rats still became obese (Han, 1968) suggested that there is nothing unique about the obesity that develops in weanling rats. A feature of the ventromedial hypothalamic lesioned rat is that when body weight reaches a stable plateau, it can he maintained on a near-normal food intake inspite of the increased size of the ventromedial hypothalamic-lesioned rat. Han (1968) showed that tube-fed VMH-lesioned rats exhibited mild hypothermia, decreased activity and a reduction in the resting rate of oxygen consumption. Thus, the ventrc~edial hypothalamic lesioned rats show features in common with the genetic model of obesity which further suggests connections between body temperature regulation and obesity. M,,cdulations in Energy Expenditure induced b~z Diet in Experimental Animals Laboratory rodents tend to eat many small meals during the day, although about two thirds of the daily food intake is taken during the dark. Body temperature of

Metabolic Aspectsof Obesity

325

mice also shows a diurnal rhythm which is in part dependent on feeding since it is largely abolished in fasted mice (Cawthorne and Arch, in prep. ). If rats or mice are forced to eat all their daily food in a short-period (2-4h), they gain considerably more body fat than if they are allowed food ad-lib (Fabry, 1969). This is in spite of the fact that total food intake in these 'meal-fed' rodents is often lower than in the animals fed ad-lib. This suggests that the meal-fed rodents have a reduced metabolic rate relative to fed rodents (Cawthorne and Arch, in prep. ). Meal-fed mice have a very different daily profile in body temperature from ad-lib fed mice, showing a marked rise in body temperature during and following the feeding period. Earlier in this chapter, the effect of experimental overfeeding on body weight gain in man was discussed. It was shown that body weight gain in these subjects did correlate well with food eaten. Animal experiments, in theory, allow the possibility of quantifying the efficiency of weight gain. Until relatively recently, the dominant view was the animals maintained energy balance by matching food intake to energy output. However, there is now considerable evidence that the quantity and type of food eaten has a major effect on the rate of energy expenditure. Perhaps the most dramatic demonstrations of this effect was in a study by Miller and Payne (1962) to compare the energy requirement for weight maintenance in weanling pigs. Weight maintenance was achieved in one pig by protein restriction and in another by energy restriction. Over a 40 day period, the protein-limited pig consumed 5 times more energy than its pair. The authors suggested that weight maintenance in the protein-restricted pig was achieved by a large increase in metabolic rate. Some workers found the magnitude of the proposed therrm~enic response difficult to accept and criticised the experiment because measurements of body composition and metabolic rate were not carried out (Blaxter, 1975). However, a recent study (Gurr, Mawson, Rothwell and Stock, 1980), which included full energy balance measurements, has confirmed that young pigs have a large capacity for thermogenesis when fed on a low protein-diet. Similar findings have been obtained in rats (McCracken and Gray, 1976) and man (Miller and Mumford, 1967). The usual diet of laboratory rodents is a pelleted grain-based diet. On this type of diet adult rats have a fat content of 10-20%, are not normally considered obese and are usually assumed to regulate energy balance by regulating food intake. However, in comparison with wild rats (Miller, 1979) they are obese. Obesity can be further induced in laboratory animals by manipulation of the diet. Thus, rats fed on a diet containing 60% by weight of fat will become obese. Rats prefer greasy diets to the pelleted grain-based diet and increase their caloric intake (Hamilton, 1964). However, the efficiency in converting dietary energy into body energy gain is also increased (Schenmel, Mickelson and Motawi, 1972) and, in some experiments animals have become obese on high fat diets even when caloric intake was not increased (Herberg, Dopper, Major and Gries, 1974; Lemonnier, 1972). Diets high in sucrose or fructose have also been found to produce obesity in rats without producing an increase in caloric intake (Allen and Leahy, 1966). In this study, rats fed on a high glucose diet ate more than the rats fed on a high sucrose diet but also gained less weight and less body fat. This differential effect of sucrose and glucose was also found by Kandarek and Hirsch (1977) in experiments in which rats were allowed access to a 32% sucrose or glucose solution in addition to their normal food and water. Sclafani and Springer (1976) produced obesity in adult rats by offering a variety of palatable snack foods in addition to their normal diet. These foods included chocholate, chips, biscuits, salami, cheese, bananas, marshmallows, candy, peanut batter and condensed milk. This type of diet has become to be known as a 'cafeteria' diet or more disrespectfully as 'junk-food' diet. In their original experiment, rats offered the cafeteria diet gained 124g in a two month period compared with 46g for control rats given the normal laboratory diet only. The excess weight produced by the cafeteria feeding did not appear to be a permanent

326

M.A. Cawthorne

effect since withdrawal of the cafeteria foods led to a return to the same weight as the control rats (Sclafani and Springer, 1976; Sclafani and Gorman, 1977). Rothwell and Stock (1979) carried out the somewhat daunting task of measuring energy balance in cafeteria-fed rats. The cafeteria-fed rats, which were adults (greater than 400g) overate by 75% bat had the same efficiency of weight-gain as the control rats. However, in similar experiments using 75 day old rats (Rothwell and Stock, 1980), the same authors showed that cafeteria rats were more efficient in converting dietary energy into body energy. Paradoxically, these rats also increased their energy expenditure so that the actual increase in body weight was considerably less than that which would be expected from measurement of their food intake. It has been well established that there are large strain differences in the ability of rats to become obese on various diets (Schenmel and Mickelsen, 1974: Miller, 1979). Rothwell and Stock, (1980) showed that such differences also existed between two different colonies of the same strain of rat, (Table 2.2). Thus in the context of energy expenditure, Sprague Dawley rats from Charles River were able to dissipate more of the excess dietary intake than rats from Tuck. These studies have parallels with the human overfeeding experiments of Sims and his colleagues cited earlier and provide evidence of a natural variation in the ability of animals and man to undertake dietary-induced thermogenesis in response to hyperphagia.

TABLE 2.2

Intra-strain Differences in Feed Efficiency

CONTROL DIET

CAFETERIA DIET

C.R. rats

Tuck rats

C.R. rats

Tuck rats

1830 ± 60%

1850 ± 20

3430 ± 40t

3500 ± 40

Body energy gain (kJ)

80 ± 30%

70 ± 10

Energy Expenditure (kJ)

1750 ± 80t

1780 ± 20

Metabolisable energy intake (kJ)

320 ± 40**

760 ± 80

3110 ± 50***

2740 ± 80

Rats from both colonies were Sprague-Dawley males of the same age (75 d) at the start of the experiment, which lasted for 8 days. (Mean values ~ s.e.m.;n=8) t n.s.,

**p< 0.01,

***p< 0.001

Taken from Rothwell, N.J. and Stock, M.J. in Recent Advances in Obesity Research, vol. 3, P.214-219, John Libbey, London, with permission.

C~nents

of the Metabolic Rate

Animal and human studies have demonstrated that there is a natural variation in metabolic rate. Metabolic rate can be divided into a number of quantifiable components which include basal metabolic rate and energy associated with heat production.

Metabolic Aspects of Obesity

327

Basal metabolic rate. This is defined as the energy expenditure under conditions of relaxation after a 12 hour fast and at an environmental temperature at which the body does not need to expend any energy to keep warm. This thermoneutral temperature is about 28°C for man, but is somewhat higher for small rodents such as rats and mice. The basal metabolic rate is composed of the energy costs of synthetic activity such as protein synthesis, maintenance of cellular integrity by maintaining ion pumping mechanisms, and mechanical work such as heart and lung function to maintain fuel supplies. Thus, basal metabolic rate is the energy cost of keeping the body alive and in man accounts for 50-60 per cent of daily energy expenditure. Basal metabolic rate is related to the mass of fat-free tissue since the fat itself is relatively metabolically inactive. However, because methods of measuring lean body mass are not always available it is customary to express results on a surface area basis (MR/m2) or on a body weight 0.75 basis. Both of these factors correlate reasonably but not perfectly with lean body mass. Obese human subjects generally have an increased basal metabolic rate since they not only have an increased fat mass but also an increased lean mass (Jung and James, 1980). Because basal metabolic rate is such a large component of the total energy expenditure it is tempting to suggest that variation in this component is sufficient to account for variations in total metabolic rate. The fundamental question is did obese subjects have a reduced basal metabolic rate prior to becoming obese? Such prospective studies have not been carried out. Jung and James (1980) found that 9 obese women, who returned to and maintained a normal weight by dietary restriction, had a significantly lower than ideal basal metabolic rate (Fleisch, 1951). HOWever, these authors did not consider that the difference was sufficient to account for the susceptibility of these subjects to gain weight. A number of workers have claimed difference in metabolic rate between the sexes (Robertson and Reid, 1952; Fleisch, 1951) and a decline in metabolic rate with age (Boothby, Berkson and Dunn, 1936). Whether such differences are real or merely a reflection of different methods of expression which would disappear if the data was normalized with respect to lean body mass is not clear. It seems likely that basal metabolic rate is also affected by a number of hormones. For example, insulin in addition to its action on glucose metabolism, has a number of actions including effects on protein synthesis and sodium transport. A reduction in the rate of these processes would result in a reduced basal metabolic rate in insulin dependent diabetics. Thyroxine and growth hormone also affect the basal metabolic rate. Physical activity. It is estimated that physical activity accounts for about 20% of the daily energy expenditure in man, but this is difficult to quantify. Obese subjects are generally less active physically than lean subjects but the energy cost of moving is much greater in the obese. Thus, Miller and Parsonage (1975) found that the energy cost of walking or just standing can be four times the cost of sitting in obese subjects. They found that this energy cost difference increased in a linear fashion with subjects weight. It seems likely that physical activity is an important determinant of body composition. Clearly, the energy expenditure involved in lumberjacking is very different from that involved sitting at a desk. However, it is also apparent that changes in physical activity have to he substantial if one is to make a major contribution to the overall energy expenditure. Thus, jogging or the occasional game of squash or golf make an insignificant contribution to energy turnover and the calorigenic effect may often be totally negated by post-exercise comsumption of alcoholic beverages. Cold- induced thermogenesis. Man and animals living in an environment below thermoneutrality produce heat to maintain body temperature. Earlier in this

328

M . A . Cawthorne

chapter, it was noted that a number of genetically obese rodents failed to maintain their body temperatures in response to cold. Experiments using ob/ob mice (Trayhurn, Thurlby, Woodward and James, 7979) and db/db mice (Trayhurn, 1979a) have shown that the genetically obese animals expend less energy than lean mice on the non-shivering thermogenesis needed to maintain homeothermy (Fig. 2.4). Thus, at 34° the ob/ob and db/db mice both have a slightly higher metabolic rate than the lean mice, presumably as a result of their greater size. However, as the environmental temperature was decreased the increment in metabolic rate between lean mice and the mutants progressively increased. This difference in metabolic rate was matched by a difference in body temperature (Fig. 2.5). These studies have shown that the metabolic rate of lean mice at 20°C is approximately twice that found at 34°C. This increase is the result of non-shivering thermogenesis since shivering is only seen with sudden exposure to severe cold. By reducing their body temperature, the mutants reduce their energy requirement for thermogenesis. Thus, if lean mice and the mutants are given the same amount of food at an environmental temperature of around 20°C, it can be shown that the increased energy deposition as fat is almost exactly matched by the decrease requirement for non-shivering thermo~enesis. However, if mice are kept at 34°C, then when hyperphagia is absent there is much smaller difference in the energy deposition between lean mice and the obese mutants (Trayhurn and Fuller, 1980; Thurlby and Trayhurn, 1979). Nevertheless, even at thermoneutrality, the mutants that were pair-fed with the lean litt~tes gained n~)re weight suggesting that thermoregulatory thermogenesis is not entirely responsible for the obesity in these mutants (Fig. 2.6). The energy needed for maintenance of body temperature is related to surface area and thus the proportion of energy turnover required for thermogenesis would be expected to decline with increasing body size. Furthermore, with the exception of some races such as Australian aborigines (Scholander, Hammel, Hart et al, 1958) and Kalahari bushman (Wyndham and Morrison, 1958), acute cold exposure is an unusual event in everyday life. Man adapts behaviourally to changes in his environment, not only by creating artificial temperature in the home, at work and in his car but also by adjusting his micro-environment through changes of clothing. These factors all tend to limit the extent to which man is exposed to cold. Thus, to be of importance in human obesity, it is necessary to consider changes in metabolic rate over a relatively small range in environmental temperatures. Dauncey (1981) has compared the metabolic rate of nine women at 28°C and 22°C and found an average 7% difference with a range of 2 to 12%. Although Occasional piloerection was reported at the lower environmental temperature, there was no overt signs of shivering and thus the increased metabolic rate was assumed to be the result of non-shivering thermogenesis. The difference in energy expenditure at the two environmental ter~peratures is not insignificant. Dauncey (1981) calculates that if all other factors being equal, subjects experienced mild cold for only 10% of each year for a 10 year period, they would have an average of 8 kg loss in body weight. Consequently, it is possible that the increase in environmental living conditions and the greater availability of warm clothing over the last 20 years in Western societies could have indirectly led to obesity. In addition, there is the possibility that some people behave like the obese mouse mutants and fail to expend as much energy on thermoregulatory thermogenesis, and therefore in response to cold, they reduce their core temperature marginally. This has been demonstrated in the Lapps (Andersen, 1963), in Australian Aborigines (Scholander et al, 1958) and Kalahari bushmen (Wyndham and Morrison, 1958). They allow their core ter~perature to fall during a cool night, and furthermore on being acculturated into an urban society with a greater availability of food, the Aborigines are prone to obesity. A number of studies have shown that some obese subjects show a greater fall in core temperature than lean subjects when exposed to reduced environmental t~nperature (Itoh, 1974; Andrews and Jackson, 1978) and Andrews (1980) has shown a smaller increase in metabolic rate in obese subjects exposed to cold than in lean subjects.

Metabolic Aspectsof Obesity

329

°°li =z

~

200

100

34 °

30 °

25 °

20 °

15 °

10 °

Environmental temperature (°C)

Fig. 2.4

Metabolic rate of lean ([]) and obese (U) mice during exposure to various environmental temperatures for 40 min. The results are the means + S.E.M. (bars) for 12 animals in each group. Reproduced from thermoregulation in genetically obese rodents by P. Trayhurn, P.L. Thurlby, C.J.H. Woodward and W.P.T. James in Animal Models of Obesity, p191-203, copyright Macmillan Press Ltd., London, with permission. m

Dietary induced therm~enesis. Earlier in this chapter, it was noted that in both experimental animals and man, a mechanism appeared to operate that tended to resist weight gain during periods of overfeeding. Furthermore, it was noted that there was individual variation in this response. In addition to the effects on energy expenditure of an elevated plane of nutrition (luxuskconsumption - Neumann, 1902), there is, as first reported by Lavoisier in 1789, an increase in metabolic rate following the consumption of a meal. Miller (1975) proposed that it was an adjustable dietary-induced thermogenesis that was the key to the preservation of energy balance in normal individuals in the face of a constantly varying food intake. This led to the proposition that obese subjects may well have defective dietaryinduced thermogenesis. Early studies on this matter provided conflicting evidence, which has been sunmarised by Miller and Mumford (1973) and Garrow (1980). However, recent work has supported Miller's suggestion. Pettet, Chapuis, ~mheson, de Techterman and Jequier (1976) measured the thermogenic response of a 50 g glucose load in lean and obese ~ m e n and found a significant reduced effect in the obese. Indeed, James, Dauncey, Jung, Shetty and Trayhurn (1979) make calculations to show that such a reduction in the thermic response could produce a net positive energy balance of 6 kg per year. Shetty, Jung, James, Barrand and Callingham (1981),

4o!

330

M.A.

Cawthorne

o

35

E

n-

30I

35

30

I

25

I

20

15

10

Environmental temperature (°C) Fig.

2.5

Rectal temperatures of adult lean ( 0 ) and obese ( O ) mice following exposure to a range of environmental temperatures for 1 h. The results are the means + S.E.M. (bars) for i0 lean and I0 obese animals. Reproduced from T h e r m o r e g u l a t i o n in genetically obese rodents by P. Trayhurn, P.L. Thurlby, C.J.H. w o o d w a r d and W.P.T. James in Animal Model of Obesity, p191-203, copyright Macmillan Press Ltd., London, with permission. 33 °

23 °

, m

c

w

Fig.

2.6

Excess energy gain of young obese mice pair-fed to the ad lib food intake of lean litter mates at thermoneutrality and at 23°C. The results are expressed as means + S.E.M. (bars) for 8 animals at each temperature The experiment was performed with males only. Reproduced from T h e r m o r e g u l a t i o n .... as above.

Metabolic Aspects of Obesity

331

using a standard test meal, showed that the diminished response to the meal was not simply due to the obese state, since post-obese subjects also had a small thermic response despite considerable weight loss. Zed and James (1980; 1981) have attempted to examine the components of the mixed meal that are responsible for the differential thermogenic response. Starch and protein did not produce lower metabolic responses in obese subjects whereas fat did. James and Trayhurn (1981a) note that in the Vermont Study (Sims, 1976) , lean men without any family history of obesity were able to resist weight gain in spite of consuming excessive amounts of a high-fat diet. From the data of Dauncey (1980), they calculate that thermogenic response to fat is inversely related to body fat content. Psychological thermogenesis. The influence of stress and various emotions such as love, hate, anger, fear and excitement on metabolic rate is largely unknown. Conmon experience suggests that such factors are important in regulating weight and that their effect is unlikely to be merely due to effects on food intake. However, it is difficult to quantify the effect of these various 6~otions on metabolic rate although Garrow (1974) notes a very pronounced increase in metabolic rate in a subject watching the first lunar landing.

Chapter 3

Thermoregulatory and Dietary-Induced Thermogenesis

In the previous chapter, arguments were developed that suggested that thermcxjenesis may be of importance in the maintenance of normal body weight in rodents and possibly also in man. Studies using genetically obese rodents suggest that the major problem is a failure to maintain a normal body temperature at any environmental temperature below thermoneutrality owing to defective thermogenesis. Other studies in rodents that have beccme obese because of dietary manipulation, point to dietary-induced thermogenesis as being an important ccmloonent of energy balance. Currently, there is a substantial body of experimental work being carried out to identify the biochemical mechanisms that are responsible for controlling both coldinduced (non-shivering) thermogenesis and dietary-induced thermogenesis and the progress of these studies will be highlighted in the present chapter. Much of our knowledge on non-shivering t h ~ e n e s i s comes frcm studies in two areas. The first is in the new-born homeothermic animal. Whilst the foetus remains within the mother, it is maintained in a constant t ~ _ r a t u r e environment and the foetus does not have to produce any heat to maintain its body tesperature. Ho%~ver, at parturition this situation changes dramatically in that the new-born marm~l is suddenly exposed to an environmental temperature below 37°C. Furthermore, this environmental temperature is no longer fixed. Since the homeothermic animal is dependent upon the maintenance of body temperature, the new-born animal needs to produce heat and to regulate its heat production. In most species, unless the environment is very cold, there is no evidem~e of shivering and maintenance of body temperature is achieved by non-shivering thermogenesis. The second area of research that has contributed to our understanding of non-shivering thermogenesis is cold-acclimation. Experiments on non-shivering thermogenesis were originally carried out by Claude Bernard in the 19th century, but it was not until 1954, that it was convincingly demonstrated that cold-acclimated rats increased their heat production in the cold without any increase in the electrical activity of the skeletal muscles; that is without shivering, (Sellers, Scott, and Thomas, 1954). In addition, it has been shown that hibernating species such as the h~mlster are able to arouse frcm a body tesloerature of around 5°C and to elevate their body t~mperature to 37°C, even when they are still in cold surroundings, without any visible signs of shivering (Cannon and Johansson, 1980). These findings contrast with those found in warm-acclimated animals that are given an acute exposure to cold. In this situation, much of the heat production arises from shivering and electrical activity can he detected by electrcmyograph recordings.

332

Metabolic Aspects of Obesity

333

Body Size and Non-Shivering Thermo~enesis The need for extra heatproduction by animals upon exposure to a temperature below thermoneutrality is dependent upon body size. It appears that the maximal capacity for heat production by non-shivering thermogenesis is also related to body size. Heldmaier (1971) found that the noradrenaline-induced increase in oxygen consumption (a measure of the maximal capacity for non-shivering thermogenesis) decreased in proportion to the logarithm of the body weight (Fig. 3.1). Thus, based on this relationship a 20g mouse would be expected to increase its metabolic rate by about 400% when exposed to a 5°C environment, whereas a 300g rat would produce a 150% increase and a 100kg man a 20% increase.

-15

15-

r=0.900 b = 0.312

10-

-10

-5 -4

5-

3-

3 2

I

2

Fig. 3.1

I

10

I

100

I

1000

10000

'

I

100000

Increase in oxygen consumption (vertical axis) due to norepinephrine infusion in mammals as a function of body weight. Reproduced from Heldmaier, G. (1971), Z.vergl. Pysiol. 7_~3, 222-248, with permission.

At tEnperatures between 5°C and thermoneutrality, the requirement for non-shivering thermogenesis in order to maintain body temperature will be correspondingly reduced. However in small laboratory rodents, such as mice, maintained at normal laboratory tenloeratures (22°C) , there is still a considerable requirement for thermogenesis. Adult man, because of his favourable surface area to volume ratio and his facility for adopting warm-clothing when faced with a cold-environment, has a low requirement for non-shivering thermogenesis. Nevertheless, as was pointed out in the previous chapter, metabolic rate increases with relatively small decreases in the environmental temperature. Similarly, infusion of noradrenaline into man produces a marked increase in oxygen consumption (Jung, Shetty, James, Barrand and Callingham, 1979) (Fig. 3.2). In other species the increase in oxygen consumption produced by noradrenaline has been taken to be equal to the animal's maximum capacity for non-shivering thermogenesis and thus the findings of Jung et al (1979) have been interpreted that man has the capability of increasing heat production by non-shivering t h ~ e n e s i s .

334

M . A . Cawthorne

0.8-

i

._= E 0.6-

r-

0.4-

G) L.

0.2-

"5 t~

0

Noradrenaline infusion I

I

I

I

0 5 10 15

f

I

I

I

30

45

60

90

Time ( m i n )

Fig. 3.2

Absolute increase in RMR of women during and after a 45 min intravenous infusion of noradrenaline. Adapted by permission from Jung, R.T. et al, Nature 279, 322-323, copyright 1979, Macmillan Journals Limited.

Noradrenaline and Non-Shivering Thermogenesis From studies carried out in the 1950's, it is known that cold adapted rats have a higher adrenergic tone than rats kept at higher environmental temperatures. This finding was not unexpected since it had long been recognised that adrenergic metabolism increased in stress conditions and cold exposure could be regarded as a stress. However Hsieh and Carlson (1957) investigated the effect of intravenously infused catecholamines on oxygen consuniotion in cold-adapted and control rats. In these experiments, the infusion of the catecholamines was carried out at room temperature to anaesthetized animals. Adrenaline increased oxygen consumption much more in cold-adapted rats than in controls. However, the authors were surprised to find that noradrenaline was a much more powerful stimulus than adrenaline. This immediately suggested that the mechanism of the themncgenic response was not hormonal but neural. The finding that noradrenaline infusion at thermoneutral temperatures produces non-shivering themnc~enesis does not necessarily suggest that noradrenaline is the effector of non-shivering therrm~enesis in the cold. However, except in birds, there is qualitative parallelism between rates of oxygen consumption in the cold and rates of oxygen consumption in the warm following a noradrenaline infusion. (In birds, noradrenaline does not appear to be the effector of non-shivering themncx/enesis). Jansky (1969) has examined the quantitative nature of the parallelism in rats (Fig. 3.3). He acclimated rats at 5°C and then measured their response to a noradrenaline infusion at an environmental temperatures of 15°C and 30o C. At these temperatures oxygen consumption will stabilize at the level required for thermobalance, but infusion of noradrenaline increases the rate of oxygen consumption so that it is equal to that obtained at 5°C.

Metabolic Aspects of Obesity

4 O rE

335

6.6% BMR

~

3-

92% BMR

2-

c~

O

I-

E

[

0

10

I

20

30°C

I I Resting metabolism •

Fig. 3.3

Metabolic increase after 0.4 mg NA/kg

Substitution of thermogenesis evoked by norepinephrine (NA) by increased metabolism in the cold in nonanaesthetised rats acclimated to 5°C. Reproduced from Jansky, L., 1969, Fed. Proc. 2~8, 1053-1058, with permission.

Location of Tissues responsible for Non-Shivering Thermogenesis Oxygen consumption measurements show that cold-exposure to 4°C of small manmals requires an increase in metabolic rate of up to 4 times the basal rate in order to maintain body temperature and even in large animals such as man the metabolic rate rises by 20%. If the increase in thermogenesis is limited to one organ or tissue, the increase in the oxygen consumption by that tissue becomes much greater. The main contenders for the sites of non-shivering thermogenesis are skeletal muscle, viscera and brown adipose tissue. Smith (1961) found that brown fat was thermogenic and thus established a function for a tissue, that had previously been without any known function since its first description in 1670. However, until recently, brown fat was only thought to be important in neonatal animals and hibernating animals. In adult animals it was thought that there was not enough tissue to be of any significant thermogenic consequence and, in these animals, skeletal muscle was thought to be the major site of thermogenesis. Studies on the localisation of the thermogenic site have concentrated on changes in blood flow to various organs in cold-exposed and noradrenaline infused animals. In order to calculate the oxygen consumption of an organ in vivo, one needs to know both the blood flow to that organ and the arterio-venous difference for oxygen across the organ. In practice to obtain an estimate of the blood flow, measurements are made of the cardiac output and the distribution of the blood to the organ. During the 1960's the principal method for measuring blood flow used the diffusable ionic tracer 86Rb. Such studies showed that in adult rats skeletal muscle was the major site of non-shivering thermcxjenesis (Evonuk and Harmon, 1963; Kuroshima, Konno and Itoh, 1967; Jansky and Hart, 1968). The radioactive rubidium method depends on the isotope being taken up by the tissues in direct relationship to the

336

M . A . Cawthorne

flow through the tissue. Unfortunately, the passage of rubidium from the blood to the tissues is by a rather slow diffusion process. Thus, it has been found that the proportion of 86Rb extracted by tissues from blood is not constant but decreases with increasing blood flow (Friedman, 1968; Sheehan and Renkin, 1972). As a result, blood flow to organs with a low flow rate will be overestimated whilst flow to organs with a high flow rate will be underestimated (Foster and Frydman, 1978a). Using the rubidium method, it was found the noradrenaline infusions altered the pattern of blood flow; more blood went to brown fat and the liver but the major effect was to increase the flow to muscles. Rudolph and Heymann (1967) introduced a different method for measuring blood flow which uses radio-actively labelled microspheres. These microspheres are introduced into either the left atrium or left ventricle and follow the distribution of the blood. The microspheres are of such a size that they are able to penetrate the microvasculature system where they become trapped causing embolism. There is no requirement with this method for diffusion and blood flow to organs with a high blood flow will be measured without the previous systematic error. However, the method is unsuitable for measuring flow to those organs (liver and pituitary) with a portal circulation. Foster and Frydman (1978b) , using the microsphere method, concluded that brown adipose tissue was the dominant site of non-shivering thermogenesis in both coldacclimated rats (Table 3. I) and in warm-acclimated rats given noradrenaline (Table 3.2). These authors showed that during noradrenaline stinmlation of nonshivering thermogenesis in cold-acclimated rats, over 30% of the total cardiac output goes to brown adipose tissue; even though this tissue accounts for only approximately 2% of the body weight. Furthermore, the extraction of oxygen from the blood by brown adipose tissue was extremely high. Foster and Fry~hnan (1978b) found that blood leaving the interscapular brown fat via Sulzer's vein was virtually oxygen depleted, and therefore calculated that more than 60% of the extra oxygen used during noradrenaline-induced non-shivering thernogenesis in adult rats is consumed by brown fat. The contribution of skeletal muscle to this nonshivering thermogenesis was no more than 12%. In another study, Foster, Depocas and Frydman (1980) showed that the increase in blood flow to brown adipose tissue was related to a sigmoid function of arterial noradrenaline concentration. The in vivo dose response curve for blood flow in cold-acclimated rats was displaced to the left compared with that obtained in warm-acclimated rats and also s h ~ a much higher Vmax (Fig. 3.4). In addition these authors showed that not all brown adipose tissue sites shared the same sensitivity with respect to noradrenaline concentration (Fig. 3.5). These results in adult rats parallel the earlier studies of Hull and his colleagues using new-horn rabbits. HeLm and Hull (1966) found that the transfer of a new-born rat from 35°C to 20°C caused a three-fold increase in the rate of oxygen consumption. After excision of 80% of the brown adipose tissue there was virtually no increase in oxygen consumption upon cold exposure or noradrenaline infusion. In other studies, noradrenaline infusion in the new-horn rabbit increased venous outflow from brown fat from 0.9 to 3.6 ml per g tissue per rain. The current concensus of opinion is that brown adipose tissue is peculiarly adapted to be a thermogenic tissue. For example, the blood flow through the tissue can increase up to seven times its own volume per min and it can develop an oxygen consumption as high as 50 ~mol 02/g min -I , which represents a rate of heat production of about 400 W/kg (whereas the general average heat production of man is about I W/kg (Cannon and Johansson, 1980). To achieve these effects the tissue is doubly innervated with sympathetic nerve fibres to both the blood vessels and adipocytes. However, in the face of this stupendous capacity of brown fat for heat production, one should not lose sight of the fact that most studies only show of the order of 60% of excess heat production arising from brown fat and thus it would be unwise totally to discount the contribution of other tissues such as muscle.

375.8 5.24 I. 39 15.9 I. 35 1.92 I. 10 144.3 2.89 !. 29 3.44 I. 38 19.3 37.0 4.02 43.9

(100.0) 2.6 +-0.1 3.9 +-0.2 2.2 -+0.1 0.37 +-0.03 1.4 +-0.04 2.9 +-0.3 11.2 +-0.5 7.4 +-0.5 1.6 +-0.I 4.0 +-0.6 0.77+-0.06 4.0 +-0.5 13.9 +-I.I 18.6 +-1.2 9.9 +-1.0

At rest

(100•0) 33.5 +-1.5 10.1 +-0.7 4.9 +-0.2 0.75+-0.07 1.4 +-0.2 2.4 +-0.4 8.4 -+0.4 5.2 +-0 2 0.95+-0 07 2.0 +-0 4 0.36+-0 01 1.9 +-0 3 6.4 +-0 5 8 . 0 +-0 1 3.2 +-0 2

With NA

Percentage of CO

-

<0.001 <0.001 <0.001 <0.001 ns ns <0.05 <0.01 <0.001 <0.02 <0.001 <0.01 <0.001 <0.001 <0.001

P

88.9 +-4.1 2.3 +-0.2 3.5 +-0.3 1.9 +-0.1 0.33+-0.03 1.2 +-0.1 2.4 +-0.3 9.9 +-1.2 6.5 +-0.3 1.4 +-0.1 3.6 +-0.7 0.67+-0.05 3.3 +-0.3 12.4 +-1.3 16.6 +-1.2 8.9 +-0.6

At rest

171.6 +-8.7 57.2 +-3.8 17.2 +-1.8 8.4 +-0.5 1.3 +-0.2 2.4 +-0.2 3.8 +-0.4 14.5 +-1.2 8.9 +-0.5 1.6 +-0.1 3.5 +-0.7 0.61+-0.03 3.2 +-0 7 10.8 +-0.5 13.7 +-0.9 5.5 +-0.3

With N A

Blood flow, ml/min

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.05 <0.02 <0.01 ns ns ns ns ns ns <0.001

P

•

2.0 +-0.1 25.5 +-2.4 5.0 +-0.7 4.4 +-0.4 4.0 +-0.3 2.0 +-0.2 1 6 +-0.2 1.5 +-0.2 1.4 +-0.04 1.2 +-0.1 0.97+-0.19 0.95+-0.09 0.95+-0.20 0.91-+0.07 0.85+-0.10 0.62+-0.04

Flow ratio

Rep~:oduced from Foster,

D.O. and Frydman,

M.L.

(1978), Can. J. Physiol.

Pharmac.

56, 110-122,

with permission•

The data are based upon the fractional distribution of 15~m microspheres. Each rat received two injections of differently labelled microspheres, the first under resting conditions, the second during infusion of noradrenaline at a dose (free base) of 12.5 ng g-0.74 min-l. The brown adipose tissue consisted of the interscapular, dorsocervical, axillary, periaortic, perirenal, iliac and inguinal sites.

Whole rat BAT Heart Rib cage Diaphragm Brain Spleen Muscle Small intestine Large intestine and caecum Lungs Stomach Liver Skeleton and spinal cord Kidneys Skin

Tissue

Average weight of tissue g

TABLE 3.1 The Fractional Distribution of Cardiac Output and the Blood Flow to Tissues of Barbital Sedated Cold-Acclimated Rats at Rest or durin~ Noradrenaline-induced Calori~enesis

OO QO "4

~-"<

m o o_~ O

o_ 5' ]>

392.5 2.94 I. 28 15.4 I. 24 I. 94 2.66 154.6 16.4 I. 15 I .41 I. 19 35.3 3.35 47. I 2.61

Average weight of tissue g

(I00.0) 0.92+-0.21 4.7 -+0.6 1.9 +-0.1 0.35±0.03 1 6 ±0.1 8 4 ±0.8 13 5 +-0.8 4 0 +-0.7 0 95±0.08 1 7 ±0.1 3 3 ±0.4 12 7 +-0.8 14 2 +-1.2 11 2 +-0.6 3 8 +-0.7

At rest

(100.0) 1 1 . 4 +-1.2 1 3 . t +-1.1 3.0 +0.3 0.50±0.03 2 . 4 ±0.1 11.6 ±1.5 1 3 . 3 -+1.0 3.5 ±0.4 0.86-+0.05 1.3 +-0.1 2.4 ±0.4 7.9 +-0.4 8.2 +-0.8 5.3 -+0.4 1.5 +-0.3

With NA

Percentage of CO

<0.001 <0.001 <0.01 <0.01 <0.01 ns ns ns ns <0.05 ns <0.001 <0.01 <0.001 <0.02

P

8 8 . 9 +-3.9 0.81+-0.18 4.3 +0.5 1 . 7 +-0.1 0.30+-0.03 1 . 4 +-0.1 7.7 ±1.4 1 2 . 0 -+1.0 3.5 -+0.6 0.86±0.08 1.5 +-0.1 2.9 +-0.4 11.3 ±0.8 12.5 +-0.7 10.0 +-1.1 3.3 +-0.4

At rest

1 1 8 . 9 +-4.4 1 3 . 5 +-1.7 15.1 + 1 . 0 3.6 +0.5 0.59+0.04 2.8 ±0.2 13.6 -+2.3 1 5 . t +-1.6 4.1 -+0.5 1.0 +-0.1 1.6 +-0.1 3.0 ±0.6 9.4 +-0.9 9.6 -+1.1 6.6 +-0.7 1.9 +-0.2

With N A

Blood flow, ml/min

<0.001 <0.001 <0.001 <0.01 <0.001 <0.001 <0.05 <0.05 ns ns ns ns ns ns <0.05 <0.05

P

1 . 3 +-0.1 2 1 . 4 +-5.2 3 . 5 +-0.3 2.1 +-0.2 2 . 0 +-0.2 2 . 0 +-0.1 1.7 +-0.2 1 . 3 +-0.1 1.2 +-0.1 1.1 +-0.05 1.0 +-0.05 1.0 +-0.1 0.83+_0.05 0.79-+0.10 0.66+-0.05 0.55+-0.04

Flow ratio

Reproduced from Foster, D.O. and Frydman, M.L.

(1978), Can. J. Physiol.

Pharmac.

5_~6, 110-122,

with permission.

The data are based upon the fractional distribution of 15~m microspheres. Each rat received two injections of differently labelled microspheres, the first under resting conditions, the second during infusion of noradrenaline at a dose (free base) of 12.5 ng g-0.74 min-l. The brown adipose tissue consisted of the interscapular, dorsocervical, axillary, periaortic, perirenal, iliac and inguinal sites.

Whole rat BAT Heart Rib cage Diaphragm Brain Small intestine Muscle Liver Stomach Large intestine and caecum Spleen Skeleton and spinal cord Kidneys Skin Lungs

Tissue

TABLE 3.2 The Fractional Distribution of Cardiac Output and the Blood Flow to Tissues of Barbital Sedated Warm-Acclimated Rats at Rest or d u r i n ~ N o r a d r e n a l i n e - i n d u c e d Calorigenesis

3

Do

C~

>

&o 00 co

Metabolic Aspects of Obesity

20

I _

ot m '-

16-

I I=

I

c'

(6)

_~o .~

- ....

,-" (5) >" (6) 4 I [ 0 8

BI

I

I

I 16

I

I

I

I

2 _- ~

0

r=0.91

I

I

I

I

I

1

I

0 4 8 12 INCREASE in Vo2(mL/min per 370 g rat) 3.2

(ng/mL) I

'''''1 oo. - ' ' CA

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,, ~= ,o-

~D.

20

.............

<~

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0.8

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ARTERIAL PLASMA NA 80 _

~;

6-

o9

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-

0[&

o~

~

~ 12-

9

I . ~

/(4) )

/ 5

o9

.E E

I

A

339

I~" 0

I

I 8

I

I 16

I

I 24

ARTERIAL PLASMA NA (ng/mL) Fig.

3.4

//

4 Y2 T~//

-4

6

I

.
~ /I PRBAT

"

~/

l ~ i i

i

10

14

l

I

..- DBATI BA"~

~

i

18

Vo2 (mL/min per 370 g rat)

Relations between plasma NA, calorigenic response and the responses of total BAT (TBAT) in barbitalsedated WA ( O ) or CA ( O ) rats infused with graded doses of NA. The reference points in this and subsequent figures represent mean values for the numbers of animals given in parenthesis in panel A; the bars indicate SEM where these are greater than the diameters of the symbols. In left to right sequence of the mean values the associated doses of NA (ng min -I g-0.74) were 0,2,3,5, and 12.5 for WA rats and 0,1,2.5,3.5,5,8.5, and 12.5 for CA rats. In panel C the slopes of the two regression lines are significantly different (p< 0.05); the values of r are correlation coefficients. Data from CA rats only are given in panel D. Reproduced from Foster et al, 1980, Can.J.Physiol. Pharmacol, 58, 915-924, with permission.

340

M . A . Cawthorne ' I WA

:t

'

I ...."":

T...~/

J..... :....'I ........... I..... ""'I"":~DBAT ~......

I,

..- ~ ~ - -

_&,-

. . . .

PAB~

.~PRE~

"..-"- . . . . . . . . . . .

_ 13")

.."/

.

C

z

E

-~"

J

E

0

0

I 4 I

o

,

I

,

8 '

I

'

I

,

I

,

12

16

~

I

'

,

I

,

I

24

IT'

28

l

I

j~DBAT

CA

LL

I

20

a o o

-

./*" r ..'"'"" I"~ABAT ...*" ~ ~PABAT

rm

...~~ ~ " ~ B A T

16°

/~.~.....7 ~ . ~ / ~

12.

_ . . - - " ' ~ PRBAT

"

l~'/ i"

.

0

'

0

I

I

I

I

4

8

12

16

A R TE R I A L P L A S M A Fig.

3.5

'

I

I

20

24

'

I

28

N A (ng/mL)

Relations between the concentration of NA in arterial plasma and the blood flows per unit mass in the differently located bodies of BAT of barbital-sedated WA or CA rats infused with graded doses of NA (see legend to Fig. 3.4). Bars indicating the SEM are not shown in all applicable cases. Abbreviations: DBAT, dorsal cervical BAT; PABAT, periaortic; IBAT, interscapular; PRBAT, perirenal; ABAT, axillary; I-IBAT, iliac and inguinal. Reproduced from Foster et al, 1980, Can. J. Physiol. Pharmacol, 58, 915-924, with permission.

Metabolic Aspects of Obesity

341

Non-Shivering Thermogenesis in Genetically Obese Rodents Since genetically obese rodents cannot maintain their normal body temperature below thermoneutrality, it would appear possible that they have a defective non-shivering thermogenic mechanism. Trayhurn and his colleagues have carried out studies in both genetically obese (ob/ob) and diabetic (db/db) mice. The administration of an intraperitoneal dose of noradrenaline (750~g/kg body weight) evoked a maxim~n elevation of the metabolic rate in both lean and obese mice after 15 mins, which was maintained for a further 5-10 mins. However, the maximum increase in metabolic rate in genetically obese (ob/ob) mice was only half that in lean mice (Trayhurn and James, 1978). similar studies in genetically diabetic (db/db) mice (Trayhurn, 1979a) indicated that these mice only gave a 64% increase in metabolic rate following noradrenaline infusion whereas lean mice produced a 125% increase in the metabolic rate (Fig. 3.6).

32-5°C

10 °C

250 ..C

200 150

0 E

100 50

i

n.db. (9X12) Fig. 3.6

n.db. (7)(7)

Non-shivering thermogenesis in normal (n) and diabetic (db) mice. The resting metabolic rate (m) was measured, noradrenaline administered, and the peak rise ([]) in metabolic rate then determined: the experiment was conducted at 32.5 and 10°C. The results, expressed per whole mouse, are mean values + SEM (bars) with the number of animals used in parentheses. At 32.5°C there was n o significant difference (p > 0.05) in the baseline metabolic rate between normal and diabetic mice, but the effect of noradrenaline was significantly different (p< 0.001). Reproduced from Trayhurn, P and Fuller, L., 1979, Pflugers Arch 380, 227-232, with permission.

In order to identify the tissue(s) responsible for the low energy expenditure on non-shivering thermogenesis in the ob/ob mouse, Thurlby and Trayhurn (1980) measured regional blood flow in lean and obese (ob/ob) mice both in the basal state and following stimulation of non-shivering thermogenesis by noradrenaline injection. Noradrenaline injection increased the cardiac output by a similar

342

M.A. Cawthorne

amount in both lean and obese mice (lean from 11.4 to 24.9 mi/min; obese from 10.1 to 24.8 mi/min.). In the basal state, blood flow to brown adipose tissue in both lean and obese mice was very low, (Table 3.3). However, the noradrenaline infusion caused a 38 fold increase in flow to brown adipose tissue in lean mice but only a 12 fold increase in the obese mice. In addition, the extraction of oxygen from the blood was lower in obese mice (13.2 ml 02 per 100 ml blood) than in the lean mice (16.3 mls 0 9 p e r 700 ml blood). Thurlby and Trayhurn (1980) calculated the contribution of Erown adipose tissue oxygen consumption to the total increase in oxygen consumption produced by noradrenaline injection (Table 3.4). In the lean mice, the dissectable brown adipose tissue accounted for 44% of the total increase in oxygen consumption, but in obese mice brown adipose tissue accounted for only 24% of the diminished total increase in metabolic rate. It was further calculated that the reduced capacity of brown adipose tissue for thermogenesis in obese mice could totally account for the reduced ability of these mice to carry out non-shivering thermogenesis (Fig. 3.7).

< z

2

D.~

7 X

~

0 c

I ~c

I

0

Whole Body Fig. 3.7

BAT

Other Tissues

The importance of brown adipose tissue in the reduced non-shivering thermogenesis of ob/ob mice. The figure shows, for both lean (~) and obese ([~) mice, the maximum increase in whole body oxygen uptake in response to noradrenaline and the presumed contribution of BAT to these increases. Reproduced from Thulby, P.L. and Trayhurn, P., 1980, Pfluger's Arch, 385, 193-201, with permission.

The work of Thurlby and Trayhurn (1980) d~aonstrated that whilst brown adipose tissue is the major site of the non-shivering thermogenesis in lean mice it only accounted for less than 50% of the total heat production. The other regions that showed an increase in flow rate following noradrenaline administration were the heart, trunk and skin, and white adipose tissue. The increased flow to the heart is a well known effect of noradrenaline and parallels the increase in mechanical work of the heart that occurs following stimulation by the catecholamine. The increase in flow to the trunk was to a greater degree than to other muscular

0.03±0.01 0.07±0 02 0.10±0 02 0.52±0 18 0.35±0 12 1.89±0 36 0.25±0 06 0.30±0.06 3.01±0.56 3.56±0.66 0.18±0.04 1.21±0.21 0.19±0.04 0.75±0.21 1.68±0.32 0.54±0.09 0.43±0.10 3.59±0.67 0.06±0.01 0.10±0.02 0.16±0.03 0.36±0.08 0.39±0.15 1.57±0.30 0.22±0.05 0.15±0.04 2.66±0.50 3.04±0.56 0.23±0.05 1.29±0.24 0.14±0.03 0.73±0.15 1.31±0.23 0.40±0.07 0.47±0.11 3.06±0.54

Obese

1.04±0.13 2.81±0.42 3.86±0.55 2.12±0.35 2.56±0.48 3.21±0.50 0.20±0.05 0.30±0.04 3.98±0.50 4.48±0.54 0.40±0.11 1.97±0.23 0.17±0.03 1.27±0.30 3.56±0.39 0.72±0.15 0.60±0.14 6.32±0.75

Lean

0.62±0.15 *a 1.36±0.30" 2.01±0.45" 1.16±0.30 1.98±0.49 3.62±0.63 0.62±0.15" 0.32±0.09 4.81±0.87 5.73±1.02 0.57±0.13 2.65±0.39 0.20±0.06 1.61±0.30 3.47±0.70 0.84±0.19 0.69±0.20 6.80±1.14

Obese

F l o w with Noradrenaline (ml/min)

* * * *

*

** * *

** ** ** ** * * *

Obese

* * *

*** *** *** ** ***

Lean

Significance of change with N A

Reproduced from Thurlby, P.L. and Trayhurn,

P.

(1980), Pfluger's Arch,

38..~5, 193-201, with permission.

The results are mean values ~ SE, for 6 animals in each group. Data for the noradrenaline treated animals was obtained during the peak metabolic response to the hormone. a Significantly different from lean mice *p<0.05, **p<0.01, ***p<0.001

Interscapular BAT Other BAT TOTAL BAT Heart (Lung) Kidneys Liver Spleen GI tract TOTAL SPLANCHNIC Brain Skin, white adipose tissue Tail Head Trunk Hind limbs Fore limbs TOTAL MUSCULAR CARCASS

Lean

Basal Flow (ml/min)

TABLE 3.3 Reqional B l o o d Flow in Lean and Obese Mice in the Presence and Absence of Noradrenaline

~.' -<

o

R

(D

>

oo

344

M . A . Cawthorne

portions of the carcass. The trunk region contained the respiratory muscles, and may contain some of the more diffuse brown adipose tissue sites. It also contains predominantly red muscles which have a high number of mitochondriawith a large capacity for fatty acid oxidation. The increase in flow to the skin and white adipose tissue may be related to the lipolytic activity of noradrenaline. TABLE 3.4 The Contribution of BAT to the Total Metabolic Response to Noradrenaline in Lean and Obese Mice

Lean

Increase in blood flow to BAT (mi/min) a Oxygen extracted by NA-stimulated BAT (ml/100ml blood) ~'c

3.76

16.3

Obese

I .85

13.2

Increase in oxygen uptake of BAT (mi 02/rain) d

0.61

0.24

Increase in total metabolic rate (ml 02/rain) b

1.38

0.98

Increase in BAT uptake as % of total increase (%) d

44

24

a

From Table 3.3 b Arterial oxygen concentration -oxygen concentration in sultzers vein c As determined for the interscapular site d A minimum value, calculated on the basis of A-V differences for BAT being as high under basal conditions as in animals receiving NA Reproduced from Thurlby, P.L. and Trayhurn, P. (1980). Pflugers Arch, 385, 193-201, with permission.

It is ter~pting to conclude that the genetic lesion in obese (ob/ob) mice is manifest as defective thernogenesis within brown adipose tissue. However, it should be noted that blood flow studies so far have only been performed on mice subsequent to the appearance of obesity and changes in brown fat thermogenesis may be a consequence of the additional insulation provided by the obesity rather than a cause of the obesity. In addition, it is clear from noradrenaline infusion experiments in both ob/ob and db/db mice that these mice, whilst having a reduced capacity for non-shivering thermogenesis relative to lean mice, nevertheless do still have considerable thermogenic capacity. Thus, whilst a defect in the total capacity of brown adipose tissue may well account for the fatal hypothermia that develops in these mutants at environmental temperatures of 4°C, it does not explain why thermoregulatory therrmxjenesis is decreased at environmental t6mlr~ratures of between 20 and 30°C since the absolute capacity for thermogenesis is much greater than that required to maintain normal body temperature. It is possible that at these environmental tenioe~atures the defect either lies in the failure to produce the mediator (noradrenaline) or that there is resistance to sub-maximal concentrations of noradrenaline with respect to thermogenesis in the tissue or with respect to vasodilation of the microvascular system within brown adipose tissue.

Metabolic Aspectsof Obesity

345

Tissue Effectors of Dietary-Induced Thermo~enesis The feeding to rats of highly palatable 'cafeteria' diets induces hyperphagia. However, the ability of this diet to induce obesity is related to the age and strain of the rats. Young rats, especially are able to resist the development of obesity and it has been suggested that this resistance may be due to dietaryinduced thermogenesis (Rothwell and Stock, 1979) or to a capacity to use energy for growth (Hervey and Tobin, 1981). Rothwell and Stock (1979, 1980) have shown that there are many similarities between cold-induced and dietary-induced thermogenesis. Using the cafeteria diet overfeeding paradigm, they showed that the rats fed on the cafeteria diet had both an increased sensitivity and a greater responsiveness with respect to whole body oxygen consumption following noradrenaline injection (Fig. 3.8). They also found that there was hypertrophy of the interscapular brown adipose in the cafeteria rats and this was not due merely to increased accumulation of triglyceride. Indeed the brown adipose tissue of the control and cafeteria-fed rats had an identical percentage fat and fat-free dry weight content. In a second study (Rothwell and Stock, 1980), cafeteria feeding and cold-acclimation were shown to produce an additive effect on brown adipose tissue hypertrophy and on the thermogenic response to noradrenaline. Interestingly, the rats fed on the cafeteria diet were able to adapt more rapidly to acute exposure to 5°C than control rats. The cafeteria-fed rats maintained a higher core temperature and shivered less than controls suggesting that their capacity for non-shivering thermogenesis was much greater than that of the control rats. This suggestion was reinforced by the demonstration that the B-receptor antagonist propranolol re-instituted shivering in the cafeteria rats and caused a fall in the core temperature to that in control rats (Fig. 3.9). *-'*

Control Cafeteria

106

50"

t

Io

20

a

|

3"0

4%

Dose (gg per 100g) Fig. 3.8

Stimulation of resting oxygen consumption by various noradrenaline. Adult female Sprague-Dawley maintained on the cafeteria diet for 15 days and during this time gained 23g excess weight compared to their free-feeding controls. 2 days after withdrawal of the mixed diet resting oxygen consumption was d e t e r m i n e d for 2h before and after administration of noradrenaline. 1 dose of noradrenaline was t e s t e d in 4 control and 4 experimental animals and the withdrawal of the c a f e t e r i a d i e t was s t a g g e r e d in order to faciliate these measurements. Mean values, bar denotes SEM, n=4, *p<0.05, **p<0.001, compared to controls. From Rothwell, N.J., Stock, M.J., Nature, 281, copyright 1979, Macmillan Journal Ltd., with permission. d o s e s of rats w e r e

346

M . A . Cawthorne - q9

Z rr" u.l

CONTROL

.....

CAFETERIA

100-

> I

I-Z

x\

i/

OO

,

,p

50-

// . .%.

LtJ

l I

,

,

09 UJ

I--

o~

0400 -

I

I

I

E iii v

200-

// .~

\ \

/J

0O

\

O.

n. uJ F.J O iaJ n-

i/

*

I

I

I

I

I

0

2

4 propranolol

6

I 8

39373533-

TIME OF COLD EXPOSURE (h)

Fig. 3.9

Shivering activity and rectal temperature of warmadapted control and cafeteria rats in response to acute cold exposure. The percentage of recorded time when shivering was observed and the frequency of spikes greater than 20 ~V are shown in the upper and middle graphs, respectively, and rectal temperature is depicted in the lower graph. Injections of propranolol were given after 5½h of cold exposure. Mean values; bar denotes SEM; *p<0.05, **p<0.01 compared with stock-fed controls; n=6. Reproduced from Rothwell, N.J. and Stock, M.J., Can. J. Physiol. Pharmacol. 5_~8, 842-848 (1980) with permission.

Metabolic Aspectsof Obesity

347

The hypothesis that brown adipose tissue is the mediator of dietary induced thermogenesis has been further developed by measurements of blood flow in rats fed on the cafeteria-diet (Rothwell and Stock, 1981). Using the microspheres technique it was found that blood flow to brown adipose tissue in cafeteria-fed rats was twice that in control rats. Treatment of the rats with noradrenaline, which remarkably did not increase cardiac output in these experiments, produced an even greater difference in the blood flow between the cafeteria and control rats (Table 3.5). On the basis of the flow measurements and the arterial-venous difference of the oxygen concentration across interscapular brown adipose tissue, it was calculated that in the cafeteria fed rats, 74% of the noradrenaline-induced increase in oxygen consumption could be accounted for by the additional oxygen consumed by brown adipose tissue whereas in control rats only 42% of the increase could be accounted in this way (Table 3.6). This strongly supports the view that the increased sensitivity of cafeteria-fed rats to the thermmgenic effects of noradrenaline is mediated through brown adipose tissue. However, if one tries to account similarly for the difference in basal oxygen consumption between rats fed on the cafeteria diet and control rats, only 26% of the difference arises from the increased uptake of oxygen by brown adipose tissue. These data suggest that cafeteria feeding increases the total capacity of brown adipose tissue for thermogenesis. However, when the overall increase in whole body oxygen consumption is small the additional oxygen consumption takes place mainly in tissues other than brown adipose tissue. Whilst the increase in blood flow to skeletal muscle in the rats fed the cafeteria diet is small (0.02ml/min/g) it may, when multiplied by the total weight of the muscle mass, be sufficient to account for the majority of the difference in oxygen consumption between cafeteria-fed and control rats. It is well-known that oxygen consumption increases following a meal and this effect (specific dynamic action) has been regarded as an expression of energy required for digestion, absorption and metabolic conversions. The organs responsible for the increased oxygen uptake following a meal have not been clearly identified but the liver has been strongly implicated (Wilhelmj, Boliman and Mann, 1928). The specific dynamic action of a meal has generally been regarded to be different from dietary-induced thermogenesis, which is a long-term adaptive response to overfeeding to produce a sustained increase in metabolic rate. Is it possible that dietary induced thermogenesis and specific dynamic action are related? From the results of Rothwell and Stock, one could envisage the possibility that the cafeteria feeding regime provides the trophic stimulus to increase the overall capacity of the animal for thermogenesis and this then allows an increase in the specific dynamic action of each meal. Rothwell, Saville and Stock (1982) have in fact found that the acute thermic response to food is increased in rats fed on a cafeteria diet. Furthermore, Glick, Teague and Bray (1981) have demonstrated that a single meal is able to increase the weight of interscapular brown adipose tissue and the rate of respiration of brown adipose tissue (Fig. 3.10). The possibility that specific dynamic action and dietary-. induced thermogenesis are related is further supported by the finding that specific dynamic action of meals is reduced in undernutrition (Rubner, 1902; Ashwerth, Brooke, Waterlow, 1973).

Thermogenesis in Hypothalamic Obesity Electrolytic lesions in the ventromedial nucleus of the hypothalaml/s (VMH) can lead to the development of obesity in the absence of hyperphagia (Han, 1968; Kennedy, 1957). In such circumstances there must be a reduction in energy expenditure. It is therefore of interest that Seydoux, Rohner-Jeaurenaud, Assimacopoulos-Jeannet, Jeanrenaud and Girardier (]981) have claimed that in VMHlesioned rats, there is a functional disconnection of brown adipose tissue. The interscapular brown adipose tissue site is supplied with a well-defined group of nerve fibres and the authors were able to isolate this tissue together with the

0.67±0.16 0.23±0.08 0.51±0.11 0.07±0.02 0.07±0.02 1.56±0.33

3.79 1.01 2.86 0.23 0.55 8.44

±0.70~ ±0.04 D ±0.60 a ±0.15 ±0.17 a ±I.03 b

118 ±6 1.97 ±0.43 3.97 ±0.71 10.41 ±1.81 0.097±0.03

Control noradrenaline

I, Interscapular;

10.05± 1.78± 7.36± 0.82± 1.89± 22.05±

1 0 0 0 0 4

07 be 05 be 86 bc i0 be 30 ad 20 bd

134 ±10 1.77± 0.24 4.10± 1.30 10.12± 1.20 0.09± 0.02

Cafeteria noradrenaline

PA, periaortic plus

e=p
d=p
1.02±0.18 0.20±0.07 1.03±0.34 0.29±0.07 e 0.54±0.19 e 3.00±0.53 c

130 ±8 1.89±0.45 3.30±1.25 9.25±1.36 0.09±0.02

Cafeteria saline

(ml/min)

(1981), Pfluger's Arch, 389, 237-242, with permission.

DC, dorsal-cervical;

Reproduced from Rothwell, N.J. and Stock, M.J.

BAT depots: thoracic.

a=p
Mean values + SEM; n=7

I DC AX PR PA Total BAT

Brown adi~0ose tissue

115 ±9 2.06±0.57 4.21±0.93 8.41±1.35 0.07±0.01

Control saline

Cardiac Output and Blood Flow to various Orqans

Cardiac output Brain Lungs Liver Skeletal muscle (g-l)

TABLE 3.5

3

O

C%

s i o o a 02

10.31 5 0.06 1.2

7.5

25

18.6 9 I .66 18.4

12.3

17.0±0.3 15.1±0.5 9.5±0.8 2.8±0.4

NA*

Reproduced from Rothwell, N.J. and Stock, M.J.

NA

62

C=p<0.01,

12.4 6 0.09 1.5

4.1

NA

13.5

11.5±0.15 17.5±0.31 b 4.6±0.06 7.0±0.12b 0.11±0.03 1.12±0.18 b 2.4 16.0 42

6.7

18.7±0.9 18.4±0.6 12 ( n = 2 ) 4.9±0.7

Saline

Control

d=p<0.001 compared to controls

37.0 18 7.58 42.0

13.3

16.8±0.3 13.4±0.3 12.7±0.4 0.1±0.04

Saline

Cold-adapted

18.7

19.8±0.8 1.1±0.I db

NA

14.5±0.21 b 27.0±0.81 b 5.8±0.08 d I0.8±0.32 b 0.42±0.07 c 4.1±1.0 ac 7.2 32.0 74

13.9

19.7±0.6 5.8±0.4

Saline

Cafeteria

Rothwell and Stock

(1981), Pfluger's Arch, 389, 237-242, with permission.

Mean values + SEM; n=7 * NA=noradrenaline a=p<0.01, b=p<0.001 compared to saline infused,

ml/min/WO'75 ml/min BAT VO 2 (ml/min) BAT % Total 902 BAT % NA response

Whole body oxygen consumption

Arterial Sulzer's vein A-VO 2 difference

(ml/100mI)

Saline

Warm-adapted

Foster and Frydman (1978a)

TABLE 3.6 Blood Oxygen Content, BAT Oxygen Consumption and Contribution t o W hole Bod[ Oxygen Consumption: Comparison with the Results of Foster and Frydman (1978a)

O-

~D

O ~r

o

O

>

o_

350

M. A, Cawthorne

P<.O01 ... 200 im E

P<.O01

P<.O01 T I100

3 Meal-depriv~ (N:13) • Meal-fed (N=12)

r- l o o

-

o

t--

50

~

0

Body weight

Fig. 3.10

BAT weight

VO2 100 mg o f BAT

902 Total BAT

Body weight, interscapular brown adipose tissue (BAT) weight, and rate of respiration (VO2). The respiratory rate is expressed as oxygen uptake per i00 mg per hour or per total tissue per hour. Data are means + standard error. Statistical analysis was done by Student's ttest. The test meal contributed 39.4 + 4.1 kcal, with protein contributing 8%, fat 23% and carbohydrate 69%. Reproduced from Glick, Teague, and Bray, Science 21__~3, 1125-1127, 4th September 1981, copyright 1980 by the American Association for the Advancement of Science; with permission.

nerve fibres to obtain a neuro-adipose preparation, which they perifused with Krebs-Ringer solution equilibrated with 95% 02:5% CO 9. The effect of electrical nerve stimulation, noradrenaline and octanoate on th~ metabolic responses of this preparation were observed via continuous monitoring NAD(P)H/NAD(P) redox state by measuring surface emitted fluorescence obtained with an excitation light of 366 nm. The responses to all three stimuli were depressed in brown adipose tissue preparations obtained from VMH-lesioned rats. The lower respOnse to octanoate indicates that the capacity for fatty acid oxidation at the Krebs cycle level is decreased and it therefore appears likely that the activities of one or more of the enzymes in this pathway is decreased in VMH-lesioned animals. It is interesting that the authors found qualitatively analogous alterations in ob/ob and db/db mice and fa/fa rats. If destruction of the ventromedial hypothalamic nucleus leads to a reduced metabolic capacity of brown adipose tissue, then it seems possible that stimulation of this nucleus will activate brown adipose tissue. Perkins, Bothwell, Stock and Stone (1981) have found that electrical stimulation of the ventromedial hypothalamus produces an increase in the temperature recorded by a thermocouple inserted

Metabolic Aspects of Obesity

351

into the interscapular brown adipose tissue and this effect was abolished by an intravenous injection of propanolol (0. In~]/kg). These findings suggest that brown adipose tissue is under the control of a specific section of the sympathetic nervous system. It is possible therefore, that the ventromedial hypothalamus serves a dual function in energy balance being involved in both an inhibitory, satiating effect on energy intake and a stimulatory effect on thermogenesis and energy output. Although experiments have not yet been reported, it seems quite likely that chemical agents that produce lesions in the ventromedial hypothalamus such as gold thioglucose and bipiperidyl mustards also produce effects on therTm~enesis. It is particularly likely to be the case for those agents such as monosodium glutamate which produce obesity without producing hyperphagia (Miller, 1979). In both the genetically obese rodents and the VMH-lesioned animals it is not clear at the present time whether defective thermogenesis is due to a reduced amount of, a reduced level of signals to or a reduction in the metabolic activity of brown adipose tissue. It is possible that a certain level of sympathetic nervous system is required in order to maintain the activity of brown adipose tissue. Thus, if VMH-animals are placed in the cold, it is claimed that brown adipose tissue can be reactivated (Assimacopoulos-Jeannet, Girardier, Rohner-Jeanrenaud and Seydoux, 1981). Similarly, acclimation of ob/ob mice to a temperature of 14°C leads to some restoration of the capacity of brown adipose tissue for thermogenesis (Hogan and Hinms-Hagen, 1980).

Non-Shivering Thermf~enesis in Man New-born infants require an effective thermogenic mechanism in order to overcome the sudden environmental temperature drop that occurs at birth. Even though the cold stress may be considerable, new-horn babies rarely shiver and it is assumed that non-shivering t h e ~ e n e s i s is responsible for the large rise in the metabolic rate. Full-term new-born babies have significant deposits of brown adipose tissue (15-40g). However, until recently it was thought that after the age of I year, non-shivering themrc~enesis was unimportant in man. Exposure to the cold is largely prevented by adjustments of the macro- and micro-environment and when such adjustments are incomplete, shivering is used to maintain heat production. However, if man is repeatedly exposed to cold, shivering ceases but the oxygen consumption is persistently maintained at an elevated level (Davis, 1961). In such conditioned subjects, the infusion of noradrenaline produces a 20% increase in resting metabolic rate (Joy, 1963). In addition, Rennie, Covino, Howell, Song, Kang and Hong (1962) showed an appreciable metabolic response to noradrenaline in cold-adapted Area ~ n e n , who are exposed regularly during pearl diving to water temperatures of I0oc. It was noted in the previous chapter that experimental evidence is available showing that obese subjects have a smaller thermic response to food and exhibit decreased heat production when exposed to cold. If these responses reflect a decreased capacity for non-shivering thermogenesis, it would be expected that obese subjects would show smaller increase in energy expenditure when given a noradrenaline infusion than lean subjects. James and his colleagues tested this hypothesis by comparing the metabolic response of women, who were lean and able to eat without concern about weight gain, with that of women who were obese and women who were previously obese but were now lean (Fig. 3.1 I). The increase in the metabolic rate in the lean group was 21% whereas in the obese and post-obese groups it was less than half this value (Jung, Shetty, James, Barrand and Callingham, 1979). The pattern of the response to noradrenaline infusion was remarkably similar to that produced by a standard meal in a similar study (Shetty, Jung, James, Barrand and

352

M . A . Cawthorne

0.8"

T

0.6 E e~ ,¢

0,4

0.2 < ! 0

, Noradrenaline in!'usion 5 10 15 30

I 45

f;',

90

Time (rain)

Fig. 3.11

and Callingham,

1981),

Absolute increase in RMR of women during and after 45 min intravenous infusion of noradrenaline. (Q), lean; (O), obese; (A), previously obese, now lean. RMR was measured by the ventilated hood technique using a Servo paramagnetic oxygen analyser and IR carbon dioxide analysis. RMR measured by this technique agrees with direct calorimetry with a difference of 0.09%. Values are means + SEM. Fully informed consent for the procedures was obtained and ethical approval was given by the unit's ethical committee. Reproduced from Jung, R.T., Shetty, P.S., James, W.P.T., Barrand, M.A. and Callingham, B.A. Nature 279, 322-323, copyright 1979, MacMillan Journals Ltd., with permission.

(Fig. 3.12).

On the basis of these studies, it has been suggested that in parallel with rodent models of obesity, obese man may have an abnormality in the brown adipose tissue therrsogenic system. Many authors have taken the view that there is too little brown adipose tissue in man to produce a thermogenic response; but, to account for the 20% increase in oxygen consumption produced by noradrenaline infusion only 30g of fully active brown adipose tissue is required (Cannon and Johansson, 1980). At autopsy, brown fat has been found in individuals of all ages. However, when replete with lipid, the microscopic differences between white and brown fat are slight and this may account for the few reports on its existence in adult man.

Metabolic Aspectsof Obesity

353

5.5

T 5.0

E 4-5 ._u o ..o

4.0

3.5

L

0

I

l

30

l

I

60

~

I

90

120

Time(min)

Fig. 3.12

Absolute metabolic rate and the response to a standard meal. (O), obese; (A), post obese, (O), lean. Reproduced from Shetty, P.S., Jung, R.T., James, W.P.T., Barrand, M.A. and Callingham, B.A. (1981). Clin. Sci. 60, 519-525, with permission.

Heaton (1972) showed that brown adipose tissue was present around the kidneys, adrenal glands and aorta and in the neck and mediastinum. She classified the brown adipose tissue samples obtained into 5 levels of structural organization based on light microscope examination. It is remarkable how many of the subjects showing the highest level of structural organization of brown adipose tissue died in extremely stressful conditions especially by fire. It is possible that brown adipose tissue can be rapidly reactivated from its quiescent state by high levels of noradrenaline. Suggestions that brown adipose tissue is involved in non-shivering thermogenesis in normal lean man (and woman) comes from t~o experimental studies. Rothwell and Stock (1979) showed that administration of the noradrenaline-releasing agent, ephedrine, produced local heating, as visualized

354

M . A . Cawthorne

by infra-red thermography, in areas ~i~ich typically contain bro~,m adipose tissue. Similarly, by using thermister probes, James and Trayhurn (1981b) measured local increases in heat production following noradrenaline infusion. These studies raise the possibility that non-shivering thermogenesis may well have a significant role in the maintenance of energy balance in man. However all of the studies in man that have been carried out so far have compared lean with obese (or post obese) subjects (see Fig. 3.11). Such studies leave open the question as to whether any defect in non-shivering therrmx/enesis (or brown adipose tissue metabolism) is a cause or a consequence of the obese state. (The post-obese subjects had been on a severely restricted diet for I to 2 years, and this is likely to reduce the ability for non-shivering thermogenesis) . Are there subjects who might possess the thrifty 'gene' and be the human equivalent of the genetically obese rodents? If these subjects exist, it would be of interest to determine if the young pre-obese subjects already had a reduced capacity for non-shivering thermogenesis. Prime candidates are the Pima Indians who have a high prevalence of obesity and have lived for generations in the relatively isolated Gila River Basin in Southwest Arizona. Periodic famines suffered by these people may well have produced the genetic pressure to be thrifty.

Chapter 4

Cellular Mechanisms of Altered Energy Efficiency

General Considerations The final energy source of all metabolic processes is the hydrolysis of adenosine triphosphate (ATP). The maintenance of adequate ATP leve]sin each and every cell of the body is of key importance in guaranteeing the continuing existence of that cell. However, the total pool of adenine nucleotides is relatively small and thus the rate of ATP generation must be constantly and rapidly adjusted to maintain adequate ATP levels in the face of changing rates of ATP utilization. Provided that substrate oxidation is tightly coupled to ATP-generation, the measurement of energy expenditure in a subject will also provide a measure of the rate of ATPproduction. On the basis of our knowledge of the pathways of substrate oxidation overall, calculation of the total yield of ATP and energy utilized can be made for the oxidation of glucose, fatty acids and amino acids:-

Glucose + 602

H

6 CO 2 + 6 H20 + 673 Kcals (+38 ATP-2 ATP = +36 ATP)

Palmitate + 2302

16 CO 2 + 16 H20 + 23980 Kcals (+131ATP-I ATP = +130 ATP)

Amino acid + 5.1 02

4.1 CO 2 = 0.7 urea + 2.8 H20 + 475 Kcals (+28.8 ATP-5.5 ATP = +23.2 ATP)

Thus the actual energy cost of production of ATP varies according to the substrate being used: Glucose

>

673 Kcals 38 ATP

=

17.7 Kcals/mol

Palmitate

~

2398 Kcals 131 ATP

=

18.3 Kcals/mol

Amino acid

>

475 28.8 Kcals ATP

=

16.5 Kcals/mol

If man lived at a thermoneutral temperature and had an abundant supply of food

355

356

M.A. Cawthorne

which he could receive by drip-feed, he would he able to regulate his rate of substrate supply to his rate of demand and thus the maintenance of energy balance would not be a problem. However, man is a periodic eater and a constant metabolizer. Thus, it is necessary to store nutrients as they become available, for later use. In addition, his choice of nutrients does not necessarily equate with his requirements and thus interconversion of nutrients may well be needed. The processing of food in the gut, its metabolic conversion and storage all require the expenditure of ATP. Based on our knowledge of metabolic pathways it is possible to calculate the minimal energy cost of processing ingested carbohydrate, fat and amino acids. This energy cost will appear in whole body measurements of energy expenditure as the specific dynamic action (SDA) of food. For carbohydrates, the SDA will be of the order of 7% of the intake if the carbohydrates are converted to liver or muscle glycogen. This value rises to 25% if the carbohydrates are converted to triglyceride. For amino acids, the SDA will be of the order of 25% whether these are converted to protein or to glucose. The smallest SDA of 4% is for the metabolic handling of dietary fat. The concept of metabolic efficiency is difficult to define. In animal husbandry, it is used to describe the yield in animal tissue in relation to the energy consumed; that is, the gain:feed ratio. In man the situation is somewhat different as in the majority of cases body weight is stable and energy balance appears to be maintained despite wide fluctutions in energy intake without the influence of large variations in visible work. To account for this it is necessary to identify some 'slippage' in the overall ATP economy. Thus either ATP synthesis must be at least partially uncoupled from substrate utilization or it must be dissipated without any net work taking place. There is little evidence to suggest that uncoupling of oxidative phosphorylation occurs as a general phenomenon under normal physiological conditions in the majority of tissues. However, Nicholls (1979) has proposed that in brown adipose tissue substrate oxidation may not necessarily be linked to the generation of ATP. He suggests the existence of a proton conductance pathway which bypasses the norreal coupling of the oxidation of reduced nucleotides to the generation of ATP in mitochondria. Several mechanisms of ATP dissipation also exist. These include the membrane-associated enzyme Na+,K+-ATP ase, and substrate (futile) cycles. The possible significance of these processes to the regulation of energy balance will be discussed.

Brown Adipose Tissue W h i l s t controversy exists on the role of brown adipose tissue in the maintenance of energy balance in man, there is general agreement that it is the only tissue known whose primary function is heat production. Brown adipose tissue obtained from animals in which there is no dispute of its thermogenic role (e.g. new-born or hibernating animals) shows, under the electron microscope, a characteristic appearance with many small multilocular triglyceride droplets and a large number of highly invaginated mitochondria (Afzelius, 1970). In contrast white adipose tissue cells, typically, contain one large triglyceride droplet and are said to be unilocular. Non-shivering thermogenesis in brown fat is initiated by the sympathetic nervous syst~: sympathetic fibres directly innervate the brown fat cells and it appears that several cells are coupled through one fibre. Separate innervation passes to the adjacent blood vessels. The nervous stimulation is mediated by noradrenaline and studies have shown that within I min of noradrenaline binding to the plasma membrane, there is an increase in beth the rate of lipolysis and the rate of respiration (Prusiner, Cannon, Ching and Lindberg, 1968; Williamson, 1970).

Metabolic Aspects of Obesity

357

.Z~

g~f f

300

~o "7

200 c) /

E c

>..

/ /o/

100

[z t

10-9

!

10-a

t

10.?

t

10a

I

10-5

Agonist concentration (M)

Fig. 4.1

Respiratory rate as a function of agonist concentration in isolated hamster brown adipocytes. ([]), L-isoprenaline; (O), L-norepinephrine; (A), L-epinephrine. Filled symbols represent D-forms of each respective agonist. Reproduced from Pettersson, B. and Vallin, I., 1976, Eur. J. Biochem. 6~, 383-390, by permission.

Noradrenaline interacts with the brown adipocytes predominantly through 8adrenoceptors. Studies using adrenergic blocking agents showed that noradrenaline stimulated non-shivering thermogenesis in vivo and increased oxygen consumption by brown adipocytes in vitro are blocked by propanolol (8-antagonist) hut not by the s-antagonist phentolamine. In addition it has been shown that the order of potency for stimulating respiration in brown fat cells is isoprenaline>noradrenaline>adrenaline (Pettersson and Vallin, 1976) (Fig. 4.1). This potency order is characteristic of a 8-receptor. Bukowiecki, Caron, Vallieres-Le Blanc (1978), using binding study techniques identified a receptor in brown adipocytes with a dissociation constant for noradrenaline of 2NM. This dissociation constant ~Duld effectively ensure that blood-borne noradrenaline has little effect on the tissue since concentrations in the blood are about InM under normal circumstances. Even so, it is clear that the administration of, or the release of relatively large doses of noradrenaline into the blood stream will initiate non-shivering thermogenesis. In a similar study, Svobada, Svartengren, SnOchowski, Houstek and Cannon (1979)

358

M . A . Cawthorne

U

lu E

~75 0.03-6 -6

KD: 1.08'O16

E ¢,

-~50o u >.~ .c I--

C5 .m. I

\

o

Bmax: 92 frnoleS~}O6ce[is

l-

lJ

g 0.01-

o m

r = 0959

"~

-\ 0.00

i

0

50 100 Bound (fmoles)

(-)- [3H]Dihydroalprenotol

Fig. 4.2

(B~

0.02-

~25" r--

nM

(nM)

(-)-(3H) Dihydroalprenolol binding to isolated brown fat cells as a function of radioligand concentration. A: Specific binding ~s a function of the total concentrations of (-)-(H) dihyd~oalprenoloi. B: Scatchard plot of specific (-)-(-H) dihydroalprenolol binding to isolated brown adipocytes. The maximum binding capacity (Bma x) was determined as the intercept with the ordinate, and is equal to 57,000 specific receptors for alprenolol per cell. Reproduced from Svoboda, P. et al (1979), Eur. J. Biochem, 102, 203-210, by permission.

used the potent B-antagonist dihydroalprenolol to measure binding. They identi fled a binding site with a Kd of 1.08 nM for dihydroalprenolol (Fig. 4.2) and were able to calculate that there were of the order of 60,000 binding sites per cell. Using competition studies they calculated that the apparent dissociation constant for noradrenaline was 176 nM (Table 4. I). However, for half-maximum respiration to occur a receptor occupancy of less than 10% was required, indic-

Metabolic Aspects of Obesity

359

ating that the brown adipocyte has spare receptors. In the absence of spare receptors, the ECs0 for noradrenaline-induced stimulation of r e s p i r a t i o n ~ u l d probably be similar to the Kd for the receptor itself: 176 nM. The existence of spare receptors leads to an apparent Kd (EC50) of about 6 nM.

TABLE 4.1 Potencies of B-~Jrenergic Agonists for competition With [3H] dih droal P{enolol Binding Sites and for Stimulation of Respiration in Intact Brown Adipocytes

Agonist

K~ (agonist)

'' EC50

K~

EC~0

nM (-)-Isoproterenol (-)-Norepinephrine (-)-Epinephrine

26 176 220

11 77 100

Occupancy for 50% stimulation of respiration %

2.4 16.9 22.0

2 6 9

8.5 8.8 9.0

K~ (agonist) = dissociation constant determined from competition studies. ,, = concentration of agonist needed for 50% stimulation of EC50 respiration in isolated cells incubated with 5 nM alprenolol. II K~' = dissociation constant determined from E C ~ , i.e. K~ = EC50/ l+(5/l.41)nM. EC~0 is derived from the results of Pettersson and Vallin (5). Reproduced from Svoboda, P. et al (1979). with permission.

Eur. J. Biochem. 102, 203-210,

Within I sac of the administration of noradrenaline and its binding to brown adipocytes, there is a change in the membrane potential and ion conductance (Seydoux and Girardier, 1978). Noradrenaline also activates adenylate cyclase in the plasma membrane and thereby raises the concentration of cyclic AMP (Pettersson and Vallin, 1976). As in other tissues, inhibition of the breakdown of cyclic AMP by methylxanthines increases the action of noradrenaline in increasing oxygen consumption and this action can be mimicked by high concentrations of dibutyryl cyclic AMP which is a lipophilic analogue of cyclic AMP that crosses the plasma membrane. The plasma membranes of brown adiix]cytes have been shown to contain nine soluble protein kinases, all of which have the ability to catalyse the transfer of the terminal phosphate from ATP to a protein acceptor (Knight and Skala, 1977). One of these is responsible for converting inactive hormone sensitive triglyceride lipase into its active form. Cyclic AMP initiates a cascade whereby inactive protein kinase is activated so that it can itself catalyse the activation of hormone sensitive triglyceride lipase. This then in turn breaks down triglycerides to diglycerides and fatty acids. Additional lipases results in the production of fatty acids and glycerol from the diglycerides. The natural substrates for the triglyceride lipase are the triglyceride droplets within the brown adilx)cytes. The lipase is located in the cytoplasmic compartment of the cell, and because of the multilocular nature of the triglyceride stores, a large surface area of triglyceride may well be available to the enzyme. This is in marked contrast to the situation in white adipose tissue.

360

M.A. Cawthorne cAMP protein kinase \ inactive HSL + ATP

-- proteinkinase+cAMF.~

©

active l

t- HSL'P+ADP

Pi HSL-phosphatas Fig. 4.3

Cyclic AMP-sensitive protein kinase. HSL = hormone sensitive lipase.

Fatty acids are the predominant fuel for thermogenesis in brown adipose tissue and oxidation of these takes place within the mitochondria. The substrate for fatty acid oxidation is fatty acyl-CoA and the fatty acyl CoA synthetase is located exclusively on the outer mitochondrial membrane (Pedersen, Slinde, Grynne and Aas, 1975). The inner mitochondrial m6mzbrane is not permeable to fatty acyl CoA and its translocation is dependent on a supply of carnitine and the activity of carnitine acyl CoA transferases I and II. (Fig. 4.4). Experiments using isolated mitochondria and palmityl carnitine as substrate indicate that oxidation capacity of the system is extremely high (Nicholls, Gray and Lindberg, 1972) and proceeds by the 8-oxidation pathway. The enzymes of the B-oxidation system form a multienzyme couplex associated with the inner mitochondrial membrane. The initial substrate is acyl CoA and this is shortened by two carbon units for each cycle of the B-oxidation system yielding acetyl CoA as well as FADH 2 and NADH. The continued metabolism of acetyl CoA can proceed by one of a number of pathways.

I]

It may enter the tricarboxylic acid cycle by condensing with oxaloacetate under the action of citrate synthase. The enzymes of the tricarboxylate cycle appear to have sufficient activity to account for the complete oxidation of acetyl groups produced by B-oxidation (Rafael, Klass and Hohorst, 1968; Rafael, Husch, Stratmann and Hohorst, 1970.)

2]

It may condense with itself under the influence of acetoacetyl CoA synthetase to form acetoacetyl CoA, which is the first step in ketone body formation.

3]

It may undergo transesterification to acetyl-carnitine, using carnitineacetyl transferase, after which it can be transported out of mitochondria.

4]

It may undergo hydrolysis by acetyl COA hydrolase to yield acetate and coenzyme A.

Only the tricarboxylate cycle pathway produces further oxidation within brown adipose tissue and thus the potential for further heat production. Since tricarboxylate cyclic intermediates are drained off for other purposes especially

Metabolic Aspects of Obesity

outer mit. membrane

361

inner mit. membrane

.ocetylCoA

~xidation ;oP FF~

¢ytol

Fig. 4.4

atrix

The fatty acid transport system across the inner mitochrondrial membrane. Taken from Cannon, B. and Johansson, B.W. (1980), Molec. Aspects of Med., ~, 119-222, with permission).

amino acid syntheses, it is important that the level of cycle i n t ~ i a t e s is maintained. This appears to be achieved in brown fat cells by the high activity of pyruvate carboxylase which catalyzes the formation of oxaloacetate from pyrurate (Cannon and Johansson, 1980). The theoretical overall reaction for the complete oxidation of palmitic acid is as follows : palmitate + 23 02 + 129 ADP + Pi

~

16 CO 2 + 16 H20 + 129 ATP

The majority of this ATP arises during the re-oxidation of NADH and reduced flavoproteins. The synthesis of ATP normally proceeds with a specific stoichiometry and the transfer of energy to ATP is highly efficient, i.e. there is coupling of phosphorylation to respiration. The concentration of adenine nucleotides is small, which means in practice that respiration only occurs and substrates are consumed when the cell has a requirement for energy which is detected by an increase in the concentration of ADP. There is normally no respiration without a requirement for chemical energy. In the situation where the require~nent is for heat, as in brown fat, a mechanism for regeneration of ADP must exist or the tight coupling of respiration of ATP

362

M.A. Cawthorne

R. CH2 "CH2 "C O S C O A

FADH2 R. CH

CH. C O S C O A

R. CHOH. CH2 "COSCOA

~

NAD+ NADH

R.CO.CH2 °COSC°A

~

R.COSCoA

Fig. 4.5

CoA. SH

+ CH3COSCoA

The reactions of ~-oxidation of fatty acids

synthesis must be reversibly uncoupled. During the re-oxidation of NADH and reduced flavoproteins, protons are pumped out across the inner mitochondrial membrane, which results in an electrochemical gradient or proton motive force across this inner membrane. (Mitchell, 1976.) The energy in the proton motive force is used to drive the synthesis of ATP from ADP and phosphate. Measurements of the ATP-synthetase activity show that it is insufficient to support the rates of respiration commonly found in brown adipose tissue mitochondria suggesting that uncoupling of ATP synthesis from respiration occurs. Nicholls has provided evidence that brown adiIx]cytes have a proton leak mechanism that allows a controlled re-entry of protons across the mitochondrial membrane. The entry of protons through this leak mechanism appears to be regulated by nucleotides, ATP, GTP, ITP, ADP, GDP and IDP all inhibit the proton conductance pathway. Although GDP is the most potent, ADP/ATP are probably the more important physiologically. Recent studies have shown that GDP binds specifically to a polypeptide of molecular weight 32,000 dalton (Heaton, Wagenvoord, Kemp and Nicholls, 1978). Their studies have shown that the concentration of this 32K polypeptide varies with alterations in the thermogenic state of the animal (Heaton, Wagenvoords, Kemp and Nicholls, 1978; Desautels, Zaror-Behrens and Hhllms-Hagen, 1978; Ricquier and Kader, 1976). This work suggests that this protein is either the 'leak' or lies very close to it and regulates it. The overall mechanism of the proton conductance pathway is summarised in Fig. 4.6. It is envisaged that in the basal state, respiration in brown adipocytes proceeds largely by a coupled mechanism and nucleotides hound to the 32K protein will keep the proton conductance pathway closed. In order to open this pathway following noradernaline stimulation, it se6m~d likely that an antagonist to nucleotide

Metabolic Aspectsof Obesity

MP

Fig. 4.6

363

~

The inner membrane of brown fat mitochondria in the thermogenic state. Purine nucleotides are removed from their binding sites by an antagonist, possibly acyl CoA. Reproduced from Cannon, B. et al, 1978, in: Strategies in Cold. Natural Torpidity and Thermogenesis, (eds. Wang, L.C.H. and Hudson, J.W.) pp. 567-594, Academic Press, New York, by permission.

binding would exist. Cannon, Sundin and Ronl~ert (1977) showed that long-chain acyl CoA were able to antagonise nucleotide binding thereby opening up the proton conductarDepathway (Fig. 4.7). The antagonism of long-chain acyl CoA could he overcome by increasing the amount of GDP and thus one can envisage that these two substances together exert control over the degree of leakage through the proton conductance pathway. This mutual antagonism hypothesis is attractive in that raised acyl COA levels which occur upon the induction of lipolysis would compete with nucleotides and open up the proton-conductance pathway. As soon as lipolysis ceased or ch~nical energy requirements increased the change in concentration of either acyl-CoA or nucleotides would close the pathway.

Provision of Suhstrates for Heat Production in Brown Adipose Tissue Experiments have shown that exposure of a fasted hamster to 4°C for 2h virtually depletes the triglyceride stores in brown adipose tissue (Lindberg, Bicker and Houstek, 1976). If brown fat is to function efficiently as a heat producing organ it is essential that it has an highly active system for the accretion of triglyceride and/or fatty acids. McCormack and Denton (1977) , using the incorporation of 3H20 into lipids, showed that brown adipose tissue has considerable capacity for fatty acid synthesis. This method measures the rate of synthesis

364

M, A. Cawthorne

~

0.21

o

D

20

3o

4b

/JM GDP

Fig. 4.7

GDP binding to brown adipose tissue mitochondria. Mitochondria 0.5mg/ml. (A) No further additions. (B) In the presence of 2~M palmitoyl CoA. (C) In the presence of 5~M palmitoyl CoA. (D) In the presence of 10~M palmitoyl CoA. Reproduced from Cannon, B. et al (1977). Febs Lett. 74, 43-46, by permission.

independent of the carbon source used for synthesis and is the method of choice for whole animal experiments since it also largely overcomes the pool-size problem that exist with physiological substrates such as glucose and lactate. The rate of fatty acid synthesis in brown adipose tissue in rats and mice is dependent on the t6~perature at which the animals are housed. The lower the environmental temperature the greater the rate of fatty acid synthesis in brown adipose tissue (Trayhurn, 1979; 1981a; Table 4.2). Trayhurn (1979) showed that acute I hour exposure of previously warm acclimated rats to the cold (4°C) did not produce an increase in the rate of fatty acid synthesis in brown adipose tissue but acute I hour exposure of cold-acclimated rats to a warm environment produced a very significant fall in the rate of synthesis (Table 4.3). In mice, in which because of the smaller body size a 4°C exposure is a more powerful thermogenic stin~lus, cold exposure for I hour produced a doubling in the rate of fatty acid synthesis in brown adipose tissue (Rath, Salmon and Hems 1979). These experiments suggest a close coupling of fatty acid synthesis with heat production. The coincidence of these effects has also been demonstrated in mice trained to eat their meals over a 4 hour period (Hollands and Cawthorne, unpublished). Feeding for 3 hours will initiate a 15 fold increase in lipogenesis in brown adipose tissue, whilst overall metabolic rate is increased some 2.5 fold. In adult rats and mice, experiments using Triton W.R. 1339 to inhibit lipoprotein lipase activity and thereby prevent uptake of blood-borne triglyceride show that

I07.9±17.2 34.9± 3.4 9.9± 1.8 16.6± 3.0 8.5± 0.7 -

22°C

376.9±56.8 44.7± 7.1 22.6± 5.8 55.3±12.4 18.7± 3.1 -

4°C

4.4± 1.0 80.5±14.6 2.1± 0.7 2.0± 0.7* 161.4±29.0 250.4±44.6

33°C

11.1± 1.6 58.9± 6.6 4.9± 0.8 4.6± 1.1" 284.1±24.9 365.6±34.4

22°C

per tissue

(zg-atoms H incorporated/h)

73.9± 11.9 91.3± 15.6 15.1± 3.9 23.5± 6.3* 629.5±110.3 833.3±142.4

4°C

Taken from P. Trayhurn

(1981), Biochem. Biophys. Acta 664, 549-560, with permission.

Mice were acclimated for 3-4 weeks at either 33 or 4°C, or were taken directly from an animal room maintained at 22°C. 3H20 (500~Ci) was injected intraperitoneally and tissues removed 60 min later. The results are expressed as mean values ~ S.E. for seven animals at 33 and 4°C, and for 15 animals at 22°C. For all tissues, except the liver, the differences between the cold- and the warm-acclimated mice (per g tissue and per tissue) were significantly different (p<0.01 or better).

*Total synthesis in the arbitrary sized sample removed.

28.4± 5.1 52.9±10.5 4.9± 1.3 8.9± 3.5 5.7± 1.0 -

33°C

per g tissue

Fatty Acid Synthesis

Fatty Acid Synthesis in Tissues of Mice Acclimated at 33p22 OR

Interscapular brown adipose tissue Liver Epididymal white adipose tissue Subcutaneous white adipose tissue Carcass Whole-body

TABLE 4.2 4oC

O1

CO

(D

o

o

>

o

o

M. A. Cawthorne

366

Table 4.3 Total Fatty Synthesis in Tissues from Warm- and Cold-Acclimated Rats

Interscapular brown adipose tissue

Temperature Group

c

Parametrial white adipose tissue

Acclim

Expt

A

28

28

9.5± 1.3 (14)

114.5±11.2 (14)

11.5±1.5 (8)

B

28

4

9.8± 2.7

131.8±15.4

10.8±1.2

(zg atoms 'H' incorporated h -I tissue -I)

(8)

a

Liver

C

4

4

191.0±19.9 b (14)

D

4

28

p<0.01, p<0.01,

b

d

(8)

(8)

286.9±36.7 (14)

5.7±0.9 a (8)

39.9i4.5 bd

105.3± 6.9 c

4.4±0.7 b

(8)

(8)

(8)

p<0.001; compared to group A p<0.001; compared to group C

The rats were acclimated for 3-4 weeks to study the acute effect of temperature change on fatty acid synthesis. The rats were moved to temperature controlled cabinets, 1 hour before the experiment. The results are expressed as mean values + SE with the number of animals in parentheses (data obtained by multiplying synthesis/g by the total tissue weight) Reproduced from P. Trayhurn (1979), Febs Letters, 104, 13-16, with permission.

the majority of the newly synthesized triglyceride found in brown adipose tissue is synthesized there (Trayhurn, 1981a). This is not the case in cold-acclimated hamsters (Trayhurn, 1980) where 50% of the newly synthesized triglyceride is imported. In suckling young mice, rates of fatty acid synthesis in brown adipose tissue are low in spite of high rates of thermogenesis (Trayhurn 1981a). Suckling mice have a high dietary intake of fat from milk and it is possible that this inhibits de novo fatty acid synthesis in brown adipose as it does in other tissues. The fatty acids released from digested dietary fat might be expected to be taken up directly by brown fat cells through the action of lipoprotein lipase. The carbon fuel(s) for the synthesis of fatty acids in brown adipose tissue have not been identified yet, although the administration of a glucose load to rats has been shown to increase the rate of lipogenesis. Insulin injections increase the rate of fatty acid synthesis whereas diabetes induced by streptozotocin decreases it (McCormack and Denton, 1977; Agius and Williamson, 1980). A number of studies have shown that glucose uptake in brown adipocytes is stimulated by insulin (Shackney and Joel, 1966; Fain, Reed and Saperstein, 1966; Czech, Lawrence and Lynn, 1973). The glycerol moiety of triglyceride comes from glycerol phosphate. In theory this could come from glycerol via glycerol kinase or from glucose. Whilst brown adipose tissue has a higher glycerol kinase activity than white

Metabolic Aspects of Obesity

367

adipose tissue (Fain et al, 1966), it seems likely that much of the glycerideglycerol arises from the metabolism of glucose within the brown adipocyte.

Defects in BrOw~] Adipose Tissue in Obese Rodents The finding that genetically obese rodents have an inability to respond to cold because of a failure of thermogenesis, when taken together with blood flow measurements, suggest that they might have defective brown adipose tissue. Both the ob/ o b m o u s e (Table 4.4) (Himms-Hagen and Desautels, 1978) and the db/dbmouse (GoodbodyandTrayhurn, 1981) show a decrease in the number of purine binding sites in mitochondria of brown adipose tissue indicating a reduced number of proton conductance channels. Furthermore, this mitcchondrial defect is present in 14 day old mice, prior to the obvious development of obesity, indicating that this change is not a consequence of obesity (Table 4.5).

Table 4.4 Atractyloside-Insensitive Binding of Purine Nucleotides (GDP and ADP) to Brown ~Ji~ose Tissue Mitochondria of Lean and ob/obMice exposed to either 28° or to 4° for 3 hours

LEAN MICE

OBESE MICE

28°

4°

28°

Body weight (g)

22.5± 0.2

21.0b±0.3

49.4a± 0.6

Interscapular brown adipose tissue (mg)

95.0± 2.0

60.0b±8.0

Binding of GDP pmoles/mg protein

83.4±12.7

120.7b±5.1

41.2a± 6.8

50.5a± 3.4

Binding of ADP pmoles/mg protein

58.8± 8.5

93.0b±5.6

42.8 ± 6.2

31.6a±11.2

404 a

±21

4°

49.4a± 0.6 443 a

±28

Mitcchondria

Values are means ~ SE of three experiments. Weights of mice and tissues are means for 12-13 animals used in the three experiments. a Significantly different from lean animals at the same temperature, p<0.05 or less b Significantly different from similar animals at 28°C, p<0.05 or less. Reproduced from Himms-Hagen, J. and Desautels, M., 1978, Biochem. Biophys. Res. Commun. 83, 628-634, with permission.

Exposure of a rat or mouse to 4°C produces an increase in the purine binding capacity, that is the number of proton conductance channels, within I hour (Desautels, Zaror-Behrens and H ~ - H a g e n , 1978). However, acute cold exposure of ob/ob mice (Hinrns-Hagen and Desautels, 1978) failed to increase GDP binding to brown adipose

368

M.A. Cawthorne Table 4.5 GDP Binding to Brown Adipose Tissue Mitochondria of Adult and 14 Day-oldLean and Diabetic-obese (db/db) Mice

GDP Binding (nmol/n~] of mitochondrial protein)

LEAN

DIABETIC-OBESE

Adult

0.257±0.009 (9)

0.151±0.010 (9)***

14-day-old

0.370±0.024 (5)

0.208±0.017 (5)***

Mitochondria were incubated with 10~M-[3H] GDP for 7 min at 20°C pH 7.1. For details see the Experimental section. The Specific activity of cytochrome oxidase was similar in the mitochondrial preparations of lean and diabetic-obese animals, indicating that differences in GDP binding between the two genotypes were not due to any difference in the purity of the preparations. The results are mean values + SEM for the numbers of groups of mice shown in parentheses. ***p<0.001 compared with lean mice. Reproduced from Goodbody, A.E. and Trayhurn, P., 1981, Biochem. J. 194, 1019-1022, with permission.

tissue mitcchondria (Table 4.4). H ~ - H a g e n (1980) claims (citing a personal conrmanication from L. Landsherg and J.B. Young) that the activation of the sympathetic nervous system in response to cold in the ob/ob mouse is relatively normal. Furthermore, ob/ob mice have normal levels of plasma free fatty acids upon cold exposure (Thenen and Cart, 1978), indicating that lipolysis is activated at least in white adipose tissue. Since brown fat in ob/ob mice is replete with triglyceride, there appears to be no defect in the stimulus to thermogenesis nor in the availability of fuel but rather an inability to switch on the thermogenic process by activating the proton conductance pathway. The effect of acute cold exposure to increase the number of GDP binding sites is probably merely due to an unmasking of those sites. It is not known if the ob/ob mouse has a smaller total number of proton conductance channels or whether these remain masked. Paradoxically, although brown adipose tissue from ob/ob mice appears to be functionally inactive with respect to heat production, it is massively hypertrophied. Whilst much of this hypertrophy is merely due to increased accumulation of lipid, there is nevertheless twice as much protein present (Hogan and Hirm~-Hagen, 1980) (Fig. 4.8). The number of proton conductance channels in the tissue can be increased by acclimating the ob/ob mice to 14°C (Fig. 4.9). This treatment also normalises the structure of brown adipose tissue mitochondria. (Electron micrograph pictures of brown adipose tissue mitochondria from ob/ob mouse show a bizarre collection of structural features and a total lack of the normal organised pattern of membranes - Hogan and Hinms-Hagen, 1980.) No studies on fatty acid synthesis in brown adipose tissue in ob/ob mice have been reported. In unpublished studies, Hollands and Cawthorne have shown similar rates

Metabolic Aspects of Obesity

369

Mg 4([

30

OBESE

LEAN

20

10 EXPOSURE 28 °

Fig.

4.8

14 °

28 °

14 °

ACCLIMATION

T o t a l p r o t e i n c o n t e n t of interscapular plus subscapular brown adipose tissue of warm-acclimated (28°C) and cold-acclimated (14°C) lean and obese mice exposed to their temperature of acclimation or to 4°C. A significant effect of acclimation to 14°C is indicated by black portions of bars. There is no significant effect of exposure to 4°C in any group. Total protein is significantly greater than in corresponding lean animals in both obese animals at 28°C (p<0.001) and obese animsla at 14Oc (p<0.001). Reproduced from Hogan, S. and Himms-Hagen, J. 1980, Am. J. Physiol. 239, E301-E309, with permission.

of synthesis in both ob/ob and lean mice. Certainly, the high triglyceride content of brown adipose tissue in these mice would not suggest that there is any defect in synthesis. Rather the reverse may he the case since an inappropriately high rate of lipogenesis will result in excess fat accumulation, causing the coalescence of fat droplets and eventually producing the unilocular appearance of ob/ob mouse brown adipocytes. This has the disadvantage that the surface area of triglyceride available to hormone sensitive lipase is decreased and this could he a limitation on the supply of substrate for heat production.

Brown Adi~ose Tissue in Cafeteria-Fed Rats In contrast to the genetically obese rodents, it might be expected that brown

370

M.A.

Cawthorne

adipose tissue of the thermogenic cafeteria-fed rat would be more active than in controls. In a study in which overfeeding was induced without any overall weight gain, the weight of the interscapular brown adipose tissue doubled and this was accompanied by a similar increase in the specific activity of a number of mitochondrial enzymes (BrOOks, Rothwell, Stock, C
LEAN O t,_

OBESE

0.2

Q. C~

E 1 1

O

0.1

E ¢..

EXPOSURE

4° 28 <

Fig. 4.9

14 ~

28 °

14 °

ACCLIMATION

Binding of GDP by mitochondria isolated from interscapular plus subscapular brown adipose tissue of warm-acclimated (28°C) and cold-acclimated (14°C) mice exposed to their temperature of acclimation or to 4°C. A significant effect of acclimation to 14°C (p<0.001) is indicated by black portions of bars. A significant effect of exposure to 4°C is observed only in lean mice acclimated to 28°C (p<0.005) and is indicated by cross-hatched portion of bar. Binding is significantly smaller (p<0.001) in mitochondria of obese mice acclimated to 28Oc than in corresponding lean mice. Binding is significantly smaller (p<0.001) in obese mice acclimated to 14°C compared with lean mice acclimated to 14°C hut is not significantly different in obese mice and lean mice acclimated to 14°C and exposed to 4°C. Bars represent means + SE for 5 (lean mice acclimated to 28°C), 7 (lean mice acclimated to 14°C), 4 (obese mice acclimated to 28°C) and 6 (obese mice acclimated to 14°C) preparations of mitochondria obtained by pooling tissue from 3-4 mice. Reproduced from Hogan, S. and Himms-Hagen, J., 1980, Am. J. Physiol. 239, E301-E309, with permission.

Metabolic Aspectsof Obesity

371

Table 4.6 Mitochondrial Enzyme Activities in Interscapular Brown Adi~ose Tissue frc~Control and Cafeteria-FedRats

CONTROL

CAFETERIA

Weight of animals (g)

196.5 ± 3.3

190.7 ± 2.5 NS

Interscapular brown adipose tissue mass (mg)

275

538

±I 0

±I 8*

Total brown adipose tissue protein (mg)

32.4 ± 2.4

58.7 +- 4.0*

Total cytochrome oxidase activity (~mol cytochrome c oxidized per min per mg tissue)

19.8 ± 3.3

44.2 ± 7.1t

Mitochondrial cytochrome oxidase activity (~mol cytochrome c oxidized per min per total mitochondrial preparation)

5.1

e-Glycerophosphate dehydrogenase activity (~mol 09 consumed per min per total mi[ochondrial preparation)

0.58± 0.03

+- 0 . 7

11.8

± 1.5t

1.62± 0.19"

The animals used were aged approximately 6 weeks. Protein content of tissue homogenates and mitochondrial preparations was determined using a protein dye reagent kit with a bovine serum albumin standard (Bio-Rad). Mitochondria were prepared using the isolation procedure described by Slinde et al. Cytochrome oxidase activity was determined spectrophotometrically using a modification of the assay described by Yonetani and Ray and ~-glycerophosphate dehydrogenase activity was assayed polarographically. The specific activities of the two enzymes (that is, activity per mg mitochondrial protein) were not different in the two groups but the amount of mitochondrial protein isolated was different - 3.5~0.3 and 7.1~0.4mg in control and cafeteria-fed groups, respectively (p<0.001). Comparison of cytochrome oxidase activities in whole tissue homogenates and mitochondrial preparations indicates that the fractional yield of mitochondria was identical in both groups. Results are mean values + SEM, n=8. NS, not significant (p>0.05). *p<0.001, tp<0.01, compared with control values. Reproduced from Nature 286, 274-276, copyright 1980, Macmillan Journals Ltd., with permission.

Cold-acclimation (as opposed to short-term cold exposure) has been shown to lead to alterations of mitochondrial structure leading to selective synthesis of the 32K polypeptide (Desautels and Hinms-Hagen, 1979), in addition to tissue hyperplasia. However, cafeteria-feeding merely produced tissue hyperplasia without any selective increase in the content of the 32K polypeptide (Hinms-Hagen, Triandafillou and Gwilliam, 1981). This difference in action between cafeteriafeeding and cold acclimation may reflect a quantitative difference in thermogenic stimulus of these two treatments.

372

M.A. Cawthorne Table 4.7 Purine Nucleotide (GDP) Binding to Interscapular Brown Adi~ose Tissue Mitochondria from Cafeteria and Control Rats Maintained at either 24oc (warm) or 4°C (cold)

Animal weights (g) Mitochondrial protein recovered (~g) GDP binding (pmol GDPpermg mitochondrial protein)

Warm control

Warm cafeteria

Cold control

Cold cafeteria

326.3±18.1

306.0±25.5

296.6±11.8

327.4±21.2

5.7± 0.9

8.7± 0.9*

17.2± 1.4¶

21.0± 1.3¶

52.6± 7.4

135.3±24.0%

249.8±17.8¶

237.5±14.6¶

7

5

5

5

*p<0.05, %p<0.01, ¶p<0.001 compared with warm controls Reproduced from Brooks et al (1980). Reprinted by permission from Nature, 28__6, 274-276, copyright 1980, Macmillan Journals Limited.

B_r_own Adipose Tissue in Man NO biochemical studies on brown adipose tissue in man are known to the author. A major problem in working with brown adipose tissue in vitro is its high oxygen requirement. Because of the high oxygen requirement, tissue ATP content falls alarmingly during the isolation procedure (McCormack and Denton, 1977) and this fact alone causes concern about the interpretation of any studies in vitro. Brown adipose tissue is not readily available from living man and it seems likely that samples obtained post-mortem would not be viable. Thus, there is a great need for a non-invasive method that gives some measure of the activity of brown adipose tissue and/or the proton conductance pathway.

Sodium-Potassium ATP-ase Na +, K+-ATP-ase is a ~embrane associated enzyme involved in the active transmembrane transport of N a (Skou, 1957). The distribution of the enzyme is ubiquitous. The importance of the enzyme is in maintaining intracellular sodium and potassium concentrations at levels widely differing from those in extracellular fluids. In addition, the resulting ion gradient is used to drive many secretory processes. Na +, K+-ATP-ase has been proposed to account for a substantial portion of the thermogenesis induced by thyroid hormones (Smith and Edelman, 1979) and it may contribute to thermogenesis in brown adipose tissue (Horwitz, 1979). However, as noted earlier, the relatively low activity of the ATP-synthetase, let alone Na t, K+-ATP-ase militates against this pathway having a major importance in brown adipose tissue. On the other hand, it may well be of significance in muscle, where because of the large mass, a relatively low activity system could make a significant contribution to overall energy balance. A number of studies have shown that Na+,K+-ATP-ase is altered in obese (ob/ob)

373

Metabolic Aspects of Obesity Table 4.8 HindlimbMuscle Weight and [3H]ouabain Binding to Muscle Preparations

33°C

14°C

HindlimbMuscle Lean

Total wt, g

Obese

Lean

Obese

1.9

1.4

1.7

0.8

Particulate protein, mg/g muscle

71.0

73.0

68.0

69.0

Nonspecific [3H]ouabain binding* pmol/mg protein

2.3

1.9

2.1

1.8

Specific [3H]ouabain binding# pmol/mg protein

1.2

1.0

2.0

2.0

K d value, ~M

0.031

0.035

0.033

0.036

[3H]ouabain binding site conen. pmol/mg protein pmol/total hindlimbmuscle

1.2 164.0

1.0 103.0

2.2 252.0

2.1 122.0

Values are means for 9 to i0 lean and obese mice housed at either 33oc or 14°C for 3 weeks. * [3H]ouabain (0 ~ M ) binding observed in the presence of excess nonlabelled ouabain (4mM). # i H]ouabain (0.4~M) binding minus [3H]ouabain (0.4~M) binding observed in the presence of excess nonlabelled ouabain (4mM). Reproduced from Lin et al, 1980, Am. J. Physiol. 238, EI93-EI99, with permission.

Km~_e. The skeletal muscle of obese (ob/ob) mice has a lower concentartion of Na +, ATP-ase enzyme units than the muscle of lean mice. (Lin, Romsos, Akera, Leveille, 1978) (Table 4.8). Since there is also a reduced muscle mass in ob/ob mice, it w a s calculated that the adult ob/ob mice had only half the total number of muscle Na+,K+-ATP-ase enzyme units as lean animals. In an attempt to demonstrate that this reduced activity occurs early in the development of the obesity, rather than being merely a secondary consequence of it (Lin, Romsos, Akera and Leveille, 1979), the number of enzyme units in muscle of 14 day-old pre-obese (ob/ob) mice was determined. These mice, which were identified on the basis of their low oxygen consumption, had 28% frewer Na+,K + ATP-ase enzyme units in skeletal muscle than lean mice. In order to truly show that a particular lesion is causal in obesity in the ob/ob mouse, it may be necessary to show the lesion is still present in animals maintained at thermoneutrality. If mice are maintained at this temperature, there is unlikely to he any difference in sympathetic tone (see chapter 5). No such studies have been carried out, but this author would predict that there would be no difference in the Na+,K+-ATP-ase activity between lean and obese mice maintained at thermoneutrality. Alterations in Na+,K+-ATP-ase activity in livers in obese (ob/ob) mice have also been found (Bray, York and Yukimura, 1978; Guernsey and Morshige, 1979; Lin, Vander, Tuig, Romsos, Akera and Leveille, 1979), although this defect appears to develop in parallel with the obesity rather than precede it. It has been sugges+ + ted that the principal defect in the liver Na ,K -ATP-ase system of obese mice is its lack of responsiveness to thyroid hormones (York, Bray and Yukimura, 1978). Treatment of lean mice with tri-iodothyronine (20pg/kg body wt. for I week)

374

M. A. Cawthorne Table 4.9 Effect of Triiod0th~ronine on Enzymatic Activity in Liver Homogenates of db/db and Lean Mice

Animals

Treatment with T3

(Na+ + K+) ATPase

Mg++-ATPase

Glycerol 3Phosphate Dehydrogenase

~mol Pi/mg prot/hr P

~i/02/mg prot/hr P

P

db/db

0 +

3.83±0.57 (5) 4.35±0.28 (5)

NS

0.69±0.26 0.82±0.27

NS

9.40±2.0 14.9 ±0.65

<.05

Lean (+/+)

0 +

3.56±0.21 (5) 3.73±0.47 (5)

NS

0.92±0.27 1.60±0.14

<.01

10.0 ±0.7 21.3 ±2.6

<.01

The activity of the (Na+ + K+)-ATPase was measured as the ouabain-suppressible production of phosphate from ATP in a whole homogenate of liver. The glycerol 3-phosphate dehydrogenase was measured manometrically. Mean + SEM (Number of animals). Reproduced from Bray, G.A., York, D.A. and Yukimura, Y. (1978). Life Sciences 22, 1637-1642, with permission.

produced a large increase in enzyme activity, but a similar treatment to obese ob/ob or db/db mice was without effect (Table 4.9). However, in other studies using thyroxine (150-200zg/kg body weight for 2 weeks), Lin, Vander-Tuig, Romsos, Akera, and Leveille (1979) found a greater increase in the number of enzyme units in obese (ob/ob) mice than in lean (Table 4.10). These apparent differences between two laboratories could be the result of the different methodologies used. In the latter study the number of enzyme units was determined by the binding of the enzyme inhibitor [3H]ouabain. In the first study, functional enzyme activity was determined. To resolve these apparent differences, dose-response curve with respgct to thyroid hormone sensitivity are required using both methods. If the NaTKT-ATPase system has an overall significance in energy balance, it might be expected that it would be increased in animals showing dietary induced thermogenesis. However, with the exception of the d(~nonstration that Na+K+-ATPase activity of brown adipose tissue measured in vitro had a positive correlation with in vivo oxygen consumption in control and cafeteria rats (Rothwell, Stock and Wyllie, 1981) (Fig. 4.10), there have been no published studies. The activity of the Na+K+-ATPase system in obese man has been studied by two groups. De Luise, Blackburn and Flier (1980) found a significant reduction in the number of enzyme units and in the maximum activity of the system in red blood cells obtained from obese subjects. Red blood cells were employed in this study because of their ready-availability and in the hope that any alterations found would reflect those occuring in other tissues such as liver and muscle. Bray, Kral and Bjorntorp (1981) measured the activity of the enzyme system in liver biopsy samples. In contrast to the work of De Luise et al (1980) , they found an increase rather than a decrease in the enzyme activity in the obese subjects. In addition, Mir et al (1980) have failed to obtain the same results as De Luise in spite of using an identical technique. The activity of the Na+K+-ATPase system is influenced by drugs, hormones (Schwartz, Cindenmayer and Allen, 1975) and nutritional status (Kapley, 1978). With such a multiplicity of controlling factors, which are

Metabolic Aspects of Obesity

375

Table 4. I0 Body Weight and Energy, Hindlimb Muscle Weight, and-[ 3H] ouabain B [ndin@ to Muscle Preparations

SALINE

THYROXINE

Lean

Obese

Lean

Obese

Body weight, g

24.3

38.5

25.3

33.8

Body energy, kcal/ carcass

50

Hindlimbmuscle total wt, g Particulate protein, mg/g muscle

2.0 66

155 1.3 66

47 2.1 65

118 1.1 66

Specific [3H]ouabain binding* pmol/m@ protein

1.6

1.1

1.9

1.8

NOn-specific [3H]ouahain binding# pmol/m@ protein

2.4

2.1

2.2

1.6

Kd value, ~M

0.036

0.033

0.032

0.028

[3H]ouabain binding site concn, pmol/mg protein

1.7

1.2

2.0

1.9

pmol/total hindlimbmuscle

217

101

274

141

Values are means for 6 pairs of saline-treated lean an~ obese mice or 9 pairs of thyroxine-treated lean and obese mice. * [ H]ouabain (0.4ZM) binding minus [3H]ouabain (0.4~M) binding observed in the presence of excess nonlabelled ouabain (4mM). +[3H]ouabain (0.4~M) binding observed in the presence of excess nonlabelled ouabain (4mM). Reproduced from Huey-Lin et al, 1979, Am. J. Physiol. 237, E265-E272, with permission.

difficult to control in human subjects, caution is needed in interpreting any changes in enzyme activity. The current studies should he regarded as interesting preliminary investigations, but many more studies will he required before the role, if any, of Na+-K+-ATPase in the pathogenesis of human obesity can be decided.

Substrate Cycles T he concept of suhstrate cycles has been developed primarily be Newsholme as a mechanism for increasing sensitivity in metabolic regulation. In its simplest form a substrate cycle is produced when a non-equilibrium reaction in the forward direction of a metabolic pathway is opposed by another non-equilibrium reaction in the reverse direction. The two reactions must be catalyzed by separate enzymes.

376

M.A. Cawthorne

18"O"

• control o cafeteria

17"0

O 16"O' E

& o

15-0, 0

o~ c (D nr

14.0-

13'0-

0.1

0:2

0~3

0'.4

Na+K ÷ATPase activity pmol Pi./mg/h

Fig. 4.10

Relationship between oxygen consumption and brown adipose tissue Na+,K+-ATPase activity in rats fed stock diet (control) or a varied and palatable diet (cafeteria). Reproduced from Rothwell, N.J., Stock, M.J. and Wythe, M.G. (1981), Biochem. Pharmacol. 30, 14091712, with permission.

In the hypothetical reaction given in Fig. 4.11 for every molecule of A converted to B and back to A again, cher~ical energy must be converted into heat i.e. ATP is consumed and the heat produced is leaked to the environment. Newsholme (1980) has suggested that a cycle consisting of fructose-6-phosphate and fructose I ,6 bisphosphate could he important in controlling the rate of degradatior

Metabolic Aspects of Obesity

377

El S

.=A

B

~P

E2 Fig. 4.11

Hypothetical substrate cycle

ATP

ADP

~spha~isphosphot e

glycogen --~ ~ "-~ fructOse 6-

//~'~"'~'~fructose

Pi

Fig. 4.12

---~

---~

~

Ioctote

H20

The Fructose 6-Phosphate-Fructose Bisphosphate Cycle in the Glycolytic Pathway from Glycogen to Lactate. PFK denotes phosphofructokinase, FBPase fructose 1,6-bisphosphatase, and Pi inorganic phosphate. Reproduced from Newsholme, E.A. (1980), Reprinted by permission of New England J. Med. 302, 400-405

of glycogen tO lactate in muscle during exercise (Fig. 4.12). He suggests that by having two reactions opposing each other, one can achieve a greater sensitivity in the control of the flux through the total pathway (Fig. 4.13). The increase in the rate of cycling prior to exercise, is the bicche~nical equivalent of revving up the engine of a car prior to the start of a race. The major product of an increased rate of cycling is heat and hence the possibility exists that such pathways could be involved in weight regulation. It is of interest that this cycle has been d6~nonstarted to be the heat producing mechanism in the flight muscle of the bumble bee (Clark, Bloxham, Holland and Lardy, 1973). This insect cannot fly if its body t6mperature falls below 30°C, thus in autumnal weather the ~nsect needs to produce heat whilst gathering pollen in order that it can take off again. The amount of energy that can be lost in any one substrate cycle is indicated by the maximum rate of cycling. For the fructose 6-phosphate:fructose 1,6-bisphosphate cycle in human muscle the maximumrate is 2~molmin-1 g-l, and leads to the hydrolysis of 2~mol ATP min-lg -I . Thus this cycle, if switched on maximally for 24h per day, would result in the consumption of 1600 kcals (Newsholme, 1980). Clearly it is extremely unlikely that this cycle ~<)uld be maximally active for 24h per day but it nevertheless illustrates that even if it is active for only a few hours per day, a significant heat production could be achieved. The fructose 6,phosphate:fructose 1,6-bisphosphate cycle is just one example of a substrate cycle. Other possibilities are the triglyceride-fatty acid, and glycogen-glucosel-phosphate cycles. If suhstrate cycles are to be of importance in energy balance, one needs to consid~

M. A. Cawthorne

378

55 Resting condition 5

200O

Anticipation of exercise

p.

~.F6 p / ~ ~ F

100 ~.

1900

~-F6P

Exercise

FBP

4 9"90011.

100 2OO0 Immediately after exercise

~F6P'/~FBP

100 L v

1900 5OO

After exercise before complete recovery

~F6P

FBP

50

450 Fig. 4.13

Hypothetical Fluxes in the Fructose 6-PhosphateFructose Bisphosphate Cycle and in Glycolysis before, during and after exercise. The numbers refer to the rates of the reactions shown, and those on the right represent glycolytic flux in nmol min -I g-l. F6P denotes fructose 6phosphate, and FBP fructose bisphosphate. The ratios of cycling rate to flux can be readily calculated from the numbers given; the highest ratio is present during anticipation of exercise, and the lowest during exercise. The rate of cycling is 90 times higher before complete recovery from exercise than it is at rest. Reproduced from Newsholme, E.A. (1980). Reprinted by permission of New England J. Med. 30__~2, 400-405.

how they might he affected by various factors known to cause modulation in energy balance. One possibility raised by Newsholme is exercise. Theoretical calculations show that in order to he of significance in producing weight reduction, the degree of exercise has to he extreme. Calculations show that one needs to run 50 miles in order to lose a kg of fat. However, in practise, moderate regular exercise can make a very significant inloact. Newsholme (1980) notes that after exercise, more oxygen is consumed than is required to support resting metabolism the so-called oxygen debt. This debt has three phases, rapid, slow and ultraslow. The rapid phase is almost certainly due to the replenishment of creatine

Metabolic Aspects of Obesity

379

phosphate stores and reoxygenation of myoglobin. In part, the slow phase can be equated with the reconversion of lactate to glucose and glycogen. However, there is a significant amount of extra oxygen consumption during this phase and during the whole of the ultra-slow phase that presently cannot be accounted for. Newsholme predicts that this extra oxygen is consumed in suhstrate cycles, and arises through a stimulation of these cycles by a prolonged elevation in the concentration of circulating catecholamines. The increase in heat production that occurs after a meal has many properties that are consistent with the sti~]lation of cycling rates (Newsholme, 1976, 1978). Earlier in this chapter, the minimal cost of processing the various dietary nutrients was noted. The addition of a degree of cycling at various points along these metabolic pathways would increase these energy costs and since this is a controllable variable, it Would open the possibility of having the thermic effect of food as a variable rather than a constant factor. The control of rates of cycling through any of the postulated suhstrate cycles requires independent control of both sides of the pathway. This could be achieved by insulin promoting the forward reaction and catecholamines promoting the reverse reaction. Thus, high rates of cycling ~auld occur when the levels of insulin and catecholamines are high. Such a situation occurs post-prandially, at times of stress, and before and after exercise. In obesity, it would be proposed that either the activity of the enzymes of the substrate cycles are low or that there is a failure in either the production or activity of the hormonal effectors.

Concludin~ Remarks NO studies have yet been carried out that demonstrate unequivocally the flux through any th~nogenic pathway. Studies in rodents suggest that brown adipose tissue is a potent thermogenic tissue in these species. However, these studies have not yet been able to show the quantitative÷importance of the proton-conductance pathway, the leakage of energy through Na K+-ATPase or the loss of energy by substate cycling. With larger species including man, the significance of brown adipose tissue as a thermogenic organ is questionable. In these species a low activity system in muscle or adipose tissue could well be quantitatively much more significant than a highly active system in brown adipose tissue.

Chapter 5

The Sympathetic Nervous System and Energy Balance

Catecholamines influence metabolism in two major ways. First, they increase the rate of cellular metabolism and secondly they stimulate the conversion of cc~plex fuels into readily usable suhstrates. Catecholamines are released frGm sympathetic neurons and the adrenal medulla. In recent years, it has become clear that many of the physiological effects of catecholamines are predcminantly mediated by noradernaline released from nerve terminals and under most circumstances the adrenal medulla reinforces the effects of the sympathetic stin~lation. The major exception to this is the response to hypoglycaemia when adrenal medullary secretion of adrenaline is preferentially activated and the sympathetic nervous system is functionally depressed (Landsberg and Young, 1981 ). In the previous chapter, various heat producing mechanisms were outlined that might have significance in the maintenance of energy balance in man. In each of these mechanisms, catecholamines have been shown to have the capability of stimulating the heat producing process. Thus, the capacity for proton conductance in brown adipose tissue is increased by noradrenaline stimulation and repeated treatment of adult rats with the synthetic 8-agonist isoprenaline produces hyperplasia of brown adipose tissue as does cold exposure (Mory, Ricquier and Hermon, 1980). The activity of Na+,K -ATPase in vitro is increased by noradrenaline (Horwitz, Horowitz and Smith, 1969; Herd, Hanmond and Hamolsky, 1973) and recent studies by Brooks (1981) have shown that the activity of the triglyceride-fatty acid substrate cycle is also stimulated. It might therefore be reasonable to propose that the activity of the sympathetic nervous system may well be influenced by dietary manipulations that are known to cause modulation of the overall energy balance. Att6~pts to define the role of the sympathetic nervous system in any experimental situation have suffered from the lack of a method for measuring the activity of the unperturbed synloathetic nervous system. This proble~n is cc~pounded by evidence that sympathetic outflow to different organs is heterogeneous and thus neither plasma nor urinary noradrenaline can be used to obtain an accurate assessment of sympathetic activity or specific organs. Also, since the level of noradrenaline within a tissue remains relatively stable despite changes in sympathetic activity, levels of noradrenaline do not allow an assessment of the functional state of the syn~oathetic nervous system to he made. Recently Landsberg and his colleagues have er~oloyed a technique of measuring [3H]noradrenaline turnover to assess sympathetic activity in individual tissues. [3H]-noradrenaline is taken up by amine transport system of the axonal membrane

380

Metabolic Aspects of Obesity

381

and is rapidly cleared from the circulation. It equilibrates with endogenous noradrenaline stores within the nerve terminal and is then released along with endogenous noradrenaline in response to sympathetic nerve impulses (Fig. 5.1).

~r

N

Fig. 5.1

Schematic representation of a sympathetic-nerve ending. Norepinephrine (NE) is synthesized within sympathetic-nerve endings from circulating tyrosine (Tyr) by means of the intermediates, dopa and dopamine (DA). NE is stored in granules and released in response to incoming nerve impulses (wavy arrow at top). Some of the NE released diffuses into the circulation, and some interacts with adrenergic receptors on the postsynaptic cell membrane (shaded area at right). Neuronal NE stores are replenished by biosynthesis and by the aminetransport system of the axonal membrane, which takes up circulating or locally released NE. The monoamine oxidase (MAO) within the nerve ending degrades a portion of the recaptured NE. Reproduced from Landsberg, L and Young, J.B. (1978) New Eng. J. Med. 29___88,1295-1301, with permission.

%~ne rate of disappearance of [3H] noradrenaline, expressed as a rate of change of the specific activity of noradrenaline gives a measure of the sympathetic activity of the tissue. Using this method, Young and Landsberg (1979) showed that when animals were placed in a cold room to stimulate the sympathetic nervous system, noradrenaline turnover was increased markedly in heart and pancreas but there was little change in the turnover in liver (Fig. 5.2). The same authors have examined the effect of nutritional status on sympathetic activity. It might be anticipated that fasting would stimulate the sympathetic nervous system since fasting leads to an enhancement of the rate of lipolysis and glycogenolysis. Both of these prOcesses are stimulated by catecholamines. However, Young and Landsberg (1979) found that fasting decreased noradrenaline turnover in heart, liver and pancreas. Refeeding led to a rapid restitution of the rate of noradrenaline turnover to that found in ad-lib fed controls (Fig. 5.3).

382

M.A. Cawthorne

4IB

'°"IA

PANCREAS

.CONTROL

C- 103

LIVER

,o3 CONTROL

hr

c°~f t~262~tr boz

I0 2

HOURS

Fig. 5.2

~

L~

~4

HOURS

Effect of cold exposure on NE turnover in rat A: pancreas and B: liver. Immediately after injection of [3H]NE (250 ~Ci/kg iv) cold-exposed animals were placed in a cold room (4oc). Data are plotted as means ~ SE for specific activity of organs from 5 animals in both groups at each time point. The slope (k) of each line is significant at P<0.001. Open circles represent control and closed circles cold-exposed animals. Reproduced from Young, J.B. and Landsberg, L, Am. J. Physiol. 236, E524-E533, 1978, with permission.

These findings on noradrenaline turnover, if replicated in heat-producing tissues, are consistent with the sympathetic nervous systems being involved in the control of energy expenditure. However, the findings also pose a question on how lipolysis and glycogenolysis are allowed to proceed at a time when sympathetic activity is depressed. Hepatic glycogenolysis is probably controlled predominatly by glucagon. With respect to lipolysis, it is possible that the balance between lipolytic hormones and insulin is more important than the actual level of lipolytic hormone. Thus, by reducing the concentration of insulin during a fast, lipolysis can proceed in spite of an actual fall in the concentration of noradrenaline at lipolytic receptor. Young and Landsberg (1979) also studied the effect of voluntary overfeeding on noradrenaline turnover. Rats were allowed access to an 8% sucrose solution in addition to their normal diet. This regime, if applied for several weeks will produce only a small increase in body weight although the hyperphagia is considerable. Voluntary overfeeding with sucrose for 3 days produced a highly significant increase in noradrenaline turnover in liver, pancreas and heart (Fig. 5.4). In another study, Landsberg, Saville, Young, Rothwell and Stock (1981), found that noradrenaline turnover in brown adipose tissue was increased in rats fed on the cafeteria diet.

AI,II

Metabolic Aspects of Obesity

,03

383

B

LIVER

T

REFED ~ " t~,2 III hr

102

FASTED

~

~ FED

~ /

t~,2 89hr

I0

~

I0

214 HOURS

Fig. 5.3

I0

~

1~)

24 HOURS

Effect of fasting and refeeding on NE turnover in rat A: pancreas and B: liver. Fasted animals had been without food for 48 hours prior to the injection of [3H]NE (235~Ci/kg) and remained fasting for the duration of the experiment; refed animals had been similarly without food for 48 hours prior to injection of tracer but were allowed access to food after injection. Data are plotted as means + SE for specific activity of organs from 5 animals in each group at each time point. Slope (k) of each line is significant at p<0.001. Open circles represent control, closed circles fasted, and X's refed animals. Reproduced from Young, J.B. and Landsberg, L., 1978, Am. J. Physiol. 236, E524-E533, with permission.

All of these changes in noradrenaline turnover induced by dietary manipulation are in the s~ne direction as the changes in energy expenditure and they raise the possibility of a cause and effect relationship. They also indicate the existence of a signal that con[manicates nutritional status to the central nervous system. Elucidation of the nature of this signal(s) is the goal of current research. The administration of glucose either orally or intravenously to man increases the plasma c o n c e n t r a t i o n of noradrenaline (Fig. 5.5), and this occurs concomitantly with an increase in oxygen consumption (Welle, Lilavivathana and Campbell, 1980). As stated earlier, changes in plasma noradrenaline concentration are a relatively insensitive index of changes in tissue turnover, and it is presumed that the glucose load produces very substantial changes in tissue turnover. It seems likely that this inlaediate response to glucose (and other foods) is distinct from the chronic increase in sympathetic nervous system activity seen in the overfeeding experiments in rats. Landsberg and Young (1981) did not distinguish between these

384

M . A . Cawthorne A

HEART

FASTED

104

I04FB

PANCREAS

I

~ ~ '°J i

-&

f-

F"STEO

tl/2 24,2 hr

,5 10 3 t=.

SUCROSE-FED/ ~ tl/2 5 8 hr

' ~

102

HOURS

"~

1041C

LIVER

~_

FASTED

LI/2 168 hr

SUCROSE-FED ~

'~

Fig. 5.4

2

"

6

12 HOURS

24

Effect of sucrose feeding and fasting on NE turnover in A: rat heart; B: pancreas; and C: liver. Sucrosefed animals had access to 8% sucrose drinking solution for 72h prior to injection of [3H]NE (250uCi/kg) and for duration of experiment; fasted animals had been without food for 48h prior to injection and remained without food during experiment. Data are plotted as means ~ SE for specific activity of organs from 4-5 animals in each group at each time point. The slope (k) of each line is significant at p<0.001. Open circles represent control (ad lib-fed) animals, closed circles fasted, closed triangles sucrose-fed. Reproduced from Young, J.B. and Landsberg, L., 1978, Am. J. Physiol. 236, E524-E533, with permission.

Metabolic Aspects of Obesity

~1,

[0

• GLUCOSE

e ~ w ~

,,of e i '2°t

385

0 WATER

80'-

,o/I2__,__

O

.

g ~o[

!~ 200 ~

zo

-{

0'

{

' 60

;o

120 '

150 '

180 '

T I M E (rain)

Fig. 5.5

Responses to ingestion of 230 ml water or glucose solution (i00 g glucose/230 ml solution) in 6 normal males. Values are means + SE. * significantly (p<0.05) greater than corresponding value on control (water) day. Reproduced from Welle et al (1980), Metabolism 29 806-809, with permission.

two responses but suggested insulin as the mechanism whereby the brain received information about the nutritional status of the animal. On theoretical grounds, it seems unlikely that plasma glucose level per se is an adequate signal. Plasma glucose concentrations are normally maintained within narrow lintits despite large changes in total caloric and carbohydrate intake. On the other hand, the plasma insulin concentration is highly responsive to changes in carbohydrate intake and the circulating level of insulin itself may serve as an index within the central nervous system of insulin-mediated glucose metabolism. Insulin might make an effective signal in the co-ordination of sympathetic activity and carbohydrate intake since insulin levels vary widely and reflect, in a general sense, the carbohydrate load assimilated. Furthermore, insulin is the major signal to tissues

386

M . A . Cawthorne

outside the central nervous system that carbohydrate (and other nutrients) are being assimilated from the gut and it would therefore be both economic and logical for insulin to serve a similar function for the central nervous system. Rowe, Young, Minaker, Steven, Pallotta and Landsherg (1981) have evaluated the role of insulin in the stimulation of sympathetic nervous system activity in man by the use of insulin and glucose clamp techniques. These techniques, which depend upon priming and variable infusions of glucose, or glucose and insulin, permit the development of either steady-state hyperglycaemia (hyperglyca~nic clamp) or hyperinsulinaemia in association with a normal plasma glucose concentration (euglycaemic clamp). Fig. 5.6 shows the effect of hyperglycaemia and two levels of euglycaemic hyperinsulinaemia on plasma noradrenaline levels. No change in plasma noradrenaline concentration was found in the control or hyperglycaemic clamp situation, but both levels of euglycaemic hyperinsulinaemia produced a significant rise in plasma noradrenaline concentration. In addition, these insulin infusions produced alterations in pulse pressure and the product of systolic blood pressure and pulse rate, which are consistent with increased sympathetic activation of the cardiovascular system. These data must be interpreted with caution because the hyperinsulinaemia produced was massive (c 600pUnits/ml). Nevertheless, they do indicate that hyperinsulinaemia without a change in blood glucose concentration has the capacity to stimulate sympathetic nervous system activity whilst steady-state hyperglycaemia per se appears to have less of an effect. It is possible that the large doses of insulin and glucose required to produce these changes are related to the unphysiological route of administration and to the poor sensitivity of plasma noradrenaline concentration as an indicator of noradrenaline turnover. The central nervous system has often been considered to be independent of insulin action, at least as far as glucose metabolism is concerned. However, Havrankova, Roth and Brownstein (1978) found insulin binding sites in many regions of the rat brain. It is now evident that there are areas of the brain which are devoid of the tight capillary junctions that elsewhere constitute the blood-brain barrier (Oldendorf, 1975). One area that is known to be sensitive to insulin is the ventro-medial nucleus of the hypothalamus. Stimulation of this brain area produces an increase in thermcgenesis in brown adipose tissue (Perkins, Rothwell, Stock and Stone, 1981). It is possible that stimulation of the VMH by blood-borne insulin is the initiating event in this process. Insulin can reach the cerebrospinal fluid (CSF) and then, presumably, the brain tissue (Margolis and Altszuler, 1967). The equilibrium between the periphery and the CNS is reached only after several hours and just a small fraction of any injected insulin can be found in the CSF (Woods and Porte, 1977). Thus, rapid fluctations of plasma insulin are damped to a great degree in CSF and the concentration of insulin in CSF is a slow intergral over time of the concentration in the plasma (Porte and Woods, 1981). It would thus represent an ideal signal of the overall level of nutrition and may well pro~Dte the long-term activation of the sympathetic nervous system produced by overfeeding. Inmediately after a meal this signal could be supplemented by blood-born insulin acting on the ventromedial hypothalarnus. If insulin has the role outlined above, it would be presumed that sympathetic nervous system activation and thermogenesis should be depressed in diabetic animals and in normal animals in which insulin secretion is blocked. As yet, studies using somatostatin to block insulin secretion have not been carried out. Preliminary studies in streptozotocin-induced diabetic rats indicate that there are smaller changes in slmpathetic activity following changes in dietary intake than Occur in control animals (Landsberg and Young, 1981). Rothwell and Stock (1981) have also shown that streptozotocin-diabetic rats failed to respond to cafeteria feeding by increasing the basal rate of thermogenesis (Table 5.1). In addition, diabetic rats showed a reduction in mass of brown adipose tissue and a reduced temperature.

Metabolic Aspectsof Obesity 600

387

o Control • G +125 Hyperglycemic 2mU Euglycemic • 5mU Euglycemic

./.--"I

500

/ ' =

./

400 LU

° /

300

t~

~o ~o

~'~o ~ e - ~ ' ' "

•/

-Q. 2 0 0

lOOz

01 Fig. 5.6

t;

I

0

0

I

I

30 60 Minutes

I

I

I

90

120

150

Increase in plasma norepinephrine level during euglycemic hyperinsulinism in human subjects. In the control group saline only was infused; in the hyperglycemic clamp (G+125) the glucose concentration was raised 125mg/dl above basal and maintained at that level for 120 min (7 subject); in the euglycemic clamp studies insulin was infused at 2mU/kg/ min in the low dose infusion (7 subjects), and at 5 mU/kg/min in an additional study (high dose insulin infusion; 7 subjects). In both the euglycemic hyperinsulinemic clamps, glucose was infused at sufficient rate to maintain plasma glucose at the basal level. The mean plasma glucose concentration was 81 mg/dl + 1.0 SEM in control study; in the hyperglycemic clamp 208 ~ 1.6; in the 2mU englycemic clamp 78.6 ~ 0.5; ~ d i n the 5mU euglycemic clamp 80.2 + 1.0. Mean plasma insulin concentration between 20 and 120 min in the control test was 7.3uU/ml + i.i SEM; in the hyperglycemic clamp 44 ~ 0.7; in the euglycemic 2mU clamp 154 ~ 32; and in the 5mU euglycemic clamp 601 + 74. Reproduced from Rowe, J.W., Young, J.B., Minaker, K.L., Stevens, A.L., Pollotta, J. and Landsberg, L., 1981. Diabetes 300, 219-225. Reproduced with permission from the American Diabetes Association, Inc.

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M.A. Cawthorne

An apparent paradox in this hypothesis is that, almost without exception, obese animals and man are hyperinsulinaemic. Thus it might be predicted that these animals would have elevated sympathetic nervous system activity and elevated rates of thermogenesis. However, as stated earlier there is substantial evidence that genetically obese rodents have a reduced capacity for thermogenesis. "If the link with the sympathetic nervous system is to be preserved it is essential that these animals also have a reduced rate of turnover of noradrenaline. Recent studies using the genetically obese (ob/ob) mouse (Knehans and Romsos, 1981) and in weanling %MH-lesionedrat (Vander Tuig, Knehans and Romsos, 1982), have demonstratedthat noradrenaline turnover in brown adipose tissue is decreased in both these animal models. However measurements of noradrenaline turnover in heart gave different results in that it was decreased in the VMH-lesioned rats but not in the ob/obmouse. Thus, it is possible that there is a specific reduction in some obese animal models in the sympathetic outflow to brown adipose tissue. However there is also another equally plausible explanation. The genetically obese mice studied differed very significantly in body weight from their lean counterparts and thus it is likely that on a per mouse basis they had a similar metabolic rate. It is possible that noradrenaline turnover in heart is linked to metabolic rate and thus the failure to find a difference between lean and genetically obese mice in noradrenaline turnover in heart is perhaps not surprising and agrees with the findings of an earlier study on gold thioglucose-induced obese mice (Young and Landsberg, 1980). In contrast, the body weight of the weanling VMH-lesioned animals studied by Vander Tuig et al, 1982, did not differ markedly from controls and they were still in a dynamic phase of obesity. Thus it seems likely that the metabolic rate per rat was lower in the VMH-lesioned animals. It is important that in future studies measurements of noradrenaline turnover are made in the hearts of pre-obese ob/obmice. Only then will it be clear whether there is a generalized reduction in sympathetic outflow or whether there is a specific reduction in the outflow to brown adipose tissue. How can this finding that sympathetic outflow to brown adipose tissue is reduced in ob/obmice and VMH-lesioned rats be rationalized with the earlier views that an increase in insulin secretion can activate the sympathetic nervous system? It is possible that as in peripheral tissues, insulin resistance can develop at central nervous system sites concerned with sympathetic nervous system activity. Cer tainly insulin infusion to aged human subjects, who tend to be insulin resistant, is less effective at stimulating sympathetic nervous system activity than in younger subjects (Landsberg and Young, 1981). An interesting experiment would be to determine the effect of hyperinsulinaemic euglycaemia on plasma noradrenaline and metabolic rate in subjects such as the Pima Indians, who are genetically predisposed to hyperinsulinaemia and obesity. Table 5.1 Resting VO 2 (ml/min/W 75 ) Before and After Injection of Noradrenaline

Control

Cafeteria

Diabetic Control

Diabetic Cafeteria

Resting VO 2

13.3±0.3

16.5±0.3"

12.8±0.5

13.0±0.6%

After noradrenaline

20.9±1.0

31.0±0.5"

16.1±0.4%

16.8±0.6%

n=8, mean values ± SEM, *p<0.001 compared to respective control group ~p<0.001 compared to respective non-diabetic group. Reproduced from Rothwell and Stock (1981), Metabolism 3__00,673-678, by permission.

Chapter 6

Concluding Remarks

Corpulence has been a part of the human state for at least 25 millenia and for the majority of this time period it has been regarded certainly in females, as a desirable physical attribute. Stone age statues such as the Venus of Willendorf which is now displayed in the Natural History Museum in Vienna, depict an artistic representation of the female form as being well-rounded. More recently, we have the paintings of the great Masters to remind us of the fact that beauty was often associated with a degree of corpulence. The problems of excessive corpulence, that is obesity, has been recognised at least for the last 2,400 years and treatments for obesity date back as far as Hippocrates. He wrote 'Fat people who want to reduce should take their exercise on an ealoty stomach and sit down to food out of breath. They should take only one meal a day, go without baths, sleep on hard beds and walk about with as little clothes as may be.' Thus, the first known treatment for obesity recognised that there were two sides to the problem of obesity namely food intake and energy expenditure. HOweVer, much more importantly he recognised that these two factors were integrated in some way in that he instructed that his patients 'should sit down to food out of breath'. Bray (1981) has recently paraphrased the words to William Shakespeare as follows: 'But be not afraid of corpulence: some are born corpulent, some achieve corpulence and some have corpulence thrust upon them.' Bray recognised three types of obesity: I] 2] 3]

genetic Obesity: some are horn corpulent, diet-induced obesity: some achieve corpulence, hypothalamic obesity: some have corpulence thrust upon them.

It seems extr~nely likely that in a number of populations around the world that the propensity to become obese is genetically inherited. Such populations include the Pima Indians living in the Gila River basin in Arizona and the Micronesian islanders. In addition, there are a number of extreme exanples of human obesity that are probably genetic. They include a set of identical twins in the U.S.A., weighing 340 kg each. Within the general population, there is evidence beth frc~ the studies on mono and dizygotic twins and from comparative studies on children with obese or lean parents to indicate a genetic involvement in obesity. The form that this genetic influence takes has not yet been defined. Most of our knowledge on genetic obesity ccmes from genetically obese rodents. These animals have a plethora of biochestical, endrocrinological and neurological abnormalities. Some of these are undoubtably a secondary consequence of the obesity. It would

389

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M . A . Cawthorne

appear that the defects in thermogenesis occur pre-we~ling and before the development of massive obesity. It is important that future research focuses on this pre-obese period with a view to unearthing the reason for the defective regulation of themnc~enesis. It has been suggested (Joosten and Van der Kroon, 1974) that o b / o b m i c e m i g h t be functionally hypothyroid. However, Ohtake and Bray (1977) could find no major abnormality of the hypothalamic-pituitary axis although they did present evidence of a decreased tissue response to thyroid hormones. Circulating levels of insulin are elevated in all genetically obese animal models and this is invariably accompanied by tissue resistance to the action of insulin. In obese man similar changes are c o ~ n . Over the last decade there has been many claims that obesity precedes hyperinsulinaemia in obese rodents and vice versa. These studies have, like many others, been thwarted by the difficulty in identifying the potentially obese rodents and by the limited quantities of blood available for the assays. In all cases, the levels of insulin have been measured in ad-lib fed or fasted animals. It could be much more ~nportant to know if these pre-obese animals produce a larger amount of insulin than lean animals following a standard dietary stimulation. Studies by Beloff-Chain, Bogdanovic and Cawthorne (1979) have shown that perifused neurointermediate lobes of the pituitary gland of young obese mice and the lean heterozygotes carrying the obese gene release much larger quantities of the potent insulin secretagogue, B-cell tropin, than lean animals, and it is possible that this may be an importance stimulus leading to hyperinsulinaemia. In the early stages of obesity, glucose disposal following a glucose load is similar in lean and obese rodents hut the total secretion of insulin is much larger in the obese mice suggesting that there is resistance to insulin action. In view of the relationship of insulin-induced glucose metabolism and sympathetic nervous system activity, one could envisage the development of hyperinsulinaemia and hence insulin resistance having a permissive if not a causal role in the development of genetic obesity. Virtually all forms of obesity could be regarded as having a dietary origin. However, a target for future research in man must be to answer the question as to why some people can indulge in overeating with impunity whilst others do so only with the unwelcome consequences of excess adiposity. Animal studies have indicate<] that there are genetic factors involved since the degrees of obesity induced in rats by feeding on a high-fat diet vary from strain to strain (Scheamel, Mickelsen and Gill, 1970). In man the development of obesity is, in general, age-related. In aniaml studies, Stock and his colleagues have shown that young animals are able to maintain a thin body composition in the face of overfeeding by the cafeteria diet, whilst older animals cannot. In an interesting recent experiment, Jeanrenaud (1981) measured the cephalic phase of insulin secretion in rats following the placing of the non-medabolized artificial sweetener saccharin into the mouth. By this simple test, a seemingly homogeneous group of rats could be separated into t~o groups - large and small responders. Subsequently, the rats were placed on a cafeteria diet and it was found that the group producing the largest cephalic phase of insulin secretion produced the greatest weight gain. This study again raises the question as to whether inappropriate insulin secretion is intimately involved in the regulation of appetite and the likelihood to gain weight. Likewise, it is well-established that insulin resistance both in animals and man tends to develop with ageing as does the loss of sympathetic tone. Such changes could result in an age-related loss of ability to activate heat producing mechanisms in brown adipose tissue. Hypothalamic obesity is a relatively rare phenomenon in man. However, its pathological similarity with that resulting from deliberate lesions in the rat allows us to investigate the nature of the hypothalamic control of energy intake and energy expenditure. Such studies have shown that hyperphagia is not essential to the development of obesity and there is independent evidence for a functional impairment of the sympathetic nervous system. Insulin secretion is also markedly

Metabolic Aspects of Obesity

391

increased in animals with ventromedial hypothalamic injury. Sub-diaphragmatic vagotomy both reverses hypothalamic obesity and the hyperinsulinaemia (Powley and Opsah, 1974; Inoue and Bray, 1977). Thus even in rats with hypothalamic obesity, the development of obesity occ[ms concomitantly with h yperinsulina~mia. As a result of all these findings, it is possible that the three types of obesity described in man all have conmon elements. These would include a tendency to hyperinsulinaemia, possibly as a result of defective pituitary/hypothalamic control, central resistance to the action of insulin on glucose sensitive neurons and reduced sympathetic nervous system activity. It is worthwhile in the current state of knowledge to re-examine the advice of Hippocrates. After 2,400 years, we are perhaps on the verge of understanding the molecular mechanisms behind his advice to the obese.

392

M. A. Cawthorne Acknowledgements

I should like to thank my colleagues, in particular Alison Levy, Stephen Smith and Jonathan Arch, at Beecham Pharmaceuticals, Great Burgh for many useful discussions during the preparation of this manuscript. I am particularly indebted to my wife Ray, not only for keeping the children occupied whilst I was working on this manuscript bat also for taking on the task of collating the references. I would like to acknowledge the work of Mr. M.J. Connors for preparing the figures, and finally, I should like to express my especial thanks to Miss Christine Saunders for the care she has taken in the preparation of the manuscript.

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393

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