The regulation of fatty acid synthesis in brown adipose tissue by insulin

Prog. Lipid Res. Vol. 21, pp. 195-223, 1982

0163-7827/82/030195 29514.50/0 Copyright © 1982 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

THE R E G U L A T I O N OF FATTY ACID SYNTHESIS IN B R O W N A D I P O S E TISSUE BY I N S U L I N JAMES G. MCCORMACK Department of Biochemistry, University of Bristol Medical School, University Walk, Bristol, BS8 1TD, United Kingdom

CONTENTS I. INTRODUCTION

195

II. THE SITES OF INSULIN ACTION IN ITS STIMULATION OF FATTY ACID SYNTHESIS

A. Target cells for insulin action B. Intracellular sites of insulin action on fatty acid synthesis I. Pyruvate kinase (EC 2.7.1.40) (a) Properties of the enzyme (b) Effects of insulin 2. The pyruvate dehydrogenase complex (a) Properties of the enzyme (b) Effects of insulin 3. Acetyl CoA carboxylase (EC 6.4.1.2) (a) Properties of the enzyme (b) Effects of insulin C. Proposed mechanisms for the action of insulin 1. Changes in cyclic nucleotide concentration 2. Redistribution of Ca 2+ 3. Hydrogen peroxide 4. Peptide mediator 5. Cyclic AMP-independent protein kinase III. INSULIN REGULATION OF FATTY ACID SYNTHESIS IN BROWN ADIPOSE TISSUE

A. Fatty acid synthesis in different preparations of brown adipose tissue 1. In vitro studies 2. In vivo studies (a) Fatty acid synthesis (b) Pyruvate dehydrogenase and acetyl CoA carboxylase B. Effects of insulin pretreatment on pyruvate dehydrogenase activity in isolated brown adipose tissue mitochondria C. The substrates for fatty acid synthesis in brown adipose tissue D. The significance of brown adipose tissue as a site for de novo fatty acid synthesis IV. BROWN ADIPOSE TISSUE AND OBESITY V. CONCLUSIONS AND SOME SPECULATION ACKNOWLEDGEMENTS REFERENCES

197

197 198 200 200 200 200 200 202 203 203 203 204 204 204 205 205 205 205

206 206 208 208 210 210 213 214 217 218 220 220

I. I N T R O D U C T I O N B r o w n a d i p o s e tissue is p r i m a r i l y r e g a r d e d as a site for the g e n e r a t i o n o f heat by n o n - s h i v e r i n g m e c h a n i s m s . M o s t of the studies carried o u t on b r o w n a d i p o s e tissue have been c o n c e r n e d with this topic, for details of which the r e a d e r ' s a t t e n t i o n is d r a w n to several recent r e v i e w s . / I ' 7 7 " I ° l ' I l s ' I l S ' l l g ' l ' ~ 3 Overall, in a n i m a l s w h e r e the tissue has been studied, g o o d c o r r e l a t i o n s exist between the o c c u r r e n c e of the tissue t o g e t h e r with its p r o p o s e d t h e r m o g e n i c p r o p e r t i e s a n d the e x p o s u r e of the a n i m a l s to different a m b i e n t t e m p e r a t u r e s , i.e. basically, these are increased in c o l d e n v i r o n m e n t s . A l t h o u g h b r o w n fat is n o t the o n l y tissue site of n o n - s h i v e r i n g t h e r m o g e n e s i s in m a m m a l s , it a p p e a r s t h a t this is the m a j o r function of the tissue a n d it m a y be the p r e d o m i n a n t site for this process u n d e r certain conditions. 21'v7'143 T h e tissue is present in all m a m m a l s which are k n o w n to be c a p a b l e of this p r o c e s s - - t h e s e include not o n l y h i b e r n a t o r s a n d o t h e r small m a m mals but also the n e w b o r n of larger m a m m a l s . 2t'ss'118 M o r e o v e r , it is present in newb o r n h u m a n s 21 a n d d e p o s i t s of b r o w n fat have also been r e p o r t e d to exist in elderly humans.21 .vo T h e i m p o r t a n c e of the t h e r m o g e n i c p r o p e r t i e s of b r o w n a d i p o s e tissue m a y not be restricted to s i t u a t i o n s o f cold-stress. Recently, the tissue has b e c o m e a focus 195

196 for the attention

James G. McCormack

of several workers who are studying the problem of obesInverse correlations have been found between the extent of obesity and the amount of brown adipose tissue present together with the degree to which its thermogenic properties are evident. These observations have led to the hypothesis that obesity could, at least in part, be the result of a reduced capacity for the non-shivering thermogenesis which would serve as a means for burning-off any excess food-intake. This concept and, in particular, the key role envisaged for brown adipose tissue in it, has aroused considerable interest and has even been the subject of a B.B.C. television program ("Horizon," December, 1979). However, it must be mentioned that other workers in this field, while not directly disagreeing with this concept, have suggested that sufficient proof is as yet lacking, and furthermore have criticized the experimental procedures in the studies leading to this hypothesis, and have also questioned the validity of the interpretations put on the results obtained. 75 Most of the biochemical investigations on the metabolism of brown adipose tissue have been directed towards reaching an understanding of the mechanisms underlying the thermogenic properties of the tissue. Progress in this interesting field has been considerable and has been reviewed recently 21'22`l°l'llsl l~,t 19 and only an outline is given here. The major physiological stimulus for thermogenesis in the tissue is generally accepted to be nor-adrenaline which is secreted, probably under neural control, from sympathetic nerve-endings of which there are many located on the tissue. The hormone's mechanism of action is thought to be mediated primarily through an increase in cyclic AMP concentrations in the cell cytoplasm as a result of its binding to fl-receptors on the cell membrane. The subsequent activation of cyclic AMP-dependent protein kinase is then thought to lead to the phosphorylation and activation of hormone-sensitive triglyceride lipase which, therefore, stimulates the provision of what is generally thought to be the major fuel for thermogenesis, namely fatty acids. The capacity of the tissue for heat production by oxidation of the released fatty acids is thought to be enhanced by some degree of uncoupling of {or excess proton-cycling by) the brown adipose tissue mitochondria. The mechanism by which this is achieved appears to be specific to brown adipose tissue mitochondria and is thought to involve a protein which seems to be uniquely present in their inner membrane: the properties of this protein and its potential physiological relevance will be discussed in more detail in Section III.B. The lipid fuel for thermogenesis is stored in the multilocular fat droplets which are noticeable in the cytoplasm of brown adipose tissue cells. Studies on the lipid metabolism of brown adipose tissue have generally been concerned with examining lipolysis and fatty acid oxidation, whereas the study of fatty acid synthesis in this tissue had been largely ignored. Until very recently the tissue was regarded to be a pool site for de not'o fatty acid synthesis and it was generally thought that it derived most of its fat from the blood stream after de not,o synthesis elsewhere. 76'86"°21°1~1s'~'.3 However. this viewpoint has been dramatically altered in the last few years as the result of observations made in this and other laboratories, and it is now thought that brown adipose tissue, where it is present, can represent a very important site for de m~co fatty acid synthesis.2 4 - , 8 2 , 1 0 " 7 , 1 0 8 . 1 4 1 , 1 S 0 152,155 This conclusion is, as yet, only derived from in cico studies on rats, rabbits, mice and hamsters: however, it is perhaps likely thal the observations made on these animals will be generally applicable to brown adipose tissue in all animals where it is present, including humans (if fed on high carbohydrate diets). The purpose of this article is to draw attention to this recent work on the de novo synthesis of fatty acids in brown adipose tissue and in particular to examine the role which insulin plays in the regulation of this process. The article begins by detailing some of the means by which insulin stimulates the fatty acid synthesis pathways of other tissues. This section is chiefly focussed on white adipose tissue from which most of out knowledge on this topic has been obtained. The resulting general concept of the mechanism of action of insulin on fatty acid synthesis is then applied to, and compared with, the recent observations on the regulation of fatty acid synthesis in brown adipose tissue by insulin. There then follows an assessment of the importance, relevance and implications ity. 2'~3"6°'7v'~8"87"135"13~'

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of de novo fatty acid synthesis in brown adipose tissue and its regulation by insulin. Wherever it was considered to be appropriate, review articles have been cited rather than a long list of original reports.

II. THE SITES OF INSULIN ACTION IN ITS STIMULATION OF FATTY ACID SYNTHESIS Broadly, the overall function of insulin is to efficiently remove and conserve excess glucose from the blood-stream. It does this by interacting with specific receptors on its target cells and so causing them to take up the glucose and store it in a suitable form, such as glycogen or triglyceride, which can then be broken down and/or exported to be used as required later. Both long and short term effects on the metabolism of target cells are caused by insulin. The long term effects are brought about by changes in both general and specific protein synthesis and breakdown and may involve the internalization and intracellular processing of the hormone. In contrast, the short-term, or acute, effects of insulin are most unlikely to involve internalization of the hormone and are brought about by changes in the activities of pre-existing enzymes and membrane transporters. The attention of this article will be largely focussed upon the short-term effects of insulin on fatty acid synthesis. Details on other aspects of the broad spectrum of cellular events with which insulin interacts can be found elsewhere. 3°'3x'35"57"128"132

A. Target Cells for Insulin Action

Insulin is known to acutely affect the carbohydrate and fat metabolism of several m a m m a l i a n tissues. 30"31'34'35'57'61'108'128'132 These include white adipose tissue, liver,

muscle, mammary gland (principally when lactating) and, as will be discussed in detail later, brown adipose tissue. In muscle cells, the function of insulin is to stimulate the uptake of glucose into the cells and its conversion into glycogen; very little fatty acid synthesis takes place in muscle cells. The fact that most of our knowledge on the insulin regulation of fatty acid synthesis is derived from studies on white adipose tissue is largely due to practical considerations. Of primary significance in this is that white adipose tissue is very easy to work with in vitro and furthermore, the properties of the tissue in vitro appear to be very close to those found in vivo; 34'145 these features are not shared by the other tissues mentioned above. Also of great importance in facilitating the study of this topic in white adipose tissue is that this tissue appears to favor the use of glucose as the sole precursor for fatty acid synthesis, and fatty acid synthesis is a major pathway in white adipose tissue. Again, this would appear not to be the case in the other tissues mentioned and, therefore, there exists in these the possible problem of several pathways operating at once which makes the study of specific effects difficult. Added to this, these other tissues also have specific problems associated with the study of the insulin regulation of fatty acid synthesis in them. The changes brought about by insulin on this process in liver are comparatively small and also substrates will be competed for by other insulin stimulated processes such as glycogen synthesis. Furthermore, experience has taught that liver enzymes are difficult to work with in tissue extracts because of the high protease activities associated with the tissue. 43'16° With mammary gland, there is the problem of choosing at which point in the reproductive cycle to work on the tissue, particularly as other hormones such as prolactin appear to alter the properties of the fatty acid synthesis pathway in the tissue. 1'54'162 As will be seen later, there are also some problems associated with the study of the effects of insulin on fatty acid synthesis in brown adipose tissue. Therefore, white adipose tissue represents the most straightforward means of studying the mechanisms underlying the stimulation by insulin of fatty acid synthesis; however, it is likely that this process will have a similar mechanism in the other tissues. The next

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James G. McCormack

section details the specific loci in the pathway of fatty acid synthesis which are influenced by insulin and is largely derived from observations made on white adipose tissue, though occasional reference to the other tissues mentioned above will be made. The specific case of brown adipose tissue will be dealt with later. B. Intraeellular Sites of Insulin Action on Fatty Acid Synthesis The effects of insulin on fatty acid synthesis in white adipose tissue are on components of the fatty acid synthesis pathway itself rather than being secondary to the hormone's effects on lipolysis. 2~'3~'~7"~ os Insulin inhibits lipolysis, most noticeably in the presence of lipolytic hormones such as adrenaline. However, other agents which inhibit lipolysis to a similar extent as insulin, such as nicotinate or prostaglandin PGE~, do not affect fatty acid synthesis. It should also be noted that, in general, adrenaline antagonizes the effects of insulin on fatty acid synthesis in white adipose tissue. The first site of action of insulin in its stimulation of fatty acid synthesis is not strictly intracellular but is the glucose carrier which resides in the plasma membrane (Fig. 1).a°'3t'ls~' The stimulation of the uptake of glucose into target cells caused by insulin binding to its receptors appears to be brought about by an increase in the maximum activity (Vm,0 of the glucose carrier. Recently, two independent research groups, using different techniques, have each put forward on the basis of work with fat cells, the hypothesis that the mechanism underlying this increase in ~,;...... may involve the stimulation by insulin of the recruitment to the plasma membrane of glucose transport moieties from some intracellular site. >~~4~' Although it is obviously very important, the increase in glucose carrier activity does not appear to be absolutely essential for the stimulation of fatty acid synthesis which is caused by insulin. "~7 All of the other known effects of insulin on the pathway of fatty acid synthesis (and including that on the actual rates obtainedt, which are described below. are still evident in white adipose tissue incubated in the absence of glucose or in the

Glucose "--"---- Gtucose ~=I transport

Cett membrane

Glucose

I

Dihydroxyacetone phosphate Phospho!notpyruvate

Pyruvote kinase

/X [~Jvo1 ePyruvatedehydr°genase Pyr5vate

Mitochondnot membrane "

"

C2ro*e Acet!t-CoA utycerct~phosphate 1 Aeety!,-CoA earboxytase Matoiyt CoA -

Trigtyceride "

F try acid

FIG. I. Outline diagram of the pathway of fatty acid synthesis from glucose in while adipose tissue. This scheme is probably also correct for other tissues which are capable of performing this process. Steps which are known to be stimulated by insulin are highlighted by the heavy arrows. Abbreviation: CoA, Coenzyme A.

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presence of a sugar, such as fructose, whose uptake is only marginally affected by insulin. These other sites of action of insulin on this pathway are all intracellular. The binding of insulin to its receptors can alter the activity of many intracellular enzymes. 3°,31'34'35'37'57'128'132 However, in the stimulation of fatty acid synthesis by insulin in white adipose tissue, there are three key enzymes of this pathway which have been identified as being regulated by the hormone (Fig. l). 34.36-38.63.64 These are pyruvate kinase (EC 2.7.1.40), the pyruvate dehydrogenase complex, and acetyl CoA carboxylase (EC 6.4.1.2). These three enzymes are all activated by insulin in parallel with the hormone's stimulation of fatty acid synthesis (Fig. 2), and other parallels can be found. For instance, the effect of insulin in stimulating fatty acid synthesis in white adipose tissue is substantial and can be observed within minutes after the addition of the hormone; the same can be said for the effects of insulin on these three enzymes. Likewise, adrenaline, which inhibits the stimulation of fatty acid synthesis resulting from insulin addition, also inhibits the activation of these enzymes which is caused by insulin. 34'37

Conditions Control

Fattyacid synthesis I

Acetyl-CoA Pyruvate dehydmgenasecarbaxylase

Pyruvate kinase ~

~

nsu,n

a

I I t I I I I 0.5 io o 05 io o 30 6o o IO 20 /~mat/g/hr ActivityatO.ImM- %presentin % presentin PEPasratioaf activeform activefarm Vmc~

FIG. 2. The stimulation by insulin of fatty acid synthesis in white adipose tissue is brought about by mechanisms which involve the parallel activation of pyruvate kinase, pyruvate dehydrogenase and acetyl CoA carboxylase. Data are taken from refs. 34 and 36. Glucose uptake into the cells of the tissue is also stimulated by insulin in parallel with the illustrated effects.31 Adrenaline inhibits these effects of insulin. 34'36 Abbreviation: PEP, phosphoenolpyruvate.

There are reports that suggest that insulin also activates some enzymes of the fatty acid esterification pathway such as fatty acyl CoA synthetase and glycerol phosphate acyltransferase (e.g. refs. 85 and 144). However, it is unlikely that the effects of insulin on the fatty acid synthesis pathway are secondary to any effects it has on the esterification pathway as frstly, the effects on the latter pathway are best seen in the presence of other hormones such as adrenaline whereas those on the former are best seen with insulin alone; as secondly, rates of esterification in isolated fat cells incubated with glucose can be either greatly increased or decreased by the addition of palmitate or bromopalmitate, respectively, whereas, under these conditions, no changes are observed in the rates of fatty acid synthesis or the activities of pyruvate dehydrogenase or acetyl CoA carboxylase. 50 The effects of insulin on the activities of pyruvate kinase, pyruvate dehydrogenase and acetyl CoA carboxylase were observed primarily because they persist into cell extracts. This means that it is probable that some form of covalent modification of each of these enzymes is caused by insulin. Therefore, a detailed study of the intrinsic properties and effectors of these enzymes in conjunction with a detailed characterization of the effects of insulin addition to tissue or cells on their activities should throw light upon the mechanism of action by which insulin stimulates fatty acid synthesis. In more general terms, such an approach should also assist in the finding of the solution to the overall problem of how insulin works and, in particular, may lead to the identification of the elusive second messenger for insulin. A detailed description of the known properties and effectors of these three enzymes together with what is known of the effects of insulin on their activities is therefore given

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James G. McCormack

below. This information is largely restricted to evidence obtained from white adipose tissue. However, there is also some evidence that the observations listed here are probably also applicable to the situation in liver and m a m m a r y gland ~'9"3'~''~43"5"~ and, as will be seen later, brown adipose tissue. ~°~

1. Pyrut:ate Kinase {EC 2.7. I.40) (a) Properties ¢~/the enzyme. In mammalian cells, pyruvate kinase is present exclusively in the cytoplasm. The enzyme catalyzes the final step in the formation of pyruvate from glucose, namely :phosphoenolpyruvate + A D P - - , pyruvate + ATP. The reaction has a large negative AGo value and measurements of the cellular contents of its substrates and products indicate that it is far from equilibrium and essentially irreversible in the cell. ss'~-~s Thus, it is a potential site of regulation. There are at least three different isoenzymes of pyruvate kinasc: ~s'~~s the M~-type which is found in muscle and brain, the L-type which is the major isoenzyme in liver and a minor one in kidney, and the M 2- (or K- or A-)type which is the major isoenzyme in kidney. The Mz-type seems to be the isoenzymc present in white adipose tissue: it is not known what type(s) is present in brown fat as yet. The Mz-type isoenzyme appears to be regulated through changes in the K,, wdue for phosphoenolpyruvate (PEP) whereas its ~m~x activity appears to be largely unaltered. 3~''38's3 The K,,, for PEP is greatly decreased by fructose l:6-biphosphate and increased by alanine. ~~ The white adipose tissue enzyme may exist in two distinct interconvertible forms: ~2~~~° one with a high K,, for PEP and one with a low K,,. Incubation of tissue extracts with fructose 1:6-biphosphate converted the high K,, t\~rm into the lov, K,,, form. There is also evidence that the M2-type isoenzyme from chicken liver can be phosphorylated by a cyclic AMP-independent protein kinase but not by cyclic AMPdependent protein kinase. '*s However. attempts to demonstrate any phosphorylation of the rat white adipose tissue enzyme have as yet proved unsuccessful (E. M. Levett and R. M. Denton, personal communicationl. (b) E~l'ects ()f insulin. Brief exposure of epididymal fat pads from fed rats to insulin leads to the appearance of a form of pyruvate kinase which has a lower K,, value for PEP than the control value m tissue extracts: 3~' this activation has been shown to occur both ill vitro and in civo and to be independent of the stimulation of glucose transport. The mechanism for this effect has yet to be established but does not appear to involve any changes in the amount of bound effectors such as fructose l:6-biphosphate or alanine. The effect persists for several hours in extracts incubated at 0 C or 3 0 C in the presence of E D T A and fluoride, -~' suggesting that insulin may cause some form of covalent modification of the enzyme. The changes in K,, for PEP observed after exposure of white adipose tissue to insulin are similar to those associated with the dephosphorylation of L-type i s o e n z y m e Y furthermore, insulin, most noticeably when glucagon is present, decreases the K,, value for PEP of rat liver pyruvate kinase. ~-' However, as mentioned above, it appears that the adipose tissue enzyme may not be phosphorylated.

2. The Pyruvate Dehydro~enase Complex (a) Properties ~['the enzyme. The pyruvate dehydrogenase complex of mammalian cells is exclusively intramitochondrial and catalyzes the essentially irreversible reaction: pyruvate + N A D + + CoA---*acetyl CoA + CO2 + N A D H + H + The complex is made up of three enzymes which catalyze the series of reactions shown in Fig. 3. The complex is composed of about 150 separate polypeptide chains with an overall molecular weight of 7 10 million. 44 The core of the complex is composed of

The regulation of fatty acid synthesis

pyruvote

~l'PP-Gm,li~,,,~,"

enz, !

201

enz2-0¢,ei'~ . ~ d ih~tdhalil:~)oCe~,~ ?oA ~ I ~ (

enz, V

I

/x. "~'lydroxye'hhY I-TPP-/ ,rI enz, ~

~AcetyI-CoA

'7' . .......... /I

CO~

/ ~ n z 2 _ oxidised

/ /~ FADf

~ / L ipoa1"e~-....--=-.-~-NA

INo'l'e: lipoate, ~

;

÷ NAD

enz3

o

dihydrolipoote, ~SH~

FIG. 3. The component subunits of the pyruvat¢ dehydrogcnase complex and the reactions which they catalyze. See text for references. Abbreviations: enzm, pyruvate decarboxylase; cnz2, dihydrolipoate acetyl transferase; enza, dihydrolipoate dehydrogenase; TPP, thiamine pyrophosphate.

dihydrolipoate acetyl transferase (enz2) units (EC 2.3.1.2) to which are bound the dihydrolipoate dehydrogenase (enz3) units (EC 1.6.4.3) and pyruvate decarboxylase (enzl) units (EC 4.1.1.1); the last of these appears to be a tetramer of subunit composition 0(2fl 2. Interaction between the three component enzymes of the complex is brought about by the "swinging-arm" of the lipoyl groups on the transferase (enz2) units which are thought to visit each active site sequentially. 44'~34 However, much controversy still exists as to the overall subunit composition and the molecular architecture and enzymology of the complex. 3s.44.65 The reaction catalyzed by the pyruvate decarboxylase (enzt) component is by far the most thermodynamically favorable and thus, the regulation of the activity of the whole complex is almost certainly brought about by changes in the activity of this component. In animal cells, there are no pathways for the conversion of acetyl CoA to carbohydrate; thus, the rate at which pyruvate is converted into acetyl CoA will be the rate of net carbohydrate utilization. Therefore, regulation of this step is of critical importance in the energy balance and fuel economy of animal cells, and hence, by inference, the animals themselves. Pyruvate dehydrogenase serves both bioenergetic and biosynthetic roles. In many tissues, including muscle, kidney and brain, the acetyl CoA formed is almost exclusively metabolized through the citrate cycle to provide energy. 34'3~ However, in other tissues such as adipose tissue (both white, and as will be seen later, brown), liver and lactating mammary gland, a large fraction of the acetyl CoA produced is exported back out of the mitochondria (probably as citrate, at least in white adipose tissue ~°4) to be used in the synthesis of fatty acids and sterols. 34'37 Given this crucial and multifunctional role, it is hardly surprising to find that pyruvate dehydrogenase activity is controlled by a series of regulatory systems which, in turn, show sensitivity to a wide variety of external stimuli such as hormones and nutritional status. The activity of pyruvate dehydrogenase is governed by two forms of regulation. 37'44 First, the complex activity is subject to end-product inhibition by high concentration ratios of acetyl CoA/CoA and NADH/NAD +.58.154 This probably involves the accumulation of acetyl dihydrolipoate, resulting in a diminution of the amount of oxidized lipoate and hence, a limiting of the rate of pyruvate decarboxylation. 3s'4'~ The enzyme is also regulated by a phosphorylation-dephosphorylation cycle (Fig. 4), 1°2'1°3 the phosphorylated form is inactive and the dephosphorylated form is active. Interconversion is brought about by pyruvate dehydrogenase kinase (Mg. ATP dependent) and pyruvate dehydrogenase phosphate phosphatase; 44'~34 both of these enzymes are also exclusively intramitochondrial. The kinase is tightly bound to the complex and transfers the terminal phosphate from ATP to serine groups (three distinct sites) on the

202

James G. McCormack

Activators Activators Mg2+(Mn2+) Ca2+ (Sr2+) Tnhibitors

p < , Phosphatase~

Pyruvate dehydrogenase (active) ~ X A TP.Mg ~/j~ina se

(Ni 2+)

(F-)

~

f Pyruvete dehydrogenose phosphate (inactive)

ADP

High concentrationratios of acetyl- CoA/CoAand NADH/NAD* Znhibitors Pyruvate (dichoroacetate) ADP Ca'' [at high Mg':*] K'

FI(;. 4. Summary of the regulatory properties of the interconvcrting enzymes of the p.~ru~atc dehydrogenase system, pyruvate dehydrogenase phosphate phosphatasc and pyru,,ate dehydrogenase kinase. See text for references. Unphysiological effectors are given in round parentheses

~-subunits of the pyruvate decarboxylase components. 14~'1~' Phosphorylation leads to inhibition of the reactions involving the enzyme bound 2-:~-hydroxyethyl-TPP intermediate (Fig. 3). I s5 The phosphorylation of only one of the sites appears to be associated with the loss of activity "14s'16~ however, no consensus has yet been reached on the role of the phosphorylation of the other two sites, s°'1'~8'16~' The phosphatase is easily dissociable from the complex. '~4"t3'~ The known regulators of the activities of the kinase and phosphatase, which are of potential physiological significance, are listed in Fig. 42s'44'4s'°°'127'~34'161 (also note that cyclic A M P or cyclic AMP-dependent protein kinase have no known effects on any part of the pyruvate dehydrogenase system). The kinase appears to be activated when the catalytic activity of the complex will be repressed by end-product inhibition and to be inhibited by the substrates of the overall reaction. The phosphatase requires Mg 2 + for activity (K,, for Mg 2+ about 1 mM); in the presence of saturating Mg 2+, it can be further activated (about five-fold) by Ca 2 + ions (K,, for Ca 2 + about 1 I~Mt. Both the kinase and phosphatase exhibit broadly similar regulatory properties when they are located within intact mitochondria to those exhibited by the isolated enzymes. 3° 42.,~4,~,.~7.90.~0,~ 13 (b) Effects of insulin. Exposure of epididymal fat pads from fed rats to insulin leads, within a few minutes, to increases in the amount of pyruvate dehydrogenase complex in its active, nonphosphorylated form subsequently found in tissuc extracts. 3437"44"s01~1 This effect is evident both in vivo and in vitro and is not dependent on the stimulation of glucose transport. Similar activations by insulin have been reported for the liver 6"43 and m a m m a r y gland 9's4 enzymes. The answer to the most intriguing question as to how insulin, acting at the cell surface, affects this intramitochondrial enzyme, is as yet unknown. The effect persists during isolation of white fat mitochondria and their incubation in the presence of a suitable respiratory substrate for 10 20 min, 34"37'81 suggesting that the factor(s) responsible must still be present in the isolated mitochondria. Hughes and Denton 81 followed the incorporation of 32p from [7-32p]ATP within intact white fat mitochondria into pyruvate dehydrogenase [-32p]phosphate as a monitor of pyruvate dehydrogenase kinase activity. They found that incorporation was faster ill mitochondria prepared from tissue previously exposed to insulin suggesting, very strongly, that it is the phosphatase which is activated by insulin. Furthermore, there is evidence that the concentrations of the effectors of kinase activity within the mitochondria do not change after insulin treatment. 34'37 As insulin treatment does not seem to cause any persistent changes in phosphatase activity in mitochondrial extracts, the most likely explanation is that insulin changes the intramitochondrial concentration of an effector(s) of the phosphatase which would not be metabolized by. or lost from, the mitochondria during incubation. Only M g 2+ and Ca 2 + are known to affect the phosphatase. There is some indirect evidence that intramitochondrial Ca 2 + may be increased by insulin. The effect of insulin

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203

on pyruvate dehydrogenase is inhibited by agents such as ruthenium red, NiC12 and MnCI2 which are inhibitors of mitochondrial or cellular CaZ+-uptake. TM Further, this insulin effect could be mimicked by ouabain or by replacing K + with Na + in the medium in which the fat pads or cells were incubated; l°s such treatments are thought to increase Ca 2 +-uptake into cells. 8"26'62 In addition, the effect in isolated white fat mitochondria could be lost by incubating them with the CaZ+-ionophore A23187, the K+-ionophore valinomycin, FCCP and EGTA (to deplete them of Ca 2+) and Mg. ATP and oligomycin (to allow kinase activity). 34"37 For many years, the idea that changes in intramitochondrial Ca 2+ concentrations could act as a physiological regulator of the phosphatase was criticized because the effective range of Ca 2+ (0.1-10/~r~) was generally considered to be much lower than the concentration of intramitochondrial Ca2+. 2°'33'~63 However, recently it has been found that two other exclusively intramitochondrial and important regulatory enzymes, NAD +-isocitrate dehydrogenase 46 and the 2-0xoglutarate dehydrogenase complex 1°9 of mammalian tissues, 112 are also activated by increasing Ca 2 + in the range 0.1-10/~M. Furthermore, it has been demonstrated that, at least for heart and white adipose tissue, all three Ca 2 +-sensitive enzymes can respond to changes in the extramitochondrial concentration of Ca 2+ in the physiological range (0.05-5 ~M 133) when they are located in intact coupled mitochondria. 39-42'113 There are two other hormonal activations of pyruvate dehydrogenase which have been postulated to involve an increase in the intramitochondrial concentration of Ca2+; these are the activation of the liver enzyme by vasopressin 72 and ~-adrenergic agents 43 and the activation of the heart enzyme by adrenaline.79,111,~ 13

3. Acetyl CoA Carboxylase (EC. 6.4.1.2) (a) Properties of the enzyme. Acetyl CoA carboxylase is located exclusively in the cytoplasm of mammalian tissues and catalyzes the irreversible reaction:acetyl CoA + HCO~- + ATP ~ malonyl CoA + ADP + H + + Pi. Unlike the other two enzymes discussed above, acetyl CoA carboxylase is not present in all mammalian cells and is only found in any quantity in tissues which are recognized to be sites for de novo fatty acid synthesis. The above reaction is regarded to be the rate-limiting step in the biosynthesis of fatty acids. The subunits of the mammalian enzyme have a molecular weight around 230,000 and contain biotin which is involved in the reaction. Two identical subunits combine to form protomers which are inactive. Activity is only achieved when the protomers combine into long filamentous polymers (4-8 million molecular weight). The enzyme is activated by citrate and CoA and inhibited by low concentrations of fatty acyl C o A . 14'32'63'64"96'157'167 There appears to be poor correlation between the activity of the enzyme and tissue citrate concentrations whereas the correlation between enzyme activity and fatty acyl CoA levels is more suggestive of some physiological relevance; the effects of CoA have only been recently described and the significance of these findings remains to be established. Recently there have been several demonstrations of the phosphorylation of acetyl CoA carboxylase.14 19,35,68 The enzyme can be phosphorylated by cyclic AMP-dependent protein kinase which results in a loss of activity; these effects are reversed by phosphoprotein phosphatase.14"t 7,68 The enzyme also acts as a substrate for a recently described cyclic AMP-independent protein kinase which is associated with the plasma membrane fraction of fat cells; 14"15 moreover, phosphorylation by this enzyme results in an activation of the enzyme. The above observations are explained by the existence of several phosphorylation sites on acetyl CoA carboxylase; the two kinases mentioned above appear to act on distinct sites. 14-19'35'68 (b) Effects of insulin. Treatment of epididymal fat pads from fed rats with insulin for a few minutes leads to about a three-fold activation of acetyl CoA carboxylJP.L,R. 21,'3

D

204

James G. McCormack

a s e . 14"16"32'34"63"~'*'99"145 This is observed both in vitro and in vivo, is opposed by adrenaline and is independent of glucose transport stimulation. These effects appear to involve changes in enzyme polymerization and persist into tissue extracts with high dilution and incubation with albumin, or even after partial puritication of the enzyme; 14'63'64 this means that the effects of insulin and adrenaline are unlikely to be explained by residual binding of allosteric ligands. As to what brings about the insulin effect, the tissue levels of citrate can be markedly raised without there being any change in enzyme activity; ~4'~9"~'3'~'4however, there is evidence that insulin causes a marked fall in the tissue concentration of fatty acyl C o A . 32'34'63'64 An activation of the liver enzyme by insulin has also been reported. 59 Although insulin and adrenaline have opposing effects on the activity of acetyl CoA carboxylase, they each cause an increase in the amount of phosphorylation of the enzyme;14 ~9,35 this is explained by their promoting the phosphorylation of different sites on the enzyme. There is good evidence that adrenaline leads to the increased phosphorylation of the same sites as those which are phosphorylated by cyclic AMPdependent protein kinase on the extracted enzyme. Both of these treatments render the enzyme inactive and resistant to citrate-induced polymerization. Considerable excitement has been generated recently by the demonstration that insulin leads to the increased phosphorylation of acetyl CoA carboxylase, perhaps at the same sites as those which are phosphorylated by the cyclic AMP-independent kinase of the plasma membrane of fat cells described by Brownsey et ,I. ~'~ ~<35 Both of these treatments lead to an activation of the enzyme which can be lost by the incubation of extracts with citrate. From these observations has emerged the exciting hypothesis that insulin actiwttes acetyl CoA carboxylase by first activating this membrane bound kinase. However, as yet, no direct effects of insulin on the activity of this kinase have been found (R. W. Brownsey, personal communicationt.

C. Proposed Mechanisms lbr the Action ~[ Insulin These are described briefly below with particular reference to the effects of insulin on fatty acid synthesis. The reader's attention is drawn to the recent review by Denton and colleagues 35 where the cases for and against the various hypotheses are discussed in more general terms and in more detail.

1. Changes in Cyclic Nucleotide Concentration Under certain conditions, insulin causes a fall in cyclic AMP, and a transient increase in cyclic G M P concentrations, in both white fat and liver cells. 12s While these changes obviously could be very important, they do not appear to be necessary for the effect of insulin on fatty acid synthesis; in particular, the fall in cyclic A M P seems to require the presence of other hormones such as adrenaline, whereas this insulin effect is best seen in their absence. Likewise, the rise in cyclic G M P can be lost under conditions where there is no impairment of the effects of insulin.

2. Redistribution of Ca 2. An increase in cytoplasmic C a 2+ concentration has been proposed to mediate the action of i n s u l i n . 2v'91 A parallel increase in intramitochondrial C a 2+ could perhaps explain the insulin activation of pyruvate dehydrogenase phosphate phosphatase. As insulin action does not appear to require extracellular C a 2 + , 35 the source for this would have to be intracellular: there is a report that insulin inhibits a high affinity Ca 2+ATPase in white fat cell membranes. 125 However, the increase in cytoplasmic Ca 2~ hypothesis is only derived from indirect measurements and furthermore, such an increase is more likely to bring about effects opposing those of insulin. 3s Nevertheless, it should be noted that intramitochondrial Ca 2+ could be increased at the same time as cyto-

The regulation of fatty acid synthesis

205

plasmic Ca 2 + decreased if, for instance, insulin altered the activity of the Ca 2 +-transport systems of the mitochondrial inner membrane.23,39-41,~ 21

3. Hydrogen Peroxide Hydrogen peroxide can bring about a range of effects similar to those caused by insulin, 1°6 including an activation of pyruvate dehydrogenase which can also be elicited in isolated mitochondria. 123 Furthermore, there is a report that insulin stimulates an N A D P H oxidase which forms H 2 O 2 in white fat cell membranes. ~4 However, there appears to be a lack of reproducibility about some aspects of this work with H 2 0 2 .35'1°6

4. Peptide Mediator Insulin has been reported to increase the intracellular concentration of a substance which is probably a small peptide in both rat muscle and white adipose tissue. 97"13v This substance causes some insulin-like effects on enzymes in extracts 9v and has also been proposed to mediate an insulin-induced activation of pyruvate dehydrogenase phosphate phosphatase in a cell-free system composed of white fat plasma membranes and mitochondria, 137 although others have questioned the relevance of the latter work on the grounds of experimental procedure, with particular reference to the viability of the mitochondria used in the experiments, a5 Another drawback for this hypothesis is that we still await the characterization of this substance even though reports of its existence have been in the literature for some time now. The proponents of this theory have also yet to adequately explain the problem of stoichiometry in their scheme. It has been estimated that only about 1,000 insulin molecules need to bind to a cell's membrane receptors to bring about a maximal response 5~'~°° (i.e. there are many spare receptors), whereas the number of intracellular molecules affected per cell will be vastly in excess of this ( > 1,000,000). Therefore, either each insulin would have to promote the release from the membrane (as the theory proposes) or production of many copies of this substance or else the substance itself would have to act in a catalytic manner.

5. Cyclic AMP-independent Protein Kinase This hypothesis has already been alluded to in Section II. B, part 3(b). It should be noted that no effects of cyclic nucleotides or Ca 2+ were observed on this kinase from white fat cell membranes.15 Insulin is known to cause the increased phosphorylation of about five different intracellular proteins, including acetyl CoA carboxylase. 35 There is evidence of increased ATP-dependent phosphorylation of some of these proteins in highspeed supernatant fractions of white fat cells previously exposed to insulin (see ref. 35). Thus, the hypothesis that insulin binding may release the kinase from the membrane, and perhaps also activate it, has been put forward. ~4 16,35 This scheme is attractive in that it circumvents the need for a second messenger and would also account for an amplified response. III. I N S U L I N

REGULATION OF FATTY ACID SYNTHESIS IN B R O W N A D I P O S E T I S S U E

The foregoing sections make it obvious that the effects of insulin are most readily observed in white adipose tissue and consequently most of the study on this phenomenon has been directed towards that tissue. This can be regarded to have taken place on two levels; first, there is the primary objective of trying to unravel the mechanisms underlying this particular effect of insulin, and second, there is the hope that a solution to this problem will contribute to a general understanding of the mechanism of insulin action. However, this research has possibly been hampered by the very nature of white adipose tissue owing to the imposition of practical limitations on experimental procedures as a

206

James G. McCormack

result of the low volume of cellular constituents which the tissue possesses. It would also be desirable to have another tissue that could be worked on in order to check, and hence substantiate, the theories put forward fi-om the study of white fat. The mechanism by which insulin stimulates ti~tty acid synthesis m the liver and man> mary gland, which are the other major recognized sites for de m~ro fatty acid synthesis, is likely to be similar to that in white adipose tissue. However, as noted in Section 11. A, the study of this phenomenon in these tissues is beset with practical difficulties. Until recently, brown adipose tissue was not considered to be an important site tk~r ,h' m~t,o fatty acid synthesis. ~~,.u~,.1,1.11 ~. ~~,,. ~,,.~ The fatty acids, which are generally thought to be the major oxidative fuel for the tissue's recognized property of nor-adrenaline induced thermogenesis, were assumed to bc largely derived from exlraccllutar sources. There had been some reports that glucose uptake by, and fatty acid synthesis in, the tissue were stimulated by insulin: however, the obscrxcd rates were low and dismissed as insignificant (e.g. see refs. 51 53, 86, 92 and 143 and refs. thereinl. This article now seeks to demonstrate that. at least in some circumstances, brown adipose tissue should be regarded as an important site for de m~t'o fatty acid synthesis. Furthermore, it will be demonstrated that insulin can stimulate this process in brown adipose tissue in a manner which appears to be very similar to that by which it stimulates the process in white adipose tissue. The potential usefulness of brown adipose tissue as an experimental model for the study of the mechanism of action of insulin on fatty acid synthesis will be discussed. This part begins with an attempt to find out reasons why the previous misconceptions about de m~ro fatty acid synthesis in brown adipose tissue, which are prevalent in the literature, arose.

A. Fatty Acid Synthesis in Di#brent Preparations o/ Brown Adipose Tissue 1. In vitro studies There are many reports that insulin added in vitro stimulates the uptake of glucose into cells and its conversion into lipid in several different brown adipose tissue preparations (see e.g. refs. 76, 86, 118 and refs. therein). These early studies invariably used small pieces of brown adipose tissue (usually around 5mg) incubated in a suitable medium, in a manner similar to that which would be used for white fat pad incubations. Experiments of this type performed in the author's laboratory are illustrated in Fig. 5. The actual parameter values for fatty acid synthesis and glucose uptake obtained Ion a tissue wet weight basist are very similar to those obtained for analogous experiments on white adipose tissue. Essentially similar rates of fatty acid synthesis are obtained when the incorporation of radioactivity from either "~H20 o r U - l a C glucose into lipid is monitored (Fig. 5): the relevance of this knowledge will become apparent later (Section III. C).

Forty acid synthesis Condition

C°rd'r°' l'nsulin

U- 4Cglucose 3H20os Gs precursur precursor ~

I I

1

5 I /zmol/g/hr o

~ 0

I

I

Pyruvote Acetyl-CoA dehydrogenose carboxylose

P

~

~ o

Glucose uptake

[H I

I t 1 0.5 I 0 i5 3I o 40I 8O 0 25 5O Fmol/g/hr /~mo{/g/hr % presentin % presentin active form activeform

F1G. 5. Summary of the effects of insulin in vitro on aspects of the fatty acid synthesis pathway of brown adipose tissue from cold-adapted rats. Data taken from refs. 107 and 108.

The regulation of fatty acid synthesis

207

It was, therefore, of interest to ascertain whether this activation of fatty acid synthesis in brown adipose tissue was accompanied by parallel activations of pyruvate dehydrogenase and acetyl CoA carboxylase, as it is in white adipose tissue. However, using the in vitro technique as described above, it was found that no alterations in activity caused by insulin were evident, and also that both enzymes appeared to be largely activated (Fig. 5). These observations first of all suggested that the effects of insulin on fatty acid synthesis in in vitro preparations of brown adipose tissue were brought about solely by the enhancement of glucose transport. Yet, the actual total amounts of pyruvate dehydrogenase and, more importantly, acetyl CoA carboxylase (as its occurrence appears to be restricted to tissues which are capable of fatty acid synthesis), in brown fat (5 10 and 1-2 units/gm wet weight, respectively 1°8) are some 5-10 times those found in white adipose tissue [a unit is defined as the amount of enzyme required to convert 1 #mol of substrate to product/min at 30~C]. It is generally agreed that the amount of acetyl CoA carboxylase found in any tissue correlates very well with the capability of the tissue to synthesize fatty acids and moreover, that the calculated flux through the enzyme, corresponding to the degree to which the enzyme is present in its active form, correlates very well with measured rates of fatty acid synthesis. This contrasts with the case of the in vitro brown fat preparation, where the expected rates of fatty acid synthesis calculated from flux through active acetyl CoA carboxylase are some ten times greater than the rates actually observed (even ignoring ~he lower temperature at which the enzyme was assayed); and furthermore, no changes in activity are seen with insulin even though it stimulates fatty acid synthesis. The enzymes' activities suggest that brown adipose tissue should be capable of sustaining high rates of fatty acid synthesis and, therefore, contradicts with the very similar rates of fatty acid synthesis which are observed in a comparison between the white and brown adipose tissue in vitro preparations. Therefore, an alternative explanation for the observations of Fig. 5 is that in ritro preparations of pieces of brown adipose tissue are insufficiently oxygenated with the consequence that there will be only a few viable cells, perhaps on the tissue's surfaces, which are able to have their metabolism influenced by insulin. This conclusion is supported by the experiment shown in Fig. 6. This demonstrates that there is a rapid (and irreversible) fall in the ATP content of brown adipose tissue when it is incubated under the in vitro conditions described above. The tissue content of ATP falls to about 10'),~ of that found in tissue freshly excised from live animals (i.e. in vivo) after only 5 10min of incubation. It should be noted that the in vivo ratio of ATP/ADP (about 2) is similar to that which is generally accepted to exist within live cells from various tissues, and that the in vivo content of ATP (about 1-2 ~mol/g wet weight) would correspond to a concentration of around 5-10 mM if the assumption is made that about 50°~,i of the tissue will be composed of fat; 8~'1t8'~43 this concentration is again similar to that which is generally expected to be found in live cells. Therefore, the observations of Fig. 6 probably mean that the bulk of the in vitro preparation of brown adipose tissue which was described is "metabolically dead." This conclusion may have the serious and far-reaching consequence of relegating to irrelevance the observations and resultant conclusions of numerous studies on many aspects of the metabolism of brown adipose tissue which used similar in vitro preparations of brown adipose tissue from rats and other animals. Many such reports, including those cited earlier, are found in, and indeed are continually appearing in, the literature, e.g. refs. 4, 7, i0, 84, 93, 94, 98, 139 and 142. The above paragraphs should also serve as a general cautionary note as to the dangers which are implicit in the extrapolation of observations made in vitro to the physiological situation. If brown adipose tissue pieces are incubated in vitro at about pH 6.6, then the fall in the tissue content of ATP could be partially prevented. ~°7 Under these circumstances, some evidence for a parallel activation of pyruvate dehydrogenase concomitant with insulin-induced enhancement of fatty acid synthesis could be obtained. However, the rates of fatty acid synthesis obtained in this case were even lower than those noted in Fig. 5, which probably reflects an overall slowing down of metabolism.

208

James G. McCormack

"6

IO

,\ •

2\:2"-,.. • 0

0

""--'.

5

I0

.... 15

."

".

20 / [ 2 5

"in vitro" t i m e (mins) ~ E x c i s s i o n and p r e p a r a t i o n of tissue (about I - 2 rains) "in vivo" ] l

FIG. 6. Effects of in ritro incubation on the adenine nucleotide content t~l brown adipose tissue from cold-adapted rats. The symbols represent: (O), ATP: ( i ) , ADP and (A) AMP. Each point shown is the mean of at least three observations. Data are taken from refs. 107 and 108.

A suitable in ritro preparation of brown adipose tissue would obviously facilitate experimentation. However. in the case of brown adipose tissue, there would also appear to be some problems associated with the use of the other available means of the in ,,itro preparation of tissue, namely the isolated cell. ~5 Principal a m o n g these is the fact that the yield of cells is very low, though their ATP content appears to be satisfactory. ~e~' Most investigators only recover less than 10",, of the tissue as cells (e.g. refs. 52, 115. 122 and 126) and, therefore, doubts must be expressed as to the suitability of such preparations in being representative of the tissue as a whole. It should also be noted that the making of cells usually involves the preliminary preparation of tissue as small pieces analagous to that already described for in citro incubation, ~15 and therefore it would appear that much of the tissue would be in a metabolically poor state even before cells were made. This could perhaps offer some explanation for the poor yields obtained. Details of the preparation and properties of brown fat cells together with a discussion on the advantages and disadvantages of their use can be found in the recent review by Nedergaard and Lindberg. i ~5

2. In vivo Studies

(a) Fatty acid synthesis. Fortunately, there are i~ rivo techniques available by which tile study of the effects of insulin on fatty acid synthesis and the degree of activation of pyruvate dehydrogenase and acetyl CoA carboxylase can be undertaken. These techniques were alluded to earlier in Section II where they had been used to demonstrate that the effects of insulin on the above parameters in white adipose tissue in rivo were essentially the same as those found in t,itro. 36"~4s They involve the creation of the conditions of the presence and absence of insulin in the animals' bloodstreams followed by the rapid removal and freeze-clamping of the appropriate tissues after a suitable incubation period: they have been found to be adaptable to the study of the effects of insulin on the pathway of fatty acid synthesis in brown adipose tissue. 1°8 Changes in the rates of fatty acid synthesis in white adipose tissue and liver of fed rats can be induced by acutely altering plasma insulin concentrations by treatment with

The regulation of fatty acid synthesis

209

either glucose or anti-insulin serum. 1*5 In this study, glucose was injected intraperitoneally to ensure high plasma insulin concentrations; this method has the advantage that the concentrations of insulin which are achieved are within the physiological range and are not accompanied by the hypoglycaemia which, for example, the administration of insulin would result in. The absence of insulin condition was created by the injection of anti-insulin serum into one of the rats' tail veins. It is worth noting that the administration of glucose together with anti-insulin serum gives results which are essentially similar to those obtained after administration of anti-insulin serum alone, i°s This indicates that the results obtained after glucose injection are, in fact, due to increased plasma insulin concentrations and are not the result of some other effect of increased glucose availability. Estimates of the rates of fatty acid synthesis in tissues in vivo can be achieved by following the incorporation of 3H into newly synthesized fatty acids in the tissues after the intraperitoneal administration of 3H20.145 The important feature to note about using this method is that, unlike the analogous techniques where, e.g. radioactive glucose is used as a precursor, the 3H20 distributes equally between the intracellular and extracellular compartments and therefore allows the easy measurement of specific activity of the precursor pool from the blood water. In both white adipose tissue and liver, it has been estimated that between 13 and 14 g-atoms of 'H' are incorporated per mole of fatty acid synthesised, ss'164.,165 For the purposes of the present discussion, this has also been assumed to be the case in brown adipose tissue and also any isotope effects have been ignored. Unlike the case for white adipose tissue, it can be seen that much higher rates of fatty acid synthesis are obtained for brown adipose tissue of the rat when it is incubated in vivo than when it is incubated in vitro (compare Fig. 7 with Fig. 5). z-4"1°~'1~° The remainder of this section will be devoted to the description of experiments which were performed principally on the cold-adapted rat. A more general discussion on aspects of fatty acid synthesis in other animals and under other conditions is given later in Section III. D. The rates of fatty acid synthesis in brown adipose tissue in vivo are found to be greatly decreased after the administration of anti-insulin serum (in the presence or absence of glucose) and greatly elevated after the administration of glucose alone (Fig. 7). Furthermore, the rates of fatty acid synthesis which are observed in the brown adipose tissue of glucose-treated rats can be calculated to be about 10 #mol of fatty acid synthesized/hr/g wet weight. This is a very high rate and is also in much better accord with the large amounts of pyruvate dehydrogenase and acetyl CoA carboxylase which are found in the

Condition

Fatty acid synthesis

Pyruvote dehydrogenase

AcetyI-CoA corboxylese

Control (saline) Anti-insulin serum

--3

Anti-insulin serum plus glucose

b

Glucose 5

/~moi/g/hr

IO

I

2O

I

40

% present in active form

0

I

20

1

40

% present in active form

FIG. 7. Summary of the effects of insulin in viva on aspects of the fatty acid synthesis pathway of brown adipose tissue of cold-adapted rats. This shows the parallel stimulation of fatty acid synthesis and of pyruvate dehydrogenase and acetyl CoA carboxylase activities. Data are taken from refs. 34, 107 and 108.

210

James G. McCormack

tissue. ~°8 The rates obtained in brown fat are in fact about 5 times the rates observed in the white adipose tissue or liver of normal fed rats. ~°8"~'*s This large capacity of brown fat for fatty acid synthesis is further emphasized by the fact that the rates of fatty acid synthesis found in the white adipose tissue and liver of the cold-adapted rats is only some 50 70°o of the corresponding rates found m normal rats. ~°s (b) Pyruvate dehydro~lenase and acetyl CoA carboxylase. The activities of pyruvate dehydrogenase and acetyl CoA carboxylase measured in extracts of brown adipose tissue after treatment of cold-adapted rats with glucose and/or anti-insulin serum are shown in Fig. 7. The response of pyruvate kinase to insulin has not yet been studied in tiffs tissue, The "'initial" activities which are shown ira Fig. 7 arc those observed immediately after extraction of the tissue under conditions thal do not allow the interconversion of the active and inactive forms of the two enzymes. ~a~' The "total'" activities of pyruxatc dehydrogenase and acetyl CoA carboxylase were dctermined after the incubation of tissue extracts under conditions where the inactive forms of the two enzymes are con> pletely converted into their respective active forms. ~'*'~ These werc found to be similar to those in in ritro preparations of the tissue and to lie within 5 10and 1 2 e n z y m e units,g wet weight, respectively.l°S It is clear from Fig. 7 that the proportions of both pyruvate dehydrogenase and acelyl CoA carboxylase present in their respective active forms change in parallel with changes in the rates of fatty acid synthesis. This parallel response is very similar to that observed in white adipose tissue (Fig. 2). No changes m the total activities of eithcr enzyme were evident in brown adipose tissue after this acute insulin treatment. Therefore, it appears that insulin stimulates fatty acid synthesis ira brown adipose tissue by a mechanism which involves the parallel activation of both pyruvate dehydrogenase and acetyl CoA carboxylase. It has also been demonstrated that insulin activates pyruvate dehydrogenase in isolated brov, n fat cells: ~ee however, neilhcr rates of fattx acid synthesis nor acetvl ('oA carboxylasc activity were measured m lhis study.

B. Effects of ln,sulin Pretreatment on f~w'ucale Dehydro.qe;m.w Activity in l,s~lated f3row;z Adipose 7i,ssue Mitochmuh'ia The discovery that the effects of insulin on white adipose tissue pyruvale dehydrogenase persist during the isolation and incubation of mitochondria has proved to be of considerable advantage. ~'*'-~7'4a's~ In particular, it allowed the elimination of several of the candidates which had previously been proposed as mediators of this effect and also directed the attention of researchers towards pyruvate dehydrogenase phosphate phosphatase as the most likely site of insulin action on the pyruvatc dchydrogcnase syslem. Therefore, recent research in this area has been governed by the idea that the hormone changes the intramitochondrial concentration of an efl'ector of the phosphatase. As .~ct, evidence for a satisfactory mechanism has not becn demonstrated and at present lhc most plausible hypothesis would appear to be that the hormone increases the conccntration of intramitochondrial Ca: + ,~.,.,~5.,~-.4~ Brown adipose tissue mitochondria are found to be uncoupled when they are isolated using conventional techniques. ~'~''55~''~' ~ ' ~ ~ They can be partially re-coupled, or energized, by incubation in the presence of albumin which serves to bind the endogenous fatty acid and fatty acyl moieties which can act as uncoupling agents. Howcvcr. to achieve full respiratory control of these mitochondria, it is also necessary to include a purine nucleoside di- or tri-phosphate such as ADP, ATP, G D P or G T P in the incubation medium. Studies by Nicholls and co-workers ~'~'11~' 119 havc revealed that the nucleotides seem to act by binding to. and hence inhibiting, a proton-leak which appcars to be associated with a 32.000 molecular weight protein which is apparently unique to brown adipose tissue mitochondria. Significantly, the amount of this protein, which is found to be present in the inner membrane of brown adipose tissue mitochondria. correlates very well with the degree of cold exposure to which the donor animal is

The regulation of fatty acid synthesis

211

subjected, 4~'77'ttS'ttS'tt9 and recently the name "thermogenin" has been proposed for this protein.~ t 5 It will be remembered from the Introduction (Section I) that the thermogenic capacity of brown adipose tissue is thought to reside in the facility of their mitochondria to become uncoupled to some degree so that the required heat can be generated; this protein would appear to offer a specificity for this process to brown adipose tissue. However, the concentrations of nucleotide required to block the proton-leak are very much lower than those found in the cytoplasm. 2t't15 Therefore, it has recently been proposed that, physiologically, the necessary degree of uncoupling of the mitochondria may be determined by the local concentration of either free fatty acid 12° or fatty acyl CoA 21 as both of these substances have been shown to competitively inhibit the effects of the nucleotides; and of course, central to the thermogenic hypothesis is that the cytoplasmic concentrations of these substances will be elevated after the nor-adrenaline enhancement of lipolysis. 21'119 Incidentally, these proposed mechanisms would have a built-in feedback system which should ensure that the whole cell is not irreversibly de-energized as the result of uncoupling impairing the production of ATP, as it is known that the nucleotide diphosphates bind to the 32,000 molecular weight protein more strongly than the triphosphates. 118,t 19 The fatty acyl CoA hypothesis would also have the feedback of ATP being required to activate the fatty acids. Preliminary attempts have been made by the author (unpublished observations) to exploit these unique properties of brown adipose tissue mitochondria as a means for investigating the mechanism by which insulin activates brown adipose tissue pyruvate dehydrogenase. Table 1 shows that this effect persists through the isolation and the incubation, under certain conditions, of brown adipose tissue mitochondria; this can be seen in mitochondria incubated in both coupling and uncoupling conditions. It should be noted that the insulin effects cannot be explained by changes in the ATP content of the mitochondria (Table 1). Incidentally, these measurements of ATP illustrate some of the known properties of these mitochondria; in particular, they served as a striking demonstration of the effects of GDP. 22"119 Table 1 also shows the effects on pyruvate dehydrogenase activity of incubating brown adipose tissue mitochondria with some of the sub-

TABLE 1. The Effects of Insulin Pretreatment on the A m o u n t s of Active Pyruvate Dehydrogenase and A T P in Isolated Brown Adipose Tissue Mitochondria from Cold-adapted Rats Incubated Under Various Conditions" Mitochondria from rats treated with anti-insulin serum

Incubation conditions Freshly isolated b 10 mM-oxoglutarate plus 1 mM-malate 10 mM-oxoglutarate plus 1 mM-malate plus 5 mMpyruvate 10 mM-oxoglutarate plus 1 mM-malate plus 0.5 mMGDP 10 mM-oxoglutarate plus 1 mM-malate plus 0.5 mMG D P plus 5 mM-pyruvate 5 mM pyruvate plus 1 mMmalate plus 0.5 mM-GDP 10 mM-succinate plus 0.5 mM-GDP

?~, of pyruvate dehydrogenase present in active form

Mitochondria from rats treated with glucose

ATP content (nmols/mg protein)

"~i of pyruvate dehydrogenase present in active form

A T P content (nmols/mg protein)

12.9 + 0.9

0.21 + 0.04

25.0 ± 1.3"**

0.19 _+ 0.04

7.4 + 0.7

0.95 + 0.11

14.0 ± 1.1"**

0.85 + 0.09

14.2 + 1.7

1.34 ± 0.13

23.7 _+ 1.7"*

1.18 ± 0.06

2.8 _+ 0.3

1.84 + 0.08

2.9 ± 0.3

1.74 ± 0.11

13.4 + 2.4

2.01 + 0.09

20.0 + 1.6"

1.94 + 0.08

19.7 ± 2.1

1.53 + 0.11

30.5 ± 2.6**

1.64 + 0.12

3.2 ± 0.6

1.73 ± 0.15

2.9 ± 0.4

1.70 ± 0.13

"Mitochondria were incubated in KCI-based medium containing 0.1°~g albumin and 2 mM-EGTA for 5 min except at h. Values are given as means + s.e.m.'s for at least six observations. *P < 0.05, **P < 0.01 and ***P < 0.001 for the effect of insulin. Data are taken from ref. 107 and unpublished observations of the author.

212

James G. McCormack

stances which are known to influence the pyruvate dehydrogenase systems of otl~er tissues. 3v.44 The effects of insulin on brown adipose tissue pyruvate dehydrogenase can be effectively lost when the isolated mitochondria are incubated under conditions which result in very low amounts of active complex (Table 1). This situation was exploited in an attempt to assay pyruvate dehydrogenase phosphate phosphatase within intact mitochondria (Fig. 8). The pyruvate analogue dichloroacetate was added, after the insulin effect was no longer evident, in order to inhibit pyruvate dehydrogenase kinase 15~ so that the phosphatase became the only interconverting enzyme working and, therefore, could be assayed by following the subsequent increases in the amount of active pyruvate dehydrogenase.l°7 Figure 8 shows that phosphatase activity appears to be enhanced in mitochondria prepared from animals pretreated with glucose compared to those pretreated with anti-insulin serum. This suggests that, as in white adipose tissue, insulin activates the phosphatase, and as such provides supporting evidence for the proposal resultant from white fat experiments, that insulin increases the amount of active pyruvate dehydrogenase in this manner. The effect of insulin on the phosphatase in brown adipose tissue mitochondria appears to diminish with time (Fig. 8). An interesting possibility is that this could perhaps be accounted for by a leakage of Ca 2 + from the mitochondria as they were incubated with EGTA. A test for this proposal would be to include Na + ions in the incubation medium as these stimulate the rate of Ca 2 + efftux from brown adipose tissue, 5 and several other m a m m a l i a n m it o c h o n d r ia , 23't2t to see if this caused the effect to diminish more rapidly: this experiment has yet to be performed. Some cautionary notes on the interpretations of the data shown in Fig. 8 should be made. First, it was assumed that the dichloroacetate inhibited the kinase completely and had equal access to it in each of the two types of mitochondria, i.e. from rats treated with glucose or anti-insulin serum. Further, it was assumed that the substrate for the phosphatase, pyruvate dehydrogenase phosphate, was present in the same amounts in each type of mitochondria just prior to the addition of dichloroacetate. This was certainly the case in terms of the amounts of active pyruvate dehydrogenase measured, However, it has

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Time of incubotion (min) FIG. 8. Evidence that insulin pre-treatment leads to an activation of pyruvatc dehydrogenase phosphate phosphatase in isolated brown adipose tissue mitochondria from cold-adapted rats. Mitochondria were incubated in the presence of 10mM-oxoglutarate, l mM-malate and 0.5 mM-GDP (see Table 1); 2 mM-dichloroacetate was added at the arrows. The symbols represent : (O) glucose pretreatment and (O) anti-insulin serum pretreatment. The points represent the means ± s.e.m, of at least three observations. Abbreviation: DCA, dichloroacetate. Data are taken from ref. 107 and unpublished observations of the attthor.

The regulation of fatty acid synthesis

213

already been noted that phosphorylation of only one of the three phosphorylation sites on the ~-subunits of the pyruvate decarboxylase components of the complex seems to be associated with the inhibition of catalytic activity. In contrast, the phosphatase appears to act indiscriminately on all three sites. 8°'166 Therefore, it is possible that there could be more phosphate on the enzyme in mitochondria prepared from rats treated with antiinsulin serum. Further study along these lines will probably require detailed phosphorylation site analysis of the ct-subunits. Preliminary to this (unpublished observations), it has been demonstrated that 3ZP i can be incorporated into the ~-subunits of the brown fat enzyme in intact coupled mitochondria incubated with oxoglutarate, or pyruvate, and malate. Furthermore, decreases in the steady-state incorporation, corresponding to higher amounts of active enzyme, were noted, either in mitochondria incubated with dichloroacetate, or in mitochondria from glucose-treated rats (when compared to controls). The pattern of phosphorylation in tryptic digests of the ct-subunits of the brown fat enzyme was also found to be very similar to that of either the heart or white adipose tissue enzyme (see ref. 80). In summary of this section, it appears that the mechanism of the regulation of fatty acid synthesis by insulin in brown adipose tissue is very similar to that in white adipose tissue. Brown adipose tissue may prove to be a useful experimental model in the continuing search for the answer to the problem of how insulin works. In particular, the tissue has a very large capacity for the de novo synthesis of fatty acids and this process is regulated by insulin; in addition, there would appear to be few competing processes occurring in the tissue which should make study easier and, of course, in comparison to white fat, brown fat offers large amounts of experimental material. However, a major drawback could be the apparent insuitability of in vitro preparations of the tissue. The unique properties of brown adipose tissue mitochondria may also prove to be advantageous in the study of the means by which insulin activates pyruvate dehydrogenase. It would also be of considerable interest to see if brown adipose tissue possesses the membrane-associated cyclic AMP-independent protein kinase, described in white adipose tissue, which phosphorylates and activates acetyl CoA carboxylase. 15,16,35 In the remaining Sections of this article, some other aspects and possible implications of the recently recognized role of brown adipose tissue as a significant site for the de novo synthesis of fatty acids, and the role that insulin may play in the regulation of this process, are discussed. C. The Substrates for Fatty Acid Synthesis in Brown Adipose Tissue

There are now four mammalian tissues which should be considered as being significant sites for the de novo synthesis of fatty acids; 34'3v'1°8 these are the liver, the (lactating) mammary gland, and both white and brown adipose tissue. In addition, there is evidence that insulin stimulates this process in each case. 34"37'1°8 In the whole animal, the extent of fatty acid synthesis taking place in and the substrates which are used or are available for use (for this process) by the various tissues will depend very much on the nutritional and hormonal status of the animal, z-4.71.73.162 However, for simplification, if only the case of the normal adult animal fed a balanced diet is considered, then it is evident that blood borne glucose is the major, if not exclusive, precursor for newly synthesized fatty acids in both white adipose tissue and the lactating mammary gland. 34,71.73.162 In contrast, plasma glucose seems to be a poor substrate for this process in liver. 71'73 It has been suggested that glucose is not a major substrate for fatty acid synthesis in brown adipose tissue. ~5° However, this conclusion was reached by comparing rates of fatty acid synthesis obtained using 3 H 2 0 as a precursor in vivo to rates obtained using radioactive glucose as a precursor in vitro. Indeed, it was further suggested that earlier underestimates of the significance of brown adipose tissue as a site for the de novo synthesis of fatty acids were because radioactive glucose was used as the precursor in the

214

James G. McCormack

early work. ts° However, from Fig. 5, it can be seen that using 3 H , O as a precursor in ~,itro gave essentially similar results as those obtained with U-l'*C glucose and, as has been explained earlier, the previous misconceptions almost certainly arose because of the unsatisfactory nature of the in citro preparations of brown adipose tissue which were used. The technique developed by Hems and co-workers 7'v-~ allows a means whereby the contribution of b l o o d - b o r n e glucose as a precursor for fatty acid synthesis in different tissues of intact animals can be estimated. This relies on the justiliable assumption that the almost exclusive source of c a r b o n to be found in the newly synthesized fatty acids in the white adipose tissue of normally fed adult rats iJ~ cico will be circulating glucose. The m e t h o d involves the administration of both 3 H 2 0 and U-~'*C glucose to intact animals and the subsequent measurement of the ratio of l'*C"3H counts incorporated into the fatty acids in the various tissues. By then expressing this ratio of counts determined in the tissues under examination as a percentage of the ratio found in white adipose tissue, an estimate of the extent to which b l o o d - b o r n e glucose can serve as a substrate for fatty acid synthesis in these other tissues can be achieved. T~v~ "I'ABIJ2. The Usc of Glucose as a Substrate for the in rico Synthesis of gatt~ Acids in the I_,iver and Brown Adipose Tissue of Coldadapted Rals." Estimation of the ",, of fatty acid synthesized using plasma glucose as substrate in" Condition Anti-insulin serum plus glucose Glucose alone

Liver

Brown adipose tissue

3.6 + 0.7 9.9 + 1.1

66.3 ± 35) 77.9 .+ 6.6

"See lext for details of method. -'~'3 bValues are given as means + s.e.m.'s for four observations. Data arc taken from ref. 107 and unpublished obserwttions of the author. The information given in Table 2 appears to indicate that plasma glucose can act as a major substrate for the de nero synthesis of fatty acids in the brown adipose tissue of fed rats in cite. This is most strikingly evident when c o m p a r e d to the situation in the liver (Table 2) where it would seem that plasma glucose does not serve as a significant substrate for this process. " : 1 , 7 3 A report has recently been published 4 which indicates that ketone bodies may serve as a substrate for the de noco synthesis of fatty acids in rat brown adipose tissue. The tissue appears to have high activities of the necessary enzymatic machinery to allow this'* and it is suggested that ketone bodies may form the p r e d o m i n a n t substrate in the starved condition where circulating levels of these substances are high and c a r b o h y d r a t e has to be conserved: and where, interestingly and in contrast to liver and white adipose tissue, fatty acid synthesis in brown adipose tissue still proceeds at signiticant rates and retains the ability to be stimulated by insulin 4 or glucose. ~47 However, no quantitative measurements of the contribution of ketone bodies as substrates for fatty acid synthesis were made.'* These authors'* also attempted to argue that ketone bodies are an important oxidative fuel for brown adipose tissue. However, this a r g u m e n t was formulated on the basis of in citro experinaents with tissue pieces and thus should be viewed (in isolation) with caution as vet. D. T h e Siqni[icance ~?l Browt7 Adipose Tissue as a Site.[br tie m~co F a t t y acid Synthesis

The information given so far in this section has adequately d e m o n s t r a t e d that brown adipose tissue is a major site for the de not,o synthesis of fatty acids in normal fed rats which have been cold-adapted. In fact, if the assumption, which would appear to be

The regulation of fatty acid synthesis

215

valid, is made that the other major sites of d e novo fatty acid synthesis in these animals are the liver and white adipose tissue, then it can be calculated that the d e n o v o fatty acid synthesis occurring in the brown adipose tissue of these animals would account for some 40-605o of that occurring in the whole animal (Table 3); this appears to be the case in both the presence and absence of circulating insulin. Trayhurn 15° has demonstrated that the brown adipose tissue of cold-adapted rats is capable of exceptionally high rates of fatty acid synthesis in vivo if the animals are kept in a cold environment while the experiment is being performed (see Table 3). The rates obtained in these experiments are even higher than those found in the presence of circulating insulin (Table 3) in experiments which were undertaken after bringing the cold-adapted animals out into a laboratory at room temperature about 1 hr before their use. 1°8 Trayhurn 15° has shown that this leads to a marked reduction in rates of fatty acid synthesis in brown adipose tissue of cold-adapted rats. Indeed the values obtained for the control situation, i.e. cold-adapted rats in a warm environment, in each of the two independent studies 1o8,15o are very similar. A similar calculation to that described above carried out on the data obtained from the cold-adapted animals in a cold environment 15° would suggest that about 70°J,, of the d e novo fatty acid synthesis occurring in the whole animal takes place in brown adipose tissue under these circumstances (Table 3). The response of brown adipose tissue to insulin in cold-adapted rats maintained in a cold environment is not yet known. An interesting possibility is that insulin may be involved in this enhancement of fatty acid synthesis in cold-adapted rats by a cold environment; it is worth noting that cold-exposure of these animals also led to a slight enhancement of fatty acid synthesis in both white adipose tissue and, more markedly, in liver 150 (see Table 3). It would be of considerable interest to determine whether pyruvate dehydrogenase and acetyl CoA carboxylase are activated by cold-exposure of these animals arrd also to see if anti-insulin serum impaired the response of fatty acid synthesis to this treatment. However, it must be mentioned that rates of fatty acid synthesis in the brown adipose tissue of warm-acclimated rats is not stimulated by (acute) cold exposure, 15o whereas they can still be stimulated by insulin (see below). Another possibility is that the proposed thermogenic instigator, nor-adrenaline, may elicit the cold-induced response of fatty acid synthesis in brown adipose tissue of cold-adapted rats. In support of this, it was reported that the stimulation of ventromedial hypothalamic nuclei (which is thought to lead to release of nor-adrenaline from nerve-endings 14°) was found to

TABLE 3. Estimation of the Contribution that the Brown Adipose Tissue of Cold-adapted Rats Makes to the de novo Fatty Acid Synthesis Occurring in the Whole Animal Approx rates of de novo fatty acid synthesis (pmol/g/hr) occurring when rats were subjected to Experiments performed at room temperature

Tissue a Brown adipose tissue Liver White adipose tissue

Approx. weight h of tissue (g)

Control

Anti-insulin serum

Glucose

Experiments performed at 4°C. Control

4~ 10

5.1 1.1

1.9 0.8

10.6 1.7

16.7 2.5

5

0.4

0.3

1.8

0.7

Estimated percentage contribution of brown adipose tissue to wholeanimal de novo fatty acid synthesis

61

44

62

70

~These have been assumed to be the major sites in which de novo fatty acid synthesis occurs in these animals. bBased on a 200 g rat. CThis is based on the knowledge that the interscapular tissue accounts for about 25% of the total brown adipose tissue in rats 116 and the assumption that fatty acid synthesis in other sites will proceed at the same rate as that in the interscapular site. Data taken from refs. 108 and 150 and unpublished observations of the author.

216

James G. McCormack

specifically enhance fatty acid synthesis in brown adipose tissue {with respect to white adipose tissue)~without the intervention of insulin secretion. T M This report also claimed that the intraperitoneal injection of nor-adrenaline caused an increase in rates of fatty acid synthesis in brown adipose tissue: however, another report is at variance with this finding. 3 The key role that fatty acid synthesis is likely to play in the brown adipose tissue metabolism of cold-adapted rats is emphasized by the very high activities of both pyruvate dehydrogenase and acetyl CoA carboxylase found in the tissue (Fig. 71.1°~ The total activity of pyruvate dehydrogenase (5 10 units/g wet weight) is higher than that found in any other mammalian tissue that has been examined, including heart mtlscle: 74'<>'' this is especially striking when it is remembered that some 50", of the brown adipose tissue mass will be fat. 8<~1~ This activity is about 5 10 times that which is found in white adipose tissue or liver, tissues which are widely regarded as being major sites for the d<, m w o synthesis of fatty acids. Moreover. the total activity of acetyl CoA carboxylase ~1 2 units/g wet weight), which is an enzyme generally agreed as being solely concerned with fatty acid synthesis, is some three to five times the corresponding activities in white adipose tissue and liver. ~°8"~'~5 The large excess of pyruvate dehydrogenase activity lo that of the carboxylase would suggest that a considerable proportion of the acelvl CoA produced by this reaction will enter the citrate cycle for further oxidation: the possiblc significance of this will be discussed in more detail later (Section V). Up to this point, the discussion of fatty acid synthesis in brown adipose tissue has been largely restricted to the tissue from cold-adapted rats. The rates of fatty acid synthesis and the total activities of pyruvate dehydrogenase and acetyl CoA carboxylase observed in the brown adipose tissue of normally fed. warm-acclimated rats are only about 50 70°. of those observed in the cold-adapted animals. 2 ,*.~,~.~.t.;~.~, In addition. there is less brown adipose tissue present in the warm-acclimated animals, perhaps about 20--30",, of that present in the cold-adapted rats. s<'~ ~s However. rates of fatty acid synthesis, and the amounts of both pyruvate dehydrogenase and acetyl ('oA carboxylase present in their respective active forms, respond to insulin in a manner similar to that shown in Fig. 7 for the cold-adapted rats. ~'1"~'~"x'1"*~ If similar calculations to those of Table 3 are performed, then it can be estimated that the brown adipose tissue of warmacclimated rats could still account for up to about 10 20!',, of the de m~co synthesis of fatty acids occurring in the whole animal. 1"" As will be discussed in the next section. fatty acid synthesis in the brown adipose tissue of normal animals could have a role t~ play in nutritional balance. Fatty acid synthesis has also been studied in the brown adipose tissue of mice. rabbits and hamsters. The properties and comparative importance of this process in the brown adipose tissue of the mouse appears to be very similar to those exhibited by the rat tissue (as compared to liver and white adipose tissuel ls2 with the possible exception that fatty acid synthesis occurring in the remaining carcass of this animal {after remowd of the lixer and both brown and white adipose tissue) appears to make a more considerable contribution to whole animal fatty acid synthesis (around 70",,}.lSS This report ~5e also demonstrated that fatty acid synthesis in the brown adipose tissue of suckling mice is considerably diminished when compared to adults. This probably reflects the high fat content of milk as the rate can also be diminished in adult mice subjected to hmg-term feeding on a high fat diet: ~55 this feature is in common with that exhibited by the other recognized major sites of de m~,o fatty acid synthesis, the liver, lactating mammary gland and white adipose tissue. ~ss The brown adipose tissue of foetal rabbits has also been shown to exhibit very high rates of fatty acid synthesis. ~2 However, in the hamster, which is a hibernator, it was found that the de mwo synthesis of fatty acids in brown adipose tissue did not play such a significant role when compared to rates occurring elsewhere in the animal (e.g. in liver or white adipose tissue). ~s~ An attempt was also made in this study ls~ to assess the contribution that fatty acids which had been synthesized de m~co in other tissues made to the pool of fatty acids found in brown adipose tissue. This involved the injection of Triton (WR 1339)into the animals' bloodstream to try and eliminate any

The regulation of fatty acid synthesis

217

transport of fatty acids via this medium by solubilizing the lipoprotein carrier molecules. It was found that the incorporation of 3H20 into brown adipose tissue lipid was indeed reduced by this treatment which suggests that the tissue in the hamster may derive a large part of its fatty acids from sources outside the tissue rather than by de novo synthesis in the tissue. However, it should be pointed out that treatment with Triton also resulted in a decrease in the apparent rates of fatty acid synthesis occurring in the other sites which were examined (the liver and white adipose tissue). 151 An analysis of the amounts of the enzymes of the fatty acid synthesis pathway in hamster brown fat would perhaps clarify the likely importance of this process in the tissue. In summary, therefore, it would appear that with the evidence which is available to date, brown adipose tissue, at least in some circumstances, should be considered to be a significant site for the de novo synthesis of fatty acids. Certainly, in cold-adapted rats and mice, the tissue makes a far greater contribution (on a whole body basis) to this process than widely recognized sites of de novo fatty acid synthesis such as liver and white adipose tissue, and even in warm-acclimated animals still makes a considerable contribution. The subsequent fate of newly synthesized fatty acids in brown adipose tissue is not yet known; however, some possibilities will be discussed later in Section V. IV. B R O W N A D I P O S E T I S S U E AND O B E S I T Y

There is a general agreement that, if animals are placed in a cold environment, they are required to produce extra heat in order to remain alive and this has been termed "cold-induced thermogenesis." There is also good evidence that, at least in some animals, the response of brown adipose tissue to the stimulus of cold offers an explanation as to how a large fraction of the excess heat which is required can be generated by the tissue's inherent capacity for non-shivering thermogenesis. This general hypothesis also dictates that, in the cold, the animals will take in more calories as food and use the excess (compared to the intake in a normal temperature environment) to produce heat in brown adipose tissue. In the last two or three years, there has been a concerted research effort which has sought to demonstrate that a parallel situation occurs when a normal animal takes in excess calories by induced overeating, but avoids obesity by burning-off the excess as heat. This has been termed "diet-induced thermogenesis" and central to the resultant hypothesis for the mechanism of this process are the thermogenic properties of brown adipose tissue. 2.13.24"vv'sv'135'136 The evidence which has been obtained to date for this hypothesis is briefly outlined below. Later reference will be made to the potential roles of insulin and fatty acid synthesis in this scheme for brown adipose tissue. When rats are offered a varied and palatable diet (the "cafeteria diet"), 135 they are induced to overeat and consume many more calories than they require. Yet, most of them do not gain excess weight when they are compared to littermates fed on a normal laboratory "chow" diet. Instead, there is a marked increase in heat production in response to the overeating, 135 which is associated with temperature increases at sites which were subsequently found to contain brown fat. More strikingly, in the long term, there is a coincident and specific increase in the tissue mass of the animals' brown fat. However, this increased heat production and brown adipose tissue hypertrophy are not found in animals which become obese on overeating. The increased capacity for thermogenesis as a result of long-term overeating also appears to be accompanied by changes in the properties of brown adipose tissue mitochondria which are similar to those observed on cold adaptation. In particular, it would appear that brown adipose tissue mitochondria from rats which exhibit "diet-induced thermogenesis" have an increased capacity for proton-cycling (i.e. they are more uncoupled). 13 This would appear to be the result of their having increased amounts of the specific, 32,000 molecular weight, nucleotide-binding protein (see Section III. B 119) which is thought to constitute the unique proton-leakage pathway of brown adipose tissue mitochondria in their inner membrane. 13 However, it should be noted that this study

218

James G. McCormack

and those described in the next paragraph ¢'°'7s only offer the indirect evidence of G D P binding to mitochondria in support of their conclusions and as yet no direct demonstrations of changes in the amount of this protein as a result of diet have been made. Brown adipose tissue mitochondria from obese rats do not exhibit these adaptations to overeating noted in the previous paragraph. In fact. it would appear that the milochondria from these animals have lower amounts of the 32,000 molecular weight protein than lean controls even when both sets of animals are on a normal diet. '~°':~ Furthcrmorc, obese animals exhibit a failure to adapt to cold stress. 24153 Such observations have led to the idea that obesity may be the result of attticted animals having a defective brown adipose tissue or a defective means for eliciting the thermogenic response of the tissue. It should be pointed out that some criticisms of the postulated role that brown adipose tissue plays in diet-induced thermogenesis have been raised. 75 There is some evidence to indicate that insulin may play a role m the reduction of diet-induced thermogenesis. Diabetic rats do not exhibit this process unless they arc subjected to acute insulin replacement, ~~" suggesting that insulin may have a pcrmissixc or facilitatory role in the dietary induction of thermogenesis. Others haxe shown thai short-term insulin deficiency prevents the marked increase m the de nozo synthesis ol fatty acids by brown adipose tissue in response to the intragastric administration of glucose or medium-chain triacylglycerol to virgin r a t s ) These workers also demonstrated that brown adipose tissue hypertrophied during pregnancy, regressed during lactation and hypertrophied again on weaning, and that the rate of fatty a.cid synthesis occurring in the tissue paralleled these changes in tissue mass. 3 Food intake during lactalion actually exceeds that in pregnancy: however, it seems that m the former condition substrates for fatty acid synthesis are preferentially directed towards the m a m m a r y gland. probably as the result of high plasma concentrations of prolactin, e3 Further, insulin levels are high in pregnancy and low during lactation and these workers have also suggested that these may be related to the changes in the mass of brown adipose tissue. ~ When lactating rats are ted a "'cafeteria-diet," fatty acid synthesis in the m a m m a r y gland is impaired whereas that in brown adipose tissue is increased suggesting thai, in this instance, substrates are preferentially directed to brown fat. 2 However, no brown adipose tissue hypertrophy was noted in these rats unless insulin was also administered. 2 The effect of "cafeteria-diet" feeding on the de noco synthesis of fatty acids in tlac brov~n adipose tissue of non-lactating rats has not yet been examined. The above paragraph provides evidence to suggest that insulin may be revolved in brown adipose tissue hypertrophy: it also suggests that some agent(sl other than insulin. which appears to be involved in eliciting thermogenesis and is released in response t¢~ overeating, can stimulate fatty acid synthesis in the tissue. This is reminiscent of the increased rates of fatty acid synthesis induced by cold-exposure Iof cold-adapted tarsi and by the stimulation of ventromedial hypothalmic nuclei which were noted earlier (Section II1. D}. l a l ' l s ° Significantly, destruction of this area of the brain leads to obesity, 14° whereas stimulation of this area results in increased heat-production by' brown adipose tissue of normal rats, ~2"* presumably via a release of nor-adrenaline from the nerve-endings in the tissue. ~'*° However, as noted earlier, therc appear to be conflicting reports as to whether or not nor-adrenaline can enhance fatty acid synthesis in brown adipose tissue. 3~'*~ A report that glucagon increases the uptake of glucose into the brown adipose tissue of cold-adapted rats should also be mentioned in the present context. ~s V. CONCLUSIONS AND SOME SPECULATION The rates of de novo fatty acid synthesis (on a wet weight basis} which can be achieved in brown adipose tissue, at least from rat, are higher than those in any other mammalian tissue so far examined. The earlier underestimates and hence, neglect of de noro fatty acid synthesis in this tissue are probably the result of the poor viability of the tissue in ~'itro. Fatty acid synthesis in brown adipose tissue can be stimulated by insulin by a mechan-

The regulation of fatty acid synthesis

219

ism involving the parallel activations of pyruvate dehydrogenase and acetyl CoA carboxylase. Experiments on isolated mitochondria suggest that an activation of pyruvate dehydrogenase phosphate phosphatase is probably the means of achieving the former activation. This mechanism matches that in white adipose tissue suggesting that a similar means of insulin action on the two tissues is involved. The substantial capacity of brown adipose tissue for de novo fatty acid synthesis and the peculiar properties of its mitochondria may afford this tissue advantages as a model in the study of the mechanism of action of insulin. A functional role for the substantial capacity of brown adipose for de novo fatty acid synthesis and its stimulation by insulin has yet to be evaluated. For instance, the subsequent fate of the newly synthesized fatty acids has yet to be determined. There is evidence that some part of them may be exported for use elsewhere. 1~.21.~5 However, the fact that changes in brown adipose tissue thermogenesis often appear to be paralleled by similarly directed changes in de novo fatty acid synthesis suggests a more intriguing possibility. First, it could be suggested that the fatty acids will form the subsequent thermogenic fuel as obviously more of this will be required to produce heat in response to cold- or diet-induced stress. However, although it is generally accepted that fatty acids are the oxidative fuel for non-shivering thermogenesis, 2~'86'118'~9 there is, in fact, no direct evidence that this is physiologically always the case. Otherwise, it is difficult to see (unless a heat-producing futile cycle is invoked) how both fatty acid synthesis and lipolysis could be stimulated simultaneously. Therefore, the opportunity of writing this article is taken to tentatively advance an alternative (and highly speculative!) scheme in an attempt to encompass the observations which have been outlined therein. Principally, this is that thermogenesis does not have to rely on lipolysis to provide an oxidative fuel, but rather that blood glucose (which would be available in animals fed a high carbohydrate diet) can also act as a substrate for this process. Thus, it is envisaged that, if glucose is available in the bloodstream and thermogenesis in brown adipose tissue as the result of cold or over-eating is required, then insulin, in conjunction with the released thermogenic stimulator (presumably nor-adrenaline) will cause the preferential direction of the glucose to brown adipose tissue for thermogenesis. Such a scheme would be analogous to the preferential directing of glucose, as a substrate for fatty acid synthesis, to the mammary gland in lactation. In this instance, insulin seems to act in conjunction with prolactin.l'5~'162 In the case of brown adipose tissue stimulated to produce heat, the excess glucose which is not oxidized would be converted to fatty acid in this tissue rather than elsewhere; this could explain why this process appears to be activated under conditions where thermogenesis is stimulated. When glucose (and insulin) are no longer present in the bloodstream but thermogenesis is still required, then this could proceed under the influence of the thermogenic stimulator (i.e. nor-adrenaline) using the enhanced levels of fatty acid generated while glucose was available, by the more generally accepted scheme of activated lipolysis and fatty acid oxidation. In other words, the function of insulin could be seen as a switch mechanism whereby the fat fuel present in the tissue for thermogenesis can be conserved and augmented when glucose, as an alternative fuel, is available in the bloodstream. A measurement of respiratory quotients for oxidation by the tissue in situ under various conditions could help to resolve which fuel is used for oxidation and when. It should be mentioned that this scheme could not readily encompass the ideas that fatty acid 12° or fatty acyl CoA 2~ moieties mediate the thermogenic response of brown adipose tissue by uncoupling the mitochondria and probably, therefore, another signal would have to be invoked. However, it could account for the observations which have been made on fatty acid synthesis in brown adipose tissue although it is freely admitted that much more evidence is required. If thermogenesis is not required and so is not stimulated, then the insulin stimulation of fatty acid synthesis could be viewed as analogous to the situation in white fat, i.e. as a storage point for glucose till required later by other tissues. Whether acetyl CoA is derived from glucose or fatty acid, thermogenesis requires its oxidation, i.e. citrate cycle activity, to be increased; and there is plentiful evidence that J.P.L.R.

21/3

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James G. McCormack

this situation causes oxygen uptake by brown fat to be markedly increased. 2~'86'~19 It was noted that pyruvate dehydrogenase is present in about five times the quantity as that of acetyl CoA carboxylase (Section III. D), suggesting that a considerable amount of the acetyl CoA formed by the former enzyme will enter the citrate cycle even when fatty acid synthesis is stimulated. There is some indirect evidence that adrenaline (acting via ,8-receptors) may activate heart pyruvate dehydrogenase by causing an increase in the intramitochondrial concentration of Ca 2 +, parallel or secondary to its increasing cytoplasmic C a 2 + . 79'111"113 It was further suggested that, in this instance, citrate cycle activity could also be stimulated by the increased intramitochondrial C a 2 + activating NAD +-isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex. 41 These two enzymes are within the citrate cycle and both are activated by increases of C a 2 + within the same concentration range as activates pyruvate dehydrogenase phosphate phosphatase. It is tempting to speculate that nor-adrenaline could achieve its stimulation of citrate cycle activity in brown adipose tissue by an analogous mechanism, i.e. increasing intramitochondrial Ca 2+. However, it must be conceded that citrate cycle activity could also be increased by more provision of substrate (enhanced lipolysis) or removal of respiratory inhibitor products (uncoupling). Acknowledgements I would like to thank Dr. Richard M. Denton, in particular, and also Dr. Roger W. Brownsey (of this department) for much useful advice and discussion and for the critical reading of the whole, and parts, respectively, of the manuscript for this article. I also thank Dr. David G. Nicholls (Department of Psychiatry, University of Dundee) for allowing me to mention some of his work prior to its being published ~zo and Dr. Barbara Cannon (Wenner-Gren Institute, University of Stockholm) for sending me several reprints and preprints of her work and that of her colleagues in Stockholm. Experiments of the author were financially supported by grants from the Medical Research Council.

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