The obese zucker rat: A choice for fat metabolism 1968–1988: Twenty years of research on the insights of the Zucker mutation

Prog. Lipid Res. Vol. 28, pp. 53-66, 1989 Printed in Great Britain. All rights rcscrv¢d

0163-7827/89/$0.00 + 0.50 © 1989 Pergamon Press plc

THE OBESE Z U C K E R RAT: A CHOICE F O R F A T M E T A B O L I S M 1968-1988: TWENTY YEARS OF RESEARCH ON THE INSIGHTS OF THE ZUCKER MUTATION JOSEP M. ARGILI~.S Departament de Bioquimica i Fisiologia, Unitat de Bioquimica i Biologia Molecular B, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08071 Barcelona, Spain CONTENTS I. INTRODUCTION: THE FA/FA GENOTYPE II. HYPERPHAGIAOF THEFAr r r RAT: A CHOICEFOR FAT

53 53

III. THE A D I ~ GROWTH A. Hypertrophy and hypcrplasia B. Lipid synthesisand mobilization

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IV. MOLECULARBASISFORALIERF_.DCELLULARENERGYEFFICIENCY A. Futile cycling B. Brown fat thermogenesis C. Protein turnover

57 57 58 59

V. TEas ENOOCmNE ENVIRONMENT OF THE OBESE ZUCI~R RAT

A. The role of insulin B. Other hormonal changes C. Neuropeptides VI. CONCLUDINGREMARKS ~NCES

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60 61 63 63 64

I. INTRODUCTION: THE FA/FA GENOTYPE Genetically obese rodent models have been widely used to understand the etiology of obesity. Although obese animals cannot serve as exact models o f the human condition, they have provided important insights into the causes o f human obesity and possible consequences and cures. The Zucker-fatty rat (Rattus norvegicus) appeared as a result of a spontaneous mutation in a cross between the Merck Stock M and Sherman rats. 12''63 The obesity is transmitted as an autosomal Mendelian recessive trait 163and the animals which are homozygous suffer the consequences of genetic obesity, accumulating fat progressively throughout their lives. The exact molecular mechanism of the f a gene is unknown. Nonetheless, it is clear the ad libitum fed homozygous recessive animal develops, in a predictable orderly sequence, an obesity syndrome that closely resembles that displayed by juvenile onset obese man. 9'19'33 The fa/fa animal cannot be visually detected until 5 weeks of age. By then, its weight and shape are significantly different from those of its lean litter-mates. However, a number of different techniques have been used for the early detection of thefa/fa genotype. These include analysis of hypodermal adipocyte morphology, 63 which allows the identification from 7 days of age, lower rectal temperatures 58 and reduced 02 consumption, 77 the latter permitting an identification 2 weeks after birth. At the molecular level, the obese rat has an important number of disturbed processes which constitute the background of this kind of obesity. This review focuses on the importance of the metabolic environment of the fatty rat in relation to the obesity syndrome. II. HYPERPHAGIA OF THE FATTY RAT: A CHOICE FOR FAT The fatty rat is hyperphagic. Both during the day and at night the obese animals eat impressively large meals '5:39 compared with the food intake o f their lean litter-mates. This feeding behaviour is one of the main characteristics of the obese Zucker rat. Lean rats will adjust their intake of calorically different diets so as to maintain a characteristic level of 53

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caloric intake. For instance, when they are submitted to chronic ethanol treatment, they adjust their food caloric intake, taking into account the calories provided by ethanol, ss Using either cellulose-diluted'43 or calorically dense diets, it has been demonstrated that there are compensatory increases or decreases in food intake. Similarly, the lean rat can control not only his caloric needs but also dietary quality. Rozin ~29 demonstrated that diluting a liquid protein source with water can promote compensatory increases in the intake of that protein source. Collier and Bolles 36 have demonstrated, using the dilution of a sucrose solution, compensatory intake within a wide range of concentrations. It has been speculated that the rat can, under certain conditions, regulate the composition of its diet. 24 The obese rat can also, to some extent, regulate its caloric intake. Castonguay et al. 25 have reported that Zucker rats, given three separate macronutrient sources, will compose a diet that provides more than twice the fat and 1.5 times the calories of the diet selected by lean litter-mates. It has been speculated that this appetite for fat may be related to the general hyperinsulinemia of the Zucker rat since the reduction of insulin levels in obese rats to levels normally found in lean rats reduces the intake of fat that characterizes the obese rat selection habits. 27''4' Although the hyperphagia is a major factor in the deposition of lipid in f a / f a rats, it is not the only factor responsible since pair-feeding obese rats with lean ones does not stop the development of the obesity. ~7,t23Additionally, food restriction before weaning does not stop the hypertrophy and hyperplasia of adipose tissue depots. 72 Although jejunoileal bypass was originally designed to produce malabsorption of ingested nutrients, its primary effect seems to be the reduction of food intake g' through alterations in satiety signals from the lower gut 8° owing to the production of visceral discomfort, m Greenwood et al. 6° studied the effects ofjejunoileal bypass on obese Zucker rats and, although they saw a reduction in body weight to normal levels, the obese rats maintained high levels of lipoprotein lipase activity in adipose tissue, hyperinsulinemia, hyperlipemia, increased fat cell and reduced carcass protein, thus proving that the hyperphagia is not the main factor involved in the development of obesity in the obese Zucker rat. III. THE ADIPOSE GROWTH Although the peripheral mechanisms which influence feeding behaviour are only partially understood, it has been suggested that the adipose tissue provides unidentified metabolic signals which interact with the central nervous system (CNS) in a negative feedback circuit. 5° However, the lack of innervation of white adipose tissue would make these signals act in an indirect way. The possible metabolic compounds that could be involved in this feeding circuit should be related to the metabolism of the adipocyte. Glycerol and fatty acids--produced by lipolysis of the triglyceride mass contained in this tissue--may be involved in signalling the state of depletion or repletion of the adipocytes. 13,23This hypothesis has been called "lipostatic" and is supported by the fact that, when the adipocyte size reaches a maximal level, an inhibitory influence upon feeding may occur, resulting in reduced levels of food intake. Conversely, when replete adipocytes have been reduced in size for any reason, a stimulating effect upon feeding may be exerted as the adipocytes start to refill with lipid. 49 A. Hypertrophy and Hyperplasia

The type of adipose cellular growth in the obese rat involves both hyperplasia and hypertrophy. In this form of obesity, there is an increase in the size of adipocytes and an increase in their number. 74'75 The number can be partially reduced but not prevented by dietary manipulations. 1°4The number of fat cells is also influenced by age and adipose cell depot. '6 The increase in fat cell size is especially striking in the subcutaneous depot. 16 Thymidine kinase and DNA polymerases--enzymes both related to proliferative activity--

The obe~ Zueker rat

55

Liver

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Adipocyte hyperpt~ia, hypertrophy FIG. 1. The 'fatty' metabolism of the obese Zucker rat.

have high activities in this tissue. 72 It has been suggested that the metabolic activity of adipocytes is directly related to their surface area so that large cells will have a higher total activity, but activity per unit mass will be reduced compared to small cells./4° The enlargement of fat cell size in the obese Zueker rat 73could represent an attempt to correct for a reduced activity by increasing the size and total surface area of the adipocytes. On the other hand, of the three major abnormalities of the genetically-obese fatty rat--hyperphagia, excess adiposity and hyperlipidemia--excess adiposity develops as early as the first week of life before any excess caloric intake is present and raises the question of whether adipose tissue growth is the result of a defect in energy expenditure or an abnormality of fat cell storage capacity or both. 2] However, lack of feedback control from adipose tissue can only explain the problem partially because, infa/fa rats prevented from expressing hyperphagia throughout life, the complete "obese syndrome" still develops) 3

B. Lipid Synthesis and Mobilization The genetically obese Zucker rat is hyperlipidemic. The concentrations of both circulating cholesterol and triglycerides are very high. 2,4~,~3 The liver of the fatty rat synthesizes an excess of triglycerides and oxidizes little fatty acid; the imbalance between lipid synthesis and oxidation results in fat infiltration of the hepatic parenchyma and liver steatosis. ~ The intense hypcrlipoproteinemia is characterized by increases in all lipoprotein classes but, in particular, by marked elevations in very low density lipoproteins (VLDL). ~31 These lipoproteins are altered in composition in comparison with VLDL isolated from the

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Zucker lean control. The fatty VLDL are characterized by enhanced triglyceride content, alterations in density distribution and changes in apoprotein composition. 152 The source of these altered lipoproteins is not certain. They could be secreted by liver ~3zor by gut TM or they could also arise from alterations in intravascular VLDL metabolism4s.t~7 or combinations of the above. However, livers from obese rats release three times more triglyceride as VLDL than do the livers of lean rats. As already pointed out, the fatty rat has a hunger for fat and its obesity is directed by a kind of "push mechanism" that forces the nutrients towards adipose tissue. It has been demonstrated that lipoprotein lipase--the enzyme which hydrolyzes plasma triglycerides prior to their entry into extrahepatic tissue--activity in the obese rat is increased in white adipose tissue and decreased in brown adipose and muscle as early as the first or second week of life.22'68Thus, the ability to synthesize and store lipid is high in the obese rat, but the ability to utilize it for energy is impaired. The fact that adipose tissue LPL remains elevated in the fatty rat even under normally catabolic conditions suggests that this animal exhibits chronically elevated clearance of circulating triglycerides into adipose tissue. Such a "shunting" of calories into adipose tissue could underlie the maintenance of its enlarged fat cell size and obese body composition despite the metabolic challenges imposed by dietary restriction, 6~ and pharmacologicaP48 and surgical intervention.6° However, the clearance of cholesteryl esters and of remnant particles--resulting from VLDL and quilomicron metabolism--is impaired in the obese Zucker rat. ~25 In relation to fatty acid synthesis, it is clear that the lipogenic capacity is largely increased in the Zucker rat. In liver, the high lipogenic rate is potentiated by a huge glycolytic flux~°°--stimulated by high concentrations of fructose-2,6-bisphosphate69--and a high glucose-6-phosphate dehydrogenase activity,8 which leads to the formation of reduction protential in the form of NADPH. The enhanced lipogenesis is a characteristic of both adipocytes and hepatocytes. Godbole and York 57measured fatty acid synthesis in obese rats in situ and reported that fatty acid accumulation was increased in both liver and adipose tissue as a result of both an increased rate of lipogenesis and an increase in tissue mass. They also demonstrated that, whereas total hepatic lipogenesis increased with age, total adipose tissue lipogenesis decreased in older fa/fa rats. Using hepatectomized rats, it was shown that the liver was the major site of the excess fatty acid synthesis in those animals. However, the enhanced rate of liver lipogenesis can be abolished by either pair-feeding or streptozotocin treatment, thus suggesting that the increased fatty acid synthesis in the Zucker rats is secondary to the hyperphagia, hyperinsulinemia and increased mass of hepatic and adipose tissues. The increased rate of fatty acid synthesis is also associated with an enhanced utilization of glycerol for triacylglycerol synthesis in young but not in olderfa/fa rats 98'j44and an enhanced rate of fatty acid desaturase, t49 Other experiments in which Zucker rats were fed a high fat diet showed that, although there is an inhibition of fatty acid synthase--the key lipogenic enzyme--the rat continued to deposit excessive amounts of body fat. 8'83 Weaning appears to be a very important regulatory signal in obese rats, since, when it is delayed, hepatic lipogenesis is normal but increases immediately after weaningJ 7 However, adipose tissue LPL is high before weaning prior to the onset of hyperinsulinemia and hyperphagia, thus suggesting that any increase in the lipoprotein substrate passing to adipose tissue is of maternal originJ 6 It can thus be concluded that lipogenesis is a secondary event in the development of the Zucker obesity syndrome. An additional mechanism that could contribute to fat deposition in adipose tissue is a reduced liver oxidation of fatty acids. This metabolic pathway appears to be inversely related to the magnitude of fatty acid synthesis in this organ. Several groups have demonstrated that carbon dioxide production and ketogenesis from fatty acids are decreased three-fold in hepatocytes and liver slices from obese rats compared with lean rats. 1'9°'t°°'146 Ketone bodies have been shown to be important substrates for energy production during nonshivering thermogenesis in cold adapted rats. a9 Ketogenesis is strongly impaired in the obese Zucker rat. ~46 Thus, it appeared possible that decreased

The obese Zuckerrat

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ketone body production might explain the inability of the obese Zucker rat to respond appropriately to cold stress and maintain a normal body temperature. The impairment of oxidation would tend to compound the effects of hyperphagia and hyperlipogenesis on the development of adipose tissue. At the molecular level, the inverse modulation of liver lipogenesis and oxidation is based on the malonyl-CoA--the initial metabolite of the lipogenic pathway via acetyl-CoA carboxylase--physiologlcal inhibition of mitochondrial long-chain carnitine acyl transferase (CAT-1) (EC 2.3.1.2 1), which, in turn, is the rate-limiting enzyme of hepatic fatty acid oxidation since it allows for the entry of long-chain fatty acids in the mitochondrial compartment where ~-oxidation takes place, m°' Indeed, a very high sensitivity of CAT-1 to malonyl-CoA inhibition has been reported in the liver of the obese rat, 35'9' this effect being reinforced by a high malonyl-CoA concentration, and eventually by the low carnitine content of mitochondria and of total tissue. 6s However, since malonyl-CoA is an intermediary metabolite in the synthesis of fatty acids, its inhibitory effect on fatty acid oxidation should be regarded rather as a consequence of the enhanced lipogenesis. The increased plasma FFA and glycerol concentrations in obese Zucker rats are the result of the increased basal lipolysis of the enlarged fat cells. 69'7°Fa/fa adipocytes contain increased intracellular concentrations of cAMP and respond to catecholamine stimulation by increasing cAMP levels although the response is minor in adult rats. It can be concluded that the liver hyperlipogenesis observed in thefa/fa rat is the normal response to an increased flux of nutrients--especially lipogenic precursors--and an altered endocrine status---elevated insulin/glucagon ratio--which favours lipid synthesis. This environment alters the fate of exogenous fatty acid within the liver cell, resulting in an increase in triglyceride synthesis and a decrease in ketogenesis. The result is an enhanced production of triglyceride-rich lipoproteins by liver, hyperlipemia and ultimately an increase in adipose tissue mass. IV. MOLECULAR BASIS FOR ALTERED CELLULAR ENERGY EFFICIENCY For an animal to be in a state of energy balance, energy output must equal energy intake. Independently of the kind of obesity, it always follows that energy intake is in excess of energy expenditure. This fact does not necessarily imply that an abnormally high food intake is always the main cause of obesity. It is possible for an animal to have a normal energy intake but a ~ow expenditure; in this case, the obesity syndrome may not primarily be the result of hyperphagla but of an increased metabolic efficiency. It is widely accepted that the efficiency of animal energy utilization is normally lower than that predicted, taking into account the adenosine triphosphate (ATP) during oxidative metabolism of the nutrients and the cost of glycogen, protein and fat synthesis. ,05 Theoretical calorimetric efficiencies of synthesis are normally in disagreement with measurements in intact animals. Genetically obese rodents have been extensively used to help determine factors that regulate efficiency of energy utilization. These animals retain dietary energy with higher efficiency than controls. This fact must be associated, at the cellular level, with changes in the rate of ATP formation or utilization.

A. Futile Cycling The so-called futile cycles (or substrate cycles) occur when, in a sequence of metabolic reactions, 2 unidirectional enzymes catalyze opposed reactions, so that while one enzyme catalyzes the conversion of A into B, the other catalyzes the reconversion of B into A. The primary role of such cycles is concerned with metabolic regulation since they provide a mechanism for increasing the sensitivity78,'°s of the regulatory mechanisms. On the other hand, they always imply energy wasting--normally through ATP hydrolysis---thus generating energy leaks in metabolic pathways. Since they are involved in energydissipating processes, they have been said to be involved in caloric balance and the JPLR 28/I--E

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pathogenesis of obesity. The major substrate cycles that can be considered are driven by the following enzymatic pairs: glycogen phosphorylase and glycogen synthase, glucokinase and glucose-6-phosphates, phosphofructokinase and fructose-l,6-diphosphatase, pyruvate kinase and phosphoenolpyruvate carboxykinase, pyruvate carboxylase and malate dehydrogenase, cytosolic reduction of dihydroxyacetone phosphate to ~t-glycerolphosphate and mitochondrial oxidation of ~t-glycerolphosphate to dihydroxyacetone phosphate. Whole metabolic pathways also constitute important futile cycles such as lipid synthesis and oxidation or protein synthesis and degradation. Newsholme ~°9 proposed that, taking into consideration only the phosphofructokinase/fructose-1,6-bisphophatase substrate cycle of human muscle, the maximum rate of heat production per day could account for 50% of the daily caloric intake. According to this author, the increase in heat production that occurs after a meal has many properties that are consistent with stimulation of cyclic rates. Indeed, there is evidence that fasting decreases and eating increases the activity of the sympathetic nervous system in the rat, thus increasing the levels of norepinephrine in tissues.~Ss.J58Thus, a catecholamine-mediated increase in the rate of many substrate cycles during the postabsorptive phase could have a major role in increasing the sensitivity of processes that control the rates of degradation and synthesis of fuel stores, and, in addition, could have a role in weight control) °9 Available data 7'll° obtained from different obesity models including fatty rats indicate that the key enzymes and substrate turnover related to several substrate cycles are increased rather than decreased, thus suggesting that the hormonal control of these futile metabolic pathways must be more important than the cycles themselves in the control of obesity in the Zucker rat. The only exception which seems to be decreased in obesity situations is the Na+-K+-ATPase. The enzyme--which is ubiquitous and essential in the maintenance of cellular homeostasis--is located in the cell membrane and catalyzes the hydrolysis of ATP to adenosine diphosphate (ADP) and inorganic phosphate, promoting the energized Na+-extrusion from the cells, and lowering the intracellular Na ÷ concentration. The re-entry--through leakage--of Na + into the cell completes the futile cycle. The energy required for the active transport of sodium and potassium across the cell membrane has been estimated to be between 20 and 50% of total basal energy expenditure) s° However, although alterations in Na+-K+-ATPase activity have been demonstrated in liver and muscle of obese (obob) mice, sr,s7 no differences in hepatic Na+-K+-ATPase activity were detected when obese and lean Zucker rats were compared) s Na+-K+-ATPase has been proposed to account for a substantial fraction of heat production in brown adipose tissue 3° but the lack of clear results reduces the importance of this proposal in the etiology of obesity. It may have an indirect role. Rothwell et al. have demonstrated that there is a linear relation between brown adipose tissue Na+-K+-ATPase activity and resting oxygen consumption in control and overfed rats, ~28 thus suggesting an enzyme induction proportional to tissue stimulation.

B. Brown Fat Thermogenesis

The role of brown adipose tissue as a major site of nonshivering thermogenesis in hibernators and other small mammals and in the newborn of larger ones is widely recognized. 53"~ In the adult lean rat, brown adipose tissue may contribute as much as 60% of the rats nonshivering heat production in response to cold exposure despite the fact that it accounts for less than 2 0 of body weight. 53 This tissue is also the main site for dietary-induced thermogenesis) 45 The control of heat production by brown adipose tissue is mediated by norepinephrine release at sympathetic nerve endings in the tissue in response to nerve impulses.~Jt The hormone binds to a fit receptor on the plasma membrane of the brown adipocyte, concomitantly activating adenyl cyclase with the production of cAMP, which, in turn, activates the triacylglycerol lipase responsible for hydrolyzing triacylglycerol to glycerol

The obese Zucker rat

59

and fatty acids. The fatty acids are oxidized in the mitochondria and protons pass across the inner mitochondrial membrane in the same way as in other tissues. However, in brown adipose tissue, the thermogenic function is related to the presence of a 32 kDa "uncoupling" protein located in the inner mitochondrial membrane, which allows for the dissipation of the proton gradient to be uncoupled from the synthesis of ATP via a proton conductance pathway. ~2 This pathway, which is inhibited by purine nucleotides binding specifically to a locus of the "uncoupling protein", can be monitored by the specific binding of [3H]GDP to brown adipose tissue mitochondria. Although the obese Zucker rat exhibits increased brown fat mass, the thermogenic capacity of this tissue is significantly reduced when compared with the lean one. The brown fat flow of the obese rat, an indicator of in vivo brown fat 02 consumption, does not increase as much in response to maximum doses of isoproterenol--a beta agonist--as in the lean rat; ~5~ in addition, the maximum activities of several brown fat oxidative enzymes ~3s and the GDP-binding of obese brown fat mitochondria92--an index of the thermogenic capacity--are lower in these animals. Brown fat in the obese Zucker rat appears to have a decreased capacity to respond thermogenically to catecholamines ~ not attributable to decreased mass. In fact, the brown fat mass is increased due to hypertrophy rather than hyperplasia. 69 Moreover, unlike the situation in white adipose tissue, brown fat from obese rats does not exhibit higher lipoprotein lipase activity than from lean rats, as so that the increased fat accumulation in this tissue must be a consequence of an enlarged lipogenesis rather than of triglyceride removal from circulation. In the obese Zucker rat, it has been reported that fatty acid synthesis is elevated in the interscapular brown adipose tissue and this may be the consequence of both hyperinsulinemia and the presence of dietary lipid, both factors having been proposed as the main regulators of lipogenesis in this tissue, m59 The role of brown adipose tissue in the development of obesity in the Zucker rat seems to have a secondary role since the young fa/fa rat has only moderate impairment in its ability to thermoregulate in cold environments s5 and can respond normally to noradrenaline, '27 but a defective diet-induced thermogenesis can be demonstrated when young obese Zucker rats are subject to a "cafeteria diet"--highly hypercaloric. In this nutritional situation, there is no increased GDP-binding in these animals. 67However, the lower energy expenditure, coupled with hyperpha#oa, contributes secondarily to the development of obesity in the Zucker rat.

C. Protein Turnover Nitrogen balance studies have shown that, when #oven the same amount of dietary protein, the obese Zucker rat tended to deposit amino acid carbon skeletons in the form of fat, rather than muscle protein? 3 This is in agreement with the enhanced energetic efficiency of the obese rat since there is a close relationship between body protein metabolism and the rate of energy expenditure in animals.'59 Pullar and Webster lu utilized data from obese (fa/fa) and lean rats to calculate that 2.25 kcal of metabolizable energy is required to deposit 1.0 kcal of protein, and that 1.36 kcal metabolizable energy is required for the same amount of fat deposition. It is then clear that the obese Zucker rat directs nutrients towards adipose tissue and away from lean tissue and this contributes to its metabolic efficiency. Although total protein deposition is normal up to 34 days of age, the adult obese Zucker rat deposits less nitrogen in skeletal muscle and more in other organs than do lean animals. Their muscles are smaller and contain less protein, DNA and RNA than those of their lean litter-mates. 32.m3' The obese rat also has less lean body m a s s , 123'126 a reduoed rate of protein deposition, 43.m26 and a decreased rate of protein synthesis in the skeletal muscle '26 compared with their nonobese counterparts; the decrease in the rate of protein synthesis--but not in carcass protein--is evident in the obese rat just before weaning. '2~ Muscle protein breakdown is also greater in obese compared with lean rats since the urinary levels of 3-methylhistidine are very high? 3

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J . M . Argiles V. T H E E N D O C R I N E

ENVIRONMENT

OF THE OBESE Z U C K E R

RAT

A. The Role o f Insulin

Pancreatic islets from obese rats are characterized by hypertrophy and hyperplasia of the fl-cells which result in a major hypersecretion of insulin, its levels being above those of the lean rat around 3 weeks of age) 62 The underlying mechanism of the hyperinsulinemia is still not clear. Food intake restriction or pair-feeding can restore the levels of insulin to normal ~7,57in adult obese rats. Glucose intolerance and genetically-obese animal models are often associated. This is indeed the case in the obese Zucker rat where this glucose handling is especially striking since it coexists with hyperinsulinemia, a metabolic situation also seen in late gestation95 and which denotes a state of resistance to insulin. 84 In vitro a clear insulin resistance 7~'79has been described which is expressed as a decreased insulin binding to plasma membranes of isolated liver, ~17 muscle37 and adipose cells. 38 Insulin resistance by peripheral tissues has also been described in vivo. Using a euglycemic clamping technique, Terretaz and Jeanrenaud ~42have demonstrated that liver is also insulin resistant since hepatic glucose production in obese rats fails to be inhibited by insulin. In the Zucker obese rat, there are two clearly different periods in relation to insulin resistance. In the first one, there is a pancreatic oversecretion of insulin with a reduced hepatic clearance of the hormone, 7° which leads to hyperinsulinemia. In this early period, white adipose tissue lipogenesis and LPL activity are very high since they are both activated by insulin and thus promote an increase in tissue mass. Czech et al. 39 have demonstrated a marked increase in insulin's stimulatory effect on glucose transport activity and transport-limited metabolism in isolated epididymal cells from very young obese Zucker rats. The stimulation effect seems to be related to a rapid, reversible and insulin concentration-dependent translocation of glucose transporters from a large intracellular pool, associated with the cell's low density microsomes, to the plasma membrane. 62 A similar stimulation mechanism has been described in brown adipose tissue o f f a / f a rats. 59 In the later period, a general state of insulin resistance---demonstrated in vivo by the failure of exogenously-administered insulin to lower glycemia--is present in the obese Zucker rat. Using the euglycemic-hyperinsulinemic clamp technique, P6nicaud et al. 115 have been able to demonstrate that, in young obese Zucker rats, there is a normal sensitivity to insulin in adipose tissue and an impaired sensitivity in skeletal muscle. They conclude that a decreased utilization of glucose in skeletal muscles preferentially channels glucose towards the liver and white adipose tissue resulting in an increase in fatty acid synthesis. In this sense, decreased insulin binding and also intracellular defects have been reported in soleus muscle, 37 thus contributing to insulin resistance. As the rats become old, a state of decreased insulin responsiveness develops both in liver and peripheral tissues, the preferential loss of insulin sensitivity enhancing the predominant role of lipogenesis for the building up of body fat. When considering insulin resistance, one should be aware that it is normally associated with a reduction or a modification of the hormone's receptors. 7~ Due to the observation of decreased insulin receptor numbers in the obese rat, insulin resistance was initially attributed primarily to decreased insulin binding. Later on, it was realized that such decreased binding was not sufficient to explain the overall state of resistance and that additional experimental evidence was needed. Insulin receptors possess an intrinsic protein kinase activity which is activated by insulin. It has been suggested that insulin resistance could be related to a reduced stimulation of this protein kinase by insulin. Debant et alfl studied the insulin receptor protein kinase activity in 30-day-old obese Zucker rats where, despite a slight insulin resistance in vivo, the insulin-stimulatory effect on glucose transport and metabolism is increased in isolated adipocytes from these animals. They showed that the kinase activity is increased for both autophosphorylation and the capability to catalyze the phosphorylation of an exogenous substrate: ~On the other hand, skeletal muscles from the same animals--that do not exhibit an increased response to insulin--showed the same insulin receptor kinase activity as that found in lean Zucker rats. From this study, it is

The obese Zucker rat

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quite clear that the action of insulin in the adipose tissue of youngfa/fa rats is modulated through an increase in insulin receptor kinase activity. This specific feature of the insulin receptor of adipoeytes could play an important role in the development of the obesity syndrome of the Zucker rat. Since hyperphagia and hyperinsulinemia are both present in the Zucker rat, it could be said that the latter is a consequence of hyperphagia. In fact, before weaning, the hyperinsulinemicfatty rat is not hyperphagic. ~39At this stage, insulin concentration may be transiently elevated either in response to the neural or gastrointestinal reflexes triggered by suckling. H9 At a later stage after suckling, the hyperinsulinemia persists even when pair-feeding thefa/fa rats with their lean litter-mates. ~53However, a 24-hr fast reduces the plasma insulin concentration in obese rats to the same levels as observed in lean controls, thus demonstrating that hyperinsulinemia is not completely refractory to the effects of nutrient intake. 52 Chronic administration of dehydroepiandrosterone was effective in lowering serum insulin concentrations in obese Zucker rats at several different ages. m36This effect was associated with decreased hepatic and adipose tissue fatty acid synthase and glucose-6-phosphate dehydrogenase and adipose tissue lipoprotein lipase. As a result, there was a decrease in weight gain, body fat and fat cell size. 34The reason for the reduced insulin concentrations is still unknown although an increase in insulin sensitivity3~ or a direct effect of dehydroepiandrosterone on the pancreatic islets3mhave both been proposed as explanations. Although insulin plays a defnite role in the hypertrophic and hyperplasic development of white adipose tissue in the Zucker rat, it is not the primary cause of fat deposition since streptozotocin treatment--which destroys the pancreatic /~-cells--followed by insulin replacement, 26 does not prevent the development of a limited obesity. Other factors such as lipoprotein lipase activity may have a more primary role in the accumulation of excess lipid by the obese Zucker rat. The hyperinsulinemia can also account for the depressed rate of hepatic gluconeogenesis 147although renal gluconeogenesis is normal, in spite of an altered renal function, m47However, total glucose production by these tissues is elevated, and this, together with increased dietary carbohydrate, results in increased accumulation of liver glycogen,s2'|47 However, a role of insulin in signalling whole-body energy balance regulation was proposed by Porte and Woods. "8 The CNS content of insulin and the binding of the hormone to brain membranes have been shown to be severely decreased in the Zuckerfatty rat, 3"5~suggesting a central defect in this obese genotype. 97 Insulin also seems to play an important role on diet-induced thermogenesis. Indeed, insulin can stimulate oxygen consumption in brown adipose tissue TM and also potentiate the response to noradrenaline. 65 In obese rats, however, the profound insulin resistance of brown adipose tissue hampers the stimulated energy dissipation associated with food intake.

B. Other Hormonal Changes The development of obesity in the Zucker rat can be retarded by adrenalectomy at an early age. In 5-week-old rats, adrenalectomy results in a reduction in weight gain 154,16°and a normalization of brown fat thermogenic capacity ~,67 and oxidative enzyme activity. 55 It also reduces the norepinephrine-induced 02 consumption and the norepinephrinestimulated blood flow to brown fat. The administration of glucocorticoids to these adrenalectomized animals reverses these effects.55 However, although corticosterone plays a certain role in the development of obesity in the Zucker rat, very recent studies 6 have demonstrated that it may only be important in adultfa/fa rats. In these animals, the turnover of this hormone seems to be increased, and adrenalectomy restores food intake and the rate of weight gain to normal? 54,t6° It also depresses hyperlipogenesis in adipose tissue and liver and hyperinsulinemia ~ but not the characteristic adipocyte hyperplasia.~6° The reduction of energy efficiency observed in adult adrenalectomized obese rats could be due to an increase in energy expenditure resulting from increased activity of brown adipose tissue. 92 This hypothesis is supported by the

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finding that thermogenesis (assessed by GDP binding to mitochondria) was impaired in brown adipose tissue of the Zucker obese rat and that adrenalectomy restored GDP binding in adrenalectomized obese rats. 6~ However, Bazin et al. 6 have shown that, when adrenalectomy is performed in Zucker f a / f a pups (4 days old), it does not restore the decreased 02 consumption or the high fatty acid synthase and lipoprotein lipase activity or the low GDP-binding encountered in the obese Zucker pups, thus demonstrating that adrenalectomy early in life does not prevent the emergence of obesity in these animals. Fatty rats are glucagon resistant: 6 In spite of normal pancreatic glucagon levels, a reduced basal plasma glucagon, a reduction in plasma glucagon concentration in response to fasting, and a subnormal rise in plasma glucagon concentration in response to arginine stimulation44have been described. It has been shown that glucagon release from pancreatic islets is substantially reduced in obese Zucker rats, although islet stores are not reduced?° Chan et al. 28 studied the effects of long-term glucagon administration in Zucker rats and reported a 2 0 0 body weight reduction, but saw no reduction of circulating triglyceride and cholesterol. The increase in the plasma insulin/glucagon ratio may be responsible for much of the hyperlipogenesis in f a / f a rats since normalization of the ratio after halothenate therapy lowers serum hypertriglyceridemia and VLDL levels: 5 In addition to the hyperinsulinemia, there is a reduced glucagon binding in the Zucker rat which favours the favoured insulin/glucagon ratio. 4 Other endocrine changes have also been demonstrated. Although the pituitary weight is reduced, the concentration of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is not decreased in fatty rats. The secretion of gonadotropines is defective and responsible for the sexual abnormalities present in these animals---delayed vaginal opening, and prolonged estrus. ~3° All this is consistent with a low level of circulating estrogen) 3° The occurrence of normal serum FSH and LH in the presence of low serum estrogen suggests that the hypothalamic-pituitary threshold to feedback inhibition of gonadotropins is defective) 3° The male rat also has steroid deficiency which can be seen as decreased testicular size4° and absence of response to female pheromones. 64 Thyroid function is altered, the concentration of thyrotropin and circulating thyroid hormones being lower and placing the obese Zucker rat in a hypothyroid state. 64 It has recently been suggested that the parasympathetic nervous system can influence dietinduced thermogenesis. The rise in metabolic rate and brown adipose tissue temperature after a single meal are potentiated by treatment with the muscarine antagonist atropine sulphate. 127 Obese Zucker rats fail to increase oxygen consumption after food, but this response can be completely restored to normal by atropine treatment, thus indicating that high parasympathetic activity may be partially responsible for the defective thermogenesis and increased fat deposition in these animals) 27The involvement of the thyroid hormones in this parasympathetic activity seems very likely. Investigation of the thyroid function showed that the uptake and turnover of ~3~Iby the thyroid was depressed i n f a / f a rats but responded normally to TSH stimulation and to cold exposure, m This low uptake was not reversed by food restriction ~6° and was only partially corrected by estrogen treatment) 4 Serum protein-bound iodine (PBI) was depressed. Although serum T3 concentration is normal, serum thyroxine levels are depressed throughout the major portion of the day. 99 The normal concentration of TSH in serum and pituitary ~Ss suggested that the defects in thyroid function were not attributable to a lack of TSH administration. A marked variation from normal in its diurnal rhythm64supports this fact. Serum TSH was enhanced by injections of the hypothalamic releasing factor TRH, but only rose slightly after treatment with propilthiouracil, thus suggesting a hypothalamic defect) 55 Somatostatin is a very interesting peptide that can be released both by the pituitary and the pancreatic D-cells: 2"~6 The concentration of this hormone in the pancreas of 16-week-old obese Zucker rats is higher than in the lean control, its hypothalamic secretion being also increased) 37 The high circulating levels may explain the reduced growth hormone secretion--observed at all times of day96---since somatostatin inhibits growth hormone release by the pituitary. The low levels of GH may account for a diminished

The obese Zucker rat

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somatomedin activity which hampers bone and muscle growth. 97 Nevertheless, muscle growth is also affected by the reduced protein intake observed infa/fa rats unlike in other models such as "cafeteria" feeding. ~'l°6'l°Ta2°-n2 Cholecystokinin has been shown to be a short-term regulgtor of food intake ~ found in high concentration in the obese Zucker rat.l°3 It is mainly prbduced by the jejunal mucosa in response to the level of fat intake and serves to accelerate the release of biliar fluid thus contributing to a rapid emulsification of dietary fat prior to its absorption. Since this peptide reduces food intake in the rat, some kind of abnormality in its binding to receptors must exist in the obese Zucker rat, which hampers its action on feeding behaviour. Neurotransmitter levels--catecholamines, dopamine and serotonin--are also altered in the obese Zucker rat. Since these molecules have been shown to be involved in the regulation of the quantity and quality of food intake, they could also be important factors in the etiology of abnormal neuroendocrine secretion and energy balance. 97

C. Neuropeptides Some neuropeptides and biogenic amines released from the neuroendocrine system-located diffusely in brain, gastrointestinal tract, pancreas, lungs sympathetic ganglia, skin and the urogenital tract--are known to be involved in the regulation of food intake. They deal with energy conservation and expenditure, coordinating the adaptation and survival of the individual and the reproduction of the species with the availability of nutrients in the environment. The cells of the neuroendocrine system can release different peptides according to their physiological situation. These cells can either be in a state of energy conservation (endophinergic or endogenous morphine-like state) or in a state of energy expenditure (endoloxonergic or endogenous naxolone-like state). In the first state of energy conservation, seen in animals in hibernation or nonhibernating animals during starvation, three main peptides are released: fl-endorphin, dynorphin and ~-neoendorphin. In the state of energy expenditure--the state of arousal from hibernation or during reproduction--MSH and calcitonin-like peptides are released. Small doses of the naloxone, a specific opioid antagonist, can abolish overeating in obese Zucker rats. 1°2 Higher doses of naloxone also suppress feeding in lean Zucker rats, but it requires ten times the dose to produce a suppressant effect equal to that found in the obese. 94 These results suggest that these animals can have excessive opioid activity. 93 With regard to peptides involved in energy expenditure, calcitonin-like peptides have been found in excess in both the pituitary and thyroid gland of obese Zucker rats? 3 Since this family of peptides is involved with energy expenditure--they actually promote an anorexic state---it has been proposed that they would either not be released from the glands or that they would be released in an inactive form. VI. CONCLUDING REMARKS Twenty years after its discovery, the fatty mutation still remains an enigma. Perhaps the only clear picture about it is that the cause of this kind of obesity is multifactorial. The increased food intake results in a large availability of excess energy in the form of substrates for adipose tissue. In addition, the hyperinsulinemia promotes fatty acid synthesis in liver and adipose tissue, thus contributing to the hypertrophy and hyperplasia of adipose tissue depots. This metabolic environment also includes a diminished sympathetic activity which results in reduced brown adipose tissue heat loss and consequently an enhanced energetic efficiency. The main defect is probably located in the central nervous system and influences food intake, hyperlipogenesis and lipid storage capacity, and endocrine environment. Besides hyperphagia and hyperinsulinemia, many detailed studies have indicated a hypothalamic dysfunction. The result is a metabolic situation characterized by a high efficiency status where the enzymatic machinery is channeled towards lipid synthesis and accumulation. Further research into the causes of this kind of obesity is particularly promising since

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it could provide some insights into the basis of genetically-inherited human obesity and may suggest new therapeutic approaches to this problem.

(Received 5 July, 1988) REFERENCES 1. AZAIN, M. J. and MARTIN, R. J. Am. J. Physiol. 244, R400-R406 (1983). 2. BARRY, W. S. and BRAY, G. A. Metabolism 18, 833-839 (1969). 3. BASKIN, D. G., STEIN, L. J., IKEDA, H., WOODS, S. C., FIGLEWICZ, D. P., PORTE, O., Jr, GREENWOOD, M. R. C. and DAISA, D. M. Life Sci. 216, 627~533 (1984). 4. BATHENA,S. J., APARICIO, P., REVETT, K., VOYLES,N. and RECANT, L. J. Nutr. 117, 1291-1297 (1987). 5. BAZIN, R., LAVAU, M. and GUICHARD, C. Biocbem. J. 216, 543-549 (1983). 6. BAZIN, R., PLANCHE, E., OuPuY, F., KRIEF, S. and LAVAU, M. Am. J. Physiol. 252, E461-E466 (1987). 7. BELFIORE, F. and IANNELLO,S. J. Mol. Med. 3, 257-270 (1979). 8. BERKE, B. M. and KAPLAN, M. L. J. Nutr. 113, 820-834 (1983). 9. BJORNTORP, P. and SJOSTROM, L. Metabolism 20, 703-713 (1971). I0. BRAY, G. A. Proc. Soc. Exp. Biol. Med. 131, l l l l - l l l 4 (1969). ll. BRAY, G. A. J. Lipid Res. 11, 517-521 (1970). 12. BRAY, G. A. Fed. Proc. 36, 148-153 (1977). 13. BRAY, G. A. and CAMPFIELD, L. A. Metabolism 24, 99-117 (1975). 14. BRAY,G. A., SAIDUDDIN,S., YORK, D. A. and SWEROLOFF,R. S. Proc. Soc. Exp. Biol. Med. 153, 88-91 (1976). 15. BRAY, G. A. and YORK, D. A. Am. J. Physiol. 223, 176-179 (1972). 16. BRAY, G. A. and YORK, D. A. Physiol. Rev. 59, 719-809 (1979). 17. BRAY, G. A., YORK, D. A. and SWERDLOFF, R. S. Metabolism 22, 435-442 (1973). 18. BRAY, G. A., YORK, D. A. and YUKIMURA,Y. Life Sci. 22, 1637-1642 (1978). 19. BROOK, C. G. D., LLOYO, J. K. and WOLE, O. H. BE. Med. J. 2, 25-27 (1972). 20. BRYCE,G. F., JOHNSOn, P. R., SULLIVAN,A. C. and STERN, J. S. Horm. Metab. Res. 9, 366-370 (1977). 21. BOULANG]~,A., PLANCHE E. and DE GASQUET, P. J. Lipid Res. 20, 857-864 (1979.) 22. BOULANGf~,A., PLANCHE, E., DE GASQUET, P. and LELIEPURE, X. Int. J. Obesity 2, 354 0978). 23. CARPENTER, R. G. and GROSSMAN, S. P. Physiol. Behav. 30, 57-63 (1983). 24. CASTONOUAY,T. W., BURmCK, S. L., GUZMAN, M. A., COLLIER, G. H. and STERN, J. S. Physiol. Behav. 33, 119-126 (1984). 25. CASTONGUAY,T. W., HARTMAN,W. J., FITZPATRICK,E. A. and STERN,J. S. J. Nutr. 112, 228-232 (1982). 26. CHAN, C. P. C. and STERN, J. S. In The Body Weight Regulatory System: Normal and Disturbed Mechanisms, pp. 65-67 (CIOFFI, L. A., ed.) Raven Press, New York, 1981. 27. CHAN, C. P. C. and STERN, J. S. Am. J. Physiol. 242, E445-E450 (1982). 28. CHAN, E. K., MACKEY, M. A., SNOVER,D. C., SCHNEIDER,P. D., RUCKER, R. D. JR, ALLEN, C. E. and BUCHWALD, H. Exp. Mol. Pathol. 40, 320-327 (1984). 29. CHANUSSOT,F., ULMER, M., RATANASAVANH,R., MAX, J. P. and DEBRY, G. Int. J. Obesity 8, 259-270 (1983). 30. CHINET, A., CLAUSEN, T. and GIRARDIER, L. J. Physiol. (London) 265, 43-61 (1977). 31. CLEARY, M. P., SHEPHERD, A. and JEMES, B. J. Nutr. 114, 1242-1251 (1984). 32. CLEARY, M. P. and VASELLI,J. R. Proc. Soc. Exp. Biol. Med. 167, 616--623 (1981). 33. CLEARY, M. P., VASELLI, J. R. and GREENWOOD, M. R. C. Am. J. Physiol. 238, E284-E292 (1980). 34. CLEARY, M. P. and ZISK, J. Fed. Proc. 42, 536 (1983). 35. CLOUET, P., HENNINGER, C., PASCAL, M. and BEZARD, J. FEBS Lett. 182, 331-334 (1985). 36. COLLIER, G. and BOLLES, R. C. J. Comp. Physiol. Psychol. 65, 379-383 (1968). 37. CRETTAZ, M., PRENTKI, M., ZANINETTI, D. and JEANRENAUD,B. Biochem. J. 186, 525-534 (1980). 38. CUSHMAN,S. W., ZARNOWSKI,M. J., FRANZNSOFF,A. J. and SALANS,L. B. Metabolism 27, 1930-1940 (1978). 39. CZECh, M. P., RICHARDSON,D. K., BECKER, S. G., WALTERS, C. G., GITOMER, N. and HEINmCH, J. Metabolism 27, 1967--1981 (1978). 40. DEB, S. J. and MARTIN, R. J. J. Nutr. 105, 543-549 (1975). 41. DEBANT, A., GUERRE-MILO, M., LE MARCHAND-BRUSTEL,Y., FREYCHET, P., LAVAU, M. and VAN OBBERGHEN, E. Am. J. Physiol. E273-E278 (1987). 42. DUBOIS, M. P. Proc. Natl. Acad. Sci. U.S.A. 72, 1340-1343 (1975). 43. DUNN, M. A. and HARTSOOK, E. W. J. Nutr. 110, 1865-1879 (1980). 44. EATON, R. P., CONWAY, M. and SCHADE, D. S. Am. J. Physiol. 230, 1336-1341 (1976). 45. EATON, R. P., OASE, R. and SCHADE, D. S. Metabolism 25, 245-249 (1976). 46. EATON, R. P., SCHADE, D. S. and CONWAY, M. Lancet 2, 1545-1547 (1974). 47. EISENBERG,S. In Lipoprotein Metabolism, pp. 32-43, Springer-Verlag, New York, 1976. 48. FAERGMAN,O., SATA, T., KANE, J. P. and HAVEL, R. J. J. Clin. Invest. 56, 1396-1403 (1975). 49. FAUST, I. M. Int. J. Obesity 4, 314-321 (1980). 50. FAUST, I. M. In The Bo.dy Weight Regulatory System: Normal and Disturbed Mechanisms, pp. 97-106 (Ciovw, L. A., JAMES, W. P. T. and VAN ITALLIE, T. B., eds) Raven Press, New York, 1981. 51. FIGLEWICZ,D. P., DORSA,D. M., SKEIN,L. J., BASKIN,D. G., PAQUETTE,T., GREENWOOD,M. R., WOODS, S. C. and PORTE, D., JR Endocrinology 117, 1537-1543 (1985).

The obese Zucker rat

65

52. FLETCHER,J. M., H^C.~ARTY, P., WAHLE, K. W. J. and REEDS, P. J. Horm. Metab. Res. 18, 290-295 (1986). 53. Fos'r~, D. O. and FgVDMXN, M. L. Can. J. Physiol. Pharmaeol. 56, 110-122 (1978). .54. FOSTER, D. O. and FRYDMAN, M. L. Can. J. Physiol. Phaemacol. ~b'7,257-270 (1979). 55. F ~ M A N , M. R., HoRwrrz, B. A. and STEaN, J. S. Am. Y. Physiol. 250, R595-R607 (1986). 56. GIBBS, J. R., YOUNG, R. C. and SMITH, G. P. J. Comp. Physiol. Psycho/. 84, 488-495 (1973). 57. GODSOLE, V. and YORK, D. A. Diabetologia 14, 191-197 (1978). 58. GOt)SOLE, V., YORK, D. A. and BLOX.HXM,D. P. Diabetolo&ia 15, 41-44 (1978). 59. GREco-P~OTTO, R., A~LMACOPOULOS.-JEANNET,F. and JEANRENAUD,B. Biochem. J. 247, 63-68 (1987). 60. GP,~mVOOD, M. C. R., MAOGIO,C. A., KOOPI~U~NS,H. S. and SCLAFANI,A. Int. J. Obesity 6, 513-525 (1982). 61. GRtrEN, R. K. and GR~rwooD, M. R. C. Am. J. Physiol. 241, E76-E83 (1981). 62. GUELXE-MILLO,M., LAVAU,M., HORNE,J. S. and WARDZAL^,L. J. J. Biol. Chem. 260, 2917-2201 (1985). 63. HAUSMAN,G. J., K~SER, T. R., Sm~pI~, J. F. and MARTIN R. J. Int. J. Obesity 7, 487-492 (1983). 64. HEMMES, R. and V~aD, J. Fed. Proc. 36, 1165 (1977). 65. HIMMs-H^GEN, J. Ann. Ray. Nutr. 2, 69-94 (1985). 66. HOLT, S. and YORK, D. A. Biochem. J. 209, 819-822 (1982). 67. HOLT, S., YORK, D. A. and FITZSIMONS,J. T. R. Biochem. J. 214, 215-223 (1983). 68. HORWITZ, B. A., INOKUeHI, T., WIeKLER, S. J. and STERN, J. S. Metabolism 33, 354-357 (1984). 69. HuE, L., VAN DEa WEaDE, G. and JEANRENAUD, J. Biochem. J. 214, 1019-1022 (1983). 70. JEANRENAUO,B. Diabetologia 17, 133-138 (1979). 71. JEXNRENAUO,B., HALIMI,S. and VAN PER WFa~E, G. In Diabetes~Metabolism Reviews, Vol. I, pp. 261-291 (DEFRoNZO, R. A., ed.) Wiley, New York, 1985. 72. JOHNSON,P. R., STERN,J. S., GREENWOOD,M. R. C., ZUeKER,L. M. and HmscH, J. J. Nutr. 103, 738-743 (1973). 73. JOHNSON, P. R., STERN, J. S., GREENWOOD,M. R. C., ZUCKER, L. M. and Hmscn, J. Metabolism 27, 1941-1954 (1978). 74. JOHNSON, P. R., ZUCKER, L. M., CRUCE, J. A. F. and HmSCH, J. J. Lipid Res. 12, 706-714 (1971). 75. KAHN, C. R. J. Cell. Biol. 70, 261-286 (1976). 76. KAPLAN, M. L., TROUT, J. R. and SMITH, P. Metabolism 29, 333-339 (1980). 77. KAPLAN, M. L. Fed. Proc. 36, 1149 (1974). 78. KATZ, J. and ROONSTAD, R. Curt. Top. Cell. Regul. 10, 237-289 (1976). 79. KEMMER,F. W., BERGER, M., HERaERG, L., GRIES, F. A., WIRDEIER, A. and BECKER, K. Biochem. J. 178, 733-741 (1979). 80. KOOPMANS,H. S. and SCLAFANI,A. Int. J. Obesity 5, 491-495 (1981). 81. KREMAN, A. J., LINNER, J. H. and NELSON, C. H. Ann. Surg. 140, 439-451 (1954). 82. LEMONNIER,D. Horm. Metab. Res. 3, 287-288 (1971). 83. LEMONNIER,D., AUBERT, R., SUQUET,J.-P. and ROSSEUN, G. Diabetologia 10, 697-701 (1974). 84. LETURQUE,A., BURNOL, A. F., FERRE, P. and GIRARD, J. Am. J. Physiol. 2,46, E25-E31 (1984). 85. LEvis, B. E., TRISCARI, J. E. and SULLIVAN,A. C. Pharmacol. Biochem. Behav. 13, 107-113 (1980). 86. LIN, M. H., ROMSOS, D. R., AKERA,T. and L~VEILLE,G. A. Proc. Soc. Exp. Biol. Med. 161, 235-238 (1979). 87. LIN, M. H., TUIG, J. G. V., ROMSOS, D. R., AKERA, T. and LEVEILLE, G. A. Am. J. Physiol. 238, E193-E199 (1980). 88. LOP~z-TEJERO, D. Ph.D. dissertation, University of Barcelona (1986). 89. MAEKUSO, H., MORIYA, K. and HIROSHIGE,T. 3'. Appl. Physiol. 42, 159-165 (1977). 90. MALEWIAK,M. I., GmGLIO, S., KALOPISSiS,A. D. and LE LIEPVRE, X. Metabolism 32, 661-667 (1983). 91. MALEWIAK,M. I., GmGLIO, S. and LE LtEPVRE, X. Metabolism 34, 604--611 (1985). 92. MARCHINCTON,D., ROTHWELL,N. J., STOCK, M. J. and YORK, D. A. J. Nutr. 113, 1345-1402 (1983). 93. MARGULES,D. L. In The Body Weight Regulatory System: Normal and Disturbed Mechanisms, pp. 361-368 (CIoFFI, L. A., ed.) Raven Press, New York, 1981. 94. MARGULES,D. L., MOISSET,B., LEWIS,M. J., SmaUYA, H. and PERT, C. B. Science 202, 988-991 (1978). 95. MARTIN, A., ZORZANO,A., CARUNCHO, I. and HF.RRERA,E. Diabet. Metab. 12, 302-307 (1986). 96. MARTIN, R. J. and GAHAGAN,J. Horm. Metab. Res. 9, 181-186 (1977). 97. MARTIN, R. J., HARRIS, R. B. S. and JONES, D. D. Proc. Soc. Exp. Biol. Med. 183, 1-10 (1986). 98. MARTIN, R. J. and LAMPREY, P. Proc. Soc. Exp. Biol. Mad. 149, 35-39 (1975). 99. MARTIN, R. J., WANGSNESS,P. J. and GAHAGAN,J. H. Horm. Metab. Res. 10, 187-192 (1978). 100. McCUNE, S., DURANT, P. J., JENKINS, P. A. and HAms, R. A. Metabolism 30, 1170-1178 (1981). 101. MCGARRY, J. D. and FOSTER, D. W. J. Biol. Chem. 253, 8294-8300 (1978). 102. MCLAUGHLIN,C. L. and BAILE, C. A. Physiol. Behav. 32, 755-761 0984). 103. McLAUGHLIN,C. L., BAILE,C. A., DELLA-FERA,M. A. and KASSER,T. G. Physiol. Behav. 35, 215-220 (1985). 104. MILLERW. H., JR, FAUST,I. M., GOLDEERGER,A. C. and HIRSCH,J. Am. J. Physiol. 245, E74-E80 0983). 105. MILLIGAN, L. P. Fed. Proc. 30, 1454-1458 (1971). 106. MONFAR, M., PRATS, E., ARGILI~S,J. M. and ALEMANY,M. Nutr. Res. 7, 871-876 (1987). 107. MONFAR, M., PRATS, E., ARGIL[S, J. M. and ALEMANY,M. Biochem. Arch. 3, 331-336 (1987). 108. NEWSHOLME,E. A. Biochem. Soc. Syrup. 43, 183-205 (1978). 109. NEWSHOLME,E. A. New Engl. J. Mad. 302, 400-405 (1980). If0. NEWSHOLME,E. A., BRAND, K., LANG, J., STANLEY,J. C. and WILLIAMS,T. Biochem. J. 182, 621-624 (1979). Ill. NICHOLLS, D. G. Biochim. Biophys. Acta 549, 1-29 (1979). 112. NICHOLLS, D. G. and LOCKE, R. M. Physiol. Rev. 64, 1-64 0984). I13. NOSADINZ,R., URSINI, F. and TESSARI,P. Fur. J. Clin. Invest. 10, ll3-119 (1980).

66

J . M . Argil6s

114. OCKNER, R. K., HUGHES, F. B. and ISSELBACHER,K. J. J. Clin. Invest. 48, 2079-2088 (1969). 115. PI~NICAUD, L., FERRIC, P., TERRETAZ, J., KINEBANYAN,M. F., LETURQUE, A., DORI~, E., GIRARD, J., JEANRENAUD, B. and PJCON, L. Diabetes 36, 6264/31 (1987). 116. POLAK, J. M., GRIMELIUS, L., PEARSE, A. G. E., BLOOM, S. R. and ARIMURA,A. Lancet i, t220-1221 (1975). 117. POOLE, G. P., POGSON, C. I., O'CONNOR, K. J. and LAZARUS,N. R. Biosci. Rep. 1, 903-910 (1981). 118. PORTE, D. and WOODS, S. C. Diabetologia 20, 274-279 (1981). 119. PORTER, R. J. and BASSETT,J. M. Diabetologia 16, 201-206 (1979). 120. PRATS, E., MONFAR, M., ARG1LI~S, J. M. and ALEMANY, M. J. Obesity Wt. Regul. 5, 77-88 (1986). 121. PRATS, E., MONFAR, M., ARG1LI~S,J. M. and ALEMANY, M. Biochem. Int. 14, 95-101 (1987). 122. PRATS, E., MONEAR, M., ARGILI~S,J. M. and ALEMANY, M. Biochem. Int. in press, 1988. 123. PULLAR,J. D. and WEBSTER, A. J. F. Br. J. Nutr. 31, 377-392 (1974). 124. PULLAR,J. D. and WEBSTER, A. J. F. Br. J. Nutr. 37, 355-363 (1977). 125. REDGRAVE,T. G. J. Lipid Res. 18, 604~12 (1977). 126. REEDS, P. J., HAGGARTY,P., WAHLE, K. W. J. and FLETCHER,J. M. Biochem. J. 204, 393-398 (1982). 127. ROTHWELL, N. J., SAVILLE, M. G. and STOCK, M. J. Pfluegers Arch. 392, 172-177 (1981). 128. ROTHWELL, N. J., STOCK, M. J. and WYLLIE, M. G. Biochem. Pharmacol. 30, 12, 1709-1712 (1981). 129. ROZlN, P. J. Comp. Physiol. Psychol. 65, 23-29 (1968). 130. SAIDUDDIN,S., BRAY, G. A., YORK, D. A. and SWERDLOEF,R. S. Endocrinology 93, 1251-1256 (1973). 131. SCHONFELD,G., FELSKI, C. and HOWALD, M. A. J. Lipid Res. 15, 457-464 (1974). 132. SCHONFELD,G. and PFLEGER, B. Am. J. Physiol. 220, 1178-1181 (1971). 133. SCLAFANI,A. and KOOPMANS, H. S. Int. J. Obesity 5, 497-500 (1981). 134. SHACKNEY,S. E. and JOEL, C. D. J. Biol. Chem. 241, 4004-4010 (1986). 135. SHAPIRO, J. F., KIRCHER, I. and MARTIN, R. J. d. Nutr. ll0, 1313-1318 (1980). 136. SFmPHERD, A. and CLEARY, M. P. Am. J. Physiol. 246, 123-128 (1984). 137. SHEPPARD,M. C., SHAPIRO,B., HUDSON,A. and PIMSTONE,B. L. Horm. Metab. Res. 12, 236-239 (1980). 138. STERN, J. S., INOKUCHI, T., CASTONGUAY,T. W., WICKLER, S. J. and HORWITZ, B. A. Am. J. Physiol. 247, R918-R926 (1984). 139. STERN, J. S. and JOHNSON, P. R. Metabolism 26, 371-380 (1977). 140. STOCK, J. J. and ROTHWELL, N. J. Obesity and Leanness, John Libbey and Co., London, 1982. 141. STOLZ, D. J. and MARTIN, R. J. J. Nutr. 112, 997 1002 (1982). 142. TERRETAZ,J. and JEANRENAUD,B. Endocrinology 112, 1346-1351 (1985). 143. TESTAR,X., L6PEZ-TF~ERO,D., LLOBERA,M. and HERRERA,E. Pharmacol. Biochem. Behav. 24, 625-630 (1986). 144. THENEN, S. and MAYER, J. Proc. Soc. Exp. Biol. Med. 148, 953-957 (1975). 145. TRAYHURN, P., JONES, P. M., McGuCKIN, M. and GOODBODY, A. E. Nature 295, 323-325 (1982). 146. TRISCARI, J., GREENWOOD, M. R. C. and SULLIVAN,A. C. Metabolism 31, 223-228 (1982). 147. TRISCARI, J., STERN, J. S., JOHNSON, P. and SULLIVAN,A. Fed. Proc. 36, I150 (1977). 148. VASELLI,J. R., HARACZKIEWICZ,E. and GREENWOOD, M. C. R. Aliment. Nutr. Metab. 1, 373 (1980). 149. WAHLE, K. W. J. Comp. Biochem. Physiol. 1348, 565-574 (1974). 150. WHITTAM, R. and BLOND, D. M. Biochem. J. 92, 147-158 (1965). 151. WICKLER, S. J., HORWITZ, B. A. and STERN, J. S. Int. J. Obesity 6, 481-490 (1982). 152. WITZTUM, J. U and SCHONFELD, G. Diabetes 28, 509-516 (1979). 153. YORK, D. A. and BRAY, G. A. Metabolism 22, 443-453 (1973). 154. YORK, D. A. and GODBOLE, V. Horm. Metab. Res. 11, 646 (1979). 155. YORK, D. A., HERSHMAN, J. M., UTIGER, R. D. and BRAY, G. A. Endocrinology 90, 67-72 (1972). 156. YORK, D. A., STEINKE, J. and BRAY, G. A. Metabolism 21, 277-284 (1972). 157. YOUNG, J. B. and LANDSBERG,L. Science 196, 1473-1475 (1977). 158. YOUNG, J. B. and LANDSBERG,L. Nature 269, 615-617 (1977). 159. YOUNG, V. R. Cancer Res. 37, 2336-2347 (1977). 160. YUKIMURA,Y. and BRAY, G. A. gndocr. Res. Commun. 5, 189 198 (1978). 161. ZUCKER, L. M. Ann. N.Y. Acad. Sci. 131, 447-458 (1965). 162. ZUCKER, L. M. and ANTONIADES,H. N. Endocrinology 90, 1320-1330 (1972). 163. ZUCKER, L. M. and ZUCKER, Z. F. J. Hered. 52, 275-278 (1968).