Maternal influence of prolyl endopeptidase on fat mass of adult progeny.

BACKGROUND: Maternal genotype has lifetime effects on progeny, but few specific genes, and no proteases, are known to underlie maternal effects. Prolyl endopeptidase (PREP) is a serine protease with putative substrates that regulate appetite or milk production. OBJECTIVE: To test effects of PREP on obesity phenotypes in mice. DESIGN: Mice with a gene trap (GT) of PREP (PREP(gt/gt)) on the C57BL/6J (B6) background were generated. Minimal PREP protein was detected by western blot. In Experiment 1, direct effects of PREP were measured in littermate mice derived from intercrosses of heterozygotes (PREP(WT/gt)). In Experiment 2, maternal effects of PREP were measured in reciprocal crosses of heterozygous (PREP(WT/gt)) and wild-type (WT) (PREP(WT/WT)) males and females. DIETS: Mice were fed either low-fat (LF, Experiments 1 and 2) or high-fat (HF, Experiment 1) defined diets. MEASUREMENTS: Adiposity index (AI) was calculated from body weight (BW) and weights of four fat depots measured in 120-day-old mice. Fasting plasma glucose, insulin and leptin were measured. In vivo plasma alpha-MSH levels were measured by targeted quantitative peptidomics. RESULTS: Experiment 1-In intercross mice, there were significant diet effects, but few genotype effects. There were no genotype effects on BW or AI in males or females on either diet. Experiment 2-In contrast, reciprocal crosses of heterozygous males or females with WT B6 revealed highly significant parent of origin effects on all traits except body length. Progeny (WT and heterozygous genotypes and both sexes) born to female PREP(WT/gt) heterozygotes had fat pads that weighed as much as -twofold more at 120 days old than progeny born to male heterozygotes. CONCLUSION: Heterozygosity for PREP GT results in highly significant maternal effects, whereas homozygosity for the PREP(gt/gt) mutation has a much more limited direct effect.

Coincidence of genetic loci for plasma cholesterol levels and obesity in a multifactorial mouse model.

We have examined backcross progeny derived from a cross of Mus spretus with C57BL/6J, that range from 1 to 50% carcass lipid (n = 215), and from 22 to 130 mg/dl plasma total cholesterol (n = 238). Statistical analysis revealed that distal mouse chromosome 7 exhibits significant linkage both to plasma total cholesterol (likelihood of the odds [LOD] 5.8) and to carcass lipid (LOD 3.8). A locus on chromosome 6 also shows significant linkage to plasma total cholesterol (LOD 5.6), but no linkage to carcass lipid. Neither chromosomal region contains any previously mapped genes likely to influence lipoprotein metabolism, indicating that novel genetic factors contributing to plasma lipoprotein levels have been identified.

Identification of four chromosomal loci determining obesity in a multifactorial mouse model.

We previously described a new mouse model for multigenic obesity, designated BSB. We now report the use of a complete linkage map approach to identify loci contributing to body fat and other traits associated with obesity in this model. Four loci exhibiting linkage with body fat, or with the weights of four different fat depots, residing on mouse chromosomes 6, 7, 12, and 15, were identified and confirmed by analysis of additional BSB mice. Each of the four loci differed with respect to their effects on the percent of body fat, specific fat depots and plasma lipoproteins. The loci exhibited allele-specific, non-additive interactions. A locus for hepatic lipase activity was co-incident with the body fat and total cholesterol loci on chromosome 7, providing a possible mechanism linking plasma lipoproteins and obesity. The chromosome 7 locus affecting body fat, total cholesterol and hepatic lipase activity was isolated in congenic strains whose donor strain regions overlap with the chromosome 7 BSB locus. These results provide candidate genes and candidate loci for the analysis of human obesity.

Identification of an obesity quantitative trait locus on mouse chromosome 2 and evidence of linkage to body fat and insulin on the human homologous region 20q.

Chromosomal synteny between the mouse model and humans was used to map a gene for the complex trait of obesity. Analysis of NZB/BINJ x SM/J intercross mice located a quantitative trait locus (QTL) for obesity on distal mouse chromosome 2, in a region syntenic with a large region of human chromosome 20, showing linkage to percent body fat (likelihood of the odds [LOD] score 3.6) and fat mass (LOD score 4.3). The QTL was confirmed in a congenic mouse strain. To test whether the QTL contributes to human obesity, we studied linkage between markers located within a 52-cM region extending from 20p12 to 20q13.3 and measures of obesity in 650 French Canadian subjects from 152 pedigrees participating in the Quebec Family Study. Sib-pair analysis based on a maximum of 258 sib pairs revealed suggestive linkages between the percentage of body fat (P < 0.004), body mass index (P < 0.008), and fasting insulin (P < 0.0005) and a locus extending approximately from ADA (the adenosine deaminase gene) to MC3R (the melanocortin 3 receptor gene). These data provide evidence that a locus on human chromosome 20q contributes to body fat and insulin in a human population, and demonstrate the utility of using interspecies syntenic relationships to find relevant disease loci in humans.

Studies of natural allele effects in mice can be used to identify genes causing common human obesity.

Although genes causing rare Mendelian forms of human obesity have provided much useful information about underlying causes of obesity, these genes do not explain significant proportions of common obesity. This review presents evidence that animal models can be used to uncover subtle genetic effects on obesity and can provide a powerful rigorous compliment to human association studies. We discuss the advantages of animal models of obesity, various approaches to discovering obesity genes, and the future of mapping and isolating naturally occurring alleles of obesity genes. We review evidence that it is important to map naturally occurring obesity genes using quantitative trait locus (QTL) mapping, instead of mutagenesis and knockout models because the latter do not allow study of interactions and because naturally occurring obesity alleles can interfere with cloning from mutagenesis projects. Because a substantial percentage of human obesity results from complex interactions, the underlying genes can only be identified by direct studies in humans, which are still very difficult, or by studies in mice that begin with QTL mapping. Finally, we emphasize that animal model studies can be used to prove that a specific gene, only associated with obesity in humans, can indeed be the underlying cause of obesity in mammals.

Cardiac effects of starvation and semistarvation diets: safety and mechanisms of action.

A major concern with the use of starvation or semistarvation diets for weight reduction in severely obese people has been the reports of sudden death due to ventricular arrhythmias. Obesity per se is associated with cardiovascular changes, including left ventricular hypertrophy and prolongation of the QT interval. With weight loss, the mass of the heart and left ventricle decrease, but some signs of left ventricular dysfunction remain. The effect of weight loss on the electrocardiogram abnormalities of obesity appears to depend upon diet duration and upon whether protein and mineral nutritional status is maintained. Copper, potassium, and magnesium deficiencies may play important roles in promoting an electrically unstable heart. Stress, by eliciting autonomic imbalance, may act upon an electrically unstable heart to provoke acute arrhythmias in a subset of the obese population with QT interval prolongation.

Calcium, magnesium, and phosphate balances during very low calorie diets of soy or collagen protein in obese men: comparison to total fasting.

In 30 obese men, calcium, magnesium, and phosphate balances were measured for 40 days using one of four weight reducing regimens: 1) 400 kcal soy protein; 2) 400 kcal collagen protein; 3) total fasting with potassium; and 4) total fasting without potassium. Relationship of the minerals to each other and to nitrogen and to the QTc interval was also examined. All groups were in negative cumulative calcium balance but the protein-fed groups lost less calcium (soy, -3.0 +/- 2.1 g; collagen -4.9 +/- 3.2 g) than the total fasting groups (with potassium supplement, -9.2 +/- 3.4 g; without potassium supplement, -5.8 +/- 2.1 g) (p less than 0.01). The soy-fed group attained positive cumulative magnesium (0.7 +/- 0.5 g) and phosphate balances (6.9 +/- 3.9 g). The other three groups had significantly more negative magnesium (p less than 0.0005) and phosphate (p less than 0.0005) balances, (collagen, magnesium balance, -1.1 +/- 1.0 g, and phosphate balance, -7.6 +/- 3.7 g; total fasting without potassium, magnesium balance, -1.4 +/- 0.6 g, and phosphate balance, -5.4 +/- 2.7 g). Potassium supplementation during fasting increased urinary losses of calcium and fecal losses of magnesium. High phosphate intake reduced urinary calcium. Nitrogen losses predicted only magnesium losses. Serum mineral levels did not reflect tissue mineral status. Shortening in the QTc interval as an indicator of reduced myocardial instability was related to the increase in serum phosphate in the protein-fed subjects.

Effects of repeated weight loss and regain on body composition in obese rats.

To test the hypothesis that repeated loss and gain of weight through dieting will result in increasing fatness, 200-g female Sprague-Dawley rats were castrated and allowed to become obese on a high-fat diet for 6 wk. Two successive periods of severe food restriction (50% maintenance for 28 d and 25% maintenance for 21 d) were each followed by ad libitum refeeding on the high-fat diet until control body weights were attained. Percent body fat was determined indirectly from body density and total-body water at the end of each cycle. Restricted rats gained weight and attained control body weights on ad libitum feeding without overshooting these weights, and percentage body fat was not different from that of controls at the end of either cycle (cycle 1 22.3 +/- 1.7 vs 23.8 +/- 1.7%; cycle 2 19.0 +/- 1.1 vs 21.6 +/- 0.8%). Repeated cycles of weight loss and regain do not produce increased body fatness or decreased rate of weight loss in ovariectomized rats.

Nitrogen economy during very low calorie reducing diets: quality and quantity of dietary protein.

Controversy exists whether protein quantity or quality affect "nitrogen sparing" or physical health while subsisting on very low calorie diets. Therefore, in 38 obese men, nitrogen economy was evaluated over 2 months periods using one of five regimens: 1) 400 kcal high quality protein: 2) 400 kcal low quality protein; 3) 500 kcal 55 g protein natural food; 4) total fasting with potassium; and 5) total fasting without potassium. Up to the 20- and 40-day intervals, mean cumulative nitrogen deficity for all three diet groups was the same but 60% lower than with total fasting. However, within groups, individual capability to conserve nitrogen varied over as much as 2.8-fold. All 10 subjects of diet groups 1 and 2 had negative nitrogen balances to day 21, and six of these subjects were still negative by day 40. The improvement in nitrogen conservation and the ability to attain nitrogen equilibrium was unrelated to the differences in protein quantity and quality. Intake of essential or branched-chain amino acids was also unrelated to the efficiency of nitrogen conservation, as were insulin, glucagon, and 3-hydroxybutyrate levels. The only indicator correlating positively with nitrogen deficit was a fall in complement C3 (r = 0.87). Despite the extent of overall nitrogen loss, no cardiac arrhythmias were observed with either the high or low quality protein diet.

Liver fatty acid composition correlates with body fat and sex in a multigenic mouse model of obesity.

To determine whether there is altered liver lipid-fraction fatty acid distribution in a multigenic obese mouse model, we examined livers from eight lean (0.2-4.2% carcass fat), seven intermediate (5.7-13.8%), and five obese (20.2-48.7%) backcross progeny [(C57BL/6J x Mus spretus) x C57BL/6J] aged 2-3 mo. Thirteen males and seven females were fed a nonpurified stock diet. Liver lipid fractions were separated and fatty acids quantitated by thin-layer and gas chromatography. There was a significant effect of obesity on 18:2 omega 6 in liver phospholipids (PL), cholesteryl esters, and triglycerides. PL 18:2 omega 6 was negatively correlated with carcass fat (r = -0.74, P < 0.001); 20:3 omega 6 was elevated in PL with increased obesity (P < 0.0001), and was correlated with carcass fat (r = 0.92, P < 0.0001); and 20:4 omega 6 in PL did not differ with obesity status. PL 20:3 omega 6 and 20:4 omega 6 were lower in males (P < 0.01 and 0.02, respectively) than in females. We conclude that obesity and sex affect distribution of omega 6 essential fatty acids in mouse liver lipid fractions.

Zinc and copper nutriture in obese men receiving very low calorie diets of soy or collagen protein.

Balance studies for Zn and Cu were conducted over 40 days in 10 obese men housed in a metabolic balance unit. Two weight reduction diets providing 400 kcal and 100 g protein daily were administered; to five subjects, a collagen diet which was severely deficient in both Zn and Cu, and to another five subjects, a soy diet which provided a marginal intake of Zn and an adequate intake of Cu. Zn and Cu content of diets, plasma, red blood cells, urine, and feces were determined during eight 5-day periods. Balances were corrected for lean tissue catabolism or deposition. Holter ECG monitoring and measurement of the QTc interval were done on days 0 and 40. Both diets resulted in elevated plasma and red blood cell concentrations of Zn and Cu and in high urinary and fecal losses of Zn. By day 40, 6 of 10 subjects were in negative Zn balance. Urinary Zn was inversely correlated with measures of lean tissue catabolism. During each period, Cu balance was markedly positive in the soy-diet group and negative in the collagen-diet group. Shortening of prolonged QTc intervals was related to the Cu but not Zn status of the individual.

Effects of oral and parenteral quinine on rats with ventromedial hypothalamic knife-cut obesity.

The addition of quinine to the food reversed the obesity in rats with hypothalamic hyperphagia induced by knife cuts. Similarly, the injection of quinine into rats with hypothalamic knife cuts reduced food intake and body weight but the effects were smaller than those observed when quinine was added to the diet. Urinary quinine excretion was similar by the oral and parenteral routes. The food intake of the knife-cut animals receiving quinine gradually fell to the same level as in the sham-operated animals receiving quinine by either route. The weights of retroperitoneal fat pads were related to the weights of the animals and were reduced in the quinine-treated groups. Plasma insulin concentrations were significantly higher in the knife-cut animals and were reduced toward control levels by quinine treatment. Gluconeogenesis, measured by incorporation of radioactivity from labeled bicarbonate into glucose, was unaffected by treatment with quinine or by knife cuts. Lipogenesis from tritiated water in vivo was not different between treatment groups in the liver or retroperitoneal fat pads. However, in vivo lipogenesis was reduced in knife-cut rats fed ad libitum compared with quinine-treated rats. The response of lipogenesis to insulin in vitro was also not different between treatment groups. These data suggest that a major part of the reduction in food intake in hyperphagic rats eating a quinine-adulterated diet is due to postingestional events.

Effect of carbohydrate refeeding on free fatty acids after a fast in obese diabetic and obese non-diabetic females.

The metabolic effects of refeeding with oral or intravenous carbohydrate were studied in obese women after ten or 14 days of fasting. Seven patients were refed with protein-free fruit juice for a total of 250 g of carbohydrate (1,000 kcal) over ten hours. The juice was sipped continuously throughout this time, causing a drop in free fatty acids (FFA) from 1.07 +/- 0.08 to 0.61 +/- 0.05 mmol/L (P less than .01) over the first four hours. Over the next four hours, despite continuous ingestion of the carbohydrate and elevated plasma glucose (132 +/- 9 mg/dL) and insulin (2.81 +/- 0.86 ng/mL) (1 ng/mL = 25 microU/mL), FFA rose to 0.99 mmol/L (P less than .01). Similar results were obtained in five patients refed with similar amounts of oral glucose and four patients who received an equivalent amount of glucose intravenously (IV). Refeeding with carbohydrate of obese diabetic and non-diabetic women after a two-week fast caused an abrupt decrease in FFA that was followed after four hours by an increase in FFA and glycerol, despite continued ingestion of carbohydrate glucose and insulin.

Metabolic and hormonal factors as predictors of nitrogen retention in obese men consuming very low calorie diets.

The ability to conserve body protein during very low calorie diets in ten obese men was observed to correlate with plasma free amino acid concentrations, urinary N tau-methylhistidine: creatinine ratios, resting oxygen consumption, and serum triiodothyronine levels. A diet consisting of only protein, 1.3 g/kg ideal body weight/24 h, was given for 40 days. Cumulative nitrogen deficit ranged from -64 g to -227 g. Nitrogen balance on days 36 to 40 ranged from + 1.37 g/24 h to -3.30 g/24h. Nitrogen balance during this period had a significant direct correlation with pre-diet concentrations of branched-chain amino acids (r = 0.69 to 0.89), methionine (r = 0.85), histidine (r = 0.66), alanine (r = 0.73), arginine (r = 0.70), ornithine (r = 0.66), total essential (r = 0.87, and nonessential (r = 0.68) amino acids, with initial serum levels of triiodothyronine (r = 0.66) and with the fall in triiodothyronine over the 40 days (r = 0.79). Initial resting oxygen consumption was directly correlated (r = 0.78) with final nitrogen balance and inversely with total nitrogen loss (r = -0.81). On day 0, triiodothyronine levels also correlated positively (r ranging from 0.71 to 0.93) with plasma concentrations of several essential and nonessential amino acids. These correlations suggest that individuals who ultimately will or will not achieve nitrogen equilibrium during very low calorie diets can be identified prior to dieting. These data are consistent with the hypothesis that individuals with higher initial protein flux and triiodothyronine levels are better able to adjust the balance between synthesis and degradation to attain nitrogen equilibrium during hypocaloric dieting.

Dietary obesity: effects of drugs on food intake in S 5B/P1 and Osborne-Mendel rats.

The effect of several drugs on food intake has been examined in two strains of rats, one (S 5B/P1) which is resistant to developing obesity when eating a high fat diet, and one (Osborne-Mendel) which readily develops obesity when eating the same diet. Insulin and 2-deoxy-D-glucose increased food intake in a dose dependent manner in both S 5B/P1 and Osborne-Mendel rats. However, the S 5B/P1 rats showed a greater response, with a shorter latency period, to both agents than did the Osborne-Mendel rats. Conversely, d-amphetamine at the higher doses produced a dose dependent suppression of food intake with maximal suppression being similar for both strains. At a lower dose, however, d-amphetamine significantly increased food intake in the Osborne-Mendel rats, but not in the S 5B/P1 rats. The S 5B/P1 rats were also slightly more sensitive to the anorexic effects of lower dose adenosine than were the Osborne-Mendel rats whereas the reverse was true following higher dose adenosine. Naloxone suppressed food intake equally in both strains and D-glucose did not alter food intake in either strain. These studies identify three drugs, all stimulatory, to which the two strains of rat respond differently.

Peripheral 3-hydroxybutyrate and food intake in a model of dietary-fat induced obesity: effect of vagotomy.

We have examined the effect of peripheral 3-hydroxybutyrate injections on food intake and the contribution of the vagus nerve in the resistance to dietary fat-induced obesity in a rodent model. S 5B/Pl rats, which are resistant to dietary-fat induced obesity, and Osborne-Mendel rats, which are sensitive, were adapted to reverse light cycle. Food intake was measured for 24 h following the injection of 3-hydroxybutyrate, lactate, or glycerol (all 5 mMol/kg0.75, SC) at the onset of dark. Three-hydroxybutyrate reduced food intake (p < 0.0001) in S 5B/Pl rats only. Lactate reduced food intake slightly (p < 0.009) in both strains and glycerol had no effect on food intake. In a second experiment, S 5B/Pl and Osborne-Mendel rats were adapted to a high-fat diet and were then subjected to either selective hepatic vagotomy or sham operation. Vagotomy had no effect on weight gain of Osborne-Mendel rats but allowed weight gain in S 5B/Pl rats (p < 0.0001). Even in vagotomized S 5B/Pl rats, however, blood 3-hydroxybutyrate levels were inversely associated (r = -0.50) with food intake. These data suggest that the hepatic vagus nerve may contribute to the resistance of S 5B/Pl rats to dietary-fat induced obesity, but the data do not rule out a strictly central role for the regulation of food intake by 3-hydroxybutyrate in this strain.

Brain 3-hydroxybutyrate, glutamate, and GABA in a rat model of dietary obesity.

Whole brain concentrations of 3-hydroxybutyrate, glutamate and gamma-aminobutyric acid (GABA) have been measured in two strains of rats with differing susceptibility to obesity. S 5B/Pl rats are resistant to developing obesity when eating a high-fat diet, whereas Osborne-Mendel rats readily develop obesity when eating the same diet. We tested the hypotheses that brain 3-hydroxybutyrate, glutamate and GABA differ between S 5B/Pl rats and Osborne-Mendel rats, and that these substrates/neuroregulators are altered when eating a high-fat diet primarily in S 5B/Pl (resistant) rats. Blood and brain 3-hydroxybutyrate concentrations were higher in S 5B/Pl rats than in Osborne-Mendel rats (p less than 0.05) but diet effects were not significant. Brain glutamate concentration, like 3-hydroxybutyrate, was higher in S 5B/Pl rats than in Osborne-Mendel rats (p less than 0.01) and was not affected by adding fat to the diet. Brain GABA differed only slightly between strains but increased after adding fat to the diet (p less than 0.05) in both strains with a greater increase occurring in S 5B/Pl rats. The brains of S 5B/Pl rats are chronically exposed to higher levels of 3-hydroxybutyrate and glutamate than are those of Osborne-Mendel rats. Thus, 3-hydroxybutyrate is a potential signal in the regulation of body weight. Brain GABA increases with fat feeding, especially in S 5B/Pl rats, suggesting that the ability to adjust to an energy dense diet may be through suppression of food intake by elevated brain GABA.

Effect of a high-fat diet on firing rate of sympathetic nerves innervating brown adipose tissue in anesthetized rats.

Three experiments have examined the effects of ad lib and forced intake of a high-fat diet on sympathetic firing rate to brown adipose tissue. Seven days after beginning of ad lib intake of either a low-fat or high-fat diet, sympathetic activity was not significantly different in either group nor was it significantly different from the values obtained in animals measured at the switch from the chow to a semisynthetic high- or low-fat diet. After 22 days on the semisynthetic diet, however, the sympathetic firing rate of animals eating the high-fat diet had decreased nearly 25% and was significantly lower than the animals maintained on the semisynthetic low-fat diet or animals studied at the transition from the chow to the low-fat diet. In a second experiment animals were tube-fed for 3, 6 or 9 weeks on a high- or low-fat diet. Sympathetic firing rate of the rats eating the low-fat diet was higher at all three times, but the difference decreased with longer feeding. To eliminate differences in food intake, animals were tube-fed a moderate- or high-fat liquid diet three times a day for six days. The 80 kcal/day intake produced a steady weight gain in both groups. Liver weight, retroperitoneal white adipose tissue weight, and interscapular brown adipose tissue weight were all significantly greater in the animals fed the high-fat diet. Sympathetic firing rate, however, was significantly lower in the animals fed the high-fat semisynthetic diet as compared to animals fed the moderate-fat diet. These data show the high-fat diets are associated with a reduction in sympathetic activity to brown adipose tissue.

Sudden cardiac arrest in morbidly obese surgical patients unexplained after autopsy.

Sixty sudden and unexpected lethal cardiac arrests, with entirely negative findings on autopsy, were reported among 50,314 morbidly obese patients in the care of surgeons performing operations to achieve weight loss. This represented an extrapolated overall annual mortality rate of 65 deaths per 100,000 patients, about 40 times higher than the rate of unexplained cardiac arrests in a matched nonobese population. Eight sudden deaths occurred while waiting for obesity surgery and 22 had cardiac arrest within 10 days after the operation. Late postoperative deaths (more than 4 weeks postoperatively) occurred in 30 instances. A possible marker of a predisposition for sudden, unexpected cardiac arrest was an electrocardiographic abnormality; namely, a Q-Tc interval prolonged to greater than 0.43 seconds. This feature, present in 29 of 38 tracings, denoted increased susceptibility to malignant ventricular arrhythmias. The perioperative clustering of arrests implicated nonspecific stresses incident to otherwise uneventful operations as triggers of lethal dysrhythmias in the absence of organic cardiac disease. Anoxemia after abdominal surgery is an added hazard. Length of postoperative survival among the late deaths was found to be unrelated to degree of initial obesity or to magnitude of weight loss. Patients who died in the late postoperative phase were still grossly obese (mean weight 114.2 kg). Cardiac weights in patients who died within 10 postoperative days (12 patients) or after an average of 103 days (20 patients) were the same (464 and 469 g, respectively), indicating that myocardial mass had remained intact. The data do not suggest nutritional depletion or lean tissue loss as plausible explanations for the cardiac arrests. Screening and postoperative monitoring for Q-T interval prolongation is indicated. Prophylactic beta-blockade in this vulnerable subset of the morbidly obese population may be instituted in anticipation of obesity surgery. The attendant physiologic stresses should be minimized by appropriate measures.

d-fenfluramine in a rat model of dietary fat-induced obesity.

d-Fenfluramine is an appetite suppressant drug that acts by releasing serotonin from axon terminals and inhibiting its reuptake. S 5B/P1 rats, which are resistant to dietary-fat induced obesity, and Osborne-Mendel rats, which are sensitive, were adapted to ad lib feeding of either a low- or high-fat diet. d-Fenfluramine (10 mg/kg, IP) was injected daily for 12 days. Other than a slightly greater suppression of food intake in Osborne-Mendel rats, there was little difference in response to d-fenfluramine between S 5B/P1 and Osborne-Mendel rats eating the low-fat diet. However, in Osborne-Mendel rats d-fenfluramine completely abolished the excess food intake and weight gain associated with the high-fat diet. Purine nucleotide (GDP) binding on day 13 was higher in S 5B/P1 rats than in Osborne-Mendel rats and was increased by d-fenfluramine in animals of both strains eating the low-fat diet. The high-fat diet increased GDP binding only in S 5B/P1 rats and blocked the fenfluramine-induced increase in GDP binding in both strains. We speculate that d-fenfluramine blocks a feeding reward system stimulated by the high-fat diet.