Decreased responsiveness of gluconeogenesis to the modulation by sulfonylureas in hepatocytes isolated from obese (fa/fa) Zucker rats

The influence of the hypoglycemic agent glipizide (0-100 microM) on the rate of gluconeogenesis from lactate, as well as on the levels of fructose 2,6-bisphosphate, has been investigated in hepatocytes isolated from genetically obese (fa/fa) Zucker rats and from their corresponding lean (Fa/-) littermates. As compared to lean rat hepatocytes, liver cells isolated from obese animals showed a lower rate of basal gluconeogenesis (0.9 +/- 0.2 vs 5.4 +/- 0.5 micromol of lactate converted to glucose/g cell x 30 min, n=4) and higher levels of fructose 2,6-bisphosphate (11.5 +/- 1.0 vs 5.9 +/- 0.4 nmol/g cell, n=8-9). In lean rat hepatocytes, the presence of glipizide in the incubation medium caused a dose-dependent inhibition of the rate of lactate conversion to glucose (maximal inhibition=46%; EC50 value=26 microM), and simultaneously raised the cellular content of fructose-2,6-bisphosphate (maximal increment=40%; EC50 value=10 microM). In contrast, in hepatocytes isolated from obese rats, the inhibition of gluconeogenesis and the increment in fructose-2,6-bisphosphate levels elicited by glipizide were significantly reduced (maximal effects of 22 and 13%, respectively). Similarly, the activation of glycogen phosphorylase and the increase in hexose 6-phosphate levels in response to glipizide were less marked in obese rat hepatocytes than in liver cells isolated from lean animals. These results demonstrate that the efficacy of sulfonylureas as inhibitors of hepatic gluconeogenesis is reduced in the genetically obese (fa/fa) Zucker rat.     

Changes of Islet Size and Islet Size Distribution Resulting from Protein-Malnutrition in Lean (Fa/Fa) and Obese (fa/fa) Zucker Rats

TSE, ELIZABETH O, FRANCINE M GREGOIRE, BRIGITTE REUSENS, CLAUDE REMACLE, JOSEPH J HOET, PATRICIA R JOHNSON, JUDITH S STERN. Changes of islet size and islet size distribution resulting from protein malnutrition in lean (Fa/Fa) and obese (fa/fa) Zucker rats. Potential alterations in islet size and islet size distribution resulting from protein malnutrition were studied in lean (Fa/Fa) and obese (fa/fa) Zucker rats. The purpose was to investigate whether the distribution of enlarged islets in obese rats was altered by low-protein feeding. Four-week-old, male, lean and obese Zucker rats were fed either a diet containing 20% (w/w) protein (control diet) or a diet containing 5% (w/w) protein (low-protein diet) for 3 weeks. Pancreata were dissected at autopsy and immunostained for insulin. Islet size and distribution were determined by morphometric analysis. Body-weight gain, food intake, and serum insulin and glucose were also measured. After 3 weeks on the diets, serum insulin was significantly lower in both lean (-75%) and obese (-54%) rats fed low protein compared with that in controls. However, obese rats were still hyperinsulinemic compared with lean rats. Protein malnutrition resulted in a shift in distribution of islets to smaller size both in lean and in obese rats, with an increase in the population of small islets (100 μm²) and a decrease in the population of large islets (>20,000 μ;m²). In lean and obese rats fed low protein, β-cell weight was significantly lower, B cell volume fraction tended to decrease, and islet number per section area was significantly elevated when compared with controls. Taken together, these results show that protein deficiency alters the endocrine pancreas in both lean and obese Zucker rats. Although the decrease in islet size and the shift in distribution to smaller islets most likely contribute to the decrease in serum insulin concentration, these changes appear insufficient to normalize hyperinsulinemia in the obese Zucker rat.     

Lipid and lipoprotein synthesis in isolated and cultured hepatocytes from lean and obese Zucker rats

Hepatocytes were isolated by EDTA perfusion of livers from lean (Fa/-) and obese (fa/fa) Zucker rats. Triacylglycerol (TG) and sn-glycerol 3-phosphate were increased in fa/fa hepatocytes, but free fatty acids, cholesterol and phospholipid concentrations were similar in both groups. In spite of an identical fatty acid uptake rate, glycerolipid synthesis was higher in obese compared to lean rat hepatocytes, and this difference remained for at least 2-3 days of culture. Triacylglycerol mass secretion was 2-fold higher in obese than in lean rat hepatocytes. This was confirmed by the higher incorporation of labeled glycerol and oleic acid into the medium TG fraction floating at density 1.006 g/ml. Density gradient ultracentrifugation of [14C]oleate-labeled lipoproteins showed that fa/fa hepatocytes secreted more TG-rich lipoproteins, and that 87% of the label was in the VLDL fraction compared with 67% in the medium of Fa/- hepatocytes. Decreased utilisation of leucine for protein synthesis in obese rat compared to lean rat hepatocytes was associated with enhanced leucine oxidation to CO2. [35S]Methionine incorporation showed an identical cell protein synthesis rate. Autoradiography after PAGE separation of secreted apolipoproteins (apoBh, Bl, apoA-VI, apoE, apoA-I, apoC) showed an identical pattern in both cell types.     

Nonreceptor-mediated responses of adenylate cyclase in membranes from liver, muscle, and white and brown adipose tissue of obese (fa/fa) and lean (Fa/) Zucker rats

Adenylate cyclase activity was determined in membranes of liver, muscle, white adipose tissue, and brown adipose tissue (BAT) of lean (Fa/) and obese (fa/fa) Zucker rats. Responses were monitored following beta-adrenergic receptor stimulation and addition of GTP, GTP gamma S, or forskolin. beta-Adrenergic responses in liver, white adipose tissue, and BAT were lower in obese than in lean animals. No such difference was observed in muscle membranes. Production of cAMP after addition of guanine nucleotides was lower in liver and white adipose tissue membranes from obese rats compared with their lean littermates. Synthesis of cAMP in muscle membranes of obese animals after addition of GTP was either not different, or slightly higher, than that observed in muscle membranes from lean animals. Furthermore, production of cAMP after forskolin addition to muscle membranes of obese rats was significantly higher than that observed from lean rats under the same conditions. Interestingly, BAT membranes of obese rats were significantly more sensitive to guanine nucleotide activation than those of lean animals. The results confirm recent findings indicating inferior function of G proteins in liver plasma membranes of obese Zucker rats, and extend this observation to adipose tissue. The present results further suggest that the "nonreceptor" components (e.g., G proteins) responsible for the activation of adenylate cyclase in BAT membranes of obese rats are more responsive to stimulation than those of lean animals. Such sensitivity may be related to and perhaps compensate for the reduced thermogenic activity in the obese Zucker rat during the development of obesity.     

Exercise training increases glucose transporter protein GLUT-4 in skeletal muscle of obese Zucker (fa/fa) rats

The present study examined the level of GLUT-4 glucose transporter protein in gastrocnemius muscles of 36 week old genetically obese Zucker (fa/fa) rats and their lean (Fa/-) littermates, and in obese Zucker rats following 18 or 30 weeks of treadmill exercise training. Despite skeletal muscle insulin resistance, the level of GLUT-4 glucose transporter protein was similar in lean and obese Zucker rats. In contrast, exercise training increased GLUT-4 protein levels by 1.7 and 2.3 fold above sedentary obese rats. These findings suggest endurance training stimulates expression of skeletal muscle GLUT-4 protein which may be responsible for the previously observed increase in insulin sensitivity with training.     

Lipid metabolism in the newborn genetically obese (fa/fa) rat

Compared to its lean litter mate (Fa/--) the Zucker rat (fa/fa) develops obesity without hyperphagia in the first week of lite. It is characterized by adipocyte hypertrophy and higher lipid content in adipose tissue. In vitro utilization as well as in vitro oxidation by diaphragm of palmitic acid was decreased in 1 week old Zucker rat.     

Differential Responsiveness of Obese (fa/fa) and Lean (Fa/Fa) Zucker Rats to Cytokine-Induced Anorexia

Pathophysiological and pharmacological concentrations of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the cerebrospinal fluid (CSF) induce anorexia in normal rats. Obesity in humans and rodents is associated with increased TNF-α messenger RNA and protein levels in various cell types. This suggests that obese individuals may have differential regulation of cytokine production and dissimilar responsiveness to cytokines. In the present study, we investigated the effects of the intracerebroventricular (ICV) microinfusion of TNF-α (50, 100, and 500 ng/rat), IL-1β (1.0, 4.0, and 8.0 ng), and TNF-α (100 ng) plus IL-1β (1.0 ng) on obese (fa/fa) and lean (Fa/Fa) Zucker rats. The results show that: TNF-α and IL-1β, and the concomitant administration of TNF-a and IL-ip decreased the short-term (4 hours), nighttime (12 hours), and total daily food intakes in obese and lean rats; IL-1β was more potent relative to TNF-α; obese rats showed greater responsiveness to IL-1β: 8.0 ng IL-1β, for example, decreased the 12-hour food intake by 52% in obese and 22% in lean rats. On the other hand, obese and lean rats did not exhibit a significantly different responsiveness to the anorexia induced by 50,100, or 500 ng TNF-α at the 4-hour period; and the concomitant ICV administration of TNF-α and IL-1β induced anorexia with additive (4-hour period) or synergistic (12-hour and 24-hour periods) effects in obese rats. The effect of TNF-α plus IL-1β in lean rats was greater than additive for the 12-hour and 24-hour periods. The difference in suppression of total daily food intake by TNF-α plus IL-1β in obese (-43%) versus lean (-23%) rats was significantly different (p<0.01). The results show that obese (fa/fa) and lean (Fa/Fa) Zucker rats have differential responsiveness to the ICV microinfusion of two different classes of cytokines.     

Lipogenesis from U14C lactate in obese Zucker rat hepatocytes. Effect of albumin-bound oleate

Lipogenesis from U(¹⁴C) lactate was studied in hepatocytes isolated from obese Zucker rats (fa/fa) their lean littermates (Fa/?) and Sprague Dawley rats. The distribution of radioactive carbon between the glycerol and the fatty acid moieties of the acylglycerols were studied. Radioactive lactate was better utilized for glycerol formation than it was for fatty acid formation in the obese rats. However, when oleate was added to the hepatocytic incubation medium, radioactive lactate was preferentially incorporated into the fatty acid moiety of the acylglycerols.     

Age-related decrease in sensitivity to glucagon and dibutyryl cyclic AMP inhibition of fatty acid synthesis in hepatocytes isolated from obese female Zucker rats

Hepatocytes were isolated from 3 and 5 month old female genetically obese Zucker rats and their lean littermate controls. An age-dependent loss in sensitivity of fatty acid synthesis to inhibition by both glucagon and dibutyryl cyclic AMP was observed with hepatocytes from the obese rats. Hepatocytes from lean animals were much more sensitive to these agents, regardless of age. Low concentrations of glucagon and dibutyryl cyclic AMP actually produced some stimulation of fatty acid synthesis with hepatocytes prepared from the older obese rats. 5-Tetradecyloxy-2-furoic acid, a compound which inhibits fatty acid synthesis, was a very effective inhibitor of fatty acid synthesis by hepatocytes isolated from all rats used in the study. An inhibition of lactate plus pyruvate accumulation and a strong stimulation of glycogenolysis occurred in response to both glucagon and dibutyryl cyclic AMP with hepatocytes from both age groups of lean and obese rats. The results suggest that with aging of the obese female Zucker rat some step of hepatic fatty acid synthesis becomes progressively less sensitive to inhibition by glucagon and dibutyryl cyclic AMP. This may play an important role in maintenance of obesity in these animals.     

The Male Obese Wistar Diabetic Fatty Rat Is a New Model of Extreme Insulin Resistance

The male obese Wistar Diabetic Fatty (WDF) rat is a genetic model of obesity and non-insulin dependent diabetes (NIDDM). The obese Zucker rat shares the same gene for obesity on a different genetic background but is not diabetic. This study evaluated the degree of insulin resistance in both obese strains by examining the binding and post binding effects of muscle insulin receptors in obese, rats exhibiting hyperinsulinemia and/or hyperglycemia. Insulin receptor binding and affinity and tyrosine kinase activity were measured in skeletal muscle from male WDF fa/fa (obese) and Fa/? (lean) and Zucker fa/fa (obese) and Fa/Fa (homozygous lean) rats. Rats were fed a high sucrose (68% of total Kcal) or Purina stock diet for 14 weeks. At 27 weeks of age, adipose depots were removed for adipose cellularity analysis and the biceps femoris muscle was removed for measurement of insulin binding and insulin-stimulated receptor kinase activity. Plasma glucose (13.9 vs. 8.4 mM) and insulin levels (14,754 vs. 7440 pmoI/L) were significantly higher in WDF obese than in Zucker obese rats. Insulin receptor number and affinity and TK activity were unaffected by diet. Insulin receptor number was significantly reduced in obese WDF rats (2.778 ± 0.617 pmol/mg protein), compared to obese Zucker rats (4.441 ± 0.913 pmol/mg potein). Both obese strains exhibited down regulation of the insulin receptor compared to their lean controls. Maximal tyrosine kinase (TK) activity was significantly reduced in obese WDF rats (505 ± 82 fmol/min/mg protein) compared to obese Zucker rats (1907 ± 610 fmol/min/mg protein). Only obese WDF rats displayed a decrease in TK activity per receptor. These observations establish the obese WDF rat as an excellent model for exploring mechanisms of extreme insulin resistance, particularly post-receptor tyrosine kinase-associated defects, in non-insulin dependent diabetes.     

Brain insulin binding is decreased in Wistar Kyoto rats carrying the 'fa' gene

We have previously reported that insulin binding is decreased in the olfactory bulb of both heterozygous (Fa/fa) and obese (fa/fa) Zucker rats. In the present study, we measured insulin binding in membranes prepared from the olfactory bulb, cerebral cortex, and hypothalamus of control (Fa/Fa) Wistar Kyoto rats; "fatty" (fa/fa) Wistar Kyoto rats; and phenotypically lean (Fa/?) Wistar Kyoto rats. Insulin binding was decreased in all brain regions, as well as the liver of the obese Wistar Kyoto fa/fa rats. Additionally, insulin binding was decreased in the liver and brain membranes from the Fa/? Wistar Kyoto rats. As most of the Fa/? rats were probably carriers of one 'fa' gene, but the population was only slightly hyperinsulinemic, we conclude that--as in the Zucker rat--it is the presence and expression of the 'fa' gene rather than downregulation which results in the decreased insulin binding. Thus, regulation of the brain insulin receptor appears to be independent of plasma or cerebrospinal fluid insulin levels.     

Subdiaphragmatic vagal deafferentation affects body weight gain and glucose metabolism in obese male Zucker (fa/fa) rats

We investigated the effect of subdiaphragmatic vagal deafferentation (SDA) on food intake, body weight gain, and metabolism in obese (fa/fa) and lean (Fa/?) Zucker rats. Before and after recovery from surgery, food intake and body weight gain were recorded, and plasma glucose and insulin were measured in tail-prick blood samples. After implantation of a jugular vein catheter, an intravenous glucose tolerance test (IVGTT) was performed, followed by minimal modeling to estimate the insulin sensitivity index. Food intake relative to metabolic body weight (g/kg(0.75)) and daily body weight gain after surgery were lower (P < 0.05) in SDA than in sham obese but not lean rats. Before surgery, plasma glucose and insulin concentrations were lower (P < 0.05) in lean than in obese rats but did not differ between surgical groups within both genotypes. Four weeks after surgery, plasma glucose and insulin were still similar in SDA and sham lean rats but lower (P < 0.05) in SDA than in sham obese rats. IVGTT revealed a downward shift of the plasma insulin profile by SDA in obese but not lean rats, whereas the plasma glucose profile was unaffected. SDA decreased (P < 0.05) area under the curve for insulin but not glucose in obese rats. The insulin sensitivity index was higher in lean than in obese rats but was not affected by SDA in both genotypes. These results suggest that elimination of vagal afferent signals from the upper gut reduces food intake and body weight gain without affecting the insulin sensitivity index measured by minimal modeling in obese Zucker rats.     

Multiple defects occur in the guanine nucleotide regulatory protein system in liver plasma membranes of obese (fa/fa) but not lean (Fa/Fa) Zucker rats: loss of functional Gi and abnormal Gs function

Hepatocyte membranes from both lean and obese Zucker rats exhibited adenylate cyclase activity that could be stimulated by glucagon, forskolin, NaF and elevated concentrations of p[NH]ppG. In membranes from lean animals, functional Gi was detected by the ability of low concentrations of p[NH]ppG to inhibit forskolin-activated adenylate cyclase. This activity was abolished by treatment of hepatocytes with either pertussis toxin or the phorbol ester TPA, prior to making membranes for assay of adenylate cyclase activity. In hepatocyte membranes from obese animals no functional Gi activity was detected. Quantitative immunoblotting, using an antibody able to detect the alpha subunit of Gi, showed that hepatocyte plasma membranes from both lean and obese Zucker rats had similar amounts of Gi-alpha subunit. This was 6.2 pmol/mg plasma membrane for lean and 6.5 pmol/mg plasma membrane for obese animals. Using thiol pre-activated pertussis toxin and [32P]-NAD+, similar degrees of labelling of the 40 kDa alpha subunit of Gi were found using plasma membranes of both lean and obese Zucker rats. We suggest that liver plasma membranes from obese Zucker rats express an inactive Gi alpha subunit. Thus lesions in liver Gi functioning are seen in insulin-resistant obese rats and in alloxan- and streptozotocin-induced diabetic rats which also show resistance as regards the acute actions of insulin. Liver plasma membranes of obese animals also showed an impairment in the coupling of glucagon receptors to Gs-controlled adenylate cyclase, with the Kd values for activation by glucagon being 17.3 and 126 nM for lean and obese animals respectively. Membranes from obese animals also showed a reduced ability for high concentration of p[NH]ppG to activate adenylate cyclase. The use of [32P]-NAD+ and thiol-preactivated cholera toxin to label the 43 kDa and 52 kDa forms of the alpha-subunit of Gs showed that a reduced labelling occurred using liver plasma membranes from obese animals. It is suggested that abnormalities in the levels of expression of primarily the 52 kDa form of alpha-Gs may give rise to the abnormal coupling between glucagon receptors and adenylate cyclase in liver membranes from obese (fa/fa) Zucker rats.     

Increased sensitivity of glycogen synthesis to phosphorylase-a and impaired expression of the glycogen-targeting protein R6 in hepatocytes from insulin-resistant Zucker fa/fa rats

Hepatic insulin resistance in the leptin-receptor defective Zucker fa/fa rat is associated with impaired glycogen synthesis and increased activity of phosphorylase-a. We investigated the coupling between phosphorylase-a and glycogen synthesis in hepatocytes from fa/fa rats by modulating the concentration of phosphorylase-a. Treatment of hepatocytes from fa/fa rats and Fa/? controls with a selective phosphorylase inhibitor caused depletion of phosphorylase-a, activation of glycogen synthase and stimulation of glycogen synthesis. The flux-control coefficient of phosphorylase on glycogen synthesis was glucose dependent and at 10 mm glucose was higher in fa/fa than Fa/? hepatocytes. There was an inverse correlation between the activities of glycogen synthase and phosphorylase-a in both fa/fa and Fa/? hepatocytes. However, fa/fa hepatocytes had a higher activity of phosphorylase-a, for a corresponding activity of glycogen synthase. This defect was, in part, normalized by expression of the glycogen-targeting protein, PTG. Hepatocytes from fa/fa rats had normal expression of the glycogen-targeting proteins G(L) and PTG but markedly reduced expression of R6. Expression of R6 protein was increased in hepatocytes from Wistar rats after incubation with leptin and insulin. Diminished hepatic R6 expression in the leptin-receptor defective fa/fa rat may be a contributing factor to the elevated phosphorylase activity and/or its high control strength on glycogen synthesis.     

Genetically obese Zucker rats have abnormally low brain insulin content

The concentration of immunoreactive insulin (IRI) extracted from the olfactory bulb, hypothalamus, hippocampus, cerebral cortex, amygdala, midbrain, and hindbrain was significantly lower in obese (fa/fa) and heterozygous (Fa/fa) Zucker rats in comparison to lean (Fa/Fa) Zucker rats. This deficit in brain IRI content was most severe in the hypothalamus and olfactory bulb and was independent of severe obesity since the marked reduction of brain IRI content was also found in heterozygous rats which possessed only one copy of the fa allele. These results demonstrate that in the 2-3 month-old female Zucker rat, the fa allele is associated with defective regulation of insulin in the brain.     

Expression of hepatic microsomal cholesterol 7 alpha-hydroxylase activity in lean and obese Zucker rats

The activity of hepatic microsomal cholesterol 7 alpha-hydroxylase was studied in genetically obese and lean Zucker rats. The liver microsomal cholesterol 7 alpha-hydroxylase activity in fatty Zucker rats (fa/fa) is about 50% to 70% lower than that of the lean (Fa/-) rats of the same sex, when animals were sacrificed at the middle of the dark cycle. When rats were sacrificed at the middle of the light cycle, cholesterol 7 alpha-hydroxylase activity was the same as in the dark cycle in obese rats of both sexes, but was 65% lower in lean rats. However, cholesterol 7 alpha-hydroxylase activity was stimulated by the treatment with cholestyramine in both obese and lean rats. Our results suggested that the diurnal regulation of cholesterol 7 alpha-hydroxylase activity is lost in obese rats but was present under cholestyramine treatment in the genetically obese strain of rats.     

Adrenergic regulation of pyruvate kinase and gluconeogenesis in hepatocytes from phosphorylase kinase-deficient (gsdgsd) rats

Hepatocytes were prepared from a strain of rats deficient in hepatic phosphorylase $\text{b}$ kinase and were used to assess the role of this enzyme in the adrenergic regulation of pyruvate kinase and gluconeogenesis. Epinephrine (10 μM) stimulated glucose output and gluconeogenesis from 1.8 mM lactate but did not significantly affect the concentration of hepatocyte glycogen. In addition epinephrine treatment led to an inhibition of pyruvate kinase. The stimulation of gluconeogenesis and the inhibition of pyruvate kinase by epinephrine were blocked by both α- and β-antagonists: similar effects with epinephrine were observed in cells from control animals. It is concluded that mechanisms for the adrenergic regulation of pyruvate kinase and gluconeogenesis are similar in hepatocytes from both phosphorylase kinase-deficient and normal rats.     

Glucose metabolism in perfused skeletal muscle. Demonstration of insulin resistance in the obese Zucker rat.

1. The effect of insulin (0.5, 10 and 50 munits/ml of perfusate) on glucose uptake and disposal in skeletal muscle was studied in the isolated perfused hindquarter of obese (fa/fa) and lean (Fa/Fa) Zucker rats and Osborne-Mendel rats. 2. A concentration of 0.5 munit of insulin/ml induced a significant increase in glucose uptake (approx. 2.5 mumol/min per 30 g of muscle) in lean Zucker rats and in Osborne-Mendel rats, and 10 munits of insulin/ml caused a further increase to approx. 6 mumol/min per 30 g of muscle; but 50 munits of insulin/ml had no additional stimulatory effect. In contrast, in obese Zucker rats only 10 and 50 munits of insulin/ml had a stimulatory effect on glucose uptake, the magnitude of which was decreased by 50-70% when compared with either lean control group. Since under no experimental condition tested was an accumulation of free glucose in muscle-cell water observed, the data suggest an impairment of insulin-stimulated glucose transport across the muscle-cell membrane in obese Zucker rats. 3. The intracellular disposal of glucose in skeletal muscle of obese Zucker rats was also insulin-insensitive: even at insulin concentrations that clearly stimulated glucose uptake, no effect of insulin on lactate oxidation (nor an inhibitory effect on alanine release) was observed; [14C]glucose incorporation into skeletal-muscle lipids was stimulated by 50 munits of insulin/ml, but the rate was still only 10% of that observed in lean Zucker rats. 4. The data indicate that the skeletal muscle of obese Zucker rats is insulin-resistant with respect to both glucose-transport mechanisms and intracellular pathways of glucose metabolism, such as lactate oxidation. The excessive degree of insulin-insensitivity in skeletal muscle of obese Zucker rats may represent a causal factor in the development of the glucose intolerance in this species.     

Hypothalamic Monoaminergic Activity in 11-Week-Old Cold-Exposed Female Lean (Fa/Fa) and Obese (fa/fa) Zucker Rats

We previously reported that serotonergic activity was reduced in the ventromedial hypothalamic nucleus (VMN) of obese vs. lean male Zucker rats. To verify that this reduction was associated with genotype rather than gender, we measured monoamines and their major metabolites in hypothalamic nuclei of ll-week-old female lean (Fa/Fb) and obese (fa/fb) Zucker rats. In addition, since the thermic response to cold is reported to differ between lean and obese rats, some rats were also exposed to 9° or 22° C for 2h to determine if cold exposure altered hypothalamic monoaminergic activity. As in males, levels of 5-hydroxyindoleacetic acid [5-HIAA; major metabolite of serotonin (5-HT)] and the ratio of 5-HIANS-HT were lower in the VMN of obese vs. lean females (P = 0.008, 0.001, respectively). S-HIANS-HT was also reduced in the paraventricular (PVN) and suprachiasmatic nuclei (SCN) of the obese compared to the lean females. Cold exposure significantly stimulated brown fat mitochondria1 GDP binding in lean but not obese rats. Similarly, levels of norepinephrine, dopamine (DA), 5-HIAA, and 5-HT in the PVN, and 5-HIAA in the SCN increased in cold-exposed lean but not obese rats. In contrast, VMN and preoptic 3,4-dihydroxyphenylacetic acid (DOPAC; major metabolite of DA) increased in the cold-exposed obese but not lean animals. We conclude that: (1) the blunted peripheral response to cold in obese vs. lean Zucker rats is accompanied by altered hypothalamic monoaminergic activity, the physiological role of which needs further evaluation; and 2) depressed VMN serotonergic activity is associated with the obese genotype (fa/fa) rather than gender and as such may contribute to the reduced sympathetic and enhanced parasympathetic outflow from the VMN .