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Background: Leptin resistance occurs in obese patients, but its independent contribution to adiposity and the accompanying metabolic diseases, i.e., diabetes, liver steatosis, and steatohepatitis, remains to be established. This study was conducted in an extreme model of leptin resistance to investigate mechanisms initiating diabetes, fat expansion, liver steatosis, and inflammatory disease, focusing on the involvement of glucose intolerance and organ-specific glucose uptake in brown and subcutaneous adipose tissues (BAT, SAT) and in the liver., Methods: We studied preobese and adult Zucker rats (fa/fa, fa/+ ) during fasting or glucose loading to assess glucose tolerance. Relevant pancreatic and intestinal hormonal levels were measured by Milliplex. Imaging of 18 F-fluorodeoxyglucose by positron emission tomography was used to quantify glucose uptake in SAT, BAT, and liver, and evaluate its relationship with adipocyte size and biopsy-proven nonalcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH)., Results: Preobese fa/fa pups showed impaired glucose tolerance, adipocyte enlargement, hepatic microsteatosis, and lobular inflammation, with elevated hepatic post-glucose load glucose uptake and production. Adult fa/fa rats had more severe glucose intolerance, fasting hyperglycemia, hormonal abnormalities, elevated glucose uptake in SAT and BAT, and more markedly in the liver, together with macrosteatosis, and highly prevalent hepatic inflammation. Organ glucose uptake was proportional to the degree of fat accumulation and tissue inflammation and was able to dissect healthy from NAFLD and NAFLD/NASH livers. Most severe NASH livers showed a decline in glucose uptake and liver enzymes., Conclusions: In fa/fa Zucker rats, leptin resistance leads to glucose intolerance, mainly due to hepatic glucose overproduction, preceding obesity, and explaining pancreatic and intestinal hormonal changes and fat accumulation in adipocytes and hepatocytes. Our data support the involvement of liver glucose uptake in the pathogenesis of liver inflammatory disease. Its potential as more generalized biomarker or diagnostic approach remains to be established outside of our leptin-receptor-deficient rat model., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Glucokinase (GK) catalyzes the initial step in glycolysis and is a key regulator of glucose homeostasis. Therefore, glucokinase activators (GKa) have potential benefit in treating type 2 diabetes. Administration of a Bristol-Myers Squibb GKa (BMS-820132) to healthy euglycemic Sprague-Dawley (SD) rats and beagle dogs in 1 mo toxicology studies resulted in marked and extended hypoglycemia with associated clinical signs of toxicity and degenerative histopathological changes in the stomach, sciatic nerve, myocardium, and skeletal muscles at exposures comparable to those expected at therapeutic clinical exposures. To investigate whether these adverse effects were secondary to exaggerated pharmacology (prolonged hypoglycemia), BMS-820132 was administered daily to male Zucker diabetic fatty (ZDF) rats for 1 mo. ZDF rats are markedly hyperglycemic and insulin resistant. BMS-820132 did not induce hypoglycemia, clinical signs of hypoglycemia, or any of the histopathologic adverse effects observed in the 1 mo toxicology studies at exposures that exceeded those observed in SD rats and dogs. This indicates that the toxicity observed in euglycemic animals was secondary to the exaggerated pharmacology of potent GK activation. This study indicates that ZDF rats, with conventional toxicity studies, are a useful disease model for testing antidiabetic agents and determining toxicities that are independent of prolonged hypoglycemia., (© 2015 by The Author(s).)

Unattended hepatic insulin resistance predisposes individuals to dyslipidemia, type 2 diabetes and many other metabolic complications. The mechanism of hepatic insulin resistance at the gene expression level remains unrevealed. To examine the effects of vitamin A (VA), total energy intake and feeding conditions on the insulin-regulated gene expression in primary hepatocytes of Zucker lean (ZL) and fatty (ZF) rats, we analyze the expression levels of hepatic model genes in response to the treatments of insulin and retinoic acid (RA). We report that the insulin- and RA-regulated glucokinase, sterol regulatory element-binding protein-1c and cytosolic form of phosphoenolpyruvate carboxykinase expressions are impaired in hepatocytes of ZF rats fed chow or a VA sufficient (VAS) diet ad libitum. The impairments are partially corrected when ZF rats are fed a VA deficient (VAD) diet ad libitum or pair-fed a VAS diet to the intake of their VAD counterparts in non-fasting conditions. Interestingly in the pair-fed ZL and ZF rats, transient overeating on the last day of pair-feeding regimen changes the expression levels of some VA catabolic genes, and impairs the insulin- and RA-regulated gene expression in hepatocytes. These results demonstrate that VA and feeding statuses modulate the hepatic insulin sensitivity at the gene expression level.

Objective: Obese and type 2 diabetic patients present metabolic disturbance-related alterations in nonspecific immunity, to which the decrease in their plasma arginine contributes. Although diabetes-specific formulas have been developed, they have never been tested in the context of an acute infectious situation as can be seen in intensive care unit patients. Our aim was to investigate the effects of a diabetes-specific diet enriched or not with arginine in a model of infectious stress in a diabetes and obesity situation. As a large intake of arginine may be deleterious, this amino acid was given in graded fashion., Design: Randomized, controlled experimental study., Setting: University research laboratory., Subjects: Zucker diabetic fatty rats., Interventions: Gastrostomized Zucker diabetic fatty rats were submitted to intraperitoneal lipopolysaccharide administration and fed for 7 days with either a diabetes-specific enteral nutrition without (G group, n=7) or with graded arginine supply (1-5 g/kg/day) (GA group, n=7) or a standard enteral nutrition (HP group, n=10)., Measurements and Main Results: Survival rate was better in G and GA groups than in the HP group. On day 7, plasma insulin to glucose ratio tended to be lower in the same G and GA groups. Macrophage tumor necrosis factor-α (G: 5.0±1.1 ng/2×10⁶ cells·hr⁻¹; GA: 3.7±0.8 ng/2×10⁶ cells·hr⁻¹; and HP: 1.7±0.6 ng/2×10⁶ cells·hr⁻¹; p<.05 G vs. HP) and nitric oxide (G: 4.5±1.1 ng/2×10⁶ cells·hr⁻¹; GA: 5.1±1.0 ng/2×10⁶ cells·hr⁻¹; and HP: 1.0±0.5 nmol/2×10⁶ cells·hr⁻¹; p<.05 G and GA vs. HP) productions were higher in the G and GA groups compared to the HP group. Macrophages from the G and GA groups exhibited increased arginine consumption., Conclusions: In diabetic obese and endotoxemic rats, a diabetes-specific formula leads to a lower mortality, a decreased insulin resistance, and an improvement in peritoneal macrophage function. Arginine supplementation has no additional effect. These data support the use of such disease-specific diets in critically ill diabetic and obese patients.

Representative animal models for diabetes-associated vascular complications are extremely relevant in assessing potential therapeutic drugs. While several rodent models for type 2 diabetes (T2D) are available, their relevance in recapitulating renal and cardiovascular features of diabetes in man is not entirely clear. Here we evaluate at the molecular level the similarity between Zucker diabetic fatty (ZDF) rats, as a model of T2D-associated vascular complications, and human disease by urinary proteome analysis. Urine analysis of ZDF rats at early and late stages of disease compared to age- matched LEAN rats identified 180 peptides as potentially associated with diabetes complications. Overlaps with human chronic kidney disease (CKD) and cardiovascular disease (CVD) biomarkers were observed, corresponding to proteins marking kidney damage (eg albumin, alpha-1 antitrypsin) or related to disease development (collagen). Concordance in regulation of these peptides in rats versus humans was more pronounced in the CVD compared to the CKD panels. In addition, disease-associated predicted protease activities in ZDF rats showed higher similarities to the predicted activities in human CVD. Based on urinary peptidomic analysis, the ZDF rat model displays similarity to human CVD but might not be the most appropriate model to display human CKD on a molecular level.

Background: The dopamine (DA) D(2) receptor (D2R) agonist bromocriptine (BC) decreases body fat in animal and human models and increases lean muscle mass, improves glucose intolerance and insulin resistance, and reduces triglycerides and free fatty acids. We have previously shown a negative correlation between D2R and body weight in obese individuals and in rodents, and that chronic food restriction increases D2R binding in genetically obese rats. The purpose of this study was to assess whether the antiobesity and metabolic effects of BC are related to changes in midbrain DA and D2R activity by measuring D2R and DA transporter (DAT) binding in a genetic (leptin-receptor-deficient) and environmental (diet-induced) rodent obesity model., Methods: Obese (fa/fa) (leptin-receptor-deficient), lean (FA/FA) Zucker rats and rats with diet-induced obesity (DIO) were treated with 10 mg/kg BC for 4 weeks. Body weight, food intake, locomotor activity and blood glucose levels were measured along with D2R- and DAT-binding levels using in vitro receptor autoradiography., Results: BC decreased food intake and body fat and increased locomotor activity in both the (fa/fa) and DIO rats. Furthermore, BC increased D2R binding in (fa/fa) but not in DIO rats. Finally, BC increased DAT binding in DIO rats but not in the (fa/fa) rats., Conclusion: These observations are all consistent with the existence of unique leptin-DA interactions and the hypothesis that there is hyposensitivity of the DA system in obesity., ((c) 2008 S. Karger AG, Basel.)

Aim: Intramyocellular triglyceride (IMTG) correlates with insulin resistance, but there is no clear causal relationship. Insulin resistance and associated hyperinsulinaemia may increase IMTG, via the insulin-regulated transcription factor, sterol regulatory element-binding protein 1 (SREBP-1). PPAR agonists may also affect IMTG via changes in insulin sensitivity, SREBP-1 or other factors., Methods: We examined skeletal muscle IMTG and SREBP-1 expression, and metabolic parameters in Zucker diabetic fatty rats (ZDF) after 25 weeks of PPAR-gamma or PPAR-alpha administration., Results: Compared with Zucker lean rats (ZL), untreated ZDF had significantly higher weights, serum glucose, insulin, free fatty acids, total cholesterol and triglycerides. IMTG and SREBP-1c messenger RNA (mRNA) were also higher in untreated ZDF; both were decreased by fenofibrate (FF). Rosiglitazone (Rosi), despite marked improvement in glycaemia, hyperinsulinaemia and hyperlipidaemia, failed to affect SREBP-1 expression, and increased body weight and IMTG. Rosi/FF combination caused less weight gain and no IMTG increase, despite metabolic effects similar to Rosi alone., Conclusions: IMTG and SREBP-1c mRNA are high in the ZDF. FF and Rosi both improved insulin sensitivity but had opposite effects on IMTG. Thus, there was a clear discordance between insulin sensitivity and IMTG with PPAR agonists, indicating that IMTG and insulin sensitivity do not share a simple relationship.

Ultra-performance liquid chromatography (UPLC) coupled to orthogonal acceleration time-of-flight mass spectrometry (oa-TOFMS) in positive electrospray ionization mode was used to obtain metabolite profiles for urine obtained from three strains of Zucker rat. These were the Zucker lean, the Zucker (fa/fa) obese and the Zucker lean/(fa) cross. Clear age- and strain-related differences were noted with the leptin-deficient (fa/fa) obese animal showing significant differences from both the other Zucker rat strains with respect to metabolite profiles., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

The aim of this article was to describe the effects of chronic fluoxetine on mu opioid receptor expression in obese Zucker rat extrahypothalamic regions. Male obese Zucker (fa/fa) rats were administered with fluoxetine (10 mg/kg; i.p.) daily for two weeks. Brain regional immunostaining for mu opioid receptor was carried out. An increase in the numbers of neural cells immunostained for mu opioid receptor in caudatus-putamen, dentate gyrus, lateral septum, amygdala, and frontal, parietal, and piriform cortices was observed. Increased mu opioid receptor expression in the central amygdaloid nuclei suggests a decreased opioidergic tone at this level that could be involved in fluoxetine anorectic action.

There is a considerable variability in the responses of Zucker fa/fa rats in metabolic studies, which could not be solely attributed to the leprfa mutation. In order to fathom the extent of this variability, we compared the response to oleoyl-estrone (OE), a powerful lipid-mobilising agent, of two strains of Zucker lean and obese rats: Harlan (H) and Charles River (CR). Rats were given an oral gavage of 10 micromol/day/kg of OE in sunflower oil, and were compared with oil-receiving controls. Body composition, energy and water balances, and plasma parameters were studied after 10 days of treatment. H rats showed a higher water turnover than CR rats; OE treatment reduced water intake, partly compensated by metabolic water, and decreased stool water. H rats accrued more cholesterol than CR animals, which showed higher cholesterolaemia. OE facilitated cholesterol disposal in lean (CR and H) and H obese rats. CR rats had higher body and liver lipids than H animals. No differences in energy balance were found. Insulin decrease following OE treatment was greater in lean CR than in H rats, but this trend was reversed in the obese rats, lacking effective responses to leptin. The red cell glucose compartment was smaller in H than in CR rats; the higher insulin levels in H rats may be partly responsible for that difference. Obese H maintained glycaemia (and liver glycogen) with higher insulin levels than CR animals. The extent to which the leprfa mutation affects the responses of Zucker fa/fa rats could not be singled out unless the metabolic environment of the batch used is known. This variability must be taken into account when developing a metabolic or hormonal study in which this model of obesity is used.

Hepatic glucose fluxes and intracellular movement of glucokinase (GK) in response to increased plasma glucose and insulin were examined in 10-wk-old, 6-h-fasted, conscious Zucker diabetic fatty (ZDF) rats and lean littermates. Under basal conditions, plasma glucose (mmol/l) and glucose turnover rate (GTR; micromol.kg(-1).min(-1)) were slightly higher in ZDF (8.4 +/- 0.3 and 53 +/- 7, respectively) than in lean rats (6.2 +/- 0.2 and 45 +/- 4, respectively), whereas plasma insulin (pmol/l) was higher in ZDF (1,800 +/- 350) than in lean rats (150 +/- 14). The ratio of hepatic uridine 5'-diphosphate-glucose 3H specific activity to plasma glucose 3H specific activity ([3H]UDP-G/[3H]G; %), total hepatic glucose output (micromol.kg(-1).min(-1)), and hepatic glucose cycling (micromol.kg(-1).min(-1)) were higher in ZDF (35 +/- 5, 87 +/- 16, and 33 +/- 10, respectively) compared with lean rats (18 +/- 3, 56 +/- 6, and 11 +/- 2, respectively). [3H]glucose incorporation into glycogen (micromol glucose/g liver) was similar in lean (1.0 +/- 0.7) and ZDF (1.6 +/- 0.8) rats. GK was predominantly located in the nucleus in both rats. With elevated plasma glucose and insulin, GTR (micromol.kg(-1).min(-1)), [3H]UDP-G/[3H]G (%), and [3H]glucose incorporation into glycogen (micromol glucose/g liver) were markedly higher in lean (191 +/- 22, 62 +/- 3, and 5.0 +/- 1.4, respectively) but similar in ZDF rats (100 +/- 6, 37 +/- 3, and 1.4 +/- 0.4, respectively) compared with basal conditions. GK translocation from the nucleus to the cytoplasm occurred in lean but not in ZDF rats. The unresponsiveness of hepatic glucose flux to the rise in plasma glucose and insulin seen in prediabetic ZDF rats was associated with impaired GK translocation.

Genetically obese Zucker rats exhibit symptoms similar to those of obese patients with insulin-resistance or Type II diabetes; therefore, they have been used as a genetic model to study obesity, as well as a pharmacological model for the discovery of new drugs for the treatment of Type II diabetes and hyperlipidemia. In the present study, we compared the pharmacokinetics of two novel peroxisome proliferator-activated receptor (PPAR) agonists, MRL-I [(2R)-7-[3-[2-chloro-4-(4-fluorophenoxy)phenoxy]propoxy]-2-ethyl-3,4-dihydro-2H-benzopyran-2-carboxylic acid] and MRL-II [(2R)-7-[3-[2-chloro-4-(2,2,2-trifluoroethoxy)phenoxy]propoxy]-3,4-dihydro-2-methyl-2H-benzopyran-2-carboxylic acid], in obese Zucker and lean Sprague-Dawley rats following a single intravenous administration. The plasma clearance of both MRL-I and MRL-II was significantly lower in obese Zucker rats (4- and 2-fold, respectively) compared with Sprague-Dawley rats, but without any significant change in the volume of distribution, which resulted in a dramatic increase in the half-life (7- and 3-fold, respectively). The reversible in vitro plasma protein binding of [(14)C]MRL-I and [(14)C]MRL-II was comparable in the two strains, approximately 96% bound. The expression levels of uridine diphosphate-glucuronosyltransferases 1A1, 1A6, 2B1, and CYP2C11 and 3A1 mRNA in liver were lower (30-50%) in Zucker compared with Sprague-Dawley rats, as were the liver glutathione S-transferases (70%), quinone reductase (30%), organic anion-transporting protein 2 (80%), and multidrug resistance-associated protein 2 (Mrp2) (50%) mRNA levels. However, Mrp3 mRNA levels were similar in both strains. Consistent with these observations, the intrinsic clearance (CL(int)), calculated from the V(max)/K(m) of glucuronidation of [(14)C]MRL-I and [(14)C]MRL-II in liver microsomes, was approximately 2-fold lower in obese Zucker rats; the K(m) values were comparable in the two strains for both compounds. In conclusion, differences in the pharmacokinetics of two novel PPAR agonists, both cleared, predominantly, by conjugation, were evident in genetically obese Zucker rats compared with Sprague-Dawley rats. These differences were consistent with changes in the mRNA levels of hepatic drug-metabolizing enzymes and transporters. This information should be considered when comparing pharmacokinetic and efficacious doses in the obese Zucker rats, used as a pharmacological model, with those in Sprague-Dawley rats, which are used widely for drug metabolism and toxicology studies.

Fatty fa/fa Zucker rats with a missense mutation in the leptin receptor (OB-R) are obese and infertile with prolonged estrous cycles. To determine whether their reproductive deficits could be corrected by OB-R installation, we employed viral vectors to introduce the OB-R gene into either the arcuate nucleus (ARC) or the paraventricular nucleus (PVN) of the hypothalamus, sites of OB-R expression in wild-type rats. Recombinant adeno-associated viral (rAAV) vectors encoding the human leptin-receptor gene (rAAV-OB-Rb) were microinjected intraparenchymally to produce doxycycline-regulatable OB-R gene expression. Expression of the OB-R gene in the ARC and PVN was verified using reverse transcription-polymerase chain reaction. Expression of OB-R in the ARC, but not in the PVN, resulted in normalization of estrous cycle length, increased ovarian follicular development, and decreased serum progesterone levels. Compared to saline-injected rats, hypothalamic expression of neuropeptide Y (NPY) and pro-opiomelanocortin were decreased in ARC rAAV-OB-Rb-injected rats. Parallel decreases were noted in NPY and beta-endorphin (beta-END) concentrations in the hypothalamus, whereas luteinizing hormone-releasing hormone (LHRH) levels increased. These studies showed that rAAV vectors can be successfully used to install functional OB-R in the hypothalamus for extended periods. The resultant stimulation of the hypothalamo-pituitary-gonadal (HPG) axis in ARC-injected rats was probably brought about by the observed decreases in NPY and beta-END, which inhibit hypothalamic LHRH. Because these changes were seen in ARC-injected, but not in PVN-injected, rats, the results suggest that the ARC may be the primary site where leptin acts to regulate the HPG axis.

Galanin-like peptide (GALP) is a 60-amino-acid peptide with structural similarities to galanin and a high affinity for galanin receptors. GALP is expressed by a discrete population of neurons in the arcuate nucleus (ARC) and median eminence of the hypothalamus of several species, including the rat. GALP neurons express leptin receptors and GALP mRNA levels are decreased slightly in fasted rats and stimulated significantly by acute leptin treatment in combination with fasting. In studies to further explore the leptin dependence of GALP expression, we examined GALP mRNA levels in the hypothalamus of obese Zucker and streptozotocin-induced diabetic (STZ-DM) rats. In leptin receptor-deficient obese Zucker rats, with 75% higher body weight than lean littermates, GALP mRNA levels in the ARC were decreased by 75%, while neuropeptide Y (NPY) mRNA levels were increased 7-fold (n = 5, p < 0.001), consistent with earlier reports. In hypoleptinemic diabetic rats with 4.5-fold higher blood glucose and 15% lower body weight than controls, GALP mRNA levels in the ARC were decreased by 90%, while NPY mRNA levels were increased 9-fold (n = 5, p < 0.001). GALP is also expressed by pituicytes in the neural lobe of the rat pituitary gland and GALP expression is increased by osmotic stimulation such as dehydration and salt loading. Thus, in STZ-DM rats that are in a hyperosmotic state with elevated plasma vasopressin levels, GALP mRNA levels were increased by approximately 20-fold in the neural lobe relative to control (n = 4, p < 0.001). The current findings are consistent with a strong tonic influence of leptin receptor signalling on hypothalamic GALP expression under normal conditions, and possible abnormalities in GALP neuronal signalling and their putative targets, thyrotropin-releasing hormone and gonadotropin hormone-releasing hormone neurons, under pathophysiological conditions such as diabetes and obesity. Our data in STZ-DM rats also clearly demonstrate that GALP gene expression is differentially regulated in neurons and pituicytes.

The effect of insulin-resistance syndrome on vascular function has been examined in isolated basilar arteries using the obese Zucker rat (OZR) and age-matched lean littermate controls (lean Zucker rat; LZR) at 36 weeks of age. The OZR showed significantly reduced oral glucose tolerance and increased body weight, blood pressure, proteinuria, plasma levels of triglycerides, cholesterol, and insulin compared with the LZR. The contractile response to serotonin was significantly increased in the OZR. Furthermore, contractions to serotonin in LZR but not OZR were enhanced in the presence of the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (NAME). Relaxations to acetylcholine (ACh), histamine, and A23187 were significantly reduced in precontracted arteries from the OZR. In the presence of NAME, histamine responses were significantly reduced whereas ACh and A23187 responses were almost abolished. Relaxations to free-radical nitric oxide (NO) and papaverine were not different in arteries from the OZR, even though responses to sodium nitroprusside were reduced in the OZR. Western blot and immunofluorescent quantitative analyses of eNOS content in cerebral microvessel fractions and basilar artery preparations, respectively, were not significantly different between OZR and LZR. The results suggest impairment in endothelial function resulting in reduced NO function in the basilar artery from the OZR.

A newly synthesized benzoic acid derivative, (+)-(S)-p-[1-(p-tert-butylphenyl)-2-oxo-4-pyrrolidinyl]methoxybenzoic acid (S-2E), has the capacity to inhibit the biosynthesis of both sterol and fatty acids. Here, we report the mechanism by which S-2E lowers blood cholesterol and triglyceride levels. In the liver, S-2E was converted into its active metabolite, S-2E-CoA. S-2E-CoA noncompetitively inhibited the enzymatic activities of both 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase and acetyl-CoA carboxylase at K(i)=18.11 microM and K(i)=69.2 microM, respectively. Interestingly, pharmacokinetic experiments in rats showed that the concentration of S-2E-CoA in the liver was sufficient to inhibit the activities of HMG-CoA reductase and acetyl-CoA carboxylase, for example, when orally given to rats at 10 mg/kg. Indeed, S-2E (3-30 mg/kg) given orally suppressed the secretion rate of very-low-density lipoprotein (VLDL)-cholesterol and triglyceride in Triton WR-1339-injected rats. Furthermore, S-2E lowered the blood total cholesterol and triglyceride levels simultaneously in Zucker fatty rats. Collectively, S-2E may be useful in the treatment of familial hypercholesterolemia and mixed hyperlipidemia.

The hypothalamic circuits controlling food intake and body weight receive and integrate information from circulating satiety signals such as leptin and insulin and also from ghrelin, the only known circulating hormone that stimulates appetite following systemic injection. Activation of arcuate neurons by ghrelin and ghrelin mimetics (the growth hormone secretagogues) is augmented in 48-h-fasted rats compared with fed rats, as reflected by a greater number of cells expressing Fos protein in response to administration of the same maximally effective dose. Here we sought to determine whether this increased responsiveness in fasting might reflect or be influenced by low levels of circulating satiety factors such as leptin or insulin. Chronic central infusion of insulin or leptin during a 48-h fast suppressed the threefold increase in the Fos response to intravenous injection of a maximally effective dose of growth hormone-releasing peptide (GHRP)-6, a synthetic growth hormone secretagogue. This appears to be a direct central action of insulin and leptin because the marked decrease in plasma levels of insulin, leptin, and glucose during fasting were unaffected by central administration of either hormone. Furthermore, the GHRP-6-induced Fos response was twofold greater in obese leptin- and insulin-resistant Zucker rats compared with lean controls. These data provide evidence that the ghrelin-sensitive circuits in the hypothalamus are dynamically regulated by central insulin and leptin action.

We investigated angiotensinogen (AGT) expression in adipose tissue and liver of Zucker rats during the onset of obesity. The developmental pattern of AGT expression (protein and mRNA) in liver was similar in both genotypes. In inguinal adipose tissue, AGT cell content was similar in suckling and weaned pups in lean rats, whereas it continuously increased with age in obese rats. AGT amount in adipocytes was unaffected by the genotype until weaning. Thereafter, adipocytes from obese rats displayed a significant increase in AGT content that was strengthened with age. Compared with the cell content, the amount of secreted AGT over 24 h was higher, and a genotype effect was observed as early as 14 days of age. Using fat cell populations differing by size, we showed that this AGT oversecretion was not solely related to adipocyte hypertrophy. Our results demonstrate that the fa genotype exerts a control on the production of AGT in a tissue-specific manner, suggesting a local role of AGT in the overdevelopment of adipose tissue.

Type 2 diabetes is caused by a combination of beta-cell dysfunction and insulin resistance. Over time, hyperglycemia worsens, a phenomenon that has been attributed to deleterious effects of chronic hyperglycemia (glucotoxicity) or chronic hyperlipidemia (lipotoxicity) on beta-cell function and is often accompanied by increased islet triacylglycerol (TAG) content and decreased insulin gene expression. To examine these two potentially pathogenic forces, we studied Zucker rats (leptin receptor wild type, +/+; heterozygous, +/-; and mutant, -/-). First, +/+ and +/- Zucker rats were compared metabolically. At 6 weeks of age, the +/- rats had a lower level of islet insulin mRNA compared with +/+. At 12 weeks of age, differences were found in body weight and islet TAG content; however, levels of insulin mRNA were equivalent. Second, we examined whether worsening of the diabetic state in the homozygous mutant (-/-) Zucker diabetic fatty (ZDF) rat is related more to chronic hyperglycemia or to hyperlipidemia. The ZDF rats were treated for 6 weeks with either bezafibrate, a lipid-lowering drug that does not affect plasma glucose levels, or phlorizin, a drug that reduces plasma glucose without lowering lipid levels. Bezafibrate treatment lessened the rise in plasma TAG observed in nontreated rats (239 +/- 16 vs. 388 +/- 36 mg/dl, treated versus nontreated; P < 0.0001) but did not prevent the rise in fasting plasma glucose. Despite lowering plasma TAG, bezafibrate was not effective in preventing an increased islet TAG content and did not prevent the associated decrease in insulin mRNA levels. Phlorizin treatment prevented hyperglycemia (61 +/- 2 vs. 145 +/- 7 mg/dl, treated versus nontreated; P < 0.0001) and lowered islet TAG content (32.7 +/- 0.7 vs. 47.8 +/- 2.7 ng/islet, treated versus nontreated; P < 0.0001) and preserved insulin mRNA levels without preventing hypertriglyceridemia. Plasma free fatty acid level did not correlate with changes in islet TAG or insulin mRNA levels. We conclude that antecedent elevated plasma glucose levels, not plasma lipid levels, are associated with elevated islet TAG content and decreased insulin mRNA levels in ZDF animals.

High-fat diet alters apo E-dependent processing of beta-amyloid precursor protein. Here we have evaluated the effects of dietary fat on brain apo E mRNA in Zucker lean and obese rats. After approximately 2 months on a high-fat diet, there was significant up-regulation of brain apo E mRNA in the Zucker lean rat in parallel with weight gain. Densitometric quantification revealed a 17% increase in apo E mRNA in the brains of lean rats fed high-fat diet compared with those of lean rats fed rat chow. No significant difference in brain apo E mRNA of Zucker obese rats fed different diets was found. These results suggest that dietary fat alters brain apo E levels, which may be regulated, in part, through the leptin receptor.

Insulin resistance and type 2 diabetes are associated with elevated circulating levels of insulin, nonesterified fatty acids (NEFAs), and lipoprotein remnants. Extracellular matrix proteoglycan (PG) alterations are also common in macro- and microvascular complications of type 2 diabetes. In liver, extracellular heparan sulfate (HS) PGs contribute to the uptake of triglyceride-rich lipoprotein remnants. We found that HepG2 cells cultured with 10 or 50 nmol/l insulin or 300 micromol/l albumin-bound linoleic acid changed their PG secretion. The glycosaminoglycans (GAGs) of the secreted PGs from insulin-treated HepG2 cells were enriched in chondroitin sulfate (CS) PGs. In contrast, cells exposed to linoleic acid secreted PGs with decreased content of CS. Insulin caused a moderate increase in mRNA for versican (secreted CS PG), whereas linoleic acid markedly decreased mRNA for versican in HepG2 cells, as did the peroxisomal proliferator-activated receptor-alpha agonist bezafibrate. The effects of insulin or linoleic acid on syndecan 1, a cell surface HS PG, were similar to those on versican, but less pronounced. The livers of obese Zucker fa/fa rats, which are insulin-resistant and have high levels of insulin, NEFAs, and triglyceride-rich remnants, showed increased expression of CS PGs when compared with lean littermates. These changes in PG composition decreased the affinity of remnant beta-VLDL particles to PGs isolated from insulin-treated HepG2 cells and obese rat livers. The results indicated that insulin and NEFAs modulate the expression of PGs in hepatic cells. We speculate that in vivo this exchange of CS for HS may reduce the clearance of remnant beta-VLDLs and contribute to the dyslipidemia of insulin resistance.

Recent studies have demonstrated a reduction in body fat in growing animals fed conjugated linoleic acid (CLA). Two experiments were conducted to extend these observations to obese rats so that the mechanism of the actions of CLA might be more easily elucidated. In experiment 1, male lean and obese Zucker rats were fed diets containing either 0 or 0.5% CLA for 5 wk. There was no effect of diet on growth rate or food intake. Dietary CLA reduced retroperitoneal and inguinal fat pad weights in the lean rats but increased fat pad weights in the obese genotype (diet x genotype interaction; P < 0.05). Determination of fat pad cellularity indicated that these changes in fat pad weight were due to a reduction or increase in average fat cell size for the lean and obese Zucker rats, respectively. In experiment 2, we sought to reproduce these effects on fat pad size, as well as to determine the effect of dietary CLA on the catabolic response to bacterial endotoxin injection in obese Zucker rats. Growing female lean and obese Zucker rats were fed diets containing 0 or 0.5% CLA for 8 wk. On d 28, each rat was injected intraperitoneally with lipopolysaccharide from Escherichia coli serotype 055:B5 (1 mg/kg body weight) and body weight was determined over the next 96 h. There was a diet x genotype interaction (P < 0.05) for the body weight response to lipopolysaccharide 24 h postinjection. Lean rats fed CLA lost less weight than did lean controls, but obese rats fed CLA lost more weight than did obese controls. As in the first experiment, there was a diet x genotype (P < 0.05) for the effect of treatment on retroperitoneal fat pad weights determined at the end of the experiment. Lean rats fed CLA had smaller RP fat pads than did lean controls, but obese rats fed CLA once again had heavier RP fat pads than did obese controls. These results indicate that CLA reduces body fat and catabolic response to endotoxin injection in lean Zucker rats but not in the obese genotype. The observed interaction between diet and genotype warrants additional investigation into the specific mechanism(s) of the biological activities of CLA.

Agonists for the retinoid X receptor (RXR), the rexinoids, and the peroxisome proliferator-activated receptor gamma (PPARgamma), the thiazolidinediones, are effective in the treatment of insulin resistance in rodent models by enhancing insulin action and improving glycemic control. In the present study, we compared the effects of rexinoids and a thiazolidinedione on body weight and mitochondrial uncoupling protein (UCP) isoform mRNA expression in the obese Zucker fa/fa rat. Long-term (2 weeks) oral treatment with the rexinoids LG100268 and LG100324 reduced food intake and body weight gain, whereas rosiglitazone (BRL49653) tended to increase both food intake and weight gain. LG100268 and LG100324 increased brown adipose tissue (BAT) UCP-1 mRNA content by 2.7-fold (P < .002) and 3.1-fold (P < .001), respectively, while BRL49653 had no effect on BAT UCP-1 mRNA content. Neither the rexinoids nor the thiazolidinedione had any effect on the level of mRNA encoding UCP-2 and the recently described PPARgamma coactivator-1 (PGC-1). LG100324 increased UCP-3 mRNA content by 3.6-fold (P < .0005) in muscle and 4.3-fold (P < .0002) in white adipose tissue (WAT). LG100268 increased UCP-3 mRNA content in WAT by 2-fold (P < .005) but was without any effect on muscle UCP-3. BRL49653 increased UCP-3 mRNA content by 2.1-fold (P < .005) in muscle and 2.7-fold (P < .003) in WAT. Thus, the rexinoids, but not the thiazolidinedione, have an antiobesity action by reducing food intake, and the increase in UCP-1 mRNA content in BAT may reflect a stimulation of BAT UCP-1 activity.

This study investigates the mechanisms responsible for glucagon-like peptide-1 (GLP-1)-induced insulin secretion in Zucker diabetic fatty (ZDF) rats and their lean control (ZLC) littermates. Glucose, and 100 nmol/L GLP-1 (7-37 hydroxide) in the presence of stimulatory glucose concentrations, induced insulin secretion in islets from ZLC animals. In contrast, ZDF islets hypersecreted insulin at low glucose (5 mmol/L) and were poorly responsive to 15 mmol/L glucose stimulation, but increased insulin secretion following exposure to GLP-1. The insulin secretory response to 100 nmol/L GLP-1 was reduced by 88% in ZLC islets exposed to exendin 9-39. The intracellular Ca2+ concentration ([Ca2+]i) increased in fura-2-loaded ZLC islets following stimulation with 12 mmol/L glucose alone or GLP-1 in the presence of 12 mmol/L glucose. The increases in [Ca2+]i and insulin secretion in ZLC islets induced by GLP-1 were attenuated by 1 micromol/L nitrendipine. In contrast, neither glucose nor GLP-1 substantially increased [Ca2+]i in ZDF islets. Furthermore, insulin secretory responses to GLP-1 were not significantly inhibited in ZDF islets by nitrendipine. However, the insulin secretory response to GLP-1 in both ZLC and ZDF islets was ablated by cholera toxin. Our findings indicate that in ZLC islets, GLP-1 induces insulin secretion by a mechanism that depends on Ca2+ influx through voltage-dependent Ca2+ channels, whereas in ZDF islets, the action of GLP-1 is mediated by Ca2+-independent signaling pathways.

A lipolytic domain (AOD9401) of human growth hormone (hGH) which resides in the carboxyl terminus of the molecule and contains the amino acid residues 177-191, has been synthesized using solid-phase peptide synthesis techniques. AOD9401 stimulated hormone-sensitive lipase and inhibited acetyl coenzyme A carboxylase (acetyl CoA carboxylase) in isolated rat adipose tissues, in a similar manner to the actions of the intact hGH molecule. The synthetic lipolytic domain mimicked the effect of the intact growth hormone on diacylglycerol release in adipocytes. Chronic treatment of obese Zucker rats with AOD9401 for 20 days reduced the body weight gain of the animals, and the average cell size of the adipocytes of the treated animals decreased from 110 to 80 microm in diameter. Unlike hGH, synthetic AOD9401 did not induce insulin resistance or glucose intolerance in the laboratory animals after chronic treatment. The results suggest that AOD9401 has the potential to be developed into a therapeutic agent for the control of obesity.

The obese Zucker rat is resistant to insulin for glucose disposal, but it is unknown whether this insulin resistance is accompanied by alterations of insulin-mediated muscle protein synthesis. We examined rates of muscle protein synthesis either with or without insulin in lean and obese Zucker rats with the use of a bilateral hindlimb preparation. Additional experiments examined insulin's effect on protein synthesis with or without rapamycin, an inhibitor of protein synthesis. Protein synthesis in red and white gastrocnemius was stimulated by insulin compared with control (no insulin) in obese (n = 10, P<0.05) but not in lean (n = 10, P>0.05) Zucker rats. In white gastrocnemius, rapamycin significantly reduced rates of protein synthesis compared with control in lean (n = 6) and obese (n = 6) rats; however, in red gastrocnemius, the attenuating effect of rapamycin occurred only in obese rats. The addition of insulin to rapamycin resulted in rates of synthesis that were similar to those for rapamycin alone for lean rats and to those for insulin alone (augmented) for obese rats in both tissues. Our results demonstrate that insulin enhances protein synthesis in muscle that is otherwise characterized as insulin resistant. Furthermore, rapamycin inhibits protein synthesis in muscle of obese Zucker rats; however, stimulation of protein synthesis by insulin is not via a rapamycin-sensitive pathway.

Co-localization of chemical messengers in the same neuron is linked to neurochemical plasticity and has been studied extensively [B. Meister, M.J. Villar, S. Ceccatelli, T. Hökfelt, Localization of chemical messengers in magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei: an immunohistochemical study using experimental manipulation, Neuroscience 37 (1990) 603-633; B. Meister, R. Cortés, M.J. Villar, M. Schalling, T. Hökfelt, Peptides and transmitter enzymes in hypothalamic magnocellular neurons after administration of hyperosmotic stimuli: comparison between messenger RNA and peptide/protein levels, Cell Tissue Res. 260 (1990) 279-297]. Obese Zucker rats display an example of such a phenomenon expressing an enzyme of catecholamine synthesis-tyrosine hydroxylase (TH)-in magnocellular neurons (MCN) of supraoptic and paraventricular nuclei of hypothalamus [S. Fetissov, F. Marsais, S. Nicolaïdis, A. Calas, Expression of tyrosine hydroxylase in magnocellular hypothalamic neurons of obese (fa/fa) and lean heterozygous (Fa/fa) Zucker rats, Mol. Brain Res. 50 (1997) 314-318]. To understand the biological role of TH in MCN of obese Zucker rat, we studied TH expression in relation to the vasopressinergic and oxytocinergic neurons. We present a protocol of double labelling including immunohistochemical for TH and in situ hybridization for OT and VP mRNA. The described protocol can be applied for detection of co-localized expressions of a broad range of chemical brain messengers and proteins.

Diabetic rats have a deficiency in their heart ATP concentrations, and although the mechanism remains to be elucidated, this deficiency may involve increased uncoupling of oxidative phosphorylation. To investigate whether heart uncoupling proteins (UCPs) are subject to transcriptional regulation in diabetes, we examined changes in UCP mRNA expression in the heart of streptozotocin-induced diabetic (STZ-DM) rats. Heart UCP3 mRNA expression significantly increased by 9.4-fold in STZ-DM rats, while levels of UCP2 mRNA expression were not significantly altered. Insulin supplementation in STZ-DM rats returned UCP3 mRNA concentrations to control levels. The expression of UCP3 mRNA was similarly elevated in the heart of fasted rats, which also have hypoinsulinemia and hyper-free fatty acidemia but, unlike the STZ-DM rats, are hypoglycemic. Since hyperinsulinemia alone was previously reported to not affect UCP3 gene expression in the muscle, these results indicate that hyper-free fatty acidemia is a potent enhancer of UCP3 gene expression in the diabetic rat heart. Interestingly, we found no changes in UCP3 mRNA levels in Zucker fatty (fa/fa) rats with excessive chronic hyper-free fatty acidemia, which suggests that upregulation of heart UCP3 mRNA may depend on an acute change in free fatty acid concentrations rather than on their sustained elevation. High-energy ATP deficiencies in the diabetic rat heart may primarily result from proton leakage due to the upregulation of UCP3 expression.

We have examined the effects of underfeeding and obesity on the density of hypothalamic melanocortin MC3 and MC4 receptors (MC3-R and MC4-R, respectively), which may mediate the hypophagic effects of alpha-melanocyte-stimulating hormone (MSH) in the rat. MC3-R and MC4-R were measured by quantitative autoradiography in brain sections using 125I-labeled Nle4-D-Phe7-alpha-MSH (125I-NDP-MSH) and discriminated by masking MC3-R with excess unlabelled gamma2-MSH. High densities of MC4-R occurred in the ventromedial (VMH) and arcuate (ARC) nuclei, median eminence (ME), and medial habenular nucleus (MHb), with lower densities in the dorsomedial hypothalamus (DMH) and forebrain regions. MC3-R were confined to the VMH, ARC, and MHb. After 10-days of food restriction (14% weight loss), density of MC4-R was significantly increased by 20-65% in the VMH, ARC, ME, and DMH, with no changes elsewhere. Similarly, obese (fa/fa) Zucker rats showed 43-98% increases in MC4-R in the same regions. By contrast, rats with diet-induced obesity (18% heavier than controls) showed significantly decreased binding to MC4-R, especially in the VMH, ARC, and ME. MC3-R showed no significant alterations in any model. We suggest that increased density of MC4-R with food restriction and in obese Zucker rats reflects receptor upregulation secondary to decreased release of alpha-MSH, consistent with increased hunger in these models. Conversely, downregulation of MC4-R in diet-induced obesity may indicate increased alpha-MSH secretion in an attempt to limit overeating. This alpha-MSH/MC4-R system may be inhibited by leptin and/or insulin. MC3-R are not apparently involved in regulating feeding.

Insulin is known to stimulate Na+/K+ ATPase and to relax vascular smooth muscle. We hypothesized that vascular tone in the obese Zucker (fa/fa) rat, a hyperinsulinemic model in which hypertension can develop, may be influenced by insulin's ability to stimulate Na+/K+ ATPase at the vascular level. We studied isometric preparations of tail and femoral arteries from 10-wk-old, male obese Zucker rats, which were hyperinsulinemic but still normotensive vs. lean controls. Sensitivity to potassium-induced relaxations, an index of vascular Na+/K+ ATPase activity was significantly greater in the obese Zucker rat than control. Sensitivity to transmural-nerve-stimulation-induced contractions was decreased in the femoral and tail arteries from obese rats as compared with lean controls. Insulin (50 to 200 mU/ml) mimicked potassium-induced relaxations in the femoral artery, an effect that was significantly greater in the obese group. These data suggest that in the young hyperinsulinemic Zucker rat, insulin has a stimulatory effect on the vascular Na+/K+ pump, which may be associated with a decreased presynaptic adrenergic influence on vascular tone. Development of resistance to these vascular relaxant effects of insulin with advancing age might contribute to the onset of hypertension in this model.

The obese Zucker fa/fa rat is characterized by hyperinsulinemia, obesity, and altered monoamine metabolism in the central nervous system (CNS). It has been proposed that the changes in monoamine metabolism may contribute to the metabolic pathophysiology of these animals. Because it has been reported that insulin may regulate the catecholamine reuptake transporters, which terminate monoaminergic synaptic signaling, in the present study we tested whether messenger ribonucleic acid (mRNA) levels for the noradrenergic (NE) or dopaminergic (DA) transporters were altered in obese fa/fa vs. lean Fa/Fa Zucker rats. We found significantly elevated DA transporter levels in both the ventral tegmental area/substantia nigra pars compacta (VTA/SNc) and zona incerta (ZI) of obese Zucker fa/fa rats (164 +/- 24% of control levels, p = .024; and 316 +/- 61% of control levels, p = .019, respectively). Measurement of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme for NE and DA synthesis revealed no effect of the fa gene in either NE or DA neurons. These findings suggest that increased DA clearance, and perhaps decreased DA signaling, may occur in the obese Zucker fa/fa rat.

The effects of inclusion of different levels of raw kidney bean (Phaseolus vulgaris) of high lectin content (27 g/kg meal) in a high-quality (lactalbumin) control diet were tested in nutritional trials on the growth and metabolism of obese Zucker (fafa) rats and their lean littermates in comparison with pair-fed controls. All diets contained 100 g total protein/kg and either 50 g lipids/kg (low fat) or 150 g lipids/kg (moderate fat). The growth of both obese and lean rats on bean diets was retarded by the daily bean intake in a dose-dependent manner. However, most of this was because bean-fed rats contained less body fat than the controls after 10 d. Thus, after feeding low-fat diets containing up to 130 g kidney bean/kg (lectin intake < or = 0.2 g/kg body weight (BW) per d) in both 10 d and 70 d trials, the bodies of obese rats contained less fat but not protein than their pair-fed controls. Moreover, by increasing the lipid content of the diet to 150 g/kg, the level of bean inclusion could be increased to 280 g/kg (lectin intake > or = 0.4 g/kg BW per d) without loss of body protein and skeletal muscle. Although these rats contained more body fat than those which were fed on low-fat diets, their weight reduction could be accounted for exclusively by reduced lipid content. In contrast, significant body protein loss occurred when the same diet of high lectin content was fed to lean littermates. Plasma insulin levels were significantly depressed in the obese Zucker rats on bean diets but the pancreas was not significantly enlarged nor its insulin content changed in 10 d trials. However, significant pancreatic growth occurred on long-term (70 d) bean feeding compared with pair-fed controls. The results suggest that, in addition to animal nutrition, it may also be possible to use the bean lectin as a dietary adjunct or therapeutic agent to stimulate gut function and ameliorate obesity if a safe and effective dose-range can be established for human subjects.

We measured the proton leak across the inner membrane of liver mitochondria isolated from six different vertebrate species and from obese and control Zucker rats. Proton leak at 37 degrees C was similar in rat and pigeon, and in obese and control Zucker rats. Compared to rat, it was lower in cane toad, shingleback lizard, and the Madeiran lizard Lacerta dugessi. Proton leak at 20 degrees C was similar in xenopus toad and higher in rainbow trout, compared to rat. In general, proton permeability and substrate oxidation activity were greater in liver mitochondria from endotherms than those from ectotherms. Analysis of this and previous data showed that proton leak per milligram of mitochondrial protein correlated with standard metabolic rate, and proton leak per milligram of inner membrane phospholipid correlated with 11 phospholipid fatty acid compositional parameters, including unsaturation index.

Clusterin is a multifunctional glycoprotein associated with development and tissue injury. Because renal function decreases with advancing age in the obese Zucker rat, clusterin mRNA expression was examined in the kidney of young adult Zucker rats and compared with age-related changes in renal clusterin mRNA expression in Fischer 344 (F344) rats. Renal clusterin mRNA levels in the obese Zucker rat were 2.5-fold higher by 3 mo of age and fourfold higher at 5 mo of age compared with the lean strain. In comparison, renal clusterin mRNA in 12-mo-old F344 rats was twofold higher than in 3-mo-old animals and was tenfold higher at 24 mo of age. Clusterin mRNA was positively correlated with urinary protein excretion and negatively correlated with creatinine clearance in Zucker rats. Clusterin was increased in select nephrons of the obese Zucker rat kidney and in 24-mo-old F344 rat kidney as assessed by in situ hybridization. Increased expression of clusterin mRNA occurred mostly in the tubular epithelium of dilated, convoluted proximal tubules. These data indicate that renal clusterin mRNA levels increase as a function of age and that age-related increases in renal clusterin and the associated tubular abnormalities are accelerated in obese Zucker rats.

Genetically obese Zucker rats exhibit mild hyperglycaemia and hyperinsulinaemia suggesting the existence of peripheral insulin resistance. We have examined the effects of YM268, an analogue of thiazolidinedione, on the content and translocation of a glucose transporter (GLUT4) in epididymal adipose tissue in 11-week-old obese and lean Zucker rats. The administration of YM268 at a dose of 10 mg/kg for 2 weeks ameliorated hyperglycaemia, hyperinsulinaemia, and impaired glucose tolerance after glucose load in obese rats. The GLUT4 content per fat pad in obese rats was reduced to 36% of that in lean littermates. Obese rats treated with YM268 increased GLUT4 concentrations in their fat pads from a basal value of 36% up to 191% of the level in lean rats. Furthermore, in adipocytes prepared from obese rats, an increase in the ratio of GLUT4 in plasma membrane to the total amount of GLUT4 (PM-GLUT4 ratio) induced by the submaximal concentration of insulin (0.3 nmol/l) was significantly attenuated compared with that in lean rats. But the maximum effect of insulin (3 nmol/l) was not attenuated. Meanwhile, YM268 had no significant effect on the attenuated PM-GLUT4 ratio in response to insulin in obese rats. These data suggested that one of the mechanisms by which YM268 improved insulin resistance in obese Zucker rats was to normalize the decreased GLUT4 content in the adipose tissue.

Because obesity, insulin resistance, and hyperlipidemia are often associated, and recent evidence suggests that the cytokine tumor necrosis factor-alpha (TNF) may influence the activity of insulin in various target tissues, the present study was designed to see whether TNF was also associated with the changes in lipid metabolism that lead to hyperlipidemia in the obese model of the Zucker rat. A polyclonal goat anti-rat TNF antibody was subcutaneously administered to Zucker rats for 4 days to block TNF actions. The results indicate that none of the alterations in lipid metabolism seen in the obese animals were reversed by the anti-TNF treatment. This was the case for the lipogenic rate in liver and adipose tissue, the disposal of an exogenous [14C]triolein load, adipose tissue lipoprotein lipase activity, and the hypertriglyceridemia. Measurements of lipolysis in adipose tissue slices from the anti-TNF-treated animals also did not show any significant effect of the treatment. In conclusion, TNF does not seem to be involved in the abnormalities of lipid metabolism observed in the obese Zucker rat.

Thirty-five years ago, Lois and Theodore Zucker reported the discovery of a genetic mutation in the rat that resulted in juvenile-onset obesity, increased food intake, decreased energy expenditure, and insulin resistance. The mutation was called fatty (fa). The fatty gene is passed on to successive generations by an autosomal recessive mode of inheritance. In the intervening years, much work has been done to characterize the many abnormalities of this animal model of obesity. Nearly 10 years ago, we reviewed the evidence for a central nervous system mechanism in the etiology of obesity in the fatty Zucker rat. Since that time, the discovery of novel peptides and genes has revolutionized the study of the etiology of genetically linked obesities. In this review, we update the evidence for a central nervous system mechanism of obesity in Zucker rats by focusing on the possible role of neuropeptide Y (NPY) and leptin in the etiology of obesity. We also discuss the role of glucocorticoids and insulin in the regulation of NPY.

Genetically obese Zucker rats, like obese humans, have normal or elevated circulating insulin-like growth factor-I (IGF-I) levels in the presence of low GH secretion. Hyperinsulinemia, increased energy status, or other nutritional factors associated with obesity could be responsible for these findings directly by increasing hepatic IGF-I production at the transcriptional or posttranscriptional level. Alternatively, circulating IGF-I could be modulated indirectly by affecting its binding proteins. To further elucidate this point, we quantitated hepatic IGF-I, IGF-binding protein-3 (IGFBP-3), and GH receptor messenger RNAs (mRNAs) expression in obese Zucker rats under different serum GH and insulin conditions using lean rats as controls. Eleven-week-old male rats were studied basally (intact) or after hypophysectomy (hx) at 9 weeks. In each condition, animals were killed before or 6 h after one dose of recombinant human GH (1.5 micrograms/g body weight ip). At this time, in addition to the mRNA expression of the above-mentioned genes, body weight, glycemia, insulinemia, serum GH (rat and human), and serum IGF-I levels were determined. Obese Zucker rats were significantly heavier than controls in all the conditions studied and did not show differences in glycemia. Severely hyperinsulinemic intact obese rats (146.9 +/- 14 vs. 46.3 +/- 3 microU/ml, P < 0.001) showed compared with intact lean rats significantly lower serum GH (2.39 +/- 0.9 vs. 4.98 +/- 0.68 ng/ml, P < 0.01), decreased hepatic IGF-I mRNA and IGFBP-3 mRNA accumulation (IGF-Ia: 79 +/- 5.9% vs. 100 +/- 0.9%, P < 0.05; IGF-Ib: 67 +/- 5.5% vs. 100.1 +/- 1.9%, P < 0.001; IGFBP-3: 54.7 +/- 2.75% vs. 100.5 +/- 1.55%, P < 0.001), and similar circulating IGF-I levels (1439 +/- 182 vs. 1516 +/- 121 ng/ml). Under comparable serum GH levels in GH-treated intact, hx, and GH-treated hx animals, hyperinsulinemia and/or increased body weight present in obese rats were not associated with increased hepatic IGF-I and IGFBP-3 mRNA amount. No differences in GH receptor/GH-binding protein mRNAs were found in any experimental condition. These results suggest that in vivo the imbalance of the serum GH/IGF-I axis present in obesity is primarily due to events distal to the hepatic IGF-I and IGFBP-3 mRNAs expression, which is tightly correlated to GH levels.

Expression of Na(+)-K(+)-adenosinetriphosphatase (ATPase) in tissues from obese and lean Zucker rats was monitored. The phosphatase activity of the sodium pump was increased in liver and intestinal mucosa from obese animals but was unaltered in skeletal muscle, brown adipose tissue, kidney, and heart. Induction of Na(+)-K(+)-ATPase activity was correlated with increased alpha 1-subunit protein amounts in liver and intestinal mucosa, although alpha 1-subunit mRNA levels were increased only in liver tissue. Neither protein nor mRNA amounts for both subunits were significantly altered in the other tissues analyzed. The only exception was a decrease in the amount of beta 1-protein in kidney from obese rats. alpha 2-Subunit protein and alpha 2- and beta 2-mRNA levels were not altered in brown adipose tissue, heart, and soleus. In summary, this study shows that in obese Zucker rats the expression of the sodium pump is enhanced in tissues that are directly involved in nutrient uptake and processing. This adaptation may be related to the ongoing hyperphagia and to tissue hypertrophia but develops in a different manner in each tissue, suggesting differential regulation of alpha 1-subunit expression.

Zucker lean and obese rats were injected under pentobarbital anesthesia with 125I-labeled insulin; at timed intervals from 30 to 120 sec, blood samples were extracted and used for the estimation of insulin levels by RIA. A group of rats from each series was maintained under a constant infusion of noradrenaline. For each insulin determination, a duplicate blood sample containing the same amount of insulin as that used in the RIA, but without the radioactive label, was used as a blank for insulin measurement. The radioactivity in these tubes was then used for the measurement of insulin label per ml blood. From plasma label decay curves and insulin concentrations, the insulin pool size, half-life, and rate of degradation were calculated. Obese rats had higher insulin levels (2.43 nM) and showed less effect of noradrenaline than their lean counterparts, in which insulin distribution volume shrank with noradrenaline treatment. The half-life of plasma insulin was similar in all groups (range, 226-314 sec). Pool size and overall degradation rates were higher in obese (198 femtokatals) than in lean rats (28 femtokatals). It is postulated that obese rats synthesize and cleave much more insulin than lean controls despite their higher circulating levels of insulin.

Studies were designed to find the molecular basis for previous observations that lipolysis is less active and A1 adenosine receptor signaling is more active in adipocytes from obese than from lean Zucker rats. With quantitative immunoblot procedures for detection, Gi alpha 1 and Gs alpha 45 levels were found anomalously low in obese compared with lean membranes (50 and 30%, respectively), but other G alpha subunit levels were normal. However, the sensitivity of the receptor-Gi protein to GTP was about 5- to 10-fold higher in obese compared with lean membranes when assessed from 1) the ability of GTP to inhibit forskolin-stimulated adenylyl cyclase in the presence of an adenosine receptor agonist and 2) the ability of a nonhydrolyzable guanine nucleotide analogue to alter A1 adenosine receptor agonist binding. Alkaline phosphatase treatment of isolated adipocyte membranes from obese but not lean animals decreased guanine nucleotide sensitivity of agonist binding. Surprisingly, solubilized adipocyte A1 adenosine receptors from all animals exhibited the same high sensitivity to guanine nucleotides as that of intact obese membranes, and this high sensitivity could be decreased 20-fold by treatment with alkaline phosphatase. These data suggest that protein phosphorylation may regulate coupling of the A1 adenosine receptor in rat adipocyte membranes.

We previously reported lower ventromedial hypothalamic nucleus (VMN) serotonergic activity in 11-wk genetically obese vs. lean Zucker rats. To determine whether the activity was secondary to metabolic alterations associated with this established obesity (e.g., significant hyperphagia and hyperinsulinemia), we examined monoaminergic activity in various brain nuclei of 12-day lean (Fa/Fa and Fa/fa) and obese (fa/fa) rats early in the development of obesity. Obese pups had greater percent carcass fat than heterozygotes, both of which were fatter than homozygous lean rats. Obese, but not heterozygous lean, pups were hyperinsulinemic vs. Fa/Fa pups. VMN 5-hydroxy-3-indoleacetic acid levels, an index of serotonin release, were lower in obese and heterozygous than in homozygous lean pups and were not correlated with plasma insulin levels. Although monoamine differences also occurred in several other nuclei, for the most part they appeared to be unrelated to the obese genotype. We conclude that blunted VMN serotonergic activity is not secondary to the obese rat's hyperinsulinemia and may play a significant role in the development of obesity.

Insulin secretion from the isolated perfused pancreas is characterized by pulses occurring every 5-15 min. The present experiments were performed to explore the role of glucose in regulating these pulses. The pancreata from 12 Wistar (W), 12 Zucker diabetic fatty (ZDF), and 6 nondiabetic lean Zucker control (ZC) male rats were isolated and perfused at 37 degrees C with an oxygenated Krebs Ringer solution containing bovine serum albumin and glucose. In W and ZDF, insulin secretion was pulsatile during constant glucose, as assessed by pulse analysis (ULTRA). The pulse period in W was significantly shorter than in ZDF (7.1 +/- 0.6 vs. 14.7 +/- 1.0 min; P < 0.001), whereas the median relative pulse amplitude was not different. When glucose was administered as a series of 10-min sine waves, spectral analysis showed that the normalized spectral power at 10 min was greater in W and ZC compared with ZDF (34.2 +/- 5.9 and 32.9 +/- 2.9 vs. 3.2 +/- 0.9; P < 0.0001), demonstrating entrainment of the insulin pulses to the exogenous glucose oscillations in W and ZC but not in ZDF. Furthermore, in ZDF, the insulin secretory rates were not higher when 28 mM rather than 7 mM glucose were used. In additional studies, islets of Langerhans from one W, three ZDF, and three ZC rats were isolated and perifused using an oscillatory glucose concentration. Single and groups of islets were studied. Islets from diabetic rats demonstrated the same lack of entrainment by glucose seen in the perfused pancreas, suggesting that the defect is at the cellular level.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of this study was to investigate the energetic metabolism in obese Zucker rats, using phosphorus nuclear magnetic resonance spectroscopy at rest and during a 2-Hz muscle stimulation and subsequent recovery. Animals were anesthetized with ketamine (150 mg/kg ip). Fed obese rats and 2-day-fasted obese rats were compared with their normally fed and 2-day-fasted lean litter mates. No differences were found between the two groups for ATP, total creatine, phosphocreatine (PCr), and intracellular pH. Starvation in lean rats resulted in a significant fall in inorganic phosphate (Pi), increased resting ADP level, and decreased PCr and ADP recovery after stimulation. The obese rats exhibited a decreased PCr/Pi and increased ADP at rest and a decreased PCr resynthesis and ADP metabolization rate after stimulation. Muscle stimulation in fasted obese rats induced higher PCr depletion and more pronounced acidosis. These results suggest an in vivo mitochondrial metabolism dysfunction in fasted lean as well as in fed and fasted obese rats.

The genetically obese Zucker rat (fa/fa) is an animal model with severe insulin resistance of the skeletal muscle. We investigated whether a defect of insulin-dependent glucose transporter (GLUT 4) translocation might contribute to the pathogenesis of the insulin-resistant state. fa/fa rats, lean controls (Fa/Fa) as well as normal Wistar rats were injected intraperitoneally with insulin and were killed after 2 or 20 min, respectively. Subcellular fractions were prepared from hind-limb skeletal muscle and were characterized by determination of marker-enzyme activities and immunoblotting applying antibodies against alpha 1 Na+/K+ ATPase. The relative amounts of GLUT 1 and GLUT 4 were determined in the fractions by immunoblotting with the respective antibodies. Insulin induced an approximately two-fold increase of GLUT 4 in a plasma membrane and transverse tubule enriched fraction and a decrease in the low density enriched membrane fraction in all three groups of rats. There was a high individual variation in GLUT 4 translocation efficiency within the groups. However, no statistically significant difference was noted between the groups. No effect of insulin was detectable on the distribution of GLUT 1 or alpha 1 Na+K+ ATPase. The data suggest that skeletal muscle insulin resistance of obese Zucker rats is not associated with a lack of GLUT 4 translocation.

N-Myristoyltransferase is the enzyme that catalyses the transfer of myristate from myristoyl-CoA to the NH2-terminal glycine residue of a number of protein of diverse functions. Many of the known myristoylated proteins are important in signal transduction. We have compared the activity of rat liver N-myristoyltransferase from lean and obese (fa/fa) Zucker rats (a model for non-insulin dependent diabetes mellitus, NIDDM). N-myristoyltransferase activity isolated from the particulate fraction of obese (fa/fa) Zucker rat liver was approximately 4.7-fold lower than the corresponding activity observed in either the controls or the vanadate-treated obese rat livers. This pattern was only observed in the particulate fraction; the homogenate and soluble N-myristoyltransferase activities were not significantly different to the control activities. N-myristoyltransferase activity isolated from the brain of the four groups showed no significant variations. These results, and previous work [King, M. J., Pugazhenthi, S., Khandelwal, R. L. and Sharma, R. K. (1993) Biochim. Biophys. Acta. 1165, 259-262], would indicate that the rat liver particulate N-myristoyltransferase activity appears to be inversely proportional to the level of plasma insulin, implicating insulin in the control of N-myristoylation. The specific activity of the particulate liver N-myristoyltransferase was approximately 10-fold higher than that of the soluble liver N-myristoyltransferase, raising the possibility that N-myristoyltransferase exists in 2 populations, with the active form of N-myristoyltransferase residing in the membranous fraction. This situation could provide a system whereby N-myristoylation is regulated by the translocation of N-myristoyltransferase from the cytosol to its active site in the membranes.

Insulin attenuates agonist-induced vascular contractility of aortic rings and decreases vasopressin (AVP)-elicited increases in vascular smooth muscle cell (VSMC) intracellular calcium ([Ca2+]i). To determine if insulin's effects on AVP-induced [Ca2+]i responses are altered in an insulin-resistant and hypertensive state, we studied vascular smooth muscle calcium responses in VSMC derived from Zucker lean and obese rats. AVP concentration-response experiments revealed that VSMC derived from obese animals exhibited exaggerated [Ca2+]i responses over the range of 1 x 10(-10) to 1 x 10(-7) M AVP compared to lean controls (P < 0.05, by multiple analysis of variance). Insulin treatment (7 x 10(-7) M) decreased the [Ca2+]i response to 1 nM AVP by 66 +/- 8% and 71 +/- 9% in lean and obese VSMC, respectively. Similar decreases were observed with the 10 nM AVP stimulus (41 +/- 9% and 61 +/- 7%, for lean and obese, respectively). AVP receptor binding studies revealed that exaggerated [Ca2+]i responses in obese VSMC were not due to alterations in AVP-binding properties (no significant differences in ID50, Kd, or binding capacity in lean and obese VSMC preparations). In addition, insulin treatment (1 x 10(-7) M) resulted in no differences in AVP receptor-binding properties in either cell line. Therefore, exaggerated [Ca2+]i responses in obese VSMC are most likely due to a postreceptor abnormality. These abnormalities in VSMC [Ca2+]i metabolism preceed and may play a role in the development of hypertension in the Zucker obese rat. Although insulin resistance in Zucker obese rats has been demonstrated in several tissues, VSMC [Ca2+]i responses to AVP are, nonetheless, similarly attenuated by insulin in obese and lean VSMC preparations.

Apolipoprotein (apo) A-IV, a structural component of chylomicrons and high-density lipoproteins, may play a role in the catabolism of triglyceride-rich lipoproteins and in reverse cholesterol transport. To study the regulation of apoA-IV gene expression by genetic and nutritional factors, we determined the effect of a fish oil-rich and a sucrose-rich diet on apoA-IV gene transcription and nuclear and total cellular apoA-IV mRNA abundance in livers of genetically obese, hyperlipoproteinemic (fa/fa) Zucker rats and their lean (Fa/-) littermates. In obese rats fed chow, hepatic apoA-IV gene expression was more than twofold higher than in lean rats because of a post-transcriptional mechanism. apoA-I gene expression and apoC-III mRNA levels, studied as controls, were similar in both groups. The fish oil-rich diet reduced total cellular apoA-IV mRNA abundance transcriptionally to 34 +/- 4% of basal values in lean rats, but did not alter apoA-IV gene expression in obese rats. In contrast, this diet reduced apoA-I gene expression in both lean and obese animals. The sucrose-rich diet increased apoA-IV gene expression twofold in both lean and obese rats. Thus, genetic obesity alters the response of hepatic apoA-IV gene expression to a lipid-lowering diet rich in fish oil by a mechanism affecting transcriptional regulation.

Galanin (GAL), a 29 aminoacid peptide, is widely distributed in the central nervous system and especially in the hypothalamus. It strongly stimulates food intake when it is injected in the paraventricular nucleus (PVN) of normal rats. The obese Zucker rat with a well-established hyperphagia is characterized by a general dysregulation of some important neuropeptides involved in the regulation of feeding behavior e.g. neurotensin, NPY or CCK and the aim of this study was to measure GAL in different microdissected brain areas in lean (Fa/Fa) and obese (fa/fa) male Zucker rats. As feeding status may modulate the central peptide concentrations, it was measured in ad libitum fed rats and in 48-h fasted rats of both genotypes. GAL was measured by a specific radioimmunoassay in the arcuate nuclei (ARC) and parvocellular (PVNp) and magnocellular (PVNm) parts of the PVN as well as in the median eminence (ME), median preoptic area (MPOA), supraoptic (SON) and dorsomedian (DMN) nuclei. Two-way analysis of variance revealed a very significant effect of genotype in the PVNp (P < 0.001), SON (P < 0.001) and in the ME (P < 0.02). No significant variations at all were noted in the ARC, PVNm, MPOA and DMN. GAL concentrations were more than doubled in the PVNp and SON of ad lib obese rats when compared to the ad lib lean rats (P < 0.005). On the other hand, in the ME where GAL concentration was about 4-fold greater than in the other areas, there was a 20 to 30% decrease in GAL concentrations in the obese rat (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated hepatocytes from obese Zucker rats showed an increased capacity for lipid synthesis measured as the incorporation of 3H2O into lipid fractions. In addition lipid synthesis from 1 mM U-14C-alanine was elevated as compared to those isolated from their lean counterparts. On these lines, the amino acid was a better lipogenic substrate than U-14C-lactate at 5 mM concentration. Insulin increased lipid synthesis from the amino acid in the lean animals while showed no effects on this parameter in the obese. While the hepatocytes from the lean animals used to a similar extent U-14C-alanine carbon from 14CO2 production, lipid synthesis and 14C-glycogen incorporation, those from the obese animals showed a decreased oxidation and glycogen incorporation while an augmented lipid synthesis. The total amount of the amino acid taken up by the obese animals was also significantly elevated. Conversely, the metabolic fate of U-14C-leucine was very similar between the two groups. It can be concluded that in the obese Zucker rat alanine is a very important amino acid which contributes to sustain the high liver hyperlipogenesis previously described.