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To study the first stages of excess fat deposition in Zucker rats, we artificially fed littermates with identical amounts of milk from 4 to 15-16 days of age while continuously recording oxygen consumption (VO2) and deep body temperature. Under intermittent cold loads simulating the periodic thermoregulatory stimulation experienced in the nest, differences between the amounts of body fat deposited by artificially reared fatty (fa/fa) and lean (Fa/-) pups were as large as those seen in mother-reared pups. The decreased VO2 of the cold-reared fatty pups could account for 90-100% of their extra fat deposition. At thermoneutrality, 16-day-old littermates reared with low feeding rates showed small but significant genotype differences in body fat that were not energetically accounted for by differences in VO2 or lean body mass. Slightly but significantly lower fecal energy losses indicated that differences in resorption might account for the positive energy balance of thermoneutrally reared fa/fa pups. Reduced energy expenditure thus efficiently fuels excess fat deposition but is not essential for the onset of excess fat deposition in fa/fa pups. Other factors than reduced sympathetic activation of brown adipose tissue must be considered as a primary cause for the development of fa/fa obesity.

Hepatic serine dehydratase activity was significantly lower in the obese Zucker rats. In both skeletal muscle and kidney adenylate deaminase showed a lower activity in the obese animals. In the small intestine the activity of glutamate dehydrogenase was increased while that of glutamine synthetase was reduced. No changes were found in the enzymatic activities of white adipose tissue while those found in brown adipose tissue were lower for glutamine synthetase. Starvation resulted in increase in liver serine dehydratase in the lean animals and in aspartate transaminase in both lean and obese. Kidney aspartate transaminase and glutamine synthetase were increased with starvation in the lean rats while kidney adenylate deaminase and small intestine glutamine synthetase and branched-chain amino acid transaminase were increased with starvation in the obese animals. In brown adipose tissue starvation caused an increase in branched-chain amino acid transaminase in the lean rats while it significantly lowered the adenylate deaminase and increased branched-chain amino acid transaminase in the obese rats.

Lean and genetically obese Zucker rats were implanted with permanent intravenous catheters and a guide cannula was aimed at the region of the ventromedial (VMH) and paraventricular (PVN) nuclei to measure immunoreactive insulin collected by means of microdialysis. Preliminary experiments assessed the validity of a novel assay of insulin in microdialysates by a sensitized radioimmunoassay technique. This method was then used to measure basal levels of insulin and those induced by i.v. infusion of 0.5 U of insulin over 30 min in both lean and obese rats. Basal hypothalamic immunoreactive insulin levels were lower in the obese rats than in the lean Zucker rats. When insulin was infused i.v. for 30 min, hypothalamic immunoreactive insulin showed an increase in the 30-60 min sample, which was twice as great in the obese rats. Two facts suggest that the insulin found in the microdialysates was of cerebral, not vascular origin: the short latency in the response and the finding that the response was greater in obese rats.

Glomerular proteoglycans (PG) are important in modulating extracellular matrix assembly and glomerular permselectivity. In the obese Zucker rat, an experimental model of non-insulin dependent diabetes mellitus, expansion of the mesangial matrix, and microalbuminuria occur before the development of overt renal disease. The in vivo incorporation of (35S)sulfate into glomerular PG in 12-wk-old obese Zucker rats at the onset of microalbuminuria was compared with that of 12-wk-old lean Zucker rats. Specific (35S)sulfate incorporation into glomerular PG over 8 h was increased by 57% in obese rats compared with lean rats, suggesting increased PG synthesis. However, at variance with the observation in experimental models of insulin-dependent diabetes mellitus, the proportion of total glomerular (35S)PG released by heparin treatment was unchanged. Heparan sulfate (HS)-PG constituted over 60% of radiolabeled de novo synthesized glomerular PG. Similar proportions of HS-PG were extracted from the glomeruli of obese and lean rats. Isolated glomeruli spontaneously released two HS-PG, which constituted approximately 30% of total glomerular (35S)PG. On the basis of their chromatographic and electrophoretic patterns, these PG were similar in obese and lean rats. Heparin treatment of isolated glomeruli released an additional HS-PG, which appeared to be derived primarily from the glomerular extracellular matrix compartment and not from the detergent soluble cell fraction. Heparin-releasable HS-PG from both the lean and obese Zucker rats eluted at a KAV of 0.31 from Sepharose CL-6B chromatographic columns, indicating a hydrodynamic size similar to that reported for glomerular basement membrane HS-PG. However, gel electrophoresis demonstrated faster migration of the HS-PG released by heparin from the glomeruli of obese Zucker rats, suggesting increased electronegativity. Thus, early in the course of nephropathy in the obese Zucker rat, there is increased glomerular PG synthesis with no change in the proportions of the constitutively releasable and heparin-releasable HS-PG. Whether electrophoretic abnormalities of the heparin-releasable HS-PG observed in the obese rats contribute to the development of albuminuria and/or mesangial matrix expansion remains to be established.

Proteins from 5- to 7-wk-old lean and obese Zucker rats were separated by one-dimensional sodium dodecyl sulfate (SDS) and two-dimensional SDS-isoelectric focusing-polyacrylamide gel electrophoresis. Laser densitometry revealed an obesity-related decrease in the concentration of a 28-kDa cytosolic adipocyte protein, the most abundant protein in adipocytes from lean Zucker rats. Microsequencing revealed the identity of this protein to be carbonic anhydrase III (CA III). The identity and obesity-related decrease was further confirmed using isoform-specific antisera and CA III enzyme activity measurements made by 18O mass spectrometry. Immunoblotting studies also revealed that CA III is present in at least two charge isoforms in adipocytes. Our data indicate that lean Zucker rat adipocytes may represent the richest source of CA III in nature (24% of the cytosolic protein content). An obesity-related decrease in both the concentration and activity of CA III was observed in two lipogenic tissues, liver and white fat, but not in soleus muscle. Adipocyte CA III activity was no longer depressed when hyperinsulinemic obese rats were made insulin deficient by streptozotocin injection. This suggests that the obesity-related decrease in CA III may be related to the hyperinsulinemia as well as to the insulin hyperresponsiveness that adipocytes from obese Zucker rats of this age display.

The local cerebral glucose utilization (LCGU) of brains from Wistar, lean Zucker (FA/FA), and obese Zucker (fa/fa) rats was investigated using the method of Sokoloff et al. (L. Sokoloff, M. Reiwich, C. Kennedy, M.H. Des Rosiers, C.S. Patlak, K.D. Pettigrew, O. Sakurada, and M. Shinohara. J. Neurochem. 28: 897-916, 1977.). The LCGU of obese Zucker (fa/fa) rats was decreased in comparison to the relatively high values obtained for the lean Zucker (FA/FA) rats in all gray matter areas studied, on average to the extent of 50%. When compared with Wistar rats, several brain areas of lean Zucker (FA/FA) animals had a normal glucose uptake. When these normal areas were assessed for common efferent and afferent pathways, it was found that many of these common connections had normal glucose utilizations. In direct comparison to the obese fa/fa rat, the LCGU rates of these areas were decreased, hinting that this would also be the case for their functional activity. Because these areas (limbic, thalamic, hypothalamic, autonomic) have been reported to be potentially relevant for bringing about the behavioral and neuroendocrine alterations known to occur in obese fa/fa rats, it is proposed that they represent dysfunctions that are partly responsible for the obesity syndrome of the fa/fa strain.

Brown adipocytes are thermogenic cells which play an important role in energy balance. Their thermogenic activity is due to the presence of a mitochondrial uncoupling protein (UCP). Until recently, it was admitted that in rodents brown adipocytes were mainly located in classical brown adipose tissue (BAT). In the present study, we have investigated the presence of UCP protein or mRNA in white adipose tissue (WAT) of rats. Using polymerase chain reaction or Northern blot hybridization, UCP mRNA was detected in mesenteric, epidydimal, retroperitoneal, inguinal and particularly in periovarian adipose depots. The uncoupling protein was detected by Western blotting in mitochondria from periovarian adipose tissue. When rats were submitted to cold or to treatment with a beta-adrenoceptor agonist, UCP expression was increased in this tissue as in typical brown fat. Moreover, the expression was decreased in obese fa/fa rats compared to lean controls. Morphological studies showed that periovarian adipose tissue of rats kept at 24 degrees C contained cells with numerous typical BAT mitochondria with or without multilocular lipid droplets. Immunocytochemistry confirmed that multilocular cells expressed mitochondrial UCP. Furthermore, the number of brown adipocytes and the density of mitochondrial cristae increased in parallel with exposure to cold. These results demonstrate that adipocytes expressing UCP are present in adipose deposits considered as white fat. They suggest the existence of a continuum in rodents between BAT and WAT, and a great plasticity between adipose tissue phenotypes. The physiological importance of brown adipocytes in WAT and the regulation of UCP expression remain open questions.

To determine whether the capacity of thyroxine (T4)-to-3,5,3'-triiodothyronine (T3) conversion was altered in Zucker fa/fa pups, we measured thyroxine 5'-monodeiodinase (T(4)5'D) activity in brown adipose tissue (BAT) and liver of suckling and weaned fa/fa and Fa/fa littermates. In suckling fa/fa compared with Fa/fa rats, T(4)5'D was reduced by 30-55% in BAT and slightly increased in liver, while serum free T3 was significantly decreased (-30%). Altered T(4)5'D activity in BAT of fa/fa pups could be corrected by adrenergic stimulation. After weaning, in fa/fa rats, the capacity for T4-to-T3 conversion was totally restored in BAT while it was dramatically reduced in liver. The concentration of serum free T3 remained lower in fa/fa than in Fa/fa rats (-40%). These data confirm that BAT is a very early site of fa gene expression and are consistent with the hypothesis that a defect in the autonomic nervous system may be a primary cause of this genetic obesity. It is also suggested that, during suckling, BAT plays an important role in systemic production of T3.

In a cross-sectional study, we evaluated the metabolic profiles of lean (Fa/?) and obese (fa/fa) Zucker male rats at 4 to 8 months of age. Although all of the obese rats (N = 108) demonstrated glucose intolerance, most of the obese rats exhibited only mild elevations of fasted and fed plasma glucose. Only 14 of the obese rats were severely hyperglycemic, which resulted in substantial elevations of glycohemoglobin (GHb) levels. The nerve and lens levels of glucose, sorbitol, and fructose were elevated, and the myo-inositol was depleted in all hyperglycemic obese rats, but not in the euglycemic obese rats. With increasing duration of hyperglycemia, the neural myo-inositol level approached normal, while the lenses became cataractous. All obese rats had increased urinary albumin excretion (UAE), which was dependent on age (r = .45, P less than .02) and independent of hyperglycemia, glucosuria, and polyuria. In conclusion, although the euglycemic obese rats exhibited some diabetic abnormalities, the hyperglycemic obese Zucker rat more closely resembled the altered metabolic profile associated with type II diabetes mellitus.

Lean and obese Zucker fatty rats were adrenalectomized or sham operated at 10 wk of age. At 15 wk one-half of each group was placed on a high-fat diet. At 32 wk of age the experiment was ended. Several conclusions can be drawn about the effects of adrenalectomy, high-fat diets, and their interaction in the Zucker fatty rat. First, adrenalectomy slowed the weight gain in both obese fatty rats and in the lean animals, although the effect was greater in the fatty rats. Second, weight gain was accelerated in intact lean and fatty rats eating a high-fat diet. Third, adrenalectomy attenuated the weight gain associated with a high-fat diet and reduced the body content of fat and protein in the lean animals and fatty rats fed the low-fat diet. Fifth, adrenalectomy significantly affected the retroperitoneal and subcutaneous fat depots but not the epididymal fat depot. Sixth, adrenalectomy decreased fat cell number in retroperitoneal and subcutaneous fat depots, but this was much less evident in the epididymal fat depot. Seventh, lipoprotein lipase activity expressed per milligram protein increased after adrenalectomy in the fatty rat but was reduced on the same basis in lean animals regardless of diet. Finally, the increase in retroperitoneal lipoprotein lipase activity expressed per fat cell observed in lean animals fed the high-fat diet was not observed in the fatty rat. These studies show that a high-fat diet and adrenalectomy interact in the development of obesity in both lean and fatty Zucker rats.

The insulin secretion rate in response to different secretagogues and neurotransmittors was studied in perifused pancreas of 5-day-old lean (Fa/Fa) and preobese (fa/fa) Zucker rats. Glucose (16.6 mM) alone or in combination with 20 mM arginine or 5 mM theophylline induced a net stimulation of insulin secretion. This effect was similar in the two groups. By contrast, the stimulatory effect of acetylcholine on glucose-induced insulin secretion was significantly higher in preobese pups than in lean rats. There was also a tendency toward a higher inhibitory effect of norepinephrine on insulin secretion in preobese than in lean rats, but this difference did not reach statistical significance. Together these results demonstrate a normal insulin secretion in response to nutrient secretagogues in preobese fa/fa rats but an enhanced effect of acetylcholine. This latter effect may be related to the changes in the autonomic nervous system activity, which is usually described in obese fa/fa rats.

Cardiovascular disease represents the major cause of morbidity and mortality in noninsulin-dependent diabetic patients. While it was once thought that atherosclerotic vascular disease was responsible for all of these adverse effects, recent studies support the notion that one of the major adverse complications of diabetes is the development of a diabetic cardiomyopathy characterized by defects in both diastolic and systolic function. Contributing to the development of the cardiomyopathy is a shift in myosin isozyme content in favor of the least active V3 form. Also defective in the noninsulin-dependent diabetic heart is regulation of calcium homeostasis. While transport of calcium by the sarcolemmal and sarcoplasmic reticular calcium pumps are minimally affected by noninsulin-dependent diabetes, significant impairment occurs in sarcolemmal Na(+)-Ca2+ exchanger activity. This defect limits the ability of of the diabetic heart to extrude calcium, contributing to an elevation in [Ca2+]i. Also promoting the accumulation of calcium by the diabetic cell is a decrease in Na+, K+ ATPase activity, which is known to increase [Ca2+]i secondary to a rise in [Na+]i. In addition, calcium influx via the calcium channel is stimulated. Although the molecular mechanisms underlying these defects are presently unknown, the possibility that they may be related to aberrations in glucose or lipid metabolism are considered. The evidence suggests that classical theories of glucose toxicity, such as excessive polyol production or glycosylation, appear to be insignificant factors in heart. Also insignificant are defects in lipid metabolism leading to accumulation of toxic lipid amphiphiles or triacylglycerol. Rather, the major defects involve membrane changes, such as phosphatidylethanolamine N-methylation and protein phosphorylation, which can be attributed to the state of insulin resistance.

The aim of the present investigation was to study the effects of fish oil feeding in obese Zucker rats to establish its suitability as an animal model of hyperlipidaemia, and to understand the possible mechanism of fish oil-induced perturbations in cell metabolism. Lean and obese Zucker rats were fed on diets containing 180 g coconut, safflower, or menhaden oil/kg for 10 weeks. Body-weights and food intakes of lean coconut (LC), safflower (LS), and menhaden (LM) groups were similar. Obese menhaden (OM) rats had lower food intakes and body-weights compared with obese coconut (OC) and obese safflower (OS) groups, but values for all obese rats were higher than those for lean rats. Liver weights were higher in obese compared with lean rats, but on a percentage body-weight basis menhaden oil rats had higher values within genotype. Serum cholesterol and triacylglycerol levels were lower in the OM group compared with the OC and OS groups, and in the LM group compared with the LC group. Glucose and insulin levels were highest in OS rats followed by OC and OM rats and then the lean rats. Serum triiodothyronine and thyroxine were lower in OM rats compared with OC and OS rats. Liver mitochondrial state 3 rates with glutamate-malate and succinate were lower; mitochondrial beta-oxidation was unaffected and peroxisomal beta-oxidation was higher in menhaden oil rats compared with both coconut and safflower oil rats. In general, consumption of menhaden oil lowered hepatic malic enzyme (EC 1.1.1.38, 1.1.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and glutathione peroxidase (EC 1.11.1.9) activities and elevated long-chain fatty acyl-CoA hydrolase (EC 3.1.2.2) activity when compared with the two other diets. It is concluded that obese Zucker rats do respond like human subjects to fish oil feeding but not to vegetable oils. The hypolipidaemic effect of fish oil appears to be mediated through a lowering of lipogenic enzymes, glucose-6-phosphate dehydrogenase and malic enzyme.

Previous studies have shown that hepatocyte and liver membranes from insulin resistant animals exhibit an impairment of inhibitory guanine nucleotide binding regulatory protein, Gi function, such that a Gi defect may contribute towards the diabetic syndrome. In the current studies, it is shown that the demonstration of Gi activity in liver and hepatocyte membranes is dependent critically on the membrane preparation technique. A technique is defined that allows functional Gi activity to be demonstrated in liver and hepatocyte membranes from both lean (Fa/?) and obese (fa/fa) Zucker rats. Consequently, previous reports on the loss of Gi function in insulin resistant states require revaluation.

Lipoprotein lipase (LPL) activity and mRNA levels were measured in cardiac muscle and adipose tissue from lean, obese, and weight-stable reduced-obese Zucker rats, both fasted and 2 h after feeding. Fasting epididymal fat LPL activity was substantially higher in obese rats relative to lean rats [6.9 vs. 0.2 nmol free fatty acid (FFA).10(6) cells-1.min-1; P = 0.0001], and was higher still in reduced-obese rats (15.7 nmol FFA.10(6) cells-1.min-1; P = 0.002). Adipose tissue LPL increased with feeding in all three groups. In marked contrast, fasting cardiac muscle LPL was lower in obese rats relative to lean (28.8 vs. 38.5 nmol FFA.g-1.min-1; P = 0.0064) and was lower still in reduced-obese rats (14.5 nmol FFA.g-1.min-1; P = 0.0001). LPL mRNA levels increased in adipose tissue along with enzyme activity; however, the magnitude of the changes were relatively small, suggesting that the primary regulatory steps are posttranslational. Weight reduction studies were also carried out in Sprague-Dawley rats with similar results. These studies show that sustained weight reduction results in coordinate changes in tissue-specific LPL, favoring delivery of lipoprotein triglyceride fatty acids to adipose tissue relative to cardiac muscle and the restoration of energy stores.

To investigate the possible role of islet amyloid polypeptide (IAPP) in the development of type 2 diabetes mellitus, we examined the IAPP content and secretion in pancreatic islets isolated from ventromedial hypothalamic (VMH)-lesioned rats and genetically obese Zucker rats, using a specific RIA for IAPP. Obesity and hyperinsulinemia were observed in rats 21 days after VMH lesioning. IAPP content was increased in the islets of VMH-lesioned rats compared with findings in the sham-operated controls (100.9 +/- 6.6 vs. 72.8 +/- 3.85 fmol/islet; P less than 0.01). Isolated islets of VMH-lesioned rats secreted larger amounts of IAPP in the presence of 2.8 mM and 16.7 mM glucose (2.99 +/- 0.98 and 11.2 +/- 1.29 fmol.islet(-1).3 h-1) than was noted in sham-operated rats (ND and 6.65 +/- 0.78 fmol.islet(-1).3 h-1). In the obese Zucker rats, aged 14 weeks, IAPP concentrations in the islets were elevated compared with lean rats (133.3 +/- 10.6 vs. 84.4 +/- 8.5 fmol/islet; P less than 0.01). The isolated islets secreted larger amounts of IAPP in response to 2.8 mM and 16.7 mM glucose (2.83 +/- 0.88 and 15.81 +/- 1.35 fmol.islet(-1).3 h-1) than did those from lean control rats (0.36 +/- 0.19 and 12.49 +/- 1.20 fmol.islet(-1).3 h-1). These results strongly suggest that overproduction and hypersecretion of IAPP occur in animals with obesity and hyperinsulinemia.

Platelet activity is controlled, in part, by cytosolic free ionized calcium concentration ([Ca++]i). Regulation of platelet thromboxane (TXB2) synthesis may be by regulation of [Ca++]i. Dietary linoleate is a regulator of TXB2 synthesis, therefore, it may act by influencing [Ca++]i. Aspirin is a regulator of TXB2 synthesis by inhibition of cyclooxygenase; ouabain and nifedipine are regulators of [Ca++]i. This study was conducted to determine whether these affectors of TXB2 synthesis and [Ca++]i cause associated responses. Male nonobese Zucker rats were fed diets supplying 30% of energy (en%) as fat. Dietary fat was a mixture of corn oil and beef tallow to provide 3.0, 4.5, 6.0, or 7.5 en% linoleic acid, with cholesterol added to provide equal cholesterol in all diets. Rats were fed for 30 days with 6 rats/diet. Isolated rat platelets were assayed for FA composition; the percentage of linoleic acid in platelet FA rose linearly with increasing dietary linoleate (r = 0.76, P less than 0.0001). Resting and thrombin-stimulated platelet [Ca++]i and TXB2 synthesis were measured in the presence or absence of extracellular calcium and aspirin, ouabain, or nifedipine. Aspirin caused reductions in both parameters; nifedipine blocked [Ca++]i, but did not affect TXB2; ouabain increased both. Changes induced by those modifiers of TXB2 and platelet [Ca++]i caused changes that were in the same direction for both. CaCl2 caused an increase in both and the [Ca++]i was correlated with the square root of the TXB2; without CaCl2 the two were negatively correlated; aspirin, ouabain, and nifedipine treatments resulted in no significant correlations. The results suggest that there is a common modifier of [Ca++]i and TXB2 synthesis.

Life-long sequential changes in glucose tolerance and insulin secretion were investigated in genetically obese Zucker rats (fa/fa) fed a diabetogenic diet rich in lard and sucrose. Comparisons were made with lean littermates (Fa/-) receiving normal chow diet. At 3-month intervals, seven to nine lean and obese rats had two permanent venous catheters implanted, allowing stress- and pain-free sampling of blood before, during, and after substrate administration. Intravenous glucose, iv arginine, and oral glucose tolerance were tested. The obese rats progressively developed hyperglycemia and severe hyperinsulinemia; their basal glycemia reached 8.8 +/- 1.1 vs. 5.8 +/- 0.2 mmol/liter in the lean rats at 46 weeks of age; respective insulinemia was 287.7 +/- 61.9 and 18.1 +/- 2.8 mU/liter (mean +/- SD). In the obese rats a distinct loss in glucose tolerance was seen with progression of age in spite of rising stimulated insulin secretion, which suggests progressive development of insulin resistance without exhaustion of B-cell secretory capacity. Absence of insulin deficiency was also suggested by immunohistochemical staining of pancreatic tissue specimens from obese rats, which showed large populations of insulin-containing cells. Like the obese animals, lean rats exhibited a decrease in insulin sensitivity with age. Relating basal individual glycemia and insulinemia, a rise by 1 mmol/liter in glycemia was associated with a 8.8-fold rise in basal insulinemia in lean rats, but only with a 1.8-fold increase in obese rats. Similar correlations for stimulated glycemia and insulinemia suggest impaired glucose sensitivity of pancreatic B-cells in obese vs. lean rats. In conclusion, hyperglycemia and hyperinsulinemia in insulin-resistant obese Zucker rats on a diabetogenic diet are not characterized by quantitatively deficient B-cell secretory capacity, but, rather, by impaired B-cell sensitivity to glucose with qualitatively intact regulation of glycemia and insulinemia at elevated plasma concentrations.

The obese Zucker rat is a classic model of non-immune-mediated spontaneous focal glomerulosclerosis. An early morphological hallmark of glomerular damage in the obese Zucker rat is a mesangial expansion, which precedes and mediates the development of glomerular damage in these animals. This study was designed to investigate whether there might be a reduced activity of glomerular proteinases in kidneys of obese Zucker rats, thereby being involved in the pathogenesis of mesangial expansion, which is mainly due to protein overloading. In fact, we found a decreased proteinase activity in ultrasonically destroyed isolated glomeruli obtained by a differential sieving technique in obese Zucker rats compared with their lean littermates. This held true at acid as well as at neutral pH and could be confirmed when proteinase activity was related to DNA instead of protein. When investigating the glomerular cathepsin B content--this is a lysosomal enzyme with acid pH optimum, which is involved in the degradation of glomerular structural as well as filtered plasma proteins--we found a significantly increased level in the kidneys of obese Zucker rats. Hence, the intraglomerular proteinase activity is reduced in the face of enhanced glomerular content of at least lysosomal proteinases. The underlying reason for this depressed activity remains to be established. We propose that reduced activities of intraglomerular proteinases may be important in the pathogenesis of mesangial matrix expansion in obese Zucker rats, an important initiating hallmark of glomerulosclerosis in this model.

Disturbances of insulin or brain monoamine metabolism may play a role in the impaired regulation of food intake and body weight in the obese Zucker rat. We investigated a possible insulin-monoamine interaction by measuring monoamine levels in the hypothalamus and striatum of obese (fa-fa) and lean (Fa-Fa and Fa-fa) Zucker rats after peripheral insulin administration. The classically reported effects of insulin, i.e., increases in tryptophan, 5-hydroxy-indolacetic acid (5-HIAA) and dihydroxyphenylacetic acid (DOPAC) levels, were observed in the hypothalamus of Fa-Fa and Fa-fa rats, but not in obese fa-fa rats. Given the mechanism of action of insulin, this lack of effect in the obese rats may be related to the peripheral insulin resistance they exhibit. Furthermore, given the role of these monoaminergic systems, this reduced effect may be related to the impaired regulation of food intake and body weight. At 8 wk of age, however, insulin restored the decreased basal 5-HIAA levels observed in the obese rats. Increase in 5-HIAA levels following insulin administration appeared in the striatum of Fa-Fa rats only, suggesting that, as for brain insulin content, other central insulin-related disturbances may be related to the presence of the "fa" gene. In addition, certain effects of insulin on striatal dopamine release were observed in only the Fa-Fa and fa-fa rats, suggesting a particular disturbance related to the heterozygous character. This latter point calls for further investigations on the central dopaminergic effects of insulin.

Treatment of intact, 32Pi-labelled hepatocytes from lean Zucker rats with a range of agents including 12-O-tetradecanoyl-phorbol 13-acetate (TPA), vasopressin, and angiotensin II elicited substantial increases in the phosphorylation of the alpha-subunit of the inhibitory G protein of adenylate cyclase (alpha Gi-2). These agonist-induced phosphorylations of alpha Gi-2 were associated with loss of Gi function as assessed by the ability of low concentrations of guanylyl 5'-[beta,gamma imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity. Hepatocytes from obese Zucker rats displayed a resistance to both agonist-induced phosphorylation of alpha Gi-2 and to p[NH]ppG-mediated inhibition of adenylate cyclase. The basal level of alpha Gi-2 phosphorylation in hepatocytes from obese Zucker rats was considerably greater at 1.06 +/- 0.09 mol phosphate/mol alpha Gi-2 than in hepatocytes from lean animals which gave 0.54 +/- 0.09 mol phosphate/mol alpha Gi-2. Incubation with TPA (10 ng/ml, 15 min) approximately doubled the level of phosphorylation of alpha Gi-2 in the hepatocytes from lean animals but had little effect on the phosphorylation of alpha Gi-2 in hepatocytes from obese animals. Incubation of hepatocytes from lean animals with ligands which lead to the phosphorylation of alpha Gi-2 abolished the ability of low concentrations of p[NH]ppG to inhibit adenylate cyclase expressed in isolated membranes. Treatment of hepatocyte plasma membranes from lean but not obese Zucker rats with pure protein kinase C led to the phosphorylation of alpha Gi-2. The resistance to protein-kinase-C-mediated phosphorylation in hepatocyte membranes from obese animals could be overcome by treatment of the membranes with alkaline phosphatase. These results indicate that the defect in guanine-nucleotide-mediated 'Gi function' seen in obese Zucker rats may be due to an inactivating phosphorylation of alpha Gi-2.

To determine the time onset of the growth hormone (GH) alteration in the genetically obese rat, we studied the in vivo and in vitro rat growth hormone releasing factor (rGRF(1-29)NH2)-induced GH secretion in 6- and 8-week-old lean and obese male Zucker rats. Under sodium pentobarbital anesthesia, rGRF(1-29)NH2 (GRF) was injected intravenously at two doses: 0.8 and 4.0 micrograms/kg b.w. Basal serum GH concentrations were similar in lean and obese age-matched animals. The GH response to both GRF doses tested was unchanged in 6-week-old obese rats as compared to their lean litter mates. In contrast, a significant decrease of the GH secretion in response to 4.0 micrograms/kg b.w. GRF was observed in the 8-week-old obese rats. The effect of GRF (1.56, 6.25 and 12.5 pM) was further studied in vitro, in a perifusion system of freshly dispersed anterior pituitary cells of lean and obese Zucker rats. Basal GH release was similar in the 6-week-old animal group. In contrast, it was significantly decreased in 8-week-old obese rats as compared to their lean litter mates. Stimulated GH response to 1.56 and 6.25 pM GRF was significantly greater in the 6-week-old obese group than in the age-matched control group. In contrast, the GH response to all GRF concentrations tested was significantly decreased in the 8-week-old obese rats as compared to their respective lean siblings. In 8-week-old obese rats, a decrease of GH pituitary content and an increase of hypothalamic somatostatin (SRIF) concentration were observed. Insulin and free fatty acid serum were significantly increased in 8-week-old obese rats. In contrast, lower insulin-like growth factor I serum levels were observed in the obese animals as compared to their lean litter mates. Finally, to further clarify the role of the periphery in the inhibition of GH secretion observed in the 8-week-old fatty rats, we exposed cultured pituitary cells of 8-week-old lean animals to 17% serum of their obese litter mates. A significant decrease of GRF-stimulated GH secretion of lean rat pituitary cells exposed to the obese serum was noted (P less than 0.05). This study demonstrates that, in the obese Zucker rat, an alteration of the GH response to GRF is evident by the 8th week of life. This defective GH secretion could be related to peripheral and central abnormalities.

It has previously been established that norepinephrine (NE) in the central nervous system is involved in feeding and the development of obesity. The present experiments were carried out to investigate the relationship between the uptake of NE by a crude hypothalamic homogenate and NE-mediated sympathetic activity in interscapular brown adipose tissue (IBAT). Sympathetic nervous system activity was assessed by measuring the binding of the purine nucleotide guanosine-5'-diphosphate (GDP) to mitochondria isolated from IBAT. Four situations known to alter food intake and sympathetic activity, namely, corticotropin releasing factor infusion, adrenalectomy, fenfluramine treatment and obesity due to genetic transmission were studied. In each case, [3H]NE uptake by the hypothalamic preparation and GDP binding to IBAT mitochondria were measured. A highly significant negative correlation between the uptake of NE by hypothalamic homogenates and the binding of GDP to IBAT mitochondria was obtained in both lean and obese animals. These findings are discussed with regard to the regulation of food intake and sympathetic nervous system mediated thermogenesis.

In order to study the metabolic effect of dietary sucrose or cornstarch on plasma total triglyceride lipase (T-TGL), hepatic triglyceride lipase (H-TGL) and monoglyceride hydrolase (MGH) activities, young male Zucker fatty and lean rats were pair-fed or fed ad libitum for 4 weeks with diets containing 68% carbohydrate as either sucrose or cornstarch. Our results show that ad libitum feeding of fatty rats with both diets produced an intensification of plasma hypertriglyceridemia which was accompanied by high levels of all plasma postheparin lipolytic activities. These diets did not affect the enzyme activities of lean littermates. Pair-feeding the fatties with sucrose-rich diet produced an increase in T-TGL, H-TGL and MGH. If cornstarch was the carbohydrate included in the diet no difference in postheparin lipolytic activities was found between phenotypes.

Normal, male Sprague-Dawley (S-D) rats and female, lean and obese Zucker rats were studied in the fed state and after 48 hours of food deprivation. Somatostatin-like immunoreactivity (SLI) was measured from acetic acid extracts of oesophagus-cardia, stomach, small and large intestine, pancreas, hypothalamus, pituitary and cerebellum. Within the CNS, the highest levels of SLI were found in the hypothalamus, while in the gut, these levels were highest in the stomach and pancreas. All Zucker rats displayed higher hypothalamic levels of SLI than did S-D rats. Obese Zucker rats in the fed state differed from their lean littermates in that SLI levels were lower in oesophagus-cardia, stomach and hypothalamus, while being higher in pancreas and pituitary. The response to starvation in both obese and lean Zucker rats was qualitatively similar, and included significant increases in stomach and oesophagus-cardia SLI, but with a significant fall hypothalamic SLI. We have concluded that the increase in gastrointestinal SLI with starvation in Zucker as well as in S-D rats may represent a significant regulatory mechanism in nutrient homeostasis. We postulate that gastric SLI may decrease the availability of intestinal insulin secretagogues in the fasting state. This adaptive mechanism appears to be intact in the obese Zucker rat.

1. The feeding pattern, growth rate and energetic metabolism were studied in obese Zucker rats of 5-12 weeks of age kept at ambient temperatures of 22 or 10 degrees C. 2. During this period, the increment in obesity of the 22 degrees C-exposed animal was found to be due to diurnal hyperphagia and not to a lowered resting metabolism. 3. In the 10 degrees C-exposed rat the development of non-shivering thermogenesis associated with a lack of enhancement of food intake leads to prevent the obesity.

The role of insulin status in the development of obesity in Zucker rats was determined by equalization of available insulin. When streptozotocin-induced diabetic obese and lean rats received the same level of exogenous insulin, body weight gain and food intake per body weight were equalized. These types of observations are usually interpreted as proof for a role of insulin in genetically induced obesity. However, a more definitive examination of the composition of gain showed that the obese rat was still gaining significantly more lipid than the lean rat in spite of the equalization of available insulin. Further examination of liver enzymes involved in de novo lipogenesis indicated that increased heptic lipogenesis was not dependent on hyperinsulinemia. Protein gain in lean and obese rats was not significantly different. This study demonstrates that excessive weight gain and food intake of genetically obese rats is dependent on the differences in insulin status between lean and obese rats. The partitioning of more dietary energy toward lipid in the obese rat is independent of both food intake and insulin.

Insulin plays a major role in the control of pancreatic amylase biosynthesis. In this study we determined glucose metabolism by pancreatic acini as well as the pancreatic content of both amylase protein and amylase mRNA during development of insulin resistance in the obese Zucker rat. At age 4 weeks there were no abnormalities detected in the above parameters, although the obese animals were already hyperinsulinaemic. At 6 weeks glucose metabolism was decreased by 50% in acini from obese rats, whereas pancreatic amylase-gene expression was only slightly impaired. At 22 weeks glucose metabolism was decreased by 50%, amylase content by 55% and amylase mRNA by 60% in acinar tissue of obese rats. As expected, hyperinsulinaemia increased markedly with age. Thus development of severe insulin resistance was associated with impairment of amylase-gene expression. To decrease insulin resistance, one group of adult obese rats was treated with Ciglitazone for 4 weeks. A lowered plasma insulin concentration without alteration of food intake was taken as evidence of decreased insulin resistance. This was associated with normalization of glucose metabolism and a marked increase of both amylase content of pancreatic tissue and amylase mRNA. In conclusion, both the increase of insulin resistance with age and its partial reversal by Ciglitazone treatment appear to modulate pancreatic amylase-gene expression in the obese Zucker rat.

Circadian rhythms were studied in ad libitum- and meal-fed Zucker rats. Lean and obese female rats were fed ad libitum or in a single meal from 1400-1800 for 12 d. Both groups of ad libitum-fed rats exhibited a nocturnal feeding pattern; obese rats ate more than the lean rats during the light and dark parts of the cycle. After 12 d, groups of rats were killed at 4-h intervals over a 24-h period, inclusively, and the presence of circadian rhythms was evaluated for the serum metabolites, liver constituents and the liver lipogenic enzymes. The obese ad libitum-fed rats demonstrated rhythms for serum triglycerides, serum cholesterol, liver protein and glycogen, and the activities of fatty acid synthetase, citrate cleavage enzyme (EC 4.1.3.8), and glucose-6-P dehydrogenase (EC 1.1.1.49). Meal feeding the obese rats modified the rhythms for serum cholesterol, liver glycogen and the activities of glucose-6-P dehydrogenase, and citrate cleavage enzyme and a rhythm was established for serum glucose. The feeding schedule did not cue a rhythm for serum triglycerides and phosphogluconate dehydrogenase (EC 1.1.1.44) or modify the rhythm for liver protein. The lean ad libitum-fed rats displayed rhythms for all the measured parameters except serum glucose. Feeding-cued rhythms for all metabolites except serum cholesterol were observed when the lean rats were meal-fed. These data suggest that the rhythms of the ad libitum-fed obese rats may not be similar to those observed in the lean rat. Yet, changing the feeding schedule may be important in establishing rhythm patterns that will alter the anabolic profile characteristic of the Zucker obese 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.

These studies were undertaken to further characterize and explain the differences in hepatic fatty acid metabolism between lean and obese Zucker rats. It was shown that the rate of palmitate or octanoate oxidation and the inhibition of palmitate oxidation by malonyl CoA in mitochondria isolated from lean and obese Zucker rats were similar. Cytochrome oxidase activity was similar in lean and obese rat livers. It was found that the addition of cytosol from the obese rat liver inhibited palmitate oxidation by 20-30% in mitochondria isolated from lean or obese rat livers and thus reproduced the conditions observed in the intact cell. Increased concentrations of metabolites such as malonyl CoA and glycerophosphate in the liver of the obese rat are likely contributors to this inhibitory effect. These results are extrapolated to the intact cell and suggest that decreased hepatic fatty acid oxidation in the obese rat can be accounted for by cytosolic influences on the mitochondria. The decreased rate of fatty acid oxidation observed in the intact hepatocyte or perfused liver cannot be explained by a defect in the capacity of mitochondria to oxidize substrate or by a decrease in mitochondrial number in the obese rat liver.

We investigated whether genetic selection for growth influences pituitary GH secretion in two strains of rats, LL (large) and SS (small). Animals were bled every 15 min for 6 h via an indwelling atrial Silastic catheter, and GH levels were determined by RIA. LL and SS males displayed a low frequency, high amplitude episodic pattern of GH secretion, with surges of GH occurring at 3- to 4-h intervals, separated by trough periods of approximately 60-120 min. In contrast, LL females showed a high frequency, low amplitude pattern of GH secretion, with GH pulses occurring every 1-2 h. The number of GH pulses in SS females was lower than that in LL females. SS males and SS females displayed lower peak amplitudes and lower baseline levels and, therefore, lower mean plasma GH levels compared to LL animals. The anterior pituitary GH content was not significantly different in LL and SS animals of either sex. Thus, the reduction of GH levels in SS animals is most likely the result of reduced release of GH-releasing factor from the hypothalamus or an attenuated pituitary sensitivity to GH-releasing factor.

In response to arginine infusion, Zucker fatty rats developed hyperglycemia accompanying hyperglucagonemia and hyperinsulinemia. The hyperglycemia could not be explained by changes in the ratio of glucagon/insulin. However, arginine infusion failed to increase plasma glucose in fatty rats pretreated with anti-glucagon serum, suggesting a hyperglycemic effect of glucagon released by arginine. Kinetic studies of glucose metabolism revealed that the rates of glucose appearance (Ra) and glucose disappearance (Rd) of lean rats rapidly and simultaneously increased to the same extent after the start of arginine infusion. By contrast, in fatty rats, Ra markedly increased immediately after the start of the infusion, but Rd did not significantly increase until hyperglycemia was established. Overall insulin sensitivity of fatty rats was markedly reduced, determined by the steady state plasma glucose method. We conclude tht arginine-induced hyperglycemia in fatty rats is caused by enhanced glucose output from the liver due to hyperglucagonemia and/or by decreased glucose utilization due to insulin resistance.

Hepatic mitochondrial function was studied in lean and obese Zucker rats in the fed state and at 3 and 6 days of starvation. No significant differences in state 3 mitochondrial oxidative rates were found due to obesity or starvation. Palmitoylcarnitine utilization rates in mitochondria were unaffected by obesity or starvation; however, when expressed per gram liver weight, they were lower in the obese rats due to the decreased amount of mitochondrial protein per gram liver. For palmitoylcarnitine oxidation and acetoacetate and citrate production, the patterns were the same: per milligram mitochondrial protein, both lean and obese rates were equivalent; per total liver, the obese rates were higher; per gram liver, the obese rates were lower. Mitochondrial carnitine palmitoyltransferase specific activity was higher in fed obese than in lean rats and remained higher during starvation. The results indicate that mitochondrial capacity to oxidize fatty acids and to produce keto acids is not affected by genetic obesity or starvation. The differences in fatty acid oxidation and keto acid production that have been observed in hepatocytes and perfused liver might be explained by decreased mitochondrial protein per unit weight of liver or hepatocytes in obese rats.

In order to determine whether there is an anomaly in the pituitary responsiveness to growth hormone (GH)-releasing factor in the genetically obese rat, we examined the in vitro and in vivo effects of rGRF(1-29)NH2 (GRF) on GH release in male and female Zucker rats. The effect of increasing GRF concentrations (1.56, 6.25, 12.5 25 and 50 pM) was first tested on GH release from freshly perifused anterior pituitary cells. In both sexes, the GH response per one pituitary equivalent to each GRF concentration tested was reduced in the obese group. However, when GH release was expressed as a percent of initial cell GH content, there was no difference between the lean and the obese groups. Furthermore, under pentobarbital anesthesia, GRF was injected intravenously at two consecutive doses of 0.8 and 4.0 microgram/kg body weight in obese and lean animals. In both sexes, the GH response to each dose of GRF tested was decreased in the obese group. Basal serum GH concentrations were similar in male and female obese rats compared to their respective lean siblings. In conclusion, this study demonstrates a decrease of the in vitro and in vivo pituitary response to GRF in the obese Zucker rat, suggesting a possible secondary defect at the pituitary level.

Regional brain glucose utilization was investigated in lean and fatty Zucker rats when feeding status was changed. Ad-lib-fed fat rats exhibited lower glucose utilization in the central amygdala than ad-lib-fed lean rats. Food deprivation for 72 h enhanced glucose utilization in the ventromedial hypothalamus, lateral hypothalamic area of both phenotypes, hippocampus of fat rats, mammillary body and ventral tegmental area of leans. These results suggest association of the central amygdala with the development of genetic obesity and of the latter 5 areas with hunger-motivated behaviors.

Imaging and quantitative analysis of insulin-receptor interaction was studied in vivo in lean and obese Zucker rats, using a recently developed technique in which purified Tyr A14 123I-monoiodoinsulin is intravenously injected and the tracer followed by scintillation scanning. The obese rats were 72% overweight, had near normal blood glucose concentrations and an 11-fold increase in plasma insulin concentration. In both groups of rats, the tracer was rapidly taken up by the liver (by a receptor mediated mechanism) and the kidneys (by a non-receptor mediated process). Past this maximum, radioactivity decreased in both organs as 123I-insulin was degraded and free 123I-iodide was released into the plasma compartment. Heart radioactivity (i.e. blood pool) mirrored that of the liver and kidneys. The rapid initial decrease of blood radioactivity was concomitant with liver and kidney uptake of 123I-insulin. Release of free iodide from these organs induced a slow secondary rise of blood radioactivity followed by a final decline corresponding to clearance of plasma iodide, mainly by urinary excretion. Liver radioactivity profiles of lean and obese rats were parallel. When expressed per g weight, liver radioactivity was significantly decreased in obese rats. However, due to hepatomegaly in obese rats, total liver radioactivity was significantly higher in homozygous fa/fa rats than in lean littermates. Furthermore, if the marked hyperinsulinaemia of the obese rats is taken into account, total bound insulin was enhanced in the liver of fa/fa rats whatever reference is used, either g weight or total liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Skeletal muscle proteins (myofibrillar, sarcoplasmic, and stromal) were assayed in the gastrocnemius muscles from Zucker obese rats (fa/fa) at 3, 8, and 52 weeks of age. Total muscle weights were similar in obese and nonobese rats at 3 weeks, but reduced in the obese rats by 8 weeks of age. When the muscle proteins were expressed per 100 mg of muscle, the obese rats had lower concentrations of all three muscle proteins, with a greater loss of sarcoplasmic proteins than the nonobese rats at 8 and 52 weeks of age. By 8 weeks of age total muscle proteins were also reduced in obese compared with nonobese rats. With increasing age, the amount of total proteins continued to rise in both obese and nonobese rats with concomitant increases in myofibrillar, sarcoplasmic, and stromal proteins. The increase in muscle proteins was greater between 3 and 8 weeks than between 8 and 52 weeks of age. It is suggested that the inactivity of the obese rat may contribute to the reduction in muscle proteins observed during their early development.

The role of the gut hormone GIP (gastric inhibitory polypeptide; glucose-dependent insulinotropic polypeptide) in the development of hyperinsulinemia of pre-obese (fa/fa) Zucker rats was investigated. Plasma GIP levels were compared in lean and fa/fa pups from 21 to 35 days of age. The onset of both basal and glucose-stimulated hyperinsulinemia was studied. Possible causal roles for glucose, GIP and acetylcholine in hyperinsulinemia were investigated in the isolated perfused pancreas preparation. Immunocytochemical studies of pancreatic islets were also carried out. Glucose-stimulated hyperinsulinemia was present in fa/fa rats at 21 days of age but fasting hyperinsulinemia did not become apparent until 35 days of age. At no time did plasma GIP levels differ between lean and fa/fa rats. Immunocytochemical analysis of the pancreas revealed enlarged islets in fa/fa rats from 7 days of age onward. In the in vitro perfused pancreas of 21 day old fa/fa pups the insulin response was not different from that of lean controls in the presence of glucose (300 mg/dl) plus GIP or acetylcholine. An increased pancreatic insulin response to glucose (300 mg/dl or 80 mg/dl) plus GIP in fa/fa compared to lean animals was observed at 35 days of age. These data suggest that defects in the beta-cell response to GIP become apparent at 35 days of age in fa/fa rats resulting in a loss of the glucose threshold for the insulinotropic action of GIP and onset of fasting hyperinsulinemia in vivo. Causal factors for glucose-stimulated hyperinsulinemia at 21 days of age appear to be complex and not easily replicated in in vitro experiments.

Previously we found that adult Zucker fatty rats have C-cell hyperplasia and increased thyroidal calcitonin (CT) compared to lean controls. In this study we have evaluated both secretion of CT and responsiveness to CT in order to see whether they, too, were altered. Fat rats and lean littermates, 13-15 months old, were used. CT secretion was provoked by (1) feeding for 2 hr after an 18-hr fast, (2) giving pentagastrin iv, and (3) injecting CaCl2 iv. CT was measured by radioimmunoassay. Responsiveness to CT was examined by giving porcine or salmon CT iv and measuring serum Ca 1-3 hr later. For CT secretion, compared to leans the fat rats showed (1) higher fasting serum Ca and CT and a greater rise in CT after feeding, (2) a similar 5- to 10-fold increase in CT after iv pentagastrin, and (3) a greater rise in both serum Ca and CT at various times between 5 min and 3 hr after iv CaCl2. For CT responsiveness, fat and lean rats were equally responsive to iv CT in terms of the fall in plasma Ca 1-3 hr later. The results show that fat rats can secrete as much or more CT in response to provocative stimuli as lean rats and that they appear normally responsive to injected CT. Therefore, inability to release CT and insensitivity to CT do not underly the C-cell hyperplasia, increased thyroidal CT, and increased circulating CT in the fat rat.

The metabolism of 125I-labelled insulin by hepatocytes isolated from 48-h-starved Zucker lean and obese rats was studied. Hepatocytes from the lean animals bound significantly more 125I-labelled insulin and had a greater receptor number per cell than did cells from obese littermates. Hepatocytes from the lean animals degraded and internalized more hormone than did those from obese ones. Increased degradation and internalization correlated with the increased receptor number.

Hepatocytes isolated from genetically obese female Zucker rats and lean female Zucker rats were compared. Hepatocytes from fed obese rats exhibited greater rates of fatty acid synthesis, more extensive accumulation of lactate and pyruvate from their glycogen stores, increased rates of net glucose utilization but produced less ketone bodies from exogenous fatty acids and had lower citrate levels than hepatocytes from lean rats. Lipogenesis was not as sensitive to dibutyryl cyclic AMP (DBcAMP) inhibition in hepatocytes from obese rats but glycogenolysis was stimulated to the same extent by this nucleotide in both preparations. Ketogenesis was less sensitive to stimulation by DBcAMP in hepatocytes from obese rats. A difference in sensitivity of lipogenesis to DBcAMP was not found when lactate plus pyruvate was added to the incubation medium, suggesting that a greater rate of glycolysis by hepatocytes from obese rats accounts for their relative insensitivity to DBcAMP. Citrate levels were elevated by DBcAMP to a greater extent in hepatocytes from obese rats. Hepatocytes prepared from lean rats starved for 48 hr were glycogen depleted and lacked significant capacity for lipogenesis and glycogen synthesis. In contrast, hepatocytes isolated from starved obese rats retained considerable amounts of liver glycogen and exhibited detectable rates of lipogenesis and glycogen synthesis. Hepatocytes prepared from starved lean rats gave faster apparent rates of lactate gluconeogenesis than hepatocytes prepared from starved obese rats. Thus, hepatocytes prepared from obese Zucker rats are more glycogenic, glycolytic, and lipogenic but less ketogenic and glucogenic than hepatocytes prepared from lean rats.

Insulin binding was measured in membrane particles prepared from the liver and several brain regions of 4-month-old female Zucker fa/fa (obese), Fa/fa (heterozygous), and Fa/Fa (lean) rats. High affinity insulin binding was decreased in the olfactory bulb of fatty (0.23 pmol bound/mg protein) and heterozygous (0.16 pmol/mg) rats compared with that in the lean controls (0.64 pmol/mg). Total binding was not changed in the cerebral cortex or hypothalamus. High affinity insulin binding was also decreased in the liver of both fatty (0.44 +/- 0.22 pmol/mg; P less than 0.01) and heterozygous (0.75 +/- 0.35 pmol/mg) animals compared with that in the lean rats (2.10 +/- 1.55 pmol/mg). This decreased binding is probably not due to down-regulation of receptors in the heterozygous rats, as they do not exhibit the hyperinsulinemia observed in the fatty rats. Rather, our findings suggest that there is a gene-related alteration in insulin binding in the Zucker rat, as low binding was observed in rats carrying either one (Fa/fa) or two (fa/fa) doses of the gene. We postulate that this central defect in insulin binding may contribute to inadequate perception of a central insulin feedback signal and to the hyperphagia observed in the obese rats.

The regulation of glucose transport in normal and insulin-resistant obese rat hearts have been studied by measuring glucose transport via the efflux of labelled 3-0-methyl-D-glucose. Glucose transporters in obese rat hearts were also investigated using the labelled cytochalasin B-binding assay. Basal, and insulin- or increasing workload-induced stimulation of glucose transport was decreased in obese rat hearts compared to those of normal ones. Total number of glucose transporters (plasma membrane plus microsomal ones) was about half that previously reported for normal rat hearts. Insulin or workload favoured the translocation of glucose transporters from an intercellular pool (microsomes) to the plasma membrane, as they do in normal rats. Due to the measured decrease in total number of transporters of obese rat hearts, those present in the plasma membrane (under basal conditions, or following stimulation by insulin or workload) were less than those previously found in normal rat hearts tested under identical conditions. In obese rat hearts, regulation of plasma membrane transporters was perturbed. The Hill coefficient (an index of positive cooperativity amongst glucose transporters) was paradoxically decreased by insulin while leaving affinity values unaltered. The Hill coefficient was unaltered by workload, although the affinity values were increased compared to respective controls. To sum up, obese rat hearts have decreased total transporter number, and although the two stimuli studied favour the translocation of available transporters, they fail to "activate" them adequately once present in the plasma membrane.

Parabiosis is the surgical union of two animals to produce a chronic blood exchange. This model has previously been used to demonstrate the involvement of a blood-borne factor in the feedback control of food intake and regulation of energy balance. It has been hypothesized that obese rats produce a humoral agent that acts centrally to inhibit food intake and accumulation of fat. In this study 50-day-old male or female Zucker rats were joined in either lean-lean pairs or lean-obese pairs. They ate ad libitum until 152 days of age when body composition was determined. Parabiosis inhibited growth in all rats compared with single controls. Lean partners of obese rats had reduced carcass weights, the same percent body protein but less fat than members of lean-lean pairs. Female rats showed larger changes in body composition than did males. These results suggest that obese Zucker rats produce the hypothesized regulatory signal but do not respond to it.

A method for the isolation of cells from lactating mammary gland tissue of rats is described. Metabolic properties of mammary cells isolated by the described method either from Wistar of Zucker rats as well as insulin binding were studied. Whereas synthesis of protein and lipid showed no significant differences, the rate of glucose oxidation is slightly increased in cells isolated from Zucker rats. Incorporation of (14)C-uridine in the RNA-fraction is stimulated by insulin; the half-maximal concentration is given by 5 x 10(-10) mol/1). Specific binding of insulin to both types of cells can be described by a nonlinear Scatchard-plot. The apparent affinity constants for the high affinity receptors (1 x 10(-10) mol/1) correlate well with the described metabolic effect of insulin on RNA-synthesis. The total capacity for insulin binding is reduced in cells isolated from Zucker rats when compared to those from Wistar rats.

Impaired protein deposition has been suggested to be a critical factor promoting the hyperphagia of the obese Zucker rat. The selection patterns of adult, male, genetically obese (fa/fa) and lean (Fa/-) Zucker rats were studied to determine if obese rats would select a diet that was higher in protein than the diet selected by lean littermates. Rats were provided ad libitum access to three macronutrient sources and were allowed to compose their own diets for 9 days. The three dietary items were: a vitamin + mineral--supplemented carbohydrate source (cornstarch), a vitamin + mineral--supplemented protein source (casein) and commercially available corn oil. Obese rats ate 43% more calories than lean littermates. Further, obese rats selected a diet that provided 12% of their total caloric intake as protein, 24% as carbohydrate and 64% as fat. Lean rats selected a diet that provided 33% of their total caloric intake as protein, 37% as carbohydrate and 30% as fat. These selection data are not consistent with the hypothesized importance of the role of dietary protein and its incorporation into lean body mass as a stimulus promoting the hyperphagia demonstrated by the genetically obese Zucker rats.