Glucose handling by hepatocytes from obese Zucker rats

Hepatocytes isolated from obese Zucker rats showed a significantly higher rate of both [U-¹⁴C]glucose and [U-¹⁴C]lactate incorporation into [¹⁴C]lipid than those from their lean counterparts. This was associated with a marked increase in the lipogenic rate measured by the incorporation of³H₂O into the cell esterified fatty acids. Although there were no changes in the incorporation of the tracer into either [¹⁴C]glycogen or¹⁴CO₂, the [¹⁴C] total uptake was significantly higher in the obese animals. The high rate of [¹⁴C]lipid synthesis from glucose was observed both at 15 and 30 mM substrate concentrations and was linked to an enhanced uptake of the tracer into the cell as measured using the decarboxilation of [1-¹⁴C]glucose in the presence of phenazine methosulphate. The presence of insulin in the incubation medium had no effect on the uptake of glucose by the liver cells. However, the large uptake of glucose by the hepatocytes from the obese animals was not related to an enhanced rate of transport as measured using 3-O-methyl[U-¹⁴C]glucose. The activity of glucose-6-phosphate dehydrogenase together with a higher [1-¹⁴C]glucose/[U-¹⁴C]glucose descarboxylation ratio indicate a predominant very active pentose phosphate pathway which may be responsible for the enhanced glucose uptake observed in the hepatocytes from the obese animals.     

Management of dietary essential metals (iron,copper, zinc,chromium and manganese) by Wistar and Zucker obese rats fed a self-selected high-energy diet

The balances and content of essential elements (iron, copper, zinc, chromium and manganese) in the body of Wistar, Zucker lean and Zucker obese rats fed a reference or cafeteria diet from day 30 to 60 after birth have been studied. Intestinal iron absorption compensated for low iron content of the cafeteria diet and the extra needs of growth and fat deposition. It can be assumed that the altered energy regulation processes that afflict the genetically obese rat are not directly related to altered iron metabolism. Obese Zucker rats had lower copper tissue concentrations than lean rats, but when fed a cafeteria diet the differences between Zucker rats strains disappear. This cannot be traced to large differences in diet copper concentration. A low diet availability of zinc—such as that of cafeteria-fed fa/fa rats—is easily compensated for by increasing absorption. So, as a consequence, we can conclude that genetic obesity did not impair zinc absorption. There was no deficit of zinc in any of the groups studied; the rats have enough capacity to extract zinc within a wide range of dietary concentrations. The absorption of dietary chromium was inversely proportional to its concentration. The ability to extract chromium from the diet and the very low urinary losses are a consequence of its scarcity in most dietary items. Despite wide variations in the manganese of the diets, the absorption rates were practically unchanged except for obese rats fed the cafeteria diet. It seems that this low absorptive capacity is enough to supply the rat with the manganese it needs, since a sizeable—but subjected to 8-fold-span variations-proportion is lost in the urine. This alone points towards a considerable excess of manganese in both diets studied. Obesity does not have a significant effect on the abilities to absorb and retain minerals, since these processes were more related to dietary availability. Management of essential metals by obese rats depends whether this condition is genetic or induced by diet. Most of the differences observed can be related to differences in diet concentration, to the excess fat content or different metabolic attitude to use substrates of obese animals. The data presented show that the cafeteria diet used adequately serves the mineral needs of the rat, since the rat adapts its absorbing and retaining strategies to match the dietary availability of these minerals.     

Individual amino acid balances in young lean and obese Zucker rats fed a cafeteria diet

The amino acid composition of the diet ingested by reference and cafeteria diet-fed lean and obese Zucker rats has been analyzed from day 30 to 60 after birth. Their body protein amino acid composition was measured, as well as the urinary and faecal losses incurred during the period studied. The protein actually selected by the rats fed the cafeteria diet had essentially the same amino acid composition as the reference diet. The mean protein amino acid composition of the rat showed only small changes with breed, age or diet.Cafeteria-fed rats had a higher dietary protein digestion/absorption efficiency than reference diet-fed rats. Obese rats wasted a high proportion of dietary amino acids when given the reference diet, but not on the cafeteria diet. In all cases, the amino acids lost as such in the urine were a minimal portion of available amino acids.In addition to breed, the rates of protein accretion are deeply influenced by diet, but even more by the age — or size — of the animals: cafeteria-fed rats grew faster, to higher body protein settings, but later protein accrual decreased considerably; this is probably due to a limitation in the blueprint for growth which restricts net protein deposition when a certain body size is attained. Obese rats, however, kept accuring protein with high rates throughout.Diet composition — and not protein availability or quality-induced deep changes in amino acid metabolism. Since the differences in the absolute levels of dietary protein or carbohydrate energy ingested by rats fed the reference or cafeteria diets were small, it can be assumed that high (lipid) energy elicits the changes observed in amino acid metabolism by the cafeteria diet. The effects induced in the fate of the nitrogen ingested were more related to the fractional protein energy proportion than to its absolute values. Cafeteria-fed rats tended to absorb more amino acids and preserve them more efficiently; these effects were shown even under conditions of genetic obesity.There were deep differences in handling of dietary amino acids by dietary or genetically obese rats. The former manage to extract and accrue larger proportions of their dietary amino acids than the latter. The effects of both models of amino acid management were largely additive, suggesting that the mechanisms underlying the development of obesity did not run in parallel to those affecting the control of amino acid utilization. Obesity may be developed in both cases despite a completely different strategy of amino acid assimilation, accrual and utilization. (Mol Cell Biochem121: 45–58, 1993)     

Primary cultures of fetal hepatocytes from the genetically obese Zucker rat: Protein synthesis

Summary Primary fetal hepatocytes derived from Zucker rats with expectedfa gene frequencies of 0.0 and 0.75 have been established and can be used to detect early effects of thefa gene on hepatocellular metabolism. Paired incubation experiments demonstrate that protein synthesis in 0.75fa gene cultures is significantly less than in 0.0fa gene cultures under basal conditions. Insulin stimulates protein synthesis in 0.0fa gene cultures but has no effect on 0.75fa gene cultures. Cycloheximide inhibits protein synthesis in both types of culture. NH₄Cl inhibits protein synthesis in 0.0 but not in 0.75fa gene cultures. These data suggest that fetal hepatocytes bearing thefa gene have in vitro a generally sluggish anabolic capacity and a blunted capacity to respond to insulin compared to fetal hepatocytes without thefa gene. These diminished capacities may be expressions of a genetic error in lysosomal function. A portion of this work was presented in preliminary form at the 1980 meeting of the Tissue Culture Association. This work was supported in part by National Institutes of Health Grants AM19382 and AM06197.     

Primary cultures of fetal hepatocytes from the genetically obese Zucker rat: Characterization and total lipogenesis

Summary Primary fetal hepatocyte cultures derived from Zucker rats and with expectedfa-gene frequencies of 0.0 and 0.75 have been established and can be used to detect early effects of thefa gene on hepatocellular metabolism. Proliferative capacity is similar in both types of culture. Changes of the growth media significantly decrease total lipogenesis in both 0.0 and 0.75fa-gene culture grown in arginine-free DME medium. Paired incubation experiments demonstrate that total lipogenesis in 0.75fa gene cultures is significantly less than in 0.0fa-gene cultures under basal conditions. Stimulation of total lipogenesis by pharmacological doses of insulin and excess substrate (glucose) is significantly less in the 0.75fa gene than in the 0.0fa-gene cultures. These data suggest that the development of obesity in the Zucker rat cannot be attributed to elevated hepatic lipogenesis in the fetus. This work constitutes part of a Ph.D. dissertation submitted to New York University by A. L. Goldstein. A portion of this work was presented in preliminary form at the 1978 meeting of the Tissue Culture Association. Supported in part by National Institutes of Health Grant AM 19382 and a Grant from the Weight Watchers Foundation, Inc.     

Splanchnic amino acid pattern in genetic and dietary obesity in the rat

The study of intestinal and hepatic uptake of amino acids by obese rats has been the main objective of this work. The obese animals used were either from genetic or from nutritional basis. In fed state, the intestinal release of amino acids was higher in obese animals than in lean ones (around the double values), but nutritionally and genetically obese rat showed a related pattern, specially for the case of alanine (increased release in relation to controls by a factor of 10). The higher alanine release by intestine is not reversed by 12-h food deprivation. The hepatic availability was also higher in obesity models than in lean animals (increases over 30%). However, the hepatic uptake was increased in genetically obese animals (more than 35%) and decreased in nutritionally obese animals (more than 40%), especially due to alanine uptake(2419, 1100 and 3794 nmols/min/g protein in lean, Diet-ob andfa/fa animals respectively). In obese animals the food deprivation tended to normalize the hepatic uptake of alanine. The differences in alanine uptake between both types of obesity may reflect the differences of urea synthesis.     

Insulin binding in the hypothalamus of lean and genetically obese Zucker rats

Recent reports have suggested that the obesity and hyperphagia of the genetically obese Zucker rat may be related to defective insulin action or binding in the hypothalamus. We used quantitative autoradiography to determine if insulin binding is altered in specific hypothalamic nuclei associated with food intake. Insulin binding was measured in the arcuate (ARC), dorsomedial (DMN), and ventromedial (VMN) hypothalamic nuclei of 3–4-month-old lean (Fa/Fa) and genetically obese (fa/fa) Zucker rats. A consistently reproducible 15% increase in the total specific binding of 0.1 nM [¹²⁵I]-insulin was found in the ARC of the obese genotype. A slight increase in insulin binding in the DMN was also found. No difference in specific insulin binding was found between genotypes in the VMN. Nonlinear least squares analysis of competitive binding studies showed that the K_d of the ARC insulin binding site was 33% higher in the lean rats than in the obese rats, indicating an increased affinity for insulin. No difference in site number (B_max) was found in the ARC, DMN or VMN, and no evidence was found for reduced insulin binding in the hypothalamus of the obese (fa/fa) genotype. The results suggest that hyperphagia and obesity of the obese (fa/fa) Zucker rat genotype may be associated with increased insulin binding in the arcuate nucleus.     

Effects of intracerebroventricular leptin administration on feeding and sexual behaviors in lean and obese female Zucker rats

Obese Zucker rats (fa/fa) are characterized by inadequate leptin signaling caused by a mutation in the leptin receptor gene. Obese Zucker females are infertile and hyporesponsive to the inductive effects of ovarian hormones on sexual behaviors. Leptin treatment reverses aspects of reproductive dysfunction due to perturbations in energy balance in other animal models. Our first experiment tested the hypothesis that intracerebroventricular (icv) leptin administration would enhance the display of sexual behaviors in obese Zucker females. A second experiment compared lean and obese Zucker females' responses to leptin, during fed and fasted conditions. Ovariectomized (OVX) Zucker rats were implanted with lateral ventricular cannulae. In Experiment 1, fasted, obese females received estradiol benzoate, progesterone, and icv injections of 3, 18, or 36 microg murine leptin or vehicle. Leptin administration reduced food intake, but did not enhance sexual behaviors. In Experiment 2, steroid-replaced, OVX lean and obese females (from a different source than those in Experiment 1) received icv injections of vehicle or 3 or 36 microg leptin under fed and fasted conditions. Leptin treatment reduced food intake and weight gain in the fed, but not the fasted, condition in both genotypes. Sexual receptivity and locomotion were not affected, but icv leptin injections reduced proceptive behaviors in ad libitum-fed rats. These data confirm previous reports that centrally administered leptin decreases food intake and weight gain in obese Zucker rats; results from Experiment 2 suggest that lean and obese females are similarly responsive to these actions of leptin. Contrary to our hypothesis, leptin treatment did not stimulate sexual behaviors; rather, the hormone appears to inhibit the display of sexual proceptivity in ad libitum-fed lean and obese Zucker female rats.     

Differences in L-alanine uptake by livers of Wistar and lean Zucker rats

The L-alanine uptake by livers of Wistar and lean Zucker rats has been studied. The hepatic uptake and fractional extraction rates of alanine were estimated in 50–55 day old rats. No significant differences in amino acid concentrations and blood flows in afferent and efferent liver vessels were seen in lean Zucker rats when compared with Wistar rats. However, the hepatic uptake (1.6±0.1 and 0.7±0.1 mol/min/100 g bw, p<0.01) and the fractional extraction (26.8±2.1 and 15.2±3.1%, p<0.05) were much lower in Zucker than in Wistar rats. The hepatic active transport of L-alanine was determinedin vitro using isolated plasma membrane vesicles. Vesicles isolated from livers of lean Zucker rats showed similar values of Km (2.5±0.7 vs 2.0±0.5 mM for Wistar and Zucker respectively, N.S.), but lower values of Vmax when compared with Wistar rats (1.1±0.1 vs 0.6±0.005 nmol/mg prot 5 s, p<0.01, for Wistar and lean Zucker rats respectively). These results indicate that, the liver of lean Zucker rats concentrates alanine less efficiently than the liver of Wistar rats. This fact correlates well with a lower capacity of the Na⁺-dependent L-alanine trasport in liver plasma membrane vesicles from lean Zucker rats.     

Effects of CCK on gastrointestinal function in lean and obese Zucker rats

Cholecystokinin (CCK), a hormone affecting several gastrointestinal functions, has also been shown to elicit satiety and affect daily meal patterns. Since Zucker obese rats are less sensitive to the satiety effects of CCK, two experiments were designed to determine if they are also less sensitive to the gastric emptying and intestinal transit rate effects of CCK. In the first experiment phenol red was administered to 5.5 hr fasted rats 15 minutes after intraperitoneal injection of CCK-8 or saline. Rats were sacrificed after 30 minutes, the stomach and small intestine were removed, and phenol red content was measured. More phenol red was in the stomach of obese but not lean rats treated with CCK-8. The rate of transit of the contents of the small intestine was increased by CCK-8 and the percent of phenol red in the fourth quarter of the small intestine was greater in obese than lean rats (91 vs 37%, p<0.05). In the second experiment gastrointestinal transit of ferric oxide was measured during the light and dark phases of the diurnal cycle, and when obese rats were ad lib or yoke-fed to lean pair-mates. Total gastrointestinal transit time of the ferric oxide was decreased 15% when CCK-8 was administered to yoke-fed obese rats in either the light or dark portions of the diurnal cycle but was not affected in ad lib-fed obese rats or lean rats. Thus, while Zucker obese rats are less sensitive to satiety effects of CCK, they appear to be more sensitive to the gastrointestinal effects of CCK, and therefore it is not clear what role these gastrointestinal responses have on the feeding behavior responses.     

Calcitonin mRNA activity in young obese (fa/fa) Zucker rats

Alterations in both calcitonin (CT) secretion and plasma calcium were recently described in adult obese Zucker rats. We have investigated the CT biosynthetic activity of thyroid glands in 30-day-old obese Zucker rats (fa/fa), and their controls (Lean). Plasma calcium level was significantly increased (+0.6 mg/dl) in obese animals, but plasma phosphate was unchanged. Plasma CT levels measured by radioimmunoassay (RIA) were significantly decreased in fatty (0.50 +/- 0.03 vs 0.68 +/- 0.03 ng/ml in Leans; P less than 0.001), but thyroidal hormone content was not different between Lean and fatty rats (68.7 +/- 5.1 in Leans vs 60.5 +/- 3.6 ng/gland in fatty rats). mRNA was extracted from 10 thyroids, and translated in a rabbit reticulocyte lysate (NEN) in the presence of [35S]methionine. After polyacrylamide gel electrophoresis, specific immunoprecipitates were autoradiographed and quantified by integration. A 50% decrease in translatable CT mRNA was observed in fatty rats. In basal conditions, the biosynthetic activity of C cells in obese rats correlates with the secretion rate of the hormone in the face of unchanged thyroidal CT contents.     

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

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

Response of genetically obese Zucker rats to ciprofibrate,a hypolipidemic agent,with peroxisome proliferation activity as compared to Zucker lean and Sprague-Dawley rats

Summary— Genetically obese Zucker (fa/fa) rats were used as an experimental model to study the effects of hypolipidemic agents on peroxisome proliferation; comparison was made with Zucker lean phenotype (Fa/?) and Sprague-Dawley strain/phenotype. The pharmacokinetics of a single administration of ciprofibrate (1 or 3 mg/kg), appeared to be similar in all strains/phenotypes. After a 2-week oral administration at the same dosages, there were dosage-related increases in hepatocellular peroxisomal yield and in the hepatic enzymes' cyanide-insensitive acyl-CoA oxidase and catalase. The peroxisomal yield was less increased in Zucker than in Sprague-Dawley rats, while the enzyme activities were similarly increased. Although the absolute specific activity of microsomal ω-lauryl hydroxylase (cytochrome P4504A1) was lower in Zucker rats, it was increased more in this strain than in Sprague-Dawley rats in response to drug exposure. The hypolipidemic effect (cholesterol and triglyceride reduction) was more pronounced in Zucker obese rats. Based on biochemical and morphological results, no major differences between strains/phenotypes in terms of peroxisome proliferation were observed following a 2-week administration of ciprofibrate.     

Fatty liver and insulin resistance in obese Zucker rats: no role for mitochondrial dysfunction

The relationship between insulin resistance and mitochondrial function is of increasing interest. Studies looking for such interactions are usually made in muscle and only a few studies have been done in liver, which is known to be a crucial partner in whole body insulin action. Recent studies have revealed a similar mechanism to that of muscle for fat-induced insulin resistance in liver. However, the exact mechanism of lipid metabolites accumulation in liver leading to insulin resistance is far from being elucidated. One of the hypothetical mechanisms for liver steatosis development is an impairment of mitochondrial function. We examined mitochondrial function in fatty liver and insulin resistance state using isolated mitochondria from obese Zucker rats. We determined the relationship between ATP synthesis and oxygen consumption as well as the relationship between mitochondrial membrane potential and oxygen consumption. In order to evaluate the quantity of mitochondria and the oxidative capacity we measured citrate synthase and cytochrome c oxidase activities. Results showed that despite significant fatty liver and hyperinsulinemia, isolated liver mitochondria from obese Zucker rats display no difference in oxygen consumption, ATP synthesis, and membrane potential compared with lean Zucker rats. There was no difference in citrate synthase and cytochrome c oxidase activities between obese and lean Zucker rats in isolated mitochondria as well as in liver homogenate, indicating a similar relative amount of hepatic mitochondria and a similar oxidative capacity. Adiponectin, which is involved in bioenergetic homeostasis, was increased two-fold in obese Zucker rats despite insulin resistance. In conclusion, isolated liver mitochondria from lean and obese insulin-resistant Zucker rats showed strictly the same mitochondrial function. It remains to be elucidated whether adiponectin increase is involved in these results.     

Developmental changes in levels of growth hormone mRNA in Zucker rats

Levels of pituitary growth hormone (GH) messenger RNA (mRNA) were compared in groups of genetically obese (fa/fa) and lean (Fa/-) littermate male Zucker rats at four different ages, 3, 5, 9, and 11 weeks, in order to determine the earliest age at which a difference between the two groups could be detected. No difference was seen in three-week-old animals. Five weeks of age was the earliest time at which the level of GH mRNA was significantly decreased in the obese rats; this decrease was present at all subsequent ages. Mean serum growth hormone levels were lower in obese animals at all ages, but the differences were not statistically significant because of the large individual variation associated with the pulsatile nature of GH release. The earliest occurrence of differences in GH mRNA level is later than some of the obesity associated abnormalities present in adipose tissue. The earliest time of the GH mRNA differences can be associated with the time when decreased protein deposition is initially seen in the obese rats. Because of this association, decreased GH mRNA may enhance the development of obesity.     

Amino acid uptake regulation by cell growth in cultured hepatocytes isolated from fetal and adult rats

Amino acid uptake mediated by system A was studied in cultured fetal and adult hepatocytes, subjected to growth stimulation by EGF and insulin, or to growth inhibition by high cell density. The mitogenic stimulation induced a strong transport increase only in fetal cells, while the cell density-dependent growth inhibition, probably mediated by molecules present on adult hepatocyte membranes, provoked the decrease of amino acid uptake only in the adult cells. The results indicate that the different modulation of amino acid transport by cell growth is dependent on the age and the differentiation stage of hepatocytes.     

CCK-resistance in Zucker obese versus lean rats

Obese Zucker rats are less sensitive to the satiety effect of CCK than lean litter mates. The present studies further characterised this CCK resistance. Subcutaneous injection of the CCK agonist caerulein dose-dependently decreased food intake in Zucker obese and lean rats whereas the CCK-B agonist gastrin-17 did not. Caerulein at 4 μg/kg, which resulted in CCK plasma bioactivity slightly above postprandial levels, decreased food intake in lean rats but not in obese rats. The decrease in food intake was also more marked at higher caerulein doses (20–100 μg/kg) in lean versus obese rats. In lean animals the satiety effects of the “near physiological” 4 μg/kg caerulein dose was abolished after blockade of vagal afferents with capsaicin, whereas the effects of higher caerulein doses were not. CCK-stimulated amylase secretion from pancreatic acini and binding capacity of ¹²⁵I- labelled CCK-8 were decreased in obese versus lean rats. The CCK-A antagonist loxiglumide at 20 mg/kg, a dose which abolished the action of all caerulein doses on food intake, failed to alter the food intake either in obese or in lean rats when given without an agonist. The results suggest that the satiety effects of “near physiological” doses of caerulein in lean rats are mediated by vagal afferents whereas pharmacological doses act via non-vagal mechanisms. The differences in CCK's satiety effect between lean and obese rats may be due to differences in CCK-receptor binding and action at peripheral vagal sites. However, the failure of the CCK-A antagonist to increase food intake questions whether any of the effects of exogenous CCK are of physiological relevance.     

Cysteine uptake and taurine biosynthesis in freshly isolated and primary cultured rat hepatocytes

Analysis of the uptake and metabolism of [14C]cysteine in rat liver was undertaken using freshly isolated hepatocytes and hepatocytes maintained in primary culture. The uptake of [14C]cysteine by freshly isolated hepatocytes was by means of both saturable and non-saturable transport systems and the former system was thought to involve facilitated diffusion. The uptake of [14C]cysteine by hepatocytes maintained in primary culture for 24 h also consisted of non-saturated and saturated transport mechanisms. The magnitude of the saturable transport system in cultured hepatocytes was, however, much greater than that found in freshly isolated hepatocytes, and was considered to be operated by active transport. Both freshly isolated and primary cultured hepatocytes had cysteine sulphinic acid decarboxylase activity, but this enzyme activity in the latter cells was noticeably reduced in comparison with that found in freshly isolated hepatocytes. Hepatocytes maintained in primary culture produced not only radiolabelled taurine, but also radiolabelled cysteine sulphinic acid, hypotaurine and alanine when incubated with [14C]cysteine. The present results indicate that cultured hepatocytes actively transport cysteine as well as metabolizing cysteine to taurine via cysteine sulphinic acid and hypotaurine.     

Dephosphorylation increases insulin-stimulated receptor kinase activity in skeletal muscle of obese Zucker rats

Serine/threonine phosphorylation of insulin receptor has been implicated in the development of insulin resistance. To investigate whether dephosphorylation of serine/threonine residues of the insulin receptor may restore the decreased insulin-stimulated receptor tyrosine kinase activity in skeletal muscle of obese Zucker rats, insulin receptor tyrosine kinase activity was measured before and after alkaline phosphatase treatment. Compared to lean controls, insulin-stimulated glucose transport was depressed by 61% (p < 0.05) in obese Zucker rats. The insulin receptor and insulin receptor substrate-1 contents were decreased by 14% (p < 0.05) and 16% (p < 0.05), respectively, in skeletal muscle of obese Zucker rats. In vivo insulin-induced tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 was depressed by 82% (p < 0.05) and 86% (p < 0.05), respectively. In the meantime, in vitro insulin-stimulated receptor tyrosine kinase activity in obese rats was decreased by 39% (p < 0.05). Dephosphorylation of the insulin receptor by prior alkaline phosphatase treatment increased insulin-stimulated receptor tyrosine kinase activity in both lean and obese Zucker rats, but the increase was three times greater in obese Zucker rats (p < 0.05). These findings suggest that excessive serine/threonine phosphorylation of the insulin receptor in obese Zucker rats may be a cause for insulin resistance in skeletal muscle.