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.     

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

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

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

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

Use of Glycated Hemoglobin to Assess Glycemic Control in Wistar Diabetic Fatty Rats and Zucker Fatty Rats

The Wistar Diabetic Fatty rat (WDF fafa) is a con-genic strain of the Wistar Kyoto rat. Studies using blood glucose reveal that only fatty male (not female) WDF rats spontaneously develop hyperglycemia when fed a stock diet Blood glucose values have not provided consistent results for evaluation of glycemic status in fatty male WDF rats. Zucker fatty (fafa) rats, while sharing the fa gene and the development of hyperinsulinemia and hyperlipemia, do not spontaneously become hyperglycemic. In order to examine strain differences and the effects of age on long-term average glycemic status in WDF and Zucker rats, glycated hemoglobin (GHb) was analyzed. Glycated hemoglobin was measured in male lean and obese WDF and Zucker rats at 2,3,6, and 12 months of age. Nonfasted plasma glucose was measured in male lean and obese WDF rats at 2, 3, 6, and 12 months of age and in lean and obese Zucker rats at 3, 6, and 12 months of age. Plasma insulin was measured in lean and obese WDF and Zucker rats at 3, 6, and 12 months of age. Obese WDF rats had significantly elevated GHb compared to lean controls at 3, 6, and 12 months of age. Glycated hemoglobin was substantially above the normal range (3.8-6.5%) at 3 months of age (14.1%). Glycated hemoglobin significantly declined in the obese WDF rats between 6 and 12 months of age. Nonfasted plasma glucose was significantly elevated in the obese WDF rats at 3 months (14.1 ± 2.1 mM/L) and 6 months of age (16.2 ± 2.3 mM/L) compared to lean controls. At 12 months of age there was no difference in plasma glucose between obese and lean WDF rats. Obese and lean Zucker rats had similar levels of GHb and plasma glucose at all ages. In conclusion, GHb provides more integrated data for classifying disease status of WDF rats and evaluation of potential long-term complications associated with hyperglycemia.     

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.     

Insulin resistance in the New Zealand obese mouse (NZO): lipolysis and lipogenesis in isolated adipocytes

The marked stimulatory effect of insulin on the metabolism of [U-¹⁴C]glucose to CO₂, glyceride-glycerol, and fatty acid observed with adipocytes from normal New Zealand yellow (NZY) mice and young (nonobese) New Zealand obese (NZO) mice was greatly diminished in cells obtained from adult obese NZO mice. Adipocytes from obese NZO mice had lower basal rates of CO₂ formation and fatty acid synthesis than cells from NZY or young NZO mice. Glyceride-glycerol was labeled to a similar extent under basal conditions in adipocytes from all three groups of mice, implying that the basal rate of glucose transport and the enzymes of the glycolytic pathway are intact in obese NZO adipocytes. Both basal and epinephrine-stimulated lipolysis were impaired in adipocytes from obese NZO mice when compared with cells from NZY and young NZO mice. Epinephrine-stimulated lipolysis was markedly less sensitive to the inhibitory effect of insulin in adipocytes from obese NZO mice than in NZY and young NZO controls. These studies suggest that adipocytes from young, nonobese NZO mice do not exhibit resistance to epinephrine and insulin, and that hormone resistance and decreased rates of metabolism accompany the onset and evolution of obesity.     

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

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

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

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

Evidence for activation of inflammatory lipoxygenase pathways in visceral adipose tissue of obese Zucker rats

Central obesity is associated with low-grade inflammation that promotes type 2 diabetes and cardiovascular disease in obese individuals. The 12- and 5-lipoxygenase (12-LO and 5-LO) enzymes have been linked to inflammatory changes, leading to the development of atherosclerosis. 12-LO has also been linked recently to inflammation and insulin resistance in adipocytes. We analyzed the expression of LO and proinflammatory cytokines in adipose tissue and adipocytes in obese Zucker rats, a widely studied genetic model of obesity, insulin resistance, and the metabolic syndrome. mRNA expression of 12-LO, 5-LO, and 5-LO-activating protein (FLAP) was upregulated in adipocytes and adipose tissue from obese Zucker rats compared with those from lean rats. Concomitant with increased LO gene expression, the 12-LO product 12-HETE and the 5-LO products 5-HETE and leukotriene B4 (LTB4) were also increased in adipocytes. Furthermore, upregulation of key proinflammatory markers interleukin (IL)-6, TNFα, and monocyte chemoattractant protein-1 were observed in adipocytes isolated from obese Zucker rats. Immunohistochemistry indicated that the positive 12-LO staining in adipose tissue represents cells in addition to adipocytes. This was confirmed by Western blotting in stromal vascular fractions. These changes were in part reversed by the novel anti-inflammatory drug lisofylline (LSF). LSF also reduced p-STAT4 in visceral adipose tissue from obese Zucker rats and improved the metabolic profile, reducing fasting plasma glucose and increasing insulin sensitivity in obese Zucker rats. In 3T3-L1 adipocytes, LSF abrogated the inflammatory response induced by LO products. Thus, therapeutic agents reducing LO or STAT4 activation may provide novel tools to reduce obesity-induced inflammation.     

Subchronic treatment with metformin produces anorectic effect and reduces hyperinsulinemia in genetically obese Zucker rats.

The effect of subchronic metformin treatment on food intake, weight gain and plasma and tissue hormone levels was investigated in genetically obese male Zucker rats and in their lean controls. Metformin hydrochloride (320 mg/kg/day for 14 days in the drinking water) significantly reduced 24 hour food intake both after one and two weeks treatment in obese rats. In contrast, metformin had only a transient effect on food intake in lean animals. The reduced food intake was associated with body weight decrease, particularly in obese rats. Metformin markedly reduced also the hyperinsulinemia of the obese animals without altering their plasma glucose or pancreatic insulin content which may reflect an improved insulin sensitivity after metformin treatment. Metformin did not change plasma corticosterone levels or insulin and somatostatin concentrations in the pancreas. Metformin reduced pyloric region somatostatin content in lean rats. It is concluded that metformin has an anorectic effect and reduces body weight and hyperinsulinemia in genetically obese Zucker rat.     

Insulin resistance in soleus muscle from obese Zucker rats. Involvement of several defective sites

1. The effect of insulin upon glucose transport and metabolism in soleus muscles of genetically obese (fa/fa) and heterozygote lean Zucker rats was investigated at 5–6 weeks and 10–11 weeks of age. Weight-standardized strips of soleus muscles were used rather than the intact muscle in order to circumvent problems of diffusion of substrates. 2. In younger obese rats (5–6 weeks), plasma concentrations of immunoreactive insulin were twice those of controls, whereas their circulating triacylglycerol concentrations were normal. Insulin effects upon 2-deoxyglucose uptake and glucose metabolism by soleus muscles of these rats were characterized by both a decreased sensitivity and a decrease in the maximal response of this tissue to the hormone. 3. In older obese rats (10–11 weeks), circulating concentrations of insulin and triacylglycerols were both abnormally elevated. A decrease of 25–35% in insulin-binding capacity to muscles of obese rats was observed. The soleus muscles from the older obese animals also displayed decreased sensitivity and maximal response to insulin. However, at a low insulin concentration (0.1m-i.u./ml), 2-deoxyglucose uptake by muscles of older obese rats was stimulated, but such a concentration was ineffective in stimulating glucose incorporation into glycogen, and glucose metabolism by glycolysis. 4. Endogenous lipid utilization by muscle was calculated from the measurements of O₂ consumption, and glucose oxidation to CO₂. The rate of utilization of fatty acids was normal in muscles of younger obese animals, but increased in those of the older obese rats. Increased basal concentrations of citrate, glucose 6-phosphate and glycogen were found in muscles of older obese rats and may reflect intracellular inhibition of glucose metabolism as a result of increased lipid utilization. 5. Thus several abnormalities are responsible for insulin resistance of muscles from obese Zucker rats among which we have observed decreased insulin binding, decreased glucose transport and increased utilization of endogenous fatty acid which could inhibit glucose utilization.     

Contraction-activated glucose uptake is normal in insulin-resistant muscle of the obese Zucker rat.

The rates of muscle glucose uptake of lean and obese Zucker rats were assessed via hindlimb perfusion under basal conditions (no insulin), in the presence of a maximal insulin concentration (10 mU/ml), and after electrically stimulated muscle contraction in the absence of insulin. The perfusate contained 28 mM glucose and 7.5 microCi/mmol of 2-deoxy-D-[3H-(G)]glucose. Glucose uptake rates in the soleus (slow-twitch oxidative fibers), red gastrocnemius (fast-twitch oxidative-glycolytic fibers), and white gastrocnemius (fast-twitch glycolytic fibers) under basal conditions and after electrically stimulated muscle contraction were not significantly different between the lean and obese rats. However, the rate of glucose uptake during insulin stimulation was significantly lower for obese than for lean rats in all three fiber types. Significant correlations were found for insulin-stimulated glucose uptake and glucose transporter protein isoform (GLUT-4) content of soleus, red gastrocnemius, and white gastrocnemius of lean (r = 0.79) and obese (r = 0.65) rats. In contrast, the relationships between contraction-stimulated glucose uptake and muscle GLUT-4 content of lean and obese rats were negligible because of inordinately low contraction-stimulated glucose uptakes by the solei. These results suggest that maximal skeletal muscle glucose uptake of obese Zucker rats is resistant to stimulation by insulin but not to contractile activity. In addition, the relationship between contraction-stimulated glucose uptake and GLUT-4 content appears to be fiber-type specific.     

Effect of adrenalectomy on in vivo glucose metabolism in insulin resistant Zucker obese rats

Lean (Fa/?) and obese (fa/fa) Zucker rats were adrenalectomized (ADX) in order to assess the contribution of adrenal hormones to insulin resistance of the obese Zucker rat. Glucose utilization was measured using an insulin suppression test. Sham-operated obese rats gained almost twice as much weight as sham-operated lean littermates. However, body weight gain of ADX animals was comparable in both genotypes. It was significantly less than that of the respective sham-operated controls. Body weight differences can be accounted for almost entirely by a marked loss of adipose tissue. Although insulin resistance may be attributable to obesity in part, steroid hormones are thought to be directly antagonistic to insulin for glucose metabolism. Adrenalectomy resulted in a decrease in serum glucose concentrations for both lean and obese Zucker rats compared with their respective sham-operated groups. Serum insulin concentration of lean ADX rats was 23% of sham-operated controls; in obese ADX rats, it was 9% of controls. Elevated levels of steady state serum glucose (SSSG) levels in sham-operated obese rats demonstrate a marked resistance to insulin induced glucose uptake compared with sham-operated lean animals. Adrenalectomy caused a marked improvement in insulin sensitivity of obese rats. The hyperglycemic SSSG levels of the obese rats were reduced 2.5 times by ADX. These results indicate that insulin resistance of Zucker obese rats can be ameliorated by ADX, suggesting adrenal hormones contribute to insulin resistance in these animals.     

Effect of dietary Platycodon grandiflorum on the improvement of insulin resistance in obese Zucker rats

The effect of dietary Platycodon grandiflorum on the improvement of insulin resistance and lipid profile was investigated in lean (Fa/-) and obese (fa/fa) Zucker rats, a model for noninsulin dependent diabetes mellitus. Dietary Platycodon grandiflorum feeding for 4 weeks resulted in a significant decrease in the concentration of plasma triglyceride in both lean and obese Zucker rats. Furthermore, dietary Platycodon grandiflorum markedly decreased both plasma cholesterol and fasting plasma insulin levels, and significantly decreased the postprandial glucose level at 30 min during oral glucose tolerance test in obese Zucker rats. Although there was no statistical significance, the crude glucose transporter 4 protein level of obese rats fed Platycodon grandiflorum tended to increase when compared with that of obese control rats. Therefore, the present results suggested that dietary Platycodon grandiflorum may be useful in prevention and improvement of metabolic disorders characterized by hyperinsulinemia states such as noninsulin dependent diabetes mellitus, syndrome X, and coronary artery disease.     

Diazoxide down-regulates leptin and lipid metabolizing enzymes in adipose tissue of Zucker rats.

We have previously reported that attenuation of hyperinsulinemia by diazoxide (DZ), an inhibitor of glucose-mediated insulin secretion, increased insulin sensitivity and reduced body weight in obese Zucker rats. These findings prompted us to investigate the effects of DZ on key insulin-sensitive enzymes regulating adipose tissue metabolism, fatty acid synthase (FAS), and lipoprotein lipase (LPL), as well as on circulating levels of leptin. We also determined the direct effects of diazoxide on FAS in 3T3-L1 adipocytes. Seven-week-old female obese and lean Zucker rats were treated with DZ (150 mg/kg/d) or vehicle (C, control) for a period of 6 wk. Changes in plasma parameters by DZ include significant decreases in triglycerides, free fatty acids, glucose, and insulin, consistent with our previous reports. DZ obese rats exhibited lower plasma leptin levels (P<0.03) compared to their C animals. DZ significantly reduced adipose tissue FAS activity in both lean (P<0.0001) and obese (P<0.01) animals. LPL mRNA content was also decreased significantly in DZ-treated obese animals (P<0.009) as compared to their respective controls without a significant effect on lean animals. The possibility that DZ exerted a direct effect on adipocytes was further tested in cultured 3T3-L1 adipocytes. Although diazoxide (5 microM) alone did not change FAS activity in cultured 3T3-L1 adipocytes, it significantly attenuated insulin's effect on FAS activity (P<0.001). We demonstrate that DZ regulates key insulin-sensitive enzymes involved in regulation of adipose tissue metabolism. These findings suggest that modification of insulin-sensitive pathways can be therapeutically beneficial in obesity management.     

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

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

Insulin resistance in fat cells from obese Zucker rats - Evidence for an impaired activation and translocation of protein kinase B and glucose transporter 4

The effect of insulin on glucose transport, glucose transporter 4 (Glut4) translocation, and intracellular signaling were measured in fat cells from lean and obese Zucker rats of different ages. Insulin-stimulated glucose transport was markedly reduced in adipocytes from old and obese animals. The protein content of Glut4 and insulin receptor substrates (IRS) 1 and 2 were also reduced while other proteins, including the p85 subunit of PI3-kinase, Shc and the MAP kinases (ERK1 and 2) were essentially unchanged. There was a marked impairment in the insulin stimulated tyrosine phosphorylation of IRS-1 and 2 as well as activation of PI3-kinase and PKB in cells from old and obese animals. Furthermore, insulin-stimulated translocation of both Glut4 and PKB to the plasma membrane was virtually abolished. The phosphotyrosine phosphatase inhibitor, vanadate, increased the insulin- stimulated upstream signaling including PI3-kinase and PKB activities as well as rate of glucose transport. Thus, the insulin resistance in cells from old and obese Zucker rats can be accounted for by an impaired translocation process, due to signaling defects leading to a reduced activation of PI3-kinase and PKB, as well as an attenuated Glut4 protein content.     

Diazoxide effects on hypothalamic and extra-hypothalamic NPY content in Zucker rats

To determine if the anorectic effects of the insulin antagonist diazoxide (DZ) are mediated by reduced central neuropeptide Y (NPY), female Zucker rats, given DZ (150 mg/kg/day) or placebo for about four weeks, were sacrificed following overnight fasting or free feeding. Several hypothalamic and extra-hypothalamic nuclei were extracted for NPY content. DZ reduced weight gain in obese rats and lowered glucose of lean and obese rats without affecting insulin. Contrary to the hypothesis, DZ increased NPY in hypothalamic nuclei of free fed lean and obese rats. DZ elevated hypothalamic NPY levels in fasted obese rats and had more diverse effects in extra-hypothalamic nuclei of lean rats.     

Fructose transport and metabolism in adipose tissue of Zucker rats: Diminished GLUT5 activity during obesity and insulin resistance

Fructose is a major dietary sugar, which is elevated in the serum of diabetic humans, and is associated with metabolic syndromes important in the pathogenesis of diabetic complications. The facilitative fructose transporter, GLUT5, is expressed in insulin-sensitive tissues (skeletal muscle and adipocytes) of humans and rodents, where it mediates the uptake of substantial quantities of dietary fructose, but little is known about its regulation. We found that GLUT5 abundance and activity were compromised severely during obesity and insulin resistance in Zucker rat adipocytes. Adipocytes from young obese (fa/fa), highly insulin-responsive Zucker rats contained considerably more plasma membrane GLUT5 than those from their lean counterparts (1.8-fold per microgram membrane protein), and consequently exhibited higher fructose transport (fivefold) and metabolism (threefold) rates. Lactate production was the preferred route for fructose metabolism in these cells. As the rats aged and become more obese and insulin-resistant, adipocyte GLUT5 surface density (12-fold) and fructose transport (10-fold) and utilisation rates (threefold) fell markedly. The GLUT5 loss was more dramatic in adipocytes from obese animals, which developed a more marked insulin resistance than lean counterparts. The decline of GLUT5 levels in adipocytes from older, obese animals was not a generalised effect, and was not observed in kidney, nor was this expression pattern shared by the 1 subunit of the Na⁺/K⁺ ATPase. Our findings suggest that plasma membrane GLUT5 levels and thus fructose utilisation rates in adipocytes are dependent upon cellular insulin sensitivity, inferring a possible role for GLUT5 in the elevated circulating fructose observed during diabetes, and associated pathological complications. (Mol Cell Biochem 261: 23–33, 2004)