Metformin, but not exercise training, increases insulin responsiveness in skeletal muscle of Sprague-Dawley rats

We assessed the effects of combined metformin treatment and exercise training on body composition, on insulin concentration following glucose loading, on insulin-stimulated glucose transport in skeletal muscle, and on muscle glycogen content. Male Sprague-Dawley rats were treated for 35 days with or without metformin (320 mg/kg/day) and/or treadmill exercise training (20 min at 20 m/min, 5 days/wk). Because metformin reduces food intake, pair-fed controls were included. Metformin, training, and pair-feeding all decreased food intake, body weight, and insulin concentration following glucose loading. Metformin and training reduced intra-abdominal fat, but pair feeding did not. In isolated strips derived from soleus, epitrochlearis and extensor carpi ulnaris muscles, metformin increased insulin-stimulated transport of [3H]-2-deoxyglucose by 90%, 89% and 125%, respectively (P < 0.02) and training increased [3H]-2-deoxyglucose transport in the extensor carpi ulnaris muscle only (66%, P < 0.05). Pair-feeding did not alter [3H]-2-deoxyglucose transport. Training increased gastrocnemius muscle glycogen by 100% (P < 0.001). Metformin and pair-feeding did not alter muscle glycogen. We conclude that metformin reverses the maturation-induced impairment of insulin responsiveness in Sprague-Dawley rats by increasing insulin-stimulated glucose transport in skeletal muscle and that this effect is not secondary to reduced food intake. We also conclude that metformin and exercise training may increase insulin sensitivity by different mechanisms, with training causing increased glucose transport only in some muscles and also causing increased muscle glycogen storage.     

Interactive effect of exercise training and growth hormone administration on glucose tolerance and muscle GLUT4 protein expression in rats

The insulin-resistance effect of growth hormone (GH) administration has been frequently reported. The present study investigated the effect of GH administration on glucose tolerance and muscle GLUT4 protein expression in exercise-trained and untrained rats. Forty-eight rats were weight-matched and assigned to the following 4 groups: control, GH, exercise training, and exercise training + GH groups. After 2 weeks of GH injections (65 µg/kg/day) and exercise training, the glucose tolerance and insulin response were measured in these rats. The GLUT4 protein level, glycogen storage, and citrate synthase activity were determined in red gastrocnemius and plantaris muscles. Daily GH administration elevated the curves of the oral glucose tolerance test and insulin response compared with those of saline-injected control rats. Furthermore, exercise training completely eliminated this GH-induced insulin resistance as determined 18 h after the last bout of exercise training. Additionally, exercise training significantly increased muscle glycogen storage and GLUT4 protein levels. GH administration did not affect the GLUT4 protein and glycogen storage increases induced by exercise training, but the citrate synthase activity in the plantaris muscle was further elevated by GH administration to a level above that induced by training. In conclusion, this is the first study that demonstrates that regular exercise training prevents GH-induced insulin-resistance side effect in rats.     

Metformin treatment enhances insulin-stimulated glucose transport in skeletal muscle of Sprague-Dawley rats

Although the glucose-lowering properties of metformin are well-established, its effects on glucose metabolism in skeletal muscle have not been clearly defined. We tested the effects of metformin in young adult male Sprague-Dawley rats, which have a documented reduced response to insulin in skeletal muscle. Rats were treated with metformin for 20 days (320 mg/kg/day) in the drinking water. During this period, metformin completely prevented the increase in food intake and decreased adiposity by 30%. Metformin also reduced insulin secretion by 37% following an intra-peritoneal injection of glucose. Finally, metformin enhanced transport of [3H]-2-deoxyglucose in isolated strips of soleus muscle. Metformin substantially increased insulin-stimulated transport, while having no effect on basal transport. In control rats, a maximal concentration of insulin stimulated transport 77% above basal. In metformin-treated rats, insulin stimulated transport 206% above basal. We conclude that in the Sprague-Dawley rat model, metformin causes a significant increase in insulin-responsiveness.     

Association of resistin with visceral fat and muscle insulin resistance

Maturing Sprague-Dawley (S-D) rats develop obesity and skeletal muscle insulin resistance. To investigate the relationship between fat mass and insulin responses, we performed surgical removal of the epididymal and retroperitoneal depots of visceral adipose tissue (VF) or sham surgery (SHAM) in male rats aged 4 months. At sacrifice, 30 days later, the mass of visceral fat was 48% lower (p<0.05) in VF- compared to SHAM, while subcutaneous fat was essentially unchanged. VF- animals displayed increased insulin responses in isolated strips of skeletal muscle. Insulin-stimulated glucose transport was increased 28% in soleus muscle (p<0.05), with a trend toward a 31% increase in extensor digitorum longus muscle (p=0.058). Glucose tolerance was not significantly affected by surgical fat removal. In VF- animals, serum resistin was reduced 26% (p<0.05) and serum adiponectin was reduced 30% (p<0.05), with trends for reductions in IL-4 (58% reduction, p=0.084) and IL-6 (56% reduction, p=0.123). TNF-alpha, leptin and free fatty acids (NEFAs) were unchanged. We conclude that in maturing S-D rats, increased visceral adiposity leads to an increase in systemic release in resistin and possibly interleukins. Elevation of circulating cytokines may play a role in the development of muscle insulin resistance.     

Effect of chronic metformin treatment of hepatic and muscle glycogen metabolism in KK mice.

Noninsulin-dependent diabetic KK mice, aged 90-100 days, with hyperinsulinemia and insulin resistance were treated with either metformin (N = 13) or water (control, N = 10) orally at a concentration of 50 mg/kg twice daily for 28 weeks. Age-matched nondiabetic Swiss Webster (SW) mice were also similarly treated. Liver and skeletal muscle glycogen synthase and phosphorylase enzymes were determined in all groups of mice. Both enzymes were significantly lower in control KK than in control SW mice. Metformin did not influence either of these enzymes in nondiabetic SW mice. However, it significantly increased the active form of glycogen synthase (a form) in both the liver and muscle of KK mice. Metformin also increased the active form of phosphorylase (a form) in the liver but not in the muscle of these mice. Hepatic glycogen content was similar in both control and metformin-treated KK mice. However, the muscle glycogen content was significantly higher in metformin-treated than in control KK mice. These data suggest that metformin preferentially stimulates glycogen synthesis in skeletal muscle, and this seems to be responsible for the observed improvement in fasting glucose and glucose response to an oral glucose load in KK mice.     

Improvement of metabolic parameters and vascular function by metformin in obese non-diabetic rats

AimsMetformin is an insulin sensitizing agent with beneficial effects in diabetic patients on glycemic levels and in the cardiovascular system. We examined whether the metabolic changes and the vascular dysfunction in monosodium glutamate-induced obese non-diabetic (MSG) rats might be improved by metformin.Main methods16 week-old MSG rats were treated with metformin for 15 days and compared with age-matched untreated MSG and non-obese non-diabetic rats (control). Blood pressure, insulin sensitivity, vascular reactivity and prostanoid release in the perfused mesenteric arteriolar bed as well as nitric oxide production and reactive oxygen species generation in isolated mesenteric arteries were analyzed.Key findings18-week-old MSG rats displayed higher Lee index, fat accumulation, dyslipidemia, insulin resistance and hyperinsulinemia. Metformin treatment improved these alterations. The norepinephrine-induced response, increased in the mesenteric arteriolar bed from MSG rats, was corrected by metformin. Indomethacin corrected the enhanced contractile response in MSG rats but did not affect metformin effects. The sensitivity to acetylcholine, reduced in MSG rats, was also corrected by metformin. Indomethacin corrected the reduced sensitivity to acetylcholine in MSG rats but did not affect metformin effects. The sensitivity to sodium nitroprusside was increased in preparations from metformin-treated rats. Metformin treatment restored both the reduced PGI2/TXA2 ratio and the increased reactive oxygen species generation in preparations from MSG rats.SignificanceMetformin improved the vascular function in MSG rats through reduction in reactive oxygen species generation, modulation of membrane hyperpolarization, correction of the unbalanced prostanoids release and increase in the sensitivity of the smooth muscle to nitric oxide.     

Low-dose growth hormone treatment with diet restriction accelerates body fat loss, exerts anabolic effect and improves growth hormone secretory dysfunction in obese adults.

Growth hormone (GH) can induce an accelerated lipolysis. Impaired secretion of GH in obesity results in the consequent loss of the lipolytic effect of GH. Dietary restriction as a basic treatment for obesity is complicated by poor compliance, protein catabolism, and slow rates or weight loss. GH has an anabolic effect by increasing insulin-like growth factor (IGF)-I. We investigated the effects of GH treatment and dietary restriction on lipolytic and anabolic actions, as well as the consequent changes in insulin and GH secretion in obesity. 24 obese subjects (22 women and 2 men; 22-46 years old) were fed a diet of 25 kcal/kg ideal body weight (IBW) with 1.2 g protein/kg IBW daily and were treated with recombinant human GH (n = 12, 0.18 U/kg IBW/week) or placebo (n = 12, vehicle injection) in a 12-week randomized, double-blind and placebo-controlled trial. GH treatment caused a 1.6-fold increase in the fraction of body weight lost as fat and a greater loss of visceral fat area than placebo treatment (35.3 vs. 28.5%, p < 0.05). In the placebo group, there was a loss in lean body mass (-2.62 +/- 1.51 kg) and a negative nitrogen balance (-4.52 +/- 3.51 g/day). By contrast, the GH group increased in lean body mass (1.13 +/- 1.04 kg) and had a positive nitrogen balance (1.81 +/- 2.06 g/day). GH injections caused a 1.6-fold increase in IGF-I, despite caloric restriction. GH response to L-dopa stimulation was blunted in all subjects and it was increased after treatment in both groups. GH treatment did not induce a further increase in insulin levels during an oral glucose tolerance test (OGTT) but significantly decreased free fatty acid (FFA) levels during OGTT. The decrease in FFA area under the curve during OGTT was positively correlated with visceral fat loss. This study demonstrates that in obese subjects given a hypocaloric diet, GH accelerates body fat loss, exerts anabolic effects and improves GH secretion. These findings suggest a possible therapeutic role of low-dose GH with caloric restriction for obesity.     

GH receptor signaling in skeletal muscle and adipose tissue in human subjects following exposure to an intravenous GH bolus

Growth hormone (GH) regulates muscle and fat metabolism, which impacts on body composition and insulin sensitivity, but the underlying GH signaling pathways have not been studied in vivo in humans. We investigated GH signaling in biopsies from muscle and abdominal fat obtained 30 (n = 3) or 60 (n = 3) min after an intravenous bolus of GH (0.5 mg) vs. saline in conjunction with serum sampling in six healthy males after an overnight fast. Expression of the following signal proteins were assayed by Western blotting: STAT5/p-STAT5, MAPK, and Akt/PKB. IRS-1-associated PI 3-kinase activity was measured by in vitro phosphorylation of PI. STAT5 DNA binding activity was assessed with EMSA, and the expression of IGF-I and SOCS mRNA was measured by real-time RT-PCR. GH induced a 52% increase in circulating FFA levels with peak values after 155 min (P = 0.03). Tyrosine-phosphorylated STAT5 was detected in muscle and fat of all subjects after GH. Activation of MAPK was observed in several lysates but without GH dependency. Neither PKB/Akt nor PI 3-kinase activity was affected by GH. GH-induced STAT5 DNA binding and expression of IGF-I mRNA were detected in fat, whereas expression of SOCS-1 and -3 tended to increase after GH in muscle and fat, respectively. We conclude that 1) STAT5 is acutely activated in human muscle and fat after a GH bolus, but additional downstream GH signaling was significant only in fat; 2) the direct GH effects in muscle need further characterization; and 3) this human in vivo model may be used to study the mechanisms subserving the actions of GH on substrate metabolism and insulin sensitivity in muscle and fat.     

Adipose tumor necrosis factor-alpha is reduced during onset of insulin resistance in Sprague-Dawley rats

Overexpression of mRNA for tumor necrosis factor-alpha (TNF-alpha) has been observed in adipose tissue in several rodent models of insulin resistance. The purpose of the present study was to examine the expression of TNF-alpha protein during the onset of insulin resistance in maturing Sprague-Dawley (S-D) rats. Compared to 2 months, rats aged 5 and 12 months were glucose intolerant and fasting glucose was elevated at 12 months (p < 0.05). Compared to 2 months, insulin concentrations following glucose loading were elevated at 5 months (p < 0.05) and also at 12 months, but to a lesser degree. In isolated strips of soleus muscle, insulin-stimulated glucose transport was reduced by 38% and 59% between 2 and 5 months and between 2 and 12 months, respectively (p < 0.05), with no changes in basal transport. Insulin resistance was associated with decreased content of TNF-alpha protein in visceral and subcutaneous fat. TNF-alpha protein content was also decreased in tibialis anterior muscle, but was unchanged in soleus and red gastrocnemius muscles. Liver was the only tissue examined that showed an increase in TNF-alpha protein content. In vitro secretion of TNF-alpha protein was markedly reduced in explants of visceral and subcutaneous fat from mature, insulin-resistant animals, but TNF-alpha bioactivity in subcutaneous fat was maintained with age. These results indicate that the onset of insulin resistance in mature S-D rats is associated with reduced adipose expression of TNF-alpha. Our findings do not support the adipose-endocrine model of TNF-alpha in insulin resistance. Our findings do support a paracrine role for TNF-alpha or for a reduction in endogenous TNF-alpha inhibitors in insulin resistance.     

Metformin increases the PGC-1alpha protein and oxidative enzyme activities possibly via AMPK phosphorylation in skeletal muscle in vivo.

AMP-activated protein kinase (AMPK), which was activated by an antihyperglycemic drug metformin, has been hypothesized to mediate metabolic adaptations. The purposes of the present study were 1) to confirm whether acute metformin administration induced AMPK phosphorylation and 2) to determine whether chronic metformin treatment increased the peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) protein expression, glycolytic and oxidative enzyme activities, and cytochrome c and glucose transporter-4 (GLUT4) protein expressions in the rat soleus and red and white gastrocnemius muscles. The single oral administration of metformin (300 mg/kg body wt) enhanced the AMPK phosphorylation at 5 and/or 6 h after treatment. In the chronic study, rats were fed either normal chow or chow containing 1% metformin for 14 days. Metformin treatment resulted in a mean daily metformin intake of 631 mg.kg body wt(-1).day(-1). Metformin increased the PGC-1alpha content in all three muscles. Metformin increased the hexokinase activity in the white gastrocnemius, the citrate synthase activity in all three muscles, and the beta-hydroxyacyl-CoA dehydrogenase activity in the soleus. The cytochrome c protein content in the soleus muscle also increased. The GLUT4 content was unchanged by metformin. These results suggest that metformin enhances the PGC-1alpha expression and mitochondrial biogenesis possibly at least in part via AMPK phosphorylation in the skeletal muscle. Metformin has thus been proposed to possibly ameliorate insulin resistance, at least partially, by means of such metabolic effects.     

The contribution of visceral fat to improved insulin signaling in Ames dwarf mice

Ames dwarf (Prop1^df, df/df) mice are characterized by growth hormone (GH), prolactin, and thyrotropin deficiency, remarkable extension of longevity and increased insulin sensitivity with low levels of fasting insulin and glucose. Plasma levels of anti‐inflammatory adiponectin are increased in df/df mice, while pro‐inflammatory IL‐6 is decreased in plasma and epididymal fat. This represents an important shift in the balance between pro‐ and anti‐inflammatory adipokines in adipose tissue, which was not exposed to GH signals during development or adult life. To determine the role of adipose tissue in the control of insulin signaling in these long‐living mutants, we examined the effects of surgical removal of visceral (epididymal and perinephric) adipose tissue. Comparison of the results obtained in df/df mice and their normal (N) siblings indicated different effects of visceral fat removal (VFR) on insulin sensitivity and glucose tolerance. The analysis of the expression of genes related to insulin signaling indicated that VFR improved insulin action in skeletal muscle in N mice. Interestingly, this surgical intervention did not improve insulin signaling in df/df mice skeletal muscle but caused suppression of the signal in subcutaneous fat. We conclude that altered profile of adipokines secreted by visceral fat of Ames dwarf mice may act as a key contributor to increased insulin sensitivity and extended longevity of these animals.     

Growth hormone-induced insulin resistance is associated with increased intramyocellular triglyceride content but unaltered VLDL-triglyceride kinetics

The ability of growth hormone (GH) to stimulate lipolysis and cause insulin resistance in skeletal muscle may be causally linked, but the mechanisms remain obscure. We investigated the impact of GH on the turnover of FFA and VLDL-TG, intramuscular triglyceride content (IMTG), and insulin sensitivity (euglycemic clamp) in nine healthy men in a randomized double-blind placebo-controlled crossover study after 8 days treatment with (A) Placebo+Placebo, (B) GH (2 mg daily)+Placebo, and (C) GH (2 mg daily)+Acipimox (250 mgx3 daily). In the basal state, GH (B) increased FFA levels (P<0.05), palmitate turnover (P<0.05), and lipid oxidation (P=0.05), but VLDL-TG kinetics were unaffected. Administration of acipimox (C) suppressed basal lipolysis but did not influence VLDL-TG kinetics. In the basal state, IMTG content increased after GH (B; P=0.03). Insulin resistance was induced by GH irrespective of concomitant acipimox (P<0.001). The turnover of FFA and VLDL-TG was suppressed by hyperinsulinemia during placebo and GH, whereas coadministration of acipimox induced a rebound increase FFA turnover and VLDL-TG clearance. We conclude that these results show that GH-induced insulin resistance is associated with increased IMTG and unaltered VLDL-TG kinetics; we hypothesize that fat oxidation in muscle tissue is an important primary effect of GH and that circulating FFA rather than VLDL-TG constitute the major source for this process; and the role of IMTG in the development of GH-induced insulin resistance merits future research.     

Aging does not contribute to the decline in insulin action on storage of muscle glycogen in rats

Increase in fat mass (FM) and changes in body composition may account for the age-associated impairment in insulin action on muscle glycogen storage. We wish to examine whether preventing the increase in FM abolishes this defect seen with aging. We studied the novel aging model of F1 hybrids of BN/F344 NIA rats fed ad libitum (AL) at 2 (weighing 259+/-17 g), 8 (459+/-17 g), and 20 (492+/-10 g) mo old. To prevent the age-dependent growth in FM, rats were caloric restricted (CR) at 2 mo by decreasing their daily caloric intake by 45% (weighing 292+/-5 g at 8 mo, 294+/-9 g at 20 mo). As designed, the lean body mass (LBM) and %FM remained unchanged through aging (8 and 20 mo old) in the CR rats and was similar to that of 2-mo-old AL rats. However, 8- and 20-mo-old AL-fed rats had three- to fourfold higher FM than both CR groups. Peripheral insulin action at physiological hyperinsulinemia was determined (by 3 mU x kg(-1). min(-1) insulin clamp). Prevention of fat accretion maintained glucose uptake (R(d); 29+/-2, 29+/-2, and 31+/-4 mg x kg LBM(-1) x min(-1)) and glycogen synthesis rates (GS, 12+/-1, 12 +/-1, and 14+/-2 mg x kg LBM(-1) x min(-1)) at youthful levels (2 mo AL) in 8- and 20-mo-old CR rats, respectively. These levels were significantly increased (P<0.001) compared with AL rats with higher %FM (R(d), 22+/-1 and 22+/-2 and GS, 7+/-1 and 8+/-2 mg x kg LBM(-1). min(-1) in 8- and 20-mo-old rats, respectively). The increase in whole body GS in age-matched CR rats was accompanied by approximately 40% increased accumulation of [(3)H] glucose into glycogen and a similar increase in insulin-induced muscle glycogen content. Furthermore, the activation of glycogen synthase increased, i.e., approximately 50% decrease in the Michaelis constant, in both CR groups (P<0.01). We conclude that chronic CR designed to prevent an increase in storage of energy in fat maintained peripheral insulin action at youthful levels, and aging per se does not result in a defect on the pathway of glycogen storage in skeletal muscle.     

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.     

Aspirin inhibits serine phosphorylation of insulin receptor substrate 1 in growth hormone treated animals

In this study, we demonstrate that pretreatment with aspirin inhibits GH-induced insulin resistance. GH was observed to lead to serine phosphorylation of IRS-1, a phenomenon which was reversed by aspirin in liver, muscle and WAT in parallel with a reduction in JNK activity. In addition, our data show an impairment of insulin activation in the IR/IRS/PI(3)kinase pathway and a reduction in IRS-1 protein levels in rats treated with GH, which was also reversed in the animals pretreated with aspirin. Overall, these results provide new insights into the mechanism of GH-induced insulin resistance.     

High-fat diet-induced muscle insulin resistance: relationship to visceral fat mass

It has been variously hypothesized that the insulin resistance induced in rodents by a high-fat diet is due to increased visceral fat accumulation, to an increase in muscle triglyceride (TG) content, or to an effect of diet composition. In this study we used a number of interventions: fish oil, leptin, caloric restriction, and shorter duration of fat feeding, to try to disassociate an increase in visceral fat from muscle insulin resistance. Substituting fish oil (18% of calories) for corn oil in the high-fat diet partially protected against both the increase in visceral fat and muscle insulin resistance without affecting muscle TG content. Injections of leptin during the last 4 days of a 4-wk period on the high-fat diet partially reversed the increase in visceral fat and the muscle insulin resistance, while completely normalizing muscle TG. Restricting intake of the high-fat diet to 75% of ad libitum completely prevented the increase in visceral fat and muscle insulin resistance. Maximally insulin-stimulated glucose transport was negatively correlated with visceral fat mass (P < 0.001) in both the soleus and epitrochlearis muscles and with muscle TG concentration in the soleus (P < 0.05) but not in the epitrochlearis. Thus we were unable to dissociate the increase in visceral fat from muscle insulin resistance using a variety of approaches. These results support the hypothesis that an increase in visceral fat is associated with development of muscle insulin resistance.     

Sensitivity of the soleus muscle to insulin in resting and exercising rats with experimental hypo- and hyper-thyroidism.

1. The effects of hypothyroidism (caused by surgical thyroidectomy followed by treatment for 1 month with propylthiouracil) and of hyperthyroidism [induced by subcutaneous administration of L-tri-iodothyronine (T3)] on glucose tolerance and skeletal-muscle sensitivity to insulin were examined in rats. Glucose tolerance was estimated during 2 h after subcutaneous glucose injection (1 g/kg body wt.). The sensitivity of the soleus muscle to insulin was studied in vitro in sedentary and acutely exercised animals. 2. Glucose tolerance was impaired in both hypothyroid and hyperthyroid rats in comparison with euthyroid controls. 3. In the soleus muscle, responsiveness of the rate of lactate formation to insulin was abolished in hypothyroid rats, whereas the sensitivity of the rate of glycogen synthesis to insulin was unchanged. In hyperthyroid animals, opposite changes were found, i.e. responsiveness of the rate of glycogen synthesis was inhibited and the sensitivity of the rate of lactate production did not differ from that in control sedentary rats. 4. A single bout of exercise for 30 min potentiated the stimulatory effect of insulin on lactate formation in hyperthyroid rats and on glycogen synthesis in hypothyroid animals. 5. The data suggest that thyroid hormones exert an interactive effect with insulin in skeletal muscle. This is likely to be at the post-receptor level, inhibiting the effect of insulin on glycogen synthesis and stimulating oxidative glucose utilization.     

Insulin enhances growth hormone induction of the MEK/ERK signaling pathway

Growth hormone (GH) plays an important role in growth and metabolism by signaling via at least three major pathways, including STATs, ERK1/2, and phosphatidylinositol 3-kinase/Akt. Physiological concentrations of insulin promote growth probably by modulating liver GH receptor (GHR) levels in vivo, but the possible effects of insulin on GH-induced post-GHR signaling have yet to be studied. We hypothesized that short-term insulin, similar to the fluctuations that occur following feeding, affects GH-induced post-GHR signaling. Our present studies suggest that, in rat H4IIE hepatoma cells, insulin (4 h or less) selectively enhanced GH-induced phosphorylation of MEK1/2 and ERK1/2, but not GH-induced activation of STAT5 and Akt. Although insulin pretreatment altered GH-induced formation of Shc.Grb2.SOS complex, it did not significantly affect GH-induced activation of other signaling intermediates upstream of MEK/ERK, including JAK2, Ras, and Raf-1. Immunofluorescent staining indicated that insulin pretreatment facilitated GH-induced cell membrane translocation of MEK1/2. Insulin pretreatment also increased the amount of MEK association with its scaffolding protein, KSR. In summary, short-term insulin treatment of cultured, liver-derived cells selectively sensitized GH-induced MEK/ERK phosphorylation independent of JAK2, Ras, and Raf-1, but likely resulted from increased cell membrane translocation of MEK1/2. These findings suggest that insulin may be necessary for sensitization of cells to GH-induced ERK1/2 activation and provides a potential cellular mechanism by which insulin promotes growth.     

Protein-tyrosine phosphatases are involved in interferon resistance associated with insulin resistance in HepG2 cells and obese mice

Insulin resistance is a risk factor for non-response to interferon/ribavirin therapy in patients with chronic hepatitis C. The aim of this study was to determine the role played by protein-tyrosine phosphatases (PTPs) in the absence of interferon-α (IFNα) response associated with insulin resistance. We induced insulin resistance by silencing IRS-2 or by treating HepG2 cells with tumor necrosis factor-α (TNFα) and analyzed insulin response by evaluating Akt phosphorylation and IFNα response by measuring Stat-1 tyrosine phosphorylation and 2',5'-oligoadenylate synthase and myxovirus resistance gene expression. The response to IFNα was also measured in insulin-resistant obese mice (high fat diet and ob/ob mice) untreated and treated with metformin. Silencing IRS-2 mRNA induces insulin resistance and inhibits IFNα response. Likewise, TNFα suppresses insulin and IFNα response. Treatment of cells with pervanadate and knocking down PTP-1B restores insulin and IFNα response. Both silencing IRS-2 and TNFα treatment increase PTP and PTP-1B activity. Metformin inhibits PTP and improves IFNα response in insulin-resistant cells. Insulin-resistant ob/ob mice have increased PTP-1B gene expression and activity in the liver and do not respond to IFNα administration. Treatment with metformin improves this response. In HepG2 cells, insulin resistance provokes IFNα resistance, which is associated with an increased PTP-1B activity in the liver. Inhibition of PTP-1B activity with pervanadate and metformin or knocking down PTP-1B reestablishes IFNα response. Likewise, metformin decreases PTP-1B activity and improves response to IFNα in insulin-resistant obese mice. The use of PTP-1B inhibitors may improve the response to IFNα/ribavirin therapy.     

Methionine restriction decreases visceral fat mass and preserves insulin action in aging male Fischer 344 rats independent of energy restriction

Reduced dietary methionine intake (0.17% methionine, MR) and calorie restriction (CR) prolong lifespan in male Fischer 344 rats. Although the mechanisms are unclear, both regimens feature lower body weight and reductions in adiposity. Reduced fat deposition in CR is linked to preservation of insulin responsiveness in older animals. These studies examine the relationship between insulin responsiveness and visceral fat in MR and test whether, despite lower food intake observed in MR animals, decreased visceral fat accretion and preservation of insulin sensitivity is not secondary to CR. Accordingly, rats pair fed (pf) control diet (0.86% methinone, CF) to match the food intake of MR for 80 weeks exhibit insulin, glucose, and leptin levels similar to control-fed animals and comparable amounts of visceral fat. Conversely, MR rats show significantly reduced visceral fat compared to CF and PF with concomitant decreases in basal insulin, glucose, and leptin, and increased adiponectin and triiodothyronine. Daily energy expenditure in MR animals significantly exceeds that of both PF and CF. In a separate cohort, insulin responses of older MR animals as measured by oral glucose challenge are similar to young animals. Longitudinal assessments of MR and CF through 112 weeks of age reveal that MR prevents age-associated increases in serum lipids. By 16 weeks, MR animals show a 40% reduction in insulin-like growth factor-1 (IGF-1) that is sustained throughout life; CF IGF-1 levels decline much later, beginning at 112 weeks. Collectively, the results indicate that MR reduces visceral fat and preserves insulin activity in aging rats independent of energy restriction.