Aerobic exercise + weight loss decreases skeletal muscle myostatin expression and improves insulin sensitivity in older adults.

OBJECTIVE: To determine whether aerobic exercise training + weight loss (AEX + WL) would affect the expression of myostatin and its relationship with insulin sensitivity in a longitudinal, clinical intervention study. DESIGN AND METHODS: Thirty-three obese sedentary postmenopausal women and men (n = 17 and 16, age: 61 ± 1 years, body mass index: 31 ± 1 kg/m(2) , VO2 max: 21.9 ± 1.0 mL/kg/min, X ± Standard error of the mean (SEM)) completed 6 months of 3 days/week AEX + WL. During an 80 mU m(-2) min(-1) hyperinsulinemic-euglycemic clamp, we measured glucose utilization (M), myostatin, myogenin, and MyoD gene expression by real-time RT-PCR in vastus lateralis muscle at baseline and 2 h. RESULTS: Body weight (-8%) and fat mass (-17%) decreased after AEX + WL (P < 0.001). Fat-free mass (FFM) and mid-thigh muscle area by computed tomography did not change but muscle attenuation increased (P < 0.05). VO2 max increased 14% (P < 0.001). AEX + WL increased M by 18% (P < 0.01). Myostatin gene expression decreased 19% after AEX + WL (P < 0.05). Basal mRNA myostatin levels were negatively associated with M before the intervention (r = -0.43, P < 0.05). Insulin infusion increased myoD and myogenin expression before and after AEX + WL (both P < 0.001) but basal levels did not change. The insulin effect on myostatin expression was associated with the change in M after AEX + WL (r = 0.56, P < 0.005). CONCLUSIONS: Exercise and weight loss results in a downregulation of myostatin mRNA and an improvement in insulin sensitivity in obese older men and women.

Prostaglandylinositol cyclic phosphate (cPIP): a novel second messenger of insulin action. Comparative analysis of two kinds of "insulin mediators".

Insulin induces a broad spectrum of effects over a wide time interval. It also stimulates the phosphorylation of some cellular proteins, while decreasing the state of phosphorylation of others. These observations indicate the presence of different, but not necessarily mutually exclusive, pathways of insulin action. One well-known pathway represents a phosphorylation cascade initiated by the tyrosine kinase activity of the insulin receptor followed by involvement of different MAP-kinases. Another pathway suggests the existence of low molecular weight insulin mediators whose synthesis and/or release is initiated by insulin. Comparable analysis of two kinds of insulin mediators, namely inositolphosphoglycans and prostaglandylinositol cyclic phosphate (cPIP), has been carried out. It has been shown that the expression of a number of enzymes, such as phospholipase A(2), phospholipase C, cyclo-oxygenase and IRS-1-like enzyme, could regulate the biosynthesis of cPIP in both normal and diabetes-related conditions. Data on the activity of a key enzyme of cPIP biosynthesis termed cPIP synthase (IRS-1-like enzyme) in various monkey tissues before and twice during an euglycemic hyperinsulinemic clamp have been presented. It has been concluded that in vivo insulin increases cPIP synthase activity in both liver and subcutaneous adipose tissue of lean normal monkeys. It has been also suggested that abnormal production of cPIP could be related to several pathologies including glucocorticoid-induced insulin resistance and diabetic embryopathy. Further studies on cPIP and other types of insulin mediators are necessary to aid our understanding of insulin action.

Prostaglandylinositol cyclic phosphate synthase activity in the liver of insulin-resistant rhesus monkeys before and after a euglycemic hyperinsulinemic clamp.

Prostaglandylinositol cyclic phosphate (cPIP), functionally a cAMP antagonist, is a novel, low-molecular weight mediator of insulin action. Both essential hypertension and type 2 diabetes may be associated with a reduction of cPIP synthesis. In intact cells and in plasma membranes, cPIP synthesis is stimulated by insulin, which activates cPIP synthase by tyrosine phosphorylation. We measured the activities of cPIP synthase in the homogenates of freeze-clamped and then lyophilized liver samples from five insulin-resistant, adult rhesus monkeys, obtained under basal fasting conditions and again under maximal insulin stimulation during a euglycemic hyperinsulinemic clamp. The mean cPIP synthase activity in basal samples (0.33 +/- 0.09 pmol/min/mg protein) was not significantly different at the end of the clamp (0.24 +/- 0.11 pmol/min/mg protein). Basal cPIP synthase activityVoL 12, No. 1, 2001 was directly related to both basal cAMP content and basal fractional activity of cAMP-dependent protein kinase (PKA): r=0.85, p<0.05 and r=0.86, p<0.05, respectively. In turn, insulin-stimulated cPIP synthase activity was inversely related to both the insulin-stimulated fractional activity of PKA (r=0.89, p<0.02) and the insulin-stimulated total PKA activity: r=0.94, p<0.005. The findings suggest that in the liver of insulin-resistant rhesus monkeys, cPIP synthase activity, which leads to the synthesis of the low-molecular weight mediator cPIP, may oppose cAMP synthesis and PKA activity.

Circulating concentrations of the adipocyte protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys.

Adiponectin is an adipose-specific plasma protein whose plasma concentrations are decreased in obese subjects and type 2 diabetic patients. This protein possesses putative antiatherogenic and anti-inflammatory properties. In the current study, we have analyzed the relationship between adiponectin and insulin resistance in rhesus monkeys (Macaca mulatta), which spontaneously develop obesity and which subsequently frequently progress to overt type 2 diabetes. The plasma levels of adiponectin were decreased in obese and diabetic monkeys as in humans. Prospective longitudinal studies revealed that the plasma levels of adiponectin declined at an early phase of obesity and remained decreased after the development of type 2 diabetes. Hyperinsulinemic-euglycemic clamp studies revealed that the obese monkeys with lower plasma adiponectin showed significantly lower insulin-stimulated peripheral glucose uptake (M rate). The plasma levels of adiponectin were significantly correlated to M rate (r = 0.66, P < 0.001). Longitudinally, the plasma adiponectin decreased in parallel to the progression of insulin resistance. No clear association was found between the plasma levels of adiponectin and its mRNA levels in adipose tissue. These results suggest that reduction in circulating adiponectin may be related to the development of insulin resistance.

In vivo insulin regulation of skeletal muscle glycogen synthase in calorie-restricted and in ad libitum-fed rhesus monkeys.

Chronic calorie restriction in primates has been shown to have profound and unexpected effects on basal and on in vivo insulin action on skeletal muscle glycogen synthase (GS) activity. The decreased ability of insulin to activate skeletal muscle GS is a hallmark of insulin resistance and type 2 diabetes. The mechanism and role of in vivo insulin regulation of skeletal muscle GS are not fully understood. Two pathways for the activation of GS by insulin have been described by Larner and others: 1) insulin activates glucose transport that results in an increase in glucose-6-phosphate (G6P), thereby activating protein phosphatase-1, which in turn dephosphorylates and activates GS, therefore, pushing substrate into glycogen; and 2) insulin activates GS (perhaps by forming low-molecular-weight mediators which may activate protein phosphatase-1 and 2C) and activated GS subsequently pulls intermediates (e.g., G6P and uridine 5'-diphosphoglucose) into glycogen. To determine whether in vivo insulin regulates glycogen synthesis primarily via a push or pull mechanism and how this mechanism might be affected by long-term calorie restriction, skeletal muscle samples were obtained before and during a euglycemic hyperinsulinemic clamp from 41 rhesus monkeys. The monkeys varied widely in their degree of insulin sensitivity and age and included chronically calorie-restricted (CR) monkeys and ad libitum-fed monkeys. The ad libitum-fed monkeys included spontaneously type 2 diabetic, prediabetic and clinically normal animals. The apparent affinity of GS for the allosteric activator G6P (G6P Ka of GS) was measured and compared with G6P content in the muscle samples. Basal G6P Ka of GS was lower in the CR monkeys compared with the 3 ad libitum-fed groups (P: < or = 0.05). Only the normal ad libitum-fed monkeys had a decrease in the G6P Ka of GS with insulin (P: < 0.005). The insulin effect (insulin-stimulated minus basal) on the G6P Ka of GS was strongly positively related to the insulin effect on G6P content (r = 0.80, P: < 0.0001) across the entire group of monkeys. This finding supports the hypothesis that activation/dephosphorylation of GS by insulin is related to a decrease in G6P content and that paradoxical inactivation/phosphorylation of GS by insulin is related to an increase in G6P content (as demonstrated in 4 of 6 CR monkeys). Therefore, during a euglycemic hyperinsulinemic clamp, insulin regulates skeletal muscle glycogen synthesis primarily via a pull mechanism in both CR and in ad libitum-fed rhesus monkeys.

A thiazolidinedione improves in vivo insulin action on skeletal muscle glycogen synthase in insulin-resistant monkeys.

Thiazolidinediones (TZD) have been shown to have anti-diabetic effects including the ability to decrease fasting hyperglycemia and hyperinsulinemia, increase insulin-mediated glucose disposal rate (M) and decrease hepatic glucose production, but the mechanisms of action are not well established. To determine whether a TZD (R-102380, Sankyo Company Ltd., Tokyo, Japan) could improve insulin action on skeletal muscle glycogen synthase (GS), the rate-limiting enzyme in glycogen synthesis, 4 insulin-resistant obese monkeys were given 1 mg/kg/day R-102380 p.o. for a 6-week period. Skeletal muscle GS activity and glucose 6-phosphate (G6P) content were compared between pre-dosing and dosing periods before and during the maximal insulin-stimulation of a euglycemic hyperinsulinemic clamp. Compared to pre-dosing, insulin-stimulated GS activity and G6P content were increased by this TZD: GS independent activity (p = 0.02), GS total activity (p = 0.005), GS fractional activity (p = 0.06) and G6P content (p = 0.02). The change in GS activity induced by in vivo insulin (insulin-stimulated minus basal) was also increased by this TZD: GS independent activity (p = 0.03) and GS fractional activity (p = 0.04). We conclude that the TZD R-102380 improves insulin action at the skeletal muscle in part by increasing the activity of glycogen synthase. This improvement in insulin sensitivity may be a key factor in the anti-diabetic effect of the thiazolidinedione class of agents.

Age-related adipose tissue mRNA expression of ADD1/SREBP1, PPARgamma, lipoprotein lipase, and GLUT4 glucose transporter in rhesus monkeys.

Aging has been shown to have an effect on the capacity to differentiate preadipocytes and on the expression of some genes expressed in adipose tissue. The mRNA levels of adipocyte differentiation-related genes were examined in rhesus monkeys (Macaca Mulatta) ranging in age from 7 to 30 years. The effect of aging on the expression of peroxisome proliferator activated receptor gamma (PPARgamma), adipocyte determination- and differentiation-dependent factor 1/sterol regulatory element binding protein 1 (ADD1/SREBP1), CCAAT/enhancer binding protein alpha (C/EBPalpha), lipoprotein lipase (LPL), GLUT4 glucose transporter, and adipsin were examined by slot blot analysis. Significant inverse correlations were observed between age and the mRNA levels of PPARgamma, ADD1/SREBP1, LPL, and GLUT4. The coordinate downregulation of these genes may be linked to the declining fat mass of senescent animals. There was no correlation between age and the mRNA levels of adipsin. The mRNA levels of these genes were not correlated to body weight orfasting plasma insulin. These findings indicate that aging may have an effect on the adipocyte differentiation program and this effect appears to be gene specific.

Paradoxical phosphorylation of skeletal muscle glycogen synthase by in vivo insulin in very lean young adult rhesus monkeys.

Calorie restriction (CR) has previously been shown to unexpectedly induce a reversal of in vivo insulin action (phosphorylation instead of dephosphorylation) on skeletal muscle glycogen synthase (GS) in four out of six long-term calorie-restricted (CR) monkeys. The purpose of the present study was to determine whether this increase in Ka (concentration of glucose 6-phosphate [G6P] at which GS activity is half-maximal) during insulin is also present in very lean (VL) young adult monkeys maintained on a controlled feeding regimen. Muscle samples from 10 VL monkeys (10 +/- 2% body fat; 7 years old) were obtained before and during a euglycemic hyperinsulinemic clamp and the Ka was determined and compared to the Ka of two other groups of monkeys, one matched in age but fully ad libitum (AL)-fed (n = 9.8 +/- 1 years old, 20 +/- 3% body fat, p = 0.01 vs. VL monkeys), and the other our previously described weight-clamped long-term CR monkeys (n = 6.20 +/- 1 years old, 21 +/- 2% body fat, p = 0.01 vs. VL monkeys). All of the AL monkeys had the expected decrease in Ka with insulin; however, similar to the 4 out of 6 CR monkeys, 7 out of 10 VL monkeys had an increase in Ka with insulin. The 11 monkeys with an increase in Ka (+Ka) (7 VL + 4 CR) were compared to the 14 monkeys with a decrease in Ka with insulin (-Ka) (3 VL + 2 CR + 9 AL). The +Ka monkeys had lower basal Ka (p = 0.0001), higher basal GS fractional activity (p = 0.0003), lower basal G6P content (p = 0.002), lower glycogen phosphorylase fractional activity (p = 0.01), and lower whole-body insulin-mediated glucose disposal rate (p < 0.05) than the -Ka monkeys. We conclude that the condition of steady-state restrained calorie intake (as in the CR monkeys and in the controlled feeding VL monkeys) produces the paradoxical action of in vivo insulin to phosphorylate muscle GS, and raises the possibility that the presence of the unusual response to insulin may serve as a marker in calorie-restrained individuals for the genotype of obesity, insulin resistance and/or Type 2 diabetes.

Calorie restriction in nonhuman primates: mechanisms of reduced morbidity and mortality.

Long term chronic calorie restriction (CR) of adult nonhuman primates significantly reduces morbidity and increases median age of death. The present review is focused upon an ongoing study of sustained adult-onset calorie restriction, which has been underway for 15 years. Monkeys, initially calorie restricted at about 10 years of age, are now approximately 25 years old. The median life span of these restricted monkeys is increasing, now exceeding that of ad libitum (AL)-fed monkeys. In our laboratory, maximum life span for AL-fed monkeys appears to be about 40 years. Thus, whether CR can also increase maximal life span, as it does in rodents, cannot be determined for at least another 15 years. The earliest detectable positive benefit on morbidity in these monkeys was previously reported as the prevention of obesity. Current evidence, as reviewed here, suggests that much obesity-associated morbidity is also mitigated by sustained calorie restraint in nonhuman primates. Furthermore, probably because of the prevention of obesity, diabetes has also been prevented. Recent findings include the identification of extraordinary changes in the glycogen synthesis pathway, and on the phosphorylation of glycogen synthase in response to insulin. This calorie restriction-induced prevention of morbidity does not require excessive leanness, but is clearly present when body fat is within the normal range of 10 to 22%, and this is likely to be true in humans as well.

Relationships of PPARgamma and PPARgamma2 mRNA levels to obesity, diabetes and hyperinsulinaemia in rhesus monkeys.

OBJECTIVE: To examine the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) together with CCAAT/enhancer binding protein alpha (C/EBPalpha), lipoprotein lipase (LPL) and glucose transporter (GLUT4) mRNA in adipose tissue of rhesus monkeys in relation to obesity. DESIGN: Cloning of the PPARgamma1 and gamma2 cDNAs and analysis of PPARgamma, C/EBPalpha, LPL and GLUT4 mRNA levels in the adipose tissue of lean and obese monkeys. SUBJECTS: 28 rhesus monkeys (Macaca mulatta) with a wide range of body weights (9.2-22.6 kg) and with or without type 2 diabetes. MEASUREMENTS: Sequence of PPARgamma1 and gamma2. Tissue distribution of PPARgamma1 and gamma2. The mRNA levels of PPARgamma, C/EBPalpha, LPL and GLUT4 in adipose tissue. The ratio of PPARgamma2 mRNA to total PPARgamma mRNA. RESULTS: The monkey PPARgamma2 protein showed 99% identity with the human protein. PPARgamma1 mRNA was shown to be expressed in various tissues and most abundantly in adipose tissue. PPARgamma2 existed mainly in adipose tissue. A significant correlation between the ratio of PPARgamma2 mRNA to total PPARgamma mRNA and obesity was observed, whereas total PPARgamma mRNA levels showed no significant relationships to obesity. There was also a significant relationship between the ratio of PPARgamma2 mRNA to total PPARgamma mRNA and fasting plasma insulin concentration. The mRNA levels of C/EBPalpha, LPL and GLUT4 were highly correlated to that of total PPARgamma mRNA. They were also significantly correlated to the mRNA levels of PPARgamma1 and PPARgamma2. CONCLUSIONS: The ratio of PPARgamma2 mRNA to total PPARgamma mRNA is related to obesity in the rhesus monkey and mRNA expression of PPARgamma1, PPARgamma2, C/EBPalpha, LPL and GLUT4 appear to be coordinated in vivo.

Monkey leptin receptor mRNA: sequence, tissue distribution, and mRNA expression in the adipose tissue of normal, hyperinsulinemic, and type 2 diabetic rhesus monkeys.

OBJECTIVE: We have cloned the rhesus monkey leptin receptor and examined its mRNA expression levels in the adipose tissue of monkeys to investigate the regulation of gene expression of the leptin receptor. RESEARCH METHODS AND PROCEDURES: Monkey leptin receptor cDNA was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR). Tissue distribution of monkey leptin receptor was examined by Northern blot analysis and RT-PCR. The mRNA levels of monkey leptin receptor in adipose tissue of normal (n=10), hyperinsulinemic obese (n=8), and type 2 diabetic monkeys (n=8) were measured by quantitative RT-PCR. RESULTS: Monkey leptin receptor cDNA had at least two alternatively spliced isoforms (long and short forms). The long form of the leptin receptor mRNA was expressed relatively highly in liver, adipose tissue, hypothalamus, and choroid plexus, whereas the total leptin receptors were expressed in every tissue examined. The mRNA levels of the long form of the leptin receptor in adipose tissue were not correlated to body weight, fasting plasma insulin, plasma glucose, or plasma leptin levels. The mRNA levels of the long form of the leptin receptor were highly correlated to that of the total leptin receptor (long and short form). DISCUSSION: The long form of leptin receptor mRNA existed in adipose tissue as well as in liver and hypothalamus, suggesting that the leptin receptor in adipose tissue may be functional in adipose tissue. The expression of the leptin receptor mRNA in adipose tissue is not affected by obesity, hyperinsulinemia, or diabetes.

Lack of defect in insulin action on hepatic glycogen synthase and phosphorylase in insulin-resistant monkeys.

It is well known that an alteration in insulin activation of skeletal muscle glycogen synthase is associated with insulin resistance. To determine whether this defect in insulin action is specific to skeletal muscle, or also present in liver, simultaneous biopsies of these tissues were obtained before and during a euglycemic hyperinsulinemic clamp in spontaneously obese insulin-resistant male rhesus monkeys. The activities of glycogen synthase and glycogen phosphorylase and the concentrations of glucose 6-phosphate and glycogen were measured. There were no differences between basal and insulin-stimulated glycogen synthase and glycogen phosphorylase activities or in glucose 6-phosphate and glycogen contents in muscle. Insulin increased the activities of liver glycogen synthase (P < 0.05) and decreased the activities of liver glycogen phosphorylase (P 0.001). Insulin also caused a reduction in liver glucose 6-phosphate (P = 0.05). We conclude that insulin-resistant monkeys do not have a defect in insulin action on liver glycogen synthase, although a defect in insulin action on muscle glycogen synthase is present. Therefore, tissue-specific alterations in insulin action on glycogen synthase are present in the development of insulin resistance in rhesus monkeys.

Insulin increases liver protein phosphatase-1 and protein phosphatase-2C activities in lean, young adult rhesus monkeys.

Liver glycogen synthase activity is increased, and glycogen phosphorylase activity and glucose 6-phosphate content reduced by in vivo insulin during a euglycemic hyperinsulinemic clamp in lean young adult rhesus monkeys. To examine the mechanism of dephosphorylation of liver glycogen synthase and glycogen phosphorylase, the enzyme activities of protein phosphatase-1, protein phosphatase-2C, cAMP-dependent protein kinase, glycogen synthase kinase-3, protein kinase C and protein tyrosine kinase were determined before and after three hours of in vivo insulin in these same monkeys. The bioactivity of an inositol phosphoglycan insulin mediator (pH 2.0) and cAMP concentrations were also measured in the liver before and after insulin administration. Insulin caused significant increases in protein phosphatase-1 (p = 0.005) and in protein phosphatase-2C activities (p = 0.001). Insulin-stimulated minus basal bioactivity of the pH 2.0 insulin mediator was strongly inversely related to the insulin-stimulated minus basal glucose 6-phosphate content (r = -0.93, p < 0.0001). These findings suggest that protein phosphatase-1 and protein phosphatase-2C may be involved in the mechanism of in vivo insulin activation of liver glycogen synthase and inactivation of liver glycogen phosphorylase.

Insulin unexpectedly increases the glucose 6-phosphate Ka of skeletal muscle glycogen synthase in calorie-restricted monkeys.

In skeletal muscle of normal subjects, the concentration of glucose 6-phosphate (G6P) at which the activity of glycogen synthase (GS) is half maximal (Ka) is decreased by in vivo insulin, and the fractional activity is increased without a change in GS maximal activity (Vmax). We have shown that moderate chronic calorie restriction, previously shown in rodents to be effective in slowing aging, resulted in the prevention of obesity and type 2 diabetes in primates (rhesus monkeys, Macaca mulatta). However, unexpectedly, in a subgroup of calorie-restricted monkeys, insulin during a euglycemic hyperinsulinemic clamp caused an unanticipated decrease in skeletal muscle GS fractional activity. These same monkeys had the lowest whole-body glucose disposal rate (M), the greatest increase in skeletal muscle G6P content and the greatest increase in skeletal muscle glycogen phosphorylase activity during the euglycemic hyperinsulinemic clamp compared to the remaining calorie-restricted monkeys with normal insulin action. To determine whether this highly unusual insulin-mediated decrease in GS fractional activity was due to increased phosphorylation (increased Ka), we measured the activity of skeletal muscle GS at 9 different G6P concentrations before and during the euglycemic hyperinsulinemic clamp in 6 calorie-restricted monkeys. G6P Ka increased (n = 4) and Vmax decreased (n = 5) during the clamp. Basal G6P Ka was inversely related to basal GSfv (r = -0.94, p < 0.002). G6P Ka and skeletal muscle G6P content were positively related under insulin-stimulated conditions (r = 0.93, p < 0.005). The change in G6P Ka (insulin-stimulated minus basal) was inversely related to M (r = -0.94, p < 0.002) and positively related to the change in skeletal muscle G6P content (r = 0.93, p < 0.005). We conclude that moderate calorie restriction results in a reversal of normal insulin action at the skeletal muscle with inactivation of glycogen synthase which is likely to be due to an increase in phosphorylation of GS together with a decrease in Vmax of GS during a euglycemic hyperinsulinemic clamp in most of the calorie-restricted monkeys. These alterations are likely to be involved in the anti-diabetogenic effects of calorie restriction.

Insulin regulates liver glycogen synthase and glycogen phosphorylase activity reciprocally in rhesus monkeys.

In skeletal muscle of both humans and monkeys, the effects of in vivo insulin during a euglycemic hyperinsulinemic clamp on the enzymes and substrates of glycogen metabolism have been well established. In liver, such effects of insulin during a clamp have not been previously studied in primates. To examine insulin action at the liver, euglycemic hyperinsulinemic clamps were performed in 10 lean young adult male rhesus monkeys. Liver biopsies were obtained at three time points: basal (fasting), that is, immediately before the onset of the clamp, and during insulin infusion at 130 and 195 min. Glycogen synthase (GS), glycogen phosphorylase (GP), glucose 6-phosphate (G-6-P), and glycogen were determined at each time point, with the greatest effects observed most frequently at 195 min. Whole body insulin-mediated glucose disposal rate was related to the change in the independent activity of GS (r = 0.63, P < 0.05). Insulin increased the GS fractional activity (P < 0.005) and decreased the activity ratio of GP (P < 0.001) compared with basal. The changes in fractional activity of GS and in activity ratio of GP were inversely related (r = - 0.68, P < 0.05), G-6-P concentration was decreased during insulin stimulation compared with basal (P = 0.01). Glycogen concentration was not significantly different between the basal and insulin-stimulated time points. We conclude that insulin during a euglycemic clamp activates liver GS while inhibiting liver GP and that insulin action on liver GS is positively related to whole body insulin-mediated glucose disposal rates in lean young adult rhesus monkeys.

Insulin decreases skeletal muscle cAMP-dependent protein kinase (PKA) activity in normal monkeys and increases PKA activity in insulin-resistant rhesus monkeys.

Insulin activation of skeletal muscle glycogen synthase and glucose disposal is defective in both prediabetic and diabetic primates. Reduction in the activation of glycogen synthase by insulin could be the cause of lower glucose disposal rates, and could be the result, at least in part, of the failure of insulin to inhibit cAMP-dependent protein kinase activity (protein kinase A, PKA). To examine this proposed mechanism, PKA activity was measured in skeletal muscle (vastus lateralis) samples freeze-clamped in situ under basal fasting conditions before, and again during a euglycemic hyperinsulinemic clamp in 27 rhesus monkeys. Nine of the monkeys were normal (normal fasting glucose and insulin), eight were prediabetic (normal fasting glucose and hyperinsulinemia) and ten had spontaneous non-insulin-dependent diabetes (hyperglycemia). Insulin lowered PKA activity ratio in normal monkeys (basal vs insulin-stimulated, 14.4 +/- 3.2 vs 8.1 +/- 1.8%, p < 0.05), but raised PKA activity ratio in prediabetic monkeys (5.4 +/- 1.4 vs 10.5 +/- 2.6%, p < 0.05). PKA activity ratio was unaffected by insulin in the diabetic monkeys (6.7 +/- 1.8 vs 7.5 +/- 1.4%). Basal PKA activity ratio was higher in normal monkeys compared to prediabetic (p < 0.05) and diabetic monkeys (p < 0.05). Basal PKA activity ratio was inversely related to the insulin-stimulated change in PKA activity ratio (r = -0.72, p < 0.001). We conclude that in vivo insulin during euglycemic hyperinsulinemic clamp decreases skeletal muscle PKA activity ratio in normal monkeys but fails to decrease the activity ratio of PKA in insulin resistant (prediabetic and diabetic) monkeys. The insulin resistant state is characterized by low basal fasting skeletal muscle PKA activity ratio.

Relationship of glycogen synthase and glycogen phosphorylase to protein phosphatase 2C and cAMP-dependent protein kinase in liver of obese rhesus monkeys.

The regulation of glycogen synthase (GS) and glycogen phosphorylase (GP) activity by phosphorylation/ dephosphorylation has been proposed to be via changes in activities of several different protein (serine/threonine) phosphatases and kinases, including protein phosphatase (PP) 1/2A, PP2C, and cAMP-dependent protein kinase (PKA). In order to determine whether PP1/2A, PP2C, and/or PKA activities are related to GS and/or GP activities, these enzymes were measured in freeze-clamped liver biopsies obtained under basal fasting conditions from 16 obese monkeys. Four monkeys were normoglycemic and normoinsulinemic, five were hyperinsulinemic, and seven had type 2 diabetes (NIDDM). Liver glycogen and glucose 6-phosphate (G6P) contents were also determine. Basal enzyme activities and basal substrate concentrations were not significantly different between the three group of obese monkeys; however, there were several significant linear relationships observed when the monkeys were treated as one group. Therefore, multiple regression was used to determine the correlation between key variables. GS fractional activity was correlated to GP fractional activity (p < 0.05) and to PP2C activity (p = 0.005) (adjusted R2, 53%). GP independent activity was correlated to GS independent activity (p < 0.07) and to PKA fractional activity (p = 0.005) (adjusted R2, 64%). PP2C activity was correlated to GS fractional activity (p < 0.0005) and to PP1/2A activity (p < 0.0001) (adjusted R2, 83%). PKA fractional activity was correlated to GP total activity (p < 0.0005) and to age (p = 0.001) (adjusted R2, 82%). G6P content was correlated to glycogen content (p < 0.05) and to PP2C activity (p = 0.0005) (adjusted R2, 73%). In conclusion, PP2C and PKA are involved in the regulation of GS and GP activity in the basal state in liver of obese monkeys with a wide range of glucose tolerance.

Hyperleptinemia: relationship to adiposity and insulin resistance in the spontaneously obese rhesus monkey.

Plasma leptin levels in normal-weight and spontaneously obese male rhesus monkeys, and the relationships of circulating leptin to beta-cell basal secretion, glucose-stimulated responsiveness and peripheral insulin sensitivity, were determined. Basal leptin in normal lean adult monkeys averaged 6.0 +/- 1.3 ng/ml and in the obese monkeys averaged 22.6 +/- 2.9 ng/ml. In all monkeys, plasma leptin concentration was significantly related to body weight, body fat, fasting plasma insulin, acute insulin response to intravenous glucose, and peripheral insulin sensitivity but not to fasting glucose or glucose tolerance. Body fat and plasma insulin concentration were the best predictors of circulating leptin levels (R2 = 62.6%) independent of peripheral insulin sensitivity. Four of 17 obese monkeys had plasma leptin concentrations in the normal range, a finding that may be related to the heterogeneity of obesity. The close association of plasma leptin to body fat and plasma insulin (both basal and glucose-stimulated) support the possibility of a role of leptin in the link between obesity and beta-cell hypersecretion. However, the potential role of leptin in the development of peripheral insulin resistance, hyperglycemia and type 2 diabetes will require further study.

Dietary myoinositol results in lower urine glucose and in lower postprandial plasma glucose in obese insulin resistant rhesus monkeys.

In a previous study, D-chiroinositol added to a meal (0.5 g/kg) resulted in significantly lower postprandial plasma glucose concentrations without an increase in insulin concentrations in obese insulin-resistant monkeys. The present report describes the effects of another isomer of inositol, myoinositol, on postprandial plasma glucose and insulin concentrations and on urine glucose concentrations in 6 similarly insulin-resistant monkeys. The three 5 day study periods included a control period (liquid diet ad libitum) and 2 experimental periods (liquid diet ad libitum with either 1.5 g/kg/day myoinositol or D-chiroinositol added). Twenty-four hour urine samples were collected during each 5 day period. On the sixth day of each period the monkeys were anesthetized 110 min after completing either the control meal (15 ml/kg) or the experimental meals (1.5 g/kg myoinositol or D-chiroinositol) and plasma samples were obtained at 120, 150, 180, 210, 240, 270 and 300 min. The plasma glucose concentration was lower after the meal with myoinositol compared to the control meal at 120, 150 and 180 min (p's < 0.05). The plasma insulin concentration was lower after the meal with myoinositol compared to the control meal at 150 and 180 min (p's < 0.05). In addition, 24 hour urine glucose concentrations were lower during the myoinositol diet compared to the control diet (p < 0.001). The plasma glucose concentration was lower after the meal with D-chiroinositol compared to the control meal at 150, 240, 270 and 300 min (p's < or = 0.05). In obese insulin-resistant monkeys, myoinositol added to the diet lowers urine glucose concentrations and both myoinositol and D-chiroinositol added to a meal lower postprandial plasma glucose concentrations without increasing postprandial insulin concentrations. Therefore, myoinositol, like D-chiroinositol, may be a useful agent for reducing meal-induced hyperglycemia without inducing hyperinsulinemia in insulin-resistant subjects.