In vivo activation of ROCK1 by insulin is impaired in skeletal muscle of humans with type 2 diabetes

To determine whether serine/threonine ROCK1 is activated by insulin in vivo in humans and whether impaired activation of ROCK1 could play a role in the pathogenesis of insulin resistance, we measured the activity of ROCK1 and the protein content of the Rho family in vastus lateralis muscle of lean, obese nondiabetic, and obese type 2 diabetic subjects. Biopsies were taken after an overnight fast and after a 3-h hyperinsulinemic euglycemic clamp. Insulin-stimulated GDR was reduced 38% in obese nondiabetic subjects compared with lean, 62% in obese diabetic subjects compared with lean, and 39% in obese diabetic compared with obese nondiabetic subjects (all comparisons P < 0.001). Insulin-stimulated IRS-1 tyrosine phosphorylation is impaired 41-48% in diabetic subjects compared with lean or obese subjects. Basal activity of ROCK1 was similar in all groups. Insulin increased ROCK1 activity 2.1-fold in lean and 1.7-fold in obese nondiabetic subjects in muscle. However, ROCK1 activity did not increase in response to insulin in muscle of obese type 2 diabetic subjects without change in ROCK1 protein levels. Importantly, insulin-stimulated ROCK1 activity was positively correlated with insulin-mediated GDR in lean subjects (P < 0.01) but not in obese or type 2 diabetic subjects. Moreover, RhoE GTPase that inhibits the catalytic activity of ROCK1 by binding to the kinase domain of the enzyme is notably increased in obese type 2 diabetic subjects, accounting for defective ROCK1 activity. Thus, these data suggest that ROCK1 may play an important role in the pathogenesis of resistance to insulin action on glucose disposal in muscle of obese type 2 diabetic subjects.     

Effects of fasting on insulin action and glucose kinetics in lean and obese men and women

The development of insulin resistance in the obese individual could impair the ability to appropriately adjust metabolism to perturbations in energy balance. We investigated a 12- vs. 48-h fast on hepatic glucose production (R(a)), peripheral glucose uptake (R(d)), and skeletal muscle insulin signaling in lean and obese subjects. Healthy lean [n = 14; age = 28.0 +/- 1.4 yr; body mass index (BMI) = 22.8 +/- 0.42] and nondiabetic obese (n = 11; age = 34.6 +/- 2.3 yr; BMI = 36.1 +/- 1.5) subjects were studied following a 12- and 48-h fast during 2 h of rest and a 3-h 40 mUxm(-2)xmin(-1) hyperinsulinemic-euglycemic clamp (HEC). Basal glucose R(a) decreased significantly from the 12- to 48-h fast (lean 1.96 +/- 0.23 to 1.63 +/- 0.15; obese 1.23 +/- 0.07 to 1.07 +/- 0.07 mgxkg(-1)xmin(-1); P = 0.004) and was equally suppressed during the HEC after both fasts. The increase in glucose R(d) during the HEC after the 12-h fast was significantly decreased in lean and obese subjects after the 48-h fast (lean 9.03 +/- 1.17 to 4.16 +/- 0.34, obese 6.10 +/- 0.77 to 3.56 +/- 0.30 mgxkg FFM(-1)xmin(-1); P < 0.001). After the 12- but not the 48-h fast, insulin-stimulated AKT Ser(473) phosphorylation was greater in lean than obese subjects. We conclude that 1) 48 h of fasting produces a marked decline in peripheral insulin action, while suppression of hepatic glucose production is maintained in lean and obese men and women; and 2) the magnitude of this decline is greater in lean vs. obese subjects.     

Insulin resistance,intramyocellular lipid content,and plasma adiponectin in patients with type 1 diabetes

Insulin resistance is a key pathogenic factor of type 2 diabetes (T2DM); in contrast, in type 1 diabetes (T1DM) it is considered a secondary alteration. Increased intramyocellular lipid (IMCL) content accumulation and reduced plasma adiponectin were suggested to be pathogenic events of insulin resistance in T2DM. This study was designed to assess whether IMCL content and plasma adiponectin were also associated with the severity of insulin resistance in T1DM. We studied 18 patients with T1DM, 7 older and overweight/obese patients with T2DM, and 15 nondiabetic, insulin-resistant offspring of T2DM parents (OFF) and 15 healthy individuals (NOR) as appropriate control groups matched for anthropometric features with T1DM patients by means of the euglycemic hyperinsulinemic clamp combined with the infusion of [6,6-2H2]glucose and 1H magnetic resonance spectroscopy of the calf muscles. T1DM and T2DM patients showed reduced insulin-stimulated glucose metabolic clearance rate (MCR: 5.1 +/- 0.6 and 3.2 +/- 0.8 ml x kg(-1) min(-1)) similar to OFF (5.3 +/- 0.4 ml x kg(-1) x min(-1)) compared with NOR (8.5 +/- 0.5 ml x kg(-1) min(-1), P < 0.001). Soleus IMCL content was increased in T1DM (112 +/- 15 AU), T2DM (108 +/- 10 AU) and OFF (82 +/- 13 AU) compared with NOR (52 +/- 7 AU, P < 0.05) and the result was inversely proportional to the MCR (R2 = 0.27, P < 0.001); an association between IMCL content and Hb A1c was found only in T1DM (R2 = 0.57, P < 0.001). Fasting plasma adiponectin was reduced in T2DM (7 +/- 1 microg/ml, P = 0.01) and OFF (11 +/- 1 microg/ml, P = 0.03) but not in T1DM (25 +/- 6 microg/ml), whose plasma level was increased with respect to both OFF (P = 0.03) and NOR (16 +/- 2 microg/ml, P = 0.05). In conclusion, in T1DM, T2DM, and OFF, IMCL content was associated with insulin resistance, demonstrating that IMCL accretion is a marker of insulin resistance common to both primary genetically determined and secondary metabolic (chronic hyperglycemia) alterations. The increased adiponectin levels in insulin-resistant patients with T1DM, in contrast to the reduced levels found in patients with T2DM and in OFF, demonstrated that the relationship of adiponectin to insulin resistance in humans is still unclear.     

Normal Insulin Sensitivity in Lean Offspring of Obese Parents

Objective: Offspring of diabetic or hypertensive patients are insulin resistant at a prediabetic/prehypertensive stage. We tested the hypothesis that insulin action may be impaired in the offspring of obese nondiabetic parents. Research Methods and Procedures: Twenty‐one lean offspring of nonobese subjects [(OL) 22 ± 3 years of age] were matched to 23 lean offspring of obese subjects (OOb) by gender distribution, age, BMI, and waist circumference. Anthropometry, oral glucose tolerance, in vivo insulin sensitivity [by a euglycemic insulin clamp (6 pmol/min per kilogram_FFM; where FFM represents fat‐free mass)], and thermogenesis (by indirect calorimetry) were measured in each subject. The study subjects were from a population of 267 nuclear families (one offspring and both his/her parents) in which there was statistically significant (χ² = 30.2, p = 0.001) concordance of BMI between parents and offspring. Results: In comparing OOb with OL, no statistically significant difference or trend toward a difference was detected in fasting plasma glucose and insulin concentrations, glucose and insulin responses to oral glucose, insulin sensitivity [metabolism value = 45 ± 12 (OOb) vs. 47 ± 17 μmol/min per kilogram_FFM (OL)], insulin‐induced inhibition of protein and lipid oxidation, stimulation of glucose oxidation and nonoxidative glucose disposal, respiratory quotient, resting energy expenditure, and glucose‐induced thermogenesis. Discussion: The metabolic similarity between lean offspring of obese parents and those of nonobese parents suggests that insulin resistance and its correlates are not co‐inherited with the predisposition to develop obesity.     

Fatty liver in type 2 diabetes mellitus: relation to regional adiposity,fatty acids,and insulin resistance

The current study was undertaken to examine metabolic and body composition correlates of fatty liver in type 2 diabetes mellitus (DM). Eighty-three men and women with type 2 DM [mean body mass index (BMI): 34 +/- 0.5 kg/m2] and without clinical or laboratory evidence of liver dysfunction had body composition assessments of fat mass (FM), visceral adipose tissue (VAT), liver and spleen computed tomography (CT) attenuation (ratio of liver to spleen), muscle CT attenuation, and thigh adiposity; these assessments were also performed in 12 lean and 15 obese nondiabetic volunteers. Insulin sensitivity was measured with a euglycemic insulin infusion (40 mU. m-2. min-1) combined with systemic indirect calorimetry to assess glucose and lipid oxidation, and with infusions of [2H2]glucose for assessment of endogenous glucose production. A majority of those with type 2 DM (63%) met CT criteria for fatty liver, compared with 20% of obese and none of the lean nondiabetic volunteers. Fatty liver was most strongly correlated with VAT (r = -0.57, P < 0.0001) and less strongly but significantly associated with BMI (r = -0.42, P < 0.001) and FM (r = -0.37, P < 0.001), but only weakly associated with subcutaneous adiposity (r = -0.29; P < 0.01). Fatty liver was also correlated with subfascial adiposity of skeletal muscle (r = -0.44; P < 0.01). Volunteers with type 2 DM and fatty liver were substantially more insulin resistant those with type 2 DM but without fatty liver (P < 0.001) and had higher levels of plasma free fatty acids (P < 0.01) and more severe dyslipidemia (P < 0.01), a pattern observed in both genders. Plasma levels of cytokines were increased in relation to fatty liver (r = -0.34; P < 0.01). In summary, fatty liver is relatively common in overweight and obese volunteers with type 2 DM and is an aspect of body composition related to severity of insulin resistance, dyslipidemia, and inflammatory markers.     

Palmitate acutely induces insulin resistance in isolated muscle from obese but not lean humans

Exposure to high fatty acids (FAs) induces whole body and skeletal muscle insulin resistance. The globular form of the adipokine, adiponectin (gAd), stimulates FA oxidation and improves insulin sensitivity; however, its ability to prevent lipid-induced insulin resistance in humans has not been tested. The purpose of this study was to determine 1) whether acute (4 h) exposure to 2 mM palmitate would impair insulin signaling and glucose transport in isolated human skeletal muscle, 2) whether muscle from obese humans is more susceptible to the effects of palmitate, and 3) whether the presence of 2 mM palmitate + 2.5 mug/ml gAd (P+gAd) could prevent the effects of palmitate. Insulin-stimulated (10 mU/ml) glucose transport was not different, relative to control, following exposure to palmitate (-10%) or P+gAd (-3%) in lean muscle. In obese muscle, the absolute increase in glucose transport from basal to insulin-stimulated conditions was significantly decreased following palmitate (-55%) and P+gAd (-36%) exposure (control vs. palmitate; control vs. P+gAd, P < 0.05). There was no difference in the absolute increase in glucose transport between palmitate and P+gAd, indicating that in the presence of palmitate, gAd did not improve glucose transport. The palmitate-induced reduction in insulin-stimulated glucose transport in muscle from obese individuals may have been due to reduced Ser Akt (control vs. palmitate; P+gAd, P < 0.05) and Akt substrate 160 (AS160) phosphorylation (control vs. palmitate; P+gAd, P < 0.05). FA oxidation was significantly increased in muscle of lean and obese individuals in the presence of gAd (P < 0.05), suggesting that the stimulatory effects of gAd on FA oxidation may not be sufficient to entirely prevent palmitate-induced insulin resistance in obese muscle.     

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.     

Insulin action in skeletal muscle from patients with NIDDM

Insulin resistance in peripheral tissues is a common feature of non insulin-dependent diabetes mellitus (NIDDM). The decrease in insulin-mediated peripheral glucose uptake in NIDDM patients can be localized to defects in insulin action on glucose transport in skeletal muscle. Following short term in vitro exposure to both submaximal and maximal concentrations of insulin, 3-O-methylglucose transport rates are 40-50% lower in isolated skeletal muscle strips from NIDDM patients when compared to muscle strips from nondiabetic subjects. In addition, we have shown that physiological levels of insulin induce a 1.6-2.0 fold increase in GLUT4 content in skeletal muscle plasma membranes from control subjects, whereas no significant increase was noted in NIDDM skeletal muscle. Impaired insulin-stimulated GLUT4 translocation and glucose transport in NIDDM skeletal muscle is associated with reduced insulin-stimulated IRS-1 tyrosine phosphorylation and PI3-kinase activity. The reduced IRS-1 phosphorylation cannot be attributed to decreased protein expression, since the IRS-1 protein content is similar between NIDDM subjects and controls. Altered glycemia may contribute to decreased insulin-mediated glucose transport in skeletal muscle from NIDDM patients. We have shown that insulin-stimulated glucose transport is normalized in vitro in the presence of euglycemia, but not in the presence of hyperglycemia. Thus, the circulating level of glucose may independently regulate insulin stimulated glucose transport in skeletal muscle from NIDDM patients via a down regulation of the insulin signaling cascade.     

Effect of acute exercise on glycogen synthase in muscle from obese and diabetic subjects

Insulin stimulates glycogen synthase (GS) through dephosphorylation of serine residues, and this effect is impaired in skeletal muscle from insulin-resistant [obese and type 2 diabetic (T2DM)] subjects. Exercise also increases GS activity, yet it is not known whether the ability of exercise to affect GS is impaired in insulin-resistant subjects. The objective of this study was to examine the effect of acute exercise on GS phosphorylation and enzyme kinetic properties in muscle from insulin-resistant individuals. Lean normal glucose-tolerant (NGT), obese NGT, and obese T2DM subjects performed 40 min of moderate-intensity cycle exercise (70% of Vo(2max)). GS kinetic properties and phosphorylation were measured in vastus lateralis muscle before exercise, immediately after exercise, and 3.5 h postexercise. In lean subjects, GS fractional activity increased twofold after 40 min of exercise, and it remained elevated after the 3.5-h rest period. Importantly, exercise also decreased GS K(m) for UDP-glucose from ≈0.5 to ≈0.2 mM. In lean subjects, exercise caused significant dephosphorylation of GS by 50-70% (Ser(641), Ser(645), and Ser(645,649,653,657)), and phosphorylation of these sites remained decreased after 3.5 h; Ser? phosphorylation was not regulated by exercise. In obese NGT and T2DM subjects, exercise increased GS fractional activity, decreased K(m) for UDP-glucose, and decreased GS phosphorylation as effectively as in lean NGT subjects. We conclude that the molecular regulatory process by which exercise promotes glycogen synthesis in muscle is preserved in insulin-resistant subjects.     

Insulin-stimulated glucose transport in muscle. Evidence for a protein-kinase-C-dependent component which is unaltered in insulin-resistant mice.

The aim of our work was to investigate a possible role of protein kinase C (PKC) in insulin-stimulated glucose uptake in mouse skeletal muscle, and to search for a defect in PKC activation in insulin resistance found in obesity. In isolated soleus muscle of lean mice, insulin (100 nM) and 12-O-tetradecanoylphorbol 13-acetate (TPA) (1 microM) acutely stimulated glucose uptake 3- and 2-fold respectively. The effects of insulin and TPA were not additive. When PKC activity was down-regulated by long-term (24 h) TPA pretreatment, before measurement of glucose transport, the TPA effect was abolished, but in addition insulin-stimulated glucose transport returned to basal values. Furthermore, polymyxin B, which inhibits PKC in muscle extracts, prevented insulin-stimulated glucose uptake in muscle. In muscle of obese insulin-resistant mice, glucose uptake evoked by insulin was decreased, whereas the TPA effect, expressed as a fold increase, was unaltered. Thus both agents stimulated glucose transport to the same extent. Furthermore, no difference was observed when PKC activation by TPA was measured in muscle from lean and obese mice. These results suggest that: (1) PKC is involved in the insulin effect on glucose transport in muscle; (2) PKC activation explains only part of the insulin stimulation of glucose transport; (3) the defect in insulin response in obese mice does not appear to be due to an alteration in the PKC-dependent component of glucose transport. We propose that insulin stimulation of glucose uptake occurs by a sequential two-step mechanism, with first translocation of transporters to the plasma membrane, which is PKC dependent, and second, activation of the glucose transporters. In obesity only the activation step was decreased, whereas the translocation step was unaltered.     

Discordant effects of a chronic physiological increase in plasma FFA on insulin signaling in healthy subjects with or without a family history of type 2 diabetes

Muscle insulin resistance develops when plasma free fatty acids (FFAs) are acutely increased to supraphysiological levels (approximately 1,500-4,000 micromol/l). However, plasma FFA levels >1,000 micromol/l are rarely observed in humans under usual living conditions, and it is unknown whether insulin action may be impaired during a sustained but physiological FFA increase to levels seen in obesity and type 2 diabetes mellitus (T2DM) (approximately 600-800 micromol/l). It is also unclear whether normal glucose-tolerant subjects with a strong family history of T2DM (FH+) would respond to a low-dose lipid infusion as individuals without any family history of T2DM (CON). To examine these questions, we studied 7 FH+ and 10 CON subjects in whom we infused saline (SAL) or low-dose Liposyn (LIP) for 4 days. On day 4, a euglycemic insulin clamp with [3-3H]glucose and indirect calorimetry was performed to assess glucose turnover, combined with vastus lateralis muscle biopsies to examine insulin signaling. LIP increased plasma FFA approximately 1.5-fold, to levels seen in T2DM. Compared with CON, FH+ were markedly insulin resistant and had severely impaired insulin signaling in response to insulin stimulation. LIP in CON reduced insulin-stimulated glucose disposal (Rd) by 25%, insulin-stimulated insulin receptor tyrosine phosphorylation by 17%, phosphatidylinositol 3-kinase activity associated with insulin receptor substrate-1 by 20%, and insulin-stimulated glycogen synthase fractional velocity over baseline (44 vs. 15%; all P < 0.05). In contrast to CON, a physiological elevation in plasma FFA in FH+ led to no further deterioration in Rd or to any additional impairment of insulin signaling. In conclusion, a 4-day physiological increase in plasma FFA to levels seen in obesity and T2DM impairs insulin action/insulin signaling in CON but does not worsen insulin resistance in FH+. Whether this lack of additional deterioration in insulin signaling in FH+ is due to already well-established lipotoxicity, or to other molecular mechanisms related to insulin resistance that are nearly maximally expressed early in life, remains to be determined.     

Triacylglycerol accumulation is not primarily affected in myotubes established from type 2 diabetic subjects

In the present study, we investigated triacylglycerol (TAG) accumulation, glucose and fatty acid (FA) uptake, and glycogen synthesis (GS) in human myotubes from healthy, lean, and obese subjects with and without type 2 diabetes (T2D), exposed to increasing palmitate (PA) and oleate (OA) concentrations with/without high glucose and/or high insulin concentrations for 4 days. We showed that these myotubes expressed an increased TAG accumulation (P<0.001) without differences between groups. Chronically high insulin, but not high glucose concentrations, increases TAG accumulation by 25% (P<0.001). Inhibition of oxidative phosphorylation by antimycin A and oligomyin was followed by a reduced lipid oxidation (P<0.05) and increased TAG accumulation (P<0.05), but only in the presence of FAs. Both chronic PA and OA exposure reduced the insulin-mediated PA and OA uptake (fold change) (P<0.001), but could not induce insulin resistance at the level of glucose uptake, whereas high insulin concentrations induced insulin resistance (P<0.001). Chronic, high PA, but not OA, induced insulin resistance at the GS level in control subjects (P<0.05). The TAG content correlated negatively with insulin-stimulated FA uptake (P<0.001), but did not correlate with insulin-stimulated glucose uptake for PA or OA (P>0.05). These results indicate that (1) TAG accumulation is not primarily affected in skeletal muscle tissue of obese and T2D; (2) induced inhibition of oxidative phosphorylation is followed by TAG accumulation; (3) increasing FA and insulin availability, and reduced oxidative phosphorylation, and to a lesser extent glucose, are determinants for differences in intramyocellular TAG accumulation; (4) quantitative TAG content may not be the best marker for insulin resistance. Thus, increased TAG content in skeletal muscle of obese and T2D subjects is adaptive.     

Increased collagen content in insulin-resistant skeletal muscle

Oversupply and underutilization of lipid fuels are widely recognized to be strongly associated with insulin resistance in skeletal muscle. Recent attention has focused on the mechanisms underlying this effect, and defects in mitochondrial function have emerged as a potential player in this scheme. Because evidence indicates that lipid oversupply can produce abnormalities in extracellular matrix composition and matrix changes can affect the function of mitochondria, the present study was undertaken to determine whether muscle from insulin-resistant, nondiabetic obese subjects and patients with type 2 diabetes mellitus had increased collagen content. Compared with lean control subjects, obese and type 2 diabetic subjects had reduced muscle glucose uptake (P<0.01) and decreased insulin stimulation of tyrosine phosphorylation of insulin receptor substrate-1 and its ability to associate with phosphatidylinositol 3-kinase (P<0.01 and P<.05). Because it was assayed by total hydroxyproline content, collagen abundance was increased in muscle from not only type 2 diabetic patients but also nondiabetic obese subjects (0.26+/-0.05, 0.57+/-0.18, and 0.67+/- 0.20 microg/mg muscle wet wt, lean controls, obese nondiabetics, and type 2 diabetics, respectively), indicating that hyperglycemia itself could not be responsible for this effect. Immunofluorescence staining of muscle biopsies indicated that there was increased abundance of types I and III collagen. We conclude that changes in the composition of the extracellular matrix are a general characteristic of insulin-resistant muscle.     

Expression and regulation by insulin of low-density lipoprotein receptor-related protein mRNA in human skeletal muscle

Evidence suggests that increased hydrolysis and/or uptake of triglyceride-rich lipoprotein particles in skeletal muscle can be involved in insulin resistance. We determined the steady state mRNA levels of the low-density lipoprotein-related receptor (LRP) and lipoprotein lipase (LPL) in skeletal muscle of eight healthy lean control subjects, eight type 2 diabetic patients and eight nondiabetic obese individuals. The regulation by insulin of LRP and LPL mRNA expression was also investigated in biopsies taken before and at the end of a 3 h euglycemic hyperinsulinemic clamp (insulinemia of about 1 nM). LRP mRNA was expressed in human skeletal muscle (1.3+/-0.1 amol/microg total RNA in control subjects). Type 2 diabetic patients, but not nondiabetic obese subjects, were characterized by a reduced expression of LRP (0.8+/-0.2 and 1.3+/-0.3 amol/microg total RNA in diabetic and obese patients, respectively; P<0.05 in diabetic vs. control subjects). Insulin infusion decreased LRP mRNA levels in muscle of the control subjects but not in muscle of type 2 diabetic and nondiabetic obese patients. Similar results were found when investigating the regulation of the expression of LPL. Taken together, these results did not support the hypothesis that a higher capacity for clearance or hydrolysis of circulating triglycerides in skeletal muscle is present during obesity- or type 2 diabetes-associated insulin resistance.     

Increased insulin receptor signaling and glycogen synthase activity contribute to the synergistic effect of exercise on insulin action.

The purpose of this study was to determine the factors contributing to the ability of exercise to enhance insulin-stimulated glucose disposal. Sixteen insulin-resistant nondiabetic and seven Type 2 diabetic subjects underwent two hyperinsulinemic (40 mU x m-2 x min-1) clamps, once without and once with concomitant exercise at 70% peak O2 consumption. Exercise was begun at the start of insulin infusion and was performed for 30 min. Biopsies of the vastus lateralis were performed before and after 30 min of insulin infusion (immediately after cessation of exercise). Exercise synergistically increased insulin-stimulated glucose disposal in nondiabetic [from 4.6 +/- 0.4 to 9.5 +/- 0.8 mg x kg fat-free mass (FFM)-1x min-1] and diabetic subjects (from 4.3 +/- 1.0 to 7.9 +/- 0.7 mg. kg FFM-1x min-1) subjects. The rate of glucose disposal also was significantly greater in each group after cessation of exercise. Exercise enhanced insulin-stimulated increases in glycogen synthase fractional velocity in control (from 0.07 +/- 0.02 to 0.22 +/- 0.05, P < 0.05) and diabetic (from 0.08 +/- 0.03 to 0.15 +/- 0.03, P < 0.01) subjects. Exercise also enhanced insulin-stimulated glucose storage (glycogen synthesis) in nondiabetic (2.9 +/- 0.9 vs. 4.9 +/- 1.1 mg x kg FFM-1x min-1) and diabetic (1.7 +/- 0.5 vs. 4.2 +/- 0.8 mg x kg FFM-1. min-1) subjects. Increased glucose storage accounted for the increase in whole body glucose disposal when exercise was performed during insulin stimulation in both groups; effects of exercise were correlated with enhancement of glucose disposal and glucose storage (r = 0.93, P < 0.001). Exercise synergistically enhanced insulin-stimulated insulin receptor substrate 1-associated phosphatidylinositol 3-kinase activity (P < 0.05) and Akt Ser473 phosphorylation (P < 0.05) in nondiabetic subjects but had little effect in diabetic subjects. The data indicate that exercise, performed in conjunction with insulin infusion, synergistically increases insulin-stimulated glucose disposal compared with insulin alone. In nondiabetic and diabetic subjects, increased glycogen synthase activation is likely to be involved, in part, in this effect. In nondiabetic, but not diabetic, subjects, exercise-induced enhancement of insulin stimulation of the phosphatidylinositol 3-kinase pathway is also likely to be involved in the exercise-induced synergistic enhancement of glucose disposal.     

Acute selective glycogen synthase kinase-3 inhibition enhances insulin signaling in prediabetic insulin-resistant rat skeletal muscle

Glycogen synthase kinase-3 (GSK3) has been implicated in the multifactorial etiology of skeletal muscle insulin resistance in animal models and in human type 2 diabetic subjects. However, the potential molecular mechanisms involved are not yet fully understood. Therefore, we determined if selective GSK3 inhibition in vitro leads to an improvement in insulin action on glucose transport activity in isolated skeletal muscle of insulin-resistant, prediabetic obese Zucker rats and if these effects of GSK3 inhibition are associated with enhanced insulin signaling. Type I soleus and type IIb epitrochlearis muscles from female obese Zucker rats were incubated in the absence or presence of a selective, small organic GSK3 inhibitor (1 microM CT118637, Ki < 10 nM for GSK3alpha and GSK3beta). Maximal insulin stimulation (5 mU/ml) of glucose transport activity, glycogen synthase activity, and selected insulin-signaling factors [tyrosine phosphorylation of insulin receptor (IR) and IRS-1, IRS-1 associated with p85 subunit of phosphatidylinositol 3-kinase, and serine phosphorylation of Akt and GSK3] were assessed. GSK3 inhibition enhanced (P <0.05) basal glycogen synthase activity and insulin-stimulated glucose transport in obese epitrochlearis (81 and 24%) and soleus (108 and 20%) muscles. GSK3 inhibition did not modify insulin-stimulated tyrosine phosphorylation of IR beta-subunit in either muscle type. However, in obese soleus, GSK3 inhibition enhanced (all P < 0.05) insulin-stimulated IRS-1 tyrosine phosphorylation (45%), IRS-1-associated p85 (72%), Akt1/2 serine phosphorylation (30%), and GSK3beta serine phosphorylation (39%). Substantially smaller GSK3 inhibitor-mediated enhancements of insulin action on these insulin signaling factors were observed in obese epitrochlearis. These results indicate that selective GSK3 inhibition enhances insulin action in insulin-resistant skeletal muscle of the prediabetic obese Zucker rat, at least in part by relieving the deleterious effects of GSK3 action on post-IR insulin signaling. These effects of GSK3 inhibition on insulin action are greater in type I muscle than in type IIb muscle from these insulin-resistant animals.     

Lactate induces insulin resistance in skeletal muscle by suppressing glycolysis and impairing insulin signaling

Elevation of plasma lactate levels induces peripheral insulin resistance, but the underlying mechanisms are unclear. We examined whether lactate infusion in rats suppresses glycolysis preceding insulin resistance and whether lactate-induced insulin resistance is accompanied by altered insulin signaling and/or insulin-stimulated glucose transport in skeletal muscle. Hyperinsulinemic euglycemic clamps were conducted for 6 h in conscious, overnight-fasted rats with or without lactate infusion (120 micromol x kg(-1) x min(-1)) during the final 3.5 h. Lactate infusion increased plasma lactate levels about fourfold. The elevation of plasma lactate had rapid effects to suppress insulin-stimulated glycolysis, which clearly preceded its effect to decrease insulin-stimulated glucose uptake. Both submaximal and maximal insulin-stimulated glucose transport decreased 25-30% (P < 0.05) in soleus but not in epitrochlearis muscles of lactate-infused rats. Lactate infusion did not alter insulin's ability to phosphorylate the insulin receptor, the insulin receptor substrate (IRS)-1, or IRS-2 but decreased insulin's ability to stimulate IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase activities and Akt/protein kinase B activity by 47, 75, and 55%, respectively (P < 0.05 for all). In conclusion, elevation of plasma lactate suppressed glycolysis before its effect on insulin-stimulated glucose uptake, consistent with the hypothesis that suppression of glucose metabolism could precede and cause insulin resistance. In addition, lactate-induced insulin resistance was associated with impaired insulin signaling and decreased insulin-stimulated glucose transport 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.     

Evidence for physiological coupling of insulin-mediated glucose metabolism and limb blood flow

We hypothesized that the vasodilation observed during insulin stimulation is closely coupled to the rate of glucose metabolism. Lean (L, n = 13), obese nondiabetic (OB, n = 13), and obese type 2 diabetic subjects (Type 2 DM, n = 16) were studied. Leg blood flow (LBF) was examined under conditions of euglycemic hyperinsulinemia (EH) and hyperglycemic hyperinsulinemia (HH), which produced a steady-state whole body glucose disposal rate (GDR) of approximately 2,000 micromol. m(-2). min(-1). At this GDR, under both conditions, subjects across the range of insulin sensitivity exhibited equivalent LBF (l/min EH: L, 0.42 +/- 0.03; OB, 0.43 +/- 0. 03; Type 2 DM, 0.38 +/- 0.07; P = 0.72 by ANOVA. HH: L, 0.44 +/- 0. 04; OB, 0.39 +/- 0.05; Type 2 DM, 0.41 +/- 0.04; P = 0.71). The continuous relationship between LBF and GDR did not differ across subject groups [slope x 10(-5) l/(micromol. m(-2). min(-1)) by ANOVA. EH: L, 8.6; OB, 9.2; Type 2 DM, 7.9; P = 0.91. HH: L, 4.2; OB, 2.5; Type 2 DM, 4.1; P = 0.77], although this relationship did differ between the EH and HH conditions (P = 0.001). These findings support a physiological coupling of LBF and insulin-mediated glucose metabolism. The mechanism(s) linking substrate delivery and metabolism appears to be intact in insulin-resistant states.     

Determinants of insulin-stimulated glucose disposal in middle-aged, premenopausal women

Controversy exists regarding the relative importance of adiposity, physical fitness, and physical activity in the regulation of insulin-stimulated glucose disposal. To address this issue, we measured insulin-stimulated glucose disposal [mg. kg fat-free mass (FFM)(-1). min(-1); oxidative and nonoxidative components] in 45 nondiabetic, nonobese, premenopausal women (mean +/- SD; 47 +/- 3 yr) by use of hyperinsulinemic euglycemic clamp (40 mU. m(-2). min(-1)) and [6,6-2H2]glucose dilution techniques. We also measured body composition, abdominal fat distribution, thigh muscle fat content, maximal oxygen consumption (VO2 max), and physical activity energy expenditure ((2)H(2)(18)O kinetics) as possible correlates of glucose disposal. VO2 max was the strongest correlate of glucose disposal (r = 0.63, P < 0.01), whereas whole body and abdominal adiposity showed modest associations (range of r values from -0.32 to -0.46, P < 0.05 to P < 0.01). A similar pattern of correlations was observed for nonoxidative glucose disposal. None of the variables measured correlated with oxidative glucose disposal. The relationship of VO2 max to glucose disposal persisted after statistical control for FFM, percent body fat, and intra-abdominal fat (r = 0.40, P < 0.01). In contrast, correlations of total and regional adiposity measures to insulin sensitivity were no longer significant after statistical adjustment for VO2 max. VO2 max was the only variable to enter stepwise regression models as a significant predictor of total and nonoxidative glucose disposal. Our results highlight the importance of VO2 max as a determinant of glucose disposal and suggest that it may be a stronger determinant of variation in glucose disposal than total and regional adiposity in nonobese, nondiabetic, premenopausal women.