Muscle Insulin Research Articles

Impaired regulation of glucose transporter 4 gene expression in insulin resistance associated with in utero undernutrition.

The aim of this study was to investigate whether insulin resistance-associated in utero undernutrition was related to changes in insulin action on gene expression of molecules involved in the insulin signaling pathway and peripheral glucose metabolism in muscle and adipose tissue. Thirteen insulin-resistant subjects born with intrauterine growth retardation (IUGR) were matched for age, gender, and body mass index to 13 controls. Gene expression of insulin receptor, insulin receptor substrate-1, p85alpha phosphatidylinositol 3-kinase, glucose transporter-4 (GLUT4), hexokinase II, and glycogen synthase was studied in skeletal muscle at baseline and after a 3-h euglycemic insulin stimulation. Target messenger ribonucleic acid (mRNA) levels were quantified using the RT-competitive PCR method. Insulin-stimulated glucose uptake was significantly lower in IUGR-born subjects than in controls (36.9 +/- 12, 7 vs. 53.9 +/- 12.7 micromol/kg.min; P = 0.007), affecting both the glucose oxidation rate and the nonoxidative glucose disposal rate. At baseline, the expression of the six genes in muscle did not significantly differ between the two groups. The insulin-induced changes over baseline were comparable in both groups for all mRNAs, except GLUT4. In contrast to what observed in the control group (mean increment, 49 +/- 23%; P = 0.0009), GLUT4 expression was not stimulated by insulin in the IUGR group (8 +/- 8%; P = 0.42). Moreover, the magnitude of the defect in GLUT4 mRNA regulation by insulin was correlated to the degree of insulin resistance (r = 0.73; P = 0.01). A similar lack of significant GLUT4 mRNA stimulation by insulin was observed in the adipose tissue of IUGR-born subjects. In conclusion, insulin resistance in IUGR-born subjects is associated with an impaired regulation of GLUT4 expression by insulin in muscle and adipose tissue. Our data provide additional information about the mechanism of insulin resistance associated with in utero undernutrition and strengthen the role of glucose transport in the control of insulin sensitivity.

Insulin-stimulated GLUT4 translocation requires the CAP-dependent activation of TC10

The stimulation of glucose uptake by insulin in muscle and adipose tissue requires translocation of the GLUT4 glucose transporter protein from intracellular storage sites to the cell surface. Although the cellular dynamics of GLUT4 vesicle trafficking are well described, the signalling pathways that link the insulin receptor to GLUT4 translocation remain poorly understood. Activation of phosphatidylinositol-3-OH kinase (PI(3)K) is required for this trafficking event, but it is not sufficient to produce GLUT4 translocation. We previously described a pathway involving the insulin-stimulated tyrosine phosphorylation of Cbl, which is recruited to the insulin receptor by the adapter protein CAP. On phosphorylation, Cbl is translocated to lipid rafts. Blocking this step completely inhibits the stimulation of GLUT4 translocation by insulin. Here we show that phosphorylated Cbl recruits the CrkII-C3G complex to lipid rafts, where C3G specifically activates the small GTP-binding protein TC10. This process is independent of PI(3)K, but requires the translocation of Cbl, Crk and C3G to the lipid raft. The activation of TC10 is essential for insulin-stimulated glucose uptake and GLUT4 translocation. The TC10 pathway functions in parallel with PI(3)K to stimulate fully GLUT4 translocation in response to insulin.

Muscle cell lipid and insulin sensitivity

Muscle cell lipid and insulin sensitivity

Activation of p38 mitogen-activated protein kinase alpha and beta by insulin and contraction in rat skeletal muscle: potential role in the stimulation of glucose transport.

The stress-activated p38 mitogen-activated protein kinase (MAPK) was recently shown to be activated by insulin in muscle and adipose cells in culture. Here, we explore whether such stimulation is observed in rat skeletal muscle and whether muscle contraction can also affect the enzyme. Insulin injection (2 U over 3.5 min) resulted in increases in p38 MAPK phosphorylation measured in soleus (3.2-fold) and quadriceps (2.2-fold) muscles. Increased phosphorylation (3.5-fold) of an endogenous substrate of p38 MAPK, cAMP response element binder (CREB), was also observed. After in vivo insulin treatment, p38 MAPKalpha and p38 MAPKbeta isoforms were found to be activated (2.1- and 2.4-fold, respectively), using an in vitro kinase assay, in immunoprecipitates from quadriceps muscle extracts. In vitro insulin treatment (1 nmol/l over 4 min) and electrically-induced contraction of isolated extensor digitorum longus (EDL) muscle also doubled the kinase activity of p38 MAPKalpha and p38 MAPKbeta. The activity of both isoforms was inhibited in vitro by 10 micromol/l SB203580 in all muscles. To explore the possible participation of p38 MAPK in the stimulation of glucose uptake, EDL and soleus muscles were exposed to increasing doses of SB203580 before and during stimulation by insulin or contraction. SB203580 caused a significant reduction in the insulin- or contraction-stimulated 2-deoxyglucose uptake. Maximal inhibition (50-60%) occurred with 10 micromol/l SB203580. These results show that p38 MAPKalpha and -beta isoforms are activated by insulin and contraction in skeletal muscle. The data further suggest that activation of p38 MAPK may participate in the stimulation of glucose uptake by both stimuli in rat skeletal muscle.

Measurement of interstitial insulin in human muscle.

Previous measurements in lymph and adipose tissue have indicated that interstitial insulin concentrations are approximately 40% lower than in plasma. Measurements of insulin in human muscle interstitial fluid have not been performed yet. We developed a new external reference technique for calibration of microdialysis catheters in situ. This technique allows correct assessments of interstitial peptide concentrations and was employed to estimate the insulin concentration in medial quadriceps femoris muscle in 11 individuals (age: 37 +/- 3 yr; body mass index: 25.2 +/- 1.2 kg/m2) during a two-step euglycemic hyperinsulinemic clamp. At steady-state insulin and glucose infusion, plasma glucose was 5.9 +/- 0.2 mmol/l, plasma insulin was 155 +/- 17 mU/l, and interstitial muscle insulin was 67 +/- 19 mU/l (n = 9; P < 0.01). At a higher insulin infusion rate, the steady-state plasma insulin concentration was 379 +/- 58 mU/l, and interstitial insulin concentration was 180 +/- 40 mU/l (P < 0.01). The data show for the first time that high physiological and supraphysiological plasma insulin levels give 30-50% lower interstitial concentrations of insulin in the muscle. The importance of capillary delivery as a rate-limiting step for the insulin effect is suggested.

Differential regulation of endothelin-1 action by insulin and vanadate in vascular smooth muscle.

The effects of insulin (10 nM) and sodium orthovanadate (SOV, 25 microM) pretreatment for 24 h on endothelin-1 (ET-1) and IRL-1620 (ETB agonist)-evoked increases in intra-cellular free calcium levels [Ca2+]i were determined by fura-2 fluorescence methodology using cultured rat aortic smooth-muscle cells (ASMCs). Whereas insulin preincubation led to an increase (p < 0.05) in [Ca2+]i responses to ET-1, SOV enhanced [Ca2+]i responses to both ET-1 (p < 0.05) and IRL-1620 (p < 0.01). Saturation binding studies revealed that insulin pretreatment led to a 2.2-fold (p < 0.01) increase in [125I]ET-1 binding sites, and SOV pretreatment led to a 2.4-fold (p < 0.01) and 6.6-fold (p < 0.01) increase in the maximal number of binding sites labeled by [125I]ET-1 and [125I]IRL-1620, respectively. Northern blot analysis revealed that insulin upregulated only ETA mRNA levels, and SOV enhanced both ETA and ETB mRNA levels. Pretreatment with the tyrosine kinase inhibitor genistein abolished these effects. These data suggest that insulin pretreatment of ASMCs selectively upregulates ETA receptor expression and ETA-mediated [Ca2+]i, signaling, and SOV upregulates both ETA and ETB receptor expression and exaggerates [Ca2+]i responses mediated by both ETA and ETB in rat ASMCs.

Muscle GLUT-4 and Insulin Binding to Erythrocytes and to Adipose, Liver and Muscle Tissue in Genetically Hypertensive Obese Rats and in Their Lean Siblings: Effect of Long Lasting Terguride Treatment

Experiments were performed in the genetically hypertensive obese rats of Koletsky type (SHR/N-cp) and in their lean siblings of both sexes. Insulin binding to erythrocytes and to adipose tissue, lever tissue and muscle tissue was monitored in the control animals and in the animals under the long lasting terguride treatment. In control animals insulin binding shows substrain and tissue dependence being elevated in lean rats except insulin binding to erythrocytes where inverse is true. Terguride increases percentage of specific insulin binding to erythrocytes in all groups except obese females, terguride increases percentage of specific binding to adipose tissue except lean females, the mentioned drug remained without effect in muscle tissue in all group except lean females where drug induced elevation was detected. The effect of terguride in liver tissue was monitored only in males of both substrains, elevation was found only in lean. GLUT-4 was analyzed only in muscle tissue. The effect of terguride was found in obese females, i.e., in the group which shows reduced GLUT-4 relative to lean females.

Selective chronic regulation of GLUT1 and GLUT4 content by insulin, glucose, and lipid in rat cardiac muscle in vivo.

The glucose transporter GLUT1 may play a more important role in cardiac than in skeletal muscle, but its regulation is unclear. During fasting, cardiac GLUT1 declines in the presence of low plasma insulin and glucose and high nonesterified fatty acid (NEFA) levels, whereas GLUT4 is unchanged. We investigated insulin, glucose, and NEFA levels as regulatory factors of cardiac GLUT content in chronically cannulated rats. Fasting rats were infused for 24 h with saline or insulin (2 rates) while plasma glucose was equalized by a glucose clamp; final transporter content was compared with a fed control group. There was a close association of GLUT1 content with insulin (r2 = 0.83, P < 0.001), with GLUT1 varying over a threefold range, under equivalent fasting glycemic conditions (plasma glucose, 5.1 +/- 0.1 mM). Maintenance of fed insulin levels during fasting prevented the GLUT1 fall (P < 0.01), whereas hyperinsulinemia (117 +/- 10 mU/l) led to significant overexpression of GLUT1 (155 +/- 12% of control, P < 0.01). When high glucose (7.6 +/- 0.1 mM) or high NEFA (0.76 +/- 0.05 mM) levels accompanied the hyperinsulinemia, upregulation of GLUT1 was blocked. GLUT1 content correlated with an estimate of cardiac glucose clearance across the groups. Cardiac GLUT4 content, hexokinase, and acyl-CoA synthase activities were unaffected by fasting, insulin, or substrate manipulation. In conclusion, insulin preferentially upregulates GLUT1 (but not GLUT4) in a dose-dependent manner in cardiac muscle in vivo, and substrate supply modulates this response, since upregulation can be effectively blocked by increased glucose or lipid availability. Therefore, both insulin exposure and energy status of cardiac muscle may be important determinants of cardiac GLUT1 expression.

Effects of acute and chronic hindlimb suspension on sensitivity and responsiveness to insulin in the rat soleus muscle

Effects of acute and chronic hindlimb suspension on sensitivity and responsiveness to insulin in the rat soleus muscle

Insulin Signalling and Insulin Actions in the Muscles and Livers of Insulin-Resistant, Insulin Receptor Substrate 1-Deficient Mice

We and others recently generated mice with a targeted disruption of the insulin receptor substrate 1 (IRS-1) gene and demonstrated that they exhibited growth retardation and had resistance to the glucose-lowering effect of insulin. Insulin initiates its biological effects by activating at least two major signalling pathways, one involving phosphatidylinositol 3-kinase (PI3-kinase) and the other involving a ras/mitogen-activated protein kinase (MAP kinase) cascade. In this study, we investigated the roles of IRS-1 and IRS-2 in the biological action in the physiological target organs of insulin by comparing the effects of insulin in wild-type and IRS-1-deficient mice. In muscles from IRS-1-deficient mice, the responses to insulin-induced PI3-kinase activation, glucose transport, p70 S6 kinase and MAP kinase activation, mRNA translation, and protein synthesis were significantly impaired compared with those in wild-type mice. Insulin-induced protein synthesis was both wortmannin sensitive and insensitive in wild-type and IRS-1 deficient mice. However, in another target organ, the liver, the responses to insulin-induced PI3-kinase and MAP kinase activation were not significantly reduced. The amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) was roughly equal to that of IRS-1 (in wild-type mice) in the liver, whereas it only 20 to 30% of that of IRS-1 in the muscles. In conclusion, (i) IRS-1 plays central roles in two major biological actions of insulin in muscles, glucose transport and protein synthesis; (ii) the insulin resistance of IRS-1-deficient mice is mainly due to resistance in the muscles; and (iii) the degree of compensation for IRS-1 deficiency appears to be correlated with the amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) relative to that of IRS-1 (in wild-type mice).

Extracellular distribution of insulin in muscle of rats exposed to long‐term hyperinsulinaemia

The importance of increased capillary density for the regulation of insulin sensitivity by transcapillary delivery of insulin to muscle cells in insulin-exposed rats was investigated by direct microdialysis measurements of interstitial (125I] insulin concentrations in the femoral muscle during an euglycaemic hyperinsulinaemic clamp. In insulin-exposed rats plasma insulin was approximately 25% (P < 0.05) higher than that in control animals during the first 100 min and reached their maximal concentrations after 100 min. After a nitroprusside infusion given at 100 min both groups had similar concentrations of insulin in plasma as well as in muscle interstitial fluid. However, mean glucose infusion rate during the first clamp hour was 20.5 +/- 2.3 and 12.6 +/- 5.2 mg kg-1 min-1 (P < 0.05) in insulin-exposed and control animals, respectively. During the second clamp hour the corresponding figures were 21.1 +/- 2.4 and 13.9 +/- 2.6 (P < 0.05). It may be concluded that capillarization and/or nitroprusside affected plasma insulin concentrations without altering either the interstitial insulin levels or the insulin effect on glucose consumption. The data suggest that the elevated insulin sensitivity after chronic insulin exposure is dependent on other than transcapillary transport events and demonstrate the different kinetics for insulin distribution in plasma and in the interstitial fluid.

Wortmannin inhibits insulin-stimulated but not contraction-stimulated glucose transport activity in skeletal muscle

In skeletal muscle, glucose transport is stimulated by insulin, contractions and hypoxia. In this study, we used the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin to examine whether (i) PI 3-kinase activity is necessary for stimulation of glucose transport by insulin in muscle, and (ii) PI 3-kinase mediates a step in the pathway by which contractions/hypoxia stimulate glucose transport. Wortmannin completely blocked insulin- and insulin-like growth factor-1-stimulated glucose transport in muscle. In contrast, wortmannin had no effect on the stimulation of glucose transport by contractions or hypoxia, providing evidence that PI 3-kinase activity is not involved in the activation of glucose transport by these stimuli.

Inhibitory Effect of Fatty Acids on Glucose and Insulin Uptake in the Perfused Rat Hindquarter

To elucidate the effects of fatty acids on the uptake of glucose and insulin in the muscle, the effects of oleic and palmitic acids on the uptake of glucose and insulin were investigated in perfused hind-quarters of rats. In the absence of insulin, glucose uptake in the hindquarter was slightly, but not significantly, decreased by the addition of oleic and palmitic acids. In the presence of 100 or 500 microU/ml insulin, glucose uptake in the hindquarter (243 +/- 58 or 282 +/- 65 mumol/30 min) was significantly decreased by the addition of 1000 microM oleic acid and 500 microM palmitic acid (175 +/- 50 or 207 +/- 47 mumol/30 min). The decrease in insulin uptake rate, although not significant at 500 microU/ml insulin, is of similar magnitude to the decrease in glucose uptake. In the presence of 1000 microU/ml insulin, glucose and insulin uptake was not significantly altered by the addition of fatty acids. These results indicate that fatty acids directly inhibit the muscular glucose uptake via the decrease in muscular insulin uptake at a physiological concentration of insulin.

Exercise training increases GLUT-4 protein concentration in previously sedentary middle-aged men

The purpose of this study was to determine if 14 wk of exercise training would increase insulin-sensitive glucose transporter protein (GLUT-4) concentration in skeletal muscle of previously sedentary middle-aged men (47.2 +/- 1.3 yr; n = 13). Muscle samples (lateral gastrocnemius) and insulin action [insulin sensitivity index (ISI), minimal model] were obtained in the sedentary condition and 48 h after the final training bout. GLUT-4 protein concentration increased (P < 0.001, 2,629 +/- 331 to 4,140 +/- 391 absorbance units/100 micrograms protein) with exercise training by 1.8-fold. ISI increased by twofold (P < 0.05, 2.1 +/- 0.5 to 3.4 +/- 0.7 SI x 10(5) min/pM) with training. The percentage of GLUT-4 rich type IIa muscle fibers increased by approximately 10% (P < 0.01), which may have contributed to the elevation in transporter protein. GLUT-4 concentration and citrate synthase activity (1.7-fold, P < 0.001) also increased by similar increments. These findings indicate that GLUT-4 protein concentration is elevated in middle-aged individuals with exercise training.

Amylin and insulin in rat soleus muscle: dose responses for cosecreted noncompetitive antagonists

Increasing concentrations of amylin progressively depressed the maximal insulin-stimulated radioglucose incorporation into soleus muscle glycogen, but did not substantively change the EC50 (range 0.78 to 1.52 nM); these findings show noncompetitive, insurmountable antagonism of insulin action by amylin. The results from 36 combinations of different insulin and amylin concentrations were used to construct a response surface that can be used to predict the response for any combination of insulin and amylin concentration. The predicted response to a constant ratio of insulin and amylin concentration is a bell-shaped curve. The experimentally determined response to increasing amounts of an amylin-insulin mixture (molar ratio of 0.14:1, within the range measured for pancreatic secretion and plasma levels) gave a bell-shaped response rather than the sigmoidal response seen with insulin alone. The amylin dose-response relation in the soleus system provides a useful bioassay for amylin agonists. The dose response for highly purified, synthetic human amylin obtained by measuring amylin concentrations by radioimmunoassay in the incubation medium gave an EC50 of 456 pM (+/- 0.18 log units). Human amylin had a potency greater than or equal to that of human insulin in this highly insulin-sensitive preparation.

Unimpaired effect of insulin on glucokinase gene expression in hepatocytes challenged with amylin

Amylin appears to interfere with the action of insulin in muscle and possibly in liver. We have attempted to detect a direct antagonism between amylin and insulin in cultured rat hepatocytes. The stimulation of glucokinase gene expression was used as a marker of insulin action. Amylin proved ineffective in suppressing subsequent accumulation of glucokinase mRNA in response to maximal or submaximal doses of insulin. When applied to cells already induced by prior incubation with insulin alone, amylin failed to reverse induction, in contrast to the effectiveness of glucagon under the same conditions. Thus, amylin is not a physiological antagonist of insulin in the control of hepatic glucokinase gene expression.

In chronic uremia, insulin activates receptor kinase but not pyruvate dehydrogenase.

We studied in vivo biochemical effects of insulin in the skeletal muscle of chronically uremic and control rats. The rate of disappearance of blood glucose (determined with a short intravenous test) was reduced by 38% in uremia (p less than 0.05). Intraperitoneal treatment with insulin plus glucose for 30 min caused a 3-fold increase in the activity of insulin receptor tyrosine kinase in the skeletal muscle of both rat groups. Conversely, pyruvate dehydrogenase activity increased by 115% in controls but only by 26% in uremics (p less than 0.01). Exercise (swimming for 30 min) increased muscle pyruvate dehydrogenase activity approximately 2-fold in both groups of animals. These experiments show that in uremic rats, insulin binds normally to its muscle receptors and adequately activates receptor tyrosine kinase but fails to activate an otherwise responsive pyruvate dehydrogenase.

Anomalous leucine metabolism in total lipoatrophic diabetes: a possible mechanism of muscle mass hypertrophy

Total acquired lipoatrophic diabetes (LD) is characterized by muscle mass hypertrophy, non-ketotic hyperglycaemia and insulin resistance with respect to glucose and lipid metabolism. To assess whether the defect in insulin action extends to leucine/protein metabolism, a female subject (age=33 years; body weight=44 kg; HbAlc=9.5%) with LD was studied twice: in study I we used a three-step euglycaemic hyperinsulinaemic clamp (40, 80 and 200 mU · m−2 · min−1) combined with [3-3H]glucose and [1-14C]leucine infusions along with indirect calorimetry. In study II we used a 40 mU · m−2 · min−1 euglycaemic hyperaminoacidaemic (plasma leucine 160 μmol/l) hyperinsulinaemic clamp. Five controls were also studieD. In the basal state the patient with LD had plasma leucine (130 μmol/l), isoleucine (63), valine (169) and phenylalanine levels (48) comparable to those of the controls. Basal hepatic glucose production (3.2 vs 2.0±0.2 mg · kg−1 · min−1), endogenous leucine flux (ELF=45.4 vs 40±1 μmol · m−2 · min−1) and non-oxidative leucine disposal (NOLD=37.2 vs 34±1 μmol · m−2 · min−1) were increased in the patient with LD, while basal leucine oxidation (LO=8.2 vs 6.0±2 μmol · m−2 · min−1) was similar in LD and controls. Following the three-step insulin infusion, insulin-stimulated glucose metabolism was defective in the subject with LD (glucose oxidation=60%, 50%, and 52% of controls; non-oxidative glucose disposal =39%, 34% and 30% of controls at 40, 80 and 200 mU · m−2 · min−1 respectively). The decrease of leucine, isoleucine, valine and phenylalanine, as well as the suppression of ELF, LO and NOLD was defective in the subject with LD at each insulin step. In vitro studies demonstrated a defect in receptor insulin binding on erythrocytes, the absence of anti-insulin receptor antibodies and the presence of insulin antibodies in the serum of the patient with LD. Following combined hyperaminoacidaemia/hyperinsulinaemia a similar stimulation of protein synthetic rate (20 vs 30%) was demonstrated in the patient with LD and controls respectively. In conclusion the patient with LD shows a reduced insulin sensitivity and a reduced maximal response to insulin in both glucose and protein metabolism. The present data support the hypothesis that in LD the defect in insulin action is both at the receptor and post-receptorial sites. The patient with LD showed a normal stimulation of protein synthesis under combined hyperinsulinaemic/hyperaminoacidaemic conditions. Our results may explain the muscle mass hypertrophy in LD.

Effect of endurance and sprint exercise on the sensitivity of glucose metabolism to insulin in the epitrochlearis muscle of sedentary and trained rats

The effects of two types of acute exercise (1 h treadmill running at 20 m.min-1, or 6 x 10-s periods at 43 m.min-1, 0 degree inclination), as well as two training regimes (endurance and sprint) on the sensitivity of epitrochlearis muscle [fast twitch (FT) fibres] to insulin were measured in vitro in rats. The hormone concentration in the incubation medium producing the half maximal stimulation of lactate (la) production and glycogen synthesis was determined and used as an index of the muscle insulin sensitivity. A single period of moderate endurance as well as the sprint-type exercise increased the sensitivity of la production to insulin although the rate of la production enhanced markedly only after sprint exercise at 10 and 100 microU.ml-1 of insulin. These effects persisted for up to 2 h after the termination of exercise. Both types of exercise significantly decreased the muscle glycogen content, causing a moderate enhancement in the insulin-stimulated rates of glycogen synthesis in vitro for up to 2 h after exercise. However, a significant increase in the sensitivity of this process to insulin was found only in the muscle removed 0.25 h after the sprint effort. Training of the sprint and endurance types increased insulin-stimulated rates of glycolysis 24 h after the last period of exercise. The sensitivity of this process to insulin was also increased at this instant. Both types of training increased the basal and maximal rates of glycogen synthesis, as well as the sensitivity of this process to insulin at the 24th h following the last training session.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C activators selectively inhibit insulin-stimulated system A transport activity in skeletal muscle at a post-receptor level

We have investigated the role of phorbol esters on different biological effects induced by insulin in muscle, such as activation of system A transport activity, glucose utilization and insulin receptor function. System A transport activity was measured by monitoring the uptake of the system A-specific analogue alpha-(methyl)aminoisobutyric acid (MeAIB), by intact rat extensor digitorum longus muscle. The addition of 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.5 microM) for 60 or 180 min did not modify basal MeAIB uptake by muscle, suggesting that insulin signalling required to stimulate MeAIB transport does not involve protein kinase C activation. However, TPA added 30 min before insulin (100 nM) markedly inhibited insulin-stimulated MeAIB uptake. The addition of polymyxin B (0.1 mM) or H-7 (1 mM), protein kinase C inhibitors, alone or in combination with TPA leads to impairment of insulin-stimulated MeAIB uptake. This paradoxical pattern is incompatible with a unique action of Polymyxin B or H-7 on protein kinase C activity. Therefore these agents are not suitable tools with which to investigate whether a certain insulin effect is mediated by protein kinase C. TPA did not cause a generalized inhibition of insulin action. Thus both TPA and insulin increased 3-O-methylglucose uptake by muscle, and their effects were not additive. Furthermore, TPA did not modify insulin-stimulated lactate production by muscle. In keeping with this selective modification of insulin action, treatment of muscles with TPA did not modify insulin receptor binding or kinase activities. In conclusion, phorbol esters do not mimic insulin action on system A transport activity; however, they markedly inhibit insulin-stimulated amino acid transport, with no modification of insulin receptor function in rat skeletal muscle. It is suggested that protein kinase C activation causes a selective post-receptor modification on the biochemical pathway by which insulin activates system A amino acid transport in muscle.