Yuichi Kawano

Insulin Receptor Substrate-1 Phosphorylation and Phosphatidylinositol 3-Kinase Activity in Skeletal Muscle From NIDDM Subjects After In Vivo Insulin Stimulation

We examined the effect of physiological hyperinsulinemia on insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and phosphatidylinositol (PI) 3-kinase activity in skeletal muscle from six lean–to–moderately obese NIDDM patients and six healthy subjects. A rise in serum insulin levels from ∼60 to ∼650 pmol/l increased IRS-1 tyrosine phosphorylation sixfold over basal levels in control muscle (P < 0.01), whereas no significant increase was noted in NIDDM muscle. The reduced IRS-1 phosphorylation in the NIDDM muscle was not related to changes in IRS-1 protein content, since IRS-1 protein expression was similar between control and NIDDM subjects (16.0 ± 1.7 vs. 22.9 ± 4.0 arbitrary units/mg protein for control and NIDDM, respectively; NS). Physiological hyperinsulinemia increased PI 3-kinase activity in control muscle twofold (P < 0.01), whereas no increase in insulin-stimulated PI 3-kinase activity was noted in the NIDDM muscle. Furthermore, in vitro insulin-stimulated (600 pmol/l) 3-O-methylglucose transport was 40% lower in isolated muscle from NIDDM subjects (P < 0.05). The present findings couple both reduced insulin-stimulated IRS-1 tyrosine phosphorylation and PI 3-kinase activity to the impaired insulin-stimulated glucose transport in skeletal muscle from lean–to–moderately obese NIDDM subjects.

Improved Glucose Tolerance Restores Insulin-Stimulated Akt Kinase Activity and Glucose Transport in Skeletal Muscle From Diabetic Goto-Kakizaki Rats

The serine/threonine kinase Akt (protein kinase B [PKB] or related to A and C protein kinase [RAC] has recently been implicated to play a role in the signaling pathway to glucose transport. However, little is known concerning the regulation of Akt activity in insulinsensitive tissues such as skeletal muscle. To explore the role of hyperglycemia on Akt kinase activity in skeletal muscle, normal Wistar rats or Goto-Kakizaki (GK) diabetic rats were treated with phlorizin. Phlorizin treatment normalized fasting blood glucose and significantly improved glucose tolerance (P < 0.001) in GK rats, whereas in Wistar rats, the compound had no effect on glucose homeostasis. In soleus muscle from GK rats, maximal insulin-stimulated (120 nmol/l) Akt kinase activity was reduced by 68% (P < 0.01) and glucose transport was decreased by 39% (P < 0.05), compared with Wistar rats. Importantly, the defects at the level of Akt kinase and glucose transport were completely restored by phlorizin treatment. There was no significant difference in Akt kinase protein expression among the three groups. At a submaximal insulin concentration (2.4 nmol/l), activity of Akt kinase and glucose transport were unaltered. In conclusion, improved glucose tolerance in diabetic GK rats by phlorizin treatment fully restored insulin-stimulated activity of Akt kinase and glucose transport. Thus, hyperglycemia may directly contribute to the development of muscle insulin resistance through alterations in insulin action on Akt kinase and glucose transport.

Muscle fiber type specificity in insulin signal transduction

We determined the muscle fiber type-specific response of intracellular signaling proteins to insulin. Epitrochlearis (Epi; 15% type I, 20% type IIa, and 65% type IIb), soleus (84, 16, and 0%), and extensor digitorum longus (EDL; 3, 57, and 40%) muscles from Wistar rats were incubated without or with 120 nM insulin (3-40 min). Peak insulin receptor (IR) tyrosine phosphorylation was reached after 6 (soleus) and 20 (Epi and EDL) min, with sustained activity throughout insulin exposure (40 min). Insulin increased insulin receptor substrate (IRS)-1 and IRS-2 tyrosine phosphorylation and phosphotyrosine-associated phosphatidylinositol (PI)-3-kinase activity to a maximal level after 3-10 min, with subsequent downregulation. Akt kinase phosphorylation peaked at 20 min, with sustained activity throughout insulin exposure. Importantly, the greatest insulin response for all signaling intermediates was observed in soleus, whereas the insulin response between EDL and Epi was similar. Protein expression of the p85α-subunit of PI 3-kinase and Akt kinase, but not IR, IRS-1, or IRS-2, was greater in oxidative versus glycolytic muscle. In conclusion, increased function and/or expression of key proteins in the insulin-signaling cascade contribute to fiber type-specific differences in insulin action in skeletal muscle.We determined the muscle fiber type-specific response of intracellular signaling proteins to insulin. Epitrochlearis (Epi; 15% type I, 20% type IIa, and 65% type IIb), soleus (84, 16, and 0%), and extensor digitorum longus (EDL; 3, 57, and 40%) muscles from Wistar rats were incubated without or with 120 nM insulin (3-40 min). Peak insulin receptor (IR) tyrosine phosphorylation was reached after 6 (soleus) and 20 (Epi and EDL) min, with sustained activity throughout insulin exposure (40 min). Insulin increased insulin receptor substrate (IRS)-1 and IRS-2 tyrosine phosphorylation and phosphotyrosine-associated phosphatidylinositol (PI)-3-kinase activity to a maximal level after 3-10 min, with subsequent downregulation. Akt kinase phosphorylation peaked at 20 min, with sustained activity throughout insulin exposure. Importantly, the greatest insulin response for all signaling intermediates was observed in soleus, whereas the insulin response between EDL and Epi was similar. Protein expression of the p85alpha-subunit of PI 3-kinase and Akt kinase, but not IR, IRS-1, or IRS-2, was greater in oxidative versus glycolytic muscle. In conclusion, increased function and/or expression of key proteins in the insulin-signaling cascade contribute to fiber type-specific differences in insulin action in skeletal muscle.

Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice

To determine the role of GLUT4 on postexercise glucose transport and glycogen resynthesis in skeletal muscle, GLUT4‐deficient and wild‐type mice were studied aftera3h swim exercise. In wild‐type mice, insulin and swimming each increased 2‐deoxyglucose uptake by twofold in extensor digitorum longus muscle. In contrast, insulin did not increase 2‐deoxyglucose glucose uptake in muscle from GLUT4‐null mice. Swimming increased glucose transport twofold in muscle from fed GLUT4‐null mice, with no effect noted in fasted GLUT4‐null mice. This exercise‐associated 2‐deoxyglucose glucose uptake was not accompanied by increased cell surface GLUT1 content. Glucose transport in GLUT4‐null muscle was increased 1.6‐fold over basal levels after electrical stimulation. Contraction‐induced glucose transport activity was fourfold greater in wild‐type vs. GLUT4‐null muscle. Glycogen content in gastrocnemius muscle was similar between wild‐type and GLUT4‐null mice and was reduced ~50% after exercise. After 5 h carbohydrate refeeding, muscle glycogen content was fully restored in wild‐type, with no change in GLUT4‐null mice. After 24 h carbohydrate refeeding, muscle glycogen in GLUT4‐null mice was restored to fed levels. In conclusion, GLUT4 is the major transporter responsible for exercise‐induced glucose transport. Also, postexercise glycogen resynthesis in muscle was greatly delayed; unlike wild‐type mice, glycogen supercompensation was not found. GLUT4 it is not essential for glycogen repletion since muscle glycogen levels in previously exercised GLUT4‐null mice were totally restored after 24 h carbohydrate refeeding.—Ryder, J. W., Kawano, Y., Galuska, D., Fahlman, R., Wallberg‐Henriksson, H., Charron, M. J., Zierath, J. R. Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4‐deficient mice. FASEB J. 13, 2246–2256 (1999)

Changes in glucose transport and protein kinase Cβ2 in rat skeletal muscle induced by hyperglycaemia

Aims/hypothesis. We have previously reported that hyperglycaemia activates glucose transport in skeletal muscle by a Ca2+-dependent pathway, which is distinct from the insulin-signalling pathway. The aim of this study was to explain the signalling mechanism by which hyperglycaemia autoregulates glucose transport in skeletal muscle. Methods. Isolated rat soleus muscle was incubated in the presence of various concentrations of glucose or 3-O-methylglucose and protein kinase C and phospholipase C inhibitors. Glucose transport activity, cell surface glucose transporter 1 and glucose transporter 4 content and protein kinase C translocation was determined. Results. High concentrations of 3-O-methylglucose led to a concentration-dependent increase in [3H]-3-O-methylglucose transport in soleus muscle. Dantrolene, an inhibitor of Ca2+ released from the sarcoplasmic reticulum, decreased the Vmax and the Km of the concentration-response curve. Protein kinase C inhibitors (H-7 and GF109203X) inhibited the stimulatory effect of high glucose concentrations on hexose transport, whereas glucose transport stimulated by insulin was unchanged. Incubation of muscle with glucose (25 mmol/l) and 3-O-methylglucose (25 mmol/l) led to a three fold gain in protein kinase Cβ2 in the total membrane fraction, whereas membrane content of protein kinase Cα, β1, δ, ɛ and ϑ were unchanged. A short-term increase in the extracellular glucose concentration did not change cell surface recruitment of glucose transporter 1 or glucose transporter 4, as assessed by exofacial photolabelling with [3H]-ATB-BMPA bis-mannose. Conclusion/interpretation. Protein kinase Cβ2 is involved in a glucose-sensitive, Ca2+-dependent signalling pathway, which is possibly involved in the regulation of glucose transport in skeletal muscle. This glucose-dependent increase in 3-0-methylglucose transport is independent of glucose transporter 4 and glucose transporter 1 translocation to the plasma membrane and may involve modifications of cell surface glucose transporter activity. [Diabetologia (1999) 42: 1071–1079]

Skeletal muscle insulin resistance after trauma: insulin signaling and glucose transport

Surgical trauma induces peripheral insulin resistance; however, the cellular mechanism has not been fully elucidated. We examined the effects of surgical trauma on insulin receptor signaling and glucose transport in skeletal muscle, a tissue that plays a predominant role in maintaining glucose homeostasis. Surgical trauma was induced by intestinal resection in the rat. Receptor phosphorylation was not altered with surgical trauma. Phosphotyrosine-associated phosphatidylinositol (PI) 3-kinase association was increased by 60 and 82% compared with fasted and fed controls, respectively (P < 0. 05). Similar results were observed for insulin receptor substrate-1-associated PI 3-kinase activity. Insulin-stimulated protein kinase B (Akt kinase) phosphorylation was increased by 2.2-fold after surgical trauma (P < 0.05). The hyperphosphorylation of Akt is likely to reflect amplification of PI 3-kinase after insulin stimulation. Submaximal rates of insulin-stimulated 3-O-methylglucose transport were reduced in trauma vs. fasted rats by 51 and 38% for 100 and 200 microU/ml of insulin, respectively (P < 0.05). In conclusion, insulin resistance in skeletal muscle after surgical trauma is associated with reduced glucose transport but not with impaired insulin signaling to PI 3-kinase or its downstream target, Akt. The surgical trauma model presented in this report provides a useful tool to further elucidate the molecular mechanism(s) underlying the development of insulin resistance after surgical trauma.Surgical trauma induces peripheral insulin resistance; however, the cellular mechanism has not been fully elucidated. We examined the effects of surgical trauma on insulin receptor signaling and glucose transport in skeletal muscle, a tissue that plays a predominant role in maintaining glucose homeostasis. Surgical trauma was induced by intestinal resection in the rat. Receptor phosphorylation was not altered with surgical trauma. Phosphotyrosine-associated phosphatidylinositol (PI) 3-kinase association was increased by 60 and 82% compared with fasted and fed controls, respectively ( P < 0.05). Similar results were observed for insulin receptor substrate-1-associated PI 3-kinase activity. Insulin-stimulated protein kinase B (Akt kinase) phosphorylation was increased by 2.2-fold after surgical trauma ( P < 0.05). The hyperphosphorylation of Akt is likely to reflect amplification of PI 3-kinase after insulin stimulation. Submaximal rates of insulin-stimulated 3- O-methylglucose transport were reduced in trauma vs. fasted rats by 51 and 38% for 100 and 200 μU/ml of insulin, respectively ( P< 0.05). In conclusion, insulin resistance in skeletal muscle after surgical trauma is associated with reduced glucose transport but not with impaired insulin signaling to PI 3-kinase or its downstream target, Akt. The surgical trauma model presented in this report provides a useful tool to further elucidate the molecular mechanism(s) underlying the development of insulin resistance after surgical trauma.

The effect of hyperglycaemia on glucose disposal and insulin signal transduction in skeletal muscle

Skeletal muscle is an important tissue for the proper maintenance of glucose homeostasis as it accounts for the major portion of glucose disposal following infusion or ingestion of glucose. Thus, cellular mechanisms regulating glucose uptake in skeletal muscle have a major impact on whole-body glucose homeostasis. Glucose transport into skeletal muscle is a rate-limiting step for glucose utilization under physiological conditions and a site of insulin resistance in patients with non-insulin-dependent diabetes mellitus (NIDDM). Defects in insulin signalling have been coupled to impaired glucose uptake in skeletal muscle from NIDDM patients. Although the exact aetiology is unclear, genetic and environmental (high-energy diets combined with a sedentary lifestyle) factors contribute to the onset of NIDDM. Furthermore, hyperglycaemia is linked with insulin resistance. This chapter will consider mechanisms for glucose disposal in skeletal muscle, potential sites of insulin resistance in skeletal muscle in NIDDM patients and the impact of hyperglycaemia on insulin action.