Up-regulation of phosphatidylinositol 3-kinase and glucose transporter 4 in muscle of rats subjected to maternal undernutrition

Early postnatal nutrition has been associated with the long-term effects on glucose homeostasis in adulthood. To elucidate the molecular mechanisms by which undernutrition during early life leads to changes in insulin sensitivity, we investigated the insulin signaling in skeletal muscle of rats during development. Offspring of dams fed with either protein-free or normal diets during the first 10 days of lactation were studied from lactation period until adulthood. Early maternal undernutrition impaired secretion of insulin but maintained normal blood glucose levels until adulthood. Insulin receptor (IR) activation after insulin stimulation was decreased during the period of protein restriction. In addition, glucose uptake, insulin receptor substrate 1 (IRS-1) phosphorylation and glucose transporter 4 (GLUT-4) translocation in muscle were reduced in response to insulin during suckling. In contrast, non- or insulin-stimulated glucose uptake and GLUT-4 translocation were found significantly increased in muscle of adult offspring. Finally, basal association of phosphatidylinositol 3-kinase (PI3-kinase) with IRS-1 was increased and was highly stimulated by insulin in muscle from adult rats. Our findings suggest that early postnatal undernutrition increases insulin sensitivity in adulthood, which appears to be directly related to changes in critical steps required for glucose metabolism.     

Exercise modulates the insulin-induced translocation of glucose transporters in rat skeletal muscle

Insulin and acute exercise (45 min of treadmill run) increased glucose uptake into perfused rat hindlimbs 5-fold and 3.2-fold, respectively. Following exercise, insulin treatment resulted in a further increase in glucose uptake. The subcellular distribution of the muscle glucose transporters GLUT-1 and GLUT-4 was determined in plasma membranes and intracellular membranes. Neither exercise nor exercise----insulin treatment altered the distribution of GLUT-1 transporters in these membrane fractions. In contrast, exercise, insulin and exercise----insulin treatment caused comparable increases in GLUT-4 transporters in the plasma membrane. The results suggest that exercise might limit insulin-induced GLUT-4 recruitment and that following exercise, insulin may alter the intrinsic activity of plasma membrane glucose transporters.     

Increased glucose transporter (GLUT4) protein expression in hyperthyroidism

We have studied skeletal muscle glucose uptake by perfused hindquarter preparations from rats treated with thyroxine. Basal glucose uptake (in the absence of insulin) was approximately 2 fold higher in muscle of hyperthyroid rats compared to controls. Insulin (10(-7) M) stimulated glucose uptake 4.0 and 6.8 fold in the 10 day and 30 day controls rats, respectively. Maximal glucose uptake (10(-7) M insulin) was not different in control and hyperthyroid rats and thus insulin responsiveness in the hyperthyroid animals was reduced to 2.5 fold stimulation. The abundance of the insulin-sensitive glucose transporter protein (muscle/fat, GLUT-4), measured by Western blot analysis using polyclonal antisera, was higher in skeletal muscle from both groups of hyperthyroid rats. These studies indicate that thyroid hormones increase basal glucose uptake in skeletal muscle and this is due, at least in part, to an increment of GLUT-4 isoform. Increased expression of muscle glucose transporter proteins may be responsible for the increased peripheral glucose utilization seen in hyperthyroidism.     

ERK1/2 activation by angiotensin II inhibits insulin-induced glucose uptake in vascular smooth muscle cells

Clinical evidence suggests a relationship between hypertension and insulin resistance, and cross-talk between angiotensin II (Ang II) and insulin signaling pathways may take place. We now report the effect of Ang II on insulin-induced glucose uptake and its intracellular mechanisms in vascular smooth muscle cells (VSMC). We examined the translocation of glucose transporter-4 (GLUT-4) and glucose uptake in rat aortic smooth muscle cells (RASMC). Mitogen-activated protein (MAP) kinases and Akt activities, and phosphorylation of insulin receptor substrate-1 (IRS-1) at the serine and tyrosine residues were measured by immunoprecipitation and immunoblotting. As a result, Ang II inhibited insulin-induced GLUT-4 translocation from cytoplasm to the plasma membrane in RASMC. Ang II induced extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) activation and IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹⁶. Ang II-induced Ser³⁰⁷ and Ser⁶¹⁶ phophorylation of IRS-1 was inhibited by a MEK inhibitor, PD98059, and a JNK inhibitor, SP600125. Ang II inhibition of insulin-stimulated IRS-1 tyrosyl phophorylation and Akt activation were reversed by PD98059 but not by SP600125. Ang II inhibited insulin-induced glucose uptake, which was also reversed by PD98059 but not by SP600125. It is shown that Ang II-induced ERK1/2 activation inhibits insulin-dependent glucose uptake through serine phophorylation of IRS-1 in RASMC.     

Role of insulin on exercise-induced GLUT-4 protein expression and glycogen supercompensation in rat skeletal muscle.

The purpose of this study was to investigate the role of insulin on skeletal muscle GLUT-4 protein expression and glycogen storage after postexercise carbohydrate supplementation. Male Sprague-Dawley rats were randomly assigned to one of six treatment groups: sedentary control (Con), Con with streptozocin (Stz/C), immediately postexercise (Ex0), Ex0 with Stz (Stz/Ex0), 5-h postexercise (Ex5), and Ex5 with Stz (Stz/Ex5). Rats were exercised by swimming (2 bouts of 3 h) and carbohydrate supplemented immediately after each exercise session by glucose intubation (1 ml of a 50% wt/vol). Stz was administered 72-h before exercise, which resulted in hyperglycemia and elimination of the insulin response to the carbohydrate supplement. GLUT-4 protein of Ex0 rats was 30% above Con in fast-twitch (FT) red and 21% above Con in FT white muscle. In Ex5, GLUT-4 protein was 52% above Con in FT red and 47% above Con in FT white muscle. Muscle glycogen in FT red and white muscle was also increased above Con in Ex5 rats. Neither GLUT-4 protein nor muscle glycogen was increased above Con in Stz/Ex0 or Stz/Ex5 rats. GLUT-4 mRNA in FT red muscle of Ex0 rats was 61% above Con but only 33% above Con in Ex5 rats. GLUT-4 mRNA in FT red muscle of Stz/C and Stz/Ex0 rats was similar but significantly elevated in Ex5/Stz rats. These results suggest that insulin is essential for the increase in GLUT-4 protein expression following postexercise carbohydrate supplementation.     

Role of PDE3B in insulin-induced glucose uptake, GLUT-4 translocation and lipogenesis in primary rat adipocytes

In adipocytes, phosphorylation and activation of PDE3B is a key event in the antilipolytic action of insulin. The role of PDE4, another PDE present in adipocytes, is not yet known. In this work we investigate the role of PDE3B and PDE4 in insulin-induced glucose uptake, GLUT-4 translocation and lipogenesis. Inhibition of PDE3 (OPC3911, milrinone) but not PDE4 (RO 20-1724) lowered insulin-induced glucose uptake and lipogenesis, especially in the presence of isoproterenol (a general beta-adrenergic agonist), CL316243, a selective beta3-adrenergic agonist, and pituitary adenylate cyclase-activating peptide. The inhibitory effect of OPC3911 was associated with reduced translocation of GLUT-4 from the cytosol to the plasma membrane. Both OPC3911 and RO 20-1724 increased protein kinase A (PKA) activity and lipolysis. H89, a PKA inhibitor, did not affect OPC3911-mediated inhibition of insulin-induced glucose uptake and lipogenesis, whereas 8-pCPT-2'-O-Me-cAMP, an Epac agonist which mediates PKA independent cAMP signaling events, mimicked all the effects of OPC3911. Insulin-mediated activation of protein kinase B, a kinase involved in insulin-induced glucose uptake, was apparently not altered by OPC3911. In summary, our data suggest that PDE3B, but not PDE4, contributes to the regulation of insulin-induced glucose uptake, GLUT-4 translocation, and lipogenesis presumably by regulation of a cAMP/Epac signalling mechanisms.     

Insulin-induced translocation of facilitative glucose transporters in fetal/neonatal rat skeletal muscle

We examined the effect of insulin on fetal/neonatal rat skeletal muscle GLUT-1 and GLUT-4 concentrations and subcellular distribution by employing immunohistochemical analysis and subcellular fractionation followed by Western blot analysis. We observed that insulin did not alter total GLUT-1 or GLUT-4 concentrations or the GLUT-1 subcellular distribution in fetal/neonatal or adult skeletal muscle in 60 min. The basal and insulin-induced changes in subcellular distribution of GLUT-4 were different between the fetal/neonatal and adult skeletal muscle. Under basal conditions, sarcolemma-associated GLUT-4 was higher in the newborn compared with the adult, translating into a higher glucose transport. In contrast, insulin-induced translocation of GLUT-4 to the sarcolemma- and insulin-induced glucose transport was lower in the newborn compared with the adult. This age-related change results in enhanced basal glucose transport to fuel myocytic proliferation and differentiation while relatively curbing the insulin-dependent glucose transport in the newborn.     

Chronic elevated calcium blocks AMPK-induced GLUT-4 expression in skeletal muscle

Muscle contraction stimulates glucose transport independent of insulin. Glucose uptake into muscle cells is positively related to skeletal muscle-specific glucose transporter (GLUT-4) expression. Therefore, our objective was to determine the effects of the contraction-mediated signals, calcium and AMP-activated protein kinase (AMPK), on glucose uptake and GLUT-4 expression under acute and chronic conditions. To accomplish this, we used pharmacological agents, cell culture, and pigs possessing genetic mutations for increased cytosolic calcium and constitutively active AMPK. In C2C12 myotubes, caffeine, a sarcoplasmic reticulum calcium-releasing agent, had a biphasic effect on GLUT-4 expression and glucose uptake. Low-concentration (1.25 to 2 mM) or short-term (4 h) caffeine treatment together with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR), had an additive effect on GLUT-4 expression. However, high-concentration (2.5 to 5 mM) or long-term (4 to 30 h) caffeine treatment decreased AMPK-induced GLUT-4 expression without affecting cell viability. The negative effect of caffeine on AICAR-induced GLUT-4 expression was reduced by dantrolene, which desensitizes the ryanodine receptor. Consistent with cell culture data, increases in GLUT-4 mRNA and protein expression induced by AMPK were blunted in pigs possessing genetic mutations for both increased cytosolic calcium and constitutively active AMPK. Altogether, these data suggest that chronic exposure to elevated cytosolic calcium concentration blocks AMPK-induced GLUT-4 expression in skeletal muscle.     

Effect of carbohydrate supplementation on postexercise GLUT-4 protein expression in skeletal muscle.

The effect of carbohydrate supplementation on skeletal muscle glucose transporter GLUT-4 protein expression was studied in fast-twitch red and white gastrocnemius muscle of Sprague-Dawley rats before and after glycogen depletion by swimming. Exercise significantly reduced fast-twitch red muscle glycogen by 50%. During a 16-h exercise recovery period, muscle glycogen returned to control levels (25.0 +/- 1.4 micromol/g) in exercise-fasted rats (24.2 +/- 0. 3 micro). However, when carbohydrate supplementation was provided during and immediately postexercise by intubation, muscle glycogen increased 77% above control (44.4 +/- 2.1 micromol/g). Exercise-fasting resulted in an 80% increase in fast-twitch red muscle GLUT-4 mRNA but only a 43% increase in GLUT-4 protein concentration. Conversely, exercise plus carbohydrate supplementation elevated fast-twitch red muscle GLUT-4 protein concentration by 88% above control, whereas GLUT-4 mRNA was increased by only 40%. Neither a 16-h fast nor carbohydrate supplementation had an effect on fast-twitch red muscle GLUT-4 protein concentration or on GLUT-4 mRNA in sedentary rats, although carbohydrate supplementation increased muscle glycogen concentration by 40% (35.0 +/- 0.9 micromol/g). GLUT-4 protein in fast-twitch white muscle followed a pattern similar to fast-twitch red muscle. These results indicate that carbohydrate supplementation, provided with exercise, will enhance GLUT-4 protein expression by increasing translational efficiency. Conversely, postexercise fasting appears to upregulate GLUT-4 mRNA, possibly to amplify GLUT-4 protein expression on an increase in glucose availability. These regulatory mechanisms may help control muscle glucose uptake in accordance with glucose availability and protect against postexercise hypoglycemia.     

Contraction-activated glucose uptake is normal in insulin-resistant muscle of the obese Zucker rat.

The rates of muscle glucose uptake of lean and obese Zucker rats were assessed via hindlimb perfusion under basal conditions (no insulin), in the presence of a maximal insulin concentration (10 mU/ml), and after electrically stimulated muscle contraction in the absence of insulin. The perfusate contained 28 mM glucose and 7.5 microCi/mmol of 2-deoxy-D-[3H-(G)]glucose. Glucose uptake rates in the soleus (slow-twitch oxidative fibers), red gastrocnemius (fast-twitch oxidative-glycolytic fibers), and white gastrocnemius (fast-twitch glycolytic fibers) under basal conditions and after electrically stimulated muscle contraction were not significantly different between the lean and obese rats. However, the rate of glucose uptake during insulin stimulation was significantly lower for obese than for lean rats in all three fiber types. Significant correlations were found for insulin-stimulated glucose uptake and glucose transporter protein isoform (GLUT-4) content of soleus, red gastrocnemius, and white gastrocnemius of lean (r = 0.79) and obese (r = 0.65) rats. In contrast, the relationships between contraction-stimulated glucose uptake and muscle GLUT-4 content of lean and obese rats were negligible because of inordinately low contraction-stimulated glucose uptakes by the solei. These results suggest that maximal skeletal muscle glucose uptake of obese Zucker rats is resistant to stimulation by insulin but not to contractile activity. In addition, the relationship between contraction-stimulated glucose uptake and GLUT-4 content appears to be fiber-type specific.     

Inhibitory effect of hyperglycemia on insulin-induced Akt/protein kinase B activation in skeletal muscle

To determine the molecular mechanism underlying hyperglycemia-induced insulin resistance in skeletal muscles, postreceptor insulin-signaling events were assessed in skeletal muscles of neonatally streptozotocin-treated diabetic rats. In isolated soleus muscle of the diabetic rats, insulin-stimulated 2-deoxyglucose uptake, glucose oxidation, and lactate release were all significantly decreased compared with normal rats. Similarly, insulin-induced phosphorylation and activation of Akt/protein kinase B (PKB) and GLUT-4 translocation were severely impaired. However, the upstream signal, including phosphorylation of the insulin receptor (IR) and insulin receptor substrate (IRS)-1 and -2 and activity of phosphatidylinositol (PI) 3-kinase associated with IRS-1/2, was enhanced. The amelioration of hyperglycemia by T-1095, a Na(+)-glucose transporter inhibitor, normalized the reduced insulin sensitivity in the soleus muscle and the impaired insulin-stimulated Akt/PKB phosphorylation and activity. In addition, the enhanced PI 3-kinase activation and phosphorylation of IR and IRS-1 and -2 were reduced to normal levels. These results suggest that sustained hyperglycemia impairs the insulin-signaling steps between PI 3-kinase and Akt/PKB, and that impaired Akt/PKB activity underlies hyperglycemia-induced insulin resistance in skeletal muscle.     

Insulin-mediated translocation of GLUT-4-containing vesicles is preserved in denervated muscles

Skeletal muscle denervation decreases insulin-sensitive glucose uptake into this tissue as a result of marked GLUT-4 protein downregulation ( approximately 20% of controls). The process of insulin-stimulated glucose transport in muscle requires the movement or translocation of intracellular GLUT-4-rich vesicles to the cell surface, and it is accompanied by the translocation of several additional vesicular cargo proteins. Thus examining GLUT-4 translocation in muscles from denervated animals allows us to determine whether the loss of a major cargo protein, GLUT-4, affects the insulin-dependent behavior of the remaining cargo proteins. We find no difference, control vs. denervated, in the insulin-dependent translocation of the insulin-responsive aminopeptidase (IRAP) and the receptors for transferrin and insulin-like growth factor II/mannose 6-phosphate, proteins that completely (IRAP) or partially co-localize with GLUT-4. We conclude that 1) denervation of skeletal muscle does not block the specific branch of insulin signaling pathway that connects receptor proximal events to intracellular GLUT-4-vesicles, and 2) normal levels of GLUT-4 protein are not necessary for the structural organization and insulin-sensitive translocation of its cognate intracellular compartment. Muscle denervation also causes a twofold increase in GLUT-1. In normal muscle, all GLUT-1 is present at the cell surface, but in denervated muscle a significant fraction (25.1 +/- 6.1%) of this transporter is found in intracellular vesicles that have the same sedimentation coefficient as GLUT-4-containing vesicles but can be separated from the latter by immunoadsorption. These GLUT-1-containing vesicles respond to insulin and translocate to the cell surface. Thus the formation of insulin-sensitive GLUT-1-containing vesicles in denervated muscle may be a compensatory mechanism for the decreased level of GLUT-4.     

Protein kinase Cdelta mediates insulin-induced glucose transport in primary cultures of rat skeletal muscle

Insulin activates certain protein kinase C (PKC) isoforms that are involved in insulin-induced glucose transport. In this study, we investigated the possibility that activation of PKCdelta by insulin participates in the mediation of insulin effects on glucose transport in skeletal muscle. Studies were performed on primary cultures of rat skeletal myotubes. The role of PKCdelta in insulin-induced glucose uptake was evaluated both by selective pharmacological blockade and by over-expression of wild-type and point-mutated inactive PKCdelta isoforms in skeletal myotubes. We found that insulin induces tyrosine phosphorylation and translocation of PKCdelta to the plasma membrane and increases the activity of this isoform. Insulin-induced effects on translocation and phosphorylation of PKCdelta were blocked by a low concentration of rottlerin, whereas the effects of insulin on other PKC isoforms were not. This selective blockade of PKCdelta by rottlerin also inhibited insulin-induced translocation of glucose transporter 4 (GLUT4), but not glucose transporter 3 (GLUT3), and significantly reduced the stimulation of glucose uptake by insulin. When overexpressed in skeletal muscle, PKCdelta and PKCdelta were both active. Overexpression of PKCdelta induced the translocation of GLUT4 to the plasma membrane and increased basal glucose uptake to levels attained by insulin. Moreover, insulin did not increase glucose uptake further in cells overexpressing PKCdelta. Overexpression of PKCdelta did not affect basal glucose uptake or GLUT4 location. Stimulation of glucose uptake by insulin in cells overexpressing PKCdelta was similar to that in untransfected cells. Transfection of skeletal myotubes with dominant negative mutant PKCdelta did not alter basal glucose uptake but blocked insulin-induced GLUT4 translocation and glucose transport. These results demonstrate that insulin activates PKCdelta and that activated PKCdelta is a major signaling molecule in insulin-induced glucose transport.     

Insulin secretion and GLUT-2 expression in undernourished neonate rats

In previous studies, we verified increased insulin sensitivity in adult male offspring of lactating rats readjusting to lack of insulin secretion reduction brought about by protein restriction during lactation. The present study aims to evaluate the effects of maternal protein undernutrition during lactation on glucose-induced insulin secretion and GLUT-2 expression in beta-cells of neonate male and female rats. Lactating Wistar rats were given a protein-free diet during the first 10 days and a normal diet (22% of protein) until weaning. The neonates were separated at birth by sex and diet and studied at 4, 8 and 21 days of lactation. Glucose-induced insulin secretion by pancreatic islets was analyzed by radioimmunoassay and GLUT-2 expression in beta-cells by Western blot. Glucose-induced insulin secretion of the undernourished groups was higher than in the control groups except among females. When comparing the male and female groups and the control and undernourished groups, female neonates showed significantly greater insulin secretion than the male group. Also it was noted that undernutrition induced greater GLUT-2 expression. For instance, comparing the undernourished male and female neonates there was an increase in female GLUT-2 expression on day 4. On the other hand, in undernourished male neonates a GLUT-2 expression increased later in lactation. In conclusion, during a short term, maternal undernutrition induces an increase of the glucose-induced insulin secretion only in male neonates and is associated with an increase in GLUT-2 expression in the beta-cell.     

Biological effects of THC and a lipophilic cannabis extract on normal and insulin resistant 3T3-L1 adipocytes

Type 2 diabetes, a chronic disease, affects about 150 million people world wide. It is characterized by insulin resistance of peripheral tissues such as liver, skeletal muscle, and fat. Insulin resistance is associated with elevated levels of tumor necrosis factor alpha (TNF-α), which in turn inhibits insulin receptor tyrosine kinase autophosphorylation. It has been reported that cannabis is used in the treatment of diabetes. A few reports indicate that smoking cannabis can lower blood glucose in diabetics. Δ⁹-tetrahydrocannabinol (THC) is the primary psychoactive component of cannabis. This study aimed to determine the effect of a lipophilic cannabis extract on adipogenesis, using 3T3-L1 cells, and to measure its effect on insulin sensitivity in insulin resistant adipocytes. Cells were cultured in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) and differentiated over a 3 day period for all studies. In the adipogenesis studies, differentiated cells were exposed to the extract in the presence and absence of insulin. Lipid content and glucose uptake was subsequently measured. Insulin-induced glucose uptake increased, while the rate of adipogenesis decreased with increasing THC concentration. Insulin-resistance was induced using TNF-α, exposed to the extract and insulin-induced glucose uptake measured. Insulin-induced glucose was increased in these cells after exposure to the extract. Semiquantitative real time polymerase chain reaction (RT-PCR) was performed after ribonucleic acid (RNA) extraction to evaluate the effects of the extract on glucose transporter isotype 4 (GLUT-4), insulin receptor substrate-1 (IRS-1) and IRS-2 gene expression.     

Effect of denervation or unweighting on GLUT-4 protein in rat soleus muscle.

The purpose of this study was to test the hypothesis that the decreased capacity for glucose transport in the denervated rat soleus and the increased capacity for glucose transport in the unweighted rat soleus are related to changes in the expression of the regulatable glucose transporter protein in skeletal muscle (GLUT-4). One day after sciatic nerve sectioning, when decreases in the stimulation of soleus 2-deoxyglucose (2-DG) uptake by insulin (-51%, P less than 0.001), contractions (-29%, P less than 0.05), or insulin and contractions in combination (-40%, P less than 0.001) were observed, there was a slight (-18%, NS) decrease in GLUT-4 protein. By day 3 of denervation, stimulation of 2-DG uptake by insulin (-74%, P less than 0.001), contractions (-31%, P less than 0.001), or the two stimuli in combination (-59%, P less than 0.001), as well as GLUT-4 protein (-52%, P less than 0.001), was further reduced. Soleus muscle from hindlimb-suspended rats, which develops an enhanced capacity for insulin-stimulated glucose transport, showed muscle atrophy similar to denervated soleus but, in contrast, displayed substantial increases in GLUT-4 protein after 3 (+35%, P less than 0.05) and 7 days (+107%, P less than 0.001). These results indicate that altered GLUT-4 expression may be a major contributor to the changes in insulin-stimulated glucose transport that are observed with denervation and unweighting. We conclude that muscle activity is an important factor in the regulation of GLUT-4 expression in skeletal muscle.     

The interrelation between aPKC and glucose uptake in the skeletal muscle during contraction and insulin stimulation

Contraction and insulin increase glucose uptake in skeletal muscle. While the insulin pathway, better characterized, requires activation of phosphoinositide 3‐kinase (PI3K) and atypical protein kinase (aPKC), muscle contraction seems to share insulin‐activated components to increase glucose uptake. This study aimed to investigate the interrelation between the pathway involved in glucose uptake evoked by insulin and muscle contraction. Isolated muscle of rats was treated with solvent (control), insulin, wortmannin (PI3K inhibitor) and the combination of insulin plus wortmannin. After treatment, muscles were electrically stimulated (contracted) or remained at rest. Glucose transporter 4 (GLUT4) localization, glucose uptake and phospho‐aPKC (aPKC activated form) were assessed. Muscle contraction and insulin increased glucose uptake in all conditions when compared with controls not stimulating an effect that was accompanied by an increase in GLUT4 and of phospho‐aPKC at the muscle membrane. Contracted muscles treated with insulin did not show additive effects on glucose uptake or aPKC activity compared with the response when these stimuli were applied alone. Inhibition of PI3K blocked insulin effect on glucose uptake and aPKC but not in the contractile response. Thus, muscle contraction seems to stimulate aPKC and glucose uptake independently of PI3K. Therefore, aPKC may be a convergence point and a rate limit step in the pathway by which, insulin and contraction, increase glucose uptake in skeletal muscle. Copyright © 2014 John Wiley & Sons, Ltd.     

Alterations in the level of insulin receptor and GLUT-4 mRNA in skeletal muscle from rats fed a kidney bean (Phaseolus vulgaris) diet.

1. A decline in the level of circulating insulin was observed in rats fed a diet containing kidney bean. 2. Consumption of a diet containing kidney bean caused an increase in the level of mRNAs for the insulin receptor (327%) and GLUT-4 (185%) in the gastrocnemius muscle. In contrast there was only a small increase in the amount of actin mRNA (125%). Since the kidney bean-fed rats are euglycaemic the results suggest that insulin receptor and GLUT-4 mRNA levels are regulated in response to circulating insulin concentrations rather than glucose. 3. No increases in the level of insulin receptor and actin mRNA were evident in the soleus muscle of rats fed the diet containing kidney bean; however a decline was observed in the level of GLUT-4 mRNA. 4. It is proposed that a component of kidney beans, most likely the lectin phytohaemagglutinin, has systemic effects which lead to changes in expression of the insulin receptor and GLUT-4 genes and to the sensitivity of muscle to insulin.     

Cross-talk mechanisms in the development of insulin resistance of skeletal muscle cells palmitate rather than tumour necrosis factor inhibits insulin-dependent protein kinase B (PKB)/Akt stimulation and glucose uptake.

Insulin resistance in skeletal muscle is one of the earliest symptoms associated with non-insulin-dependent diabetes mellitus (NIDDM). Tumour necrosis factor (TNF) and nonesterified fatty acids have been proposed to be crucial factors in the development of the insulin-resistant state. We here show that, although TNF downregulated insulin-induced insulin receptor (IR) and IR substrate (IRS)-1 phosphorylation as well as phosphoinositide 3-kinase (PI3-kinase) activity in pmi28 myotubes, this was, unlike in adipocytes, not sufficient to affect insulin-induced glucose transport. Rather, TNF increased membrane expression of GLUT1 and glucose transport in these muscle cells. In contrast, the nonesterified fatty acid palmitate inhibited insulin-induced signalling cascades not only at the level of IR and IRS-1 phosphorylation, but also at the level protein kinase B (PKB/Akt), which is thought to be directly involved in the insulin-induced translocation of GLUT4, and inhibited insulin-induced glucose uptake. Palmitate also abrogated TNF-dependent enhancement of basal glucose uptake, suggesting that palmitate has the capacity to render muscle cells resistant not only to insulin but also to TNF with respect to glucose transport by GLUT4 and GLUT1, respectively. Our data illustrate the complexity of the mechanisms governing insulin resistance of skeletal muscle, questioning the role of TNF as a direct inhibitor of glucose homoeostasis in this tissue and shedding new light on an as yet unrecognized multifunctional role for the predominant nonesterified fatty acid palmitate in this process.     

Recruitment of GLUT-4 glucose transporters by insulin in diabetic rat skeletal muscle

The cause of reduced insulin-stimulated glucose transport in skeletal muscle of diabetic rats was investigated. Basal and insulin-stimulated glucose uptake into hindquarter muscles of 7-day diabetic rats were 70% and 50% lower, respectively, than in nondiabetic controls. Subcellular fractionation of hindquarter muscles yielded total crude membranes, plasma membranes and intracellular membranes. The number of GLUT-4 glucose transporters was lower in crude membranes, plasma membranes and intracellular membranes, relative to non-diabetic rat muscles. These results were paralleled by reductions in D-glucose-protectable binding of cytochalasin B. Insulin caused a redistribution of GLUT-4 transporters from intracellular membranes to plasma membranes, in both control and diabetic rat muscles. This redistribution was also recorded using binding of cytochalasin B. The insulin-dependent decrement in glucose transporters in intracellular membranes was similar for both animal groups, but the gain and final amount of transporters in the plasma membrane were 50% lower in the diabetic group. The results suggest that insulin signalling and recruitment of GLUT-4 glucose transporters occur in diabetic rat muscle, and that the diminished insulin response may be due to fewer glucose transporters operating in the muscle plasma membrane.