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.     

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.     

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.     

Insulin-induced translocation of glucose transporters in rat hindlimb muscles

Insulin causes a translocation of glucose transporters from intracellular microsomes to the plasma membrane in adipocytes. To determine whether insulin has a similar effect in rat hindlimb muscles, we used glucose-inhibitable cytochalasin B binding to estimate the number of glucose transporters in membrane fractions from insulinized and control muscles. Insulin treatment caused an approx. 2-fold increase in cytochalasin B-binding sites in a plasma membrane fraction and an approx. 70% decrease in cytochalasin B-binding sites in an intracellular membrane fraction. In order to detect this effect of insulin, it was necessary to develop a procedure for isolating a plasma membrane fraction and an intracellular membrane fraction that were not contaminated with sarcoplasmic reticulum. Our results show that, as in adipocytes, insulin stimulates translocation of glucose transporters from an intracellular membrane pool to the plasma membrane in hindlimb skeletal muscles.     

Identification of an intracellular pool of glucose transporters from basal and insulin-stimulated rat skeletal muscle

The purpose of this study was to simultaneously isolate skeletal muscle plasma and microsomal membranes from the hind limbs of male Sprague-Dawley rats perfused either in the absence or presence of 20 milliunits/ml insulin and to determine the effect of insulin on the number and distribution of glucose transporters in these membrane fractions. Insulin increased hind limb glucose uptake greater than 3-fold (2.4 +/- 0.7 versus 9.2 +/- 1.0 mumol/g x h, p less than 0.001). Plasma membrane glucose transporter number, measured by cytochalasin B binding, increased 2-fold (9.1 +/- 1.0 to 20.4 +/- 3.1 pmol/mg protein, p less than 0.005) in insulin-stimulated muscle while microsomal membrane transporters decreased significantly (14.8 +/- 1.6 to 9.8 +/- 1.4 pmol/mg protein, p less than 0.05). No change in the dissociation constant (Kd approximately 120 nm) was observed. K+-stimulated-p-nitrophenol phosphatase, 5'-nucleotidase, and galactosyltransferase specific activity, enrichment, and recovery in the plasma and microsomal membrane fractions were not altered by insulin treatment. Western blot analysis using the monoclonal antibody mAb 1F8 (specific for the insulin-regulatable glucose transporter) demonstrated increased glucose transporter densities in plasma membranes from insulin-treated hind limb skeletal muscle compared with untreated tissues, while microsomal membranes from the insulin-treated hind limb skeletal muscle had a concomitant decrease in transporter density. We conclude that the increase in plasma membrane glucose transporters explains, at least in part, the increase in glucose uptake associated with insulin stimulation of hind limb skeletal muscle. Our data further suggest that these recruited transporters originate from an intracellular microsomal pool, consistent with the translocation hypothesis.     

Impaired insulin signaling in endothelial cells reduces insulin-induced glucose uptake by skeletal muscle

In obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.     

Identification of the glucose transporter in rat skeletal muscle

The glucose transporter in the plasma membrane of rat skeletal muscle has been identified by two approaches. In one, the transporter was detected as the polypeptide that was differentially labeled by photolysis with [³H]cytochalasin B in the presence of l- and d-glucose. [³H]Cytochalasin B is a high-affinity ligand for the transporter that is displaced by d-glucose. In the other, the transporter was detected by means of its reaction with rabbit antibodies against the purified glucose transporter from human erythrocytes. By both procedures, the transporter was found to be a polypeptide with a mobility corresponding to a molecular weight of 45,000–50,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Leucine reduces the duration of insulin-induced PI 3-kinase activity in rat skeletal muscle

Leucine (Leu) is known to stimulate translation initiation of protein synthesis at mammalian target of rapamycin (mTOR) in the insulin signaling pathway. However, potential feedback from mTOR to upstream aspects of the insulin signaling pathway remains controversial. This study evaluates the impact of a physiological oral dose of Leu and/or carbohydrate (CHO) on upstream elements of the insulin signaling pathway using phosphatidylinositol 3-kinase (PI 3-kinase) activity and glucose uptake as markers for insulin sensitivity and glucose homeostasis. Rats (approximately 200 g) were fasted 12 h and administered oral doses of CHO (1.31 g glucose, 1.31 g sucrose), Leu (270 mg), or CHO plus Leu. Animals were killed at 15, 30, 60, and 90 min after treatment. Plasma and gastrocnemius muscles were collected for analyses. Treatments were designed to produce elevated blood glucose and insulin with basal levels of Leu (CHO); elevated Leu with basal levels of glucose and insulin (Leu); or a combined increase of glucose, insulin, and Leu (CHO + Leu). The CHO treatment stimulated PI 3-kinase activity and glucose uptake with no effect on the downstream translation initiation factor eIF4E. Leu alone stimulated the release of the translation initiation factor eIF4E from 4E-BP1 with no effects on PI 3-kinase activity or glucose uptake. The CHO + Leu treatment reduced the magnitude and duration of the PI 3-kinase response but maintained glucose uptake similar to the CHO treatment and eIF4E levels similar to the Leu treatment. These findings demonstrate that Leu reduces insulin-stimulated PI 3-kinase activity while increasing downstream translation initiation and with no effect on net glucose transport in skeletal muscle.     

Contraction-associated translocation of protein kinase C in rat skeletal muscle

Electrical stimulation of the sciatic nerve of the anaesthetized rat in vivo led to a time-dependent translocation of protein kinase C from the muscle cytosol to the particulate fraction. Maximum activity of protein kinase C in the particulate fraction occurred after 2 min of intermittent short tetanic contractions of the gastrocnemius-plantaris-soleus muscle group and coincided with the loss of activity from the cytosol. Translocation of protein kinase C may imply a role for this kinase in contraction-initiated changes in muscle metabolism.     

Acute effects of cycloheximide on the translocation of glucose transporters in rat adipose cells

Insulin's rapid action to increase glucose transport is believed to occur primarily through the translocation of glucose transporters from an intracellular pool to the plasma membrane. To better understand the mechanism involved, we studied the role of protein synthesis in glucose transporter translocation by using the protein synthesis inhibitor, cycloheximide. Isolated rat epididymal adipose cells were incubated in the presence or absence of cycloheximide (10 micrograms/ml) for a total of 120 min. Insulin (7 nM) was added to half of the cells from both groups for the final 30 min. Protein synthesis was inhibited by approximately 90%, as measured by [14C]leucine incorporation, in the cells exposed to cycloheximide. The 3-O-methylglucose uptake in intact cells was slightly increased in the basal state with cycloheximide treatment, but the insulin-stimulated 3-O-methylglucose uptake was unchanged by cycloheximide. The distribution of glucose transporters in the different subcellular membrane fractions, as measured by the cytochalasin B binding assay, was unchanged by cycloheximide. These results suggest that insulin's stimulation of glucose transport and translocation of glucose transporters can occur without acute protein synthesis.     

Reconstitution of the glucose transporter from rat skeletal muscle

As a step in the purification and characterization of the glucose transporter from rat skeletal muscle, we have reconstituted glucose transport activity in liposomes. Plasma membranes were prepared from skeletal muscle which display D-glucose reversible binding of cytochalasin B (10 pmol sites/mg protein; KD = 0.3 microM). Older rats gave a slightly lower specific activity and much lower yield of sites per g muscle than young rats. Glucose transport activity was reconstituted into liposomes by the freeze-thaw procedure using either plasma membranes directly or cholate-extracted membrane proteins; the latter gave a 50% higher specific activity. The reconstituted transport activity was stereospecific, saturable, and inhibited by cytochalasin B, phloretin, and mercuric chloride. The optimum cholate concentration for extraction and reconstitution of transport activity was about 1.5%, and the highest specific activity of reconstituted transport was seen only at low ratios of protein to lipid in the reconstitution. Chromatography on agarose lentil lectin and agarose ethanethiol doubled both the specific activity of reconstituted transport and the fraction of glucose uptake which was stereospecific. In all of these respects the results were similar to our results with the bovine heart transporter (T. J. Wheeler and M. A. Hauck, Biochim. Biophys. Acta 818, 171-182 (1985)). Our findings suggest that further purification procedures developed for the heart transporter may be applicable to the skeletal muscle transporter as well.     

Effects of selective hyperglycemia and hyperinsulinemia on glucose transporters in fetal ovine skeletal muscle

We measured net fetal glucose uptake rate from the placenta, shown previously to be equal to total fetal glucose utilization rate (GUR(f)) and proportional to fetal hindlimb skeletal muscle glucose utilization, under normal conditions and after 1, 2.5, and 24 h of selective hyperglycemia increasing G or selective hyperinsulinemia increasing I. We simultaneously measured the amount of Glut 1 and Glut 4 glucose transporter proteins in fetal sheep skeletal muscle. With increasing G , GUR(f) was increased approximately 40% at 1 and 2.5 h but returned to the control rate by 24 h. This transient increasing G -specific increasing GUR(f) was associated with increased plasma membrane-associated Glut 1 (4-fold) and intracellular Glut 4 (3-fold) protein beginning at 1 h. With increasing I, GUR(f) was increased approximately 70% at 1, 2.5, and 24 h. This more sustained increasing I-specific increasing GUR(f) was associated with a significant increase in Glut 4 protein (2-fold) at 2.5 h but no change in Glut 1 protein. These results show that increasing G and increasing I have independent effects on the amount of Glut 1 and Glut 4 glucose transporter proteins in ovine fetal skeletal muscle. These effects are time dependent and isoform specific and may contribute to increased glucose utilization in fetal skeletal muscle. The lack of a sustained temporal correlation between the increase in transporter proteins and glucose utilization rates indicates that subcellular localization and activity of a transporter or tissues other than the skeletal muscle contribute to net GUR(f).     

Opening of potassium channels modulates mitochondrial function in rat skeletal muscle

We have investigated the presence of diazoxide- and nicorandil-activated K+ channels in rat skeletal muscle. Activation of potassium transport in the rat skeletal muscle myoblast cell line L6 caused a stimulation of cellular oxygen consumption, implying a mitochondrial effect. Working with isolated rat skeletal muscle mitochondria, both potassium channel openers (KCOs) stimulate respiration, depolarize the mitochondrial inner membrane and lead to oxidation of the mitochondrial NAD-system in a strict potassium-dependent manner. This is a strong indication for KCO-mediated stimulation of potassium transport at the mitochondrial inner membrane. Moreover, the potassium-specific effects of both diazoxide and nicorandil on oxidative phosphorylation in skeletal muscle mitochondria were completely abolished by the antidiabetic sulfonylurea derivative glibenclamide, a well-known inhibitor of ATP-regulated potassium channels (K(ATP) channels). Since both diazoxide and nicorandil facilitated swelling of de-energised mitochondria in KSCN buffer at the same concentrations, our results implicate the presence of a mitochondrial ATP-regulated potassium channel (mitoK(ATP) channel) in rat skeletal muscle which can modulate mitochondrial oxidative phosphorylation.     

Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: Characterization of subcellular fractions

Insulin stimulates glucose transport in rat adipose cells through the translocation of glucose transporters from an intracellular pool to the plasma membrane. A detailed characterization of the morphology, protein composition and marker enzyme content of subcellular fractions of these cells, prepared by differential ultracentrifugation, and of the distribution of glucose transporters among these fractions is now described. Glucose transporters were measured using specific d-glucose-inhibitable [³H]cytochalasin B binding. In the basal state, roughly 90% of the cells' glucose transporters are associated with a low-density microsomal, Golgi marker enzyme-enriched membrane fraction. However, the distributions of glucose transporters and Golgi marker enzyme activities over all fractions are clearly distinct. Incubation of intact cells with insulin increases the number of glucose transporters in the plasma membrane fraction 4–5-fold and correspondingly decreases the intracellular pool, without influencing any other characteristics of the subcellular fractions examined or the estimated total number of glucose transporters (3.7·10⁶/cell). Insulin does not influence the K_d of the glucose transporters in the plasma membrane fraction for cytochalasin B binding (98 nM), but lowers that in the intracellular pool (from 141 to 93 nM). The calculated turnover numbers of the glucose transporters in the plasma membrane vesicles from basal and insulin-stimulated cells are similar (15·10³ mol of glucose/min per mol of transporters at 37°C), whereas insulin appears to increase the turnover number in the plasma membrane of intact cells roughly 4-fold. These results suggest that (1) the intracellular pool of glucose transporters may comprise a specialized membrane species, (2) intracellular glucose transporters may undergo conformational changes during their cycling to the plasma membrane in response to insulin, and (3) the translocation of glucose transporters may represent only one component in the mechanism through which insulin regulates glucose transport in the intact cell.     

Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: characterization of subcellular fractions

Insulin stimulates glucose transport in rat adipose cells through the translocation of glucose transporters from an intracellular pool to the plasma membrane. A detailed characterization of the morphology, protein composition and marker enzyme content of subcellular fractions of these cells, prepared by differential ultracentrifugation, and of the distribution of glucose transporters among these fractions is now described. Glucose transporters were measured using specific D-glucose-inhibitable [3H]cytochalasin B binding. In the basal state, roughly 90% of the cells' glucose transporters are associated with a low-density microsomal, Golgi marker enzyme-enriched membrane fraction. However, the distributions of glucose transporters and Golgi marker enzyme activities over all fractions are clearly distinct. Incubation of intact cells with insulin increases the number of glucose transporters in the plasma membrane fraction 4-5 fold and correspondingly decreases the intracellular pool, without influencing any other characteristics of the subcellular fractions examined or the estimated total number of glucose transporters (3.7 X 10(6)/cell). Insulin does not influence the Kd of the glucose transporters in the plasma membrane fraction for cytochalasin B binding (98 nM), but lowers that in the intracellular pool (from 141 to 93 nM). The calculated turnover numbers of the glucose transporters in the plasma membrane vesicles from basal and insulin-stimulated cells are similar (15 X 10(3) mol of glucose/min per mol of transporters at 37 degrees C), whereas insulin appears to increase the turnover number in the plasma membrane of intact cells roughly 4-fold. These results suggest that (1) the intracellular pool of glucose transporters may comprise a specialized membrane species, (2) intracellular glucose transporters may undergo conformational changes during their cycling to the plasma membrane in response to insulin, and (3) the translocation of glucose transporters may represent only one component in the mechanism through which insulin regulates glucose transport in the intact cell.     

Induction of amino acid transporters expression by endurance exercise in rat skeletal muscle

We here investigated whether an acute bout of endurance exercise would induce the expression of amino acid transporters that regulate leucine transport across plasma and lysosomal membranes in rat skeletal muscle. Rats ran on a motor-driven treadmill at a speed of 28 m/min for 90 min. Immediately after the exercise, we observed that expression of mRNAs encoding l-type amino acid transporter 1 (LAT1) and CD98 was induced in the gastrocnemius, soleus, and extensor digitorum longus (EDL) muscles. Sodium-coupled neutral amino acid transporter 2 (SNAT2) mRNA was also induced by the exercise in those three muscles. Expression of proton-assisted amino acid transporter 1 (PAT1) mRNA was slightly but not significantly induced by a single bout of exercise in soleus and EDL muscles. Exercise-induced mRNA expression of these amino acid transporters appeared to be attenuated by repeated bouts of the exercise. These results suggested that the expression of amino acid transporters for leucine may be induced in response to an increase in the requirement for this amino acid in the cells of working skeletal muscles.     

Exercise alters the profile of phospholipid molecular species in rat skeletal muscle.

We have determined the effect of two exercise-training intensities on the phospholipid profile of both glycolytic and oxidative muscle fibers of female Sprague-Dawley rats using electrospray-ionization mass spectrometry. Animals were randomly divided into three training groups: control, which performed no exercise training; low-intensity (8 m/min) treadmill running; or high-intensity (28 m/min) treadmill running. All exercise-trained rats ran 1,000 m/session for 4 days/wk for 4 wk and were killed 48 h after the last training bout. Exercise training was found to produce no novel phospholipid species but was associated with significant alterations in the relative abundance of a number of phospholipid molecular species. These changes were more prominent in glycolytic (white vastus lateralis) than in oxidative (red vastus lateralis) muscle fibers. The largest observed change was a decrease of approximately 20% in the abundance of 1-stearoyl-2-docosahexaenoyl-phosphatidylethanolamine [PE(18:0/22:6); P < 0.001] ions in both the low- and high-intensity training regimes in glycolytic fibers. Increases in the abundance of 1-oleoyl-2-linoleoyl phopshatidic acid [PA(18:1/18:2); P < 0.001] and 1-alkenylpalmitoyl-2-linoleoyl phosphatidylethanolamine [plasmenyl PE (16:0/18:2); P < 0.005] ions were also observed for both training regimes in glycolytic fibers. We conclude that exercise training results in a remodeling of phospholipids in rat skeletal muscle. Even though little is known about the physiological or pathophysiological role of specific phospholipid molecular species in skeletal muscle, it is likely that this remodeling will have an impact on a range of cellular functions.