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Insulin-stimulated glucose uptake in skeletal muscle is mediated through the GLUT4 glucose transporter. Transgenic (TG) mice overexpressing human GLUT4 in skeletal muscle show an increased ability to handle a glucose [...]

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. [...]

Stress-activated protein kinases (SAPKs) are involved in cellular responses to heat shock, inflammatory cytokines, irradiation, and certain toxins. Recent work has implicated p38 stress-activated protein kinase in the regulation of [...]

We have previously shown that SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38MAPK), attenuated insulin-stimulated glucose uptake in L6 skeletal muscle cells and 3T3-L1 adipocytes. Here we further explore [...]

The membrane potential of L6 muscle cells was measured with the fluorescent dye bis-oxonol. Hyperpolarizations of up to 15 mV were caused by gramicidin (in N-methyl-D-glucamine +medium), or by monensin or ionomycin. Depolarization was achieved with gramicidin (in Na +medium), or with K +. Insulin did not change the resting membrane potential of − 70 mV, yet it effectively stimulated 2-deoxy-D-glucose uptake. Conditions that hyperpolarize the cells did not alter the basal rate of hexose uptake. Moreover, insulin was still capable of stimulating hexose uptake in depolarized cells. It is concluded that modulation of the membrane potential is probably not a signalling event in insulin stimulaton of hexose uptake.

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.

Insulin releases inositol phosphoglycans from myocytes in culture [(1986) Science 233, 967–972], which display insulinomimetic activity. Because 5′‐nucleotidase is anchored to the membrane through inositol‐containing phospholipid glycans, we investigated whether insulin could release the enzyme from the membrane. Membranes prepared from hindquarter muscles of rats perfused with insulin showed a 23% decrease in 5′‐nucleotidase activity. Isolated membranes from muscle exposed to insulin in vitro also showed a small but reproducible decrease (9%) in 5′‐nucleotidase activity relative to unexposed controls. Phospholipase C from Staphylococcus aureusreleased 60% of the membrane‐bound 5′‐nucleotidase. We propose that insulin may activate an endogenous phospholipase C that cleaves phospholipid‐glycan‐anchored proteins.

The GLUT4 glucose transporter and the α2subunit of the Na+,K+‐ATPase of rat skeletal muscle are two proteins which redistribute from intracellular membranes to plasma membranes following in vivo insulin stimulation. Here we show that although both proteins co‐segregate after subcellular fractionation of unstimulated rat hindlimb muscles, they do not share the same intracellular residence inside the muscle fibre. By immunogold single‐ and double‐labeling on ultrathin muscle cryosections with specific antibodies, the GLUT4 glucose transporter and the Na+,K+‐ATPase α2subunit were observed on different vesicular structures within the cell. GLUT4 was detected on subsarcolemmal and perinuclear membranes, and at the junction between myofibrillar A and I bands where triads are localized. The α2subunit of the Na+,K+‐ATPase was observed at the plasma membrane and in distinct subsarcolemmal vesicles and intermyofibrillar membranes. Quantitative analysis of double‐labeling of GLUT4 and Na+,K+‐ATPase α2subunit revealed that less than 6% of the two proteins co‐localize in the same continuous vesicular structures. The differential intracellular localization of the two proteins was further confirmed by immunopurification of GLUT4‐containing membranes from muscle homogenates, in which the α2subunit of the Na+,K+‐ATPase was found only at the same extent as the α1subunit of the enzyme, a protein exclusively present at the plasma membrane.