Asia Bak

Activation of Protein Kinase Cζ Induces Serine Phosphorylation of VAMP2 in the GLUT4 Compartment and Increases Glucose Transport in Skeletal Muscle

ABSTRACT Insulin stimulates glucose uptake into skeletal muscle tissue mainly through the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The precise mechanism involved in this process is presently unknown. In the cascade of events leading to insulin-induced glucose transport, insulin activates specific protein kinase C (PKC) isoforms. In this study we investigated the roles of PKCζ in insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of rat skeletal muscle. We found that insulin initially caused PKCζ to associate specifically with the GLUT4 compartments and that PKCζ together with the GLUT4 compartments were then translocated to the plasma membrane as a complex. PKCζ and GLUT4 recycled independently of one another. To further establish the importance of PKCζ in glucose transport, we used adenovirus constructs containing wild-type or kinase-inactive, dominant-negative PKCζ (DNPKCζ) cDNA to overexpress this isoform in skeletal muscle myotube cultures. We found that overexpression of PKCζ was associated with a marked increase in the activity of this isoform. The overexpressed, active PKCζ coprecipitated with the GLUT4 compartments. Moreover, overexpression of PKCζ caused GLUT4 translocation to the plasma membrane and increased glucose uptake in the absence of insulin. Finally, either insulin or overexpression of PKCζ induced serine phosphorylation of the GLUT4-compartment-associated vesicle-associated membrane protein 2. Furthermore, DNPKCζ disrupted the GLUT4 compartment integrity and abrogated insulin-induced GLUT4 translocation and glucose uptake. These results demonstrate that PKCζ regulates insulin-stimulated GLUT4 translocation and glucose transport through the unique colocalization of this isoform with the GLUT4 compartments.

Rat skeletal muscle in culture expresses the ?1 but not the ?2 protein subunit isoform of the Na+/K+ pump

Studies from this laboratory have shown that the physiological expression of the Na+/K+ pump in primary cultures of rat skeletal muscle increases with development. The molecular mechanisms underlying these changes are not known. Therefore, we have examined the expression of α and β subunits of the Na+/K+ pump at both the protein and mRNA levels during myogenesis of primary skeletal muscle cell cultures obtained from newborn rats. Protein isoforms were identified by Western blotting techniques with specific monoclonal and polyclonal antibodies and subunit mRNA was studied with specific cDNA probes. Freshly isolated skeletal muscle from newborn rats expressed both α1 and α2 protein subunits. From day 1 after plating, primary cultures expressed only the α1 protein isoform. In contrast, both β1 and β2 isoforms were expressed in freshly isolated muscle and in primary cultures, with β1 expression being stronger in both preparations. Studies on RNA expression showed that mRNA for α1, α2, β1, and β2 isoforms was identified both in freshly isolated muscle and after plating of cells in culture. These findings indicate that the lack of α2 protein expression in primary muscle cell cultures reflects a form of posttranscriptional regulation. There did not appear to be a quantitative difference in isoform expression as a function of age or of fusion in spite of developmental increases in Na+/K+ pump activity and its dependence on cell fusion. The lack of expression of the α2 subunit isoform suggests that the developmental changes in physiological expression of the Na+/K+ pump in primary cultures of skeletal muscle may be attributable either to the changes in activity of the α1 subunit or to differential activities of αβ complexes involving either of the β subunits. J. Cell. Physiol. 180:236–244, 1999.

Dependence of Na+,K+-ATPase and electrogenic component of Em in cultured myotubes on cell fusion.

This study was undertaken in order to determine the relation among cell fusion, [3H]ouabain binding and the membrane potential (Em) of cultured rat skeletal muscle. The amount of ouabain bound and the Em both increased with age, the increases being most dramatic following fusion. Inhibition of fusion prevented the developmental increases in both properties of cultured muscle. After fusion, the size of the electrogenic component of Em, determined by the decrease in Em produced by ouabain within 5-10 min, increased independent of the age at which fusion occurred. It is concluded that the increase in Em with age depends on postfusion appearance and activity of Na,K-ATPase.

Na+/K+ pump expression in the L8 rat myogenic cell line: Effects of heterologous α subunit transfection*

We have characterized the physiological and biochemical properties of the Na+/K+ pump and its molecular expression in L8 rat muscle cells. Pump properties were measured by [3H]ouabain binding and 86Rb uptake. Scatchard plot analysis of specific ouabain binding indicated the presence of a single family of binding sites with a Bmax of ∼135 fmol/ mg P and a KD of 3.3 × 10−8. 86Rb uptake due to specific pump activity was found to be 20% of the total in L8 cells. The results indicated lower affinity of L8 cells for ouabain and lower activity of the pump than that reported for chick or rat skeletal muscle in primary culture. Both the α1 and β1 protein and mRNA isoforms were expressed in myoblasts and in myotubes, while the α2, α3, and β2 isoforms were not detectable. We attempted to overcome low physiological expression of the Na+/K+ pump by employing a vector expressing an avian high affinity α subunit. This allowed identification of the transfected subunit separate from that endogenously expressed in L8 cells. Successful transfection into L8 myoblasts and myotubes was recognized by anti‐avian α subunit monoclonal antibodies. Fusion index, Na+/K+ pump activity, and the level of the transmembrane resting potential were all significantly greater in transfected L8 (tL8) cells than in non‐tL8. The total amount of α subunit (avian and rat) in tL8 cells was greater than that (only rat) in non‐tL8 cells. This relatively high abundance of the Na+/K+ pump in transfected cells may indicate that avian and rat α subunits hybridize to form functional pump complexes.

Some electrophysiological properties of cultured rat cerebral cortical neurons dissociated from fetuses at various gestational ages.

Neurons from dissociated cerebral cortex of fetal rat of different gestational ages were grown in culture for up to 4 weeks. Studies of membrane and action potentials, input resistance, neuron size and neurite outgrowth showed that neurons from 7‐day fetuses develop rapidly both electrophysiologically and morphologically, but are maintained for only about 2 weeks. In contrast, neurons from 14 to 17 day fetuses mature slowly, but can be maintained for at least 4 weeks. Neurons from both young and old fetuses show an increase in resting Em with age, the maximum value of −60 to −65 mV being attained by about 2 weeks. While neurons can be maintained in serum‐free medium, their membrane electrical properties deteriorate with time. Thus, appropriate development of neurons in culture depends upon both the age of the starting tissue and the presence of adequate, but still undefined factors found in animal serum for at least the first several days in culture.