Contraction-induced glucose transport into skeletal muscle : the involvement of protein kinase C

The role of PKc as a possible intracellular mediator of exercise-induced increase in skeletal muscle glucose transport was investigated. Two skeletal systems were used, the in vivo rat gastrocnemius-plantaris-soleus muscle preparation, and the in vitro isolated soleus muscle preparation. PKc was partially purified from gastrocnemius-plantarissoleus skeletal muscle preparations by DE52 anion exchange chromatography. The skeletal muscle content of PKc was found to be higher than previously reported. Levels of PKc activity were found to be comparable to those of published PKa values, which suggest an equally important function for PKc in cellular events. The complete extraction of PKc from skeletal muscle membranes required a high concentration of Triton X100. This suggested that the hydrophobic domain of the enzyme was tightly bound to the membrane bilayer. Analysis of the intracellular distribution showed that a large proportion of the enzyme activity (60%) was associated with the membrane component of resting muscle. Electrical-induced contraction of the gastrocnemius' - plantaris-soleus muscle group resulted in a time-dependent translocation of PKc and a 2-fold increase in the concentration of both diacylglycerol and phosphatidic acid. The maximal values for the latter were reached at 2 min and preceded the maximum translocation of PKc (10 min). No PKm formation was detected by the production of a Ca 2+ -, phospholipid-independent histone kinase activity. No reversal of translocated PKc or decrease in the concentrations of diacyglycerol and phosphatidic acid occurred after 30 min of rest following a 5 min period of stimulation in vivo. The translocation of PKc was not influenced by variations in applied load at maximal fiber recruitment, but was dependent on the frequency of non-tetanic stimuli reaching a maximum at 4 Hz. Thus, it is concluded that electrical-induced muscle contraction increased the production of diacylglycerol and the translocation of PKc. This is indictative of enzyme activation. The relationship between PKc and glucose transport was explored, in vivo, by varying the number of tetanic stimuli. Only one train of stimuli (200 ms, 100 Hz) was required for maximum translocation of PKc and for diacylglycerol and phosphatidic acid production. However, more than 35 trains of stimuli were required to activate glucose transport. The electrical stimulation of isolated soleus muscle in vitro increased the rate of glucose transport, but this required 20 min to reach maximum. Therefore, the time course for the activation of glucose transport is slower than PKc translocation. Thus, it is concluded that a \causal\" relationship between electrical stimulation and the increase in glucose transport involving PKc activation may exist. In an attempt to further clarify a role for PKc in the mechanism of glucose transport activation isolated soleus muscle was depleted (\"downregulated\") of PKc activity by prolonged TPA treatment. The response to both insulin and contraction in terms of glucose transport was assessed. Muscles treated with TPA showed an increased basal rate of 3-0-methylglucose uptake responded partially to insulin but did not respond to contraction. The TPA treated and non-treated muscles were indistinguishable in terms of pre-contraction content of adenine nucleotide creatine phosphate lactate and glycogen as well as contractile performance and contraction-induced glycogenolysis. Thus it is concluded that treatment of isolated soleus muscle with TPA gives rise to a marked loss of contraction-induced glucose transport. Overall the work embodied by this thesis supports a case for the involvement of PKc in contraction-mediated uptake of glucose by skeletal muscle."