Involvement of phosphoinositide 3-kinase in insulin stimulation of MAP-kinase and phosphorylation of protein kinase-B in human skeletal muscle: implications for glucose metabolism.

Isolated skeletal muscle from healthy individuals was used to evaluate the role of phosphoinositide 3-kinase (PI 3-kinase) in insulin signalling pathways regulating mitogen activated protein kinase (MAP-kinase) and protein kinase-B and to investigate whether MAP-kinase was involved in signalling pathways regulating glucose metabolism. Insulin stimulated glycogen synthase activity (approximately 1.7 fold), increased 3-o-methylglucose transport into human skeletal muscle strips (approximately 2 fold) and stimulated phosphorylation of the p42 ERK-2 isoform of MAP-kinase. This phosphorylation of p42 ERK2 was not blocked by the PI 3-kinase inhibitors LY294002 and wortmannin although it was blocked by the MAP-kinase kinase (MEK) inhibitor PD 98059. However, PD98059 (up to 20 micromol/l) did not block insulin activation of glycogen synthase or stimulation of 3-o-methylglucose transport. Wortmannin and LY294002 did block insulin stimulation of protein kinase-B (PKB) phosphorylation and stimulation of 3-o-methylglucose transport was inhibited by wortmannin (IC50 approximately 100 nmol/l). These results indicate that MAP-kinase is activated by insulin in human skeletal muscle by a PI 3-kinase independent pathway. Furthermore this activation is not necessary for insulin stimulation of glucose transport or activation of glycogen synthase in this tissue.

Compartment-specific regulation of phosphoinositide 3-kinase by platelet-derived growth factor and insulin in 3T3-L1 adipocytes.

To understand how the stimulation of phosphoinositide 3-kinase (PI 3-kinase) by different growth factors can activate different subsets of downstream responses, growth-factor regulation of PI 3-kinase activity at different intracellular locations was investigated in 3T3-L1 adipocytes. Insulin caused a large stimulation of glucose transport and stimulated recruitment of transferrin receptors to the plasma membrane (PM) in these cells, whereas platelet-derived growth factor (PDGF)-bb was virtually without effect on these responses. Subcellular fractionation studies after stimulation with PDGF-bb or insulin revealed a differential effect of these growth factors on subcellular localization of PI 3-kinase activity. PDGF was more effective than insulin in stimulating PI 3-kinase activity and recruiting the p85 alpha PI 3-kinase adaptor subunit in the fraction containing the PM. However, in the microsomal fraction insulin significantly increased PI 3-kinase activity and p85 alpha levels, whereas PDGF was almost without effect. In the microsomal membrane fraction the insulin-stimulated recruitment of p85 alpha closely matched the increase PI 3-kinase activity, indicating that insulin stimulation of PI 3-kinase in this fraction is largely due to recruitment of PI 3-kinase enzyme rather than alterations in specific activity. Insulin-stimulated recruitment of p85 alpha to the microsomal membranes was not inhibited by wortmannin, indicating that PI 3-kinase activity was not required for this process. A further level of compartment-specific regulation of PI 3-kinase in response to PDGF was revealed by the finding that tyrosine phosphorylation of the p85 alpha adaptor was restricted to the PM-containing fraction. Insulin had no effect on p85 tyrosine phosphorylation in either fraction. In summary, these results suggest a basis by which insulin and PDGF could both use PI 3-kinase signalling cascades but achieve different signalling outcomes.

The measurement of drug-induced calcium changes in vascular smooth muscle using X-ray microanalysis of pyroantimonate precipitates.

A method is described for the X-ray microanalysis of calcium pyroantimonate precipitates in smooth muscle using an electron microscope. Stimulation of calcium influx through voltage-operated channels using KCl and blockade of this effect with nifedipine were used to validate the method. Significant changes in the calcium content of rat renal artery were detected when osmium tetroxide-pyroantimonate were used as fixatives, whereas addition of glutaraldehyde yielded unsatisfactory data. Some effects of hydralazine are also briefly described.

Penile verrucous carcinoma.

Eleven patients with verrucous carcinoma of the penis are presented. The age range was from 41 to 80 years with a mean of 59 years. The pathological features of these tumours are described and the aetiology of penile verrucous carcinoma is discussed.