Regulation of protein kinase C by short term hyperglycaemia in human platelets in vivo and in vitro

Aims/hypothesis. Postprandial hyperglycaemia carries an increased risk of m acrovascular disease even without Type II (non-insulin-dependent) diabetes mellitus. Chronic hyperglycaemia activates protein kinase C (PKC) in vitro and in vivo but it is not known whether PKC is regulated by short-term post prandial hyperglycaemia in vivo in humans. We investigated whether PKC is r egulated in vivo in hyperglycaemic and hyperinsulinaemic infusion tests and correlated the results to stimulations in vitro. Methods. Protein kinase C regulation was measured in platelets obtained fro m 8 healthy subjects who were infused with glucose and insulin for 2 h atta ining peak concentrations of 16 mmol/l glucose and in platelets from 8 heal thy young subjects, 8 older subjects without diabetes, and 10 older subject s with Type II diabetes after incubation in vitro with 16 mmol/l glucose or glucose and insulin. For precise quantification, a shortened PKC beta1 sta ndard protein was generated by bacterial expression and PKC alpha, beta1, b eta2 and delta isoenzyme values were measured by immunoblot analyses. Results. Hyperglycaemic and hyperinsulinaemic in vivo tests increased the a mounts of PKC alpha, beta1 and beta2 in the membrane fraction of platelets to 225 +/- 87%, 164 +/- 22% and 302 +/- 135%, respectively, when compared w ith the baseline values in young healthy volunteers (n = 8, p < 0.05). The expression of PKC <delta> did not change. In comparison to the recombinant PKC beta1 standard protein, 5 ng PKC beta1/ mug protein was measured before the test and 2 ng/mug were translocated to the membrane fraction after the infusion. No change in the absolute amount of PKC beta1 was detected. In c ontrast, after incubation in vitro PKC was not regulated by glucose or gluc ose and insulin in 8 young healthy subjects (age 26 +/- 0.7 years) and in 8 older, healthy subjects (age 64,8 +/- 4 years) although 100 nmol/l 12-O-te tradecanoylphorbol 13-acetate caused maximal activation. In marked contrast , PKC beta1 and PKC beta2, but not PKC alpha or PKC delta, were increased i n vitro in the membrane fraction by 292 +/- 61% and 432 +/- 88% (p < 0.05) in 10 subjects with Type II diabetes mellitus matched for age, sex and BMI. Conclusion/interpretation. We found that short-term hyperglycaemia activate s PKC <alpha>, beta1 and beta2 in platelets of healthy persons making them potential candidates for mediating the increased cardiovascular risk of pos tprandial hyperglycaemia. Hyperglycaemia and hyperinsulinaemia did not caus e short-term activation of PKC in platelets in vitro suggesting the existen ce of additional stimuli. Subjects with Type II diabetes showed a markedly altered reactivity of platelet PKC beta in vitro indicating some diabetes-r elated regulation.