Insulin and high glucose modulation of phosphatase and reductase enzymes in the human erythrocytes: a comparative analysis in normal and diabetic states

Abstract

The ability of insulin to influence activities of various protein kinases and protein phosphatases, that are thought to mediate insulin action, are limited in patients with insulin resistance. Because numerous responses to insulin are affected, we undertook studies to determine whether protein tyrosine phosphatases (PTPs) activities are altered in patients with diabetes syndrome. In order to evaluate abnormal PTP activities, we done a comparative study using erythrocytes from normal and diabetic patients. We determined the activity of the cytosolic acid PTP in basal and insulin-dependent states. Mean basal PTP activities, were found to be significantly higher in diabetics than in normal subjects (type 1 diabetics: 0.36±0.01 vs 0.28±0.01 mmol p-nitrophenolate/h per g hemoglobin (Hb), P<0.001; type 2 diabetics: 0.35±0.01 vs 0.28±0.01 mmol p-nitrophenolate/h per g Hb, P<0.001). Insulin, at concentrations above physiological levels (1 mIU/ml), inhibited the PTP activities in erythrocytes from normal subjects (−15±4.1%, P<0.01). Insulin could also modulate glycolysis, probably as a consequence of receptor tyrosine kinase activation, inducing phosphorylation of protein band 3 and hence the release of glycolytic enzymes. We have previously reported that a reductase enzyme in human erythrocytes is dependent on glycolysis being significantly activated (+28±3.1%, P<0.001) by high insulin levels (1 mIU/ml). Mean basal reductase activities were found to be significantly lower in diabetics than in normal subjects (type 1 diabetics: 0.77±0.03 vs 0.97±0.02 mmol ferrocyanide/20 min per l cells, P<0.001; type 2 diabetics: 0.77±0.04 vs 0.97±0.02 mmol ferrocyanide/20 min per l cells, P<0.001), indicating altered erythrocyte metabolism in the diabetic patients. High glucose levels were used to mimic hyperglycemia condition, using erythrocytes from normal subjects. At 30 mM glucose, erythrocytic phosphatase activity was stimulated (+32±4.2%, P<0.0001), although no effect was observed on the reductase enzyme at the same glucose levels. Results indicated that diabetic disorders appear to be associated with quantitative alterations of erythrocyte acid phosphatase activity and other enzymes that depend on the glycolytic rate (reductase). The overall data suggest that erythrocyte acid phosphatase may have a role in the modulation of glycolytic rates through the control of insulin receptor phosphorylation.