Evidence for mechanistic alterations of Ca²⁺ homeostasis in Type 2 diabetes mellitus

Abstract

Altered cytosolic Ca²⁺ is implicated in the aetiology of many diseases including diabetes but there are few studies on the mechanism(s) of the altered Ca²⁺ regulation. Using human lymphocytes, we studied cytosolic calcium (Cai) and various Ca2+ transport mechanisms in subjects with Type 2 diabetes mellitus and control subjects. Ca²⁺-specific fluorescent probes (Fura-2 and Fluo-3) were used to monitor the Ca²⁺ signals. Thapsigargin, a potent and specific inhibitor of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA), was used to study Ca²⁺-store dependent Ca2+ fluxes. Significant (P<0.05) elevation of basal Ca, levels was observed in lymphocytes from diabetic subjects. Cai levels were positively correlated with fasting plasma glucose and HbA_1c. There was also a significant (P<0.05) reduction in plasma membrane calcium (PMCA) ATPase activity in diabetic subjects compared to controls. Cells from Type 2 diabetics exhibited an increased Ca²⁺ influx (as measured both by Fluo-3 fluorescence and 45Ca assays) as a consequence of thapsigargin-mediated Ca²⁺ store depletion. Upon addition of Mn²⁺ (a surrogate of Ca²⁺), the fura-2 fluorescence decayed in an exponential fashion and the rate and extent of this decline was steeper and greater in cells from type 2 diabetic patients. There was also a significant (P<0.05) difference in the Na⁺/Ca²⁺ exchange activity in Type 2 diabetic patients, both under resting conditions and after challenging the cells with thapsigargin, when the internal store Ca²⁺ sequestration was circumvented. Pharmacological activation of protein kinase C (PKC) in cells from patients resulted in only partial inhibition of Ca²⁺ entry. We conclude that cellular Ca²⁺ accumulation in cells from Type 2 diabetes results from (a) reduction in PMCA ATPase activity, (b) modulation of Na⁺/Ca²⁺ exchange and (3) increased Ca²⁺ influx across the plasma membrane.