Calcium homeostasis and glucose uptake of murine myotubes exposed to insulin,caffeine and 4‐chloro‐m‐cresol

The modulation of glucose uptake by cytosolic calcium and the role of insulin on calcium homeostasis in insulin‐target cells are incompletely understood and results are contradictory. To address this issue, we used the C2C12 murine skeletal muscle cell line model and examined the influence of caffeine and 4‐chloro‐m‐cresol, two ryanodine receptor agonists known to mobilize intracellular calcium stores and increase cytosolic free calcium concentration. We followed ⁴⁵calcium efflux, a validated indicator of cytosolic calcium concentration, and 3‐O‐methyl‐[1–³H]‐d ‐glucose uptake in parallel. We also determined if insulin incubation affected ⁴⁵calcium influx rate. A 30‐min treatment by 1 μm insulin highly significantly increased ⁴⁵calcium efflux by 8.5% (P = 0.0014), despite a significant reduction of ⁴⁵Ca²⁺ influx already measurable after 20 and 30 min of insulin stimulation (?16.6%, P = 0.0119 and ?21.3%, P = 0.0047, respectively). Caffeine (1–20 mm ) and 4‐chloro‐m‐cresol (0.05–10 mm ) concentration‐dependently increased ⁴⁵calcium efflux, the latter being more potent and efficacious. These agents, in a concentration‐dependent manner, inhibited both basal and, more potently, insulin‐stimulated glucose uptake. This resulted in a negative correlation of glucose uptake and ⁴⁵calcium efflux (r > 0.95, P < 0.001). This effect was ～5 times greater for caffeine than for 4‐chloro‐m‐cresol, suggesting a calcium‐independent part of the glucose uptake inhibition by caffeine. In our in vitro model of cultured muscle cells, insulin appears to prevent calcium overload by both stimulating efflux and inhibiting cell storage. This effect, taken together with the observed inhibitory, inverse relationship between ⁴⁵calcium efflux and glucose uptake, contributes to describing the complex insulin–calcium interplay involved in target cells.