Insulin regulation of sugar transport in giant muscle fibres of the barnacle

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Abstract
1. Sugar transport in the giant muscle cells of Balanus nubilus is accelerated during contractile activity and exposure to porcine insulin. The characteristics of hexose‐transfer regulation in the giant muscle cells have been examined by studying the transport of 3‐O‐methylglucose (a non‐metabolized sugar) in both intact giant fibres and fibres subjected to internal solute control by internal dialysis.2. Sugar transport in barnacle muscle is mediated by a saturable process which is inhibited by both phloretin and cytochalasin B. Insulin increases the capacity of the transport system with little effect on its apparent affinity for sugar. Under the same conditions insulin increases 3‐O‐methylglucose‐displaceable cytochalasin B binding. The effects of insulin on transport are half‐maximal at 5 μM‐insulin and are abolished by both insulin antibody and phloretin. The intact barnacle releases an insulin‐like material in response to a rise in blood glucose levels.3. Insulin increases the cyclic GMP (cGMP) content and reduces the cyclic AMP (cAMP) content of barnacle muscle. Experiments with fibres injected with aequorin show that insulin also lowers cytosolic ionized Ca levels. The changes in cyclic nucleotide levels induced by insulin precede the effects on sugar transport and cytosolic ionized Ca. During repetitive contractile activity, cAMP, cGMP and ionized Ca levels are raised.4. Agents which raise the cAMP content of barnacle muscle normally inhibit sugar transport. Dibutyryl cAMP also inhibits transport. Alterations in cytosolic ionized Ca levels in intact fibres are without effect on sugar transport. Nevertheless, stimulation of transport by insulin is blunted when cytosolic ionized Ca is lowered by intracellular injection of the Ca‐chelating agent, EGTA.5. Sugar uptake in the internally dialysed fibre is inhibited by intracellular application of cAMP. Internal application of Ca and cGMP stimulate sugar uptake in the dialysed fibre. Cyclic AMP reduces the capacity of the transport system whereas Ca and cGMP increase the capacity of the saturable transfer system. Cyclic AMP and cGMP act at kinetically independent sites. Internal ATP (2 mM) inhibits sugar uptake in the dialysed fibre by some 40%, possibly through the production of cAMP.6. External insulin stimulates sugar uptake in the dialysed fibre even when ionized Ca levels are buffered using EGTA. Stimulation by insulin requires the presence of cytosolic ATP and is potentiated by internal application of 1 mM‐GTP. In the dialysed fibre stimulation of transport by insulin is greater than that brought about by Ca and cGMP.7. The stimulation of transport by insulin in the intact fibre and its inhibition by dibutyryl cAMP are abolished by intracellular injection of Gpp(NH)p. Injection of intact fibres with GTPγS potentiates the stimulation of transport by insulin and renders insulin‐activation of transport irreversible. Injection of intact fibres with ATPγS leads to the irreversible inhibition of transport.8. Injection of intact fibres with cAMP phosphodiesterase lowers cAMP levels close to zero and stimulates sugar transport. Application of insulin to diesterase‐injected fibres still stimulates transport in the absence of altered cytosolic cAMP.