The mechanism of insulin stimulation of (Na+,K+)-ATPase transport activity in muscle.

N K Rosić,M L Standaert,R J Pollet

doi:10.1016/s0021-9258(18)88958-x

Abstract

Since the mechanism underlying the insulin stimulation of (Na+,K+)-ATPase transport activity observed in multiple tissues has remained undetermined, we have examined (Na+,K+)-ATPase transport activity (ouabain-sensitive 86Rb+ uptake) and Na+/H+ exchange transport (amiloride-sensitive 22Na+ influx) in differentiated BC3H-1 cultured myocytes as a model of insulin action in muscle. The active uptake of 86Rb+ was sensitive to physiological insulin concentrations (1 nM), yielding a maximum increase of 60% without any change in 86Rb+ permeability. In order to determine the mechanism of insulin stimulation of (Na+,K+)-ATPase activity, we demonstrated that insulin also stimulates passive 22Na+ influx by Na+/H+ exchange transport (maximal 200% increase) and an 80% increase in intracellular Na+ concentration with an identical time course and dose-response curve as insulin-stimulated (Na+,K+)-ATPase transport activity. Incubation of the cells with high [Na+] (195 mM) significantly potentiated insulin stimulation of ouabain-inhibitable 86Rb+ uptake. The ionophore monensin, which also promotes passive Na+ entry into BC3H-1 cells, mimics the insulin stimulation of ouabain-inhibitable 86Rb+ uptake. In contrast, incubation with amiloride or low [Na+] (10 mM), both of which inhibit Na+/H+ exchange transport, abolished the insulin stimulation of (Na+,K+)-ATPase transport activity. Furthermore, each of these insulin-stimulated transport activities displayed a similar sensitivity to amiloride. These results indicate that insulin stimulates a large increase in Na+/H+ exchange transport and that the resulting Na+ influx increases the intracellular Na+ concentration, thus activating the internal Na+ transport sites of the (Na+,K+)-ATPase. This Na+ influx is, therefore, the mediator of the insulin-induced stimulation of membrane (Na+,K+)-ATPase transport activity classically observed in muscle.

Highlights

Since the mechanism underlying the insulin stimu- chemical gradient maintained by this active cation transport lation of (Na+,K+)-ATPasetransport activity observed significantly influences cell volumeand osmotic pressure and in multiple tissues hasremained undetermined, we provides the driving force for the inward cotransport of amino sshye(troueniaaeunlvtniliasnedasibtitpneaieaovdigxcnreatt-Biamso(Ctenaoipnmn3mhsHeiaiyindtxlm-sio1viiumro(eicNslduBocuealmgl6e-+tisR.uc,eKTriabnnel+h+usdcienip)rt-emsitAvauaayekslcTioeetn"Pic)Nvaoyeascfta+eeonstu6ndrpNa0icansten%aafnsa+lkupt/weromxHoaf)irto+tti"heaiodRnxocenbtculs(d+ihtov1iaffimwaftnieyngna)ryes-,pceaa(Nascnotlidrlacdty+isuopt,afmoKrnoH+mdct+e)ome-in,mAnCocTbaens2rnoPya+tsannra,saatechetncip,etdohhosnaeil(Ks,arr4imerd+i,fzi5eoooas)drnt,e(uies2,oll)ani(en3tc,efa)tdls.rnuroTcdiegvtehnhelerclenosegiutoscrrguoaaihtwnlnwltsyttrheahacrpe,coddsienteiclhtflromfueieullbreianuaecrtlntheepstarsiHotnarmdtiaisiaonoumnndsm-sd
Insulin stimulation of (Na+,K+)-ATPasewas proposed (19, 20) as the basis for the insulin-induced hyperpolarization observed in this tion of (Na+,K+)-ATPasetransport activity
The continuously cultured mouse musclecell line BC3H-1, upon obtaining cell confluence, spontaneously differentiates from logarithmically growing myoblasts to differentiated myocyte~, as demonstrated by the appearance of membrane acetylcholine receptors and by the synthesis of muscle-specific proteins (37). This differentiation process is accompanied by a 3-5-fold increase in the number of cell membrane insulin receptors andthe development of physiologic insulin responses, including stimulation of glucose and amino acid transport,as well as receptor down-regulation (34)

Summary

EXPERIMENTAL PROCEDURES

Materkb"RbC1 and 22NaC1 werepurchased from New England Nuclear. Insulin was purchased from Lilly. Measurement of @Rb+and *'Na+ Uptake-For uptake experiments, the media was aspirated and the cells were washed 2-3 times with 138 mM NaC1, 8.1 mM Na2HP0,, 0.5 mMMgC12, 1.5 mM KH2P04, 2.5 mMKC1, 0.1 mM CaCl', 0.1% bovine serum albumin (pH 7.3) at 37 "C and equilibrated with 900 pl of this buffer at 37 "C for 30 min in the presence or absence of 2 mM ouabain and/or 0.3 mM amiloride. After incubating the BC3H-1 myocytes in 0.5 ml of buffer containing 1 pCiof =Na+ at 37 "C in the absence of ouabain, the cells reached equilibrium with respect to =Na+ content within 10 min. After incubation for 30 min with or without 0.3 mM amiloride, insulin (200 nM) was added, and the subsequent =Na+cellular content was determined at thiendicated times. Protein concentrations were determined according to the method of Lowry et al (36)

RESULTS

AML AML MMOONN

DISCUSSION