Further evidence for the involvement of a membrane proteolytic step in insulin action

Abstract

The hypothesis that insulin action involves a membrane proteolytic step was further explored, by using isolated rat adipocytes and liver plasma membranes. (1) The maximal insulin stimulation of 2-deoxyglucose transport and lipogenesis in fat-cells was selectively inhibited (73-88%) by N alpha-p-tosyl-L-lysine chloromethyl ketone (Tos-Lys-CH2Cl; active-site inhibitor of trypsin; 30-125 microM), p-nitrophenyl p'-guanidinobenzoate (active-site inhibitor of serine proteinases; 30-125 microM) and p-tosyl-L-arginine methyl ester (arginine ester substrate analogue of proteinases; 1-2 mM), under conditions where neither the basal rate of each metabolic process nor insulin binding nor cellular ATP content were affected. In contrast, N-acetyl-L-alanyl-L-alanyl-L-alanine methyl ester (alanine ester substrate analogue of proteinases; 1-2 mM) was ineffective. (2) Endoproteinase Arg-C (0.25-40 micrograms/ml) exerted dose-dependent insulin-like effects on both 2-deoxyglucose transport and lipogenesis in fat-cells, whereas endoproteinase Lys-C (5-100 micrograms/ml) was ineffective. The maximal activation by endoproteinase Arg-C of both processes (200 and 177% of control values respectively) was shown to occur under conditions where membrane integrity (assessed by measurement of lactate dehydrogenase leakage and passive glucose diffusion) was preserved. This effect was inhibited by Tos-Lys-CH2Cl (125 microM) and was not additive with the maximal insulin effect. (3) Insulin (1-100 ng/ml) produced a dose-dependent increase in the trichloroacetic acid-soluble 125I radioactivity released after a 30 min incubation at 37 degrees C of 125I-labelled liver plasma membranes, but was ineffective on 125I-labelled bovine serum albumin. Insulin effects on both radio-labelled proteins were reproduced by wheat-germ agglutinin (20 micrograms/ml), an insulin mimicker shown to act through the insulin receptor. These data provide further evidence for the hypothesis that insulin bioeffects involve the activation of a membrane serine proteinase with arginine specificity.

Involvement of hormone processing in insulin-activated glucose transport by isolated cardiac myocytes

“…Thus not insulin degradation but a degradative step in general may be involved in mediating steady-state insulin action. Specifically, since the action of chloroquine is not limited to the lysosome, but may also affect endosomes (Posner et al, 1982), our data would also be consistent with the involvement of a membrane-associated degradative, most probably proteolytic, step in the transmission of the insulin signal (Seals & Czech, 1980;Cherqui et al, 1985).…”

Phosphatidylinositol-Glycan Anchors of Membrane Proteins: Potential Precursors of Insulin Mediators

“…The involvement of proteases in the early the activity of phospholipase C. For this stges of insulin action has been suggested purpose, we studied the effects of insulin on by several investigators (1,(17)(18)(19)(20)(21). To test the production of dimyristoylglycerol on the the possible participation of a proteolytic assumption that it reflects, as suggested by reaction on the release ofAP, we studied the Saltiel t al.…”

“…However, it is purified (22). The involvement of proteases generally admitted that insulin generates a in the action of insulin has also becn report-cascade ofevents involving phosphorylation ed by direct demonstration ofinsulin-stimu-and dephosphorylation of polypeptides and lated proteolync activity (18) and by the the generation of second messengers by blockade of insulin-mediated effects, indud-mechanisms that are related to insulin receping mediator production, by certain protetor phosphorylation (13). Mediator generaase inhibitors (17,(19)(20)(21).…”

Insulin Action is Mimicked by Polyclonal Antireceptor Antibodies that Activate the Insulin Receptor Tyrosine Kinase

“…Partial purification of rat liver membrane insulin receptors was carried out as previously described {Caron, Lascols, Picard, Cherqui and Capeau 1983). Rat adipocytes and ZHC cells were obtained as described (Cherqui, Caron, Capeau and Picard 1985;Capeau, Flaig-Staedel, Beck and Picard 1982). Insulin receptors from ZHC cells were partially purified on wheat germ-agarose column in the presence of protease inhibitors (0.2 mM PMSF and 1200 U/ml aprotinin).…”

“…2-deoxy-D[l-l4 C]glucose transport (0.25 mM) and lipogenesis from [3-3 H]glucose (0.5 mM) in rat adipocytes were measured as previously described (Cherqui, Caron, Capeau and Picard 1985). In experiments where the effects of insulin, P95 Ab or control IgG were studied, the adipocytes (approx.…”