Tumour-promoting phorbol esters have insulin-like effects on glucose transport and lipogenesis in adipocytes and myocytes. It is believed that insulin activates the glucose-transport system through translocation of glucose transporters from subcellular membranes to the plasma membrane. The aim of the present study was to investigate if phorbol esters act through the same mechanism as insulin on glucosetransport activity of rat adipocytes. We compared the effects of the tumour-promoting phorbol ester tetradecanoylphorbol acetate (TPA) and of insulin on 3-O-methylglucose transport and on the distribution of D-glucose-inhibitable cytochalasin-B binding sites in isolated rat adipocytes. Insulin (100 /tunits/ml) stimulated 3-O-methylglucose uptake 9-fold, whereas TPA (1 nM) stimulated the uptake only 3-fold (mean values of five experiments, given as percentage of equilibrium reached after 4 s: basal 7+ 1.3%, insulin 60 + 3.1 %, TPA 22 + 2.3%). In contrast, both agents stimulated glucose-transporter translocation to the same extent [cytochalasin B-binding sites (pmol/mg of protein; n = 7): plasma membranes, basal 6.2 + 1.0, insulin 13.4+2.0, TPA 12.7+2.7; low-density membranes, basal 12.8+2.1, insulin 6.3 +0.9, TPA 8.9+0.7; high-density membranes, 6.9 + 1.1; insulin 12.5 + 1.0, TPA 8.1 + 0.9]. We conclude from these data: (1) TPA stimulates glucose transport in fat-cells by stimulation of glucose-carrier translocation; (2) insulin and TPA stimulate the carrier translocation to the same extent, whereas the stimulation of glucose uptake is 3-fold higher with insulin, suggesting that the stimulatory effect of insulin on glucose-transport activity involves other mechanisms in addition to carrier translocation.

(1) the data show that AlCl3, probably through activation of a pertussis-toxin-inhibitable G protein, and PLC are able to modulate the intrinsic glucose carrier activity; (2) as pertussis toxin did not modify the effect of insulin, it seems unlikely that the insulin signal on glucose transport involves activation of this specific G protein.

The insulin effect on glucose uptake is not sufficiently explained by a simple glucose-carrier translocation model. Recent studies rather suggest a two-step model of carrier translocation and carrier activation. We used several pharmacological tools to characterize the proposed model further. We found that inositol phosphate (IP)-oligosaccharides isolated from the drug Actovegin, as well as the alkaloid vinblastine, show a partial insulin-like effect on glucose-transport activity of fat-cells (3-O-methylglucose uptake, expressed as % of equilibrium value per 4 s: basal 5.8%, insulin 59%, IP-oligosaccharides 30%, vinblastine 29%) without inducing carrier translocation. On the other hand, two newly developed anti-diabetic compounds (alpha-activated carbonic acids, BM 130795 and BM 13907) induced carrier translocation to the same extent as insulin and phorbol esters [cytochalasin-B-binding sites in plasma membranes: basal 5 pmol/mg of protein, insulin 13 pmol/mg of protein, TPA (12-O-tetradecanoylphorbol 13-acetate) 11.8 pmol/mg of protein, BM 130795 10.8 pmol/mg of protein], but produce also only 40-50% of the insulin effect on glucose-transport activity (basal 5.8%, insulin 59%, TPA 23%, BM 130795 35%). Almost the full insulin effect was mimicked by a combination of phorbol esters and IP-oligosaccharides (basal 7%, insulin 50%, IP-oligosaccharides 30%, TPA 23%, IP-oligosaccharides + TPA 45%). None of these substances stimulated insulin-receptor kinase in vitro or in vivo, suggesting a post-kinase site of action. The data confirm the following aspects of the proposed model: (1) carrier translocation and carrier activation are two independently regulated processes; (2) the full insulin effect is mimicked only by a simultaneous stimulation of carrier translocation and intrinsic carrier activity, suggesting that insulin acts through a synergism of both mechanisms; (3) IP-oligosaccharides might be involved in the transmission of a stimulatory signal on carrier activity.

Insulin resistance of the skeletal muscle is a key feature of Type 2 (non-insulin-dependent) diabetes mellitus. To determine whether a decrease of glucose carrier proteins or an altered subcellular distribution of glucose transporters might contribute to the pathogenesis of the insulin resistant state, we measured glucose transporter numbers in membrane fractions of gastrocnemius muscle of 14 Type 2 diabetic patients and 16 non-diabetic control subjects under basal conditions. Cytochalasin-B binding and immunoblotting with antibodies against transporter-subtypes GLUT 1 and GLUT 4 were applied. The cytochalasin-B binding values (pmol binding sites/g muscle) found in a plasma membrane enriched fraction, high and low density membranes of both groups (diabetic patients and non-diabetic control subjects) suggested a reduced number of glucose transporters in the plasma membranes of the diabetic patients compared to the control subjects (diabetic patients: 1.47 +/- 1.01, control subjects: 3.61 +/- 2.29, p less than or equal to 0.003). There was no clear difference in cytochalasin-B binding sites in high and low density membranes of both groups (diabetic patients: high density membranes 3.76 +/- 1.82, low density membranes: 1.67 +/- 0.81; control subjects: high density membranes 5.09 +/- 1.68, low density membranes 1.45 +/- 0.90). By Western blotting analysis we determined the distribution of the glucose transporter subtypes GLUT 1 and GLUT 4 in the plasma membrane enriched fraction and low density membranes of seven patients of each group.(ABSTRACT TRUNCATED AT 250 WORDS)

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