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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 glucose-transport 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 mu units/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.

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.

Recently it was speculated that activation of GTP-binding proteins and of phospholipase is involved in the transmission of a signal from the insulin-receptor kinase to effector systems in the cell. To confirm this hypothesis, we have tested the effect of AlCl3, which has been recently used as an experimental tool to activate GTP-binding proteins, on glucose transport in fat-cells. We found that AlCl3 has a partial insulin-like effect on glucose transport activity (3-O-methylglucose uptake, expressed as % of equilibrium value per 4 s: basal 9.6 +/- 2, AlCl3 29.6 +/- 4, insulin 74.0 +/- 3). The AlCl3 effect is totally blocked by pertussis toxin, whereas the insulin effect was not altered. The effect starts at [AlCl3] greater than 1 fM and reaches its maximum at 0.1 nM. Addition of phospholipase C (PLC; 50 munits/ml) also stimulated glucose transport (maximal 53.0 +/- 5%). Both substances acted faster than insulin itself (maximal values within 1 min for PLC, 2 min for AlCl3 and 5-10 min for insulin). Using the cytochalasin-B-binding assay to determine the effects of AlCl3 and PLC on the distribution of glucose carrier sites in subcellular fractions, we found that their glucose-transport-stimulating effect does not occur through an increase in glucose carrier sites in the plasma-membrane fraction. When PLC was combined with the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate), which increases glucose carrier sites in the plasma membrane, an additive effect on glucose transport was found [PLC (50 munits/ml), 53.0 +/- 5%, TPA (1 nM), 17.3 +/- 2%; PLC + TPA, 68.0 +/- 3%]. In conclusion: (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.