The inhibitory effect of staurosporine on insulin action is prevented by okadaic acid. Evidence for an important role of serine/threonine phosphorylation in eliciting insulin-like effects.

The serine/threonine phosphatase inhibitor, okadaic acid (OA), exerted several insulin-like effects in rat adipose cells and was, in part, synergistic with insulin. OA stimulated glucose transport activity, altered the electrophoretic mobility of IRS-1, increased the phosphorylation of the MAP-kinases ERK 1 and 2 on tyrosine sites, markedly increased MAP kinase activity and also acted synergistically with insulin in activating these enzymes. However, OA did not increase PI 3-kinase activity or the tyrosine phosphorylation of key upstream proteins in insulin's signaling cascade. Staurosporine virtually completely inhibited the insulin-stimulated glucose transport and MAP kinase activation in spite of a maintained high PI 3-kinase activity. In contrast, the effects of OA alone or in the presence of insulin were less, or not at all, affected. These data suggest that OA exerts an insulin-like effect through a serine/threonine-related pathway which is distinct from, but converges with, that of insulin downstream PI 3-kinase and upon which staurosporine exerts an inhibitory effect.

Measurement by microdialysis of the insulin concentration in subcutaneous interstitial fluid. Importance of the endothelial barrier for insulin.

To evaluate the interstitial insulin and inulin concentrations, 20-min microdialysis samples from the abdominal subcutaneous tissue were obtained by using two 45-mm polypropylene dialyzing tubes (o.d. approximately 0.5 mm, pore size 0.2 micron) during a euglycemic hyperinsulinemic (120 mU.m-2 x min-1) clamp (n = 9) or during a constant inulin infusion (n = 5). After in situ calibration of the microdialysis catheters during steady-state conditions, interstitial and plasma insulin concentrations were estimated to 654 +/- 102 and 1176 +/- 66 pM, respectively, i.e., a 44% difference (P < 0.001). A doubling of the insulin infusion rate (240 mU.m-2 x m-1), leading to supraphysiological plasma insulin levels, raised the interstitial insulin concentrations markedly slower (approximately 20 min) than in plasma. Moreover, at steady state the concentration difference in the two compartments prevailed even during the high insulin infusion rate (55% difference, P < 0.01). In contrast, the interstitial inulin levels were similar to the plasma concentrations in subjects given a constant inulin infusion. Thus, the data suggest the presence of an endothelial barrier for insulin in the subcutaneous tissue. This barrier, in combination with tissue clearance of insulin, leads to lower insulin levels and altered kinetics with a slower rise in the interstitial fluid compared with plasma.

Impaired glucose transport and protein kinase B activation by insulin, but not okadaic acid, in adipocytes from subjects with Type II diabetes mellitus.

AIMS/HYPOTHESIS: To study the effects of insulin and okadaic acid, a serine/threonine phosphatase inhibitor which does not increase PI3-kinase activity, on the rate of glucose transport and protein kinase B activation in adipocytes from healthy subjects and subjects with Type II (non-insulin-dependent) diabetes mellitus. METHODS: Adipocytes were incubated with or without insulin or okadaic acid or both and glucose transport, protein kinase B activity, phosphorylation and protein expression measured. RESULTS: Insulin and okadaic acid alone increased glucose uptake to a similar degree in adipocytes from healthy subjects and, when combined, exerted a partial additive effect. The effect of insulin was reduced by about 60% in adipocytes from Type II diabetic patients, whereas the effect of okadaic acid was essentially unchanged and no further increase was seen when okadaic acid and insulin were combined. Okadaic acid increased protein kinase B activity to a greater extent (two to threefold) than insulin but only slightly increased the serine phosphorylation of protein kinase B. Adipocytes from Type II diabetic subjects exhibited both an impaired sensitivity as well as a reduced total serine phosphorylation and activation of protein kinase B in response to insulin but protein kinase B activity in response to okadaic acid was intact. CONCLUSION/INTERPRETATION: These results show that the ability of insulin to increase glucose transport and activate protein kinase B is reduced in fat cells from Type II diabetic subjects. Protein kinase B can, however, be activated by agents like okadaic acid which bypass the upstream defects in the insulin signalling pathway in Type II diabetic cells and, thus, increase glucose uptake.

Phosphorylation of PDE3B by phosphatidylinositol 3-kinase associated with the insulin receptor.

Phosphatidylinositol 3-kinase mediates several actions of insulin including its antilipolytic effect. This effect is elicited by the insulin-stimulated serine phosphorylation and activation of cGMP-inhibited phosphodiesterase (PDE3B). In human adipocytes, we found that insulin differentially stimulated phosphatidylinositol 3-kinase activity; the lipid kinase activity was associated with IRS-1, whereas the serine kinase activity was associated with the insulin receptor and phosphorylated a number of proteins including p85, p110, and a 135-kDa protein identified as PDE3B. PDE3B phosphorylation was associated with enzyme activation, thus initiating the antilipolytic effect of insulin. These results show a novel pathway for intracellular signaling through the insulin receptor leading to the serine phosphorylation of key proteins involved in insulin action.

A stable peroxovanadium compound with insulin-like action in human fat cells.

Aqueous solutions of peroxovanadium (pV) compounds are potent insulin-mimics in various types of cell. Since chemical instability is a problem with these agents, we studied the insulin-like action in human fat cells of a stable pV complex, bpV(pic). It enhanced 14C-U-glucose uptake in a dose-dependent manner by approximately twofold which was slightly less than the effect of insulin (approximately threefold). The pV complex did not alter cell-surface insulin binding and submaximal concentrations did not influence cellular sensitivity to insulin action on glucose uptake. The bpV(pic) inhibited the lipolytic effect of isoprenaline to the same extent as insulin; however, when the cGMP-inhibitable low-K(m) phosphodiesterase (cGI-PDE) was blocked with the specific inhibitor OPC 3911, the antilipolytic effect of insulin, but not that of bpV(pic), was completely prevented. Moreover, when lipolysis was stimulated by the non-hydrolysable cAMP analogue N6-monobutyryl cAMP, bpV(pic), in contrast to insulin, maintained an antilipolytic effect. These findings indicate that bpV(pic) exerts its antilipolytic effect not only through cGI-PDE activation, similar to the effect of insulin, but also by means of other mechanisms. The tyrosine kinase activity of insulin receptors from human placenta was not altered by the pV compound itself, whereas bpV(pic) clearly enhanced insulin-stimulated activity. In contrast, in situ tyrosine phosphorylation of the insulin receptor beta-subunit as well as that of several other proteins was clearly increased in cells which were treated with bpV(pic), whereas vanadate only amplified insulin-stimulated tyrosine phosphorylation. In conclusion, bpV(pic) exerts powerful insulin-like effects in human fat cells and may be a new and potentially useful agent in the management of insulin-resistant states.