Abstract
The effect of okadaic acid, a serine/threonine phosphatase inhibitor, was analyzed in two insulin-responsive systems, the isolated mouse soleus muscle and 3T3-L1 adipocytes. While okadaic acid alone was a potent stimulator of glucose transport in both systems, it prevented transport stimulation by insulin. To gain insight into this inhibitory action, the activation of phosphatidylinositol 3-kinase (PI3-kinase), one of the earliest postreceptor steps identified so far, was studied. In 3T3-L1 adipocytes and muscle, insulin increased PI3-kinase activity in immunoprecipitates obtained with antibodies to phosphotyrosine. Okadaic acid alone had no effect but strongly inhibited this hormonal action. Okadaic acid treatment did not interfere with insulin-induced receptor autophosphorylation or with its tyrosine kinase activity toward artificial substrates. In contrast, in the presence of the phosphatase inhibitor, we did not observe tyrosine phosphorylation of the insulin receptor cellular substrate p185 (IRS-1) or immunoprecipitation of PI3-kinase by antibodies to phosphotyrosine. These results suggest that okadaic acid interferes with insulin's stimulation of glucose transport by inhibiting IRS-1 phosphorylation and its association with PI3-kinase and/or other signaling molecules. However, okadaic acid did not block the insulin stimulation of aminoisobutyric acid uptake in muscle. This would indicate that IRS-1 phosphorylation and PI3-kinase activation are not required for all the effects of insulin and that the serine/threonine phosphorylation events implicated in the translocation of glucose transporters are not controlling amino acid transport in muscle.
Highlights
The effect of okadaic acid, a serinelthreonine phos- insulin receptor substrate 1 (IRS-1)’ has been cloned, sephatase inhibitor, was analyzed in two insulin-respoqnu-enced, and characterized in detail [2]
To better understand the potential role of IRS-1 and PI3kinase in insulin action, we have studied the effects of okadaic acid either alone or in combination with insulin on rapid tion and PI3-kinase activationare not required forall metabolic effects in two different insulin-sensitive systems, theeffects of insulin and that the serinelthreonine the isolated soleus muscle and the 3T3-Laldipocytes
Effectof Okadaic Acidon Glucose Transport and AminAocid Uptake inSoleus Muscle and in 3T3-Ll Adipocytes-We first looked at the effect of okadaic acid on deoxyglucose uptake, in the
Summary
Effectof Okadaic Acidon Glucose Transport and AminAocid Uptake inSoleus Muscle and in 3T3-Ll Adipocytes-We first looked at the effect of okadaic acid on deoxyglucose uptake (an index of glucose transport and phosphorylation), in the PI3-kinAacstieoannd Insulin absence and presence of insulin, in the isolated soleus muscle 70%the 20-fold stimulation of a-Tyr(P)-PIS-kinase activity (Fig. 1) and in 3T3-Ll adipocytes (Table I). Insulin was able in 3T3-Ll with observations made in othersystems [13,14,15,28] we found adipocytes to stimulate the total P13-kinase activity in antithat okadaic acid stimulated deoxyglucose uptake in muscle PIS-kinase immunoprecipitates by2-%fold (Table 111); this in a dose-dependent fashion (Fig. 1, left panel). P13-kinase was present in okadaic acid per se at 0.5 W M (panelA ) or 2 p~ (panel B ) had immunoprecipitates from cells that have been stimulated with no effect, while insulin induced a severalfold stimulation of insulin, as evidenced by the presence of a band at 85 kDa the c~-Tyr(P)-PI3-kinaseactivity. Neither insulin nor okadaic tation with anti-P13-kinase immunoglobulins (Fig., panel acid modified the total PI3-kinaseactivity (measured in anti- B ). Results are expressed as a percentage of basal uptake and are presented as means f S.E. of 3-5 values
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