Abstract
The effect of okadaic acid, an inhibitor of protein phosphatases-1 and -2A, was studied on glucose transport and metabolism in soleus muscles isolated from lean and insulin-resistant obese mice. In muscles from lean mice, the uptake of 2-deoxyglucose, an index of glucose transport and phosphorylation, was increased by okadaic acid in a concentration-dependent manner. At 5 microM, okadaic acid was as efficient as a maximally effective insulin concentration. Glucose metabolism (glycolysis and glycogen synthesis) was also measured. Whereas glycolysis was stimulated by okadaic acid, glycogen synthesis was unchanged. When okadaic acid and insulin were added together in the incubation medium, the rates of glucose transport, glycolysis, and glycogen synthesis were similar to those obtained with insulin alone, whether maximal or submaximal insulin concentrations were used. Furthermore, okadaic acid did not activate the kinase activity of the insulin receptor studied in an acellular system or in intact muscles. These results indicate that a step in the insulin-induced stimulation of muscle glucose transport involves a serine/threonine phosphorylation event that is regulated by protein phosphatases-1 and/or -2A. In muscles of insulin-resistant obese mice, the absolute values of deoxyglucose uptake stimulated by okadaic acid were lower than in muscles from lean mice. However, the okadaic acid effect, expressed as a fold stimulation, was normal. These observations suggest that in the insulin-resistant state linked to obesity, the serine/threonine phosphorylation event is likely occurring normally, but a defect at the level of the glucose transporter itself would prevent a normal response to insulin or okadaic acid.
Highlights
From the Institut National dela Santk etde la Recherche Medicale, Unit6 145,Faculte deMedecine, Avenue de Valombrose, 06034 Nice Ceder, France
When okadaic acid and insulin were added together in theincubation meformed to investigate whether okadaic acid was exerting insulin-like effects in normal skeletal muscle, a major site of glucose utilizationandinsulinaction.We looked at whether an alteration in this pathwcaoyuld contribute to the insulin resistanceof glucose uptake in skeletamluscle of obese dium, the rates of glucose transport, glycolysis, and mice
T o answerthesequestions, we studied okadaicacid glycogen synthesis were similar tthoose obtained with effects in soleus muscles of mice rendered obese and insulin insulin alone, whether maximal or submaximal insulin resistant by gold thioglucoseinjection and which display concentrations were used
Summary
Stimulation of muscleglucose transport involves a serinelthreonine phosphorylation event that is regulated by protein phosphatases-1 and/or -2A. Preincubated for 1 h (unless otherwise indicated) in the absence or the okadaic acid effect, expressed as a fold stimulation, was normal. These observations suggest that in the insulin-resistantstate linked to obesity, the serinelthreonine phosphorylation event is likeolyccurring normally, but a defect at the level of the glucose transporter itself would prevent a normalresponse to insulin or okadaic acid. It is Insulin Receptor Autophosphorylation in an Acellular Systemwell established thata very early step after insulin binding toPartially purified insulin receptorswere prepared from skeletal musreceptor is the activation of the receptor kinase and its auto- cle as described previously[8].Briefly, 5 g of muscle was homogenized phosphorylation [1, 2]. The copolymer polyglutamate tyrosine (0.25 mg/ml, final concentration) was added, and itsphosphorylation was measured as described previously [8]
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