okadaic-acid and Diabetes-Mellitus--Type-2

We previously identified and characterized a glutamate- and magnesium-sensitive PP2A-like phosphatase (GAPP), which dephosphorylated and activated acetyl-CoA carboxylase (ACC) in the islet beta cell. Herein, we studied potential regulatory mechanisms by which GAPP is activated by glutamate and magnesium, and also quantitated the degree of activation, by glutamate- and magnesium, of ACC in normal rat islets and islets derived from the diabetic Goto-Kakizaki (GK) rat, a model for type 2 diabetes in humans. Our findings indicate that magnesium, but not glutamate, specifically activates the post-translational carboxylmethylation (CML) of the 36 kDa catalytic subunit of GAPP. Okadaic acid (OKA), which inhibits GAPP-mediated activation of ACC, also reduced the magnesium-stimulated CML of the catalytic subunit of GAPP in all the beta cell preparations studied. These data suggest that the CML step may be necessary for magnesium- and glutamate-mediated activation of ACC. We also observed a marked attenuation in magnesium- and glutamate-facilitated activation of ACC activity in islets derived from the GK rat. Together, our findings raise an interesting possibility that inhibition of GAPP-catalyzed inactivation of ACC (and subsequent reduction in the generation of long-chain fatty acids) could contribute toward the abnormalities in insulin secretion demonstrable in this animal model for type 2 diabetes.

Topics: Acetyl-CoA Carboxylase; Animals; Diabetes Mellitus, Type 2; Disease Models, Animal; Enzyme Activation; Enzyme Inhibitors; Glutamic Acid; Islets of Langerhans; Magnesium; Male; Okadaic Acid; Phosphoprotein Phosphatases; Rats; Rats, Sprague-Dawley; Rats, Wistar

Insulin resistance is a primary characteristic of type 2 diabetes and likely causally related to the pathogenesis of the disease. It is a result of defects in signal transduction from the cell surface receptor of insulin to target effects. We found that insulin-stimulated phosphorylation of serine 307 (corresponding to serine 302 in the murine sequence) in the immediate downstream mediator protein of the insulin receptor, insulin receptor substrate-1 (IRS1), is required for efficient insulin signaling and that this phosphorylation is attenuated in adipocytes from patients with type 2 diabetes. Inhibition of serine 307 phosphorylation by rapamycin mimicked type 2 diabetes and reduced the sensitivity of IRS1 tyrosine phosphorylation in response to insulin, while stimulation of the phosphorylation by okadaic acid, in cells from patients with type 2 diabetes, rescued cells from insulin resistance. EC(50) for insulin-stimulated phosphorylation of serine 307 was about 0.2 nM with a t(1/2) of about 2 min. The amount of IRS1 was similar in cells from non-diabetic and diabetic subjects. These findings identify a molecular mechanism for insulin resistance in non-selected patients with type 2 diabetes.

Topics: Adipocytes; Aged; Diabetes Mellitus, Type 2; Dose-Response Relationship, Drug; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Female; Humans; Immunoblotting; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Kinetics; Male; Middle Aged; Okadaic Acid; Phosphoproteins; Phosphorylation; Serine; Signal Transduction; Sirolimus; Time Factors; Tyrosine

In this study, we examined the molecular mechanism of myosin-bound protein phosphatase (MBP) regulation by insulin and evaluated the role of MBP in insulin-mediated vasorelaxation. Insulin rapidly stimulated MBP in confluent primary vascular smooth muscle cell (VSMC) cultures. In contrast, VSMCs isolated from diabetic and hypertensive rats exhibited impaired MBP activation by insulin. Insulin-mediated MBP activation was accompanied by a rapid time-dependent reduction in the phosphorylation state of the myosin-bound regulatory subunit (MBS) of MBP. The decrease observed in MBS phosphorylation was due to insulin-induced inhibition of Rho kinase activity. Insulin also prevented a thrombin-mediated increase in Rho kinase activation and abolished the thrombin-induced increase in MBS phosphorylation and MBP inactivation. These data are consistent with the notion that insulin inactivates Rho kinase and decreases MBS phosphorylation to activate MBP in VSMCs. Furthermore, treatment with synthetic inhibitors of phosphatidylinositol-3 kinase (PI3-kinase), nitric oxide synthase (NOS), and cyclic guanosine monophosphate (cGMP) all blocked insulin's effect on MBP activation. We conclude that insulin stimulates MBP via its regulatory subunit, MBS partly by inactivating Rho kinase and stimulating NO/cGMP signaling via PI3-kinase as part of a complex signaling network that controls 20-kDa myosin light chain (MLC20) phosphorylation and VSMC contraction.

Topics: Androstadienes; Animals; Aorta; Cells, Cultured; Diabetes Mellitus, Type 2; Enzyme Activation; Enzyme Inhibitors; Hypertension; Insulin; Intracellular Signaling Peptides and Proteins; Kinetics; Male; Muscle Contraction; Myosin-Light-Chain Phosphatase; Okadaic Acid; Phosphatidylinositol 3-Kinases; Phosphoprotein Phosphatases; Phosphorylation; Protein Serine-Threonine Kinases; Rats; Rats, Inbred WKY; rho-Associated Kinases; Signal Transduction; Thrombin; Tunica Media; Wortmannin

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.. Adipocytes were incubated with or without insulin or okadaic acid or both and glucose transport, protein kinase B activity, phosphorylation and protein expression measured.. 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.. 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.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Adipocytes; Biological Transport; Diabetes Mellitus, Type 2; Glucose; Humans; Insulin; Middle Aged; Okadaic Acid; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt