Semester

Summer

Date of Graduation

2006

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Biochemistry

Committee Chair

Lisa M Salati

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of pentose phosphate pathway and provides NADPH for de novo fatty acid biosynthesis. For the later reason, G6PD is known as a lipogenic enzyme. Insulin induces expression of G6PD mRNA via activating the PI3 kinase pathway in primary rat hepatocytes. Addition of arachidonic acid, a polyunsaturated fatty acid, in the medium interferes with the PI3 kinase pathway, and thus inhibits the insulin-mediated induction of G6PD. Arachidonic acid in the presence of insulin induces p38 MAP kinase pathway over insulin treatment alone. Activation of p38 MAP kinase by arachidonic acid interferes with the insulin signal transduction pathway by phosphorylating IRS-1 at Ser-307 and preventing PI3 kinase activation and Akt-phosphorylation. Inhibition of the p38 MAP kinase pathway by using a specific inhibitor, SB203580, attenuates the inhibitory effect of arachidonic acid on G6PD and at the same time reverses its effect on IRS-1 and Akt-phosphorylation.;Arachidonic acid also activates AMP-activated protein kinase (AMPK) in primary rat hepatocytes by phosphorylating it at Thr-172. Activators of AMPK, AICAR and metformin inhibit the induction of G6PD mRNA by insulin. AICAR induces phosphorylation of p38 MAP kinase in primary rat hepatocytes. AICAR-mediated inhibition of G6PD mRNA expression is dependent on the activation of the p38 MAP kinase pathway, as in the presence of the p38 MAP kinase inhibitor the inhibitory effect of AICAR on G6PD mRNA expression is abolished. AICAR also interferes with PI3 kinase pathway by inducing Ser-307 phosphorylation of IRS-1 and inhibiting Akt phosphorylation. Thus, this AMPK activator mimics the effect of arachidonic acid both on insulin signaling pathway and on G6PD mRNA expression.;Activation of the mammalian target of rapamycin (mTOR) pathway by insulin is required for the insulin-mediated induction of G6PD mRNA expression. Arachidonic acid inhibits the phosphorylation of the downstream effector protein of mTOR, S6K-1 at Thr-389 which suggests it interferes with activation of mTOR. This provides an indirect evidence of the activation of AMPK by arachidonic acid in primary rat hepatocytes, as activation of AMPK results in the inhibition of mTOR/S6K-1 pathway. However, inhibition of mTOR, rather than activation, rules out the possibility of involvement of this pathway in the interference of the PI3 kinase activation upon arachidonic acid treatment. We also have ruled out the possibility of involvement of protein kinase C (PKC), as neither activators of PKC mimic the action of arachidonic acid on G6PD nor inhibitors of PKC can abolish the effect of arachidonic acid on G6PD mRNA expression.

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