Acetyl CoA/Carboxylase (ACC) is a rate limiting enzyme for fatty acid synthesis and is regulated through phosphorylation by 5 -AMP-activated protein kinase (AMPK) which inactivates ACC. Work over the past cycle by the PI and his collaborators has resulted in the cloning of a second isoform of ACC (ACC Beta) and AMPK. AMPK is comprised of one alpha catalytic subunit and Beta and gamma non-catalytic subunits, all of which are required for optimal enzyme activity. The subunits each may be members of a differentially regulated family of proteins, since alpha, and alpha 2 plus gamma1, gamma2, and gamma3 subunits have been described, and there are homologies to the glucose sensing SNF1 kinase complex in yeast. The PI believes that the AMPK/ACC system functions as a glucose sensor in mammalian tissues to regulate FA synthesis and oxidation and relative metabolism of carbohydrates versus fat: low glucose activates AMPK due to an increase in 5' AMP addition to phosphorylation by a putative AMPK kinase; AMPK phosphorylates and inactivates ACC decreasing formation of malonyl CoA; less malonyl CoA relaxes inhibition of CPT1 allowing FA oxidation in mitochondria. Work over the past cycle has been enhanced by a close working relationship with Dr. Bruce Kemp in Melbourne; the Kemp laboratory is specialized in protein chemistry, microsequencing, and protein translation systems which complement the cellular and molecular biology expertise of the PI. In the current application, the PI will study AMPK structure, function, and regulation in more detail, and identify the upstream AMPK kinase.
In Specific Aim 1, the PI will first try to create a constitutively active alpha subunit (independent of beta-gamma) by either truncating the C-terminal half of alpha polypeptide which may function to inhibit the N-terminal catalytic half based on a model proposed for its homolog SNF1 kinase in yeast, or by mutating a Thr 172 phosphorylation site to a charged amino acid (Asp or Glu) to mimic an activating phosphorylation event, or both. These constructs will be transfected into PS120 cells and ACC activity and Ka for citrate will be assessed. Structure/function studies relevant to the beta subunit will also be performed. An N-terminal myristoylation site of the beta subunit will be mutated to determine whether myristoylation affects localization of AMPK in membranes versus cytosol, activation of AMPK activity, and regulation of ACC.
In Specific Aim 2, AMPK kinase and other AMPK-interacting proteins will be identified and cloned. AMPK heterotrimer with a GST-tagged alpha subunit are expressed in COS cells, harvested by glutathione-agarose absorption, and resolved by SDS-PAGE for staining of AMPK and associated proteins. Protein bands will be microsequenced and this information used to clone full length cDNAs. Similar experiments will be conducted using N-terminal alpha subunit as bait, using both the GST/glutathione system and the yeast 2-hybrid system.
Specific Aim 3 entails a search for non-ACC targets of AMPK action in cells. Constitutively active AMPK-alpha subunits (generated in specific aim 1) and dominant negative constructs (point mutation at phosphorylation site and N-terminal deletion previously published) will be transfected into: (i) clone 9 cells (liver epitheliod cells) for measurements of GLUT1 expression, glucose oxidation, Fatty Acid Synthase, and ATP-citrate lyase; (ii) into INS-1 cells for assessment of insulin secretion; and (iii) into CHO-IR and 3T3-IR cells for measurement of insulin receptor and IRS phosphorylation, GS activation, MAPK pathway activation, and 3H-thymidine incorporation.
In Specific Aim 4, the PI will examine AMPK regulation in fed/fasted rats and in rats with streptozotocin-induced diabetes. AMPK activity, AMPK subcellular distribution, ACC activity and expression, and ACC and AMPK phosphorylation will be examined.
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