The long-term objective of this proposal continues to be the delineation of the mechanism by which acetyl-CoA carboxylase (ACC), a major rate-limiting enzyme for fatty acid biosynthesis, is regulated by hormones, especially insulin, with a particular focus on the regulation of its phosphorylation state in intact cells and in vitro by several protein kinases and phosphatases. The regulation of this enzyme by insulin has proven to be an excellent """"""""reporter"""""""" system, which may yield more general insight into the mechanism of insulin action. In extension of these studies, three major specific aims are proposed. First, we will explore the mechanism by which insulin and other ACC stimulators promote the dephosphorylation of ACC coincident with its activation in rat hepatoma cells. In these studies, we plan to determine site- specific alterations in ACC phosphorylation after stimulation, to purify a major ACC regulatory kinase and to characterize the regulation of ACC kinases/phosphatases in hepatoma cells. Second, we plan to detail the mechanism underlying differences in ACC specific activity in varying clones of Reuber hepatoma cells and to select and subclone other lines with alterations in ACC activity or content. Differences in ACC phosphorylation and kinase/phosphatase activity will be compared; cells will be selected for subcloning based on Nile Red 0 fluorescence and cell sorting and after cell killing with an ACC inhibitor, TOFA. Third, we plan to purify and characterize an insulinomimetic oligosaccharide autocrine factor derived from hepatoma cells in order to determine its structure, its potential loci of action in intact cells and in vitro and whether the factor is generated in response to insulin and other stimulators, the latter to address the important question of its role in insulin-stimulated signal transduction in hepatoma and other cells. The results of these investigations will further our understanding of the regulation of fatty acid synthesis and may provide important insight into the mechanism of insulin action. This would supply information needed to understand abnormalities in cellular function in diabetes mellitus with respect to fatty acid and lipoprotein metabolism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK035712-05
Application #
3233961
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1985-01-01
Project End
1993-12-31
Budget Start
1989-01-01
Budget End
1989-12-31
Support Year
5
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Dartmouth College
Department
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Iseli, Tristan J; Oakhill, Jonathan S; Bailey, Michael F et al. (2008) AMP-activated protein kinase subunit interactions: beta1:gamma1 association requires beta1 Thr-263 and Tyr-267. J Biol Chem 283:4799-807
Anderson, Kristin A; Ribar, Thomas J; Lin, Fumin et al. (2008) Hypothalamic CaMKK2 contributes to the regulation of energy balance. Cell Metab 7:377-88
Folmes, Karalyn D; Witters, Lee A; Allard, Michael F et al. (2007) The AMPK gamma1 R70Q mutant regulates multiple metabolic and growth pathways in neonatal cardiac myocytes. Am J Physiol Heart Circ Physiol 293:H3456-64
Barre, Laura; Richardson, Christine; Hirshman, Michael F et al. (2007) Genetic model for the chronic activation of skeletal muscle AMP-activated protein kinase leads to glycogen accumulation. Am J Physiol Endocrinol Metab 292:E802-11
Ho, Richard C; Fujii, Nobuharu; Witters, Lee A et al. (2007) Dissociation of AMP-activated protein kinase and p38 mitogen-activated protein kinase signaling in skeletal muscle. Biochem Biophys Res Commun 362:354-9
Rockl, Katja S C; Hirshman, Michael F; Brandauer, Josef et al. (2007) Skeletal muscle adaptation to exercise training: AMP-activated protein kinase mediates muscle fiber type shift. Diabetes 56:2062-9
Hallows, Kenneth R; Fitch, Adam C; Richardson, Christine A et al. (2006) Up-regulation of AMP-activated kinase by dysfunctional cystic fibrosis transmembrane conductance regulator in cystic fibrosis airway epithelial cells mitigates excessive inflammation. J Biol Chem 281:4231-41
Hurley, Rebecca L; Barre, Laura K; Wood, Sumintra D et al. (2006) Regulation of AMP-activated protein kinase by multisite phosphorylation in response to agents that elevate cellular cAMP. J Biol Chem 281:36662-72
Iseli, Tristan J; Walter, Mark; van Denderen, Bryce J W et al. (2005) AMP-activated protein kinase beta subunit tethers alpha and gamma subunits via its C-terminal sequence (186-270). J Biol Chem 280:13395-400
Konrad, D; Rudich, A; Bilan, P J et al. (2005) Troglitazone causes acute mitochondrial membrane depolarisation and an AMPK-mediated increase in glucose phosphorylation in muscle cells. Diabetologia 48:954-66

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