Diabetes mellitus may be caused by an inadequate level of circulating insulin or by a decrease in the ability of cells to respond to the hormone. Long term objectives of this project are to define mechanisms involved in insulin action and insulin resistance. Insulin binds to a receptor in the plasma membrane; however, subsequent events in the signal transduction pathways have not been fully defined. There is evidence that increased protein phosphorylation is involved. Insulin activates several protein Ser/Thr kinases; however, it has not been possible to determine the roles of these enzymes, in part because of difficulties in modulating the activities of the kinases independently of insulin. A unique approach is proposed to address this problem. Microinjection will be used to introduce protein kinases, phosphatases, and inhibitors of these enzymes into cells. Microanalytical methods will then be used to measure 2-deoxyglucose uptake and glycogen synthase activity (-/+ glucose-6-P) in individual cells that have been injected. By determining which injections mimic or block insulin action, we hope to be able to define steps in the pathways leading to activation of glycogen synthase and glucose transport. Epinephrine and agents that increase cAMP inhibit insulin-stimulated glucose transport. Identifying the mechanism responsible for this inhibition is another objective of this proposal. The role of cAMP-dependent protein kinase (cAdPK) will be investigated by determining whether injecting cells with the catalytic subunit of cAdPK inhibits transport, and whether injecting inhibitors of the kinase blocks the inhibitory effects of agents which increase cAMP. We also plan to determine whether down-regulation of cAdPK prevents the inhibitory effects associated with increased cAMP. Depletion of cAdPK will be accomplished by expressing a mutant regulatory subunit that is unable to bind cAMP. Recent findings have shown that Glut-4, the major insulin-regulatable glucose transporter in muscle and fat cells, is phosphorylated in adipocytes in response to agents that increase cAMP. The general hypothesis that phosphorylation of Glut-4 affects the ability of the protein to transport glucose and/or its subcellular distribution will be tested. Site-directed mutagenesis will be performed to change the Ser residues that are phosphorylated to Ala residues. Mutant transporters will be expressed in cells, and experiments will be performed to determine whether deleting the phosphorylation sites affects either the inhibition of transport by agents which increase cAMP or the distribution of transporters between plasma membrane and intracellular compartments.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Physiological Chemistry Study Section (PC)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Washington University
Anatomy/Cell Biology
Schools of Medicine
Saint Louis
United States
Zip Code
Zhang, Chongben; Wendel, Angela A; Keogh, Matthew R et al. (2012) Glycerolipid signals alter mTOR complex 2 (mTORC2) to diminish insulin signaling. Proc Natl Acad Sci U S A 109:1667-72
Kumar, Anil; Lawrence Jr, John C; Jung, Dae Young et al. (2010) Fat cell-specific ablation of rictor in mice impairs insulin-regulated fat cell and whole-body glucose and lipid metabolism. Diabetes 59:1397-406
Péterfy, Miklós; Harris, Thurl E; Fujita, Naoya et al. (2010) Insulin-stimulated interaction with 14-3-3 promotes cytoplasmic localization of lipin-1 in adipocytes. J Biol Chem 285:3857-64
Liu, Guang-Hui; Qu, Jing; Carmack, Anne E et al. (2010) Lipin proteins form homo- and hetero-oligomers. Biochem J 432:65-76
Blancquaert, Sara; Wang, Lifu; Paternot, Sabine et al. (2010) cAMP-dependent activation of mammalian target of rapamycin (mTOR) in thyroid cells. Implication in mitogenesis and activation of CDK4. Mol Endocrinol 24:1453-68
Kim, Hyun Bae; Kumar, Anil; Wang, Lifu et al. (2010) Lipin 1 represses NFATc4 transcriptional activity in adipocytes to inhibit secretion of inflammatory factors. Mol Cell Biol 30:3126-39
Gropler, Matthew C; Harris, Thurl E; Hall, Angela M et al. (2009) Lipin 2 is a liver-enriched phosphatidate phosphohydrolase enzyme that is dynamically regulated by fasting and obesity in mice. J Biol Chem 284:6763-72
Wang, Lifu; Lawrence Jr, John C; Sturgill, Thomas W et al. (2009) Mammalian target of rapamycin complex 1 (mTORC1) activity is associated with phosphorylation of raptor by mTOR. J Biol Chem 284:14693-7
Chen, Zhouji; Gropler, Matthew C; Norris, Jin et al. (2008) Alterations in hepatic metabolism in fld mice reveal a role for lipin 1 in regulating VLDL-triacylglyceride secretion. Arterioscler Thromb Vasc Biol 28:1738-44
Wang, Lifu; Harris, Thurl E; Lawrence Jr, John C (2008) Regulation of proline-rich Akt substrate of 40 kDa (PRAS40) function by mammalian target of rapamycin complex 1 (mTORC1)-mediated phosphorylation. J Biol Chem 283:15619-27

Showing the most recent 10 out of 72 publications