Dysregulation of hepatic de novo lipogenesis is prevalent in obesity and closely associates with insulin resistance, Type 2 Diabetes and cardiovascular disease. The regulation of de novo lipogenesis in the liver is a complex process that is dependent upon the nutrient/hormone state, availability of substrate precursors, allosteric regulation of key enzymati activity steps, and the transcriptional control of lipogenic gene expression through the transcripton factors ChREBP and in particular for this application the sterol response element-binding protein-1 (SREBP-1c) transcription factor. SREBP-1c is a pivotal activator of rate-limiting enzymes that are responsible for hepatic biosynthesis of fatty acids and triglycerides and it is the primary effecto of insulin-induced de novo lipogenesis in hepatocytes. Insulin and feeding acutely regulates SREBP-1c transcriptional activity at three distinct levels by 1) increasing SREBP-1c transcription;2) protelytic maturation from its precursor that is initially located in endoplasmic reticulum membrane, and 3) increased nuclear SREBP-1c protein stability. Recently, we have uncovered a novel insulin/nutrient regulated signaling pathway that is primarily responsible for the stability of the nuclear SREBP-1c protein. We have found that in the basal (fasted state) cyclin- dependent kinase-8 (CDK8) in comple with its activator cyclin C (CycC) phosphorylates nuclear SREBP-1c that induces nuclear SREBP-1c ubiquitination and rapid proteasome mediated degradation. In contrast, refeeding results in the dow-regulation of CDK8/CycC protein complex thereby stabilizing nuclear SREBP-1c protein to enhance lipogenic gene expression and de novo lipogenesis in conjunction with other actions of insulin. Thu, the nutrient/hormone down regulation of the CDK8/CycC complex functionally results in the stabilizaion of the nuclear SREBP-1c protein as part of the complex mechanisms driving hepatic lipogenic gene expression. More recently, we have observed that that CDK8 protein stability is inversely related t the mTORC1 activation state. Based upon these recent findings, we propose the following working hypothesis: Activation of the mTORC1-signaling pathway rapidly down regulates the CDK8/CycC protein complex through either a direct or indirect phosphorylation, that in turn leads to dissociaion of the CDK8/CycC complex, subsequent CDK8 and CycC ubiquitination and proteasome-mediated degradation. In this proposal we propose to determine the initial nutrient signaling pathways and molecular mechanisms regulating the CDK8/CycC protein complex and nuclear SREBP-1c stability in the control of lipogenic gene expression and de novo lipogenesis. This will be accomplished by determining hormone/nutrient down regulation of CDK8/CycC complex occurs through 1) activation of mTORC1 and subsequent proteasome-mediated degradation of the CDK8 and CycC proteins;mTORC1-dependent regulation CDK8/CycC complex assembly state;and 3) ubiquitination of lysine residues in CDK8 and CycC. We will then determine whether CDK8, CycC or both are the targets of hormone/nutrient stimulation of mTORC1 signaling by 1) analyzing CDK8 and/or CycC phosphorylation/dephosphorylation;2) identifying the mTORC1-dependent kinase(s) responsible for CDK8/CycC down regulation;3) mapping the specific phosphorylation sites in CKD8 and/or CycC;and 4) analyzing the effect of phosphorylation defective mutations on CDK8/CycC stability, nuclear SREB- 1c stability and lipogenic gene expression. Lastly, we will determine if the mTORC1 regulatory mechanism contributes to the normal physiologic and pathophysiologic regulation of de novo lipogenesis in insulin resistant states by 1) examining the CDK8/CycC complex, nuclear SREBP-1c stability, lipogenic gene expression and de novo lipogenesis in liver-specific Raptor and LKB1 knocout mice;2) determining the function of CDK8/CycC degradation-resistant mutants in mouse liver;and 3) analyzing the regulation of the CDK8/CycC complex in high fat diet-induced insulin resistant and genetically diabetic db/db mice.

Public Health Relevance

It is well-known that dysregulation of lipid homeostasis is a risk factor for major human diseases, including type 2 diabetes, obesity and cardiovascular diseases, which constitute the leading cause of death in US. According to the CDC data, nearly 24 million people inUS had diabetes in 2007, and strikingly, up to 70% of those diabetic patients were also diagnosed with non-alcoholic fatty liver disease (NAFLD), which is tightly associated with hepatic insulin resistace. The most common feature of NAFLD is excessive fat accumulation in hepatocytes primarily due to increased triglyceride synthesis. Although lipolysis of peripheral fat and dietary fatty acids can upply free fatty acids for hepatic triglyceride synthesis, about 30% of hepatic fatty acids are from de nvo lipogenesis in NAFLD, while the normal contribution is 3-5%. Moreover, increased hepatic lipogenesi also leads to dyslipidemia and atherosclerosis, the primary risk factors for heart disease. Therefoe, understanding the regulation of hepatic de novo lipogenesis is highly important and clearly relevan to public health. Previous studies have demonstrated that the SREBP-1c transcription factor is a key lipogenic activator in hepatocytes, as it can stimulate the transcription of a series of rate-limitng enzymes in the pathway of fatty acid as well as triglyceride biosynthesis. Understanding the normaland pathophysiologic regulation of the SREBP-1c transcriptional activity will improve our understandingof how hepatic de novo lipogenesis is controlled. In this regard, we have recently demonstrated that te CDK8/CycC complex plays an important role in the regulation of nuclear SREBP-1c stability and therefore SREBP-1c dependent lipogenic gene transcription. The current proposal is directed at understanding the molecular mechanisms regulating CDK8/CycC function and its dysregulation in states of insulin resistance and NAFLD.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK098439-02
Application #
8664843
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Pawlyk, Aaron
Project Start
2013-06-01
Project End
2017-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
2
Fiscal Year
2014
Total Cost
$417,166
Indirect Cost
$167,366
Name
Albert Einstein College of Medicine
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461