1. Anti-Lipemic Mechanism of (R)-α-Lipoic Acid (LA) Regis Moreau, Ph.D. Assistant Professor Department of Nutrition and Health Sciences University of Nebraska-Lincoln LA Zucker Diabetic Fatty (ZDF) rats, model of hypertriglyceridemia

2. Fit with the COBRE Theme Controlling blood lipid abnormalities is a major public health challenge Hypertriglyceridemia affects 65 million Americans Major risk factor for cardiovascular disease and type-2 diabetes Dietary molecule: (R)-  -lipoic acid (LA) –Naturally occurring molecule synthesized by plants and animals –Used therapeutically in diabetics –Safe in humans –Lipid-lowering properties have recently been recognized LA Zucker Diabetic Fatty (ZDF) rats, model of hypertriglyceridemia

3. Goals of the Project Identify the molecular targets and signaling mechanism of LA Generate interest in the therapeutic use of LA Central Hypothesis LA corrects hypertriglyceridemia by the combined stimulation of triglyceride (TG) clearance mediated by FGF21 and inhibition of hepatic TG synthesis mediated by mTORC1

4. Novel Molecular Targets of LA Fibroblast growth factor-21 (FGF21) mediates the lipolytic properties of LA (Tf = Transcription factor) LA downregulates SREBP1c-mediated transcription of lipogenic genes through inactivation mTORC1 (mammalian target of rapamycin complex 1) β-oxidation genes Tf FGFR1 β-Klotho Fgf21 Tf X5X5X5X5 FGF21 LA X6X6X6X6 SREBP1c LA ChREBP Lipogenic genes ACC, FAS, GPAT1, DGAT2 mTORC1 Rheb TSC1/2

5. Preliminary Studies A) Feeding LA to obese rats corrects hypertriglyceridemia (–70%). B) Feeding LA to obese rats increases blood FGF21 levels (+600%). AB C) LA (50 µM) added to liver cells inactivates mTORC1 via dephosphorylation of mTOR and loss of Raptor. Ctl = vehicle control. D) LA lowers hepatic levels of transcription factors SREBP1c and ChREBP downstream of mTORC1. C D Butler et al. 2009 ABB; Finlay et al. 2011 AJP

6. Experimental Models Fgfr1 f/f /Cre mice lacking fibroblast growth factor-21 (FGF21) receptor Tsc1 f/f /Cre mice constitutively expressing mammalian target of rapamycin complex 1 (mTORC1) Gt(ROSA)26Sor Cre ESR Fgfr 1 loxP Cre / ESR + Tamoxife n Fgfr1 KO Gt(ROSA)26Sor Cre ESR Tsc1 loxP Cre / ESR + Tamoxife n Tsc1 KO

7. Innovation Shift from the current paradigm that LA acts as an antioxidant Novel lipid-lowering mechanisms of LA –Stimulation of lipid clearance (FGF21) –Downregulation of triglyceride synthesis (mTORC1) Outcome Safe and economical alternative to current lipid-lowering therapies Mechanism-based rationale for clinical trials with LA

8. Timetable of the Proposed Studies The project will span 3 years Two Specific Aims –Specific Aim 1. Define the role of FGF21 in the mechanism of LA 1A. Elucidate the mechanism by which LA up-regulates FGF21 gene expression 1B. Assess the FGF21 sensitizing properties of LA and consequences on lipolysis –Specific Aim 2. Define the role of mTORC1 in the mechanism of LA 2A. Elucidate the mechanism by which LA represses mTORC1 activity 2B. Ascertain SREBP1c requirement in mTORC1 repression by LA 2C. Evaluate the consequences of mTORC1 repression by LA on lipogenesis Year 1: Initiate Specific Aims 1A, 1B and 2A, complete Specific Aim 1A Year 2: Complete Specific Aims 1B and 2A, initiate Specific Aims 2C October of Year 2: Submission date for an R01 (NHLBI, NIDDK, NCCAM) Year 3: Complete Specific Aims 2B and 2C

9. Mentorship Work Plan Primary mentor –Dr. Janos Zempleni (NHS, UNL) Published work on LA Collection of chemically defined LA metabolites (structure-function studies) Secondary mentors –Dr. Edgar Cahoon (Biochemistry, UNL) Expert in secondary plant metabolites Strategies to increase LA content in plants –Dr. Mark Wilson (Biochemistry, UNL) Expert in X-ray crystallography Structure-signaling relationships of LA and signaling protein

10. Use of UNL Core Research Facilities NPOD Epigenetics Core –Chromatin immunoprecipitation (ChIP) assay –Antibody validation NPOD Computational and Data Sharing Core –Statistical data analysis Genomics Core –DNA sequencing –Primer validation –RNA integrity Animal Research Facility –Breeding colonies –Feeding trials

11. NPOD Scientific Collaborations Dr. Dmitri Fomenko (Project Leader 2) –Thiol disulfide exchange between LA and protein sulfhydryls (COBRE-supported Redox Biology Center) Dr. Saraswathi Viswanathan (Project Leader 5) –LA-omega 3 fatty acids synergies Existing Collaborations Dr. Qingsheng Li (COBRE-supported Nebraska Center for Virology) –Recombinant lentivirus gene knockdown Dr. David Wasserman (Vanderbilt-NIDDK Mouse Metabolic Phenotyping Center) –Metabolic studies in Fgfr1 f/f /Cre mice