Wu, Hoi Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 92-104). / Abstracts in English and Chinese. / Thesis/ Assessment Committee --- p.i / Declaration --- p.ii / Acknowledgments --- p.vi / Table of Content --- p.vii / List of Abbreviations --- p.x / List of Figures --- p.xiv / List of Tables --- p.xv / Chapter 1. --- Literature Review --- p.1 / Chapter 1.1 --- Introduction of diabetes mellitus --- p.1 / Chapter 1.1.1 --- Definition and prevalence --- p.1 / Chapter 1.1.2 --- Diagnosis and classification --- p.2 / Chapter 1.1.3 --- Symptoms and complications --- p.4 / Chapter 1.1.4 --- Cause and risk factors --- p.5 / Chapter 1.1.5 --- Prevention and treatment --- p.9 / Chapter 1.2 --- The role of adipose tissue in pathophysiology of T2DM --- p.10 / Chapter 1.2.1 --- Randle's glucose-fatty acid hypothesis --- p.11 / Chapter 1.2.2 --- Ectopic fat storage hypothesis --- p.12 / Chapter 1.2.3 --- Adipose tissue as an endocrine organ --- p.13 / Chapter 1.2.4 --- Low-grade inflammation --- p.15 / Chapter 1.2.5 --- Endoplasmic reticulum (ER) stress --- p.17 / Chapter 1.3 --- Use of berberine in the treatment of T2DM --- p.18 / Chapter 1.3.1 --- Efficacy of berberine in treating diabetes --- p.18 / Chapter 1.3.2 --- Berberine on glucose and lipid metabolism of animals --- p.19 / Chapter 1.3.3 --- Inhibition of adipogenesis --- p.20 / Chapter 1.3.4 --- Activation of AMP-Activated Protein Kinase (AMPK) --- p.20 / Chapter 1.3.5 --- Mitochondrial inhibition --- p.21 / Chapter 1.4 --- Introduction of proteomics --- p.21 / Chapter 1.4.1 --- Why proteomics? --- p.22 / Chapter 1.4.2 --- Gel-based proteomics: Two-Dimensional Gel Electrophoresis --- p.23 / Chapter 1.4.3 --- Gel-free proteomics --- p.25 / Chapter 1.4.4 --- Mass spectrometry --- p.26 / Chapter 1.4.5 --- Proteomics as tool for diabetes research --- p.27 / Chapter 1.5 --- Objectives and significance --- p.32 / Chapter 2. --- Materials and Methods --- p.34 / Chapter 2.1 --- Drug preparation --- p.34 / Chapter 2.2 --- Animal experiment --- p.34 / Chapter 2.3 --- Comparison of proteome of visceral white adipose tissue: obese db/db micevs lean m+/db mice and BBR-treated vs control db/db mice --- p.36 / Chapter 2.3.1 --- Protein sample preparation from adipose tissue --- p.36 / Chapter 2.3.2 --- Protein quantitation --- p.37 / Chapter 2.3.3 --- 2D Gel electrophoresis --- p.37 / Chapter 2.3.4 --- Image analysis --- p.39 / Chapter 2.3.5 --- In-gel digestion and MALDI-ToF MS --- p.39 / Chapter 2.4 --- Cell culture experiment --- p.40 / Chapter 2.5 --- Oil Red O staining --- p.42 / Chapter 2.6 --- Glycerol determination --- p.42 / Chapter 2.7 --- Comparison of proteomes of BBR-treated and control 3T3-L1 adipocytes..… --- p.43 / Chapter 2.7.1 --- Protein sample preparation from 3T3-L1 cells --- p.43 / Chapter 2.7.2 --- Protein quantitation --- p.43 / Chapter 2.7.3 --- 2D Gel electrophoresis --- p.44 / Chapter 2.7.4 --- Image analysis --- p.44 / Chapter 2.7.5 --- In-gel digestion and MALDI-ToF MS --- p.44 / Chapter 2.8 --- Western Immunoblotting --- p.44 / Chapter 2.8.1 --- Protein sample preparation of BBR-treated and control 3T3-L1 --- p.44 / Chapter 2.8.2 --- SDS-PAGE --- p.44 / Chapter 2.8.3 --- Protein blotting --- p.45 / Chapter 2.8.4 --- Membrane blocking and antibody incubations --- p.45 / Chapter 2.8.5 --- Detection of Proteins --- p.46 / Chapter 2.9 --- Statistical analysis --- p.46 / Chapter 3. --- Results --- p.47 / Chapter 3.1 --- Comparison of total protein profiles of visceral adipose tissue of obese db/db and lean m+/db mice --- p.47 / Chapter 3.2 --- Effect of berberine on glucose metabolism of obese db/db mice --- p.53 / Chapter 3.3 --- Comparison of the protein profiles of visceral adipose tissue of BBR-treated and control db/db mice --- p.55 / Chapter 3.4 --- Effect of berberine treatment on 3T3-L1 adipocytes --- p.61 / Chapter 3.4.1 --- Berberine treatment inhibited intracellular triglyceride accumulation in both mature and pre-mature 3T3-L1 adipocytes --- p.61 / Chapter 3.4.2 --- Berberine treatment enhanced lipolysis in mature 3T3-L1 adipocytes but inhibited lipolysis in pre-mature 3T3-L1 adipocytes --- p.65 / Chapter 3.4.3 --- Color change in culture media after berberine treatment --- p.65 / Chapter 3.4.4. --- Comparison of protein profiles between berberine-treated and control 3T3-L1 adipocytes --- p.67 / Chapter 3.4.5 --- Western blotting --- p.73 / Chapter 4. --- Discussion --- p.75 / Chapter 4.1 --- Comparison of total protein profiles of visceral adipose tissue of obese db/db and lean m+/db mice --- p.75 / Chapter 4.2 --- "Berberine lowers body weight, reduces fasting blood glucose level and improves glucose-lowering ability of db/db mice" --- p.78 / Chapter 4.3 --- Comparison of the protein profiles of visceral adipose tissue of BBR-treated and control db/db mice --- p.79 / Chapter 4.4 --- Berberine inhibited lipid accumulation in mature and pre-mature 3T3-L1 adipocytes --- p.84 / Chapter 4.5 --- Berberine enhanced lipolysis in mature 3T3-L1 adipocytes but inhibited lipolysis in pre-mature 3T3-L1 adipocytes --- p.84 / Chapter 4.6 --- Comparison of the protein profiles of BBR-treated and control 3T3-L1 adipocytes --- p.85 / Chapter 4.7 --- Western blotting --- p.88 / Chapter 4.8 --- General discussion --- p.89 / Chapter 5. --- References --- p.92

Indiana University-Purdue University Indianapolis (IUPUI) / Excess caloric intake and/or obesity currently remain the largest predisposing risk factors for the development of type 2 diabetes. Discerning the cellular and molecular mechanisms responsible and amendable to therapy represents a growing challenge in medicine. At a cellular level, increased activity of the hexosamine biosynthesis pathway (HBP), a sensor of excess energy status, has been suggested to promote the exacerbation of insulin resistance through increasing adipose tissue and skeletal muscle membrane cholesterol content. This in turn compromises cortical filamentous actin structure necessary for proper incorporation of the insulin-sensitive glucose transporter GLUT4 into the plasma membrane. The current studies attempted to elucidate the mechanism by which hexosamines provoke membrane cholesterol toxicity and insulin resistance. In 3T3-L1 adipocytes cultured with pathophysiologic hyperinsulinemia to induce insulin resistance, increased HBP flux was observed. This occurred concomitant with gains in the mRNA and protein levels of HMG-CoA reductase (HMGR), the rate limiting enzyme in cholesterol synthesis. Mechanistically, immunoprecipitation demonstrated increased HBP-induced N-acetylglucosamine (O-GlcNAc) modification of specificity protein 1 (Sp1), a regulator of HMGR synthesis. This was associated with increased affinity toward and activity of Hmgcr, the gene encoding HMGR. Global HBP inhibition or Sp1 binding to DNA prevented membrane cholesterol accrual, filamentous actin loss, and glucose transport dysfunction. Furthermore, hyperinsulinemia and HBP activation impaired cholesterol efflux in adipocytes, exacerbating cholesterol toxicity and potentially contributing to cardiovascular disease. In this regard, chromium picolinate (CrPic), known to have beneficial effects on glucose and lipoprotein metabolism, improved cholesterol efflux and restored membrane cholesterol content. To test the role of membrane cholesterol accumulation in vivo, studies were conducted on C57Bl/6J mice fed a low or high fat diet. High fat feeding promoted increased HBP activity, membrane cholesterol accumulation, and insulin resistance. Supplementation of mice with CrPic in their drinking water (8µg/kg/day) countered these derangements and improved insulin sensitivity. Together, these data provide mechanistic insight for the role of membrane cholesterol stress in the development of insulin resistance, as well as cardiovascular disease, and highlight a novel therapeutic action of chromium entailing inhibition of the HBP pathway.