published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

Free radicals are thought to be involved in the destructive process of beta cells in Type 1 diabetes mellitus. Studies were performed to test the hypotheses (1) that malondialdehyde (MDA), a by-product of lipid peroxidation, affects $ beta$-cell function and integrity in vitro and (2) that such effects might be prevented in the BB rat (a model of spontaneous autoimmune diabetes) in vivo by administration of $ alpha$-phenyl-N-tert-butylnitrone (PBN), a free radical spin trap. First, islets of Wistar-Furth rats were studied at 12, 24 and 40 hr of culture in either 5.5, 11 or 16.5 mM glucose, and MDA at a range of concentrations ($6 times10 sp{-12}$-10$ sp{-3}$M). High concentrations of MDA inhibited glucose-stimulated insulin release without corresponding decreases in islet insulin content, suggesting that in situations with high predicted islet free radical content (e.g., autoimmune insulitis) beta cell function may be affected even before the cells are destroyed. Second, 28 diabetes-prone (BBdp) and 13 non diabetes-prone (BBn) rats were given PBN (20 mg/kg) s.c. 2x/day and 27 BBdp and 12 BBn rats received an equal volume of saline. PBN was able to decrease MDA in the absence of the autoimmune process and is remarkably non-toxic. However, it did not prevent diabetes for reasons which may include its concentration at the site of the inflammatory process or specificity to types of radicals trapped. Because it did decrease MDA, either a higher dose or a combination of PBN with other agents may hold promise for disease prevention.

<p>  </p> <p>The disruption of homeostasis in tendon extracellular matrix and altered biomechanical properties lead to poor tendon healing, creating a significant clinical challenge for millions of diabetics. Furthermore, improving blood glucose levels doesn't normalize tendon properties in diabetics. Diabetes-related tendon complications are often associated with advanced glycation end products (AGEs) crosslinking with collagen. However, recent studies have found no evidence of higher collagen crosslinking in diabetics and no correlation between tendon AGE content and tensile strength. The interaction between serum AGEs and AGE receptors (RAGE) is a less explored mechanism of AGE-mediated effects. People with diabetes are more likely to accumulate AGEs in their serum as a result of hyperglycemia, the consumption of AGE-rich foods, and diminished kidney clearance of AGEs. In previous studies, advanced glycation end-products (AGEs) have been shown to inhibit cell proliferation and migration, both of which are critical to tendon healing. We hypothesized that serum AGEs and activation of RAGE represent a mechanism underlying impaired tendon properties with diabetes. The increasing serum AGE levels would impair tendon biomechanical properties and tendon healing, while inhibition of RAGE [Azeliragon (AZ)] would improve tendon mechanics.</p> <p>Db/db mice with naturally elevated serum AGEs and impaired tendon function were treated daily with a RAGE inhibitor [Azeliragon (AZ), n=9] or vehicle (n=10) for three weeks. Patellar tendon stiffness and modulus were greater (p<0.05) in mice receiving AZ (stiffness: 9.6±1.2 N/mm, modulus: 78.2±8.2 MPa) compared to vehicle (5.8±0.9 N/mm, modulus: 49.0±8.3 MPa). Maximum strain (vehicle: 0.9±0.1, AZ: 0.8±0.05) and toughness (vehicle: 6.1±1.4, AZ: 6.5±1.2 J·m−3) were not different between groups (p>0.05). Maximum stress tended to be greater in the AZ group (vehicle: 14.6±2.4, AZ: 23.3±2.9 N/mm2, p=0.156).</p> <p>Ten-week-old non-diabetic mice were assigned to receive daily injections of bovine serum albumin (BSA-only, n=6), BSA and AZ (BSA-AZ, n=5), 200 mg/ml glycated BSA (AGE-BSA, n=4), and AGE with AZ (AGE-AZ, n=6). A full-thickness, partial-width defect was created in both patellar tendons. Treatments were started one week before surgery and continued for three weeks after surgery. Three Tendon stiffness was lower in mice treated with AGEs (p<0.05, 10.8±1.4 N/mm) compared to BSA-only (17.6±1.3 N/mm). Further, tendon stiffness in AGE-treated mice given AZ was not different from AGE-BSA (p<0.05, 12.7±1.8 N/mm). Tendon modulus was lower in mice treated with AGEs (p<0.05, 28.0±7.0 MPa) compared to BSA-only (63.5±9.0 MPa). Additionally, modulus in AGE-treated mice given AZ was not different from AGE-BSA (p>0.05, 47.6±10.4 N/mm). </p> <p>We demonstrate that administering a RAGE inhibitor improves tendon properties in an established mouse model for type 2 diabetes. In healthy mice, serum AGE levels inhibit the recovery of tendon biomechanical properties after injury; RAGE inhibitors did not have an effect on mice given AGEs. Based on these data, we suggest elevated serum AGEs, as seen with diabetes, are associated with poor mechanical properties and delayed tendon healing.</p>

No description available.

This thesis project will investigate the effects of fasting on the serum levels of two key regulatory hormones, insulin and thyroid hormone (T3) and the effects of these hormones on the trafficking of Glut4 on soleus muscle.

Wong, Wing Tak Jack. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 204-227). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Poon, Chui Wa Christina. / "August 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 123-131). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 論文摘要 --- p.vi / ACKNOWLEDGEMENTS --- p.ix / PUBLICATIONS --- p.x / Abstracts --- p.x / ABBREVIATIONS --- p.xii / Chapter 1. --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1. --- Diabetes --- p.1 / Chapter 1.1.1. --- Overview --- p.1 / Chapter 1.1.2. --- Diagnostic Criteria of Type-2 Diabetes --- p.2 / Chapter 1.1.3. --- Type-2 Diabetes (T2DM) --- p.3 / Chapter 1.1.3.1. --- Impaired Insulin Synthesis and Insulin Secretory Defects in Type-2 Diabetes --- p.3 / Chapter 1.1.3.2. --- β-Cell Dysfunction --- p.5 / Chapter 1.1.3.3. --- Insulin Resistance --- p.5 / Chapter 1.1.4. --- Glucose Toxicity --- p.6 / Chapter 1.1.4.1. --- Fasting Hyperglycemia --- p.8 / Chapter 1.1.4.2. --- Postprandial Hyperglycemia --- p.8 / Chapter 1.2. --- Oxidative Stress --- p.8 / Chapter 1.2.1. --- ROS and Mitochondria --- p.8 / Chapter 1.2.2. --- ROS Production by Mitochondria --- p.9 / Chapter 1.2.3. --- The Relationship of Glucose Recognition by β-cells and Oxidative Stress --- p.11 / Chapter 1.2.4. --- Important Roles of Glutathione in Pancreatic β-cells and Glutathione Synthesis --- p.14 / Chapter 1.2.5. --- N-acetyl-L-cysteine - A Potential Drug Treatment for Type-2 Diabetes? --- p.17 / Chapter 1.3. --- Role of F-actin Cytoskeleton on Glucose-induced Insulin Secretion --- p.18 / Chapter 1.4. --- Current Clinical Treatments for Type-2 Diabetes Mellitus --- p.21 / Chapter 1.4.1. --- Metformin --- p.22 / Chapter 1.4.2. --- Sulfonylureas --- p.22 / Chapter 1.4.3. --- Thiazolidinediones --- p.23 / Chapter 1.4.4. --- Glinides (Meglitinide Analogues) --- p.23 / Chapter 1.4.5. --- α-Glucosidase (AG) Inhibitors --- p.24 / Chapter 1.4.6. --- Dipeptidyl Peptidase-4 (DPP-4) Inhibitors --- p.24 / Chapter 1.4.7. --- (Clinical) Antioxidant Treatment --- p.24 / Chapter 1.5. --- Animal Models Used in Type-2 Diabetes Research --- p.25 / Chapter 1.6. --- Aims of Study --- p.27 / Chapter 2. --- RESEARCH DESIGN & METHODS --- p.28 / Chapter 2.1. --- Materials --- p.28 / Table 1. Sources and concentrations of drugs tested in this study: --- p.28 / Culture Medium - --- p.29 / General Reagents --- p.29 / Chapter 2.2. --- Isolation of Islets of Langerhans and Single Pancreatic β-Cells --- p.31 / Chapter 2.3. --- Measurement of Mitochondrial ROS Levels --- p.32 / Chapter 2.4. --- Measurement of Islets Insulin Release and Insulin Content --- p.34 / Chapter 2.4.1. --- Preparation of Samples --- p.34 / Chapter 2.4.2. --- Enzyme-Link Immunosorbent Assay (ELISA) --- p.35 / Chapter 2.5. --- Immunocytochemistry --- p.35 / Chapter 2.6. --- Data and Statistical Analysis --- p.37 / Chapter 3. --- RESULTS --- p.38 / Chapter 3.1. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on Releasable Insulin Levels and Insulin Contents in Response to Low Glucose (5 mM) and High Glucose (15 mM) of Isolated Pancreatic Islets of (db+/m+) and (db+/db+) Mice" --- p.38 / Chapter 3.1.1. --- Effect of L-NAC on Insulin Secretion and Insulin Contents --- p.38 / Chapter 3.1.2. --- Effect of Cytochalasin B on Insulin Secretion and Insulin Contents --- p.39 / Chapter 3.1.3. --- Effect of 4-Phenyl Butyric Acid on Insulin Secretion and Insulin Contents --- p.43 / Chapter 3.1.4. --- Effect of Ursodeoxycholic Acid on Insulin Secretion and Insulin Contents --- p.46 / Chapter 3.1.5. --- Effect of Hydrogen Peroxide on Insulin Secretion and Insulin Contents --- p.49 / Chapter 3.1.6. --- Effect of Jasplakinolide on Insulin Secretion and Insulin Contents --- p.53 / Chapter 3.1.7. --- Effect of Thapsigargin on Insulin Secretion and Insulin Contents --- p.57 / Chapter 3.1.8. --- Effect of BSO on Insulin Secretion and Insulin Contents --- p.61 / Chapter 3.2. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on Mitochondrial ROS Levels in Response to High Glucose (15 mM) Challenge in Isolated Single Pancreatic β-Cells of (db +/m+) and (db +/db +) Mice" --- p.65 / Chapter 3.2.1. --- "Effects of L-NAC (20 mM), 4-Phenyl Butyric Acid (4-PBA) (1 mM), Ursodeoxycholic Acid (UA) (500 μg/ml), H202 (200 μM), Thapsigargin (0.5 μM) and DL-Buthionine-[S,R]-Sulfoximine (BSO) (0.1 μM) Pre-treatments on Mitochondrial ROS Level in Response to High Glucose (15 mM) Challenge" --- p.65 / Chapter 3.2.2. --- "Effects of L-NAC (20 mM), Cytochalasin B (10 μM) and Jasplakinolide (5 μM) Pre-treatments on Mitochondrial ROS Level in Response to High Glucose (15 mM) Challenge_" --- p.76 / Chapter 3.3. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on F-actin Cytoskeleton Levels Incubated in Low Glucose (5 mM) and High Glucose (15 mM) Medium in Single Pancreatic β-Cells of Non-Diabetic (db +/m+) and Diabetic (db +/db +) Mice" --- p.81 / Chapter 4. --- DISCUSSION --- p.100 / Chapter 4.1. --- General Discussion --- p.100 / Chapter 5. --- SUMMARY --- p.120 / Chapter 6. --- FUTURE PERSPECTIVES --- p.121 / Chapter 7. --- REFERENCES --- p.123

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.

Indiana University-Purdue University Indianapolis (IUPUI) / Glucose homeostasis is maintained through the coordinated actions of insulin secretion from pancreatic beta cells and insulin action in peripheral tissues. Dysfunction of insulin action yields insulin resistance, and when coupled with altered insulin secretion, results in type 2 diabetes (T2D). Exocytosis of intracellular vesicles, such as insulin granules and glucose transporter (GLUT4) vesicles is carried out by similar SNARE (soluble NSF attachment receptor) protein isoforms and Munc18 proteins. An additional regulatory protein, Doc2b, was implicated in the regulation of these particular exocytosis events in clonal cell lines, but relevance of Doc2b in the maintenance of whole body glucose homeostasis in vivo remained unknown. The objective of my doctoral work was to delineate the mechanisms underlying regulation of insulin secretion and glucose uptake by Doc2b in effort to identify new therapeutic targets within these processes for the prevention and/or treatment of T2D. Towards this, mice deficient in Doc2b (Doc2b-/- knockout mice) were assessed for in vivo alterations in glucose homeostasis. Doc2b knockout mice were highly susceptible to preclinical T2D, exhibiting significant whole-body glucose intolerance related to insulin secretion insufficiency as well as peripheral insulin resistance. These phenotypic defects were accounted for by defects in assembly of SNARE complexes. Having determined that Doc2b was required in the control over whole body glycemia in vivo, whether Doc2b is also limiting for these mechanisms in vivo was examined. To study this, novel Doc2b transgenic (Tg) mice were engineered to express ~3 fold more Doc2b exclusively in pancreas, skeletal muscle and fat tissues. Compared to normal littermate mice, Doc2b Tg mice had improved glucose tolerance, related to concurrent enhancements in insulin mumsecretion from beta cells and insulin-stimulated glucose uptake in the skeletal muscle. At the molecular level, Doc2b overexpression promoted SNARE complex assembly, increasing exocytotic capacities in both cellular processes. These results unveiled the concept that intentional elevation of Doc2b could provide a means of mitigating two primary aberrations underlying T2D development.