Type 2 Diabetes Mellitus (T2DM) is a metabolic disorder of global concern that is primarily characterized by insulin resistance, relative insulin deficiency, and hyperglycemia. G- Protein Coupled Receptors (GPCRs), important mediators of cellular signaling responses, have been prime targets of drug discovery efforts in various therapeutic areas. Human G-Protein Receptor 40 (HGPR40) is highly expressed in the pancreas and has been implicated in the regulation of glucose metabolism and pathophysiology of T2DM. For effective control of diabetes, combination therapy is being considered because no single drug can completely control diabetes and its associated complications. This necessitates the identification of novel drug targets including HGPR40 which might permit development of drugs which function differently from existing drugs. This project focused on the construction of two chimeric receptor proteins named HGPR40.1-715_43.709-1013 (Chimera I) and HGPR40.1-431_43.389-1013 (Chimera II) which were made by exchanging domains within trans-membranes regions 6 and 4, of HGPR40 and HGPR43 DNA, homologous receptors which vary in amino acid sequence but which have the same three-dimensional structure. After PCR amplification of sufficient quantities of the desired gene fragments they were ligated together to form the desired recombinant chimeric proteins which were cloned into the expression vector pcDNA3.1 in two successive cloning steps. The vector contains CMV promoter, multiple cloning sites, neomycin resistance gene etc for high-level expression in a wide range of mammalian cells. The two full-length chimeras were designed to be 6452 bp and 6483 bp by exchanging either the first or the first two external domains of HGPR40 with those of HGPR43. The creation of the correct chimeras was verified by both agarose gel electrophoresis and PCR analysis. Then chimeric DNA was transformed into Escherichia coli to obtain sufficient amounts of DNA for sequencing verification of the desired construct. Upon verification, the cloned DNA was to be transfected into cultured mammalian kidney (HEK293) cells for expression of the chimeric proteins. A (FLIPR) Fluorometric Imaging Plate Reader analysis by our collaborators at Eli Lilly would have been used to measure the extent of Ca2+ efflux from the endoplasmic reticulum testing a variety of stimulatory molecules to obtain an indication of which would activate the receptor. Activation would initiate the internal (second messenger cascade) G-protein signaling pathway and result in the secretion of insulin. However, transformation of the chimeric membrane receptors into E. coli resulted in altered sequences which could not be used for the FLIPR analysis. Expression of membrane proteins in E. coli can lead to such gene rearrangements. These experiments will be repeated in the future using another type of competent cell designed to prevent genetic re-arrangements. Construction of stable clones will permit us to test numerous potential ligands to aid in development of novel therapeutic drugs targeting HGPR40 to aid in combating diabetes. / Department of Biology

This study was conducted to determine if improvements in glucose tolerance could be demonstrated following an acute bout of progressive resistance exercises. Fourteen individuals, not currently weight training, were assigned to two groups using the guidelines established by the WHO for NIDDM and normal (CON), based on the results of a three hour 75 g (-1.2M) load oral glucose tolerance test (OGTT). Eight blood samples were collected during the OGTT and assayed for glucose, insulin, and C-peptide. Each subject from the NIDDM (n=7) and CON (n=7) groups participated in a familiarization period, including a IRM, with eight different Nautilus selectorized exercise machines utilizing both the upper and lower body. A 3 set x 10 repetition exercise protocol based on the IRM was conducted and followed 18 hours later by another OGTT. Two day diets were replicated from the prior OGTT. Analysis of variance failed to demonstrate significant differences in the total responses or at any specific sampling points from pre to postprotocol for glucose (p=0.53), C-peptide (p=0.07) or the C-peptide:insulin ratio (p=0.16) in either group. Blood insulin levels from pre to postprotocol were significantly reduced (p=0.001) by 24% and 22% for the NIDDM and CON groups, respectively. These data suggest that a single series of progressive resistance exercises improve insulin uptake by the tissues without augmenting glucose disposal. / School of Physical Education

Patients with non-insulin-dependent diabetes mellitus (NIDDM) usually exhibit a marked disturbance of lipid metabolism which is reflected in high levels of plasma total cholesterol, low-density lipoproteins (LDL), and triglycerides, along with low levels of high-density lipoproteins (HDL). Elevated triglycerides are the major contributor to diabetic hyperlipidemia. These plasma lipid concentrations and the fatty acid composition of these lipids are clearly influenced by the type of diet consumed along with the proportion of dietary fatty acids. Therefore, it was the purpose of this investigation to examine the relationships between glycemic control, serum lipid levels of total cholesterol, HDL, LDL and triglycerides to the amounts and types of fats in the typical diets of NIDDM patients as compared to non-diabetic individuals. The dietary fats were also compared with the distribution of fatty acids found in their total lipids and free (in vivo) fatty acids. The relationship between dietary fat intake and serum total lipid levels along with total and free fatty acid distributions was the primary focus.This investigation found that NIDDM subjects have significantly greater triglyceride levels (200 ± 18.4 mg/dL) than non-diabetic controls (93 ± 13.2 mg/dL). Total and LDL cholesterol levels of the NIDDM group were elevated from the control group while HDL levels were depressed, but these differences were of nonsignificant proportions. The NIDDM group typically consumed significantly lowered amounts of teal, saturated, and monounsaturated dietary fatty acids (46.7 ± 7.1 grams, 114.0 ± 2.9 grams, and 16.8 ± 2.5 grams, respectively) compared to the control group (80.0 ± 10.9 grams, 26.7 ± 4.5 grams, and 30.2 ± 4.5 grams, respectively). Even so, the percentage of kilocalories from total fat in the NIDDM vs. the control group diets was not statistically different which may explain the lack of significance between groups with regard to distribution of serum fatty acids. On an individual basis, the types of fat that predominated in the diet were also found in a large percentage in the serum lipid distributions. Positive correlations between saturated fat intake and the blood serum stearic free fatty acid along with polyunsaturated fat intake and linoleic free fatty acid supported this observation. Other investigators (6,62) have reported that dietary intake does indeed contribute to the percentage of fatty acids distributed in the plasma lipids. To determine if a particular dietary fatty acid contributes more significantly to hyperlipidemia, the diet needs to be controlled.On an individualized basis, it was also noted that the diabetics with the lowest amount and percentage of fat in their diets, also had the lowest serum lipid levels. Besides diet, other influential factors which may have contributed to the lipid levels of these NIDDM patients are genetic predisposition, environmental influences, and the stage and progression of each individual's disease. Thus, due to the underlying metabolic impairments which are exacerbated by genetic and/or environmental influences, it is of vital importance to recognize how essential diet manipulation is with regards to lipid control in the treatment of NIDDM patients. / Department of Biology

Autoimmune diabetes is a disorder in which the (3-cells in the pancreatic islets of Langerhans are specifically destroyed resulting in absolute insulin deficiency; typically this is a childhood-onset disease, Type 1 Diabetes (T1D). Type 2 Diabetes (T2D) is a metabolic disorder usually developing in adults resulting from defects in insulin secretion and action. Latent Autoimmune Diabetes in Adults (LADA) is a form of diabetes that shares autoimmune disease pathology with T1D but a clinical presentation similar to T2D; LADA patients develop diabetes as adults (>25 years) and do not immediately require insulin treatment for survival. They are therefore often misdiagnosed with T2D. The aims of this work were to characterise immunological and genetic aspects of LADA using a large cohort collected from various patient repositories the United Kingdom to determine if it is a separate disease entity or an age-related extension of T1D. Both T1D and LADA are characterised by autoantibodies to the islet cell protein glutamic acid decarboxylase 65 (GADA) at diagnosis. The persistence and titre of GADA post-diagnosis in LADA was examined at 0.5, 3 and 6 years. GADA persisted in 93% of patients for 6 years; GADA litres decreased between 0.5 and 3 years post-diagnosis and either stabilised or increased again between 3 and 6 years. GADA titre was not associated with age at diagnosis, glycaemic control, β-cell function or other clinical features. GADA titre at 0.5 years was associated with a greater likelihood of requiring more intensive antihyperglycaemic therapy but did not predict therapy or insulin requirement at 3 and 6 years. Autoantibodies against IA-2 plus GADA compared to GADA alone at diagnosis predicted increased therapy requirement by 3 and 6 years and insulin requirement by 3 years postdiagnosis. Variants of the Human Leukocyte Antigen (HLA) genes DRB1 and DQB1, are associated with susceptibility for T1 D. An analysis of these variants in LADA (n = 378) revealed that the predisposing and protective variants in LADA are similar to those reported in T1D; DR3 (in linkage disequilibrium, LD with DQ2) and DR4 (in LD with DQ8) were the main predisposing variants whereas DR2 (in LD with DQ6) was most the protective against LADA. 85% of LADA patients possessed the DR3 and DR4 specificities, compared with 95% seen in T1D, suggesting a reduced predisposition in LADA compared with T1D. Synergistic effects of the DR3 and DR4 specificities occurred in LADA and the DRB1*0401 allele within the DR4 specificity was predisposing to disease, as seen in T1D. No other predisposing variants were identified in LADA. As reported for T1D, DR11, DR13, DQ5, DQ7 and DQ9 were protective against LADA; DQ6 was positively correlated with age at diagnosis. Association analysis of the insulin gene region in LADA (n = 400) showed that the variable number of tandem repeats (VNTR) locus primarily confers susceptibility to disease. Overall, the short Class I alleles predisposed to disease whereas longer Class III alleles conferred dominant protection, as reported in T1D. Fine-structure analysis showed that the Class I haplotypes 'IC+/ID+' and 'ID-' both conferred susceptibility for LADA - unlike in T1D, where the ID- haplotype has been reported to have protective effects. The Class III 'Protective' and Very Protective' haplotypes, conferred equal protection in LADA, as reported forTID. In conclusion; GADA persist post-diagnosis but are not markers for disease progression of LADA. Patterns of susceptibility at the HLA and insulin gene regions in LADA are similar to that reported for T1 D. LADA is likely to represent an age-related extension of T1D rather than a separate disease entity.

Pregestational and gestational diabetes (GDM) places the mother and her offspring at an increased risk for later development of insulin resistance and type 2 diabetes. Oxidative stress may mediate long-term disturbances in glucose homeostasis associated with type 2 diabetes and the metabolic syndrome. This thesis describes a cross-sectional study examining serum concentrations of free radical generated oxysterols as markers of oxidative stress in a cohort of teenage daughters from pregnancies with and without GDM. Daughters of GDM-pregnancies had a tendency of higher levels of serum oxysterols (7beta-hydroxycholesterol); however, this difference was not statistically significant after adjustment for total cholesterol. Serum oxysterols were significantly correlated with obesity measures such as waist circumference and BMI, which likely accounted for the tendency for higher measures of oxysterol concentrations in the GDM daughters. Oxysterols represent potentially important biomarkers for oxidative stress in adolescent girls as their levels track with the metabolic syndrome risk factors. / Le diabète pré-gestationnel et le diabète de gestation (DG) augmentent le risque dedéveloppement d'une future résistance à l'insuline et de diabète de type 2 autant pourla mère que pour l'enfant. Le stress oxydatif est un facteur potentiel impliqué dans ledéséquilibre du glucose sanguin associé au diabète de type 2 et au syndromemétabolique. La présente thèse est une étude sectionnelle croisée, ayant pour but demesurer des marqueurs du stress oxidatif, notamment la concentration des oxystérolsgénérés par les radicaux libres dans le sérum d'adolescentes, nées de mères ayantprésenté ou non un diabète de gestation. Nos résultats montrent des concentrationsd'oxystérols (7P-hydroxycholesterol) plus élevées dans le sérum de filles issues degestations diabétiques à comparer aux filles de mères n'ayant pas eu de DG.Cependant, la différence entre les deux groupes n'était pas statistiquementsignificative après un ajustement au cholestérol total. La concentration d'oxystérolsétait significativement corrélée aux marqueurs d'obésité, notamment la circonférencede la taille et l'index de masse corporelle, possiblement à l'origine de la tendance desoxystérols à être plus élevés dans le cas des adolescentes issues de gestationsdiabétiques.

Although insulin resistance of glucose and fat metabolism in type 2 diabetes mellitus (T2DM) is firmly established, that of protein remains controversial for methodological reasons. A hyperinsulinemic (40MU/m2·min) euglycemic (5.5 mmol/L) isoaminoacidemic (postabsorptive concentrations) clamp was combined with [3-3H]glucose and [1-13C]leucine kinetics to concurrently assess protein and glucose metabolism in 10 hyperglycemic men with T2DM and 11 men without (all BMI=29+/-kg/m2), matched also for age, body composition, and waist circumference. In response to hyperinsulinemia, protein turnover and synthesis were stimulated in controls, but not in T2DM. Both insulin-stimulated total and non-oxidative glucose disposal were diminished in T2DM vs. controls. There was a robust positive correlation between the change in synthesis and glucose disposal. Hence, there is an additive effect of T2DM, beyond that of having excess fat, on insulin resistance of whole body protein turnover and synthesis. Furthermore, protein sensitivity to insulin parallels that of glucose, establishing this as an important concern in T2DM management.

We questioned whether improved glycemic control achieved by oral agent (gliclazide) would correct the altered protein metabolism during an iso-energetic (ISO) and a low energetic (50% of ISO) diets. Seven diabetic (DM) and 7 matched obese control (OB) subjects were give ISO for 14 (DM) or 7 (OB) days, followed by 28 days of the low energetic diet with constant 1.5 g protein/kg BMI$ sb{25}$/d. Giclazide (+ metformin in 4 DM) was given during days 8-14 of ISO and the low energety diet to DM. With ISO and gliclazide, fasting plasma glucose decreased and plasma insulin and nitrogen retention increased while 3-methylhistidine excretion and resting metablic rate decreased to levels no longer different from OB. With moderate energy restriction, weight decreased in all subjects and glycemia normalized in DM. Nitrogen equilibrium was maintained and 3-methylhistidine excretion did not change. The altered protein metabolism observed during hyperglycemia can be improved with oral hypoglycemic agent therapy $ pm$ the low energy diet. Moderate energy restriction with oral hypoglycemic agent therapy achieves diabetes control, nitrogen equilibrium, and a modest decrease in resting metabolic rate.

To examine the effects of Momordica charantia (MC) and cinnamon on glucose uptake and adiponectin secretion (AS) fat cells, 3T3-L1 adipocytes were treated with a water extract of cinnamon (CE) and three concentrations of MC water and ethanol extracts. The treatment combination of 0.2 mg/ml MC water extract and 0.5 nM insulin was associated with an increased glucose uptake into the cells (61%) and increased AS from the cells (75%). Without insulin, 0.2 mg/ml of CE increased glucose uptake (100%) and completely inhibited AS from the cells. Sub-optimal concentrations of insulin did not further enhance the CE activity and, in combination with 50 nM insulin, a dose-dependent decrease in glucose uptake was observed. The present results indicate that preferentially water-soluble component(s) in MC enhance the glucose uptake action of sub-optimal concentrations of insulin in 3T3-L1 adipocytes. This effect is accompanied by and may be a result of increased AS. CE increases glucose uptake in these adipocytes but inhibits AS.

Thesis (M.S.)--Indiana University, 2006. / Includes bibliographical references. Also available online (PDF file) by a subscription to the set or by purchasing the individual file.

Thesis (M.S.)--Indiana University, 2006. / Includes bibliographical references.