Indiana University-Purdue University Indianapolis (IUPUI) / Type 2 diabetes mellitus is a chronic disorder characterized by hyperglycemia, insulin resistance, and insufficient insulin secretion from the pancreatic β cells. To maintain adequate levels of insulin secretion, β cells rely on highly coordinated control of luminal ER Ca2+. Stromal Interaction Molecule 1 (STIM1) is an ER Ca2+ sensor that serves to replenish ER Ca2+ stores in response to depletion by gating plasmalemmal Orai1 channels in a process known as store-operated calcium entry (SOCE). We developed a method for the direct measurement of SOCE in pancreatic β cells and found that deletion of STIM1 in INS-1 cells (STIM1KO) is sufficient to block Ca2+ influx in response to store-depletion. To determine the physiological importance of β cell STIM1, we created mice with pancreatic β cell specific deletion of STIM1 (STIM1Δβ) and placed them on a high fat diet (HFD) with 60% of kilocalories derived from fat. After 8 weeks of HFD, female, but not male, STIM1Δβ mice exhibited increased body weight and fat mass as well as significant glucose intolerance and impaired insulin secretion without observable differences in insulin tolerance. Immunohistochemical analysis revealed a reduction of β cell mass and an increase of α cell mass; ELISA of islet lysates revealed a similar significant reduction in insulin content and increased glucagon content. RNA-sequencing performed on STIM1Δβ islets revealed differentially expressed genes for functions related to apoptosis, lipid metabolism, and epithelial cell differentiation, as well as loss of β cell identity. Proteomics analysis of STIM1KO cells phenocopied the metabolic findings, revealing significantly increased glucagon expression. Analysis of islet RNA-sequencing results showed modulation of pathways related to 17-β estradiol (E2) signaling, with notable downregulation of G-protein coupled estrogen receptor 1 (GPER1) expression. Consistently, treatment of female wild-type islets with pharmacological SOCE inhibitors led to reduced expression GPER1, while STIM1KO cells showed lower mobilization of intracellular cAMP levels in response to GPER agonist treatment. Taken together, these findings identify a novel interaction between SOCE and E2 signaling in the female islet and suggest that loss of STIM1 and impairments in SOCE may contribute to diabetes pathophysiology through loss of β cell identity. / 2022-12-28

Mitochondrial dynamics includes the processes of fusion, fission, and motility. These processes form interdependent adaptive mechanisms that, together with autophagy, maintain mitochondrial function to meet cellular needs. Mitochondrial dynamics control function directly by inducing bioenergetic remodeling or indirectly by promoting turnover of mitochondria via autophagy. Importantly, mitochondrial dysfunction has been implicated in beta-cell failure during type 2 diabetes. This thesis will investigate the role of dynamics and autophagy in regulating mitochondrial and pancreatic beta-cell function during chronic exposure to excess glucose and fatty acids, termed glucolipotoxicity (GLT). It remains ill-defined what role fusion and motility play in determining mitochondrial turnover, as current methodologies to assess turnover lack subcellular resolution. To address this need we developed the use of MitoTimer, a mitochondrial fluorescent probe that undergoes a time-dependent green-to-red transition. Turnover was revealed by the integrated proportions of young (green) and old (red) MitoTimer protein. The results demonstrate that mitochondrial fusion and motility regulate turnover by promoting the distribution of newer protein to subsets of mitochondria in the network. GLT inhibits mitochondrial fusion and networking in pancreatic beta-cells. Since fusion is dependent on motility we tested the hypothesis that GLT impairs fusion by affecting motility. We determined that GLT arrests motility, which may contribute to mitochondrial and beta-cell dysfunction. We show that excess nutrients increase O-linked β-N-acetyl glucosamine (O-GlcNAc) modification of mitochondrial motor adaptor Milton1, which decreases its activity and results in arrest of motility and increased fission. Thus Milton1 O-GlcNAc modification acts as a nutrient-sensor linking fusion, fission, and motility to nutrient supply in the beta-cell. Finally, GLT inhibits autophagic flux with concurrent lysosomal pH increase in beta-cells. To address the hypothesis that impaired lysosomal acidification is a causative event inhibiting autophagic flux and beta-cell function, we developed lysosome-localizing nanoparticles that expand and acidify upon UV photo-activation. Increasing lysosomal acidity with the nanoparticles increased autophagic flux and restored beta-cell function under GLT, establishing lysosomal pH as a key mediator of nutrient-induced beta-cell dysfunction. In summary the work elucidates the interdependence and specific roles of mitochondrial fusion, fission, motility, and autophagy in dictating beta-cell responses to excess nutrient environment. / 2017-06-15T00:00:00Z

Diabetes mellitus is a complex disorder characterized by chronic hyperglycemia and vascular complications leading to significant morbidity and mortality. The common feature in all forms of diabetes is the insufficient functional β-cell mass to maintain euglycemia; therefore, the promotion of β-cell survival and growth is a fundamental goal for diabetes prevention and treatment. Evidence has suggested that erythropoietin (EPO) exerts cytoprotective effects on non-erythroid cells. However, the in vivo role of EPO on the pancreatic β cells has not been evaluated to date. We hypothesized that EPO would have direct cytoprotective effects on the pancreatic β cells and provide protection against experimental models of diabetes. In Chapter IV, we report that recombinant human erythropoietin (rHuEPO) administration provided protection against diabetes development in the streptozotocin (STZ)-induced and db/db mice, models of type 1 and type 2 diabetes, respectively, through anti-apoptotic, proliferative and angiogenic effects within the islets. Next, we show in Chapter V, using β cell-specific EPO-R and JAK2 knockout (KO) mice, that these cytoprotective effects of EPO resulted from direct biological effects on the β cells, and that JAK2 is its essential intracellular mediator. We also show that endogenous EPO or JAK2 in β cells had no essential role in determining β-cell development or homeostasis. Given that epo is a target gene of the hypoxia inducible factor (HIF) pathway, we hypothesized that deletion of von Hippel Lindau (VHL), a negative regulator of this pathway, in the β cells would lead to enhanced transcription of HIF-target genes, which are largely pro-survival, and lead to enhanced β-cell mass and function. Contrary to our hypothesis, in Chapter VI, our results show that the epo gene is not expressed in islets. Furthermore, β cell-specific VHL KO mice were glucose intolerant due to impaired β-cell function and mass, which we were able to rescue with rHuEPO treatment. Our results demonstrate that EPO exerts direct biological effects on the pancreatic β cells. Further understanding of the biology of EPO may hold promise for the development of a potential novel strategy for diabetes prevention and treatment.

In order to elucidate how uncoupling protein 2 (UCP2) influences pancreatic β cells and glucose homeostasis, I have generated and characterized an inducible β cell-specific UCP2 deletion model,MIPCreER×loxUCP2 mice. Male littermates were injected with tamoxifen to induce UCP2 deletion(UCP2 iBKO) or with corn oil (CO). The phenotypes of both short-term (3-4 weeks after the last injection) and long-term (8-9 weeks after the last injection) were determined: Short-term iBKO mice displayed no differences in glucose or insulin tolerance, but enhanced in vivo and in vitro insulin secretion and suppressed islet reactive oxygen species (ROS) levels; while long-term iBKO mice displayed no difference in glucose tolerance, but impaired in vivo and in vitro insulin secretion and enhanced islet ROS levels. In conclusion, short-term UCP2 deletion in β cells promotes insulin secretion, while long-term UCP2 deletion impairs insulin secretion, possibly due to the opposite background of islet ROS.

In order to elucidate how uncoupling protein 2 (UCP2) influences pancreatic β cells and glucose homeostasis, I have generated and characterized an inducible β cell-specific UCP2 deletion model,MIPCreER×loxUCP2 mice. Male littermates were injected with tamoxifen to induce UCP2 deletion(UCP2 iBKO) or with corn oil (CO). The phenotypes of both short-term (3-4 weeks after the last injection) and long-term (8-9 weeks after the last injection) were determined: Short-term iBKO mice displayed no differences in glucose or insulin tolerance, but enhanced in vivo and in vitro insulin secretion and suppressed islet reactive oxygen species (ROS) levels; while long-term iBKO mice displayed no difference in glucose tolerance, but impaired in vivo and in vitro insulin secretion and enhanced islet ROS levels. In conclusion, short-term UCP2 deletion in β cells promotes insulin secretion, while long-term UCP2 deletion impairs insulin secretion, possibly due to the opposite background of islet ROS.

Diabetes mellitus is a complex disorder characterized by chronic hyperglycemia and vascular complications leading to significant morbidity and mortality. The common feature in all forms of diabetes is the insufficient functional β-cell mass to maintain euglycemia; therefore, the promotion of β-cell survival and growth is a fundamental goal for diabetes prevention and treatment. Evidence has suggested that erythropoietin (EPO) exerts cytoprotective effects on non-erythroid cells. However, the in vivo role of EPO on the pancreatic β cells has not been evaluated to date. We hypothesized that EPO would have direct cytoprotective effects on the pancreatic β cells and provide protection against experimental models of diabetes. In Chapter IV, we report that recombinant human erythropoietin (rHuEPO) administration provided protection against diabetes development in the streptozotocin (STZ)-induced and db/db mice, models of type 1 and type 2 diabetes, respectively, through anti-apoptotic, proliferative and angiogenic effects within the islets. Next, we show in Chapter V, using β cell-specific EPO-R and JAK2 knockout (KO) mice, that these cytoprotective effects of EPO resulted from direct biological effects on the β cells, and that JAK2 is its essential intracellular mediator. We also show that endogenous EPO or JAK2 in β cells had no essential role in determining β-cell development or homeostasis. Given that epo is a target gene of the hypoxia inducible factor (HIF) pathway, we hypothesized that deletion of von Hippel Lindau (VHL), a negative regulator of this pathway, in the β cells would lead to enhanced transcription of HIF-target genes, which are largely pro-survival, and lead to enhanced β-cell mass and function. Contrary to our hypothesis, in Chapter VI, our results show that the epo gene is not expressed in islets. Furthermore, β cell-specific VHL KO mice were glucose intolerant due to impaired β-cell function and mass, which we were able to rescue with rHuEPO treatment. Our results demonstrate that EPO exerts direct biological effects on the pancreatic β cells. Further understanding of the biology of EPO may hold promise for the development of a potential novel strategy for diabetes prevention and treatment.

Cancer cachexia is a complex syndrome that can affect up to 80% of cancer patients. Among the symptoms involved in cancer cachexia progression, the establishment of a systemic inflammation and the imbalance in glucose metabolism homeostasis take an important part in this profile. The aim of the present study was to further evaluate the role of cancer-induced inflammation in the impairment of pancreatic beta cell function in solid Ehrlich carcinoma-bearing mice. For that, we have focused the study in the pro-inflammatory mechanisms involved on β-cell death. We have observed that tumor-bearing animals developed an aggressive pancreatic inflammatory status 14 days after tumor cells inoculation. The increase of pro-inflammatory cytokines followed by an up-regulation of important transcription factors such as NF-κB and STAT-1 and its related genes, reveled a similar outline for β-cell death found in type 1 diabetes. Furthermore, expression of pro-apoptotic Bcl-2 family members followed by caspases activation was increased in pancreatic islets of tumor-bearing animals and the expression of anti-apoptotic members was decreased. We have also observed an increase in β-cell death and ER stress components, as well as a decrease in insulin content cells together with an increase in alpha cells content. Overall, our results provide strong evidences that pancreatic β-cells in tumor-bearing animals are widely affected by tumor presence and systemic inflammation establishment. Interestingly, it was shown a similarity with mechanisms of β-cell death found in type 1 diabetes. Although the exactly mechanisms behind the changes found in carbohydrate metabolism in cancer cachexia is still unclear, our data can help to clarify, at least in part, this profile and would serve as a basis for development of new strategies to prevent cachexia progression and to improve the quality of life of cancer patients. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Abstract The purpose of this work was to assess the value of humoral and genetic risk markers in the prediction of type 1 diabetes in siblings of children with type 1 diabetes, to characterise preclinical course of beta-cell autoimmunity in siblings, and to investigate the frequency of autoantibodies and their relations to genetic markers, beta-cell function and progression to type 1 diabetes in a schoolchild population. The prevalence and predictive value of autoantibodies was studied in 755 initially unaffected siblings, and the combination of genetic markers and autoantibodies in 701 of these siblings. Islet cell autoantibodies (ICA), insulin autoantibodies (IAA), glutamic acid decarboxylase antibodies (GADA) and IA-2 antibodies (IA-2A) were all shown to be of value in the prediction of type 1 diabetes in siblings initially tested at or close to the diagnosis of type 1 diabetes in the index case in the family. The risk of progression to type 1 diabetes was related to the number of autoantibodies detected, and the PPV of multiple autoantibodies was 55% over a period of 8 years. Autoantibodies were closely associated with HLA risk markers. A combination of the genetic markers and autoantibodies increased the PPVs of all autoantibodies substantially but also markedly reduced the sensitivity. The preclinical course of type 1 diabetes was investigated in 39 initially unaffected siblings who progressed to clinical disease during the follow-up. These individuals were characterised by the high-risk genetic markers, decreased beta-cell function and humoral autoimmunity against multiple beta-cell targets. However, all measures implied a remarkable individual variation in the rate of the disease process and the pattern of humoral beta-cell autoimmunity. Furthermore, the autoimmune process resulting in clinical presentation of type 1 diabetes could not be unambiguously distinguished from autoimmunity not leading to clinical disease within almost 10 years of follow-up. The frequencies of ICA, IA-2A, GADA and IAA in 3652 healthy Finnish schoolchildren were 2.8%, 0.6%, 0.5% and 0.9%, respectively, and multiple antibodies were detected in 0.6% of these children. GADA and multiple antibodies were related to the DQB1*0302 allele and the DQB1*02/0302 genotype. A reduced first-phase insulin reponse (FPIR) was associated with IA-2A, GADA, IAA and multiple antibodies, but not with ICA or any specific DQB1 allele or genotype. Four subjects progressed to type 1 diabetes, all of them having multiple autoantibodies and those two who underwent an intravenous glucose tolerance test had also a reduced FPIR. None of the progressors carried the high risk DQB1*0302 allele and two of them even carried the protective DQB1*0602 or *0603 allele. In conclusion, autoantibodies alone are recommended as first-line screening in siblings, whereas subsequent determination of HLA-DQB1 markers and their combination with autoantibodies provides a valuable tool for more precise risk assessment. Wide heterogeneity in the course of preclinical type 1 diabetes complicates an accurate estimation of the individual risk of progression to type 1 diabetes among siblings of children with type 1 diabetes. Combined screening for autoantibodies is recommended for the assessment of the risk of progression to type 1 diabetes in schoolchild populations, whereas the present observations challenge the value of current genetic risk markers in predictive strategies targeting schoolchildren.

Diabetes is a life-long chronic disease that affects more than 24 million Americans. Loss of pancreatic beta-cell mass and function is central to the development of both type 1 (T1D) and type 2 diabetes (T2D). Therefore, preservation or regeneration of functional beta-cell mass is one of the essential strategies to treat diabetes [1]. In my study, I tested if ranolazine, a synthetic compound, has potential to prevent or treat diabetes. Diabetes were induced in mice by giving multiple low-doses of streptozotocin (STZ). Ranolazine was given twice daily via an oral gavage (20 mg/kg) for 5 weeks. blood levels of glucose, insulin, and glycosylated hemoglobin (HbA1c) were measured. Glucose tolerance test was performed in control and treated mice. pancreatic tissues were stained with hematoxylin and eosin or stained with insulin antibody for islet mass evaluation. INS1-832/13 cells and human islets were further used to evaluate the effect of ranalozine on beta-cell survival and related signaling pathway. Fasting blood glucose levels after the fourth week of STZ injections were lower in ranolazine treated group (199.1 mg/dl) compared to the vehicle group (252.1 mg/dl) (p<0.01). HbA1c levels were reduced by ranolozine treatment (5.33%) as compared to the control group (7.23%) (p<0.05%). Glucose tolerance was improved in ranolazine treated mice (p<0.05). Mice treated with ranolazine had higher beta-cell mass (0.25%) than the vehicle group (0.07%)(p<0.01). In addition, ranolazine improved survival of human islets exposed to high levels of glucose and palmitate, whereas cell proliferation was not altered. In addition, ranolazine slightly increased the cAMP in MIN-6 cell and human islets. In conclusion, ranolazine may have therapeutic potential for diabetes by preserving beta-cell mass. / Master of Science

Statins are widely used in the treatment of atherosclerosis and hypercholesterolemia, both of which are comorbidities of obesity. However, the effects statins have on insulin homeostasis are relatively unknown and may increase one’s risk for type-II diabetes mellitus. INS-1 pancreatic β-cells, were cultured in 11 mM glucose with 25, 50, 100, 200 nM statin or without. Specifically, this study observed the effects that pitavastatin, simvastatin, lovastatin, and pravastatin have on insulin secretion, insulin content, and ROS levels. GSIS was measured after statin and non-statin exposed cells were incubated in 12 mM glucose KRB. Insulin content was measured after trypsinization and subsequent lysing of cells. Both were analyzed via FRET based HTRF assay. ROS levels within cells were measured following statin exposure during a 2-hour period of 12 mM glucose oxidation after DCF was added. Analysis was done using a Tecan™ fluorescent microplate reader. Pitavastatin, simvastatin, and lovastatin decrease glucose stimulated insulin secretion and insulin content as compared to control. All concentrations of pitavastatin reduced insulin secretion proportionally to insulin content, suggesting it does so through impairment of insulin synthesis or storage. Simvastatin reduced insulin secretion and content in a dose dependent manner, however when secretion was adjusted for % content, data showed that high doses of simvastatin reduced insulin content in a greater fashion than insulin secretion, suggesting both secretory mechanisms and storage/synthesis were impaired. Lovastatin reduced insulin secretion by a greater amount than its reduction of insulin content, suggesting that it impaired insulin secretion via secretory mechanism impairment. Pravastatin did not have an effect on either insulin secretion or insulin content at any concentration. Cells were also tested to determine if pitavastatin, simvastatin, or lovastatin induced a change in ROS levels within the cell. None of the three statins tested caused a statistically significant change in ROS levels at all concentrations. These results suggest that pitavastatin, simvastatin, and lovastatin may impair insulin secretion in patients with high blood glucose. As such, clinical guidelines for statin therapy use in those who are at risk, or suffer from, diabetes may need to be reevaluated.