Type 1 diabetes is an autoimmune disease in which patients' insulin-secreting beta cells in pancreatic islets are destroyed by their own immune system, leading to unregulated blood glucose levels and severe complications. Its only treatment is intensive insulin therapy, which carries the risk of hypoglycemic episodes and can result in seizures, coma, and even death. Islet transplantation has recently become an alternative, albeit experimental, treatment for type 1 diabetes patients. More than one donor graft is usually required to render recipients insulin independent, making the shortage of donor tissue an extremely important challenge in islet transplantation. Identifying the cell type that has the ability to differentiate into islet-like tissue is an important area of study. In this study, I hypothesized that insulin secreting human islet-like clusters could be generated from pancreatic ductal cells, a potential pancreatic progenitor cell type. Islet-like clusters were generated using crude exocrine tissue from human cadaveric donors. This crude exocrine tissue contained a large number of ductal cells, as well as other pancreatic cell types. To evaluate insulin secretion by human islet-like clusters, a static incubation system was set up and tested using Min6 cells, a known insulin-secreting cell line. Using static incubation, significant increases in insulin secretion by islet-like clusters were observed when the clusters were exposed to higher glucose levels and GLP-1, a known insulin secretagogue. Presence of corresponding C-peptide secretion demonstrated that de novo insulin secretion occurred. Furthermore, basal insulin secretion increased as culture stages progressed. An attempt was made to generate islet-like clusters using ductal cells purified by fluorescent activated cell sorting or magnetic activated cell sorting. Nevertheless, it was difficult to ensure survival and proliferation of purified ductal cells. Further studies will be necessary to confirm the role of ductal cells in the generation of islet-like clusters using the crude exocrine tissue, as well as to identify factors that can promote ductal cells proliferation after cell sorting.

Thesis (Ph.D.)--Aberdeen University, 2008. / Title from web page (viewed on July 14, 2009). Includes bibliographical references.

The goal of this research project is to understand how a high sugar diet may affect pancreatic beta cell function. High glucose concentrations lead to an increase in lipid droplets and TORC1 in beta cells, which promote high basal secretion of insulin (Erion K.A. et al., JBC, 2015). SREBP is a key regulator of cholesterol and lipid synthesis and depends on TORC1 activity. The active form of SREBP is located in the nucleus. Does glucose-induced lipid synthesis in beta cells increase via SREBP? To answer this question, we propose: 1) To test the effect of high glucose (11mM) on nuclear SREBP in INS-1 cells in comparison to physiological glucose (4mM). 2) To determine if nuclear SREBP is affected when PIP4Kgamma (a regulator of TORC1) is suppressed. SREBP translocation from the cytosol to the nucleus was measured by immunofluorescence. SREBP processing was measured by western blot. SREBP1 activation increased in response to prolonged exposure to excess glucose after at least 48hrs. Both translocation and processing increased in 11mM glucose compared to 4mM glucose. When PIP4Kgamma was suppressed in INS-1 cells, SREBP translocation was inhibited. Lipid droplet accumulation was measured by nile red staining and it was found that de novo lipid synthesis only contributes to a small fraction of total lipid droplets. In conclusion, SREBP is activated in beta cells when in excess glucose. This may allow for lipid accumulation and basal hypersecretion of insulin due to over nutrition.

Diabetes is a metabolic disease characterized by the loss of functional pancreatic beta cells. The incidence of diabetes has risen rapidly in recent decades, which has been attributed at least partially to alterations in host-associated microbial communities, or microbiota. It is hypothesized that the loss of important microbial functions from the microbiota of affected host populations plays a role in the mechanism of disease onset. Because the immune system also plays a causative role in diabetes progression, and it is well documented that immune cell development and function are regulated by the microbiota, most diabetes microbiota research has focused on the immune system. However, microbial regulation is also required for the development of many other important tissues, including stimulating differentiation and proliferation. We therefore explored the possibility that the microbiota plays a role in host beta cell development. Using the larval zebrafish as a model, we discovered that sterile or germ free (GF) larvae have a depleted beta cell mass compared to their siblings raised in the presence of bacteria and other microbes. This dissertation describes the discovery and characterization of a rare and novel bacterial gene, whose protein product is sufficient to rescue this beta cell developmental defect in the GF larvae. Importantly, these findings suggest a possible role for the microbiota in preventing or prolonging the eventual onset of diabetes through induction of robust beta cell development. Furthermore, the loss of rare bacterial products such as the one described herein could help to explain why low diversity microbial communities are correlated with diabetes.

Dietary antioxidant curcumin derived from turmeric has been suggested to decrease the risk of many chronic diseases. Much of the existing data for curcumin stem from experiments performed at supra-physiological concentrations (μM-mM) that are impossible to attain through oral ingestion. It was therefore hypothesized that curcumin at low plasma achievable concentration, though itself not acting as a direct antioxidant might up-regulate the intracellular antioxidants and thus helping combat oxidative stress and protect β-islet cells. The results indicated that Curcumin, DMC and BDMC were able to scavenge hydroxyl radicals, but showed little scavenging ability against superoxide and nitric oxide radicals. Nanomolar concentrations of curcuminoids easily prevented the deleterious effects of H₂O₂ in pancreatic <i>β</i>-islet RINm5F cells. Non of the curcuminoids showed a detrimental effect on insulin secretion, but the model did not allow assessment of any potential positive effect on insulin secretion. The findings confirmed that nanomolar concentrations of curcumin offered protection in pancreatic <i>β</i>-islet cells against H₂O₂-indicated damage by modulating the proportion of oxidised GSH (GSSG): reduced GSH in the favour of GSH and the increasing the activity of SOD. This increase in GSH and SOD levels was, at least in part, on account of an increase in GR, SOD-1 and SOD-2 gene expression. The intracellular mechanism driving this modulation of antioxidant gene was, by virtue of blocking the H₂O₂ induced NF-κB activation.

Type 1 diabetes is an autoimmune disease in which patients' insulin-secreting beta cells in pancreatic islets are destroyed by their own immune system, leading to unregulated blood glucose levels and severe complications. Its only treatment is intensive insulin therapy, which carries the risk of hypoglycemic episodes and can result in seizures, coma, and even death. Islet transplantation has recently become an alternative, albeit experimental, treatment for type 1 diabetes patients. More than one donor graft is usually required to render recipients insulin independent, making the shortage of donor tissue an extremely important challenge in islet transplantation. Identifying the cell type that has the ability to differentiate into islet-like tissue is an important area of study. In this study, I hypothesized that insulin secreting human islet-like clusters could be generated from pancreatic ductal cells, a potential pancreatic progenitor cell type. Islet-like clusters were generated using crude exocrine tissue from human cadaveric donors. This crude exocrine tissue contained a large number of ductal cells, as well as other pancreatic cell types. To evaluate insulin secretion by human islet-like clusters, a static incubation system was set up and tested using Min6 cells, a known insulin-secreting cell line. Using static incubation, significant increases in insulin secretion by islet-like clusters were observed when the clusters were exposed to higher glucose levels and GLP-1, a known insulin secretagogue. Presence of corresponding C-peptide secretion demonstrated that de novo insulin secretion occurred. Furthermore, basal insulin secretion increased as culture stages progressed. An attempt was made to generate islet-like clusters using ductal cells purified by fluorescent activated cell sorting or magnetic activated cell sorting. Nevertheless, it was difficult to ensure survival and proliferation of purified ductal cells. Further studies will be necessary to confirm the role of ductal cells in the generation of islet-like clusters using the crude exocrine tissue, as well as to identify factors that can promote ductal cells proliferation after cell sorting. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic insulin-secreting beta cell failure is central to the development of diabetes. Therapeutic applications targeted at understanding and manipulating beta cell destruction mechanisms should enhance the preservation of functional beta cell mass and prevent diabetes. To this end, we have demonstrated that diabetogenic assaults (e.g., endoplasmic reticulum stress, glucolipotoxicity, and pro-inflammatory cytokines) attenuate the activation of beta cell pro-survival signaling pathways via a stress-inducible molecule called Mitogen-inducible gene 6 (Mig6). We discovered that the overabundance of Mig6 exacerbates stress-induced beta cell apoptosis and inhibits insulin secretion. Conversely, the deficiency of Mig6 partially protected beta cells from DNA damage-induced cell death. Further, we established that Mig6 haploinsufficient mice retained islet integrity and function and exhibited greater beta cell mass recovery following treatment with multiple low doses of the beta cell toxin streptozotocin. These data suggest that Mig6 may be a therapeutic target for beta cell preservation in diabetes.

We hypothesized that with exercise training and the subsequent attenuation of hyperglycemia, β-cell adaptation to worsening insulin resistance would be maintained. Also, because classical stress-activated systems and oxidative stress are involved in hepatic insulin resistance we examined if exercise would be associated with improvements in hepatic markers of oxidative stress and inflammation. Exercise maintained fasted hyperinsulinemia and preserved normoglycemia in male Zucker diabetic fatty (ZDF) rats. β-cell function calculations indicate prolonged β-cell adaptation in exercised animals. Such improved β-cell function was associated with increased β-cell mass. Hypertrophy and replication contributed to expansion of β-cell mass; exercised animals had increased β-cell size and bromodeoxyuridine (BrdU) incorporation rates versus controls. Furthermore, we observed augmented β-cell-specific immunohistochemical staining of GLUT2 and Akt/PKB in exercised versus sedentary controls. We also observed large cytoplasmic ubiquitinated structures which form in response to oxidative stress in pancreatic tissue samples from hyperglycemic ZDF rats. In the exercised groups such aggregate numbers were reduced to numbers compared to those seen in younger non-diabetic basal ZDF animals and age-matched lean Zucker rats. With respect to the liver we investigated whether exercise alters kinases such as c-Jun NH2-terminal kinase (JNK) and IKKβ (as evidenced by IκBα levels) and related insulin receptor substrate-1 (IRS-1) serine phosphorylation which are associated with hepatic insulin resistance in obesity. On average, exercised animals ran 5250m/day which improved insulin sensitivity based on the homeostasis model assessment for insulin resistance (HOMA-IR) calculations, and maintained fed and fasted glucoregulation and glucose tolerance. Ten weeks of running decreased whole-body markers of inflammation and oxidative stress in the blood and in the liver. Exercise lowered circulating interleukin-6 (IL-6), haptoglobin, malondialdehyde (MDA) levels, and protein oxidation in the liver. Exercise reduced phosphorylated JNK (pJNK) indicating decreased JNK activity; in accordance serine phosphorylated IRS-1 was reduced in exercised rats. In conclusion, improvements in glucoregulation were associated with increased β-cell compensation at least in part due to a reduction in oxidative stress. Furthermore, we show exercise attenuates development of hyperglycemia in ZDF rats in association with decreases in plasma and hepatic markers of inflammation, oxidative stress, JNK activation, and serine phosphorylation of IRS-1.

Embryonic stem (ES) cells were used as a model system to understand the signalling events in pancreas development. ES cells were differentiated through known precursor stages towards the tissue of interest in order to recapitulate development in vitro. Thus, protocols directing differentiation of mouse ES cells towards definitive endoderm and pancreatic β-cells were developed. A combination of activin A and bone morphogenic protein 4 resulted in a population of enriched cells expressing genetic markers of definitive endoderm. In vitro differentiation of ES cells into functional pancreatic β-cells has only been partially successful, as it results in cells that are not fully differentiated or functional. This might be due to a lack of cues emanating from surrounding cells present in the developing pancreas. Conditioned media from the mouse MIN6 β-cell line were used on the basis that differentiated β- cells might send out signals affecting the differentiation of the surrounding islet cells. Mouse ES cells were enriched in definitive endoderm and then treated with MIN6 conditioned medium. Gene expression of the β-cell markers Insulin1, Insulin2, and Glucose transporter 2 was significantly increased relative to the untreated control group after 10 days of treatment with conditioned medium. This result was specific for conditioned medium from MIN6 cells as conditioned medium from a kidney-, a neuronal-, and an exocrine pancreatic cell line had no effect. In order to characterise the secreted factor(s) the conditioned medium was subjected to protein precipitation. The pancreatic differentiation factor was present in a protein fraction, suggesting that the factor(s) was proteinaceous. The protein in question was neither proinsulin nor insulin. This knowledge will support the efficient generation of insulin-secreting cells for diabetes therapy.