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Item Biomarkers of islet beta cell stress and death in type 1 diabetes(Springer Nature, 2018-11) Sims, Emily K.; Evans-Molina, Carmella; Tersey, Sarah A.; Eizirik, Decio L.; Mirmira, Raghavendra G.; Pediatrics, School of MedicineRecent work on the pathogenesis of type 1 diabetes has led to an evolving recognition of the heterogeneity of this disease, both with regards to clinical phenotype and responses to therapies to prevent or revert diabetes. This heterogeneity not only limits efforts to accurately predict clinical disease but also is reflected in differing responses to immunomodulatory therapeutics. Thus, there is a need for robust biomarkers of beta cell health, which could provide insight into pathophysiological differences in disease course, improve disease prediction, increase the understanding of therapeutic responses to immunomodulatory interventions and identify individuals most likely to benefit from these therapies. In this review, we outline current literature, limitations and future directions for promising circulating markers of beta cell stress and death in type 1 diabetes, including markers indicating abnormal prohormone processing, circulating RNAs and circulating DNAs.Item F-Actin regulation of SNARE-mediated insulin secretion(2013-10-07) Kalwat, Michael Andrew; Thurmond, Debbie C.; Atkinson, Simon; Hudmon, Andy; Mirmira, Raghavendra G.In response to glucose, pancreatic islet beta cells secrete insulin in a biphasic manner, and both phases are diminished in type 2 diabetes. In beta cells, cortical F-actin beneath the plasma membrane (PM) prevents insulin granule access to the PM and glucose stimulates remodeling of this cortical F-actin to allow trafficking of insulin granules to the PM. Glucose stimulation activates the small GTPase Cdc42, which then activates p21-activated kinase 1 (PAK1); both Cdc42 and PAK1 are required for insulin secretion. In conjunction with Cdc42-PAK1 signaling, the SNARE protein Syntaxin 4 dissociates from F-actin to allow SNARE complex formation and insulin exocytosis. My central hypothesis is that, in the pancreatic beta cell, glucose signals through a Cdc42-PAK1-mediated pathway to remodel the F-actin cytoskeleton to mobilize insulin granules to SNARE docking sites at the PM to evoke glucose stimulated second phase insulin secretion. To investigate this, PAK1 was inhibited in MIN6 beta cells with IPA3 followed by live-cell imaging of F-actin remodeling using the F-actin probe, Lifeact-GFP. PAK1 inhibition prevented normal glucose-induced F-actin remodeling. PAK1 inhibition also prevented insulin granule accumulation at the PM in response to glucose. The ERK pathway was implicated, as glucose-stimulated ERK activation was decreased under PAK1-depleted conditions. Further study showed that inhibition of ERK impaired insulin secretion and cortical F-actin remodeling. One of the final steps of insulin secretion is the fusion of insulin granules with the PM which is facilitated by the SNARE proteins Syntaxin 4 on the PM and VAMP2 on the insulin granule. PAK1 activation was also found to be critical for Syntaxin 4-F-actin complex dynamics in beta cells, linking the Cdc42-PAK1 signaling pathway to SNARE-mediated exocytosis. Syntaxin 4 interacts with the F-actin severing protein Gelsolin, and in response to glucose Gelsolin dissociates from Syntaxin 4 in a calcium-dependent manner to allow Syntaxin 4 activation. Disrupting the interaction between Syntaxin 4 and Gelsolin aberrantly activates endogenous Syntaxin 4, elevating basal insulin secretion. Taken together, these results illustrate that signaling to F-actin remodeling is important for insulin secretion and that F-actin and its binding proteins can impact the final steps of insulin secretion.Item Heat shock protein 90, a potential biomarker for type I diabetes: mechanisms of release from pancreatic beta cells(2016-05-23) Ocaña, Gail Jean; Blum, Janice Sherry, 1957-; Kaplan, Mark H.; Serezani, C. Henrique; Sun, JieHeat shock protein (HSP) 90 is a molecular chaperone that regulates diverse cellular processes by facilitating activities of various protein clients. Recent studies have shown serum levels of the alpha cytoplasmic HSP90 isoform are elevated in newly diagnosed type I diabetic patients, thus distinguishing this protein as a potential biomarker for pre-clinical type I diabetes mellitus (TIDM). This phase of disease is known to be associated with various forms of beta cell stress, including endoplasmic reticulum stress, insulitis, and hyperglycemia. Therefore, to test the hypothesis that HSP90 is released by these cells in response to stress, human pancreatic beta cells were subjected to various forms of stress in vitro. Beta cells released HSP90 in response to stimulation with a combination of cytokines that included IL-1β, TNF-α, and IFN-γ, as well as an agonist of toll-like receptor 3. HSP90 release was not found to result from cellular increases in HSP90AA1 gene or HSP90 protein expression levels. Rather, cell stress and ensuing cytotoxicity mediated by c-Jun N-terminal kinase (JNK) appeared to play a role in HSP90 release. Beta cell HSP90 release was attenuated by pre-treatment with tauroursodeoxycholic acid (TUDCA), which has been shown previously to protect beta cells against JNK-mediated, cytokine-induced apoptosis. Experiments here confirmed TUDCA reduced beta cell JNK phosphorylation in response to cytokine stress. Furthermore pharmacological inhibition and siRNA-mediated knockdown of JNK in beta cells also attenuated HSP90 release in response to cytokine stress. Pharmacological inhibition of HSP90 chaperone function exacerbated islet cell stress during the development of TIDM in vivo; however, it did not affect the overall incidence of disease. Together, these data suggest extracellular HSP90 could serve as a biomarker for preclinical TIDM. This knowledge may be clinically relevant in optimizing treatments aimed at restoring beta cell mass. The goal of such treatments would be to halt the progression of at-risk patients to insulin dependence and lifelong TIDM.Item High-throughput screening for insulin secretion modulators(Springer, 2021) Kalwat, Michael A.; Medicine, School of MedicineThe application of forward chemical genetics to insulin secretion in high-throughput has been uncommon because of high costs and technical challenges. However, with the advancement of secreted luciferase tools, it has become feasible for small laboratories to screen large numbers of compounds for effects on insulin secretion. The purpose of this chapter is to outline the methods involved in high-throughput screening for small molecules that chronically impact pancreatic beta cell function. Attention is given to specific points in the protocol that help to improve the dynamic range and reduce variability in the assay. Using this approach in 384-well format, at least 48 and as many as 144 plates can theoretically be processed per week. This protocol serves as a guideline and can be modified as required for alternate stimulation paradigms and improved upon as new technologies become available.Item Mechanisms of transcriptional regulation in the maintenance of β cell function(2015-05-08) Maganti Vijaykumar, Aarthi; Mirmira, Raghavendra G.; Thurmond, Debbie C.; Herring, Paul B.; Evans-Molina, Carmella; Mosley, Amber L.The islet β cell is central to the maintenance of glucose homeostasis as the β cell is solely responsible for the synthesis of Insulin. Therefore, better understanding of the molecular mechanisms governing β cell function is crucial to designing therapies for diabetes. Pdx1, the master transcription factor of the β cell, is required for the synthesis of proteins that maintain optimal β cell function such as Insulin and glucose transporter type 2. Previous studies showed that Pdx1 interacts with the lysine methyltransferase Set7/9, relaxing chromatin and increasing transcription. Because Set7/9 also methylates non-histone proteins, I hypothesized that Set7/9-mediated methylation of Pdx1 increases its transcriptional activity. I showed that recombinant and cellular Pdx1 protein is methylated at two lysine residues, Lys123 and Lys131. Lys131 is involved in Set7/9 mediated augmented transactivation of Pdx1 target genes. Furthermore, β cell-specific Set7/9 knockout mice displayed glucose intolerance and impaired insulin secretion, accompanied by a reduction in the expression of Pdx1 target genes. Our results indicate a previously unappreciated role for Set7/9 in the maintenance of Pdx1 activity and β cell function. β cell function is regulated on both the transcriptional and translational levels. β cell function is central to the development of type 1 diabetes, a disease wherein the β cell is destroyed by immune cells. Although the immune system is considered the primary instigator of the disease, recent studies suggest that defective β cells may initiate the autoimmune response. I tested the hypothesis that improving β cell function would reduce immune infiltration of the islet in the NOD mouse, a mouse model of spontaneous type 1 diabetes. Prediabetic NOD mice treated with pioglitazone, a drug that improves β cell function, displayed an improvement in β cell function, a reduction in β cell death, accompanied by reductions in β cell autoimmunity, indicating that β cell dysfunction assists in the development of type 1 diabetes. Therefore, understanding the molecular mechanisms involved in β cell function is essential for the development of therapies for diabetes.Item Regulation of endoplasmic reticulum calcium homeostasis in pancreatic β cells(2016-06-21) Tong, Xin; Evans-Molina, Carmella; Day, Richard; Tune, Johnathan; Fueger, Patrick T.; Dong, X. CharlieDiabetes mellitus is a group of metabolic diseases characterized by disordered insulin secretion from the pancreatic β cell and chronic hyperglycemia. In order to maintain adequate levels of insulin secretion, the β cell relies on a highly developed and active endoplasmic reticulum (ER). Calcium localized in this compartment serves as a cofactor for key proteins and enzymes involved in insulin production and maturation and is critical for ER health and function. The ER Ca2+ pool is maintained largely through activity of the sarco-endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) pump, which pumps two Ca2+ ions into the ER during each catalytic cycle. The goal of our research is to understand the molecular mechanisms through which SERCA2 maintains β cell function and whole body glucose metabolism. Our previous work has revealed marked dysregulation of β cell SERCA2 expression and activity under diabetic conditions. Using a mixture of pro-inflammatory cytokines to model the diabetic milieu, we found that SERCA2 activity and protein stability were decreased through nitric oxide and AMP-activated protein kinase (AMPK)mediated signaling pathways. Moreover, SERCA2 expression, intracellular Ca2+ storage, and β cell death under diabetic conditions were rescued by pharmacologic or genetic inhibition of AMPK. These findings provided novel insight into pathways leading to altered β cell Ca2+ homeostasis and reduced β cell survival in diabetes. To next define the role of SERCA2 in the regulation of whole body glucose homeostasis, SERCA2 heterozygous mice (S2HET) were challenged with high fat diet (HFD). Compare to wild-type controls, S2HET mice had lower serum insulin and significantly reduced glucose tolerance with similar adiposity and systemic and tissue specific insulin sensitivity, suggesting an impairment in insulin secretion rather than insulin action. Consistent with this, S2HET mice exhibited reduced β cell mass, decreased β cell proliferation, increased ER stress, and impaired insulin production and processing. Furthermore, S2HET islets displayed impaired cytosolic Ca2+ oscillations and reduced glucose-stimulated insulin secretion, while a small molecule SERCA2 activator was able to rescue these defects. In aggregate, these data suggest a critical role for SERCA2 and the maintenance of ER Ca2+ stores in the β cell compensatory response to diet induced obesity.Item Regulation of glucose homeostasis by Doc2b and Munc18 proteins.(2014-01) Ramalingam, Latha; Thurmond, Debbie C.; Elmendorf, Jeffrey S.; Mirmira, Raghavendra G.; Roach, Peter J.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.Item The roles of pancreatic hormones in regulating pancreas development and beta cell regeneration(2015-06-16) Ye, Lihua; Anderson, Ryan M.; Mirmira, Raghu G.; Roach, Peter J.; Fueger, Patrick T.; Skalnik, David G.Diabetes mellitus is a group of related metabolic diseases that share a common pathological mechanism: insufficient insulin signaling. Insulin is a hormone secreted from pancreatic β cells that promotes energy storage and consequently lowers blood glucose. In contrast, the hormone glucagon, released by pancreatic α cells, plays a critical complementary role in metabolic homeostasis by releasing energy stores and increasing blood glucose. Restoration of β cell mass in diabetic patients via β cell regeneration is a conceptually proven approach to finally curing diabetes. Moreover, in situ regeneration of β cells from endogenous sources would circumvent many of the obstacles encountered by surgical restoration of β cell mass via islet transplantation. Regeneration may occur both by β cell self-duplication and by neogenesis from non-β cell sources. Although the mechanisms regulating the β cell replication pathway have been highly investigated, the signals that regulate β cell neogenesis are relatively unknown. In this dissertation, I have used zebrafish as a genetic model system to investigate the process of β cell neogenesis following insulin signaling depletion by various modes. Specifically, I have found that after their ablation, β cells primarily regenerate from two discrete cellular sources: differentiation from uncommitted pancreatic progenitors and transdifferentiation from α cells. Importantly, I have found that insulin and glucagon play crucial roles in controlling β cell regeneration from both sources. As with metabolic regulation, insulin and glucagon play counter-balancing roles in directing endocrine cell fate specification. These studies have revealed that glucagon signaling promotes β cell formation by increasing differentiation of pancreas progenitors and by destabilizing α cell identity to promote α to β cell transdifferentiation. In contrast, insulin signaling maintains pancreatic progenitors in an undifferentiated state and stabilizes α cell identity. Finally, I have shown that insulin also regulates pancreatic exocrine cell development. Insufficient insulin signaling destabilized acinar cell fate and impairs exocrine pancreas development. By understanding the roles of pancreatic hormones during pancreas development and regeneration can provide new therapeutic targets for in vivo β cell regeneration to remediate the devastating consequences of diabetes.Item Step-growth thiol-ene photopolymerization to form degradable, cytocompatible and multi-structural hydrogels(2014-01-17) Shih, Han; Lin, Chien-Chi; Xie, Dong; Bottino, MarcoHydrogels prepared from photopolymerization have been used for a variety of tissue engineering and controlled release applications. Polymeric biomaterials with high cytocompatibility, versatile degradation behaviors, and diverse material properties are particularly useful in studying cell fate processes. In recent years, step-growth thiol-ene photochemistry has been utilized to form cytocompatible hydrogels for tissue engineering applications. This radical-mediated gelation scheme utilizes norbornene functionalized multi-arm poly(ethylene glycol) (PEGNB) as the macromer and di-thiol containing molecules as the crosslinkers to form chemically crosslinked hydrogels. While the gelation mechanism was well-described in the literature, the network properties and degradation behaviors of these hydrogels have not been fully characterized. In addition, existing thiol-ene photopolymerizations often used type I photoinitiators in conjunction with an ultraviolet (UV) light source to initiate gelation. The use of cleavage type initiators and UV light often raises biosafety concerns. The first objective of this thesis was to understand the gelation and degradation properties of thiol-ene hydrogels. In this regard, two types of step-growth hydrogels were compared, namely thiol-ene hydrogels and Michael-type addition hydrogels. Between these two step-growth gel systems, it was found that thiol-ene click reactions formed hydrogels with higher crosslinking efficiency. However, thiol-ene hydrogels still contained significant network non-ideality, demonstrated by a high dependency of hydrogel swelling on macromer contents. In addition, the presence of ester bonds within the PEGNB macromer rendered thiol-ene hydrogels hydrolytically degradable. Through validating model predictions with experimental results, it was found that the hydrolytic degradation of thiol-ene hydrogels was not only governed by ester bond hydrolysis, but also affected by the degree of network crosslinking. In an attempt to manipulate network crosslinking and degradation rate of thiol-ene hydrogels, different macromer contents and peptide crosslinkers with different amino acid sequences were used. A chymotrypsin-sensitive peptide was also used as part of the hydrogel crosslinkers to render thiol-ene hydrogels enzymatically degradable. The second objective of this thesis was to develop a visible light-mediated thiol-ene hydrogelation scheme using a type II photoinitiator, eosin-Y, as the only photoinitiator. This approach eliminates the incorporation of potentially cytotoxic co-initiator and co-monomer that are typically used with a type II initiator. In addition to investigating the gelation kinetics and properties of thiol-ene hydrogels formed by this new gelation scheme, it was found that the visible light-mediated thiol-ene hydrogels were highly cytocompatible for human mesenchymal stem cells (hMSCs) and pancreatic MIN6 beta-cells. It was also found that eosin-Y could be repeatedly excited for preparing step-growth hydrogels with multilayer structures. This new gelation chemistry may have great utilities in controlled release of multiple sensitive growth factors and encapsulation of multiple cell types for tissue regeneration.Item Stress-inducible Mig6 promotes pancreatic beta cell destruction in the pathogenesis of diabetes(2014-12-08) Chen, Yi-Chun; Fueger, Patrick T.; Day, Richard N.; Elmendorf, Jeffrey S.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.