Browsing by Author "Tong, Xin"
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Item Cigarette smoke exposure impairs β-cell function through activation of oxidative stress and ceramide accumulation(Elsevier, 2020-07) Tong, Xin; Chaudhry, Zunaira; Lee, Chih-Chun; Bone, Robert N.; Kanojia, Sukrati; Maddatu, Judith; Sohn, Paul; Weaver, Staci A.; Robertson, Morgan A.; Petrache, Irina; Evans-Molina, Carmella; Kono, Tatsuyoshi; Medicine, School of MedicineObjectives Epidemiological studies indicate that first- and second-hand cigarette smoke (CS) exposure are important risk factors for the development of type 2 diabetes (T2D). Additionally, elevated diabetes risk has been reported to occur within a short period of time after smoking cessation, and health risks associated with smoking are increased when combined with obesity. At present, the mechanisms underlying these associations remain incompletely understood. The objective of this study was to test the impact of CS exposure on pancreatic β-cell function using rodent and in vitro models. Methods Beginning at 8 weeks of age, C57BL/6 J mice were concurrently fed a high-fat diet (HFD) and exposed to CS for 11 weeks, followed by an additional 11 weeks of smoking cessation with continued HFD. Glucose tolerance testing was performed during CS exposure and during the cessation period. Cultured INS-1 β-cells and primary islets were exposed ex vivo to CS extract (CSE), and β-cell function and viability were tested. Since CS increases ceramide accumulation in the lung and these bioactive sphingolipids have been implicated in pancreatic β-cell dysfunction in diabetes, islet and β-cell sphingolipid levels were measured in islets from CS-exposed mice and in CSE-treated islets and INS-1 cells using liquid chromatography-tandem mass spectrometry. Results Compared to HFD-fed, ambient air-exposed mice, HFD-fed and CS-exposed mice had reduced weight gain and better glucose tolerance during the active smoking period. Following smoking cessation, CS-mice exhibited rapid weight gain and had accelerated worsening of their glucose tolerance. CS-exposed mice had higher serum proinsulin/insulin ratios, indicative of β-cell dysfunction, significantly lower β-cell mass (p = 0.017), reduced β-cell proliferation (p = 0.006), and increased islet ceramide content compared to non-smoking control mice. Ex vivo exposure of isolated islets to CSE was sufficient to increase islet ceramide levels, which was correlated with reduced insulin gene expression and glucose-stimulated insulin secretion, and increased β-cell oxidative and endoplasmic reticulum (ER) stress. Treatment with the antioxidant N-acetylcysteine markedly attenuated the effects of CSE on ceramide levels, restored β-cell function and survival, and increased cyclin D2 expression, while also reducing activation of β-cell ER and oxidative stress. Conclusions Our results indicate that CS exposure leads to impaired insulin production, processing, secretion and reduced β-cell viability and proliferation. These effects were linked to increased β-cell oxidative and ER stress and ceramide accumulation. Mice fed HFD continued to experience detrimental effects of CS exposure even during smoking cessation. Elucidation of the mechanisms by which CS exposure impairs β-cell function in synergy with obesity will help design therapeutic and preventive interventions for both active and former smokers.Item Endoplasmic reticulum stress alters ryanodine receptor function in the murine pancreatic β cell(American Society for Biochemistry and Molecular Biology, 2018-11-12) Yamamoto, Wataru R.; Bone, Robert N.; Sohn, Paul; Syed, Farooq; Reissaus, Christopher A.; Mosley, Amber L.; Wijeratne, Aruna B.; True, Jason D.; Tong, Xin; Kono, Kono; Evans-Molina, Carmella; Biochemistry and Molecular Biology, School of MedicineAlterations in endoplasmic reticulum (ER) calcium (Ca2+) levels diminish insulin secretion and reduce β-cell survival in both major forms of diabetes. The mechanisms responsible for ER Ca2+ loss in β cells remain incompletely understood. Moreover, a specific role for either ryanodine receptor (RyR) or inositol 1,4,5-triphosphate receptor (IP3R) dysfunction in the pathophysiology of diabetes remains largely untested. To this end, here we applied intracellular and ER Ca2+ imaging techniques in INS-1 β cells and isolated islets to determine whether diabetogenic stressors alter RyR or IP3R function. Our results revealed that the RyR is sensitive mainly to ER stress–induced dysfunction, whereas cytokine stress specifically alters IP3R activity. Consistent with this observation, pharmacological inhibition of the RyR with ryanodine and inhibition of the IP3R with xestospongin C prevented ER Ca2+ loss under ER and cytokine stress conditions, respectively. However, RyR blockade distinctly prevented β-cell death, propagation of the unfolded protein response (UPR), and dysfunctional glucose-induced Ca2+ oscillations in tunicamycin-treated INS-1 β cells and mouse islets and Akita islets. Monitoring at the single-cell level revealed that ER stress acutely increases the frequency of intracellular Ca2+ transients that depend on both ER Ca2+ leakage from the RyR and plasma membrane depolarization. Collectively, these findings indicate that RyR dysfunction shapes ER Ca2+ dynamics in β cells and regulates both UPR activation and cell death, suggesting that RyR-mediated loss of ER Ca2+ may be an early pathogenic event in diabetes.Item Human adipose derived stromal/stem cells (hASCs) protect against STZ-induced hyperglycemia; analysis of hASC-derived paracrine effectors(Wiley, 2014-07) Kono, Tatsuyoshi M.; Sims, Emily K.; Moss, Dan R.; Yamamoto, Wataru; Ahn, Geonyoung; Diamond, Julie; Tong, Xin; Day, Kathleen H.; Territo, Paul R.; Hanenberg, Helmut; Traktuev, Dmitry O.; March, Keith L.; Evans-Molina, Carmella; Department of Medicine, IU School of MedicineAdipose-derived stromal/stem cells (ASCs) ameliorate hyperglycemia in rodent models of islet transplantation and autoimmune diabetes, yet the precise human ASC (hASC)-derived factors responsible for these effects remain largely unexplored. Here, we show that systemic administration of hASCs improved glucose tolerance, preserved β cell mass, and increased β cell proliferation in streptozotocin-treated nonobese diabetic/severe combined immunodeficient mice. Coculture experiments combining mouse or human islets with hASCs demonstrated that islet viability and function were improved by hASCs following prolonged culture or treatment with proinflammatory cytokines. Analysis of hASC-derived factors revealed vascular endothelial growth factor and tissue inhibitor of metalloproteinase 1 (TIMP-1) to be highly abundant factors secreted by hASCs. Notably, TIMP-1 secretion increased in the presence of islet stress from cytokine treatment, while TIMP-1 blockade was able to abrogate in vitro prosurvival effects of hASCs. Following systemic administration by tail vein injection, hASCs were detected in the pancreas and human TIMP-1 was increased in the serum of injected mice, while recombinant TIMP-1 increased viability in INS-1 cells treated with interleukin-1beta, interferon-gamma, and tumor necrosis factor alpha. In aggregate, our data support a model whereby factors secreted by hASCs, such as TIMP-1, are able to mitigate against β cell death in rodent and in vitro models of type 1 diabetes through a combination of local paracrine as well as systemic effects.Item Impaired Store-Operated Calcium Entry and STIM1 Loss Lead to Reduced Insulin Secretion and Increased Endoplasmic Reticulum Stress in the Diabetic β-Cell(American Diabetes Association, 2018-11) Kono, Tatsuyoshi; Tong, Xin; Taleb, Solaema; Bone, Robert N.; Iida, Hitoshi; Lee, Chih-Chun; Sohn, Paul; Gilon, Patrick; Roe, Michael W.; Evans-Molina, Carmella; Medicine, School of MedicineStore-operated Ca2+ entry (SOCE) is a dynamic process that leads to refilling of endoplasmic reticulum (ER) Ca2+ stores through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor Stromal Interaction Molecule 1 (STIM1). Pathogenic reductions in β-cell ER Ca2+ have been observed in diabetes. However, a role for impaired SOCE in this phenotype has not been tested. We measured the expression of SOCE molecular components in human and rodent models of diabetes and found a specific reduction in STIM1 mRNA and protein levels in human islets from donors with type 2 diabetes (T2D), islets from hyperglycemic streptozotocin-treated mice, and INS-1 cells (rat insulinoma cells) treated with proinflammatory cytokines and palmitate. Pharmacologic SOCE inhibitors led to impaired islet Ca2+ oscillations and insulin secretion, and these effects were phenocopied by β-cell STIM1 deletion. STIM1 deletion also led to reduced ER Ca2+ storage and increased ER stress, whereas STIM1 gain of function rescued β-cell survival under proinflammatory conditions and improved insulin secretion in human islets from donors with T2D. Taken together, these data suggest that the loss of STIM1 and impaired SOCE contribute to ER Ca2+ dyshomeostasis under diabetic conditions, whereas efforts to restore SOCE-mediated Ca2+ transients may have the potential to improve β-cell health and function.Item MicroRNA 21 targets BCL2 mRNA to increase apoptosis in rat and human beta cells(Springer, 2017-06) Sims, Emily K.; Lakhter, Alexander; Anderson-Baucum, Emily; Kono, Tatsuyoshi; Tong, Xin; Evans-Molina, Carmella; Pediatrics, School of MedicineAIMS/HYPOTHESIS: The role of beta cell microRNA (miR)-21 in the pathophysiology of type 1 diabetes has been controversial. Here, we sought to define the context of beta cell miR-21 upregulation in type 1 diabetes and the phenotype of beta cell miR-21 overexpression through target identification. METHODS: Islets were isolated from NOD mice and mice treated with multiple low doses of streptozotocin, as a mouse model of diabetes. INS-1 832/13 beta cells and human islets were treated with IL-1β, IFN-γ and TNF-α to mimic the milieu of early type 1 diabetes. Cells and islets were transfected with miR-21 mimics or inhibitors. Luciferase assays and polyribosomal profiling (PRP) were performed to define miR-21-target interactions. RESULTS: Beta cell miR-21 was increased in in vivo models of type 1 diabetes and cytokine-treated cells/islets. miR-21 overexpression decreased cell count and viability, and increased cleaved caspase 3 levels, suggesting increased cell death. In silico prediction tools identified the antiapoptotic mRNA BCL2 as a conserved miR-21 target. Consistent with this, miR-21 overexpression decreased BCL2 transcript and B cell lymphoma 2 (BCL2) protein production, while miR-21 inhibition increased BCL2 protein levels and reduced cleaved caspase 3 levels after cytokine treatment. miR-21-mediated cell death was abrogated in 828/33 cells, which constitutively overexpress Bcl2. Luciferase assays suggested a direct interaction between miR-21 and the BCL2 3' untranslated region. With miR-21 overexpression, PRP revealed a shift of the Bcl2 message towards monosome-associated fractions, indicating inhibition of Bcl2 translation. Finally, overexpression in dispersed human islets confirmed a reduction in BCL2 transcripts and increased cleaved caspase 3 production. CONCLUSIONS/INTERPRETATION: In contrast to the pro-survival role reported in other systems, our results demonstrate that miR-21 increases beta cell death via BCL2 transcript degradation and inhibition of BCL2 translation.Item Pancreatic and duodenal homeobox protein 1 (Pdx-1) maintains endoplasmic reticulum calcium levels through transcriptional regulation of sarco-endoplasmic reticulum calcium ATPase 2b (SERCA2b) in the islet β cell(American Society for Biochemistry and Molecular Biology, 2014-11-21) Johnson, Justin S.; Kono, Tatsuyoshi; Tong, Xin; Yamamoto, Wataru R.; Zarain-Herzberg, Angel; Merrins, Matthew J.; Satin, Leslie S.; Gilon, Patrick; Evans-Molina, Carmella; Department of Medicine, IU School of MedicineAlthough the pancreatic duodenal homeobox 1 (Pdx-1) transcription factor is known to play an indispensable role in β cell development and secretory function, recent data also implicate Pdx-1 in the maintenance of endoplasmic reticulum (ER) health. The sarco-endoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) gradient between the cytosol and ER lumen. In models of diabetes, our data demonstrated loss of β cell Pdx-1 that occurs in parallel with altered SERCA2b expression, whereas in silico analysis of the SERCA2b promoter revealed multiple putative Pdx-1 binding sites. We hypothesized that Pdx-1 loss under inflammatory and diabetic conditions leads to decreased SERCA2b levels and activity with concomitant alterations in ER health. To test this, siRNA-mediated knockdown of Pdx-1 was performed in INS-1 cells. The results revealed reduced SERCA2b expression and decreased ER Ca(2+), which was measured using fluorescence lifetime imaging microscopy. Cotransfection of human Pdx-1 with a reporter fused to the human SERCA2 promoter increased luciferase activity 3- to 4-fold relative to an empty vector control, and direct binding of Pdx-1 to the proximal SERCA2 promoter was confirmed by chromatin immunoprecipitation. To determine whether restoration of SERCA2b could rescue ER stress induced by Pdx-1 loss, Pdx1(+/-) mice were fed a high-fat diet. Isolated islets demonstrated an increased spliced-to-total Xbp1 ratio, whereas SERCA2b overexpression reduced the Xbp1 ratio to that of wild-type controls. Together, these results identify SERCA2b as a novel transcriptional target of Pdx-1 and define a role for altered ER Ca(2+) regulation in Pdx-1-deficient states.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 SERCA2 Deficiency Impairs Pancreatic β-Cell Function in Response to Diet-Induced Obesity(American Diabetes Association, 2016-10) Tong, Xin; Kono, Tatsuyoshi; Anderson-Baucum, Emily K.; Yamamoto, Wataru; Gilon, Patrick; Lebeche, Djamel; Day, Richard N.; Shull, Gary E.; Evans-Molina, Carmella; Cellular and Integrative Physiology, School of MedicineThe sarcoendoplasmic reticulum (ER) Ca2+ ATPase 2 (SERCA2) pump is a P-type ATPase tasked with the maintenance of ER Ca2+ stores. Whereas β-cell SERCA2 expression is reduced in diabetes, the role of SERCA2 in the regulation of whole-body glucose homeostasis has remained uncharacterized. To this end, SERCA2 heterozygous mice (S2HET) were challenged with a high-fat diet (HFD) containing 45% of kilocalories from fat. After 16 weeks of the HFD, S2HET mice were hyperglycemic and glucose intolerant, but adiposity and insulin sensitivity were not different between HFD-fed S2HET mice and HFD-fed wild-type controls. Consistent with a defect in β-cell function, insulin secretion, glucose-induced cytosolic Ca2+ mobilization, and the onset of steady-state glucose-induced Ca2+ oscillations were impaired in HFD-fed S2HET islets. Moreover, HFD-fed S2HET mice exhibited reduced β-cell mass and proliferation, altered insulin production and proinsulin processing, and increased islet ER stress and death. In contrast, SERCA2 activation with a small molecule allosteric activator increased ER Ca2+ storage and rescued tunicamycin-induced β-cell death. In aggregate, these data suggest a critical role for SERCA2 and the regulation of ER Ca2+ homeostasis in the β-cell compensatory response to diet-induced obesity.Item SERCA2 regulates proinsulin processing and processing enzyme maturation in pancreatic beta cells(Springer, 2023) Iida, Hitoshi; Kono, Tatsuyoshi; Lee, Chih‑Chun; Krishnan, Preethi; Arvin, Matthew C.; Weaver, Staci A.; Jarvela, Timothy S.; Branco, Renato C. S.; McLaughlin, Madeline R.; Bone, Robert N.; Tong, Xin; Arvan, Peter; Lindberg, Iris; Evans‑Molina, Carmella; Medicine, School of MedicineAims/hypothesis: Increased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in type 1 and type 2 diabetes. Previous studies have suggested that Ca2+ signalling within beta cells regulates insulin processing and secretion; however, the mechanisms that link impaired Ca2+ signalling with defective insulin maturation remain incompletely understood. Methods: We generated mice with beta cell-specific sarcoendoplasmic reticulum Ca2+ ATPase-2 (SERCA2) deletion (βS2KO mice) and used an INS-1 cell line model of SERCA2 deficiency. Whole-body metabolic phenotyping, Ca2+ imaging, RNA-seq and protein processing assays were used to determine how loss of SERCA2 impacts beta cell function. To test key findings in human model systems, cadaveric islets were treated with diabetogenic stressors and prohormone convertase expression patterns were characterised. Results: βS2KO mice exhibited age-dependent glucose intolerance and increased plasma and pancreatic levels of proinsulin, while endoplasmic reticulum (ER) Ca2+ levels and glucose-stimulated Ca2+ synchronicity were reduced in βS2KO islets. Islets isolated from βS2KO mice and SERCA2-deficient INS-1 cells showed decreased expression of the active forms of the proinsulin processing enzymes PC1/3 and PC2. Additionally, immunofluorescence staining revealed mis-location and abnormal accumulation of proinsulin and proPC2 in the intermediate region between the ER and the Golgi (i.e. the ERGIC) and in the cis-Golgi in beta cells of βS2KO mice. Treatment of islets from human donors without diabetes with high glucose and palmitate concentrations led to reduced expression of the active forms of the proinsulin processing enzymes, thus phenocopying the findings observed in βS2KO islets and SERCA2-deficient INS-1 cells. Similar findings were observed in wild-type mouse islets treated with brefeldin A, a compound that perturbs ER-to-Golgi trafficking. Conclusions/interpretation: Taken together, these data highlight an important link between ER Ca2+ homeostasis and proinsulin processing in beta cells. Our findings suggest a model whereby chronic ER Ca2+ depletion due to SERCA2 deficiency impairs the spatial regulation of prohormone trafficking, processing and maturation within the secretory pathway. Data availability: RNA-seq data have been deposited in the Gene Expression Omnibus (GEO; accession no.: GSE207498).Item Sirt6 Regulates Insulin Secretion from the Pancreatic Beta Cells(Office of the Vice Chancellor for Research, 2015-04-17) Xiong, Xiwen; Wang, Gaihong; Tao, Rongya; Wu, Pengfei; Kono, Tatsuyoshi; Tong, Xin; Tersey, Sarah A.; Harris, Robert A.; Evans-Molina, Carmella; Mirmira, Raghavendra G.; Dong, X. CharlieSirt6 is an NAD-dependent histone deacetylase, which is involved in multiple biological processes, including aging, DNA repair, and metabolism; however, it is unclear what its functions in pancreatic beta-cells are. The beta cells play an essential role in metabolic regulation by secreting insulin in response to an elevated glucose concentration in the circulation. To examine the role of Sirt6 in beta cells, we initially used adenovirus-mediated shRNA to knock down the Sirt6 gene expression in a mouse pancreatic beta cell line - MIN6. Knockdown of the Sirt6 gene significantly reduced glucose-stimulated insulin secretion. To further validate this phenotype in vivo, we generated pancreatic beta-cell-specific Sirt6 knockout mice (bKO) using mouse genetic approach. Indeed, the bKO mice showed remarkable impairment in both first and second phases of insulin secretion in response to a glucose load. While morphometric analyses did not reveal significant difference in islet area between wild-type and bKO mice, biochemical analysis of ATP concentrations showed a 22% decrease in bKO mouse islets relative to control wild-type islets after glucose stimulation. To assess mitochondrial function in Sirt6-deficient beta cells, we also performed Seahorse bioenergetics assays in MIN6 cells after the Sirt6 gene was knocked down. Glucose oxidation in mitochondria was decreased 20-30% in Sirt6- knockdown MIN6 cells as compared to the control cells. Since calcium signaling is critical to insulin secretion, we also measured intracellular calcium concentrations using a fluorescent imaging approach. The results showed a significant decrease in cytoplasmic calcium in the bKO islets as compared to the wild-type controls. Overall, our data demonstrate that Sirt6 plays a critical role in the regulation of pancreatic insulin secretion. This work was supported in part by the NIDDK grant R01DK091592.