Induce Beta Cell Research Articles

Long-term c-Kit overexpression in beta cells compromises their function in ageing mice.

c-Kit signalling regulates intracellular pathways that enhance beta cell proliferation, insulin secretion and islet vascularisation in mice up to 28weeks of age and on short-term high-fat diet. However, long-term c-Kit activation in ageing mouse islets has yet to be examined. This study utilises beta cell-specific c-Kit-overexpressing transgenic (c-KitβTg) ageing mice (~60weeks) to determine the effect of its activation on beta cell dysfunction and insulin secretion. Wild-type and c-KitβTg mice, aged 60weeks, were examined using metabolic tests to determine glucose tolerance and insulin secretion. Pancreas histology and proteins in isolated islets were examined to determine the expression of beta cell transcription factors, proliferation and intracellular signalling. To determine the role of insulin receptor signalling in ageing c-KitβTg mice, we generated beta cell-specific inducible insulin receptor knockout in ageing c-KitβTg mice (c-KitβTg;βIRKO mice) and examined the ageing mice for glucose tolerance and islet histology. Ageing c-KitβTg mice progressively developed glucose intolerance, compared with age-matched wild-type littermates, due to impaired insulin secretion. Increased beta cell mass, proliferation and nuclear forkhead box transcription factor O1 (FOXO1) expression and reduced exocytotic protein levels were detected in ageing c-KitβTg mouse islets. Protein analyses of isolated islets showed increased insulin receptor, phosphorylated IRS-1Ser612 and cleaved poly(ADP-ribose) polymerase levels in ageing c-KitβTg mice. Ageing c-KitβTg mouse islets treated ex vivo with insulin demonstrated reduced Akt phosphorylation, indicating that prolonged c-Kit induced beta cell insulin insensitivity. Ageing c-KitβTg;βIRKO mice displayed improved glucose tolerance and beta cell function compared with ageing c-KitβTg mice. These findings indicate that long-term c-Kit overexpression in beta cells has a negative impact on insulin exocytosis and that temporally dependent regulation of c-Kit-insulin receptor signalling is important for optimal beta cell function.

Bromodomain inhibition instructs macrophages to promote beta cell proliferation and suppress autoimmune diabetes

Abstract A hallmark of autoimmune type 1 diabetes (T1D) is the decline of beta cell mass. Any regimen to promote beta cell expansion will have a great therapeutic promise. We have previously demonstrated that an epigenetic inhibitor targeting BET proteins (I-BET) promotes beta cell proliferation in an animal model of T1D. However, the underlying mechanisms remains unknown. Using both gain-of and loss-of function assays, we demonstrate that macrophages play an indispensable role in I-BET-elicited beta cell proliferation. In vivo ablation of macrophages significantly abolishes, while the supplementation of I-BET preconditioned macrophages enhances, I-BET induced beta cell proliferation. On a molecular level, elevated production of platelet-derived growth factor (PDGF) in I-BET treated macrophages mobilizes PDGFR signaling in beta cells. In summary, these findings reveal a novel strategy to expand beta cells by harnessing macrophages.

Nuclear receptor TLX regulates islet beta cell proliferation via E2F6

Both type 1 and type 2 diabetes are associated with loss of functional beta cell mass, and strategies to restore beta cells are urgently needed. We reported previously that overexpression of the nuclear receptor TLX induces beta cell proliferation, but the underlying molecular mechanism has not been defined. Here, we identified direct targets of TLX in beta cells at the genome-wide level by ChIP-Seq. These targets include a cadre of regulators that are known to be critical for proliferation. Among these ChIP targets, E2F6 was tightly associated with the cell cycle modules, and thus, we further analyzed E2F6 expression and function in beta cells. We showed that E2F6 is strongly downregulated by TLX, and its expression inhibits beta cell proliferation. Moreover, coexpression of E2F6 with TLX partially abrogated the proliferative effects of TLX. These results strongly suggest that TLX acts through E2F6 to regulate beta cell proliferation. Together, the results of this study reveal a direct interaction between TLX and E2F6 and suggest new targets for the expansion of functional beta cell mass.

DPP4 inhibitor induces beta cell regeneration and DDR-1 protein expression as an endocrine progenitor cell marker in neonatal STZ-diabetic rats.

We aim to investigate the effects of dipeptidyl-peptidase-4 inhibitor (Vildagliptin-VG) on DDR-1 as a marker for endocrine progenitor cells, β-cell regeneration, and apoptosis in neonatal streptozotocin (n2-STZ) diabetics. Neonatal rats were divided into two main groups as short- and long-term treatment, each consisted of four groups; (1) Control, (2) n2-STZ diabetic (single dose of 100 mg/kg STZ at 2nd day of birth), (3) n2-STZ + VG (60 mg/kg/day VG orally; for 8 and 28 days), (4) VG (60 mg/kg/day orally; for 8 and 28 days). Blood glucose levels and body weights were measured, and the tissue sections were immunostained using insulin, glucagon, somatostatin, PCNA, Pdx-1 and DDR-1 antibodies. The TUNEL method was used for apoptosis. The number of β cells in islets of the n2-STZ + VG group increased compared to the n2-STZ group; insulin (+) cells were observed individually or as small clusters in exocrine tissue, between pancreatic duct epithelial cells, and around the ducts. The number of Pdx-1 and DDR-1 positive cells in islet and extra-islet pancreas tissue was elevated as a result of VG application compared to the STZ diabetic group; the number of double positive cells for DDR-1 and insulin increased in n2-STZ + VG rats. We showed that vildagliptin promotes β cell neogenesis and regeneration, stimulates DDR-1 expression as an endocrine cell progenitor marker, suppresses apoptosis, induces islet cell proliferation and rearranges islet morphology in the n2-STZ diabetes model.

The Role of Glucagon-Like Peptide-1 Receptor Agonists in Type 2 Diabetes in Asia

The improvement of glycemic control in patients with type 2 diabetes (T2D) is an urgent need in Asia. East Asian T2D patients have distinct characteristics, including relatively low body weight, early impairment of islet beta cell function with reduced insulin secretion and a marked increase in postprandial blood glucose levels. Control of postprandial hyperglycemia and beta cell preservation are key elements of the therapeutic strategy for these patients. Glucagon-like peptide-1 receptor agonists (GLP-1RAs) reduce HbA1c, fasting plasma glucose, and (in particular) postprandial glucose levels, and slow gastric emptying, while minimizing the risk of hypoglycemia and weight gain. Compared with long-acting GLP-1RAs, short-acting GLP-1RAs produce greater slowing of gastric emptying (which is a key factor driving the reduction of postprandial glycemia) and induce beta cell rest rather than promoting postprandial insulin secretion when used under physiological conditions. GLP-1RAs have greater efficacy in Asian than Caucasian patients. GLP-1RA add-on therapy provides clinically meaningful reductions in HbA1c and postprandial glucose in Asians with T2D inadequately controlled by oral antidiabetic drugs (OADs) or basal insulin ± OADs. Premixed insulin is often prescribed for T2D patients in China. A short-acting GLP-1RA plus basal insulin is an alternative to premixed insulin, resulting in better efficacy and a lower risk of hypoglycemia and weight gain. In conclusion, GLP-1RAs, especially short-acting GLP-1RAs, are a practical treatment option for East Asian patients with T2D inadequately controlled by OADs or basal insulin.Funding: Sanofi.

Autologous Peripheral Blood Mononuclear Cells can Lead to Pancreatic Beta Cell Regeneration

Introduction: Type-1 diabetes (T1D) is a disease that leads to pancreatic beta cells death, resulting in complete insulin loss. T1D occurs as a result of autoimmunity which takes insulin producing β cells as a target and is considered as a metabolic disorder causing hyperglycemia resulting from a serious loss of endogenous insulin roduction. Using cadaveric islets as a replacement therapy was done to reverse diabetes. Many various progenitor cell types have been found in the peripheral blood mononuclear cell (PBMC) fraction denoting hose PBMCs may have the ability to differentiate into many mature functional cell types in certain microenvironments. It is settled that circulating peripheral blood mononuclear cells (PBMC) contain germinal cell population which can share in the regeneration of tissues in different organs. Materials and Methods: 2 groups of type I diabetes patients had been monitored in a private clinic, number 40 each group, with 35 females and 45 males. It lied between 8-25 years old with 4-7 years of diabetes onset. The first group was on insulin therapy and they received peripheral blood mononuclear cells concentrate directly in the dorsal pancreatic artery. The second group was only on insulin injection. Results: The results show a significant increase in c peptide levels after direct injection of peripheral blood mononuclear cells in the dorsal pancreatic artery with p-value less than 0.0001. Conclusion: The peripheral blood mononuclear cells can induce beta cell regeneration and increase beta cell mass which is detected by an increase in c peptide levels.

Growth Factors in Platelet Rich Plasma can Regenerate Pancreatic Beta Cells in Type 2 Diabetes

Introduction: 387 million people are considered to have diabetes all over the world, and the number is expected to increase miserably to 592 million by 2035. Alternative ways to create β-cells from endogenous sources should be found as a way for the evolution of treatment. This is to bypass the complication of tissue matching and surgical procedures. To date several rebuilding approaches have been developed to stimulate β-cells regeneration through the induction of the proliferation of remaining β-cells, neo-genesis; de novo islet formation from pancreatic progenitor cells, and trans-differentiation; converting non-β-cells within the pancreas to β-cells. Platelet Rich Plasma (PRP) contains various growth factors which can be used for tissue regeneration including pancreatic beta cells. Materials and methods: 2 groups of Type-2 diabetes patients had been monitored in a private clinic, number 40 each group, with 30 females and 50 males. The first group patients relieved oral hypoglycemic drugs as usual but injected by PRP weekly by subcutaneous injection of 3 ml, the second group received oral drugs only. Results: There were significant increases in c-peptide levels in patients with PRP injection with dpp4 inhibitors and metformin for 3 months with p-value less than 0.0001. In the second group patients on oral therapy only there were no significant change in c-peptide levels after 3 months of oral hypoglycemic drugs. Conclusion: As Growth factors (GFs) are considered as a natural biological mediators which control growth, differentiation, and have a role in the process of tissue reform and regeneration. The growth factors in platelet-rich plasma can induce beta cell regeneration and increase beta cell mass by stimulating β-cell neo-genesis and through ductal cell differentiation into β-cells which is detected by an increase in c-peptide levels which may add to Type-2 diabetes treatment.

Effect of N-acyl-dopamines on beta cell differentiation and wound healing in diabetic mice

N-acyl-dopamines are endolipids with neuroprotective, antiinflammatory and immunomodulatory properties. Previously, we showed the ability of these compounds to induce HIF-1α stabilization. Hypoxia and HIF-1α play an important role in the most relevant stages of diabetic pathogenesis. This work analyzes the possible role of these molecules on beta cell differentiation, insulin production and diabetic foot ulcer. Hypoxia response pathway has been characterized in beta-cell differentiation in rat pancreatic acinar cell line and human islet-derived precursor cells. Protein and mRNA expression of key proteins in this process have been analyzed, as well as those involved in beta cells reprogramming. The effect of N-acyl-dopamines on hypoxia response pathway, beta cells reprogramming and insulin production have been studied in both cell types, as well as its role in angiogenesis models in vitro and wound closure in type 2 diabetic mice. Our results show how the hypoxia response pathway is altered during beta cells differentiation, accompanied by an induction of the transcription factor HIF-1α. We demonstrate how some N-acyl-dopamines induce beta cell differentiation and insulin production in two different cell models. In parallel, these endolipids promote angiogenesis in vitro and wound closure in type 2 diabetic mice. These results provide a biological mechanism through which some endolipids could induce beta cell differentiation. We demonstrate how N-acyl-dopamines can modulate insulin production and, in parallel, reverse HIF-1α inhibition in a wound healing model in diabetic mice. Therefore, the potential use of the pharmacological modulation of N-acyl-dopamines may have implications for diabetes prevention and treatment strategies.

Gastrin—A Potential Predictor of Glucoregulation in Newly Diagnosed Type 2 Diabetes Patients

Background and Aims: Experimental data demonstrated that activation of GLP-1 and gastrin signaling induces beta cell neogenesis, resulting in a promotion of glucose-induced insulin secretion. In addition, treatment with proton pump inhibitors is associated with greater glycemic control in patients with type 2 diabetes (T2DM), particularly in those on insulin- or GLP-1-based therapy. The aim of this study was to assess gastrin as a potential predictor of beta cell function and glucoregulation in newly diagnosed T2DM patients. Materials and Methods: In this cross sectional study 190 patients (64 males and 126 females) with new onset T2DM were included. Patients treated with IPPs were excluded. Fasting plasma glucose (FPG), postprandial PG, HbA1c, fasting insulin, pancreatic B cell function (HOMA-B), insulin resistance index (HOMA-IR), fasting c-peptide and gastrin levels were measured at the time of diagnosis. Results: Baseline HbA1c was 7.53±2.08%, average age of patients was 61.8±10.years and body mass index (BMI) was 31.25±5.73 kg/m2. Parameters of glucoregulation were not significantly correlated with gastrin (all p>0.05), while there was moderate negative correlation with HOMA-B (HbA1c, FPG and PPG; p <0.01 for all) and positive correlation with HOMA-IR (HbA1c, FPG and PPG; p<0.01 for all). Patients with higher baseline measures of glucose regulation had lower HOMA-B and higher HIMA-IR as was expected. There was no association between gastrin and HOMA-B (p>0.05) or HOMA-IR. Furthermore, no association was established between c-peptide, insulin levels and gastrin (p>0.05). Conclusion: Baseline gastrin levels are not sufficient to have a significant effect on glucoregulation or HOMA-B and HOMA-IR in newly diagnosed T2DM, therefore it could be postulated that further stimulation of gastrin secretion (e.g., with IPPs or GLP-1 based therapy) is needed in order to influence beta cell function and glycemic control. Disclosure M. Cigrovski Berkovic: None. D. Herman Mahecic: None. I. Bilic-Curcic: None.

The Expression Differences of Cyclin Dependent Kinase Inhibitors In Aged and Young Pancreatic Beta Cells

The number of people affected by diabetes is continually rising, affecting over 400 million people worldwide. The insulin secreting pancreatic beta cells are essential to control proper glucose absorption and storage in insulin sensitive peripheral tissue. Both Type 1 and Type 2 diabetes are characterized by decreased functional beta cell mass and, consequently, decreased insulin production. One potential intervention is the use of beta cell transplantation from cadaveric donors. A major impediment to greater application of this treatment is the scarcity of transplant ready beta cells. Increasing the amount of functional beta cells will lead to increased insulin production and better management or a possible treatment strategy for the disease. Various genes have been defined that can induce beta cell replication. A major caveat of these findings, however, is that these factors induce replication in young beta cells and not aged beta cells. Given that the majority of beta cells that will be used for transplant will come from aged donors, it is imperative to understand why aged beta cells are refractory to these proliferative mechanisms. We aimed to determine why aged beta cells do not replicate as well as young beta cells. We hypothesized that one reason for why aged beta cells are refractory to genes that induce proliferation is greater expression of cell cycle inhibitors. The cell cycle is tightly regulated by cyclin‐dependent kinases. Cells also produce proteins known as cyclin‐dependent kinase inhibitors(CDKI's), which bind to cyclin dependent kinases, effectively shutting down cell proliferation. Here we demonstrate the expression of the INK4 and Cip/Kip family of CDKI's by mRNA, protein and histological expression in 5 week old and 5 month old primary rat beta cells. These results begin to define the functional changes that result in the inability of aged beta cells to replicate.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High Throughput Pancreatic Islet Microtissue 3D Characterization for Compound Screening

In the last few years several companies have developed 3D in vitro cell culture models and have demonstrated that these 3D models more accurately depict in vivo characteristics compared to traditional 2D monolayer cell culture. Because of this improved in vivo relevancy, these models are being rapidly adopted in the drug discovery space as they can more accurately predict in vivo efficacy and toxicological characteristics. Of the different types of 3D cell culture models being used today, pancreatic islet microtissues have been shown to be a powerful tool for assessing the effectiveness of compounds on diabetes. However, current imaging approaches (e.g. confocal microscopy, wide‐field microscopy) are limited in their ability to completely characterize these 3D models as light attenuation limits imaging depth to approx. 1–3 cell layers. Therefore, current imaging techniques are not able to completely characterize the effect of compounds on beta cell proliferation. Through this work, pancreatic islet microtissues of approx. 250 μm diameter were exposed to several compounds that induce beta cell proliferation and the microtissues were then stained for EdU or Ki67, glucagon, insulin and DAPI. The labeled microtissues were then rendered transparent with a rapid and plate‐compatible tissue clearing technique that allows for the microtissues to be imaged in their entirety using high content confocal imaging. It was shown through this work that the addition of a tissue clearing technique resulted in a 3‐fold increase in the number of cells characterized in the microtissues compared to traditional wide‐field or confocal microscopy.Support or Funding InformationThis work was support by Visikol Inc.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The role of glucokinase and insulin receptor substrate-2 in the proliferation of pancreatic beta cells induced by short-term high-fat diet feeding in mice

ObjectiveWe investigated whether glucokinase and insulin receptor substrate-2 were required for beta cell proliferation induced by short-term high-fat (HF) diet feeding, as has been shown for long-term HF diet. MethodsEight-week-old C57BL/6J mice were exposed to either a standard chow (SC) or HF diet. After 1 week on the diet, histopathological beta cell proliferation and gene expression in isolated islets were examined. Additionally, 8-week-old beta cell-specific glucokinase haploinsufficient (Gck+/−) and Irs2 knockout (Irs2−/−) mice were exposed to either an SC or HF diet. ResultsImmunohistochemical analysis revealed that short-term HF diet feeding resulted in a significant increase in BrdU incorporation rate compared with SC consumption in wild-type mice. Western blot analysis demonstrated that Irs2 expression levels did not differ between the two diets. Moreover, there was a significant increase in the BrdU incorporation rate in the HF diet group compared with the SC group in both Gck+/− and Irs2−/− mice. Gene expression profiling of isolated islets from mice fed an HF diet for 1 week revealed that the expression levels of downstream genes of Foxm1 were coordinately upregulated. One week of HF diet feeding stimulated beta cell proliferation with Foxm1 upregulation in 48-week-old mice as well as in 8-week-old. ConclusionsThe mechanism of pancreatic beta cell proliferation induced by short-term HF diet feeding in mice could involve a glucokinase- and Irs2-independent pathway. Our results suggest that the pathways that induce beta cell proliferation in response to short-term HF diet feeding may differ from those in response to sustained HF diet feeding.

Apollo-NADP+ Expressed in the Pancreatic Beta-Cells of Living Zebrafish Responds to Glucose and Diamide to Reveal NADPH Dynamics

We recently developed a family of genetically-encoded homoFRET sensors to measure NADPH/NADP+ redox state based on homo-dimerization of inactive glucose-6-phosphate dehydrogenase (Apollo-NADP+). Our aim is to use Apollo-NADP+ to study NADPH dynamics of pancreatic beta cells in vivo during redox stress. Redox stress induces beta cell failure, ultimately resulting in Type 2 Diabetes. We previously showed that light-scattering in cell aggregates caused a shift in absolute fluorescence anisotropy values, but the relative differences between samples were maintained. We now investigate zebrafish as a low-scatter alternative platform for 3D, in vivo fluorescence anisotropy imaging. Zebrafish are an ideal platform for this work due to their optical transparency for imaging, genetic tractability, and ability to model many pathological diseases. Here, we generated a transgenic zebrafish with stable Apollo-NADP+ expression targeted to the insulin-secreting beta cells of the zebrafish pancreas and show that Apollo-NADP+ responds to glucose and diamide treatments. Both 2-photon microscopy and microfluidics will be explored to track cellular NADPH dynamics and optimize imaging conditions for live transgenic zebrafish. Overall, this project aims to determine whether homoFRET sensors such as Apollo-NADP+ can be translated in vivo to study living tissue.

Gastrin - A Potential Predictor of Response to Incretin Therapy in Diabetes Type 2 Patients.

Personalized management of diabetes has become an imperative since majority of monotherapy fails within 3 years of its use. Identifying responders from nonresponders for a certain type of therapy would reduce a period of unsuccessful treatment and minimize health care costs. Incretin therapies, mainly glucagon-like peptide (GLP)-1 receptor agonists (GLP- 1RA) are relatively new glucose-lowering agents which increase insulin and lower glucagon response as well as slow down glucose absorption by acting on gastric emptying. However, problem with incretin- based therapy is distinguishing responders from non-responders and currently lack of specific predictors of treatment response. Experimental data demonstrated that activation of GLP-1 and gastrin signaling induces beta cell neogenesis, leading to glucose-dependent insulin secretion. Several studies demonstrated better glycemic control in patients with type 2 diabetes (DMT2) co-treated with proton pump inhibitors (PPI) and incretin based therapy agents. Higher gastrin levels in patients with diabetes prior to initiation of treatment with incretin mimetics could suggest a better potential for reversible human β-cell reprogramming with concomitant incretin therapy. Therefore, baseline levels of endogenous gastrin could be used as a predictor of response to GLP-1 therapy. In addition, treatment with PPI could also raise gastrin levels and in patients treated with GLP-1RA, lead to better glycemic control by initiating β-cell neogenesis and proliferation of pancreatic β-cells.

Circulating microRNA-375 as biomarker of pancreatic beta cell death and protection of beta cell mass by cytoprotective compounds

ObjectivePrevious studies demonstrated that circulating microRNA-375 (miR-375) is a suitable plasma biomarker for real-time detection of beta cell death. The present study evaluated the use of this biomarker to assess the beta cytoprotective effect of phenylpropenoic acid glucoside (PPAG), which was previously demonstrated to protect beta cells against various types of injury, and of exendin-4, which is an established antidiabetic drug.MethodsPPAG or exendin-4 were administered in mice treated with streptozotocin (STZ) to acutely induce beta cell death. Beta cell mass and apoptotic death were measured in pancreatic tissue sections. Circulating miR-375 was measured in blood plasma by RT-qPCR. The release of miR-375 was also measured in vitro by MIN-6 beta cells.ResultsAdministration of STZ resulted in measurable circulating levels of miR-375, a decrease in beta cell mass and increase in frequency of apoptotic beta cells. In vitro, there was a good correlation between miR-375 release and the extent of beta cell death. Treatment of mice with PPAG or exendin-4 significantly attenuated STZ-induced loss of beta cell mass and beta cell apoptosis, and normalized the blood level of miR-375.ConclusionsThese findings show the potential use of serological miR-375 measurements to evaluate the beta cytoprotective effect of (potential) antidiabetic drugs in vivo.

Insights into beta cell regeneration for diabetes via integration of molecular landscapes in human insulinomas

Although diabetes results in part from a deficiency of normal pancreatic beta cells, inducing human beta cells to regenerate is difficult. Reasoning that insulinomas hold the “genomic recipe” for beta cell expansion, we surveyed 38 human insulinomas to obtain insights into therapeutic pathways for beta cell regeneration. An integrative analysis of whole-exome and RNA-sequencing data was employed to extensively characterize the genomic and molecular landscape of insulinomas relative to normal beta cells. Here, we show at the pathway level that the majority of the insulinomas display mutations, copy number variants and/or dysregulation of epigenetic modifying genes, most prominently in the polycomb and trithorax families. Importantly, these processes are coupled to co-expression network modules associated with cell proliferation, revealing candidates for inducing beta cell regeneration. Validation of key computational predictions supports the concept that understanding the molecular complexity of insulinoma may be a valuable approach to diabetes drug discovery.

Kinetics of functional beta cell mass decay in a diphtheria toxin receptor mouse model of diabetes

Functional beta cell mass is an essential biomarker for the diagnosis and staging of diabetes. It has however proven technically challenging to study this parameter during diabetes progression. Here we have detailed the kinetics of the rapid decline in functional beta cell mass in the RIP-DTR mouse, a model of hyperglycemia resulting from diphtheria toxin induced beta cell ablation. A novel combination of imaging modalities was employed to study the pattern of beta cell destruction. Optical projection tomography of the pancreas and longitudinal in vivo confocal microscopy of islets transplanted into the anterior chamber of the eye allowed to investigate kinetics and tomographic location of beta cell mass decay in individual islets as well as at the entire islet population level. The correlation between beta cell mass and function was determined by complementary in vivo and ex vivo characterizations, demonstrating that beta cell function and glucose tolerance were impaired within the first two days following treatment when more than 50% of beta cell mass was remaining. Our results illustrate the importance of acquiring quantitative functional and morphological parameters to assess the functional status of the endocrine pancreas.

Levothyroxine enhances glucose clearance and blunts the onset of experimental type 1 diabetes mellitus in mice.

Thyroid hormones induce several changes in whole body metabolism that are known to improve metabolic homeostasis. However, adverse side effects have prevented its use in the clinic. In view of the promising effects of thyroid hormones, we investigated the effects of levothyroxine supplementation on glucose homeostasis. C57BL/6 mice were treated with levothyroxine from birth to 24weeks of age, when mice were killed. The effects of levothyroxine supplementation on metabolic health were determined. C57BL/6 mice treated with levothyroxine for 2weeks and then challenged with streptozotocin to monitor survival. Mechanistic experiments were conducted in the pancreas, liver and skeletal muscle. RIP-B7.1 mice were treated with levothyroxine for 2weeks and were subsequently immunized to trigger experimental autoimmune diabetes (EAD). Metabolic tests were performed. Mice were killed and metabolic tissues were extracted for immunohistological analyses. Long-term levothyroxine supplementation enhanced glucose clearance and reduced circulating glucose in C57BL/6 mice. Levothyroxine increased simultaneously the proliferation and apoptosis of pancreatic beta cells, promoting the maintenance of a highly insulin-expressing beta cell population. Levothyroxine increased circulating insulin levels, inducing sustained activation of IRS1-AKT signalling in insulin-target tissues. Levothyroxine-treated C57BL/6 mice challenged with streptozotocin exhibited extended survival. Levothyroxine blunted the onset of EAD in RIP-B7.1 mice by inducing beta cell proliferation and preservation of insulin-expressing cells. Interventions based on the use of thyroid hormones or thyromimetics could be explored to provide therapeutic benefit in patients with type 1 diabetes mellitus.

Combined Oral Administration of GABA and DPP-4 Inhibitor Prevents Beta Cell Damage and Promotes Beta Cell Regeneration in Mice.

γ-aminobutyric acid (GABA) or glucagon-like peptide-1 based drugs, such as sitagliptin (a dipeptidyl peptidase-4 inhibitor), were shown to induce beta cell regenerative effects in various diabetic mouse models. We propose that their combined administration can bring forth an additive therapeutic effect. We tested this hypothesis in a multiple low-dose streptozotocin (STZ)-induced beta cell injury mouse model (MDSD). Male C57BL/6J mice were assigned randomly into four groups: non-treatment diabetic control, GABA, sitagliptin, or GABA plus sitagliptin. Oral drug administration was initiated 1 week before STZ injection and maintained for 6 weeks. GABA or sitagliptin administration decreased ambient blood glucose levels and improved the glucose excursion rate. This was associated with elevated plasma insulin and reduced plasma glucagon levels. Importantly, combined use of GABA and sitagliptin significantly enhanced these effects as compared with each of the monotherapies. An additive effect on reducing water consumption was also observed. Immunohistochemical analyses revealed that combined GABA and sitagliptin therapy was superior in increasing beta cell mass, associated with increased small-size islet numbers, Ki67+ and PDX-1+ beta cell counts; and reduced Tunel+ beta cell counts. Thus, beta cell proliferation was increased, whereas apoptosis was reduced. We also noticed a suppressive effect of GABA or sitagliptin on alpha cell mass, which was not significantly altered by combining the two agents. Although either GABA or sitagliptin administration delays the onset of MDSD, our study indicates that combined use of them produces superior therapeutic outcomes. This is likely due to an amelioration of beta cell proliferation and a decrease of beta cell apoptosis.

Intracellular methylglyoxal induces oxidative damage to pancreatic beta cell line INS-1 cell through Ire1α-JNK and mitochondrial apoptotic pathway

An increased intracellular methylglyoxal (MGO) under hyperglycemia led to pancreatic beta cell death. However, its mechanism in which way with MGO induced beta cell death remains unknown. We investigated both high glucose and MGO treatment significantly inclined intracellular MGO concentration and inhibited cell viability in vitro. MGO treatment also triggered intracellular advanced glycation end products (AGEs) formation, declined mitochondrial membrane potential (MMP), increased oxidative stress and the expression of ER stress mediators Grp78/Bip and p-PERK; activated mitochondrial apoptotic pathway, which could mimic by Glo1 knockdown. Aminoguanidine (AG), a MGO scavenger, however, prevented AGEs formation and MGO-induced cell death by inhibiting oxidative stress and ER stress. Furthermore, both antioxidant N-acetylcysteine (NAC) and ER stress inhibitor 4-phenylbutyrate (4-PBA) could attenuate MGO-induced cell death through ameliorating ER stress. MGO treatment down-regulated Ire1α, a key ER stress mediator, increased JNK phosphorylation and activated mitochondrial apoptosis; down-regulated Bcl-2 expression which could be attenuated by the JNK inhibitor SP600125 and further inhibited cytochrome c leakage from mitochondria and blocked the conversion of pro caspase 3 into cleaved caspase 3, all these might contribute to the inhibition of INS-1 cell apoptosis. Ire1α down-regulation by Ire1α siRNAs mimicked MGO-induced cytotoxicity by activating the JNK phosphorylation and mitochondrial apoptotic pathway. In summary, we demonstrated that increased intracellular MGO induced cytotoxicity in INS-1 cells primarily by activating oxidative stress and further triggering mitochondrial apoptotic pathway, and ER stress-mediated Ire1α-JNK pathway. These findings may have implication on new mechanism of glucotoxicity-mediated pancreatic beta-cell dysfunction.