Liganded Thyroid Hormone Receptor-α Enhances Proliferation of Pancreatic β-Cells

Failure of the functional pancreatic β-cell mass to expand in response to increased metabolic demand is a hallmark of type 2 diabetes. Lineage tracing studies indicate that replication of existing β-cells is important for β-cell proliferation in adult animals. In rat pancreatic β-cell lines (RIN5F), treatment with 100 nm thyroid hormone (triiodothyronine, T₃) enhances cell proliferation. This result suggests that T₃ is required for β-cell proliferation or replication. To identify the role of thyroid hormone receptor α (TRα) in the processes of β-cell growth and cell cycle regulation, we constructed a recombinant adenovirus vector, AdTRα. Infection with AdTRα to RIN5F cells increased the expression of cyclin D1 mRNA and protein. Overexpression of the cyclin D1 protein in AdTRα-infected cells led to activation of the cyclin D1/cyclin-dependent kinase/retinoblastoma protein/E2F pathway, along with cell cycle progression and cell proliferation following treatment with 100 nm T₃. Conversely, lowering cellular cyclin D1 by small interfering RNA knockdown in AdTRα-infected cells led to down-regulation of the cyclin D1/CDK/Rb/E2F pathway and inhibited cell proliferation. Furthermore, in immunodeficient mice with streptozotocin-induced diabetes, intrapancreatic injection of AdTRα led to the restoration of islet function and to an increase in the β-cell mass. These results support the hypothesis that liganded TRα plays a critical role in β-cell replication and in expansion of the β-cell mass during postnatal development. Thus, liganded TRα may be a target for therapeutic strategies that can induce the expansion and regeneration of β-cells.     

Differential regulation of embryonic and adult β cell replication

Diabetes results from an inadequate functional β cell mass, either due to autoimmune destruction (Type 1 diabetes) or insulin resistance combined with β cell failure (Type 2 diabetes). Strategies to enhance β cell regeneration or increase cell proliferation could improve outcomes for patients with diabetes. Research conducted over the past several years has revealed that factors regulating embryonic β cell mass expansion differ from those regulating replication ofβ cells post-weaning. This article aims to compare and contrast factors known to control embryonic and postnatal β cell replication. In addition, we explore the possibility that connective tissue growth factor (CTGF) could increase adult β cell replication. We have already shown that CTGF is required for embryonicβ cell proliferation and is sufficient to induce replication of embryonic β cells. Here we examine whether adult β cell replication and expansion of β cell mass can be enhanced by increased CTGF expression in mature β cells.     

The Immunoregulation Effect of Alpha 1-Antitrypsin Prolong β-Cell Survival after Transplantation

Islet transplantation has considerable potential as a cure for diabetes. However, the difficulties that arise from inflammation and the immunological rejection of transplants must be addressed for islet transplantation to be successful. Alpha 1-antitrypsin (AAT) inhibits the damage on β cells caused by inflammatory reactions and promotes β-cell survival and proliferation. This protein also induces specific immune tolerance to transplanted β cells. However, whether the expression of AAT in β cells themselves could eliminate or decrease immunological rejection of transplants is not clear. Therefore, we established a β cell line (NIT-hAAT) that stably expresses human AAT. Interestingly, in a cytotoxic T lymphocyte (CTL)-killing assay, we found that hAAT reduced apoptosis and inflammatory cytokine production in NIT-1 cells and regulated the Th1/Th2 cytokine balance in vitro. In vivo transplantation of NIT-hAAT cells into mice with diabetes showed hAAT inhibited immunological rejection for a short period of time and increased the survival of transplanted β cells. This study demonstrated that hAAT generated remarkable immunoprotective and immunoregulation effects in a model of β cell islet transplantation for diabetes model.     

Human Islets Have Fewer Blood Vessels than Mouse Islets and the Density of Islet Vascular Structures Is Increased in Type 2 Diabetes

Human and rodent islets differ substantially in several features, including architecture, cell composition, gene expression and some aspects of insulin secretion. Mouse pancreatic islets are highly vascularized with interactions between islet endothelial and endocrine cells being important for islet cell differentiation and function. To determine whether human islets have a similar high degree of vascularization and whether this is altered with diabetes, we examined the vascularization of islets from normal human subjects, subjects with type 2 diabetes (T2D), and normal mice. Using an integrated morphometry approach to quantify intra-islet capillary density in human and mouse pancreatic sections, we found that human islets have five-fold fewer vessels per islet area than mouse islets. Islets in pancreatic sections from T2D subjects showed capillary thickening, some capillary fragmentation and had increased vessel density as compared with non-diabetic controls. These changes in islet vasculature in T2D islets appeared to be associated with amyloid deposition, which was noted in islets from 8/9 T2D subjects (and occupied 14% ± 4% of islet area), especially around the intra-islet capillaries. The physiological implications of the differences in the angioarchitecture of mouse and human islets are not known. Islet vascular changes in T2D may exacerbate β cell/islet dysfunction and β cell loss.     

Extreme Beta-Cell Deficiency in Pancreata of Dogs with Canine Diabetes

The pathophysiology of canine diabetes remains poorly understood, in part due to enigmatic clinical features and the lack of detailed histopathology studies. Canine diabetes, similar to human type 1 diabetes, is frequently associated with diabetic ketoacidosis at onset or after insulin omission. However, notable differences exist. Whereas human type 1 diabetes often occurs in children, canine diabetes is typically described in middle age to elderly dogs. Many competing theories have been proposed regarding the underlying cause of canine diabetes, from pancreatic atrophy to chronic pancreatitis to autoimmune mediated β-cell destruction. It remains unclear to what extent β-cell loss contributes to canine diabetes, as precise quantifications of islet morphometry have not been performed. We used high-throughput microscopy and automated image processing to characterize islet histology in a large collection of pancreata of diabetic dogs. Diabetic pancreata displayed a profound reduction in β-cells and islet endocrine cells. Unlike humans, canine non-diabetic islets are largely comprised of β-cells. Very few β-cells remained in islets of diabetic dogs, even in pancreata from new onset cases. Similarly, total islet endocrine cell number was sharply reduced in diabetic dogs. No compensatory proliferation or lymphocyte infiltration was detected. The majority of pancreata had no evidence of pancreatitis. Thus, canine diabetes is associated with extreme β-cell deficiency in both new and longstanding disease. The β-cell predominant composition of canine islets and the near-total absence of β-cells in new onset elderly diabetic dogs strongly implies that similar to human type 1 diabetes, β-cell loss underlies the pathophysiology of canine diabetes.     

Insulin Secretion and Ca2+ Dynamics in β-Cells Are Regulated by PERK (EIF2AK3) in Concert with Calcineurin

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) (EIF2AK3) is essential for normal development and function of the insulin-secreting β-cell. Although genetic ablation of PERK in β-cells results in permanent neonatal diabetes in humans and mice, the underlying mechanisms remain unclear. Here, we used a newly developed and highly specific inhibitor of PERK to determine the immediate effects of acute ablation of PERK activity. We found that inhibition of PERK in human and rodent β-cells causes a rapid inhibition of secretagogue-stimulated subcellular Ca²⁺ signaling and insulin secretion. These dysfunctions stem from alterations in store-operated Ca²⁺ entry and sarcoplasmic endoplasmic reticulum Ca²⁺-ATPase activity. We also found that PERK regulates calcineurin, and pharmacological inhibition of calcineurin results in similar defects on stimulus-secretion coupling. Our findings suggest that interplay between calcineurin and PERK regulates β-cell Ca²⁺ signaling and insulin secretion, and that loss of this interaction may have profound implications in insulin secretion defects associated with diabetes.     

Novel Role for Matricellular Proteins in the Regulation of Islet β Cell Survival: THE EFFECT OF SPARC ON SURVIVAL,PROLIFERATION, AND SIGNALING*

Understanding the mechanisms regulating islet growth and survival is critical for developing novel approaches to increasing or sustaining β cell mass in both type 1 and type 2 diabetes patients. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that is important for the regulation of cell growth and adhesion. Increased SPARC can be detected in the serum of type 2 diabetes patients. The aim of this study was to investigate the role of SPARC in the regulation of β cell growth and survival. We show using immunohistochemistry that SPARC is expressed by stromal cells within islets and can be detected in primary mouse islets by Western blot. SPARC is secreted at high levels by pancreatic stellate cells and is regulated by metabolic parameters in these cells, but SPARC expression was not detectable in β cells. In islets, SPARC expression is highest in young mice, and is also elevated in the islets of non-obese diabetic (NOD) mice compared with controls. Purified SPARC inhibits growth factor-induced signaling in both INS-1 β cells and primary mouse islets, and inhibits IGF-1-induced proliferation of INS-1 β cells. Similarly, exogenous SPARC prevents IGF-1-induced survival of primary mouse islet cells. This study identifies the stromal-derived matricellular protein SPARC as a novel regulator of islet survival and β cell growth.     

Conditional Expression of Smad7 in Pancreatic β Cells Disrupts TGF-β Signaling and Induces Reversible Diabetes Mellitus

Identification of signaling pathways that maintain and promote adult pancreatic islet functions will accelerate our understanding of organogenesis and improve strategies for treating diseases like diabetes mellitus. Previous work has implicated transforming growth factor-β (TGF-β) signaling as an important regulator of pancreatic islet development, but has not established whether this signaling pathway is required for essential islet functions in the adult pancreas. Here we describe a conditional system for expressing Smad7, a potent inhibitor of TGF-β signaling, to identify distinct roles for this pathway in adult and embryonic β cells. Smad7 expression in Pdx1 ⁺ embryonic pancreas cells resulted in striking embryonic β cell hypoplasia and neonatal lethality. Conditional expression of Smad7 in adult Pdx1 ⁺ cells reduced detectable β cell expression of MafA, menin, and other factors that regulate β cell function. Reduced pancreatic insulin content and hypoinsulinemia produced overt diabetes that was fully reversed upon resumption of islet TGF-β signaling. Thus, our studies reveal that TGF-β signaling is crucial for establishing and maintaining defining features of mature pancreatic β cells.     

Ciliary Neurotrophic Factor Protects Mice Against Streptozotocin-induced Type 1 Diabetes through SOCS3: THE ROLE OF STAT1/STAT3 RATIO IN β-CELL DEATH*

Type 1 diabetes is characterized by a loss of islet β-cells. Ciliary neurotrophic factor (CNTF) protects pancreatic islets against cytokine-induced apoptosis. For this reason, we assessed whether CNTF protects mice against streptozotocin-induced diabetes (a model of type 1 diabetes) and the mechanism for this protection. WT and SOCS3 knockdown C57BL6 mice were treated for 5 days with citrate buffer or 0.1 mg/kg CNTF before receiving 80 mg/kg streptozotocin. Glycemia in non-fasted mice was measured weekly from days 0–28 after streptozotocin administration. Diabetes was defined as a blood glucose > 11.2 mmol/liter. Wild-type (WT) and SOCS3 knockdown MIN6 cells were cultured with CNTF, IL1β, or both. CNTF reduced diabetes incidence and islet apoptosis in WT but not in SOCS3kd mice. Likewise, CNTF inhibited apoptosis in WT but not in SOCS3kd MIN6 cells. CNTF increased STAT3 phosphorylation in WT and SOCS3kd mice and MIN6 cells but reduced STAT1 phosphorylation only in WT mice, in contrast to streptozotocin and IL1β. Moreover, CNTF reduced NFκB activation and required down-regulation of inducible NO synthase expression to exert its protective effects. In conclusion, CNTF protects mice against streptozotocin-induced diabetes by increasing pancreatic islet survival, and this protection depends on SOCS3. In addition, SOCS3 expression and β-cell fate are dependent on STAT1/STAT3 ratio.     

Alteration of the Thymic T Cell Repertoire by Rotavirus Infection Is Associated with Delayed Type 1 Diabetes Development in Non-Obese Diabetic Mice

Rotaviruses are implicated as a viral trigger for the acceleration of type 1 diabetes in children. Infection of adult non-obese diabetic (NOD) mice with rotavirus strain RRV accelerates diabetes development, whereas RRV infection in infant NOD mice delays diabetes onset. In this study of infant mice, RRV titers and lymphocyte populations in the intestine, mesenteric lymph nodes (MLN) and thymus of NOD mice were compared with those in diabetes-resistant BALB/c and C57BL/6 mice. Enhanced intestinal RRV infection occurred in NOD mice compared with the other mouse strains. This was associated with increases in the frequency of CD8αβ TCRαβ intraepithelial lymphocytes, and their PD-L1 expression. Virus spread to the MLN and T cell numbers there also were greatest in NOD mice. Thymic RRV infection is shown here in all mouse strains, often in combination with alterations in T cell ontogeny. Infection lowered thymocyte numbers in infant NOD and C57BL/6 mice, whereas thymocyte production was unaltered overall in infant BALB/c mice. In the NOD mouse thymus, effector CD4⁺ T cell numbers were reduced by infection, whereas regulatory T cell numbers were maintained. It is proposed that maintenance of thymic regulatory T cell numbers may contribute to the increased suppression of inflammatory T cells in response to a strong stimulus observed in pancreatic lymph nodes of adult mice infected as infants. These findings show that rotavirus replication is enhanced in diabetes-prone mice, and provide evidence that thymic T cell alterations may contribute to the delayed diabetes onset following RRV infection.     

1H NMR Based Serum Metabolic Profiles Associated with Pathological Progression of Pancreatic Islet β Cell Tumor in Rip1-Tag2 Mice

Pancreatic islet β cell tumor is the most common islet cell tumor. A well-characterized tumor progression in Rip1-Tag2 mice undergoes five stages, involving normal, hyperplasia, angiogenic islets, tumorigenesis and invasive carcinoma. ¹H NMR based metabonomics was applied to identify potential biomarkers for monitoring pancreatic islet β cell tumor progression in Rip1-Tag2 mice. Multivariate analysis results showed the serum metabonome at hyperplasia stage shared the similar characteristics with the ones at normal stage as a result of slight proliferation of pancreatic islet β cells. At angiogenic islets stage, the up-regulated glycolysis, disturbed choline and phospholipid metabolism composed the metabolic signature. In addition to the changes mentioned above, several metabolites were identified as early biomarkers for tumorigenesis, including increased methionine, citrate and choline, and reduced acetate, taurine and glucose, which suggested the activated energy and amino acid metabolism. All the changes were aggravated at invasive carcinoma stage, coupled with notable changes in alanine, glutamate and glycine. Moreover, the distinct metabolic phenotype was found associated with the implanting of SV40 large T antigen in Rip1-Tag2 mice. The combined metabolic and multivariate statistics approach provides a robust method for screening the biomarkers of disease progression and examining the association between gene and metabolism.     

Dipeptidyl Peptidase IV Inhibition Activates CREB and Improves Islet Vascularization through VEGF-A/VEGFR-2 Signaling Pathway

Substitution of pancreatic islets is a potential therapy to treat diabetes and it depends on reconstitution of islet’s capillary network. In this study, we addressed the question whether stabilization of Glucagon-Like-Peptide-1 (GLP-1) by inhibiting Dipeptidyl Peptidase-IV (DPP-IV) increases β-cell mass by modulating vascularization. Mouse or porcine donor islets were implanted under kidney capsule of diabetic mice treated with DPP-IV inhibitor sitagliptin. Grafts were analyzed for insulin production, β-cell proliferation and vascularization. In addition, the effect of sitagliptin on sprouting and Vascular Endothelial Growth Factor (VEGF)-A expression was examined ex vivo. The cAMP response element-binding (CREB) and VEGF-A/ Vascular Endothelial Growth Factor Receptor (VEGFR)-2 signaling pathway leading to islet vascularization was explored. Sitagliptin increased mean insulin content of islet grafts and area of insulin-positive tissue as well as β-cell proliferation. Interestingly, sitagliptin treatment also markedly increased endothelial cell proliferation, microvessel density and blood flow. Finally, GLP-1 (7-36) stimulated sprouting and VEGF expression, which was significantly enhanced by sitagliptin- mediated inhibition of DPP-IV. Our in vivo data demonstrate that sitagliptin treatment phosphorylated CREB and induced islet vascularization through VEGF-A/VEGFR-2 signaling pathway. This study paves a new pathway for improvement of islet transplantation in treating diabetes mellitus.     

Transient overexpression of cyclin D2/CDK4/GLP1 genes induces proliferation and differentiation of adult pancreatic progenitors and mediates islet regeneration

The molecular mechanism of β-cell regeneration remains poorly understood. Cyclin D2/CDK4 expresses in normal β cells and maintains adult β-cell growth. We hypothesized that gene therapy with cyclin D2/CDK4/GLP-1 plasmids targeted to the pancreas of STZ-treated rats by ultrasound-targeted microbubble destruction (UTMD) would force cell cycle re-entry of residual G₀-phase islet cells into G₁/S phase to regenerate β cells. A single UTMD treatment induced β-cell regeneration with reversal of diabetes for 6 mo without evidence of toxicity. We observed that this β-cell regeneration was not mediated by self-replication of pre-existing β cells. Instead, cyclin D2/CDK4/GLP-1 initiated robust proliferation of adult pancreatic progenitor cells that exist within islets and terminally differentiate to mature islets with β cells and α cells.Key words: cell cycle regulation, adult pancreatic progenitor cells, proliferation, differentiation, islets regeneration, diabetes     

Insulin-like Growth Factor 2 Overexpression Induces β-Cell Dysfunction and Increases Beta-cell Susceptibility to Damage

The human insulin-like growth factor 2 (IGF2) and insulin genes are located within the same genomic region. Although human genomic studies have demonstrated associations between diabetes and the insulin/IGF2 locus or the IGF2 mRNA-binding protein 2 (IGF2BP2), the role of IGF2 in diabetes pathogenesis is not fully understood. We previously described that transgenic mice overexpressing IGF2 specifically in β-cells (Tg-IGF2) develop a pre-diabetic state. Here, we characterized the effects of IGF2 on β-cell functionality. Overexpression of IGF2 led to β-cell dedifferentiation and endoplasmic reticulum stress causing islet dysfunction in vivo. Both adenovirus-mediated overexpression of IGF2 and treatment of adult wild-type islets with recombinant IGF2 in vitro further confirmed the direct implication of IGF2 on β-cell dysfunction. Treatment of Tg-IGF2 mice with subdiabetogenic doses of streptozotocin or crossing these mice with a transgenic model of islet lymphocytic infiltration promoted the development of overt diabetes, suggesting that IGF2 makes islets more susceptible to β-cell damage and immune attack. These results indicate that increased local levels of IGF2 in pancreatic islets may predispose to the onset of diabetes. This study unravels an unprecedented role of IGF2 on β-cells function.