Islet cell replacement and transplantation immunology in a mouse strain with inducible diabetes.

Improved models of experimental diabetes are needed to develop cell therapies for diabetes. Here, we introduce the B6 RIP-DTR mouse, a model of experimental diabetes in fully immunocompetent animals. These inbred mice harbor the H2b major histocompatibility complex (MHC), selectively express high affinity human diphtheria toxin receptor (DTR) in islet ß-cells, and are homozygous for the Ptprca (CD45.1) allele rather than wild-type Ptprcb (CD45.2). 100% of B6 RIP-DTR mice rapidly became diabetic after a single dose of diphtheria toxin, and this was reversed indefinitely after transplantation with islets from congenic C57BL/6 mice. By contrast, MHC-mismatched islets were rapidly rejected, and this allotransplant response was readily monitored via blood glucose and graft histology. In peripheral blood of B6 RIP-DTR with mixed hematopoietic chimerism, CD45.2 BALB/c donor blood immune cells were readily distinguished from host CD45.1 cells by flow cytometry. Reliable diabetes induction and other properties in B6 RIP-DTR mice provide an important new tool to advance transplant-based studies of islet replacement and immunomodulation to treat diabetes.

Pancreatic Ppy-expressing γ-cells display mixed phenotypic traits and the adaptive plasticity to engage insulin production.

The cellular identity of pancreatic polypeptide (Ppy)-expressing γ-cells, one of the rarest pancreatic islet cell-type, remains elusive. Within islets, glucagon and somatostatin, released respectively from α- and δ-cells, modulate the secretion of insulin by ß-cells. Dysregulation of insulin production raises blood glucose levels, leading to diabetes onset. Here, we present the genetic signature of human and mouse γ-cells. Using different approaches, we identified a set of genes and pathways defining their functional identity. We found that the γ-cell population is heterogeneous, with subsets of cells producing another hormone in addition to Ppy. These bihormonal cells share identity markers typical of the other islet cell-types. In mice, Ppy gene inactivation or conditional γ-cell ablation did not alter glycemia nor body weight. Interestingly, upon ß-cell injury induction, γ-cells exhibited gene expression changes and some of them engaged insulin production, like α- and δ-cells. In conclusion, we provide a comprehensive characterization of γ-cells and highlight their plasticity and therapeutic potential.

Combined inhibition of menin-MLL interaction and TGF-ß signaling induces replication of human pancreatic beta cells.

Both type 1 and type 2 diabetes are associated with hyperglycemia and loss of functional beta cell mass. Inducing proliferation of preexisting beta cells is an approach to increase the numbers of beta cells. In this study, we examined a panel of selected small molecules for their proliferation-inducing effects on human pancreatic beta cells. Our results demonstrated that a small molecule inhibitor of the menin-MLL interaction (MI-2) and small molecule inhibitors of TGF-ß signaling (SB431542, LY2157299, or LY364947) synergistically increased ex vivo replication of human beta cells. We showed that this increased proliferation did not affect insulin production, as a pivotal indication of beta cell function. We further provided evidence which suggested that menin-MLL and TGF-ß inhibition cooperated through downregulation of cell cycle inhibitors CDKN1A, CDKN1B, and CDKN2C. Our findings might provide a new option for extending the pharmacological repertoire for induction of beta cell proliferation as a potential therapeutic approach for diabetes.

Diabetes relief in mice by glucose-sensing insulin-secreting human α-cells.

Cell-identity switches, in which terminally differentiated cells are converted into different cell types when stressed, represent a widespread regenerative strategy in animals, yet they are poorly documented in mammals. In mice, some glucagon-producing pancreatic α-cells and somatostatin-producing δ-cells become insulin-expressing cells after the ablation of insulin-secreting ß-cells, thus promoting diabetes recovery. Whether human islets also display this plasticity, especially in diabetic conditions, remains unknown. Here we show that islet non-ß-cells, namely α-cells and pancreatic polypeptide (PPY)-producing γ-cells, obtained from deceased non-diabetic or diabetic human donors, can be lineage-traced and reprogrammed by the transcription factors PDX1 and MAFA to produce and secrete insulin in response to glucose. When transplanted into diabetic mice, converted human α-cells reverse diabetes and continue to produce insulin even after six months. Notably, insulin-producing α-cells maintain expression of α-cell markers, as seen by deep transcriptomic and proteomic characterization. These observations provide conceptual evidence and a molecular framework for a mechanistic understanding of in situ cell plasticity as a treatment for diabetes and other degenerative diseases.

ß Cell-Specific Deletion of the IL-1 Receptor Antagonist Impairs ß Cell Proliferation and Insulin Secretion.

Interleukin-1 receptor antagonist (IL-1Ra) is elevated in the circulation during obesity and type 2 diabetes (T2D) but is decreased in islets from patients with T2D. The protective role of local IL-1Ra was investigated in pancreatic islet ß cell (ßIL-1Ra)-specific versus myeloid-cell (myeloIL-1Ra)-specific IL-1Ra knockout (KO) mice. Deletion of IL-1Ra in ß cells, but not in myeloid cells, resulted in diminished islet IL-1Ra expression. Myeloid cells were not the main source of circulating IL-1Ra in obesity. ßIL-1Ra KO mice had impaired insulin secretion, reduced ß cell proliferation, and decreased expression of islet proliferation genes, along with impaired glucose tolerance. The key cell-cycle regulator E2F1 partly reversed IL-1ß-mediated inhibition of potassium channel Kir6.2 expression and rescued impaired insulin secretion in IL-1Ra knockout islets. Our findings provide evidence for the importance of ß cell-derived IL-1Ra for the local defense of ß cells to maintain normal function and proliferation.

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.

Lack of Prox1 Downregulation Disrupts the Expansion and Maturation of Postnatal Murine ß-Cells.

Transcription factor expression fluctuates during ß-cell ontogeny, and disruptions in this pattern can affect the development or function of those cells. Here we uncovered that murine endocrine pancreatic progenitors express high levels of the homeodomain transcription factor Prox1, whereas both immature and mature ß-cells scarcely express this protein. We also investigated if sustained Prox1 expression is incompatible with ß-cell development or maintenance using transgenic mouse approaches. We discovered that Prox1 upregulation in mature ß-cells has no functional consequences; in contrast, Prox1 overexpression in immature ß-cells promotes acute fasting hyperglycemia. Using a combination of immunostaining and quantitative and comparative gene expression analyses, we determined that Prox1 upregulation reduces proliferation, impairs maturation, and enables apoptosis in postnatal ß-cells. Also, we uncovered substantial deficiency in ß-cells that overexpress Prox1 of the key regulator of ß-cell maturation MafA, several MafA downstream targets required for glucose-stimulated insulin secretion, and genes encoding important components of FGF signaling. Moreover, knocking down PROX1 in human EndoC-ßH1 ß-cells caused increased expression of many of these same gene products. These and other results in our study indicate that reducing the expression of Prox1 is beneficial for the expansion and maturation of postnatal ß-cells.

A standardized method for in vivo mouse pancreas imaging and semiquantitative ß cell mass measurement by dual isotope SPECT.

PURPOSE: In order to evaluate future ß cell tracers in vivo, we aimed to develop a standardized in vivo method allowing semiquantitative measurement of a prospective ß cell tracer within the pancreas. PROCEDURES: 2-[(123)I]Iodo-L-phenylalanine ([(123)I]IPA) and [Lys(40)([(111)In]DTPA)]exendin-3 ([(111)In]Ex3) pancreatic uptake and biodistribution were evaluated using SPECT, autoradiography, and an ex vivo biodistribution study in a controlled unilaterally nephrectomized mouse ß cell depletion model. Semiquantitative measurement of the imaging results was performed using [(123)I]IPA to delineate the pancreas and [(111)In]Ex3 as a ß cell tracer. RESULTS: The uptake of [(123)I]IPA was highest in the pancreas. Aside from the kidneys, the uptake of [(111)In]Ex3 was highest in the pancreas and lungs. Autoradiography showed only uptake of [(111)In]Ex3 in insulin-expressing cells. Semiquantitative measurement of [(111)In]Ex3 in the SPECT images based on the delineation of the pancreas with [(123)I]IPA showed a high correlation with the [(111)In]Ex3 uptake data of the pancreas obtained by dissection. A strong positive correlation was observed between the relative insulin positive area and the pancreas-to-blood ratios of [(111)In]Ex3 uptake as determined by counting with a gamma counter and the semiquantitative analysis of the SPECT images. CONCLUSIONS: [(123)I]IPA is a promising tracer to delineate pancreatic tissue on SPECT images. It shows a high uptake in the pancreas as compared to other abdominal tissues. This study also demonstrates the feasibility and accuracy to measure the ß cell mass in vivo in an animal model of diabetes.

Diabetes recovery by age-dependent conversion of pancreatic δ-cells into insulin producers.

Total or near-total loss of insulin-producing ß-cells occurs in type 1 diabetes. Restoration of insulin production in type 1 diabetes is thus a major medical challenge. We previously observed in mice in which ß-cells are completely ablated that the pancreas reconstitutes new insulin-producing cells in the absence of autoimmunity. The process involves the contribution of islet non-ß-cells; specifically, glucagon-producing α-cells begin producing insulin by a process of reprogramming (transdifferentiation) without proliferation. Here we show the influence of age on ß-cell reconstitution from heterologous islet cells after near-total ß-cell loss in mice. We found that senescence does not alter α-cell plasticity: α-cells can reprogram to produce insulin from puberty through to adulthood, and also in aged individuals, even a long time after ß-cell loss. In contrast, before puberty there is no detectable α-cell conversion, although ß-cell reconstitution after injury is more efficient, always leading to diabetes recovery. This process occurs through a newly discovered mechanism: the spontaneous en masse reprogramming of somatostatin-producing δ-cells. The juveniles display 'somatostatin-to-insulin' δ-cell conversion, involving dedifferentiation, proliferation and re-expression of islet developmental regulators. This juvenile adaptability relies, at least in part, upon the combined action of FoxO1 and downstream effectors. Restoration of insulin producing-cells from non-ß-cell origins is thus enabled throughout life via δ- or α-cell spontaneous reprogramming. A landscape with multiple intra-islet cell interconversion events is emerging, offering new perspectives for therapy.

Glycoprotein 130 receptor signaling mediates α-cell dysfunction in a rodent model of type 2 diabetes.

Dysregulated glucagon secretion accompanies islet inflammation in type 2 diabetes. We recently discovered that interleukin (IL)-6 stimulates glucagon secretion from human and rodent islets. IL-6 family cytokines require the glycoprotein 130 (gp130) receptor to signal. In this study, we elucidated the effects of α-cell gp130 receptor signaling on glycemic control in type 2 diabetes. IL-6 family cytokines were elevated in islets in rodent models of this disease. gp130 receptor activation increased STAT3 phosphorylation in primary α-cells and stimulated glucagon secretion. Pancreatic α-cell gp130 knockout (αgp130KO) mice showed no differences in glycemic control, α-cell function, or α-cell mass. However, when subjected to streptozotocin plus high-fat diet to induce islet inflammation and pathophysiology modeling type 2 diabetes, αgp130KO mice had reduced fasting glycemia, improved glucose tolerance, reduced fasting insulin, and improved α-cell function. Hyperinsulinemic-euglycemic clamps revealed no differences in insulin sensitivity. We conclude that in a setting of islet inflammation and pathophysiology modeling type 2 diabetes, activation of α-cell gp130 receptor signaling has deleterious effects on α-cell function, promoting hyperglycemia. Antagonism of α-cell gp130 receptor signaling may be useful for the treatment of type 2 diabetes.

Loss of prohibitin induces mitochondrial damages altering ß-cell function and survival and is responsible for gradual diabetes development.

Prohibitins are highly conserved proteins mainly implicated in the maintenance of mitochondrial function and architecture. Their dysfunctions are associated with aging, cancer, obesity, and inflammation. However, their possible role in pancreatic ß-cells remains unknown. The current study documents the expression of prohibitins in human and rodent islets and their key role for ß-cell function and survival. Ablation of Phb2 in mouse ß-cells sequentially resulted in impairment of mitochondrial function and insulin secretion, loss of ß-cells, progressive alteration of glucose homeostasis, and, ultimately, severe diabetes. Remarkably, these events progressed over a 3-week period of time after weaning. Defective insulin supply in ß-Phb2(-/-) mice was contributed by both ß-cell dysfunction and apoptosis, temporarily compensated by increased ß-cell proliferation. At the molecular level, we observed that deletion of Phb2 caused mitochondrial abnormalities, including reduction of mitochondrial DNA copy number and respiratory chain complex IV levels, altered mitochondrial activity, cleavage of L-optic atrophy 1, and mitochondrial fragmentation. Overall, our data demonstrate that Phb2 is essential for metabolic activation of mitochondria and, as a consequence, for function and survival of ß-cells.

UCP2 regulates the glucagon response to fasting and starvation.

Glucagon is important for maintaining euglycemia during fasting/starvation, and abnormal glucagon secretion is associated with type 1 and type 2 diabetes; however, the mechanisms of hypoglycemia-induced glucagon secretion are poorly understood. We previously demonstrated that global deletion of mitochondrial uncoupling protein 2 (UCP2(-/-)) in mice impaired glucagon secretion from isolated islets. Therefore, UCP2 may contribute to the regulation of hypoglycemia-induced glucagon secretion, which is supported by our current finding that UCP2 expression is increased in nutrient-deprived murine and human islets. Further to this, we created α-cell-specific UCP2 knockout (UCP2AKO) mice, which we used to demonstrate that blood glucose recovery in response to hypoglycemia is impaired owing to attenuated glucagon secretion. UCP2-deleted α-cells have higher levels of intracellular reactive oxygen species (ROS) due to enhanced mitochondrial coupling, which translated into defective stimulus/secretion coupling. The effects of UCP2 deletion were mimicked by the UCP2 inhibitor genipin on both murine and human islets and also by application of exogenous ROS, confirming that changes in oxidative status and electrical activity directly reduce glucagon secretion. Therefore, α-cell UCP2 deletion perturbs the fasting/hypoglycemic glucagon response and shows that UCP2 is necessary for normal α-cell glucose sensing and the maintenance of euglycemia.

The zinc finger protein ZBTB20 regulates transcription of fructose-1,6-bisphosphatase 1 and ß cell function in mice.

BACKGROUND & AIMS: Fructose-1,6-bisphosphatase (FBP)-1 is a gluconeogenic enzyme that regulates glucose metabolism and insulin secretion in ß cells, but little is known about how its transcription is controlled. The zinc finger protein ZBTB20 regulates glucose homeostasis, so we investigated its effects on expression of FBP-1. METHODS: We analyzed gene expression using real-time reverse-transcription polymerase chain reaction, immunoblotting, and immunohistochemistry. We generated mice with ß cell-specific disruption of Zbtb20 using Cre/LoxP technology. Expression of Zbtb20 in ß cells was reduced using small interfering RNAs, and promoter occupancy and transcriptional regulation were analyzed by chromatin immunoprecipitation and reporter assays. RESULTS: ZBTB20 was expressed at high levels by ß cells and other endocrine cells in islets of normal mice; expression levels were reduced in islets from diabetic db/db mice. Mice with ß cell-specific knockout of Zbtb20 had normal development of ß cells but had hyperglycemia, hypoinsulinemia, glucose intolerance, and impaired glucose-stimulated insulin secretion. Islets isolated from these mice had impaired glucose metabolism, adenosine triphosphate production, and insulin secretion after glucose stimulation in vitro, although insulin secretion returned to normal levels in the presence of KCl. ZBTB20 knockdown with small interfering RNAs impaired glucose-stimulated insulin secretion in the ß cell line MIN6. Expression of Fbp1 was up-regulated in ß cells with ZBTB20 knockout or knockdown; impairments to glucose-stimulated insulin secretion were restored by inhibition of FBPase activity. ZBTB20 was recruited to the Fbp1 promoter and repressed its transcription in ß cells. CONCLUSIONS: The transcription factor ZBTB20 regulates ß cell function and glucose homeostasis in mice. It might be a therapeutic target for type 2 diabetes mellitus.

Normal glucagon signaling and ß-cell function after near-total α-cell ablation in adult mice.

OBJECTIVE: To evaluate whether healthy or diabetic adult mice can tolerate an extreme loss of pancreatic α-cells and how this sudden massive depletion affects ß-cell function and blood glucose homeostasis. RESEARCH DESIGN AND METHODS: We generated a new transgenic model allowing near-total α-cell removal specifically in adult mice. Massive α-cell ablation was triggered in normally grown and healthy adult animals upon diphtheria toxin (DT) administration. The metabolic status of these mice was assessed in 1) physiologic conditions, 2) a situation requiring glucagon action, and 3) after ß-cell loss. RESULTS: Adult transgenic mice enduring extreme (98%) α-cell removal remained healthy and did not display major defects in insulin counter-regulatory response. We observed that 2% of the normal α-cell mass produced enough glucagon to ensure near-normal glucagonemia. ß-Cell function and blood glucose homeostasis remained unaltered after α-cell loss, indicating that direct local intraislet signaling between α- and ß-cells is dispensable. Escaping α-cells increased their glucagon content during subsequent months, but there was no significant α-cell regeneration. Near-total α-cell ablation did not prevent hyperglycemia in mice having also undergone massive ß-cell loss, indicating that a minimal amount of α-cells can still guarantee normal glucagon signaling in diabetic conditions. CONCLUSIONS: An extremely low amount of α-cells is sufficient to prevent a major counter-regulatory deregulation, both under physiologic and diabetic conditions. We previously reported that α-cells reprogram to insulin production after extreme ß-cell loss and now conjecture that the low α-cell requirement could be exploited in future diabetic therapies aimed at regenerating ß-cells by reprogramming adult α-cells.

In vivo conditional Pax4 overexpression in mature islet ß-cells prevents stress-induced hyperglycemia in mice.

OBJECTIVE: To establish the role of the transcription factor Pax4 in pancreatic islet expansion and survival in response to physiological stress and its impact on glucose metabolism, we generated transgenic mice conditionally and selectively overexpressing Pax4 or a diabetes-linked mutant variant (Pax4R129W) in ß-cells. RESEARCH DESIGN AND METHODS: Glucose homeostasis and ß-cell death and proliferation were assessed in Pax4- or Pax4R129W-overexpressing transgenic animals challenged with or without streptozotocin. Isolated transgenic islets were also exposed to cytokines, and apoptosis was evaluated by DNA fragmentation or cytochrome C release. The expression profiles of proliferation and apoptotic genes and ß-cell markers were studied by immunohistochemistry and quantitative RT-PCR. RESULTS: Pax4 but not Pax4R129W protected animals against streptozotocin-induced hyperglycemia and isolated islets from cytokine-mediated ß-cell apoptosis. Cytochrome C release was abrogated in Pax4 islets treated with cytokines. Interleukin-1ß transcript levels were suppressed in Pax4 islets, whereas they were increased along with NOS2 in Pax4R129W islets. Bcl-2, Cdk4, and c-myc expression levels were increased in Pax4 islets while MafA, insulin, and GLUT2 transcript levels were suppressed in both animal models. Long-term Pax4 expression promoted proliferation of a Pdx1-positive cell subpopulation while impeding insulin secretion. Suppression of Pax4 rescued this defect with a concomitant increase in pancreatic insulin content. CONCLUSIONS: Pax4 protects adult islets from stress-induced apoptosis by suppressing selective nuclear factor-κB target genes while increasing Bcl-2 levels. Furthermore, it promotes dedifferentiation and proliferation of ß-cells through MafA repression, with a concomitant increase in Cdk4 and c-myc expression.

Alpha cell-specific Men1 ablation triggers the transdifferentiation of glucagon-expressing cells and insulinoma development.

BACKGROUND & AIMS: The tumor suppressor menin is recognized as a key regulator of pancreatic islet development, proliferation, and beta-cell function, whereas its role in alpha cells remains poorly understood. The purpose of the current study was to address this issue in relation to islet tumor histogenesis. METHODS: We generated alpha cell-specific Men1 mutant mice with Cre/loxP technology and carried out analyses of pancreatic lesions developed in the mutant mice during aging. RESULTS: We showed that, despite the alpha-cell specificity of the GluCre transgene, both glucagonomas and a large amount of insulinomas developed in mutant mice older than 6 months, accompanied by mixed islet tumors. Interestingly, the cells sharing characteristics of both alpha and beta cells were identified shortly after the appearance of menin-deficient alpha cells but well before the tumor onset. Using a genetic cell lineage tracing analysis, we demonstrated that insulinoma cells were directly derived from transdifferentiating glucagon-expressing cells. Furthermore, our data indicated that the expression of Pdx1, MafA, Pax4, and Ngn3 did not seem to be required for the initiation of this transdifferentiation. CONCLUSIONS: Our work shows cell transdifferentiation as a novel mechanism involved in islet tumor development and provides evidence showing that menin regulates the plasticity of differentiated pancreatic alpha cells in vivo, shedding new light on the mechanisms of islet tumorigenesis.

Pancreatic inactivation of c-Myc decreases acinar mass and transdifferentiates acinar cells into adipocytes in mice.

BACKGROUND & AIMS: The pancreatic mass is determined by the coordinated expansion and differentiation of progenitor cells and is maintained via tight control of cell replacement rates. The basic helix-loop-helix transcription factor c-Myc is one of the main regulators of these processes in many organs. We studied the requirement of c-Myc in controlling the generation and maintenance of pancreatic mass. METHODS: We conditionally inactivated c-Myc in Pdx1+ pancreatic progenitor cells. Pancreata of mice lacking c-Myc (c-Myc(P-/-) mice) were analyzed during development and ageing. RESULTS: Pancreatic growth in c-Myc(P-/-) mice was impaired starting on E12.5, in early primordia, because of decreased proliferation and altered differentiation of exocrine progenitors; islet progenitors were spared. Acinar cell maturation was defective in the adult hypotrophic pancreas, which hampered exocrine mass maintenance in aged animals. From 2 to 10 months of age, the c-Myc(P-/-) pancreas was progressively remodeled without inflammatory injury. Loss of acinar cells increased with time, concomitantly with adipose tissue accumulation. Using a genetic cell lineage tracing analysis, we demonstrated that pancreatic adipose cells were derived directly from transdifferentiating acinar cells. This epithelial-to-mesenchyme transition was also observed in normal aged specimens and in pancreatitis. CONCLUSIONS: These results provide evidence indicating that c-Myc activity is required for growth and maturation of the exocrine pancreas, and sheds new light on the ontogeny of pancreatic adipose cells in processes of organ degenerescence and tissue involution.

Deletion of glutamate dehydrogenase in beta-cells abolishes part of the insulin secretory response not required for glucose homeostasis.

Insulin exocytosis is regulated in pancreatic ss-cells by a cascade of intracellular signals translating glucose levels into corresponding secretory responses. The mitochondrial enzyme glutamate dehydrogenase (GDH) is regarded as a major player in this process, although its abrogation has not been tested yet in animal models. Here, we generated transgenic mice, named betaGlud1(-/-), with ss-cell-specific GDH deletion. Our results show that GDH plays an essential role in the full development of the insulin secretory response. In situ pancreatic perfusion revealed that glucose-stimulated insulin secretion was reduced by 37% in betaGlud1(-/-). Furthermore, isolated islets with either constitutive or acute adenovirus-mediated knock-out of GDH showed a 49 and 38% reduction in glucose-induced insulin release, respectively. Adenovirus-mediated re-expression of GDH in betaGlud1(-/-) islets fully restored glucose-induced insulin release. Thus, GDH appears to account for about 40% of glucose-stimulated insulin secretion and to lack redundant mechanisms. In betaGlud1(-/-) mice, the reduced secretory capacity resulted in lower plasma insulin levels in response to both feeding and glucose load, while body weight gain was preserved. The results demonstrate that GDH is essential for the full development of the secretory response in beta-cells. However, maximal secretory capacity is not required for maintenance of glucose homeostasis in normo-caloric conditions.

Deletion of the von Hippel-Lindau gene in pancreatic beta cells impairs glucose homeostasis in mice.

Defective insulin secretion in response to glucose is an important component of the beta cell dysfunction seen in type 2 diabetes. As mitochondrial oxidative phosphorylation plays a key role in glucose-stimulated insulin secretion (GSIS), oxygen-sensing pathways may modulate insulin release. The von Hippel-Lindau (VHL) protein controls the degradation of hypoxia-inducible factor (HIF) to coordinate cellular and organismal responses to altered oxygenation. To determine the role of this pathway in controlling glucose-stimulated insulin release from pancreatic beta cells, we generated mice lacking Vhl in pancreatic beta cells (betaVhlKO mice) and mice lacking Vhl in the pancreas (PVhlKO mice). Both mouse strains developed glucose intolerance with impaired insulin secretion. Furthermore, deletion of Vhl in beta cells or the pancreas altered expression of genes involved in beta cell function, including those involved in glucose transport and glycolysis, and isolated betaVhlKO and PVhlKO islets displayed impaired glucose uptake and defective glucose metabolism. The abnormal glucose homeostasis was dependent on upregulation of Hif-1alpha expression, and deletion of Hif1a in Vhl-deficient beta cells restored GSIS. Consistent with this, expression of activated Hif-1alpha in a mouse beta cell line impaired GSIS. These data suggest that VHL/HIF oxygen-sensing mechanisms play a critical role in glucose homeostasis and that activation of this pathway in response to decreased islet oxygenation may contribute to beta cell dysfunction.

Role of furin in granular acidification in the endocrine pancreas: identification of the V-ATPase subunit Ac45 as a candidate substrate.

Furin is a proprotein convertase which activates a variety of regulatory proteins in the constitutive exocytic and endocytic pathway. The effect of genetic ablation of fur was studied in the endocrine pancreas to define its physiological function in the regulated secretory pathway. Pdx1-Cre/loxP furin KO mice show decreased secretion of insulin and impaired processing of known PC2 substrates like proPC2 and proinsulin II. Both secretion and PC2 activity depend on granule acidification, which was demonstrated to be significantly decreased in furin-deficient beta cells by using the acidotrophic agent 3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine (DAMP). Ac45, an accessory subunit of the proton pump V-ATPase, was investigated as a candidate substrate. Ac45 is highly expressed in islets of Langerhans and furin was able to cleave Ac45 ex vivo. Furthermore, the exact cleavage site was determined. In addition, reduced regulated secretion and proinsulin II processing could be obtained in the insulinoma cell line betaTC3 by downregulation of either furin or Ac45. Together, these data establish an important role for furin in regulated secretion, particularly in intragranular acidification most likely due to impaired processing of Ac45.