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Gordon C Weir

Publications and source records attributed to Gordon C Weir.

At least 19 recordsLinked to original sources
PubMed2007

NeuroD and reaggregation induce beta-cell specific gene expression in cultured hepatocytes.

BACKGROUND: Our goal was to convert adult mouse hepatocytes to pancreatic beta-cells. METHODS AND RESULTS: To facilitate conversion, cultured primary hepatocytes were dedifferentiated by the removal of dexamethasone (Dex) from the culture media. Removal of Dex caused detachment of hepatocytes from the culture dish, but the addition of betacellulin prevented this from happening. With the combination of lack of Dex and addition of betacellulin, albumin mRNA levels decreased. Cultured hepatocytes had a faint expression of insulin 2 mRNA, Nkx 6.1 and Pax 6 mRNA. Dedifferentiated hepatocytes were transduced with adenoviruses expressing NeuroD1, Ngn 3, or Pax 4. NeuroD1 transduction increased the insulin 2 mRNA but caused detachment of cells. However, when hepatocytes were allowed to reaggregate for 4 and 6 days in hydrophobic plates after transduction with NeuroD1, further increases of insulin 2 mRNA were found along with induction of PDX-1, IAPP, NeuroD1, Ngn3, Pax 4, Isl-1, PC1, PC2 and islet glucokinase. Additionally, glucagon, pancreatic polypeptide and somatostatin expression were induced, but neither elastase 1 nor insulin 1 mRNA could be detected. Ngn 3 and Pax 4 had effects similar to NeuroD1, but did not increase insulin 2 mRNA as much as NeuroD1. CONCLUSION: We conclude that the combination of NeuroD1 and reaggregation promotes cultured dedifferentiated hepatocytes to differentiate towards a pancreatic beta-cell phenotype.

Animals↗
PubMed2007

A dominant role for glucose in beta cell compensation of insulin resistance.

Increased insulin secretion and expansion of pancreatic beta cell mass work together to maintain normal glucose levels when insulin resistance develops. Changes in glucose concentration have long been known to have profound effects upon the rates of insulin secretion and beta cell mass, but various other agents can also cause changes, raising questions about which mechanisms are dominant. Evidence favoring a dominant role for glucose is provided by Terauchi et al. in this issue of the JCI (see the related article beginning on page 246). Mice haploinsufficient for beta cell glucokinase (Gck) were unable to increase their beta cell mass in response to insulin resistance produced by high-fat feeding. Gck is known to be the glucose sensor for glucose metabolism in beta cells. The study also provides strong evidence that insulin receptor substrate 2 (Irs2), which is known to have major effects on beta cell growth and survival, is a key downstream mediator of the effects of glucose found in this study.

Cell Division↗
PubMed2006

Evidence for a role of the ubiquitin-proteasome pathway in pancreatic islets.

The ubiquitin-proteasome pathway is crucial for protein turnover. Part of the pathway involves deubiquitination, which is carried out by cystein proteases known as ubiquitin COOH-terminal hydrolases. The isoform Uch-L1 was found to be present in large amounts in rat islets by immunostaining, Western blot analysis, and RT-PCR. Culturing islets in high glucose concentrations (16.7 mmol/l) for 24 h led to decreased gene expression. Exposure to chronic hyperglycemia following 90% partial pancreatectomy also led to reduced Uch-L1 expression. Expression of other members of the ubiquitin-proteasome pathway studied after culturing islets at high glucose concentrations revealed little change except for modest declines in parkin, human ubiquitin-conjugating enzyme 5 (UbcH5), and beta-TRCP (transducin repeat-containing protein). With the pancreatectomy model, expression of polyubiquitin-B and c-Cbl were increased and E6-associated protein was reduced. Further insight about the proteasome pathway was obtained with the proteasome inhibitor lactacystin, which in short-term 2-h experiments enhanced glucose-induced insulin secretion. An important role for the ubiquitin-proteasome pathways in beta-cells is suggested by the findings that changes in glucose concentration influence expression of genes in the pathway and that blockade of the proteasome degradation machinery enhances glucose-stimulated insulin secretion.

Base Sequence↗
PubMed2005

The Nkx6.1 homeodomain transcription factor suppresses glucagon expression and regulates glucose-stimulated insulin secretion in islet beta cells.

We have previously described rat insulinoma INS-1-derived cell lines with robust or poor glucose-stimulated insulin secretion (GSIS). In the current study, we have further resolved these lines into three classes: class 1, glucose-unresponsive/glucagon-expressing; class 2, glucose-unresponsive/glucagon-negative; and class 3, glucose-responsive/glucagon-negative. The transcription factor Nkx2.2 was expressed with relative abundance of 3.3, 1.0, and 1.0 in class 1, class 2, and class 3 cells, respectively, whereas Nkx6.1 expression had the opposite trend: 1.0, 2.6, and 6.4 in class 1, class 2, and class 3 cells, respectively. In class 1 cells, overexpressed Nkx6.1 suppressed glucagon expression but did not affect the levels of several other prominent beta cell transcription factors. RNA interference (RNAi)-mediated suppression of Nkx6.1 in class 3 cells resulted in a doubling of glucagon mRNA, with no effect on Pdx1 levels, whereas suppression of Pdx1 in class 3 cells caused a 12-fold increase in glucagon transcript levels, demonstrating independent effects of Nkx6.1 and Pdx1 on glucagon expression in beta cell lines. RNAi-mediated suppression of Nkx6.1 expression in class 3 cells also caused a decrease in GSIS from 13.9- to 3.7-fold, whereas suppression of Pdx1 reduced absolute amounts of insulin secretion without affecting fold response. Finally, RNAi-mediated suppression of Nkx6.1 mRNA in primary rat islets was accompanied by a significant decrease in GSIS relative to control cells. In sum, our studies have revealed roles for Nkx6.1 in suppression of glucagon expression and control of GSIS in islet beta cells.

Adenoviridae↗
PubMed2005

Induced ICER Igamma down-regulates cyclin A expression and cell proliferation in insulin-producing beta cells.

We have previously found that cyclin A expression is markedly reduced in pancreatic beta-cells by cell-specific overexpression of repressor inducible cyclic AMP early repressor (ICER Igamma) in transgenic mice. Here we further examined regulatory effects of ICER Igamma on cyclin A gene expression using Min6 cells, an insulin-producing cell line. The cyclin A promoter luciferase assay showed that ICER Igamma directly repressed cyclin A gene transcription. In addition, upon ICER Igamma overexpression, cyclin A mRNA levels markedly decreased, thereby confirming an inhibitory effect of ICER Igamma on cyclin A expression. Suppression of cyclin A results in inhibition of BrdU incorporation. Under normal culture conditions endogenous cyclin A is abundant in these cells, whereas ICER is hardly detectable. However, serum starvation of Min6 cells induces ICER Igamma expression with a concomitant very low expression level of cyclin A. Cyclin A protein is not expressed unless the cells are in active DNA replication. These results indicate a potentially important anti-proliferative effect of ICER Igamma in pancreatic beta cells. Since ICER Igamma is greatly increased in diabetes as well as in FFA- or high glucose-treated islets, this effect may in part exacerbate diabetes by limiting beta-cell proliferation.

Animals↗
PubMed2005

Mechanism of insulin gene regulation by the pancreatic transcription factor Pdx-1: application of pre-mRNA analysis and chromatin immunoprecipitation to assess formation of functional transcriptional complexes.

The homeodomain factor Pdx-1 regulates an array of genes in the developing and mature pancreas, but whether regulation of each specific gene occurs by a direct mechanism (binding to promoter elements and activating basal transcriptional machinery) or an indirect mechanism (via regulation of other genes) is unknown. To determine the mechanism underlying regulation of the insulin gene by Pdx-1, we performed a kinetic analysis of insulin transcription following adenovirus-mediated delivery of a small interfering RNA specific for pdx-1 into insulinoma cells and pancreatic islets to diminish endogenous Pdx-1 protein. insulin transcription was assessed by measuring both a long half-life insulin mRNA (mature mRNA) and a short half-life insulin pre-mRNA species by real-time reverse transcriptase-PCR. Following progressive knock-down of Pdx-1 levels, we observed coordinate decreases in pre-mRNA levels (to about 40% of normal levels at 72 h). In contrast, mature mRNA levels showed strikingly smaller and delayed declines, suggesting that the longer half-life of this species underestimates the contribution of Pdx-1 to insulin transcription. Chromatin immunoprecipitation assays revealed that the decrease in insulin transcription was associated with decreases in the occupancies of Pdx-1 and p300 at the proximal insulin promoter. Although there was no corresponding change in the recruitment of RNA polymerase II to the proximal promoter, its recruitment to the insulin coding region was significantly reduced. Our results suggest that Pdx-1 directly regulates insulin transcription through formation of a complex with transcriptional coactivators on the proximal insulin promoter. This complex leads to enhancement of elongation by the basal transcriptional machinery.

Adenoviridae↗
PubMed2005

Long-term normoglycemia in rats receiving transplants with encapsulated islets.

BACKGROUND: To follow up on previously successful transplantation of encapsulated islets in mice, the present study was performed in rats to determine the effects of several factors, including alginate composition and concentration of cross-linking agent and capsule size on the effectiveness of encapsulated islets. METHODS: Highly purified alginate of either high guluronic acid or high mannuronic acid (M) with low endotoxin content was used. Regular-size (0.8-1.1 mm) or small microcapsules (0.5-0.7 mm) were produced by cross-linking with BaCl2 without additional poly-L-lysine coating and were transplanted into abdominal cavity of normoglycemic (empty capsules) or streptozotocin induced diabetic Lewis rats (islet containing capsules). RESULTS: Empty regular-size capsules made of different alginate compositions had similar biocompatibility and stability results. Compared with empty capsules, regular-size capsules made of high-M alginate containing syngeneic islets had inferior stability indicated with lower fractional volume retrieved. Islet-containing smaller-size microcapsules made of high-M alginate were more stable and had less cellular attachment compared with the regular-size capsules, although the normoglycemic period was comparable between two groups of rats receiving transplants with smaller-size microcapsules (48+/-8 days, n=8) or regular-size capsules (59+/-11 days, n=4) in allogeneic experiments. In syngeneic experiments, all of the rats (n=4) maintained normoglycemia up to 210 days after transplantation. CONCLUSION: These results indicate that regular-size alginate capsules do less well in rats than in our previous experiments with mice. Smaller capsules made of alginate cross-linked with barium appear to provide better stability and may be a useful strategy for use in larger recipients.

Animals↗
PubMed2005

The HNF-1 target collectrin controls insulin exocytosis by SNARE complex formation.

Defective glucose-stimulated insulin secretion is the main cause of hyperglycemia in type 2 diabetes mellitus. Mutations in HNF-1alpha cause a monogenic form of type 2 diabetes, maturity-onset diabetes of the young (MODY), characterized by impaired insulin secretion. Here we report that collectrin, a recently cloned kidney-specific gene of unknown function, is a target of HNF-1alpha in pancreatic beta cells. Expression of collectrin was decreased in the islets of HNF-1alpha (-/-) mice, but was increased in obese hyperglycemic mice. Overexpression of collectrin in rat insulinoma INS-1 cells or in the beta cells of transgenic mice enhanced glucose-stimulated insulin exocytosis, without affecting Ca(2+) influx. Conversely, suppression of collectrin attenuated insulin secretion. Collectrin bound to SNARE complexes by interacting with snapin, a SNAP-25 binding protein, and facilitated SNARE complex formation. Therefore, collectrin is a regulator of SNARE complex function, which thereby controls insulin exocytosis.

Animals↗
PubMed2005

Establishment of a diabetic mouse model with progressive diabetic nephropathy.

Although diabetic animal models exist, no single animal model develops renal changes identical to those seen in humans. Here we show that transgenic mice that overexpress inducible cAMP early repressor (ICER Igamma) in pancreatic beta cells are a good model to study the pathogenesis of diabetic nephropathy. Although ICER Igamma transgenic mice exhibit extremely high blood glucose levels throughout their lives, they survive long enough to develop diabetic nephropathy. Using this model we followed the progress of diabetic renal changes compared to those seen in humans. By 8 weeks of age, the glomerular filtration rate (GFR) was already increased, and glomerular hypertrophy was prominent. At 20 weeks, GFR reached its peak, and urine albumin excretion rate was elevated. Finally, at 40 weeks, diffuse glomerular sclerotic lesions were prominently accompanied by increased expression of collagen type IV and laminin and reduced expression of matrix metalloproteinase-2. Nodular lesions were absent, but glomerular basement membrane thickening was prominent. At this point, GFR declined and urinary albumin excretion rate increased, causing a nephrotic state with lower serum albumin and higher serum total cholesterol. Thus, similar to human diabetic nephropathy, ICER Igamma transgenic mice exhibit a stable and progressive phenotype of diabetic kidney disease due solely to chronic hyperglycemia without other modulating factors.

Albumins↗
PubMed2005

New sources of pancreatic beta-cells.

Two major initiatives are under way to correct the beta-cell deficit of diabetes: one would generate beta-cells ex vivo that are suitable for transplantation, and the second would stimulate regeneration of beta-cells in the pancreas. Studies of ex vivo expansion suggest that beta-cells have a potential for dedifferentiation, expansion, and redifferentiation. Work with mouse and human embryonic stem (ES) cells has not yet produced cells with the phenotype of true beta-cells, but there has been recent progress in directing ES cells to endoderm. Putative islet stem/progenitor cells have been identified in mouse pancreas, and formation of new beta-cells from duct, acinar and liver cells is an active area of investigation. Peptides, including glucagon-like peptide-1/exendin-4 and the combination of epidermal growth factor and gastrin, can stimulate regeneration of beta-cells in vivo. Recent progress in the search for new sources of beta-cells has opened promising new opportunities and spawned clinical trials.

Cell Differentiation↗
PubMed2004

Can we make surrogate beta-cells better than the original?

Insufficient pancreatic beta-cell mass is fundamental to the pathogenesis of both types 1 and 2 diabetes and constitutes the basis for the goal of beta-cell replacement therapy. Current methods for isolating islets from organ donor pancreases do not come close to supplying all in need, thus providing a compelling need to find new sources of insulin-producing cells. Possible sources include generation of cells from embryonic stem cells (ESC), adult stem/precursor cells, transdifferentiation of other cell types and xenodonors. Bioengineering can be used to improve secretory performance and strengthen cells to better withstand the challenges of transplantation. Strategies include protection against hypoxia, inflammation, and immune attack.

Animals↗
PubMed2004

Transplantation of islets transduced with CTLA4-Ig and TGFbeta using adenovirus and lentivirus vectors.

BACKGROUND: A major problem facing islet transplantation is immune destruction of grafts by transplant rejection and autoimmunity. Some success in prolonging graft rejection has been obtained by transducing islets prior to transplantation with adenoviral (Ad) vectors containing CTLA4-Ig and TGFbeta. The purpose of this study was to see if lentiviral (LV) vectors would provide superior results compared with adenoviral vectors. METHODS: Islets were isolated from Sprague-Dawley rats and transduced with Ad or LV vectors containing LacZ, CTLA4-Ig, CTLA4, and TGFbeta1 using various MOIs. Islets transduced with LV were healthy as judged by DNA and insulin content, and insulin secretion. Using the kidney capsule transplant site, 500 transduced rat islets were transplanted into streptozotocin diabetic B6AF1 mice. RESULTS: Maintenance of normoglycemia was prolonged in recipient mice carrying islets transduced with Ad vectors containing CTLA4-Ig, CTLA4, and TGFbeta1. Return of hyperglycemia in controls was 17-18 days while loss of function for the experimental groups occurred at 20-27 days. For the lentivirus transduced islets, rejection of controls was 20+/-1.6 days, for CTLA4-Ig was 42+/-21 days and for TGFbeta was 28+/-3.2 days. CONCLUSIONS: Although islets transduced with either adenovirus or lentivirus containing CTLA4-Ig, CTLA4, and TGFbeta1 could prolong graft survival in a rat to mouse transplantation model, with the conditions of this study lentivirus provided no advantage over adenovirus vectors.

Abatacept↗
PubMed2004

Survival of microencapsulated adult pig islets in mice in spite of an antibody response.

The aim of this study was to assess the capacity of simple alginate capsules to protect adult pig islets in a model of xenotransplantation. Adult pig islets were microencapsulated in alginate, with either single alginate coats (SAC) or double alginate coats (DAC), and transplanted into the streptozotocin-induced diabetic B6AF1 mice. Normalization of glucose levels was associated with an improvement of the glucose clearance during intravenous glucose tolerance tests. After explantation, all mice became hyperglycemic, demonstrating the efficacy of the encapsulated pig islets. Explanted capsules were mainly free of fibrotic reaction and encapsulated islets were still functional, responding to glucose stimulation with a 10-fold increase in insulin secretion. However, a significant decrease in the insulin content and insulin responses to glucose was observed for encapsulated islets explanted from hyperglycemic mice. An immune response of both IgG and IgM subtypes was detectable after transplantation. Interestingly, there were more newly formed antibodies in the serum of mice transplanted with SAC capsules than in the serum of mice transplanted with DAC capsules. In conclusion, alginate capsules can prolong the survival of adult pig islets transplanted into diabetic mice for up to 190 days, even in the presence of an antibody response.

Animals↗
PubMed2004

Exercise induces hypoglycemia in rats with islet transplantation.

Recently, islet transplantation in patients with type 1 diabetes has had greater success than in the past, but the important question of whether the kinetics of islet secretion are able to accommodate the metabolic demands of special conditions such as exercise remains unanswered. Syngeneic rat islets (4,000 islet equivalents/rat) were transplanted into the liver, kidney, and peritoneal cavity (encapsulated or nonencapsulated) of rats with streptozocin-induced diabetes. Normoglycemic transplanted rats and age-matched controls were subjected to 30 min of moderate exercise on a treadmill 5 weeks after transplantation. Although control rats maintained near normoglycemia during and after exercise, the rats with islet transplants had significantly lower blood glucose levels. For the rats with islets in the liver, increased C-peptide levels were found at 30 min (790 +/- 125 and 1,450 +/- 250 pmol/l at 0 and 30 min, respectively; P < 0.01), whereas a decrease was found in controls and in rats with islets transplanted into the peritoneal cavity or under the kidney capsule. Moreover, increased glucagon levels were found after exercise in the rats with islets transplanted into the liver (62 +/- 6, 165 +/- 29, 155 +/- 27, and 97 +/- 13 pg/ml at 0, 30, 60, and 90 min, respectively; P < 0.05), whereas no changes in glucagon levels were observed in controls. In conclusion, moderate exercise caused hypoglycemia in rats with islet transplants in different sites including liver, kidney, and peritoneal cavity. C-peptide and glucagon responses to exercise were very different in rats with transplanted islets compared with controls. This islet dysfunction led to exercise-induced hypoglycemia.

Animals↗
PubMed2004

Five stages of evolving beta-cell dysfunction during progression to diabetes.

This article proposes five stages in the progression of diabetes, each of which is characterized by different changes in beta-cell mass, phenotype, and function. Stage 1 is compensation: insulin secretion increases to maintain normoglycemia in the face of insulin resistance and/or decreasing beta-cell mass. This stage is characterized by maintenance of differentiated function with intact acute glucose-stimulated insulin secretion (GSIS). Stage 2 occurs when glucose levels start to rise, reaching approximately 5.0-6.5 mmol/l; this is a stable state of beta-cell adaptation with loss of beta-cell mass and disruption of function as evidenced by diminished GSIS and beta-cell dedifferentiation. Stage 3 is a transient unstable period of early decompensation in which glucose levels rise relatively rapidly to the frank diabetes of stage 4, which is characterized as stable decompensation with more severe beta-cell dedifferentiation. Finally, stage 5 is characterized by severe decompensation representing a profound reduction in beta-cell mass with progression to ketosis. Movement across stages 1-4 can be in either direction. For example, individuals with treated type 2 diabetes can move from stage 4 to stage 1 or stage 2. For type 1 diabetes, as remission develops, progression from stage 4 to stage 2 is typically found. Delineation of these stages provides insight into the pathophysiology of both progression and remission of diabetes.

Animals↗
PubMed2003

Importance of hyperglycemia on the primary function of allogeneic islet transplants.

BACKGROUND: Hyperglycemia has been shown to influence primary function of islet isografts. In this study, we investigated the influence of hyperglycemia on primary function of allogeneic islets transplanted into spontaneously diabetic recipients (NOD) or streptozotocin-induced diabetic mice (BALB/c). METHODS: Mice with moderate, severe, or very severe hyperglycemia underwent transplantation with a marginal number of islets (350 into BALB/c mice and 700 into NOD mice). To prevent the alloimmune response, we used blockade of CD28:B7 and CD40L:CD40 costimulatory signaling pathways to determine the effect of hyperglycemia alone. Blood glucose levels of the mice were monitored after transplantation, and the grafts were assessed morphologically. RESULTS: Transplantation of allogeneic islets into moderately hyperglycemic BALB/c mice or severely diabetic NOD mice normalized the blood glucose levels in all mice within 3 days after transplantation, demonstrating the primary function of the graft. However, primary nonfunction was observed in all animals when islet transplantation was performed into severely diabetic BALB/c mice or very severely diabetic NOD mice. When mice were treated with costimulation blockade, reversal of diabetes was observed in severely diabetic BALB/c mice 15 days after transplantation, showing that the islets could adapt to the environment and function. However, transplantation of islets into NOD mice with very severe diabetes treated with costimulation blockade did not reverse diabetes, showing that even in the absence of alloimmune responses and given an adaptation period, the islets could not function. CONCLUSIONS: This study demonstrates that severe hyperglycemia impairs islet allograft function in BALB/c and NOD mice and that successful islet allotransplantation depends on the degree of hyperglycemia in the recipient.

Animals↗