Biomedical subjects

Gordon C Weir

Publications and source records attributed to Gordon C Weir.

32 records · Page 2Linked to original sources

PubMed2003

Induction of Mad expression leads to augmentation of insulin gene transcription.

Insulin gene transcription is critical for the maintenance of pancreatic beta-cell differentiation and insulin production. In this study, we found that the basic helix-loop-helix transcription factor Mad, which usually acts as a repressor to c-Myc, enhances insulin gene transcription. In isolated rat islets adenoviral overexpression of Mad augmented insulin mRNA expression and insulin protein content, as well as glucokinase and GLUT2 mRNA expression. Also, Mad overexpression upregulated insulin promoter activity in beta-cell-derived cell lines, MIN6 and betaTC1, as well as in non-insulin producing liver cell line, HepG2. Mad overexpression in rat islets enhanced PDX-1 expression and its DNA binding activity. We found that Mad mediated increased PDX-1 expression by an E-box dependent transcriptional regulation of the PDX-1 gene. That the effects of Mad on insulin expression were mediated through PDX-1 was further substantiated by studies showing inhibition of insulin promoter activation by Mad in the presence of mutated PDX-1 binding site. Although Mad functions as a negative regulatory factor for multiple target genes, these studies establish the fact that Mad can also function as a positive regulatory factor for insulin gene transcription. Such regulation of insulin expression by Mad with modulation of PDX-1 expression and DNA binding activity could offer useful therapeutic and/or experimental tools to promote insulin production in appropriate cell types.

Adenoviridae↗

PubMed2003

Macrophage depletion improves survival of porcine neonatal pancreatic cell clusters contained in alginate macrocapsules transplanted into rats.

BACKGROUND: Macrophages can accumulate on the surface of empty and islet-containing alginate capsules, leading to loss of functional tissue. In this study, the effect of peritoneal macrophage depletion on the biocompatibility of alginate macrocapsules and function of macroencapsulated porcine neonatal pancreatic cell clusters (NPCCs) was investigated. METHODS: Clodronate liposomes were injected into the peritoneal cavities of normoglycemic Lewis rats 5 and 2 days before the transplantation. Empty or NPCC-containing Ca-alginate poly L-lysine (PLL)-coated macrocapsules were transplanted into the peritoneal cavities of rats injected with either clodronate liposomes or saline. On days 7, 14 and 21, samples were evaluated by immunohistochemistry for cellular immune responses on the surface of the macrocapsules and for macrophage populations in omental tissue. To assess the function of macroencapsulated NPCCs, insulin secretory responses to glucose and theophylline were measured after capsule retrieval. RESULTS: In saline-injected control groups, all of the empty and NPCC-containing macrocapsules were overgrown with macrophages, this being especially severe on NPCC-containing macrocapsules. In the clodronate liposomes-injected group, the majority of the empty macrocapsules were free of macrophage accumulation and the NPCC-containing macrocapsules were less overgrown than in control animals. Higher insulin responses to glucose and theophylline were observed in NPCCs retrieved from rats injected with clodronate liposomes. CONCLUSION: We conclude that depletion of peritoneal macrophages with clodronate liposomes improve the survival of macroencapsulated NPCCs.

Alginates↗

PubMed2003

Development and retroviral transduction of porcine neonatal pancreatic islet cells in monolayer culture.

To learn more about the potential of neonatal porcine pancreatic duct and islet cells for xenotransplantation, the development of these cells when cultured as monolayers was evaluated. Immunostaining for islet hormones and cytokeratin-7 revealed that day eight monolayers consisted of approximately 70% duct cells and less than 10% beta cells. Using Ki-67 immunostaining as a proliferation marker, the fraction of beta cells in the cell cycle was shown to decrease from 20% at day three to 10% at day eight, and for duct cells from 36 to 19%. Insulin secretion increased 2.4-fold upon glucose stimulation, and 38-fold when 10 mm theophylline was added, showing the responsiveness of the neonatal beta cells. Reaggregated monolayers consisted mostly of duct cells, but 4 weeks after transplantation, grafts contained predominantly endocrine cells, with duct cells being almost absent, suggesting in vivo differentiation of duct cells to endocrine cells. Monolayer susceptibility to retroviral transduction was also investigated using a Moloney Murine Leukemia Virus-based vector. Approximately 60% of duct cells but less than 5% of beta cells expressed the transgene, indicating that precursor duct cells are better targets for transgene expression. These results show that porcine neonatal pancreatic cells can be cultured as monolayers in preparation for transplantation. Furthermore, in such a culture setting, precursor duct cells have a high rate of proliferation and are more efficiently transduced with a retrovirus-based reporter gene than are beta cells.

Animals↗

PubMed2003

Survival and maturation of microencapsulated porcine neonatal pancreatic cell clusters transplanted into immunocompetent diabetic mice.

Differentiation and maturation of porcine neonatal pancreatic cell clusters (NPCCs) microencapsulated in barium alginate were assessed after transplantation into immunocompetent mice. Microencapsulated NPCCs were transplanted into the peritoneal cavity of streptozocin-induced diabetic B6AF1 mice (n = 32). The microcapsules were removed at 2, 6, and 20 weeks and examined for cellular overgrowth, insulin content, and insulin secretory responses to glucose and glucose with theophylline. The differentiation, maturation, and proliferation of the beta-cells in the NPCCs were assessed by immunohistochemistry. Blood glucose levels were normalized in 81% of the animals that received a transplant and remained normal until termination of the experiments at 20 weeks. Hyperglycemic blood glucose levels after explantation of the capsules confirmed the function of the encapsulated NPCCs. Insulin content of the encapsulated NPCCs was increased 10-fold at 20 weeks after transplantation compared with pretransplantation levels. A 3.2-fold increase of the ratio of the beta-cell area to the total cellular area was observed at 20 weeks, demonstrating the maturation of NPCCs into beta-cells. In conclusion, NPCCs encapsulated with simple barium alginate can differentiate into beta-cells and reverse high blood glucose levels in immunocompetent mice without immunosuppression for >20 weeks.

Alginates↗

PubMed2003

PDX-1 protein containing its own antennapedia-like protein transduction domain can transduce pancreatic duct and islet cells.

The pancreatic and duodenal homeobox factor-1 (PDX-1), also known as IDX-1/STF-1/IPF1, a homeodomain-containing transcription factor, plays a central role in regulating pancreatic development and insulin gene transcription. Furthermore, even in adults, PDX-1 is associated with islet neogenesis and differentiation of insulin-producing cells from progenitor cells. Here, we report for the first time that PDX-1 protein can permeate cells due to an Antennapedia-like protein transduction domain sequence in its structure and that transduced PDX-1 functions similarly to endogenous PDX-1; it binds to the insulin promoter and activates its expression. PDX-1 protein can also permeate into isolated pancreatic islets, which leads to stimulation of insulin gene expression. Moreover, PDX-1 protein transduced into cultures of pancreatic ducts, thought to be islet progenitor cells, induces insulin gene expression. These data suggest that PDX-1 protein transduction could be a safe and valuable strategy for enhancing insulin gene transcription and for facilitating differentiation of ductal progenitor cells into insulin-producing cells without requiring gene transfer technology.

Amino Acid Sequence↗

PubMed2002

Critical reduction in beta-cell mass results in two distinct outcomes over time. Adaptation with impaired glucose tolerance or decompensated diabetes.

We have proposed that hyperglycemia-induced dedifferentiation of beta-cells is a critical factor for the loss of insulin secretory function in diabetes. Here we examined the effects of the duration of hyperglycemia on gene expression in islets of partially pancreatectomized (Px) rats. Islets were isolated, and mRNA was extracted from rats 4 and 14 weeks after Px or sham Px surgery. Px rats developed different degrees of hyperglycemia; low hyperglycemia was assigned to Px rats with fed blood glucose levels less than 150 mg/dl, and high hyperglycemia was assigned above 150 mg/dl. beta-Cell hypertrophy was present at both 4 and 14 weeks. At the same time points, high hyperglycemia rats showed a global alteration in gene expression with decreased mRNA for insulin, IAPP, islet-associated transcription factors (pancreatic and duodenal homeobox-1, BETA2/NeuroD, Nkx6.1, and hepatocyte nuclear factor 1 alpha), beta-cell metabolic enzymes (glucose transporter 2, glucokinase, mitochondrial glycerol phosphate dehydrogenase, and pyruvate carboxylase), and ion channels/pumps (Kir6.2, VDCC beta, and sarcoplasmic reticulum Ca(2+)-ATPase 3). Conversely, genes normally suppressed in beta-cells, such as lactate dehydrogenase-A, hexokinase I, glucose-6-phosphatase, stress genes (heme oxygenase-1, A20, and Fas), and the transcription factor c-Myc, were markedly increased. In contrast, gene expression in low hyperglycemia rats was only minimally changed at 4 weeks but significantly changed at 14 weeks, indicating that even low levels of hyperglycemia induce beta-cell dedifferentiation over time. In addition, whereas 2 weeks of correction of hyperglycemia completely reverses the changes in gene expression of Px rats at 4 weeks, the changes at 14 weeks were only partially reversed, indicating that the phenotype becomes resistant to reversal in the long term. In conclusion, chronic hyperglycemia induces a progressive loss of beta-cell phenotype with decreased expression of beta-cell-associated genes and increased expression of normally suppressed genes, these changes being present with even minimal levels of hyperglycemia. Thus, both the severity and duration of hyperglycemia appear to contribute to the deterioration of the beta-cell phenotype found in diabetes.

Adaptation, Physiological↗

PubMed2002

Involvement of c-Jun N-terminal kinase in oxidative stress-mediated suppression of insulin gene expression.

Oxidative stress, which is found in pancreatic beta-cells in the diabetic state, suppresses insulin gene transcription and secretion, but the signaling pathways involved in the beta-cell dysfunction induced by oxidative stress remain unknown. In this study, subjecting rat islets to oxidative stress activates JNK, p38 MAPK, and protein kinase C, preceding the decrease of insulin gene expression. Adenovirus-mediated overexpression of dominant-negative type (DN) JNK, but not the p38 MAPK inhibitor SB203580 nor the protein kinase C inhibitor GF109203X, protected insulin gene expression and secretion from oxidative stress. Moreover, wild type JNK overexpression suppressed both insulin gene expression and secretion. These results were correlated with changes in the binding of the important transcription factor PDX-1 to the insulin promoter; adenoviral overexpression of DN-JNK preserved PDX-1 DNA binding activity in the face of oxidative stress, whereas wild type JNK overexpression decreased PDX-1 DNA binding activity. Furthermore, to examine whether suppression of the JNK pathway can protect beta-cells from the toxic effects of hyperglycemia, rat islets were infected with DN-JNK expressing adenovirus or control adenovirus and transplanted under renal capsules of streptozotocin-induced diabetic nude mice. In mice receiving DN-JNK overexpressing islets, insulin gene expression in islet grafts was preserved, and hyperglycemia was ameliorated compared with control mice. In conclusion, activation of JNK is involved in the reduction of insulin gene expression by oxidative stress, and suppression of the JNK pathway protects beta-cells from oxidative stress.

Adenoviridae↗

PubMed2002

Induction of c-Myc expression suppresses insulin gene transcription by inhibiting NeuroD/BETA2-mediated transcriptional activation.

Insulin biosynthesis and secretion are critical for pancreatic beta-cell function, but both are impaired under diabetic conditions. We have found that hyperglycemia induces the expression of the basic helix-loop-helix transcription factor c-Myc in islets in several different diabetic models. To examine the possible implication of c-Myc in beta-cell dysfunction, c-Myc was overexpressed in isolated rat islets using adenovirus. Adenovirus-mediated c-Myc overexpression suppressed both insulin gene transcription and glucose-stimulated insulin secretion. Insulin protein content, determined by immunostaining, was markedly decreased in c-Myc-overexpressing cells. In gel-shift assays c-Myc bound to the E-box in the insulin gene promoter region. Furthermore, in betaTC1, MIN6, and HIT-T15 cells and primary rat islets, wild type insulin gene promoter activity was dramatically decreased by c-Myc overexpression, whereas the activity of an E-box mutated insulin promoter was not affected. In HeLa and HepG2 cells c-Myc exerted a suppressive effect on the insulin promoter activity only in the presence of NeuroD/BETA2 but not PDX-1. Both c-Myc and NeuroD can bind the E-box element in the insulin promoter, but unlike NeuroD, the c-Myc transactivation domain lacked the ability to activate insulin gene expression. Additionally p300, a co-activator of NeuroD, did not function as a co-activator of c-Myc. In conclusion, increased expression of c-Myc in beta-cells suppresses the insulin gene transcription by inhibiting NeuroD-mediated transcriptional activation. This mechanism may explain some of the beta-cell dysfunction found in diabetes.

Adenoviridae↗

PubMed2002

Genetic regulation of metabolic pathways in beta-cells disrupted by hyperglycemia.

In models of type 2 diabetes the expression of beta-cell genes is altered, but these changes have not fully explained the impairment in beta-cell function. We hypothesized that changes in beta-cell phenotype and global alterations in both carbohydrate and lipid pathways are likely to contribute to secretory abnormalities. Therefore, expression of genes involved in carbohydrate and lipid metabolism were analyzed in islets 4 weeks after 85-95% partial pancreatectomy (Px) when beta-cells have impaired glucose-induced insulin secretion and ATP synthesis. Px rats after 1 week developed mild to severe hyperglycemia that was stable for the next 3 weeks, whereas neither plasma triglyceride, non-esterified fatty acid, or islet triglyceride levels were altered. Expression of peroxisome proliferator-activated receptors (PPARs), with several target genes, were reciprocally regulated; PPARalpha was markedly reduced even at low level hyperglycemia, whereas PPARgamma was progressively increased with increasing hyperglycemia. Uncoupling protein 2 (UCP-2) was increased as were other genes barely expressed in sham islets including lactate dehydrogenase-A (LDH-A), lactate (monocarboxylate) transporters, glucose-6-phosphatase, fructose-1,6-bisphosphatase, 12-lipoxygenase, and cyclooxygenase 2. On the other hand, the expression of beta-cell-associated genes, insulin, and GLUT2 were decreased. Treating Px rats with phlorizin normalized hyperglycemia without effecting plasma fatty acids and reversed the changes in gene expression implicating the importance of hyperglycemia per se in the loss of beta-cell phenotype. In addition, parallel changes were observed in beta-cell-enriched tissue dissected by laser capture microdissection from the central core of islets. In conclusion, chronic hyperglycemia leads to a critical loss of beta-cell differentiation with altered expression of genes involved in multiple metabolic pathways diversionary to normal beta-cell glucose metabolism. This global maladaptation in gene expression at the time of increased secretory demand may contribute to the beta-cell dysfunction found in diabetes.

Adenosine Triphosphate↗

PubMed2002

Increased expression of antioxidant and antiapoptotic genes in islets that may contribute to beta-cell survival during chronic hyperglycemia.

Hypertrophy is one mechanism of pancreatic beta-cell growth and is seen as an important compensatory response to insulin resistance. We hypothesized that the induction of protective genes contributes to the survival of enlarged (hypertrophied) beta-cells. Here, we evaluated changes in stress gene expression that accompany beta-cell hypertrophy in islets from hyperglycemic rats 4 weeks after partial pancreatectomy (Px). A variety of protective genes were upregulated, with markedly increased expression of the antioxidant genes heme oxygenase-1 and glutathione peroxidase and the antiapoptotic gene A20. Cu/Zn-superoxide dismutase (SOD) and Mn-SOD were modestly induced, and Bcl-2 was modestly reduced; however, several other stress genes (catalase, heat shock protein 70, and p53) were unaltered. The increases in mRNA levels corresponded to the degree of hyperglycemia and were reversed in Px rats by 2-week treatment with phlorizin (treatment that normalized hyperglycemia), strongly suggesting the specificity of hyperglycemia in eliciting the response. Hyperglycemia in Px rats also led to activation of nuclear factor-kappaB in islets. The profound change in beta-cell phenotype of hyperglycemic Px rats resulted in a reduced sensitivity to the beta-cell toxin streptozotocin. Sensitivity to the toxin was restored, along with the beta-cell phenotype, in islets from phlorizin-treated Px rats. Furthermore, beta-cells of Px rats were not vulnerable to apoptosis when further challenged in vivo with dexamethasone, which increases insulin resistance. In conclusion, beta-cell adaptation to chronic hyperglycemia and, hence, increased insulin demand is accompanied by the induction of protective stress genes that may contribute to the survival of hypertrophied beta-cells.

Animals↗

PubMed2002

Overexpression of c-Myc in beta-cells of transgenic mice causes proliferation and apoptosis, downregulation of insulin gene expression, and diabetes.

To test the hypothesis that c-Myc plays an important role in beta-cell growth and differentiation, we generated transgenic mice overexpressing c-Myc in beta-cells under control of the rat insulin II promoter. F(1) transgenic mice from two founders developed neonatal diabetes (associated with reduced plasma insulin levels) and died of hyperglycemia 3 days after birth. In pancreata of transgenic mice, marked hyperplasia of cells with an altered phenotype and amorphous islet organization was displayed: islet volume was increased threefold versus wild-type littermates. Apoptotic nuclei were increased fourfold in transgenic versus wild-type mice, suggesting an increased turnover of beta-cells. Very few cells immunostained for insulin; pancreatic insulin mRNA and content were markedly reduced. GLUT2 mRNA was decreased, but other beta-cell-associated genes (IAPP [islet amyloid pancreatic polypeptide], PDX-1 [pancreatic and duodenal homeobox-1], and BETA2/NeuroD) were expressed at near-normal levels. Immunostaining for both GLUT2 and Nkx6.1 was mainly cytoplasmic. The defect in beta-cell phenotype in transgenic embryos (embryonic days 17-18) and neonates (days 1-2) was similar and, therefore, was not secondary to overt hyperglycemia. When pancreata were transplanted under the kidney capsules of athymic mice to analyze the long-term effects of c-Myc activation, beta-cell depletion was found, suggesting that, ultimately, apoptosis predominates over proliferation. In conclusion, these studies demonstrate that activation of c-Myc in beta-cells leads to 1) increased proliferation and apoptosis, 2) initial hyperplasia with amorphous islet organization, and 3) selective downregulation of insulin gene expression and the development of overt diabetes.

Animals↗

PubMed2002

Islet transplantation under the kidney capsule corrects the defects in glycogen metabolism in both liver and muscle of streptozocin-diabetic rats.

Insulin-deficient rats are characterized by multiple defects in the pathway of glycogen synthesis and breakdown in both liver and skeletal muscle. The aim of this study was to clarify whether islet transplantation under the kidney capsule, which is associated with delivery of insulin into the peripheral circulation, is able to normalize glycogen metabolism in liver and muscle of streptozotocin-diabetic rats. Three groups of male Lewis rats were studied under fasting condition: controls, untreated diabetics, and islet transplanted diabetics. Glycogen content, glucose-6-phosphate concentration, and glycogen synthase activity were measured in both liver and skeletal muscle. Untreated diabetic rats were characterized by an increase in glycogen content of 178% and a reduction of glucose-6-phosphate level of 50%. Both glycogen and glucose-6-phosphate contents were restored to normal in transplanted diabetic rats. Active glycogen synthase (0.35 +/- 0.1 nmol/min/mg) and activity ratio (0.22 +/- 0.04) were significantly impaired compared with controls (0.99 +/- 0.2 nmol/min/mg and 0.43 +/- 0.06, respectively) and were normalized by islet transplantation. In the skeletal muscle, glycogen content was similar in the three groups of animals, whereas muscle glucose-6-phosphate level was reduced by 28% and glycogen synthase was in a less active state in the untreated diabetic rats. Both the glucose-6-phosphate concentration and the kinetic profile of glycogen synthase were normalized by islet transplantation. In conclusion, islet transplantation under the kidney capsule corrects the diabetes-induced abnormalities in glycogen and glucose-6-phosphate content and glycogen synthase activity in both liver and skeletal muscle.

Animals↗

PubMed2001

Involvement of protein kinase C beta 2 in c-myc induction by high glucose in pancreatic beta-cells.

The expression of the basic helix-loop-helix transcription factor c-Myc is induced in pancreatic islets of several different diabetic model animals and is possibly involved in suppression of the insulin gene transcription. In this study, we found that activity of protein kinase C is increased by high glucose, preceding the induction of c-myc expression and that PKC beta2 specifically regulates c-myc expression in pancreatic beta-cells. Since PKC alpha, beta2, delta, epsilon, and zeta were expressed in rat pancreatic islets, we prepared each wild type (WT) and dominant negative type (DN) PKC isoform (alpha, beta2, delta, epsilon, and zeta)-expressing adenovirus to examine the effect of each PKC isoform on c-myc expression. In isolated rat pancreatic islets, adenovirus-mediated overexpression of WT PKC beta2, but not other PKC isoforms, markedly increased c-myc expression. Moreover, c-myc induction by high glucose was suppressed by adenovirus-mediated overexpression of DN PKC beta2 but not by other DN PKC isoforms. Finally, adenovirus-mediated overexpression of WT PKC beta2, but not of other PKC isoforms, leads to suppression of the insulin gene transcription in pancreatic islets. These results suggest that at least some of the reduction of insulin gene transcription found in the diabetic state is mediated by PKC beta2 regulation of c-myc expression.

Adenoviridae↗

PubMedDate not supplied

Islet transplantation: progress and challenge.

For over 30 years, investigators have explored islet transplantation as a logical approach to restoring glucose homeostasis in persons with diabetes. Islet transplantation can currently provide improved glycemic control, relief from recurrent severe hypoglycemia, and potentially insulin independence. In this review, we describe details of the evolution of modern islet transplantation and provide insight into ongoing clinical and basic research efforts to overcome current obstacles for this promising therapy.

Animals↗