Downregulation of GLP-1 and GIP receptor expression by hyperglycemia: possible contribution to impaired incretin effects in diabetes.

Stimulation of insulin secretion by the incretin hormones glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) has been found to be diminished in type 2 diabetes. We hypothesized that this impairment is due to a defect at the receptor level induced by the diabetic state, particularly hyperglycemia. Gene expression of incretin receptors, GLP-1R and GIPR, were significantly decreased in islets of 90% pancreatectomized (Px) hyperglycemic rats, with recovery when glucose levels were normalized by phlorizin. Perifused islets isolated from hyperglycemic Px rats showed reduced insulin responses to GLP-1 and GIP. To examine the acute effect of hyperglycemia on incretin receptor expression, a hyperglycemic clamp study was performed for 96 h with reduction of GLP-1 receptor expression but increase in GIP receptor expression. Similar findings were found when islets were cultured at high glucose concentrations for 48 h. The reduction of GLP-1 receptor expression by high glucose was prevented by dominant-negative protein kinase C (PKC)alpha overexpression, whereas GLP-1 receptor expression was reduced with wild-type PKCalpha overexpression. Taken together, GLP-1 and GIP receptor expression is decreased with chronic hyperglycemia, and this decrease likely contributes to the impaired incretin effects found in diabetes.

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.

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.

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.

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.

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.

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.

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.