Aileen King

The use of animal models in diabetes research

Diabetes is a disease characterized by a relative or absolute lack of insulin, leading to hyperglycaemia. There are two main types of diabetes: type 1 diabetes and type 2 diabetes. Type 1 diabetes is due to an autoimmune destruction of the insulin‐producing pancreatic beta cells, and type 2 diabetes is caused by insulin resistance coupled by a failure of the beta cell to compensate. Animal models for type 1 diabetes range from animals with spontaneously developing autoimmune diabetes to chemical ablation of the pancreatic beta cells. Type 2 diabetes is modelled in both obese and non‐obese animal models with varying degrees of insulin resistance and beta cell failure. This review outlines some of the models currently used in diabetes research. In addition, the use of transgenic and knock‐out mouse models is discussed. Ideally, more than one animal model should be used to represent the diversity seen in human diabetic patients.

Somatostatin Secreted by Islet δ-Cells Fulfills Multiple Roles as a Paracrine Regulator of Islet Function

OBJECTIVE— Somatostatin (SST) is secreted by islet δ-cells and by extraislet neuroendocrine cells. SST receptors have been identified on α- and β-cells, and exogenous SST inhibits insulin and glucagon secretion, consistent with a role for SST in regulating α- and β-cell function. However, the specific intraislet function of δ-cell SST remains uncertain. We have used Sst−/− mice to investigate the role of δ-cell SST in the regulation of insulin and glucagon secretion in vitro and in vivo. RESEARCH DESIGN AND METHODS— Islet morphology was assessed by histological analysis. Hormone levels were measured by radioimmunoassay in control and Sst−/− mice in vivo and from isolated islets in vitro. RESULTS— Islet size and organization did not differ between Sst−/− and control islets, nor did islet glucagon or insulin content. Sst−/− mice showed enhanced insulin and glucagon secretory responses in vivo. In vitro stimulus-induced insulin and glucagon secretion was enhanced from perifused Sst−/− islets compared with control islets and was inhibited by exogenous SST in Sst−/− but not control islets. No difference in the switch-off rate of glucose-stimulated insulin secretion was observed between genotypes, but the cholinergic agonist carbamylcholine enhanced glucose-induced insulin secretion to a lesser extent in Sst−/− islets compared with controls. Glucose suppressed glucagon secretion from control but not Sst−/− islets. CONCLUSIONS— We suggest that δ-cell SST exerts a tonic inhibitory influence on insulin and glucagon secretion, which may facilitate the islet response to cholinergic activation. In addition, δ-cell SST is implicated in the nutrient-induced suppression of glucagon secretion.

TRANSPLANTATION OF ALGINATE MICROCAPSULES: GENERATION OF ANTIBODIES AGAINST ALGINATES AND ENCAPSULATED PORCINE ISLET-LIKE CELL CLUSTERS

BACKGROUND Microencapsulation of islets of Langherhans in alginate poly-L-lysine capsules provides an effective protection against cell-mediated immune destruction, and ideally should allow the transplantation of islets in the absence of immunosuppression. It has previously been suggested that alginate rich in mannuronic acid (high M) is more immunogenic than alginate rich in guluronic acid (high G). The ability of these alginates to induce an antibody response in the recipient or act as an adjuvant to antibody responses against antigens leaked from the capsule was investigated in the present study. METHODS Empty capsules made from these different types of alginate were transplanted intraperitoneally to Wistar rats or Balb/c mice. In addition, some animals were also injected with bovine serum albumin to assess the ability of the alginates to act as an adjuvant to this antigen. Antibody responses to intraperitoneally transplanted free and microencapsulated fetal porcine islet like cell clusters (ICC) were also evaluated, in animals treated with or without cyclosporine. RESULTS Antibodies against high M-alginate capsules were detected in the sera of mice transplanted with this capsule type. However, this response was not seen after the transplantation of high G capsules. When Wistar rats were used as recipients, no antibody responses were detected against any type of alginate capsules. Neither type of capsule acted as an adjuvant. Antibodies against ICC were present, in rats transplanted with both nonencapsulated and encapsulated ICCs. Administration of cyclosporine could abolish this production of antibodies against ICC. CONCLUSIONS High G-alginate capsules are less immunogenic than high M capsules. Because encapsulation did not protect against the generation of antibodies against ICC, it can be assumed that antigen leakage from the capsules occurs, as no evidence was found for capsules breaking in vivo.

The effect of host factors and capsule composition on the cellular overgrowth on implanted alginate capsules

Microencapsulation of islets of Langerhans in alginate/poly-L-lysine (PLL)/alginate capsules may provide a method for transplantation in the absence of immunosuppression. The aim of this study was to investigate the problem of overgrowth on implanted capsules with regard to the composition of the capsules and host factors such as cytokine and nitric oxide production. Empty capsules were implanted to C57BL/6 mice for 1, 3, 7, or 28 days. Glucose oxidation rates showed the metabolic activity of the cellular overgrowth on retrieved capsules. DNA content, histological score, and retrieval rates were also measured to assess the overgrowth. It was noted that the pericapsular host reaction arose by day 7 and had not increased further by day 28. Capsules of varying alginate compositions and different concentrations of PLL were implanted for 7 days to either C57BL/6 or Balb/c mice. Capsules were also implanted to mice lacking the inducible nitric oxide synthase enzyme. Glucose oxidation rates, DNA content, and histological score were positively correlated to each other and negatively correlated to retrieval rates. The pericapsular reaction was reduced if PLL was omitted from the capsule or if a high mannuronic acid alginate was used. Balb/c mice had reduced cellular overgrowth on implanted capsules and had reduced mRNA expression of interleukin-1 beta and tumor necrosis factor-alpha in their peritoneal macrophages. The capsular overgrowth seemed more severe in animals lacking inducible nitric oxide synthase compared with wild-type controls. It is concluded that alginate composition, PLL, and recipient factors such as nitric oxide production and cytokine expression affect the cellular overgrowth on implanted alginate capsules.

Metabolic phenotyping guidelines: Assessing glucose homeostasis in rodent models

The pathophysiology of diabetes as a disease is characterised by an inability to maintain normal glucose homeostasis. In type 1 diabetes, this is due to autoimmune destruction of the pancreatic β-cells and subsequent lack of insulin production, and in type 2 diabetes it is due to a combination of both insulin resistance and an inability of the β-cells to compensate adequately with increased insulin release. Animal models, in particular genetically modified mice, are increasingly being used to elucidate the mechanisms underlying both type 1 and type 2 diabetes, and as such the ability to study glucose homeostasis in vivo has become an essential tool. Several techniques exist for measuring different aspects of glucose tolerance and each of these methods has distinct advantages and disadvantages. Thus the appropriate methodology may vary from study to study depending on the desired end-points, the animal model, and other practical considerations. This review outlines the most commonly used techniques for assessing glucose tolerance in rodents and details the factors that should be taken into account in their use. Representative scenarios illustrating some of the practical considerations of designing in vivo experiments for the measurement of glucose homeostasis are also discussed.

Co-transplantation of islets with mesenchymal stem cells in microcapsules demonstrates graft outcome can be improved in an isolated-graft model of islet transplantation in mice

BACKGROUND AIMS Co-transplantation of islets with mesenchymal stem cells (MSCs) has been shown to improve graft outcome in mice, which has been partially attributed to the effects of MSCs on revascularization and preservation of islet morphology. Microencapsulation of islets provides an isolated-graft model of islet transplantation that is non-vascularized and prevents islet aggregation to preserve islet morphology. The aim of this study was to investigate whether MSCs could improve graft outcome in a microencapsulated/isolated-graft model of islet transplantation. METHODS Mouse islets and kidney MSCs were co-encapsulated in alginate, and their function was assessed in vitro. A minimal mass of 350 syngeneic islets encapsulated alone or co-encapsulated with MSCs (islet+MSC) were transplanted intraperitoneally into diabetic mice, and blood glucose concentrations were monitored. Capsules were recovered 6 weeks after transplantation, and islet function was assessed. RESULTS Islets co-encapsulated with MSCs in vitro had increased glucose-stimulated insulin secretion and content. The average blood glucose concentration of transplanted mice was significantly lower by 3 weeks in the islet+MSC group. By week 6, 71% of the co-encapsulated group were cured compared with 16% of the islet-alone group. Capsules recovered at 6 weeks had greater glucose-stimulated insulin secretion and insulin content in the islet+MSC group. CONCLUSIONS MSCs improved the efficacy of microencapsulated islet transplantation. Using an isolated-graft model, we were able to eliminate the impact of MSC-mediated enhancement of revascularization and preservation of islet morphology and demonstrate that the improvement in insulin secretion and content is sustained in vivo and can significantly improve graft outcome.

Normal Relationship of β- and Non–β-Cells Not Needed for Successful Islet Transplantation

Islets are composed mostly of β-cells, and therefore stem cell research has concentrated on generating purified β-cells, neglecting the other endocrine cell types in the islet. We investigated the presence of endocrine non–β-cells after islet transplantation. In addition, we studied whether the transplantation of pure β-cells, in volumes similar to that used in islet transplantation, would suffice to reverse hyperglycemia in diabetic mice. Rat islets were dispersed and β-cells were purified by fluorescence-activated cell sorting according to their endogenous fluorescence. After reaggregation, 600 islet equivalents of the purified β-cell aggregates were implanted into diabetic SCID mice. In mice implanted with β-cell–enriched aggregates, the hyperglycemia was reversed and good graft function over a 12-week period was observed with regard to glucose and insulin levels, glucose tolerance tests, and graft insulin content. The endocrine cell composition of the β-cell–enriched aggregates remained constant; before and 12 weeks after transplantation, the β-cell–enriched aggregates comprised 95% β-cells and 5% endocrine non–β-cells. However, islet grafts, despite originally having comprised 75% β-cells and 25% endocrine non–β-cells, comprised just 5% endocrine non–β-cells after transplantation, indicating a loss of these cells. β-Cell–enriched aggregates can effectively reverse hyperglycemia in mice, and transplanted intact islets are depleted in non–β-cells. It is therefore likely that islet non–β-cells are not essential for successful islet transplantation.

The effect of capsule composition in the reversal of hyperglycemia in diabetic mice transplanted with microencapsulated allogeneic islets

The transplantation of microencapsulated islets may allow reversal of hyperglycemia in the absence of immunosuppression. Poly-L-lysine (PLL) on capsules may potentiate the fibrotic reaction against implanted capsules. The aims of this study were to investigate how the biocompatibility of such capsules affects their function in vivo and to compare their efficacy relative to naked islets after intraperitoneal transplantation to nude or immune competent mice. Alloxan-diabetic C57BL/6 wild-type or nude (nu/nu) mice were transplanted with naked BALB/c islets, empty capsules, or microencapsulated BALB/c islets. Three types of capsules were used, one containing a high guluronic acid (G) alginate and PLL, one with a high mannuronic acid (M) alginate and PLL, and one high M alginate capsule with no PLL. Hyperglycemia in nude mice was reversed after transplantation of naked islets or islets encapsulated in a capsule containing high M alginate. Nude mice transplanted with islets encapsulated in the high G capsules showed only a transient reversal of hyperglycemia. In an allogeneic system, naked BALB/c islets were rejected by day 10 after transplantation, whereas the islets encapsulated in high M capsules continued to function for at least a month. When PLL was excluded from the capsules, the grafts functioned for up to 8 weeks. Islets microencapsulated in high G alginate capsules fail to reverse hyperglycemia for more than a few days in nude mice. However, islets in high M alginate capsules can reverse hyperglycemia in nude and immune competent mice. Islets microencapsulated in PLL-free high M alginate capsules function for 8 weeks in immune competent mice.

Anti-apoptotic effects of arachidonic acid and prostaglandin E2 in pancreatic beta-cells.

Background/Aims: The polyunsaturated fatty acid arachidonic acid (AA) has been implicated in β-cell defence mechanisms and prostaglandin (PG) products of cyclooxygenase (COX) 2 action confer resistance to alloxan-induced apoptosis in insulin-secreting RIN cells. We have now investigated the anti-apoptotic effects of AA and its metabolite, PGE2, in the MIN6 mouse insulin-secreting β-cell line and mouse islets. Methods: Apoptosis was determined in MIN6 β-cell and mouse islet extracts by measurement of capase-3 activity, and COX2 mRNA levels were quantified by real-time RT-PCR. Results: Exposure of MIN6 cells to AA (3.1-12.5µM) caused concentration-dependent reductions in apoptosis, and similar results were obtained when endogenous AA levels were elevated in cytosolic phospholipase A2-overexpressing MIN6 cells. 25mM glucose caused both a significant up-regulation of MIN6 cell COX2 mRNA levels and a decrease in apoptosis. Inhibition of MIN6 cell COX2 activity with a selective inhibitor, NS-398 (10-100µM), increased apoptosis and exogenous PGE2 (0.2-5µM) reduced NS-398-induced apoptosis in a concentration-dependent manner. The protective effects of AA and PGE2 were also observed in primary mouse islets. Conclusion: These data show that AA and its COX2-generated metabolite, PGE2, can protect β-cells from apoptosis.

Cytokine-induced functional suppression of microencapsulated rat pancreatic islets in vitro.

BACKGROUND It is likely that inflammatory cytokines are released near microencapsulated islets in vivo. METHODS Rates of insulin release or glucose oxidation were measured after culture of microencapsulated rat islets with interleukin (IL)-1beta and tumor necrosis factor-(TNF-alpha). Their ability to recover from IL-1beta-induced suppression was also investigated. RESULTS Microencapsulated islets were suppressed after exposure to IL-1beta, which was potentiated by TNF-alpha. After exposure to lower IL-1beta concentrations, microencapsulated islets had similar oxidation rates as corresponding controls. At higher concentrations, microencapsulated islets were more suppressed than nonencapsulated islets. Microencapsulated and control islets were able to recover from suppression after exposure to 2.5 U/ml of IL-1beta. CONCLUSIONS Microencapsulation using the present alginate/poly-L-lysine/alginate capsules does not protect islets against the detrimental effects of IL-1beta and TNF-alpha. Indeed, microencapsulated islets seem to be more susceptible to suppression at higher concentrations of IL-1beta. However, after exposure to a lower concentration of IL-1beta, microencapsulated islets can recover.