Alberto M. Davalli

Vulnerability of Islets in the Immediate Posttransplantation Period: Dynamic Changes in Structure and Function

To learn more about islet vulnerability in the immediate posttransplant period, 400 syngeneic islets were transplanted under the kidney capsule of B6AF1 mice. Three groups of recipients were used: normal mice (normal), streptozotocin (STZ)-diabetic (diabetic), and STZ-diabetic kept hypo- or normoglycemic with insulin pellets (diabetic-normalized). Normoglycemia was achieved in all three groups 14 days after transplantation; however, in the diabetic and diabetic-normalized groups, blood glucose levels throughout the posttransplantation period were respectively higher and lower than in the normal group. Grafts were harvested 1, 3, 7, and 14 days after transplantation and analyzed for morphology, β-cell death, β-cell mass, insulin content, and insulin mRNA expression. In all groups, substantial damage in islet grafts was found on days 1 and 3 with apoptotic nuclei and necrotic cores; on day 3, β-cell death was significantly higher in the diabetic group than in the other groups. Tissue remodeling occurred in all groups with stable graft appearance on day 14; the actual β-cell mass of the grafts was lowest in the diabetic group. Graft insulin content decreased in all groups on day 1 and fell even further on days 3 and 7. Insulin mRNA levels of grafts retrieved from both the diabetic and diabetic-normalized group were lower than those from the normal group already by day 1 and remained lower on day 14. In conclusion, the first few days of islet transplantation, even under the most advantageous circumstances of excellent metabolic control, are characterized by dynamic changes, with substantial islet cell dysfunction and death followed by tissue remodeling and then stable engraftment.

The Role of Oxidative Stress in the Pathogenesis of Type 2 Diabetes Mellitus Micro- and Macrovascular Complications: Avenues for a Mechanistic-Based Therapeutic Approach

A growing body of evidence suggests that oxidative stress plays a key role in the pathogenesis of micro- and macrovascular diabetic complications. The increased oxidative stress in subjects with type 2 diabetes is a consequence of several abnormalities, including hyperglycemia, insulin resistance, hyperinsulinemia, and dyslipidemia, each of which contributes to mitochondrial superoxide overproduction in endothelial cells of large and small vessels as well as the myocardium. The unifying pathophysiological mechanism that underlies diabetic complications could be explained by increased production of reactive oxygen species (ROS) via: (1) the polyol pathway flux, (2) increased formation of advanced glycation end products (AGEs), (3) increased expression of the receptor for AGEs, (4) activation of protein kinase C isoforms, and (5) overactivity of the hexosamine pathway. Furthermore, the effects of oxidative stress in individuals with type 2 diabetes are compounded by the inactivation of two critical anti-atherosclerotic enzymes: endothelial nitric oxide synthase and prostacyclin synthase. Of interest, the results of clinical trials in patients with type 2 diabetes in whom intensive management of all the components of the metabolic syndrome (hyperglycemia, hypercholesterolemia, and essential hypertension) was attempted (with agents that exert a beneficial effect on serum glucose, serum lipid concentrations, and blood pressure, respectively) showed a decrease in adverse cardiovascular end points. The purpose of this review is (1) to examine the mechanisms that link oxidative stress to micro- and macrovascular complications in subjects with type 2 diabetes and (2) to consider the therapeutic opportunities that are presented by currently used therapeutic agents which possess antioxidant properties as well as new potential antioxidant substances.

Function, mass, and replication of porcine and rat islets transplanted into diabetic nude mice

Well-characterized aliquots of adult porcine and rat islets of comparable β-cell mass were transplanted under the kidney capsule of streptozotocin-induced diabetic nude mice. In both porcine and rat islet grafts, β-cell mass decreased significantly in the first 2 months and stabilized thereafter. As with β-cell mass, insulin content decreased significantly in the first 2 months to almost 40% of that originally implanted. In porcine grafts, however, insulin content at 4 months was significantly higher than at 2 months. The endocrine non-β-cell mass of grafts also decreased significantly after transplantation: in porcine grafts, the decrease was less than in rat and was limited to the first 2 months. β-cell replication of engrafted islets was significantly lower in porcine than in rat grafts. Although β-cell mass of porcine and rat grafts was similar at all time periods, recipients of porcine islets required a significantly longer time to reach normal glucose levels; nonetheless, their blood glucose levels continued to decrease and stabilized at levels significantly lower than those of normal mice. During oral and intraperitoneal glucose tolerance tests, blood glucose increased only slightly in both the recipients of porcine and rat grafts. When graft-bearing kidneys were perfused in situ, porcine islet grafts showed a 20-fold increase in insulin release in response to both glucose and arginine. In conclusion, this evidence that adult porcine islet grafts can bring glucose levels to those that are normal for humans provides further support of their potential for human islet replacement therapy.

Pancreatic islet amyloidosis, β-cell apoptosis, and α-cell proliferation are determinants of islet remodeling in type-2 diabetic baboons

β-Cell dysfunction is an important factor in the development of hyperglycemia of type-2 diabetes mellitus, and pancreatic islet amyloidosis (IA) has been postulated to be one of the main contributors to impaired insulin secretion. The aim of this study was to evaluate the correlation of IA with metabolic parameters and its effect on islets of Langerhans remodeling and relative endocrine-cell volume in baboons. We sequenced the amylin peptide, determined the fibrillogenic propensities, and evaluated pancreatic histology, clinical and biochemical characteristics, and endocrine cell proliferation and apoptosis in 150 baboons with different metabolic status. Amylin sequence in the baboon was 92% similar to humans and showed superimposable fibrillogenic propensities. IA severity correlated with fasting plasma glucose (FPG) (r = 0.662, P < 0.001) and HbA1c (r = 0.726, P < 0.001), as well as with free fatty acid, glucagon values, decreased homeostasis model assessment (HOMA) insulin resistance, and HOMA-B. IA severity was associated with a decreased relative β-cell volume, and increased relative α-cell volume and hyperglucagonemia. These results strongly support the concept that IA and β-cell apoptosis in concert with α-cell proliferation and hypertrophy are key determinants of islets of Langerhans “dysfunctional remodeling” and hyperglycemia in the baboon, a nonhuman primate model of type-2 diabetes mellitus. The most important determinants of IA were age and FPG (R2 = 0.519, P < 0.0001), and different FPG levels were sensitive and specific to predict IA severity. Finally, a predictive model for islet amyloid severity was generated with age and FPG as required variables.

Effects of Balloon Injury on Neointimal Hyperplasia in Streptozotocin-Induced Diabetes and in Hyperinsulinemic Nondiabetic Pancreatic Islet–Transplanted Rats

Background—The mechanisms of increased neointimal hyperplasia after coronary interventions in diabetic patients are still unknown. Methods and Results—Glucose and insulin effects on in vitro vascular smooth muscle cell (VSMC) proliferation and migration were assessed. The effect of balloon injury on neointimal hyperplasia was studied in streptozotocin-induced diabetic rats with or without adjunct insulin therapy. To study the effect of balloon injury in nondiabetic rats with hyperinsulinemia, pancreatic islets were transplanted under the kidney capsule in normal rats. Glucose did not increase VSMC proliferation and migration in vitro. In contrast, insulin induced a significant increase in VSMC proliferation and migration in cell cultures. Furthermore, in VSMC culture, insulin increased MAPK activation. A reduction in neointimal hyperplasia was consistently documented after vascular injury in hyperglycemic streptozotocin-induced diabetic rats. Insulin therapy significantly increased neointimal hyperplasia in these rats. This effect of hyperinsulinemia was totally abolished by transfection on the arterial wall of the N17H-ras–negative mutant gene. Finally, after experimental balloon angioplasty in hyperinsulinemic nondiabetic islet-transplanted rats, a significant increase in neointimal hyperplasia was observed. Conclusions—In rats with streptozotocin-induced diabetes, balloon injury was not associated with an increase in neointimal formation. Exogenous insulin administration in diabetic rats and islet transplantation in nondiabetic rats increased both blood insulin levels and neointimal hyperplasia after balloon injury. Hyperinsulinemia through activation of the ras/MAPK pathway, rather than hyperglycemia per se, seems to be of crucial importance in determining the exaggerated neointimal hyperplasia after balloon angioplasty in diabetic animals.

Secretory defects induced by immunosuppressive agents on human pancreatic β-cells

Abstract. Despite the considerable interest for islet and pancreas transplantation, remarkably little is known about the direct effects of immunosuppressive drugs on human β-cell function. We measured different insulin secretory parameters and insulin gene expression of human islets cultured for 5 days in the presence of mycophenolate mofetil (MMF), cyclosporin A (CsA), tacrolimus (FK506) or a mixture of 3 cytokines. Basal insulin release after exposure to cytokines and FK506 was significantly higher than in control islets. Responsiveness to an acute glucose stimulus did not differ significantly between control and treated islets. However, absolute incremental insulin responses (Δ-AUCs) of islets exposed to cytokines or FK506 were significantly higher compared to islets exposed to CsA or MMF, mainly because of the higher basal release. Indeed, maximal over basal release (stimulation index, SI) tended to be lower in islets exposed to FK506 than in control islets. Insulin gene expression was significantly reduced only in islets exposed to CsA. FK506 was, among those tested, the immunosuppressive drug that most profoundly altered the normal insulin secretory pattern of human β-cells, whereas CsA was the only inhibiting insulin gene expression. Although the abnormalities induced by the immunosoppressive drugs utilized in this study were modest, these in vitro data are consistent with the reported in vivo diabetogenicity of CsA and FK506 and point to MMF as the ideal immunosuppressive agent from a pancreatic β-cell point of view.

Loss of glucose-induced insulin secretion and GLUT2 expression in transplanted β-cells

Either 200 or 400 syngeneic islets were transplanted under the kidney capsule of normal or streptozocin-induced diabetic B6/AF1 mice. The diabetic mice with 400 islets became normoglycemic, but those with 200 islets, an insufficient number, were still diabetic after the transplantation (Tx). Two weeks after Tx, GLUT2 expression in the islet grafts was evaluated by immunofluorescence and Western blots, and graft function was examined by perfusion of the graft-bearing kidney. Immunofluorescence for GLUT2 was dramatically reduced in the β-cells of grafts with 200 islets exposed to hyperglycemia. However, it was plentiful in grafts with 400 islets in a normoglycemic environment. Densitometric analysis of Western blots on graft homogenates demonstrated that GLUT2 protein levels in the islets, when exposed to chronic hyperglycemia for 2 weeks, were decreased to 16% of those of normal recipients. Moreover, these grafts had defective glucose-induced insulin secretion, while the effects of arginine were preserved. We conclude that GLUT2 expression in normal β-cells is promptly down-regulated during exposure to hyperglycemia and may contribute to the loss of glucose-induced secretion of diabetes.

Energy expenditure evaluation in humans and non-human primates by SenseWear Armband. Validation of energy expenditure evaluation by SenseWear Armband by direct comparison with indirect calorimetry.

Introduction The purpose of this study was to compare and validate the use of SenseWear Armband (SWA) placed on the arm (SWA ARM) and on the back (SWA BACK) in healthy humans during resting and a cycle-ergometer exercise and to evaluate the SWA to estimate Resting Energy Expenditure (REE) and Total Energy Expenditure (TEE) in healthy baboons. Methods We studied 26 (15F/11M) human subjects wearing SWA in two different anatomical sites (arm and back) during resting and a cycle-ergometer test and directly compared these results with indirect calorimetry evaluation (IC), performed at the same time. We then inserted the SWA in a metabolic jacket for baboons and evaluated the TEE and REE in free living condition for 6 days in 21 (8F/13M) non-human primates. Results In humans we found a good correlation between SWA place on the ARM and on the BACK with IC during the resting experiment (1.1±0.3 SWAs, 1±0.2 IC kcal/min) and a slight underestimation in the SWAs data compared with IC during the cycle-ergometer exercise (5±1.9 SWA ARM, 4.5±1.5 SWA BACK and 5.4±2.1 IC kcal/min). In the non-human primate (baboons) experiment SWA estimated a TEE of 0.54±0.009 kcal/min during free living and a REE of 0.82±0.06 kcal/min. Conclusion SWA, an extremely simple and inexpensive apparatus, provides quite accurate measurements of energy expenditure in humans and in baboons. Energy expenditure data obtained with SWA are highly correlated with the data obtained with “gold standard”, IC, in humans.

Predictive models of insulin resistance derived from simple morphometric and biochemical indices related to obesity and the metabolic syndrome in baboons

BackgroundNon-human primates are valuable models for the study of insulin resistance and human obesity. In baboons, insulin sensitivity levels can be evaluated directly with the euglycemic clamp and is highly predicted by adiposity, metabolic markers of obesity and impaired glucose metabolism (i.e. percent body fat by DXA and HbA1c). However, a simple method to screen and identify obese insulin resistant baboons for inclusion in interventional studies is not available.MethodsWe studied a population of twenty baboons with the euglycemic clamp technique to characterize a population of obese nondiabetic, insulin resistant baboons, and used a multivariate linear regression analysis (adjusted for gender) to test different predictive models of insulin sensitivity (insulin-stimulated glucose uptake = Rd) using abdominal circumference and fasting plasma insulin. Alternatively, we tested in a separate baboon population (n = 159), a simpler model based on body weight and fasting plasma glucose to predict the whole-body insulin sensitivity (Rd/SSPI) derived from the clamp.ResultsIn the first model, abdominal circumference explained 59% of total insulin mediated glucose uptake (Rd). A second model, which included fasting plasma insulin (log transformed) and abdominal circumference, explained 64% of Rd. Finally, the model using body weight and fasting plasma glucose explained 51% of Rd/SSPI. Interestingly, we found that percent body fat was directly correlated with the adipocyte insulin resistance index (r = 0.755, p < 0.0001).ConclusionIn baboons, simple morphometric measurements of adiposity/obesity, (i.e. abdominal circumference), plus baseline markers of glucose/lipid metabolism, (i.e. fasting plasma glucose and insulin) provide a feasible method to screen and identify overweight/obese insulin resistant baboons for inclusion in interventional studies aimed to study human obesity, insulin resistance and type 2 diabetes mellitus.

Further Evidence for Amyloid Deposition in Clinical Pancreatic Islet Grafts

Background. The reasons for the long-term complete or partial loss of islet graft function are unknown, but there are obviously other reasons than just pure allogeneic graft rejection. Earlier studies have shown that deposition of islet amyloid polypeptide amyloid in transplanted islets may indicate a mechanism for loss of &bgr; cells. Materials and Methods. Sections from liver material from four deceased islet-bearing recipients have been scrutinized for the presence of amyloid. Clinical data and certain aspects of the islet graft pathology of these patients have been published previously. Result. With this extended histological analysis, we demonstrate the occurrence of amyloid deposits in islets transplanted into the liver in three of four patients with type 1 diabetes. Conclusion. The finding adds evidence to the assumption that aggregation of islet amyloid polypeptide might be an important cause of progressing &bgr;-cell dysfunction in clinically transplanted islets.