Terms Of Insulin Secretion Research Articles

Visceral fat correlates with insulin secretion and sensitivity independent of BMI and subcutaneous fat in Chinese with type 2 diabetes.

Clinical heterogeneity exists in overall obesity and abdominal obesity in terms of insulin secretion and sensitivity. Further, the impact of visceral fat (VF) on the first- and second-phase insulin secretion (FPIS and SPIS) is controversial. We aim to investigate insulin secretion and sensitivity in Chinese patients with T2DM according to different BMI and VF levels. This study enrolled 300 participants. A dual bioelectrical impedance analyzer was used to assess the visceral and subcutaneous fat area (VFA and SFA). VF levels were categorized as normal or high, with the cutoff value of 100 cm2. FPIS and SPIS were evaluated by arginine stimulation test and standardized steamed bread meal tolerance test, respectively. β-cell function (HOMA2-β), insulin resistance (HOMA2-IR), and Gutt's insulin sensitivity index (Gutt-ISI) were also calculated. Spearman's correlation analysis and multivariate linear regression analysis were adopted for statistical analysis. Participants were categorized into four groups: normal weight-normal VF, normal weight-high VF, overweight/obese-normal VF and overweight/obese-high VF. Multivariate linear regression showed that both VFA and SFA were correlated with FPIS, HOMA2-IR and Gutt-ISI after controlling for gender and diabetes duration. After further adjustment for BMI and VFA, some associations of SFA with insulin secretion and sensitivity disappeared. After adjustment for gender, diabetes duration, BMI and SFA, VFA was positively correlated with FPIS, SPIS and HOMA2-IR. Subjects with overweight/obese-high VF were more likely to have higher FPIS, HOMA2-IR and lower Gutt-ISI (all p < 0.05). VF affects both FPIS and SPIS, and worsens insulin sensitivity independent of BMI and subcutaneous fat in Chinese patients with T2DM. http://www.chictr.org.cn, identifier ChiCTR2200062884.

Three-dimensional wet electrospun scaffold system for the differentiation of adipose-derived mesenchymal stem cells to islet-like clusters.

Stem cell-derived islet-like clusters (ILCs) are an alternative source of pancreatic beta cells for the treatment of diabetic mellitus. An ideal 3D culture platform for the generation of ILCs of desired cluster size is a challenge due to the clustering of islet cells in the 2D culture systems. The islet cells cultured in 2D conditions produce clusters of large size, which are less efficient in terms of insulin secretion and viability. In this study, we report that ILCs formed on a PCL-based wet electrospun fibrous scaffold with larger pore size produced clusters of the desired size, compared to that cultured on a conventional electrospun sheet. The collagen functionalization on this wet electrospun polycaprolactone (PCL) scaffold showed enhanced insulin secretion and cell viability compared to the non-functionalized or conventionally electrospun PCL scaffold. The collagen-coated wet electrospun 3D scaffold produced ILCs of cluster diameter 70±20μm and the conventionally electrospun PCL sheet produced larger ILC clusters of diameter 300±10μm. Hence the results indicate the collagen-functionalized wet electrospun scaffold system could be a potential scaffold for islet tissue engineering.

Reversibility of pancreatic β-cells dysfunction after vitamin D and calcium supplementation: a pilot study in a population of obese and prepubescent North-African children

ABSTRACT The mechanisms of diabetogenesis in children remain largely obscure. This study aimed to determine the impact of vitamin D and calcium supplementation on pancreatic β-cells function in terms of insulin secretion and sensitivity. This was a quasi-experimental study involving 30 obese and prepubescent Tunisian children (57% boys). During three months, the children received calcium and vitamin D supplementation at therapeutic doses. An oral glucose tolerance test (OGTT) was performed at the beginning and at the end of the study. The following metabolic definitions were applied: i) hyperinsulinism: insulinemia sum > 300 μ UI/ml during OGTT, ii) insulin-resistance: homeostatic model assessment of insulin-resistance > 2, iii) normal glycaemic profile: normal plasma levels during OGTT without any spike, and iv) pancreatic β-cells dysfunction reversibility: disappearance of the aforementioned disorders. The means ± standard-deviation of age and body mass index were 10.87 ± 1.9 years, and 30.17 ± 4.99 kg/m2, respectively. All children were at the stage of hyperinsulinism associated with insulin-resistance. These disturbances were noted even in children having a normal glycaemic profile at OGTT. After calcium and vitamin D supplementation, glycaemic profile as well as insulin-secretion improved significantly (p < 0.0001). Hyperinsulinism and insulin-resistance decreased significantly by 56.67% (p < 0.0001) and 70.00% (p < 0.0001), respectively. Complete reversibility of these two disorders was noted in 26.6% of children. To conclude, in obese and prepubescent children, vitamin D and calcium supplementation led to the reversibility of the pancreatic β-cells dysfunction.

The Role of CD36 in Type 2 Diabetes Mellitus: β-Cell Dysfunction and Beyond.

Impaired β-cell function is the key pathophysiology of type 2 diabetes mellitus, and chronic exposure of nutrient excess could lead to this tragedy. For preserving β-cell function, it is essential to understand the cause and mechanisms about the progression of β-cells failure. Glucotoxicity, lipotoxicity, and glucolipotoxicity have been suggested to be a major cause of β-cell dysfunction for decades, but not yet fully understood. Fatty acid translocase cluster determinant 36 (CD36), which is part of the free fatty acid (FFA) transporter system, has been identified in several tissues such as muscle, liver, and insulin-producing cells. Several studies have reported that induction of CD36 increases uptake of FFA in several cells, suggesting the functional interplay between glucose and FFA in terms of insulin secretion and oxidative metabolism. However, we do not currently know the regulating mechanism and physiological role of CD36 on glucolipotoxicity in pancreatic β-cells. Also, the downstream and upstream targets of CD36 related signaling have not been defined. In the present review, we will focus on the expression and function of CD36 related signaling in the pancreatic β-cells in response to hyperglycemia and hyperlipidemia (ceramide) along with the clinical studies on the association between CD36 and metabolic disorders.

Mechanism and Potential of Egg Consumption and Egg Bioactive Components on Type-2 Diabetes.

Type-2 diabetes (T2D) is one of the major global health challenges and a substantial economic burden. Egg and egg-derived components have been indicated to possess antioxidant, anti-inflammatory, anti-hypertensive, immunomodulatory, and anti-cancer activities. However, the scientific evidence about the benefits of egg on T2D is debatable. The relationship between egg consumption and the risk of T2D from observational epidemiological studies is not consistent. Interventional clinical studies, however, provide promising evidence that egg consumption ameliorates the risk of T2D. Current research progress also indicates that some egg components and egg-derived peptides might be beneficial in the context of T2D, in terms of insulin secretion and sensitivity, oxidative stress, and inflammation, suggesting possible application on T2D management. The current review summarizes recent clinical investigations related to the influence of egg consumption on T2D risk and in vivo and in vitro studies on the effect and mechanism of egg components and egg-derived peptides on T2D.

Differentiating neural crest stem cells induce proliferation of cultured rodent islet beta cells

Efficient stimulation of cycling activity in cultured beta cells would allow the design of new strategies for cell therapy in diabetes. Neural crest stem cells (NCSCs) play a role in beta cell development and maturation and increase the beta cell number in co-transplants. The mechanism behind NCSC-induced beta cell proliferation and the functional capacity of the new beta cells is not known. We developed a new in vitro co-culture system that enables the dissection of the elements that control the cellular interactions that lead to NCSC-dependent increase in islet beta cells. Mouse NCSCs were cultured in vitro, first in medium that stimulated their proliferation, then under conditions that supported their differentiation. When mouse islet cells were cultured together with the NCSCs, more than 35% of the beta cells showed cycle activity. This labelling index is more than tenfold higher than control islets cultured without NCSCs. Beta cells that proliferated under these culture conditions were fully glucose responsive in terms of insulin secretion. NCSCs also induced beta cell proliferation in islets isolated from 1-year-old mice, but not in dissociated islet cells isolated from human donor pancreas tissue. To stimulate beta cell proliferation, NCSCs need to be in intimate contact with the beta cells. Culture of islet cells in contact with NCSCs induces highly efficient beta cell proliferation. The reported culture system is an excellent platform for further dissection of the minimal set of factors needed to drive this process and explore its potential for translation to diabetes therapy.

Coculture of Insulin-Producing RIN5AH Cells With Neural Crest Stem Cells Protects Partially Against Cytokine-Induced Cell Death

Patients with type-1 diabetes lose their β-cells after autoimmune attack. Islet transplantation is a co-option for curing this disease, but survival of transplanted islets is poor. Thus, methods to enhance β-cell viability and function as well as methods to expand β-cell mass are required. The work presented in this thesis aimed to study the roles of neural crest stem cells or their derivatives in supporting β-cell proliferation, function, and survival.In co-culture when mouse boundary cap neural crest stem cells (bNCSCs) and pancreatic islets were in direct contact, differentiating bNCSCs strongly induced β-cell proliferation, and these proliferating β-cells were glucose responsive in terms of insulin secretion. Moreover, co-culture of murine bNCSCs with β-cell lines RIN5AH and β-TC6 showed partial protection of β-cells against cytokine-induced β-cell death. Direct contacts between bNCSCs and β-cells increased β-cell viability, and led to cadherin and β-catenin accumulations at the bNCSC/β-cell junctions. We proposed that cadherin junctions supported signals which promoted β-cell survival. We further revealed that murine neural crest stem cells harvested from hair follicles were unable to induce β-cell proliferation, and did not form cadherin junctions when cultured with pancreatic islets. Finally, we discovered that the presence of bNCSCs in co-culture counteracted cytokine-mediated insulin-producing human EndoC-βH1 cell death. Furthermore, these two cell types formed N-cadherin, but not E-cadherin, junctions when they were in direct contact. In conclusion, the results of these studies illustrate how neural crest stem cells influence β-cell proliferation, function, and survival which may improve islet transplantation outcome.

Experimental Microencapsulation of Porcine and Rat Pancreatic Islet Cells With Air-Driven Droplet Generator and Alginate

Transplantation of microencapsulated islets is proposed as an ideal therapy for the treatment of type 1 diabetes mellitus without immunosuppression. This strategy is based on the principle that foreign cells are protected from the host immune system by an artificial membrane. The aim of this study was to establish an ideal condition of microencapsulation using an air-driven droplet generator and alginate in vitro. The optimal conditions for islet encapsulation were an alginate inflow rate of 10 mL/h, CO 2 flow rate of 2.0 L/min in a concentration of 2% alginate. For 2.5% alginate, the alginate inflow rate of 20 mL/h, CO 2 flow rate 3.0 L/min was ideal; alginate inflow rate of 40 mL/h, CO 2 flow rate of 4.0 L/min showed good microcapsules at 3% alginate. Viability of encapsulated islets was greater than 90%. In terms of insulin secretion, encapsulated islets secreted insulin in response to glucose in static culture medium. However, there was no normal response to low or high glucose challenge with a stimulation index less than 2.0. Microencapsulation of pig islets was successfully performed with air-driven droplet generator and alginate in vitro. Further studies about biocompatibility and glucose control in vivo may provide a useful tool for treatment of patients with diabetes mellitus.

Insulin secretion from human beta cells is heterogeneous and dependent on cell-to-cell contacts

We assessed the heterogeneity of insulin secretion from human isolated beta cells and its regulation by cell-to-cell contacts. Insulin secretion from single and paired cells was assessed by a reverse haemolytic plaque assay. The percentage of plaque-forming cells, the mean plaque area and the total plaque development were evaluated after 1 h of stimulation with different secretagogues. Not all beta cells were surrounded by a haemolytic plaque under all conditions tested. A small fraction of the beta cell population (20%) secreted more than 90% and 70% of total insulin at 2.2 and 22.2 mmol/l glucose, respectively. Plaque-forming cells, mean plaque area and total plaque development were increased at 12.2 and 22.2 compared with 2.2 mmol/l glucose. Insulin secretion of single beta cells was similar at 12.2 and 22.2 mmol/l glucose. Insulin secretion of beta cell pairs was increased compared with that of single beta cells and was higher at 22.2 than at 12.2 mmol/l glucose. Insulin secretion of beta cells in contact with alpha cells was also increased compared with single beta cells, but was similar at 22.2 compared with 12.2 mmol/l glucose. Delta and other non-beta cells did not increase insulin secretion of contacting beta cells compared with that of single beta cells. Differences in insulin secretion between 22.2 and 12.2 mmol/l glucose were observed in murine but not in human islets. Human beta cells are highly heterogeneous in terms of insulin secretion so that a small fraction of beta cells contributes to the majority of insulin secreted. Homologous and heterologous intercellular contacts have a significant impact on insulin secretion and this could be related to the particular architecture of human islets.

Orchestration of Glucose Homeostasis

Type 2 diabetes is increasing at alarming rates in the U.S., in Westernized countries, and in the third world. The increasing costs in terms of human suffering as well as economics are well recognized (1). Improved understanding of the pathogenesis of diabetes should lead to better approaches to predict, forestall, or even prevent diabetes and to treat extant cases. Yet, the precise causes of this disease remain to be totally explained. There is little question that obesity per se is a primary contributor, but the causes of the so-called “obesity epidemic” are in debate (2), as are the mechanisms by which obesity itself is linked to diabetes (3,4). For some years, our laboratory has been engaged in efforts to understand causation of type 2 diabetes in terms of insulin secretion and resistance and to attempt to account for the role of obesity. The Banting Lecture provided me with a rare opportunity to describe our research efforts done over a prolonged history in a single presentation. It was my goal to address the several areas of diabetes research we have done. At first glance these different areas may appear unrelated. But these lines of work are closely linked within an overall effort to understand diabetes. I am deeply grateful to the members of the American Diabetes Association for the great privilege of making this presentation. I hope I will be able to emphasize the wonderful opportunity that I and my colleagues have had to pursue original research in a highly deserving and important cause. One can scarcely ask for more in one's professional life. For several decades it has been popular to pursue a “reductionist” approach to studying disease (Fig. 1). The underlying concept is that understanding of disease will result from describing events at increasingly more microscopic …

A theoretical study of oxygen transfer including cell necrosis for the design of a bioartificial pancreas

In an extravascular bioartificial pancreas (BAP), islet functions are probably limited by diffusive mass transfer and local consumption, leading to low oxygenation. A mathematical model based on finite elements and focusing on local oxygen transport in both the alginate core and the islets of Langerhans has been proposed to help design an efficient pancreas supply. It was possible to randomly localize islets in a hollow fiber at different densities, and the effects of hypoxia and necrosis were included in the mass transfer simulations. Thorough study of the numerical results first led to the analysis of several relevant parameters, such as necrosis factor and efficacy in terms of insulin secretion, as a way to optimize fiber geometry. The approach was then to calculate the number of islets that needed to be implanted in order to obtain a correct response in terms of insulin secretion. In most configurations, it was found to be much higher than that of ultimately functional islets, because of hypoxia and necrosis. Fiber length should thus be adjusted accordingly. Finally, we demonstrated that the compromise to be found between the reduction of the number of implanted islets and fiber length and diameter did not correspond to realistic hollow fiber systems. The alternative of using flat geometry was also envisaged with more optimistic feasibility assessments.

Biological activity and antidiabetic potential of synthetic fragment peptides of glucose-dependent insulinotropic polypeptide, GIP(1-16) and (Pro 3)GIP(1-16)

Glucose-dependent insulinotropic polypeptide (GIP) is a key physiological insulin releasing peptide and potential antidiabetic agent. The present study was undertaken in an attempt to develop small molecular weight GIP agonist and antagonist molecules. The bioactivity of two modified C-terminally truncated fragment GIP peptides, GIP(1-16) and (Pro 3)GIP(1-16), was examined in terms of insulin secretion and glucose homeostasis using BRIN-BD11 cells and type 2 diabetic mice. In vitro insulin release studies demonstrated that GIP(1-16) and (Pro 3)GIP(1-16) possessed weak GIP-receptor agonist and antagonistic properties, respectively. Intraperitoneal administration of GIP(1-16) in combination with glucose to obese diabetic ( ob/ ob) mice did not effect the glycaemic excursion and had a marginal effect on insulin release. GIP(1-16) was substantially less effective than the native GIP(1-42). (Pro 3)GIP(1-16) administration significantly curtailed ( P < 0.05) the insulinotropic and glucose lowering effects of native GIP, but was significantly less effective than (Pro 3)GIP. Based on the established concept of a therapeutic benefit of GIP receptor antagonism in obesity–diabetes, ob/ ob mice received once daily injection of (Pro 3)GIP(1-16) for 14 days. No significant effects were observed on food intake, body weight, HbA 1c, glucose tolerance, metabolic response to feeding and either insulin secretion or insulin sensitivity following prolonged (Pro 3)GIP(1-16) treatment. These data demonstrate that C-terminal truncation of GIP or (Pro 3)GIP yields small molecular weight GIP molecules with significantly reduced biological activity that precludes therapeutic utility.

Engineered β-cells secreting dipeptidyl peptidase IV-resistant glucagon-like peptide-1 show enhanced glucose-responsiveness

Type 2 diabetes is a polygenic disorder characterized by increased insulin resistance, and impaired insulin secretion leading to abnormalities of glucose and lipid metabolism. Reduced responsiveness of the β-cells to glucose is a critical feature of this syndrome. Glucagon-like peptide 1, a product of the pro-glucagon gene makes β-cells competent and has many other anti-diabetic properties. We speculated whether GLP-1-based gene therapy could be an approach for treatment of type 2 diabetes. We started with a clone of rat insulinoma cells (S4 cells), which showed reduced responsiveness to glucose in terms of insulin secretion. We transfected these cells with a plasmid encoding a mutated form of GLP-1 (GLP-1-Gly8), which is resistant to the degrading enzyme dipeptidyl-peptidase IV. Activity of secreted GLP-1-Gly8 was assayed using Chinese hamster lung fibroblasts (CHL) cells that expressed cloned GLP-1 receptor and that were transfected with CRE-Luc. Stable cell lines (Glipsulin cells) obtained by this means produced and stored immunoreactive GLP-1-Gly8. In addition to insulin, the Glipsulin cells secreted the GLP-1-Gly8. The secreted GLP-1-Gly8 was active as evidenced by the ability of the conditioned media to elevate cAMP levels in CHL cells expressing GLP-1 receptors. Glipsulin cells responded to glucose with a 6.8 fold increase in insulin secretion compared to a 2.2 fold increase in the control cells. Our results demonstrate that prolonged exposure to GLP-1-Gly8 secreted by increases glucose-responsiveness of these cells. We speculate that engineering GLP-1-Gly8 secretion by β-cells is a potential gene therapeutic strategy to treat diabetes.

Insulin secretion: mechanisms of regulation

REGULATION OF INSULIN SECRETION: Beta cells are unique endocrine cells. They respond positively, in terms of insulin secretion, not only to changes in the extracellular glucose concentration, but also to activators of the phospholipase C (cholecystokinin or acetylcholine), and to activators of adenylate cyclase (glucagon, glucagon-like peptide-1, or gastric inhibitory polypeptide). Major messengers which mediate glucose action for insulin release are Ca2+, adenosine triphosphate (ATP) and diacylglycerol (DAG). MAJOR PATHWAYS OF INSULIN RELEASE STIMULATION: There are four major pathways involved in stimulation of insulin release. The first pathway is KATP channel-dependent pathway in which increased blood glucose concentrations and increased b-cell metabolism result in a change in intracellular ATP/ADP ratio. This is a contributory factor in closure of ATP-dependent K+ channels, depolarization of b-cell membrane, in increased voltage-dependent L-type Ca2+ channel activity. Increased Ca2+ influx results in increased intracellular Ca2+ and stimulated insulin release. KATP channel-independent pathway augments Ca(2+) -stimulated insulin secretion of KATP channel-dependent pathway. Major potentiation of release results from hormonal and peptidergic activation of receptors linked to adenylyl cyclase. Adenylyl cyclase activity is stimulated by hormones such as vasoactive intestinal peptide (VIP), glucagon-like peptide-1 (GLP-1), and so on. These hormones, acting via G protein, stimulate adenylyl cyclase, thus causing a rise in cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA). Increased activity of PKA results in potentiation of insulin secretion.

Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion.

The biochemical mechanisms involved in regulation of insulin secretion are not completely understood. The rat INS-1 cell line has been used to gain insight in this area because it secretes insulin in response to glucose concentrations in the physiological range. However, the magnitude of the response is far less than that seen in freshly isolated rat islets. In the current study, we have stably transfected INS-1 cells with a plasmid containing the human proinsulin gene. After antibiotic selection and clonal expansion, 67% of the resultant clones were found to be poorly responsive to glucose in terms of insulin secretion (< or =2-fold stimulation by 15 mmol/l compared with 3 mmol/l glucose), 17% of the clones were moderately responsive (2- to 5-fold stimulation), and 16% were strongly responsive (5- to 13-fold stimulation). The differences in responsiveness could not be ascribed to differences in insulin content. Detailed analysis of one of the strongly responsive lines (832/13) revealed that its potent response to glucose (average of 10-fold) was stable over 66 population doublings (approximately 7.5 months of tissue culture) with half-maximal stimulation at 6 mmol/l glucose. Furthermore, in the presence of 15 mmol/l glucose, insulin secretion was potentiated significantly by 100 pmol/l isobutylmethylxanthine (320%), 1 mmol/l oleate/palmitate (77%), and 50 nmol/l glucagon-like peptide 1 (60%), whereas carbachol had no effect. Glucose-stimulated insulin secretion was also potentiated by the sulfonylurea tolbutamide (threefold at 3 mmol/l glucose and 50% at 15 mmol/l glucose) and was abolished by diazoxide, which demonstrates the operation of the ATP-sensitive K+ channel (K(ATP)) in 832/13 cells. Moreover, when the K(ATP) channel was bypassed by incubation of cells in depolarizing K+ (35 mmol/l), insulin secretion was more effectively stimulated by glucose in 832/13 cells than in parental INS-1 cells, which demonstrates the presence of a K(ATP) channel-independent pathway of glucose sensing. We conclude that clonal selection of INS-1 cells allows isolation of cell lines that exhibit markedly enhanced and stable responsiveness to glucose and several of its known potentiators. These lines may be attractive new vehicles for studies of beta-cell function.

Mechanisms of glucose hypersensitivity in beta-cells from normoglycemic, partially pancreatectomized mice.

Increased beta-cell sensitivity to glucose precedes the loss of glucose-induced insulin secretion in diabetic animals. Changes at the level of beta-cell glucose sensor have been described in these situations, but it is not clear whether they fully account for the increased insulin secretion. Using a euglycemic-normolipidemic 60% pancreatectomized (60%-Px) mouse model, we have studied the ionic mechanisms responsible for increased beta-cell glucose sensitivity. Two weeks after Px (Px14 group), Px mice maintained normoglycemia with a reduced beta-cell mass (0.88 +/- 0.18 mg) compared with control mice (1.41 +/- 0.21 mg). At this stage, the dose-response curve for glucose-induced insulin release showed a significant displacement to the left (P < 0.001). Islets from the Px14 group showed oscillatory electrical activity and cytosolic Ca2+ ([Ca2+]i) oscillations in response to glucose concentrations of 5.6 mmol/l compared with islets from the control group at 11.1 mmol/l. All the above changes were fully reversible both in vitro (after 48-h culture of islets from the Px14 group) and in vivo (after regeneration of beta-cell mass in islets studied 60 days after Px). No significant differences in the input resistance and ATP inhibition of ATP-sensitive K+ (K(ATP)) channels were found between beta-cells from the Px14 and control groups. The dose-response curve for glucose-induced MTT (C,N-diphenyl-N''-4,5-dimethyl thiazol 2 yl tetrazolium bromide) reduction showed a significant displacement to the left in islets from the Px14 group (P < 0.001). These results indicate that increased glucose sensitivity in terms of insulin secretion and Ca2+ signaling was not due to intrinsic modifications of K(ATP) channel properties, and suggest that the changes are most likely to be found in the glucose metabolism.

The alginate‐poly‐L‐lysine‐alginate membrane: Evidence of a protective effect on microencapsulated islets of Langerhans following exposure to cytokines

Abstract: In past experiments, we demonstrated that allografts and xenografts of pancreatic islets encapsulated in alginate‐polylysine‐alginate (APA) microcapsules were protected from immunorejection and that in both streptozotocin‐induced and spontaneously diabetic animals diabetes was reversed for extended periods of time. Because of growing evidence that cytokines may play an important role in graft rejection, it is important to determine whether the APA capsular membrane can also provide a protective barrier against cytotoxic actions of cytokines. In this study, free and encapsulated rat islets were exposed in vitro to interleukin 1, 2, and 6 (IL‐1, IL‐2, and IL‐6) to tumor necrosis factor (TNF) and interferon (INT), then challenged with 20 mM glucose for 24 hr to evaluate physiological response in terms of insulin secretion. Although IL‐2 and 6 were found to have no effect on the pancreatic islets (free or encapsulated), IL‐1, TNF, and INT were shown to interfere with the physiological pattern of insulin secretion from the free islets; microencapsulated islets' function was not affected. Transmission electron microscopy (TEM) of the rat islets exposed to the five types of cytokines revealed that IL‐1, TNF, and INF exerted morphological changes to the free islets while encapsulated islets were not affected. No changes were detected in free or encapsulated islets exposed to IL‐6 or IL‐2. In addition, encapsulated islets were transplanted into normal BALB‐c mice and into spontaneously diabetic NOD mice for 3 to 5 months and then recovered. The recovered islets were shown to continue to respond to glucose challenge in a physiological manner. The results of this study showed that, although exposure of some cytokines to free islets results in changes in the normal physiological response of the islets to glucose challenge as well as in ultrastructural changes within the cells, microencapsulated islets were not impaired by the exposure to the same cytokines.

Prolonged Reversal of Diabetic State in NOD Mice by Xenografts of Microencapsulated Rat Islets

Transplantation of the islets of Langerhans could be the most promising approach to the clinical treatment of insulin-dependent (type I) diabetes mellitus. In this study, we report on a modified encapsulation technique that produces small alginate-polylysine capsules (0.25-0.35 mm diam). In an in vitro study, both encapsulated and unencapsulated islets showed comparable responses to glucose challenge in terms of insulin secretion. With the new capsules, 16 spontaneously diabetic NOD mice received transplants of 800 encapsulated rat islets/animal. Nonfasting blood glucose concentration decreased from 24.4 +/- 1.4 to 4.0 +/- 1.3 mM. At 4 and 5 mo posttransplantation, the capsules were removed from 2 recipients. Both animals regressed to a hyperglycemic state after capsule removal. However, after another islet transplantation, normoglycemia was again restored in these 2 animals. In control mice, which received unencapsulated islets, the xenografts remained functional for less than 10 days. A high mortality rate was observed among these animals within 2 mo of the recurrence of the hyperglycemic state. Our results clearly indicate that encapsulation of pancreatic islets in the improved capsules can effectively prolong xenograft survival without immunosuppression in an animal model that mimics human type I diabetes mellitus.

Stimulus-response coupling in insulin-secreting HIT cells. Effects of secretagogues on cytosolic Ca2+, diacylglycerol, and protein kinase C activity.

The hamster islet B cell line HIT retains the ability to secret insulin in response to glucose and several receptor agonists. We used HIT cells to study the initial signaling events in glucose or receptor agonist-stimulated insulin secretion. Glucose stimulated insulin release from HIT cells in a dose-dependent manner with a half-maximal effect seen already at 1 mM. Insulin release was also stimulated by carbachol in a glucose-dependent manner. Glucose depolarized the HIT cell membrane potential as assessed with the fluorescent probe bisoxonol and raised intracellular Ca2+ as revealed by fura-2 measurements. Using a Mn2+ fura-2 quenching technique, we could show that the rise in intracellular Ca2+ was due to Ca2+ influx following opening of voltage-gated Ca2+ channels. Glucose is thought to increase the diacylglycerol (DAG) content of insulin-secreting cells. However, although HIT cells respond to glucose in terms of insulin secretion, membrane depolarization, and Ca2+ rise, the hexose was unable to increase the proportion of protein kinase C activity associated with membranes. In contrast, the membrane-associated protein kinase C activity increased in HIT cells exposed to the two receptor agonists carbachol and bombesin. Bombesin was shown to generate DAG with the expected fatty acid composition of activators of phospholipase C. Glucose, in contrast, only caused minor increases in DAG containing myristic and palmitic acid without affecting total DAG mass. The failure to detect stimulation of protein kinase C by glucose could be due to both the limited amount and to the different fatty acid composition of the metabolically generated DAG. The latter was in part supported by experiments performed on protein kinase C partially purified from HIT cells. Indeed, 1,2-dipalmitoylglycerol, presumed to be the main DAG species generated by glucose, was only one-third as active as 1,2-dioleoylglycerol and 1-stearoyl-2-arachidonylglycerol in stimulating the isolated enzyme at physiological Ca2+ concentration. It is therefore unlikely that DAG and protein kinase C play a major role in glucose-stimulated insulin secretion.

Interactions of Cholecystokinin and Glucose in Rat Pancreatic Islets

The effects of sulfated cholecystokinin (CCK-8S) and glucose on insulin secretion and polyphosphoinositide (PPI) metabolism were studied in isolated rat islets. Both agonists stimulate PPI hydrolysis, inositol phosphate accumulation, 3H efflux from [3H]inositol-prelabeled tissue, and 45Ca efflux from prelabeled cells. However, the effects of CCK-8S on PPI metabolism are considerably greater than those of glucose. Furthermore, the effects of CCK-8S on PPI and Ca2+ metabolism are observed whether islets are incubated in either 2.75 or 7 mM glucose, but CCK-8S only stimulates insulin secretion (a biphasic response) when the higher glucose concentration is present. Addition of 1 microM forskolin to islets incubated in media containing 2.75 mM glucose does not influence basal insulin secretion but sensitizes the islets to the action of CCK-8S. In the presence of forskolin, CCK-8S induces a very marked first phase but no second phase of insulin secretion. We postulate that CCK-8S acts in this tissue via receptor-linked PPI hydrolysis, leading to an inositol trisphosphate-induced Ca2+ efflux. These receptor-mediated effects of CCK-8S are not altered either by the ambient glucose concentration or the cAMP content of the islets, but these two factors determine the responsiveness of the islets (in terms of insulin secretion) to a given CCK-8S signal.