Cellular Insulin Content Research Articles

Acute effects of insulin on beta-cells from transplantable human islets

The functional role of autocrine insulin signaling remains unclear despite considerable investigation. In the present study, we tested the effects of high and low doses of exogenous insulin on Ca 2+ signaling, insulin synthesis and insulin secretion in dispersed human islet cells using a combination of imaging, radioimmunoassay and patch-clamp electrophysiology. Although 200 nM insulin stimulated Ca 2+ signals with larger amplitudes, the percentage of responding cells was lower when compared with 0.2 nM insulin. However, both 0.2 nM insulin and 200 nM insulin led to a transient increase in accessible cellular insulin content under conditions that glucose did not. This pool of insulin likely reflected de novo synthesis as it could be blocked by cyclohexamide or actinomycin D. Blocking endogenous autocrine insulin signaling in quiescent β-cells with the insulin receptor inhibitor HMNPA led to a reduction in insulin synthesis, suggesting some degree of basal activity of this positive feed-forward loop. Unlike exposure to high glucose, acute treatment with insulin did not stimulate robust insulin exocytosis, as estimated by C-peptide release and capacitance measurements from single β-cells. Together these data provide further evidence that autocrine insulin signaling can regulate the function of human pancreatic β-cells. Our findings suggest autocrine insulin signaling directly controls insulin protein levels, but not exocytosis, in β-cells and demonstrate the functional specificity of insulin signaling and glucose signaling in human islet cells.

Long-term beneficial effects of vanadate, tungstate, and molybdate on insulin secretion and function of cultured beta cells.

The ultratrace elements vanadate, tungstate, and molybdate exhibit significant antihyperglycemic effects in both type 1 and 2 diabetic animals, but possible effects on the function of pancreatic beta cells are understudied. In the present study, clonal BRIN BD11 cells were cultured for 3 days with each ultratrace element to establish doses lacking detrimental effects on viable beta cell mass. Vanadate treatment (4 micromol/L) had no effect on cellular insulin content but improved glucose-induced insulin secretory responsiveness. However, insulin secretion mediated by PKA and PKC activation was desensitized in vanadate-treated cells. Culture with tungstate (300 micromol/L) and molybdate (1 mmol/L) increased cellular insulin content and enhanced basal insulin release and the responsiveness to glucose and a wide range of other secretagogues. These observations suggest significant effects of ultratrace elements on pancreatic beta cells that may contribute to their antihyperglycemic action.

Chronic exposure to tolbutamide and glibenclamide impairs insulin secretion but not transcription of K ATP channel components

Clonal insulin-secreting BRIN-BD11 cells were used to examine effects of chronic 72–144 h exposure to the sulphonylureas tolbutamide and glibenclamide on insulin release, cellular insulin content, and mRNA levels of the Kir6.2 and SUR1 subunits of the beta-cell K ATP channel. Chronic exposure for 72–144 h to 5–100 μM tolbutamide and glibenclamide resulted in a time- and concentration-dependent irreversible decline in sulphonylurea-induced insulin secretion. In contrast, the decline in cellular insulin content induced by chronic exposure to high concentrations of sulphonylureas was readily reversible. Chronic exposure to tolbutamide or glibenclamide had no effect upon transcription of the Kir6.2 or SUR1 subunits of the pancreatic beta-cell K ATP channel. Whilst further studies are required to understand the precise nature of the chronic interactions of sulphonylurea with the insulin exocytotic mechanism, these observations may partially explain the well-known progressive failure of sulphonylurea therapy in type 2 diabetes.

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.

Genetic manipulations of fatty acid metabolism in beta-cells are associated with dysregulated insulin secretion.

Triacylglyceride (TG) accumulation in pancreatic beta-cells is associated with impaired insulin secretion, which is called lipotoxicity. To gain a better understanding of the pathophysiology of lipotoxicity, we generated three models of dysregulated fatty acid metabolism in beta-cells. The overexpression of sterol regulatory element binding protein-1c induced lipogenic genes and TG accumulation. Under these conditions, we observed a decrease in glucose oxidation and upregulation of uncoupling protein-2, which might be causally related to the decreased glucose-stimulated insulin secretion. The overexpression of AMP-activated protein kinase was accompanied by decreased lipogenesis, increased fatty acid oxidation, and decreased glucose oxidation; insulin secretions to glucose and depolarization stimuli were decreased, probably because of the decrease in glucose oxidation and cellular insulin content. It was notable that the secretory response to palmitate was blunted, which would suggest a role of the fatty acid synthesis pathway, but not its oxidative pathway in palmitate-stimulated insulin secretion. Finally, we studied islets of PPAR-gamma(+/-) mice that had increased insulin sensitivity and low TG content in white adipose tissue, skeletal muscle, and liver. On a high-fat diet, glucose-stimulated insulin secretion was decreased in association with increased TG content in the islets, which might be mediated through the elevated serum free fatty acid levels and their passive transport into beta-cells. These results revealed some aspects about the mechanisms by which alterations of fatty acid metabolism affect beta-cell functions.

Nicotinic acid-adenine dinucleotide phosphate-sensitive calcium stores initiate insulin signaling in human beta cells.

Recent studies suggest a role for autocrine insulin signaling in beta cells, but the mechanism and function of insulin-stimulated Ca(2+) signals is uncharacterized. We examined Ca(2+)-dependent insulin signaling in human beta cells. Two hundred nanomolar insulin elevated [Ca(2+)](c) to 284 +/- 27 nM above baseline in approximately 30% of Fura-4F-loaded cells. Insulin evoked multiple Ca(2+) signal waveforms, 60% of which included oscillations. Although the amplitude of Ca(2+) signals was dose-dependent between 0.002 and 2,000 nM, the percentage of cells responding was highest at 0.2 nM insulin, suggesting the interaction of stimulatory and inhibitory pathways. Ca(2+)-free solutions did not affect the initiation of insulin-stimulated Ca(2+) signals, but abolished the second phase of plateaus/oscillations. Likewise, inositol 1,4,5-trisphosphate (IP(3)) receptor antagonists xestospongin C and caffeine selectively blocked the second phase, but not the initiation of insulin signaling. Thapsigargin and 2,5-di-tert-butylhydroquinone (BHQ) blocked insulin signaling, implicating sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA)-containing Ca(2+) stores. Insulin-stimulated Ca(2+) signals were insensitive to ryanodine. Injection of the CD38-derived Ca(2+) mobilizing metabolite, nicotinic acid-adenine dinucleotide phosphate (NAADP), at nanomolar concentrations, evoked oscillatory Ca(2+) signals that could be initiated in the presence of ryanodine, xestospongin C, and Ca(2+)-free solutions. Desensitizing concentrations of NAADP abolished insulin-stimulated Ca(2+) signals. Insulin-stimulated Ca(2+) signals led to a Ca(2+)-dependent increase in cellular insulin contents, but not secretion. These data reveal the complexity of insulin signal transduction and function in human beta cells and demonstrate functional NAADP-sensitive Ca(2+) stores in a human primary cultured cell type.

Fatty acids potentiate interleukin-1β toxicity in the β-cell line INS-1E

Evidence for “lipotoxicity,” i.e., negative effects of fatty acids (FA) on pancreatic β-cells is incomplete. Here, we tested whether non-toxic concentrations of FA potentiate toxic effects of interleukin-1β (IL-1β). Culture of INS-1E clonal β-cells for 2–6 days with 70 μ M docosahexaenoic acid (DHA), eicosapentaenoic acid, arachidonic acid, 0.1 mM linoleic acid, or 0.1–0.2 mM oleic acid exerted no or minor negative effects on cell viability (MTT assay). Viability was reduced by 0.5 ng/ml IL-1β. All tested FA significantly aggravated this effect after 6 days of culture. IL-1β also exerted negative effects on cellular insulin content and DHA and oleic acid aggravated these effects. l-NAME, an inhibitor of constitutive nitric oxide (NO) synthase, reduced the negative effects of IL-1β per se but did not abolish the potentiating effects of FA. Conclusion: FA potentiate toxic effects of IL-1β on β-cells by mechanisms that include NO-independent ones.

CFLIP protein prevents tumor necrosis factor-alpha-mediated induction of caspase-8-dependent apoptosis in insulin-secreting betaTc-Tet cells.

Type 1 diabetes is characterized by the infiltration of activated leukocytes within the pancreatic islets, leading to beta-cell dysfunction and destruction. The exact role played by interferon-gamma, tumor necrosis factor (TNF)-alpha, and interleukin-1beta in this pathogenic process is still only partially understood. To study cytokine action at the cellular level, we are working with the highly differentiated insulin-secreting cell line, betaTc-Tet. We previously reported that it was susceptible to apoptosis induced by TNF-alpha, in combination with interleukin-1beta and interferon-gamma. Here, we report that cytokine-induced apoptosis was correlated with the activation of caspase-8. We show that in betaTc-Tet cells, overexpression of cFLIP, the cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein, completely abolished cytokine-dependent activation of caspase-8 and protected the cells against apoptosis. Furthermore, cFLIP overexpression increased the basal and interleukin-1beta-mediated transcriptional activity of nuclear factor (NF)-kappaB, whereas it did not change cytokine-induced inducible nitric oxide synthase gene transcription and nitric oxide secretion. The presence of cFLIP prevented the weak TNF-alpha-induced reduction in cellular insulin content and secretion; however, it did not prevent the decrease in glucose-stimulated insulin secretion induced by the combined cytokines, in agreement with our previous data demonstrating that interferon-gamma alone could induce these beta-cell dysfunctions. Together, our data demonstrate that overexpression of cFLIP protects mouse beta-cells against TNF-alpha-induced caspase-8 activation and apoptosis and is correlated with enhanced NF-kappaB transcriptional activity, suggesting that cFLIP may have an impact on the outcome of death receptor-triggered responses by directing the intracellular signals from beta-cell death to beta-cell survival.

Syntaxin-3 and syntaxin-1A inhibit L-type calcium channel activity, insulin biosynthesis and exocytosis in beta-cell lines.

Syntaxin-1A (Syn-1A) is known to play a negative regulatory role in insulin secretion but the precise mechanisms for its action are not clear. Syn-2, -3 and -4 are also present in islet beta cells but their functions are not known. Here, we investigated the role of these syntaxins in the insulin secretory process. We examined the following effects of Syn-1, -2, -3 and -4 expression in insulinoma beta-cell lines. Endogenous insulin secretion was measured by batch radioimmunoassay (RIA) and single cell patch clamp capacitance measurements. The L-type Ca(2+) channel activity was studied by patch clamp electrophysiology. Insulin gene transcription was examined by Northern blotting and measurement of insulin gene promoter activity by the co-expression of cyan fluorescent protein-labelled rat insulin promoter. Syn-1A or -3, but not Syn-2 or -4 overexpression, inhibited K(+)-induced insulin release as determined by RIA (49.7 +/- 5.5 % and 49.1 +/- 6.2 %, respectively) and electrophysiologic membrane capacitance measurements (68.0 +/- 21.0 % and 58.0 +/- 13.2 %, respectively). Overexpressed Syn-1A and -3, but not Syn-2, inhibited Ca(2+) channel current amplitude by 39.5 +/- 11.6 % and 52.7 +/- 6.0 %, respectively. Of note, overexpression of Syn-1A and -3 also reduced single cell (by confocal microscopy) and total cellular endogenous insulin content (by RIA) by 24.8 +/- 4.2 % and 31.8 +/- 3.9 %, respectively. This correlated to a reduction in endogenous insulin mRNA by 24.5 +/- 4.2 % and 25.7 +/- 4.2 %, respectively. This inhibition of insulin biosynthesis is mainly at the level of insulin gene transcription as demonstrated by an inhibition of insulin gene promoter activity (53.3 +/- 9.15 % and 39.0 +/- 6.8 %, respectively). These results demonstrate that Syn-1A and -3 possess strong inhibitory actions on both insulin exocytosis and insulin biosynthesis whereas Syn-2 and -4 do not inhibit the insulin secretory process.

Serine-protease inhibition during islet isolation increases islet yield from human pancreases with prolonged ischemia.

Islet isolation from the pancreatic tissue matrix remains highly variable. Recent evidence suggests that intrinsic human pancreatic proteases, including trypsin, may inhibit effective collagenase enzymatic activity during islet isolation, thereby impairing the isolation success. In this study we have hypothesized that serine protease inhibition applied during pancreatic digestion, could improve yield and/or functional viability of islets isolated from human pancreases. Twelve organ donor pancreases with 12.9+/-0.6 hr cold storage (mean+/-SEM) were perfused via their ducts with Liberase-HI enzyme in the presence (n=6) or absence (n=6) of 0.4 mM Pefabloc. All were then gently dissociated and their purified islets separated with Ficoll density gradient centrifugation. Donor-related factors (age, gender, cold storage time, body mass index, and pancreas weight) did not differ significantly between the two experimental groups. Pefabloc supplementation did not affect the digestion time, islets remaining trapped in exocrine tissue, or final islet purity. Islet recovery was increased in the Pefabloc-treated group (mean+/-SEM yield 323.8+/-80.8 x 10(3) islet equivalents vs. 130.8+/-13.6 x 10(3) islet equivalents, P<0.05). Cellular composition, DNA and insulin content, and insulin secretory activity of the isolated islets was similar. Inhibition of intrinsic protease activity within pancreases after prolonged cold storage improves isolation of viable islets.

Dehydroepiandrosterone sulfate and beta-cell function: enhanced glucose-induced insulin secretion and altered gene expression in rodent pancreatic beta-cells.

Administration of dehydroepiandrosterone (DHEA), or its sulfated form (DHEAS), controls hyperglycemia in diabetic rodents without directly altering insulin sensitivity. We show that DHEAS enhanced glucose-stimulated insulin secretion when administered in vivo to rats or in vitro to beta-cell lines, without changing cellular insulin content. Insulin secretion increased from 3 days of steroid exposure in vitro, suggesting that DHEAS did not directly activate the secretory processes. DHEAS selectively increased the beta-cell mRNA expression of acyl CoA synthetase-2 and peroxisomal acyl CoA oxidase in a time-dependent manner. Although DHEAS is a peroxisomal proliferator, it did not alter the mRNA expression of peroxisomal proliferator-activated receptor (PPAR) alpha or beta, or enhance the activity of transfected PPAR alpha, beta, or gamma in vitro. Thus, DHEAS directly affected the beta-cell to enhance glucose-stimulated insulin secretion and increased the mRNA expression of specific beta-cell mitochondrial and peroxisomal lipid metabolic enzymes. This effect of DHEAS on insulin secretion may contribute to the amelioration of hyperglycemia seen in various rodent models of diabetes.

Dominant Negative MyD88 Proteins Inhibit Interleukin-1β/Interferon-γ-mediated Induction of Nuclear Factor κB-dependent Nitrite Production and Apoptosis in β Cells

Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic islet beta cells are destroyed by a combination of immunological and inflammatory mechanisms. In particular, cytokine-induced production of nitric oxide has been shown to correlate with beta cell apoptosis and/or inhibition of insulin secretion. In the present study, we investigated whether the interleukin (IL)-1beta intracellular signal transduction pathway could be blocked by overexpression of dominant negative forms of the IL-1 receptor interacting protein MyD88. We show that overexpression of the Toll domain or the lpr mutant of MyD88 in betaTc-Tet cells decreased nuclear factor kappaB (NF-kappaB) activation upon IL-1beta and IL-1beta/interferon (IFN)-gamma stimulation. Inducible nitric oxide synthase mRNA accumulation and nitrite production, which required the simultaneous presence of IL-1beta and IFN-gamma, were also suppressed by approximately 70%, and these cells were more resistant to cytokine-induced apoptosis as compared with parental cells. The decrease in glucose-stimulated insulin secretion induced by IL-1beta and IFN-gamma was however not prevented. This was because these dysfunctions were induced by IFN-gamma alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1beta is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse beta cells but does not contribute to impaired glucose-stimulated insulin secretion. Furthermore, our data show that IL-1beta cellular actions can be blocked by expression of MyD88 dominant negative proteins and, finally, that cytokine-induced beta cell secretory dysfunctions are due to the action of IFN-gamma.

Desensitization of sulphonylurea- and nutrient-induced insulin secretion following prolonged treatment with glibenclamide

Functional effects of prolonged exposure to the sulphonylurea glibenclamide were examined in a popular clonal pancreatic β-cell line, denoted as BRIN-BD11. In acute 20-min incubations, 200 μM of tolbutamide or glibenclamide stimulated insulin release from non-depolarized and depolarized cells, which was dramatically reduced following 18-h culture with 100 μM glibenclamide. Sulphonylurea desensitization in non-depolarized cells was reversed following 6–36-h subsequent culture in the absence of glibenclamide. However, desensitization of insulinotropic effects of sulphonylureas in depolarized cells following glibenclamide culture and associated decline in cellular insulin content was not fully reversible. Culture with 100 μM glibenclamide also markedly reduced the acute insulinotropic actions of glucose, l-alanine, l-arginine, 2-ketoisocaproic acid (KIC) and KCl. These effects were almost completely reversed following 18-h culture in the absence of the sulphonylurea.

Induced desensitization of the insulinotropic effects of antidiabetic drugs, BTS 67 582 and tolbutamide.

Acute and chronic mechanisms of action of novel insulinotropic antidiabetic drug, BTS 67 582 (1, 1-dimethyl-2-(2-morpholinophenyl)guanidine fumarate), were examined in the stable cultured BRIN-BD11 cell line. BTS 67 582 (100 - 400 microM) stimulated a concentration-dependent increase (P<0.01) in insulin release at both non-stimulatory (1.1 mM) and stimulatory (8. 4 mM) glucose. Long-term exposure (3 - 18 h) to 100 microM BTS 67 582 in culture time-dependently decreased subsequent responsiveness to acute challenge with 200 microM BTS 67 582 or 200 microM tolbutamide at 12 - 18 h (P<0.001). Similarly 3 - 18 h culture with the sulphonylurea, tolbutamide (100 microM), also effectively suppressed subsequent insulinotropic responses to both BTS 67 582 and tolbutamide. Culture with 100 microM BTS 67 582 or 100 microM tolbutamide did not affect basal insulin secretion, cellular insulin content, or cell viability and exerted no influence on the secretory responsiveness to 200 microM of the imidazoline, efaroxan. While 18 h BTS 67 582 culture did not affect the insulin-releasing actions (P<0.001) of 16.7 mM glucose, 10 mM arginine, 30 mM KCl, 25 microM forskolin or 10 nM phorbol-12-myristate 13-acetate (PMA), significant inhibition (P<0.001) of the insulinotropic effects of 10 mM 2-ketoisocaproic acid (KIC) and 10 mM alanine were observed. These data suggest that BTS 67 582 shares a common signalling pathway to sulphonylurea but not imidazoline drugs. Desensitization of drug action may provide an important approach to dissect sites of action of novel and established insulinotropic antidiabetic agents.

Microencapsulation of neonatal porcine islets: protection from human antibody/complement-mediated cytolysis in vitro and long-term reversal of diabetes in nude mice.

Recently, we have developed a simple and reliable method to efficiently isolate large numbers of neonatal porcine islets (NPI). We and others have shown that NPI are susceptible to cytolysis by the activation of human complement in vitro. Microencapsulation of islets may be one strategy to protect NPI from this form of rejection. We examined whether microencapsulation can prevent lysis of NPI induced by human antibody and complement in vitro and also assessed their ability to reverse hyperglycemia in diabetic nude mice. NPI were microencapsulated with purified alginate, cultured for 2 days, then tested for sensitivity to fresh human serum using an established in vitro cytotoxicity assay or transplanted into alloxan-induced diabetic nude mice. Incubation of nonencapsulated NPI for 24 hr in the presence of fresh human serum resulted in a 53% loss of cellular insulin content, a 51% reduction in recoverable DNA content, and a marked reduction of insulin secretory responsiveness when compared with controls cultured in heat-inactivated human serum. In contrast, exposure of encapsulated islets to fresh human serum had no cytotoxic effect on the islets. Transplantation of 2000 encapsulated NPI i.p. into diabetic nude mice (n=16) corrected hyperglycemia in all mice within 8 weeks. Similar results were obtained when 2000 nonencapsulated NPI were implanted under the kidney capsule (n=10); however recipients of nonencapsulated NPI placed i.p. failed to obtain euglycemia and survived for only 3 weeks posttransplantation. Microencapsulation protects NPI from the cytotoxic effects of human antibody and complement and allows for long-term reversal of diabetes in nude mice.

Prolonged exposure of human beta-cells to high glucose increases their release of proinsulin during acute stimulation with glucose or arginine.

The disproportionate hyperproinsulinemia in type 2 diabetes has been attributed to either a primary beta-cell defect or a secondary dysregulation of beta cells under sustained hyperglycemia. This study examines the effect of a 10- to 13-day exposure to 20 mmol/L glucose on subsequent proinsulin and insulin release by human islets isolated from nondiabetic donors. Compared to control preparations kept at 6 mmol/L glucose, the high glucose cultured beta-cells released more proinsulin and less insulin during perifusion at 5, 10, or 20 mmol/L glucose. The lower amounts of secreted insulin resulted from a marked reduction in cellular insulin content (5-fold lower than in controls). The higher amount of secreted proinsulin is attributed to the sustained state of cellular activation that is known to occur after prolonged exposure to high glucose levels. This activated state of the beta-cell population is also held responsible for its higher secretory responsiveness to 5 mmol/L arginine at a submaximal (5 mmol/L) glucose concentration (8-fold higher proinsulin levels than in the control population). It results, together with the reduction in cellular insulin content, in 7- to 10-fold higher proinsulin over insulin ratios in the medium; at 5 mmol/L glucose, this extracellular ratio is similar to that in the cells. These data add direct support to the view that a disproportionate hyperproinsulinemia can result from a sustained activation of human beta-cells after prolonged exposure to elevated glucose levels.

Prolonged exposure of pancreatic beta cells to raised glucose concentrations results in increased cellular content of islet amyloid polypeptide precursors.

Most non-insulin dependent diabetic patients have amyloid deposits in their pancreatic islets. It is not known whether chronic hyperglycaemia contributes to the formation of amyloid fibrils from the islet amyloid polypeptide that is produced by the pancreatic beta cells. Since islet amyloid exhibits islet amyloid polypeptide precursors immunoreactivity, we examined whether sustained in vitro exposure to raised glucose increases the abundance of these precursors in human beta cells. After 6 days stimulation with 20 mmol/l glucose the cellular content of insulin but not islet amyloid polypeptide was decreased leading to an increase in the ratio of the latter over insulin (3.0 +/- 0.6 vs 1.8 +/- 0.3 after 6 mmol/l glucose culture, p < 0.05). Similar changes occurred in rat beta cells cultured for 3 days in the presence of 20 mmol/l glucose plus 3-isobutyl-1-methylxanthine. Western blot analysis of cellular islet amyloid polypeptide after prolonged exposure to high glucose indicated the presence of higher proportions of its precursor- and intermediate forms. In human beta cells cultured in 20 mmol/l glucose, the major form corresponds to an intermediate species which exhibits an immunoreactivity for the N-flanking peptide, as is also the case in islet amyloid. We concluded that prolonged in vitro exposure of beta cells to raised glucose concentrations increases the relative proportion of islet amyloid polypeptide over insulin, as well as of its precursors over the mature form of islet amyloid polypeptide.

Insulin receptor signaling in the beta-cell influences insulin gene expression and insulin content: evidence for autocrine beta-cell regulation.

The insulin receptor (IR) is expressed by insulin-secreting beta-cells, but its cellular function is unknown. We transfected betaTC6-F7 beta-cells with cDNAs encoding either wild-type or mutant kinase-inactive (A/K1018) IRs, and by fluorescence-activated cell sorting generated polyclonal beta-cell lines that overexpressed each receptor type at levels two- to fourfold higher than the parental cells. Beta-cells overexpressing wild-type IRs had a proportional increase in insulin-stimulated tyrosine kinase activity; no change occurred in beta-cells expressing the mutant IR. We observed a threefold increase in cellular insulin content in beta-cells that overexpressed the wild-type IR, as determined by radioimmunoassay. This increase occurred despite a fivefold elevated rate of both basal and 10 mmol/l glucose-induced insulin secretion. Fractional insulin secretion (percentage of total cell insulin releasable at 10 mmol/l glucose) was unchanged in beta-cells overexpressing the wild-type IR compared with the parental beta-cell line. Insulin content and insulin secretion were unaffected by overexpression of kinase-inactive IRs. Steady-state insulin mRNA levels were elevated twofold in the beta-cells overexpressing the wild-type IR and unchanged in the beta-cells expressing the kinase-inactive receptor, as determined by Northern blot analysis. The rate of insulin mRNA degradation measured in the presence of 5 microg/ml actinomycin D was not significantly affected in either cell line. In the absence of glucose, the basal level of (pro)insulin biosynthesis in the beta-cells overexpressing the wild-type IR increased significantly (61%) compared with the beta-cells transfected with the kinase-inactive IR. These data indicate that IR signaling can regulate insulin gene transcription and can modulate the steady-state insulin content of beta-cells.

Survival and function of syngeneic rat islet grafts placed within the thymus versus under the kidney capsule

The role of the thymus in the ongoing acquisition of tolerance to self antigens has made it an attractive site for islet transplantation. Several studies have reported survival of rodent islet allografts in the thymus without requiring the long-term use of immunosuppressive agents; however, the degree of glucose homeostasis in the intrathymic islet transplant recipients has not been examined. We transplanted 500, 1000, or 2000 syngeneic islets into the thymus of streptozotocin-induced diabetic Wistar-Furth rats, and compared the metabolic response of these recipients with animals receiving 2000 syngeneic islets under the kidney capsule. Three of four recipients which received 2000 islets under the kidney capsule achieved normoglycemia (≤8.4 mmol/L) within 1 wk and all animals became normoglycemic within 2 wk posttransplantation. In contrast, intrathymic implantation of 2000 islets induced normoglycemia in only one of six recipients during the same time interval, and when this number was reduced to 1000 or 500 islets, none of the recipients (n = 6) normalized within 1 wk posttransplantation. Animals that received an intrathymic transplant were glucose intolerant compared to normal controls and animals with subcapsular islet transplant. Removal of the graft-bearing organs resulted in hyperglycemia in all cases, and examination of the grafts revealed the presence of numerous well-granulated insulincontaining cells in both sites. The cellular insulin content of the subcapsular grafts (67.4 ± 12.1 μg; n = 4) was significantly higher (p ≤0.05) than what was extracted from intrathymic grafts (9.5 ± 1.2 μg from 1000 islets; n = 3 and 20.0 ± 4.6 μg from 2000 islets; n = 3). We conclude that 2000 syngeneic islets implanted either in the thymus or beneath the kidney capsule can normalize hyperglycemia in streptozotocin-diabetic rats; however, normal glucose tolerance was not established in intrathymic islet recipients, suggesting that a higher number of islets may be necessary to achieve normal glucose homeostasis.

Survival and function of syngeneic rat islet grafts placed within the thymus versus under the kidney capsule.

The role of the thymus in the ongoing acquisition of tolerance to self antigens has made it an attractive site for islet transplantation. Several studies have reported survival of rodent islet allografts in the thymus without requiring the long-term use of immunosuppressive agents; however, the degree of glucose homeostasis in the intrathymic islet transplant recipients has not been examined. We transplanted 500, 1000, or 2000 syngeneic islets into the thymus of streptozotocin-induced diabetic Wistar-Furth rats, and compared the metabolic response of these recipients with animals receiving 2000 syngeneic islets under the kidney capsule. Three of four recipients which received 2000 islets under the kidney capsule achieved normoglycemia (< or =8.4 mmol/L) within 1 wk and all animals became normoglycemic within 2 wk posttransplantation. In contrast, intrathymic implantation of 2000 islets induced normoglycemia in only one of six recipients during the same time interval, and when this number was reduced to 1000 or 500 islets, none of the recipients (n = 6) normalized within 1 wk posttransplantation. Animals that received an intrathymic transplant were glucose intolerant compared to normal controls and animals with subcapsular islet transplant. Removal of the graft-bearing organs resulted in hyperglycemia in all cases, and examination of the grafts revealed the presence of numerous well-granulated insulin-containing cells in both sites. The cellular insulin content of the subcapsular grafts (67.4 +/- 12.1 microg; n = 4) was significantly higher (p < or =0.05) than what was extracted from intrathymic grafts (9.5 +/- 1.2 microg from 1000 islets; n = 3 and 20.0 +/- 4.6 microg from 2000 islets; n = 3). We conclude that 2000 syngeneic islets implanted either in the thymus or beneath the kidney capsule can normalize hyperglycemia in streptozotocin-diabetic rats; however, normal glucose tolerance was not established in intrathymic islet recipients, suggesting that a higher number of islets may be necessary to achieve normal glucose homeostasis.