Islet Beta-cell Line Research Articles

Modeling islet enhancers using deep learning identifies candidate causal variants at loci associated with T2D and glycemic traits

Genetic association studies have identified hundreds of independent signals associated with type 2 diabetes (T2D) and related traits. Despite these successes, the identification of specific causal variants underlying a genetic association signal remains challenging. In this study, we describe a deep learning (DL) method to analyze the impact of sequence variants on enhancers. Focusing on pancreatic islets, a T2D relevant tissue, we show that our model learns islet-specific transcription factor (TF) regulatory patterns and can be used to prioritize candidate causal variants. At 101 genetic signals associated with T2D and related glycemic traits where multiple variants occur in linkage disequilibrium, our method nominates a single causal variant for each association signal, including three variants previously shown to alter reporter activity in islet-relevant cell types. For another signal associated with blood glucose levels, we biochemically test all candidate causal variants from statistical fine-mapping using a pancreatic islet beta cell line and show biochemical evidence of allelic effects on TF binding for the model-prioritized variant. To aid in future research, we publicly distribute our model and islet enhancer perturbation scores across ~67 million genetic variants. We anticipate that DL methods like the one presented in this study will enhance the prioritization of candidate causal variants for functional studies.

Sustained high glucose intake accelerates type 1 diabetes in NOD mice.

Epidemiological studies have suggested that dietary factors, especially high consumption of high glycaemic index carbohydrates and sugars, may trigger or exacerbate the progression of type 1 diabetes. We aimed to provide experimental evidence to confirm this relevance and to explore the underlying mechanisms. NOD mice were given sustained high-glucose drinking or glucose-free water and observed for the incidence of type 1 diabetes and islet inflammation. RNAseq was performed to detect the transcriptome changes of the NOD islet beta cell line NIT-1 after high glucose treatment, and mass spectrometry was performed to detect the proteome changes of NIT-1-cells-derived sEVs. Sustained high glucose drinking significantly aggravates islet inflammation and accelerates the onset of type 1 diabetes in NOD mice. Mechanistically, high glucose treatment induces aberrant ER stress and up-regulates the expression of autoantigens in islet beta cell. Moreover, high glucose treatment alters the proteome of beta-cells-derived sEVs, and significantly enhances the ability of sEVs to promote DC maturation and stimulate immune inflammatory response. This study provides evidence for negative effect of high glucose intake as a dietary factor on the pathogenesis of type 1 diabetes in genetically predisposed individuals. Therefore, avoiding high sugar intake may be an effective disease prevention strategy for children or adults susceptible to type 1 diabetes.

Exenatide improves antioxidant capacity and reduces the expression of LDL receptors and PCSK9 in human insulin-secreting 1.1E7 cell line subjected to hyperglycemia and oxidative stress

Abstract Introduction GLP-1 receptor agonists (e.g., exenatide) are novel drugs used in the treatment of diabetes. These drugs, working with other mechanisms of action, improve glycemic control by increasing secretion of insulin and improving survival of pancreatic islet beta cells. Alterations in the oxidative stress level or the expression of proteins associated with cholesterol uptake might be responsible for those findings. Currently, there are few in vitro studies on the impact of exenatide antioxidant capacity in human islet beta cell lines and none that assess the influence of exenatide on LDL receptors and PCSK9 under hyperglycemia and oxidative stress. Therefore, we evaluated the impact of exenatide on antioxidant capacity, insulin secretion, and proteins involved in cholesterol metabolism. Materials and Method An in vitro culture of insulin-secreting cells 1.1E7 was subjected to hyperglycemia and oxidative stress. Assessment was made of the expression of enzymes associated with oxidative stress (NADPH oxidase, catalase, glutathione peroxidase, superoxide dismutase, iNOS) and cholesterol uptake (LDL receptors, PCSK9). Additionally, insulin and nitrite levels in culture media were quantified. Results We showed that exenatide improves expression of catalase and reduces the amount of nitrite in cell cultures in a protein kinase A–dependent manner. Those results were accompanied by a drop in the expression of LDL receptors and PCSK9. Insulin secretion was modestly increased in the culture condition. Conclusions Our findings show potential protective mechanisms exerted by exenatide in human insulin-secreting pancreatic beta cell line (1.1E7), which may be exerted through increased antioxidant capacity and reduced accumulation of cholesterol.

Effect of diabetic induced thioredoxin interacting protein (TXNIP) on islet cell senescence

Objective: To investigate whether the increased expression of thioredoxin interacting protein (TXNIP) in diabetes affects the senescence of islet β cells. Methods: Six normal mice (db/m) and six diabetic mice (db/db) were randomly selected. Fasting blood glucose was measured by blood sugar meter, the expression levels of TXNIP protein, p16, p21 and Rb in pancreatic tissues were detected by Western blot, senescence-associated beta-galactosidase activity in pancreatic tissue was determined by immunochemical staining. INS-1 islet beta cells were randomly divided into 7 groups (n=6), and transfected with lentiviruses (30 μl) for 4 to 6 hours, then was screened with puromycin (PM, 3 μg/m) for 7 days to construct normal group, scramble ShRNA group (interference with airborne poison group), TXNIP-ShRNA-1 group (TXNIP silence group-1), TXNIP-ShRNA-2 group (TXNIP silence group 2), TXNIP-ShRNA-3 group (TXNIP silence group 3), Ad-GFP group (overexpression of the air virus group), Ad-TXNIP-GFP group (TXNIP overexpression group) stably transferred INS-1 islet beta cell line. TXNIP protein expression was detected by Western blot, aging-related beta-galactosidase activity was detected by immunochemical staining, the changes of expression of p16, p21 and Rb was determined by Western blot. Results: Compared with normal mice, the fasting blood glucose of db/db group was increased significantly (P＜0. 01), the expression of TXNIP protein was increased significantly in pancreatic tissues(P＜0. 05), positive staining rate of β- galactosidase was increased significantly in pancreatic tissues, p16/p21/Rb protein expression levels were increased significantly (P＜0. 05). Compared with Ad-GFP group, the positive staining rate of β- galactosidase in Ad-TXNIP-GFP group was increased significantly, p16/p21/Rb protein expression levels were increased significantly (P＜0. 01). Compared to the scramble ShRNA group, the positive staining rate of β- galactosidase in TXNIP-ShRNA group was decreased, p16/p21/Rb protein expression levels were decreased significantly (P＜0. 05). Conclusion: Diabetes can induce islet β-cell senescence by up-regulating TXNIP expression.

Concurrently using rosiglitazone prevents glucosamine‐induced islet β‐Cell apoptosis and dysfunction

Diabetes has merged as a significant health problem. This study aims to examine the effect of concurrently using rosiglitazone (RSG) on inhibiting glucosamine (GlcN)-induced islet beta cell apoptosis and dysfunction. Using an islet beta cell line, HIT-T15 cells, as a study platform, the inhibitory effect of RSG on GlcN-induced pathophysiological changes in islet beta cells was examined. The results showed that treatment with GlcN induced HIT-T15 cell death via apoptotic pathway, inhibited the expression of Bcl-2 and Bcl-xL, enhanced the expression of Bax, Bid and caspase-3, reduced the production of ATP and decreased in insulin secretion. The changes were in a GlcN dose-dependent manner. Concurrently using RSG with GlcN, the induced pathogenic changes in HIT-T15 cells were abrogated. We conclude that concurrently using RSG can be useful in reducing the GlcN-induced side effects on islet beta cells that has potential to prevent the complications caused by GlcN in the treatment of diabetes.

Enhanced insulin production from murine islet beta cells incubated on poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate)

Islet transplantation represents an important alternative for the treatment of diabetes. However, the selection of suitable materials is critical for the success of such an implantation application. In this study, cellular migration, aggregation, and insulin production of a murine islet beta-cell line, NIT-1 cells on microbially produced polyesters poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB) or polylactic acid (PLA) films were investigated. Spherical islet-like structures were only detected on PHBHHx films after 48 h cultivation. To understand the mechanism underlying the formation of cell aggregates, NIT-1-GFP, a stable transfectant of the green fluorescent protein was used in a time-lapse imaging study. Cell aggregation began on PHBHHx at 2 h, and became obvious at 4 h. Furthermore, cells on PHBHHx displayed higher metabolic activities measured by MTT assay than that on tissue culture plate. More importantly, insulin gene expression as well as extracellular secretion was upregulated after growth on PHBHHx for 72 h. Thus, PHBHHx can be a strong candidate for islet transplantation.

WNK1 Is a Novel Regulator of Munc18c-Syntaxin 4 Complex Formation in Soluble NSF Attachment Protein Receptor (SNARE)-mediated Vesicle Exocytosis

Defects in soluble NSF attachment protein receptor (SNARE)-mediated granule exocytosis occur in islet beta cells, adipocytes, and/or skeletal muscle cells correlate with increased susceptibility to insulin resistance and diabetes. The serine/threonine kinase WNK1 (with no K (lysine)) has recently been implicated in exocytosis and is expressed in all three of these cell types. To search for WNK1 substrates related to exocytosis, we conducted a WNK1 two-hybrid screen, which yielded Munc18c. Munc18c is known to be a key regulator of accessibility of the target membrane (t-SNARE) protein syntaxin 4 to participate in SNARE core complex assembly, although a paucity of Munc18c-binding factors has precluded discovery of its precise functions. To validate WNK1 as a new Munc18c-interacting partner, the direct interaction between WNK1 and Munc18c was confirmed using in vitro binding analysis, and endogenous WNK1-Munc18c complexes were detected in the cytosolic and plasma membrane compartments of the islet beta cell line MIN6. This binding interaction is mediated through the N-terminal 172 residues of Munc18c and the kinase domain residues of WNK1 (residues 159-491). Expression of either of these two minimal interaction domains resulted in inhibition of glucose-stimulated insulin secretion, consistent with a functional importance for the endogenous WNK1-Munc18c complex in exocytosis. Interestingly, Munc18c failed to serve as a WNK1 substrate in kinase activity assays, suggesting that WNK1 functions in SNARE complex assembly outside its role as a kinase. Taken together, these data support a novel role for WNK1 and a new mechanism for the regulation of SNARE complex assembly by WNK1-Munc18c complexes.

Genetic association of single nucleotide polymorphisms in endonuclease G-like 1 gene with type 2 diabetes in a Japanese population

In order to identify type 2 diabetes disease susceptibility gene(s) in a Japanese population, we applied a region-wide case-control association test to the 20.4 Mb region between D3S1293 and D3S2319 on chromosome 3p24.3-22.1, supported by linkage to type 2 diabetes and its related traits in Japanese and multiple populations. We performed a two-stage association test using 1,762 Japanese persons with 485 gene-centric, evenly spaced, common single nucleotide polymorphism (SNP) markers with minor allele frequency >0.1. For mouse studies, total RNA was extracted from various organs of BKS.Cg-+Lepr(db)/+Lepr(db) and control mice, and from MIN6, NIH3T3 and C2C12 cell lines. We detected a landmark SNP375 (A/G) (rs2051211, p = 0.000046, odds ratio = 1.33, 95% CI 1.16-1.53) in intron 5 of the endonuclease G-like 1 (ENDOGL1) gene. Systematic dense SNPs approach identified a susceptibility linkage disequilibrium (LD) block of 116.5 kb by |D'|, an LD units map and a critical region of 2.1 kb by r (2) in ENDOGL1. A haplotype-based association test showed that an at-risk haplotype is associated with disease status (p = 0.00001). The expression of ENDOGL1 was rather ubiquitous with relatively abundant expression in the brain and also in a pancreatic islet beta cell line. Mouse Endogl1 expression increased in pancreatic islets of hyperglycaemic BKS.Cg-+Lepr(db)/+Lepr(db) mice compared with that in control mice. Based on the population genetics, fine mapping of LD block and haplotype analysis, we conclude that ENDOGL1 is a candidate disease-susceptibility gene for type 2 diabetes in a Japanese population. Further analysis in a larger sample size is required to substantiate this conclusion.

Kinectin participates in microtubule-dependent hormone secretion in pancreatic islet β-cells

Kinectin (KNT) is a candidate membrane receptor for kinesin in the movement of intracellular organelles along microtubules. Isoforms of KNT exist containing different combinations of six small (residues 23-33) variable domains (vd) vd1-6 within the C-terminus. Here we investigate a role for KNT and its isoform KNTvd4(-) in the transport of amylin and insulin-containing secretory vesicles in the pancreatic islet beta-cell line RINm5F. KNTvd4(-) lacks vd4 that forms the kinesin-binding domain, and hence its role in the cell is an enigma. We report that amylin-containing vesicles also contained insulin, and exhibited microtubule, and small G-protein-dependent secretion. Knockdown of KNT by small interference RNA (siRNA) inhibited amylin expression and secretion. In contrast, recombinant KNTvd4(-) overexpressed in RINm5F cells associated with amylin-containing vesicles and inhibited amylin secretion, but had no discernible affect on amylin expression. The data suggests that both KNT and KNTvd4(-) participate in microtubule-dependent secretion of amylin in islet beta-cells.

Inhibition of membrane depolarisation-induced transcriptional activity of cyclic AMP response element binding protein (CREB) by the dual-leucine-zipper-bearing kinase in a pancreatic islet beta cell line

The activation of the transcription factor cyclic AMP response element binding protein (CREB) by protein kinase A is inhibited by the human orthologue of the mitogen-activated protein kinase, dual-leucine-zipper-bearing kinase (DLK) in teratocarcinoma cells. However, pancreatic beta cells are electrically excitable and a major pathway regulating CREB in these cells is membrane depolarisation, leading to calcium influx and activation of the calcium/calmodulin-dependent protein phosphatase calcineurin. Therefore, the effect of DLK on CREB activity induced by membrane depolarisation was investigated in the beta cell line HIT. Reporter gene assays and biochemical techniques were used. RT-PCR, Western blot analysis and immunohistochemistry demonstrated the expression of DLK in HIT cells and primary mouse islets. In transient transfection experiments, DLK inhibited both GAL4-CREB activity induced by membrane depolarisation, and transcription directed by the CREB binding site, the cyclic AMP response element. Furthermore, DLK inhibited the transcriptional activity conferred by the CREB coactivator, CREB binding protein, both under basal conditions and after membrane depolarisation. DLK was also effective in response to glucose, the most potent physiological stimulus and known to cause membrane depolarisation of beta cells. Inhibition of calcineurin enhanced DLK activity, whereas overexpression of calcineurin reduced the inhibition by DLK of transcription directed by cyclic AMP response element after membrane depolarisation. These results demonstrate a calcineurin-sensitive inhibition by DLK of CREB activity after membrane depolarisation in pancreatic islet beta cells. This inhibition may, at least partially, be mediated at the coactivator level. The results thus suggest that DLK plays a role in the regulation of beta cell function, including insulin gene transcription and beta cell apoptosis.

Recombinant fowlpox virus for in vitro gene delivery to pancreatic islet tissue

The feasibility of using avipox virus as a vector for gene delivery to islet tissue (adult islets and fetal proislets) was examined using a recombinant fowlpox virus (FPV) engineered to express the reporter gene LacZ (FPV-LacZ). The efficiency of in vitro transduction was dose-dependent and influenced by the donor species and maturation status of the islet tissue. Reporter gene expression in FPV-LacZ-transduced islet grafts was transient (3-7 days) in immunoincompetent nude mice and was not prolonged by in vivo treatment with anti-IFN-gamma mAb. In contrast, FPV-LacZ-transduced NIT-1 cells (a mouse islet beta cell line) expressed the LacZ gene beyond 18 days in vitro. Silencing of transgene expression therefore appeared to occur in vivo and was T cell- and IFN-gamma-independent. Isografts of FPV-LacZ-transduced islets in immunocompetent mice underwent immunological destruction by 7 days, suggesting that either FPV proteins or the reporter protein beta-galactosidase induced an adaptive immune response. Co-delivery of the rat bioactive immunoregulatory cytokine gene TGF-beta to islets using FPV-TGF-beta led to enhanced expression of TGF-beta mRNA in isografts but no long-term protection. Nevertheless, compared to control islet isografts at 5 days, FPV-transduced islets remained embedded in the clotted blood used to facilitate implantation. This phenomenon was TGF-beta transgene-independent, correlated with lack of cellular infiltration, and suggested that the FPV vector transformed the blood clot into a temporary immunological barrier.

3,5,3′‐triiodothyronine (T3) is a survival factor for pancreatic β‐cells undergoing apoptosis

3,5,3'-triiodothyronine (T3) is essential for the growth and the regulation of metabolic functions, moreover, the growth-stimulatory effect of T3 has largely been demonstrated and the pathways via which T3 promotes cell growth have been recently investigated. Type 1 diabetes (T1D) is due to the destruction of beta-cells, which occurs even through apoptosis. Aim of our study was to analyze whether T3 could have an antiapoptotic effect on cultured beta-cells undergoing apoptosis. We have demonstrated that T3 promotes cell proliferation in islet beta-cell lines (rRINm5F and hCM) provoking an increment in cell number (up to 55%: rRINm5F and 45%: hCM), cell viability, and BrdU incorporation, and regulating the cell cycle-related molecules (cyc A, D1, E, and p27(kip1)). T3 inhibited the apoptotic process induced by streptozocin, S-Nitroso-N-Acetylpenicylamine (SNAP), and H2O2 via regulation of the pro- and anti-apoptotic factors Bcl-2, Bcl-XL, Bad, Bax, and Caspase 3. The T3 protective effect was PI-3 K-, but not MAPK- or PKA-mediated, involving pAktThr308. Thus, T3 could be considered a survival factor protecting islet beta-cells from apoptosis.

Reactive oxygen species inhibit calcineurin phosphatase activity in a pancreatic islet beta cell line

Reactive oxygen species inhibit calcineurin phosphatase activity in a pancreatic islet beta cell line

The endoplasmic reticulum stress response is stimulated through the continuous activation of transcription factors ATF6 and XBP1 in Ins2+/Akita pancreatic beta cells.

The dominant C96Y mutation of one of the two murine insulin genes, Ins2, causes diabetes mellitus in 'Akita' mice. Here we established pancreatic islet beta cell lines from heterozygous mice (Ins2+/Akita). Western blot analysis of endoplasmic reticulum (ER) molecular chaperones indicated that Grp78, Grp94 and Orp150 are significantly increased in Ins2+/Akita cells compared with wild-type (Ins2+/+) cells. Reporter gene assays using the human GRP78 promoter with or without the ER stress response element (ERSE) showed that Ins2+/Akita cells exhibit significantly stronger ERSE-dependent transcriptional activity than Ins2+/+ cells. Transient over-expression of the Ins2 C96Y mutant in wild-type beta cells induces a stronger ERSE-dependent stress response than does wild-type Ins2 over-expression. The ERSE-binding transcription factor ATF6 is strongly activated in Ins2+/Akita cells. The activity of a reporter containing the specific binding sequence of another ERSE-binding transcription factor, XBP1, is also enhanced in Ins2+/Akita cells. Levels of active forms of XBP1 mRNA and protein are both markedly elevated in Ins2+/Akita cells. These results indicate that this cell line is subject to continuous ER stress and that the Ins2 C96Y mutation induces the expression of ER chaperones through the activation of ATF6 and XBP1.

Isolation and characterization of an alternatively spliced variant of transcription factor Islet-1.

The LIM homeodomain protein Islet-1 (Isl1), one of the earliest markers for motor neuron differentiation, is also expressed in all classes of islet cells in the pancreas. Isl1 is known to bind and regulate the promoters of the insulin, glucagon and somatostatin genes. In this study, we describe isolation of a novel isl1 cDNA species from the mouse islet beta cell line betaTC6, which arose from the utilization of an alternative splicing acceptor site in the fifth exon. This shorter cDNA encodes an Isl1 isoform (Isl1-beta) lacking the carboxy-terminal 23 amino acids of the previously reported product Isl1-alpha. Although the level of isl1-beta mRNA is much lower than that of isl1-alpha, isl1-beta is preferentially expressed in murine insulinoma cell lines but not in glucagonoma cell line. Upon transient transfection, both Isl1-alpha and Isl1-beta accumulate in the nuclei of murine insulinoma cells. We found that Isl1-beta is a relatively more potent transcriptional activator of the insulin promoter than Isl1-alpha and that the Isl1-alpha isoform undergoes phosphorylation. Therefore, the transcriptional activity of Isl1 is potentially regulated by the alternative splicing of its mRNA and by phosphorylation.

Evidence for recognition of novel islet T cell antigens by granule-specific T cell lines from new onset type 1 diabetic patients.

Type 1 diabetes is a T cell-mediated autoimmune disease where a number of islet beta-cell target autoantigens have been characterized on the basis of reactivity with autoantibodies. Nevertheless, there remains uncertainty of the nature of another group of autoantigens associated with the secretory granule fraction of islet beta-cells that appear to be targeted predominantly by autoreactive T cells. We have previously characterized CD4+, HLA-DR-restricted T cell lines from new onset type 1 diabetic patients that are specific for the secretory granule fraction of rat tumour insulinoma, RIN. The T cell line from the first patient, HS, proliferates in response to crude microsomal membranes prepared from a recently established, pure human islet beta-cell line NES2Y. In addition, the HS line also responds to secretory granule fractions prepared from a murine tumour insulinoma grown in RIP-Tag mice, showing the recognition of species-conserved antigen(s) in beta-cells. Using partially matched antigen-presenting cells, the HS T cells and another line derived from a second patient, MR, were shown to be restricted by disease-associated DRB1*0101 and DRB1*0404 alleles, respectively. Neither the HS or MR T cell lines proliferate in response to a large panel of candidate islet cell antigens, including insulin, proinsulin, glutamic acid decarboxylase, the protein tyrosine phosphatase IA-2/phogrin, imogen-38, ICA69 or hsp60. Our data provide compelling evidence of the presence of a group of antigens associated with the secretory granule fraction of islet beta-cells recognized by the T cell lines, whose definition may contribute to our knowledge of disease induction as well as to diagnosis.

Expression of calbindin-D(28k) in a pancreatic islet beta-cell line protects against cytokine-induced apoptosis and necrosis.

Cytokines produced by immune system cells that infiltrate pancreatic islets are candidate mediators of islet beta-cell destruction in autoimmune (type 1) diabetes mellitus. Because the calcium binding protein, calbindin-D(28k), can prevent apoptotic cell death in different cell types, we investigated the possibility that calbindin-D(28k) may prevent cytokine-mediated islet beta-cell destruction. Using the expression vector BSRalpha, rat calbindin-D(28k) was stably expressed in the pancreatic islet beta-cell line, betaTC-3. Calbindin-D(28k) expression resulted in increased cell survival in the presence of the cytotoxic combination of the cytokines IL-1beta (30 U/ml), TNFalpha (10(3) U/ml), and interferon gamma (10(3) U/ml). The greatest protection was observed in the betaTC-3 cell clone expressing the highest concentration of calbindin-D(28k). Apoptotic cell death was detected by annexin V staining and by the TdT-mediated dUTP-X nick end labeling assay in vector-transfected betaTC-3 cells incubated with cytokines (14-15% apoptotic cells). The number of apoptotic cells was significantly decreased in calbindin-D(28k)-overexpressing betaTC-3 cells incubated with cytokines (5-6% apoptotic cells). To address the mechanism of the antiapoptotic effects of calbindin, studies were done to examine whether calbindin inhibits free radical formation. The stimulatory effects of the cytokines on lipid hydroperoxide, nitric oxide, and peroxynitrite production were significantly decreased in the calbindin-D(28k)-expressing betaTC-3 cells. Our findings indicate that calbindin-D(28k), by inhibiting free radical formation, can protect against cytokine-mediated apoptosis and destruction of beta-cells. These findings suggest that calbindin-D(28k) may be an important regulator of cell death that can protect pancreatic islet beta-cells from autoimmune destruction in type 1 diabetes.

Interferon-gamma induces interleukin-1 converting enzyme expression in pancreatic islets by an interferon regulatory factor-1-dependent mechanism.

Whereas nitric oxide (NO) production is associated with the toxic effect of cytokines on rodent pancreatic beta-cells, cytokine-induced apoptosis in human islets may occur independently of NO. The cysteine protease interleukin (IL)-1 converting enzyme (ICE) is a key proapoptotic caspase. Our aim was therefore to analyze the effect of cytokines on ICE expression in human, rat, and mouse islets and rat insulinoma cells. ICE messenger RNA (mRNA) expression was highly up-regulated after 6-, 24-, and 72-h exposure of human islets to interferon (IFN)gamma, tumor necrosis factor (TNF)alpha + IFNgamma or IL-1beta + TNFalpha + IFNgamma, paralleled by increased iNOS (the inducible form of NO synthase) expression and NO production after exposure to the combined cytokines but not to IFNgamma or TNFalpha + IFNgamma. Cytokine-induced NO-independent ICE transcription was confirmed using iNOS inhibitors. Exposure of rat and mouse islets, or rat insulinoma cells, for 24 h to IFNgamma alone or in combination with the two other cytokines also resulted in a highly significant ICE mRNA expression. ICE transcription was not inducible in islets from IFN regulatory factor-1 knock-out mice, suggesting a key-role of this transcription-factor in cytokine-mediated ICE expression in pancreatic islets. In conclusion, cytokines and IFNgamma in particular increase ICE mRNA expression in pancreatic islet cells and beta-cell lines, independently of NO synthesis, suggesting that ICE up-regulation may be involved in cytokine-induced NO-independent apoptosis of human islets.

In vivo prevention of hyperglycemia also prevents glucotoxic effects on PDX-1 and insulin gene expression.

Chronic exposure of pancreatic islet beta-cell lines to supraphysiologic glucose concentrations causes defects in insulin gene expression and insulin secretion. To determine whether these in vitro phenomena have pathophysiologic relevance in vivo, we studied the Zucker diabetic fatty (ZDF) rat, an animal model of type 2 diabetes. The ZDF animals had relatively higher levels of glycemia and islet insulin mRNA at 6 weeks of age than age-matched Zucker lean control (ZLC) rats. As glycemia increased in 12- and 16-week-old ZDF rats, we observed decrements in glucose-induced insulin secretion during static incubations of pancreatic islets and in insulin mRNA levels, PDX-1 mRNA levels, and PDX-1 protein binding to the insulin promoter compared with age-matched ZLC rats and 6-week-old ZDF rats. To determine whether normalization of blood glucose levels would prevent these defects, ZDF rats were treated with troglitazone beginning at 6 weeks of age. Troglitazone prevented ZDF rats from becoming hyperglycemic and preserved glucose-induced insulin responses. Furthermore, troglitazone-treated ZDF animals had greater levels of insulin and PDX-1 mRNAs compared with untreated ZDF animals of the same ages at 12 and 16 weeks. Our results demonstrate that chronic and progressive hyperglycemia resulting from type 2 diabetes in ZDF rats is associated with loss of insulin and PDX-1 mRNAs and loss of glucose-stimulated insulin secretion. Prevention of hyperglycemia prevented the associated defects in insulin and PDX-1 gene expression and improved insulin secretion. These findings provide the first in vivo evidence that prevention of progressive hyperglycemia in a model of type 2 diabetes preserves insulin and PDX-1 gene expression.

Modulation and detection of IDDM by membrane associated antigens from the islet beta cell line NIT-1.

We have utilized the NOD islet beta-cell line NIT-1 to monitor beta-cell specific autoantibodies and to investigate the modulation of IDDM in NOD mice by NIT-1 membrane associated antigens. The sera from diabetic but not from pre-diabetic or protected NOD mice strongly stained NIT-1 cells in FACS analysis. The cell surface antigens on NIT-1 cells were trypsin-sensitive. NIT-1 cells could not be stained by anti-mouse GAD67 antibody; however, we could demonstrate the presence of GAD65 and GAD67 mRNA by RT-PCR. Longitudinal analysis of anti-NIT-1 antibodies showed that these antibodies were present in the neonates but disappeared after weaning. Sonicated NIT-1 cell membrane preparations protected NOD mice from diabetes when injected intravenously in 5 week old mice. The protection was associated with reduced cytotoxic activity and elevated Th2-like responses as indicated by IgG1 antibodies against the NIT-1 cells. Subcutaneous injection of sonicated NIT-1 membranes or the injection of control red blood cell membranes failed to induce protection. We conclude that NIT-1 cell membranes do not express GAD but contain other antigens that are important in the development and prevention of IDDM. These antigens could be useful for the diagnosis of diabetes by monitoring autoantibody levels and for the modulation of IDDM by immunotherapy.