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Background Hypoxia can occur in pancreatic islets in type 2 diabetes mellitus. Pancreatic stellate cells (PSCs) are activated during hypoxia. Here we aimed to investigate whether PSCs within the islet are also activated in hypoxia, causing β-cell injury. Methods Islet and primary PSCs were isolated from Sprague Dawley rats, and cultured in normoxia (21% O2) or hypoxia (1% O2). The expression of α-smooth muscle actin (α-SMA), as measured by immunostaining and Western blotting, was used as a marker of PSC activation. Conditioned media (hypoxia-CM) were obtained from PSCs cultured in hypoxia. Results Islets and PSCs cultured in hypoxia exhibited higher expressions of α-SMA than did those cultured in normoxia. Hypoxia increased the production of reactive oxygen species. The addition of N-acetyl-L-cysteine, an antioxidant, attenuated the hypoxia-induced PSC activation in islets and PSCs. Islets cultured in hypoxia-CM showed a decrease in cell viability and an increase in apoptosis. Conclusion PSCs within the islet are activated in hypoxia through oxidative stress and promote islet cell death, suggesting that hypoxia-induced PSC activation may contribute to β-cell loss in type 2 diabetes mellitus.

Many stresses induce the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum, a phenomenon known as ER stress. In response to ER stress, cells initiate a protective response, known as unfolded protein response (UPR), to maintain cellular homeostasis. The UPR sensor, inositol-requiring enzyme 1 (IRE1), catalyzes the cytoplasmic splicing of bZIP transcription factor-encoding mRNAs to activate the UPR signaling pathway. Recently, we reported that pretreatment of Arabidopsis thaliana plants with tunicamycin, an ER stress inducer, increased their susceptibility to bacterial pathogens; on the other hand, IRE1 deficient mutants were susceptible to Pseudomonas syringae pv. maculicola (Psm) and failed to induce salicylic acid (SA)-mediated systemic acquired resistance. However, the functional relationship of IRE1 with the pathogen and TM treatment remains unknown. In the present study, we showed that bacterial pathogen-associated molecular patterns (PAMPs) induced IRE1 expression; however, PAMP-triggered immunity (PTI) response such as callose deposition, PR1 protein accumulation, or Pst DC3000 hrcC growth was not altered in ire1 mutants. We observed that IRE1 enhanced plant immunity against the bacterial pathogen P. syringae pv. tomato DC3000 (Pst DC3000) under ER stress. Moreover, TM-pretreated ire1 mutants were more susceptible to the avirulent strain Pst DC3000 (AvrRpt2) and showed greater cell death than wild-type plants during effector-triggered immunity (ETI). Additionally, Pst DC3000 (AvrRpt2)-mediated RIN4 degradation was reduced in ire1 mutants under TM-induced ER stress. Collectively, our results reveal that IRE1 plays a pivotal role in the immune signaling pathway to activate plant immunity against virulent and avirulent bacterial strains under ER stress.

Hexokinase1 (HXK1) is an Arabidopsis glucose sensor that has a variety of roles during plant growth and devlopment, including during germination, flowering, and senescence. HXK1 also acts as a positive regulator of plant immune responses. Previous research suggested that HXK1 might influence plant immune responses via responses to glucose. Plant immune responses are governed by two main pathways: PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI involves the recognition of Pathogen-Associated Molecular Patterns (PAMPs) and leads to increased callose formation and accumulation of pathogenesis response (PR) proteins. ETI acts in response to effectors secreted by Gram-negative bacteria. During ETI, the membrane-localized protein RPM1-interacting protein 4 (RIN4) becomes phosphorylated in reponse to interactions with effectors and mediates the downstream response. In this study, the effects of glucose on plant immune responses against infection with Pseudomonas syringae pv. tomato DC3000 and other P. syringae strains were investigated in the presence and absence of HXK1. Infiltration of leaves with glucose prior to infection led to decreases in bacterial populations and reductions in disease symptoms in wild-type Arabidopsis plants, indicating that glucose plays a role in plant immunity. Both PTI and ETI responses were affected. However, these effects were not observed in a hxk1 mutant, indicating that the effects of glucose on plant immune responses were mediated by HXK1-related pathways.

LONG HYPOCOTYL5 (HY5) and STF1 (Soybean TGACG-motif binding Factor 1) are two related bZIP transcription factors that play a positive role in photomorphogenesis and hormonal signaling. In this study, we compared full length STF1 and truncated STF1 overexpression lines and found that the C-terminal 133 amino acids (194-306) possess all the HY5-like function in Arabidopsis. The STF1-DC1 mutant (1-306), with a 20 amino acid deletion at the carboxy terminus, failed to complement the hy5 mutant phenotype, which suggests an intact C-terminus is required for STF1 function. To understand the role of the C-terminal domain in photomorphogenesis we used a yeast two-hybrid screen to isolate proteins that bind to the STF1 C-terminus. We isolated three soybean cDNAs encoding the zinc-finger proteins GmSTO, GmSTH, and GmSTH2, which interact with STF1. These proteins belong to a family of B-box zinc finger proteins that include Arabidopsis SALT TOLERANCE (STO) and STO HOMOLOG (STH) and STH2, which play a role in light-dependent development and gene expression. The C-terminal 63 amino acids of STF1, containing a leucine zipper and the two N-terminal B-boxes, contains the domain involved in interactions between STF1 and GmSTO. In addition, we identified an interaction between soybean COP1 (GmCOP1) and GmSTO and GmSTH, as well as STF1, which strongly suggests the presence of a similar regulatory circuit for light signaling in soybean as in Arabidopsis. This study shows that photomorphogenic control requires complex molecular interactions among several different classes of transcription factors such as bZIP, B-box factors, and COP1, a ubiquitin ligase.

Various subtypes of enteroendocrine cells (EECs) are present in the gut epithelium. EECs and pancreatic β-cells share similar pathways of differentiation during embryonic development and after birth. In this study, similarities between EECs and β-cells were evaluated in detail. To obtain specific subtypes of EECs, cell sorting by flow cytometry was conducted from STC-1 cells (a heterogenous EEC line), and each single cell was cultured and passaged. Five EEC subtypes were established according to hormone expression, measured by quantitative RT-PCR and immunostaining: L, K, I, G and S cells expressing glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, cholecystokinin, gastrin and secretin, respectively. Each EEC subtype was found to express not only the corresponding gut hormone but also other gut hormones. Global microarray gene expression profiles revealed a higher similarity between each EEC subtype and MIN6 cells (a β-cell line) than between C2C12 cells (a myoblast cell line) and MIN6 cells, and all EEC subtypes were highly similar to each other. Genes for insulin secretion-related proteins were mostly enriched in EECs. However, gene expression of transcription factors crucial in mature β-cells, such as PDX1, MAFA and NKX6.1, were remarkably low in all EEC subtypes. Each EEC subtype showed variable methylation in three cytosine-guanosine dinucleotide sites in the insulin gene (Ins2) promoter, which were fully unmethylated in MIN6 cells. In conclusion, our data confirm that five EEC subtypes are closely related to β-cells, suggesting a potential target for cell-based therapy in type 1 diabetes.

Objectives To investigate whether the activation of pancreatic stellate cells (PSCs) leads to pancreatic β-cell dysfunction in type 2 diabetes mellitus (T2DM). Methods The pancreases of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of T2DM, and patient with T2DM were analyzed. And the in vitro and in vivo effects of pirfenidone, an antifibrotic agent, on PSC activation, islet fibrosis, and β-cells were studied. Results The extent of islet fibrosis and the percentage of activated PSCs, positive for α-smooth muscle actin, in the islets were significantly greater in OLETF rats compared with non-diabetic rats. Also, the extent of islet fibrosis in patients with T2DM was slightly greater compared with age- and BMI-matched non-diabetic patients. In rat PSCs cultured with high glucose for 72 h, pirfenidone produced decreases in cell proliferation, release of collagen, and the expression of fibronectin and connective tissue growth factor. Treatment of OLETF rats with pirfenidone for 16 weeks decreased the activation of PSCs and the extent of islet fibrosis, but did not enhance glucose tolerance, pancreatic insulin content, or β-cell mass. Conclusions Activated PSCs in islets might lead to islet fibrosis in T2DM. However, PSC activation itself might not contribute significantly to progressive β-cell failure in T2DM.

Glucagon-like peptide-1 (GLP-1) and its potent agonists have been widely studied in pancreatic islet β-cells. However, GLP-1 receptors are present in many extrapancreatic tissues including macrophages, and thus GLP-1 may have diverse actions in these tissues and cells. Therefore, we examined the mechanism by which exendin-4 (EX-4), a potent GLP-1 receptor agonist, inhibits lipopolysaccharide (LPS)-induced iNOS expression in Raw264.7 macrophage cells. EX-4 significantly inhibited LPS-induced iNOS protein expression and nitrite production. However, Northern blot and promoter analyses demonstrated that EX-4 did not inhibit LPS-induced iNOS mRNA expression and iNOS promoter activity. Electrophoretic mobility shift assay (EMSA) showed that EX-4 did not alter the binding activity of NF-κB to the iNOS promoter. Consistent with the result of EMSA, LPS-induced IκBα phosphorylation and nuclear translocation of p65 were not inhibited by EX-4. Also, actinomycin D chase study and the promoter assay using the construct containing 3'-untranslated region of iNOS showed that EX-4 did not affect iNOS mRNA stability. Meanwhile, cycloheximide chase study demonstrated that EX-4 significantly accelerated iNOS protein degradation. The EX-4 inhibition of LPS-induced iNOS protein was significantly reversed by adenylate cyclase inhibitors (MDL-12330A and SQ 22536), a PKA inhibitor (H-89) and PKAα gene silencing. These findings suggest that EX-4 inhibited LPS-induced iNOS expression at protein level, but not at transcriptional mechanism of iNOS gene and this inhibitory effect of EX-4 was mainly dependent on cAMP/PKA system.

Islet fibrosis could be important in the progression of pancreatic beta cell failure in type 2 diabetes. It is known that oxidative stress is involved in the pancreatic fibrosis through the activation of pancreatic stellate cells. However, no study has investigated the in vivo effects of antioxidants on islet fibrogenesis in type 2 diabetes. In this study, antioxidants (taurine or tempol) were administered in drinking water to Otsuka Long-Evans Tokushima Fatty rats, an animal model of type 2 diabetes, for 16 weeks. An intraperitoneal glucose tolerance test revealed that the blood glucose levels after the glucose injection were decreased by the antioxidants. The insulin secretion after the glucose injection, which was markedly reduced in the rats, was also restored by the antioxidants. Beta cell mass and pancreatic insulin content were greater in the rats treated with the antioxidants than in the untreated rats. Beta cell apoptosis was attenuated in the rats by the antioxidants. Finally, islet fibrosis and the activation of pancreatic stellate cells were markedly diminished in the rats by the antioxidants. Our data suggest that antioxidants may protect beta cells through the attenuation of both islet fibrosis and beta cell apoptosis in type 2 diabetes.

Background: Type 2 diabetes mellitus (T2DM) is often accompanied by increased levels of circulating fatty acid. Elevations in fatty acids and glucose for prolonged periods of time have been suggested to cause progressive dysfunction or apoptosis of pancreatic beta cells in T2DM. However, the precise mechanism of this adverse effect is not well understood. Methods: INS-1 rat-derived insulin-secreting cells were exposed to 30 mM glucose and 0.25 mM palmitate for 48 hours. Results: The production of reactive oxygen species increased significantly. Pancreatic and duodenal homeobox 1 (Pdx1) expres sion was down-regulated, as assessed by reverse transcription-polymerase chain reaction and Western blot analyses. The promoter activities of insulin and Pdx1 were also diminished. Of note, there was nucleocytoplasmic translocation of Pdx1, which was partially prevented by treatment with an antioxidant, N-acetyl-L-cysteine. Conclusion: Our data suggest that prolonged exposure of beta cells to elevated levels of glucose and palmitate negatively affects Pdx1 expression via oxidative stress.

Background Islet transplantation is a potential strategy to cure type 1 diabetes mellitus. However, a substantial part of the islet graft becomes nonfunctional due to several factors including hypoxia. However, the precise mechanism of cell damage is largely unknown in hypoxic exposure to pancreatic beta cells. The aim of the present study was to investigate whether acute and severe hypoxic injury could involve inducible nitric oxide synthase (iNOS)-nitric oxide (NO) signaling in beta cells. Methods The rat beta cell line (INS-1) and primary rat islets were incubated in an anoxic chamber. Cell viability was determined by propium iodide staining or cell counting kit. The expression of iNOS mRNA and protein was examined using reverse-transcription polymerase chain reaction and Western blot analysis. NO production was measured as nitrite accumulation by Griess reagent method. Results After hypoxic exposure, marked cell death occurred in INS-1 cells and rat islets, accompanied by increase in activated caspase-3 expression. NO production was increased in the culture medium in a time-dependent manner. Increase in expression of iNOS mRNA and protein was found. Pretreatment with a selective iNOS inhibitor, 1400W, significantly prevented cell death during hypoxia. In addition, hypoxia activated c-Jun N-terminal kinase (JNK) significantly, but the addition of 1400W inhibited hypoxia-induced JNK phosphorylation. Conclusions Our data suggest that iNOS-NO plays an important role in acute and severe hypoxic injury to pancreatic beta cells. Therefore, iNOS-NO might be a potential therapeutic target for preserving beta cell survival in islet transplantation through prevention of hypoxia-mediated cell death.

Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone and is a new clinically available class of agents for improving of insulin resistance in both animals and humans with type 2 diabetes. These studies aimed to determine whether long-term treatment with a long-acting GLP-1 analog, exendin-4, delayed the progression of diabetes. Male db/db mice and db/m mice at 8 wk of age were treated with exendin-4 for 8 wk, whereas the control db/db mice received only vehicle. Urinary albumin excretion was significantly decreased in db/db mice that were treated with 1 nmol/kg exendin-4 compared with those in db/db mice that were treated with 0.5 nmol/kg exendin-4 and control db/db mice (P < 0.005). Intraperitoneal glucose tolerance test was improved in db/db mice that were treated with 1 nmol/kg exendin-4 compared with other groups (P < 0.05). Despite this, fasting blood glucose, glycated hemoglobin, and creatinine concentrations were not significantly different among db/db mice. Renal histology studies further demonstrated that glomerular hypertrophy, mesangial matrix expansion, TGF-beta1 expression, and type IV collagen accumulation and associated glomerular lipid accumulation were significantly decreased in db/db mice that were treated with 1 nmol/kg exendin-4. Furthermore, there were fewer infiltrating inflammatory cells and apoptotic cells in the glomeruli of db/db mice that were treated with 1 nmol/kg exendin-4 compared with those in the other groups accompanied by an increase in the renal immunoreactivity of peroxisome proliferator-activated receptor alpha and GLP-1 receptor-positive cells and a decrease in 24-h urinary 8-hydroxy-deoxyguanosine levels (P < 0.01, respectively) along with decreases in lipid content. Taken together, exendin-4 treatment seems to ameliorate diabetic nephropathy together with improvement of the metabolic anomalies. These results suggest that exendin-4 could provide a therapeutic role in diabetic nephropathy that results from type 2 diabetes.

Cytokines that are released by infiltrating inflammatory cells around the pancreatic islets are involved in the pathogenesis of type 1 diabetes mellitus. Specifically, interleukin-1beta (IL-1beta) stimulates inducible nitric oxide synthase (iNOS) expression and nitric oxide overproduction, leading to the beta-cell damage. In activating this pathway, nuclear factor-kappaB (NF-kappaB) plays a crucial role, and many of the IL-1beta-sensitive genes contain NF-kappaB binding sites in their promoter regions. We have recently shown that epicatechin, which is a flavonoid, had a protective effect on pancreatic beta-cells in both streptozotocin-treated rats and islets. In the present study, the effects of epicatechin on IL-1beta-induced beta-cell damage were examined. RINm5F cells and islets were pretreated with epicatechin and next incubated with IL-1beta. The released nitrite, iNOS protein and mRNA expression levels were then measured. IkappaBalpha protein, nuclear translocation of NF-kappaB, and NF-kappaB DNA binding activity were also determined. Following the transient transfection of an iNOS promoter into the cells, the iNOS promoter activity was measured. ATP- or D-glucose-induced insulin release was measured in RINm5F cells and islets, respectively. Epicatechin significantly reduced IL-1beta-induced nitrite production, iNOS protein and mRNA expressions, and it also inhibited IL-1beta-induced IkappaBalpha protein degradation, NF-kappaB activation, and iNOS promoter activity. Epicatechin partly restored the IL-1beta-induced inhibition of insulin release. These results suggest that epicatechin inhibits the IL-1beta-induced iNOS expression by down-regulating NF-kappaB activation, and protecting beta-cells from IL-1beta.

Introduction Green tea catechins have diverse pharmacological effects such as anticarcinogenic and antioxidant activities. Aim To study the protective effects of green tea (-)-epicatechin (EC) against the toxic effects of streptozotocin (STZ), a selective beta cell toxin, on pancreatic islets in vivo and in vitro. Methodology Rats were randomly divided into four groups: control, EC (30 mg/kg)-treated, STZ (60 mg/kg)-treated, and EC plus STZ (same doses; EC+STZ)-treated rats. EC was administered twice a day for 6 days, and a single injection of STZ was used. In EC+STZ-treated rats, EC was administered 6 hours prior to STZ since posttreatment with EC had no beneficial effects on fully developed diabetes in our unpublished study. Insulin and insulin mRNA were detected by immunohistochemical analysis and in situ hybridization, respectively, and physiologic parameters including blood glucose concentration were measured daily. Following isolation of the islets, insulin release, nitrite levels, and islet morphology were observed in the four groups: control, EC (0.8 mM)-treated, STZ (5 mM)-treated, and EC+STZ (same doses)-treated islets. Results In EC+STZ-treated rats, hyperglycemia and weight loss were not observed and islet morphology was well preserved compared with STZ-treated rats. Compared with STZ treatment alone, insulin release was increased and nitrite production was decreased in EC+STZ-treated islets. Conclusion EC appears to be helpful in protecting pancreatic islets against exposure to STZ in both in vivo and in vitro systems.

Phospholipase D (PLD), one of the signal transducing enzymes, is recognized to play a role in the pancreatic exocrine and endocrine secretion, and yet the distribution of PLD in the pancreas has remained unclarified. We investigated the expression and localization of PLD isoforms, PLD1 and PLD2, in the rat pancreas. Western blot analysis showed that PLD1-immunoreactive band was observed in the islets, but not in the acinar cells. In contrast, PLD2-immunoreactive band was observed in both the islets and the acinar cells. In the immunohistochemistry, PLD1 was evenly distributed throughout the islets while PLD2 immunoreactivity was more intense in the periphery than in the central portion of the islets. To further elucidate the cell-specific localization of PLD2 in the islets, double immunofluorescent staining was performed. PLD2 was mainly localized in A and PP cells, which secrete glucagon and pancreatic polypeptide, respectively. PLD2 was also detected in acinar cells, ductal epithelial cells and intrapancreatic nerve fibers. Meanwhile, PLD1 was detected in ductal epithelial cells and intrapancreatic ganglia, but not in acinar cells. On the basis of this heterogeneity in the distribution of PLD isoforms, it is suggested that PLD might play a specific role in the pancreatic exocrine and endocrine function.

Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic β-cells in a process called cell reprogramming. Enteroendocrine cells and β-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in β-cells. Thus, K cells could be reprogrammed to β-cells under certain conditions. However, there is no clear evidence on whether these cells convert to β-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1(+)-K cells) was established. Induction of Neurogenin3 expression in Nkx6.1(+)-K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1(+)-K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture.

The activation of pancreatic stellate cells (PSCs) is thought to be a potential mechanism underlying islet fibrosis, which may contribute to progressive β-cell failure in type 2 diabetes. Recently, we demonstrated that antioxidants reduced islet fibrosis in an animal model of type 2 diabetes. However, there is no in vitro study demonstrating that high glucose itself can induce oxidative stress in PSCs. Thus, PSCs were isolated and cultured from Sprague Dawley rats, and treated with high glucose for 72 h. High glucose increased the production of reactive oxygen species. When treated with high glucose, freshly isolated PSCs exhibited myofibroblastic transformation. During early culture (passage 1), PSCs treated with high glucose contained an increased number of α-smooth muscle actin-positive cells. During late culture (passages 2-5), PSCs treated with high glucose exhibited increases in cell proliferation, the expression of fibronectin and connective tissue growth factor, release of interleukin-6, transforming growth factor-β and collagen, and cell migration. Finally, the treatment of PSCs with high glucose and antioxidants attenuated these changes. In conclusion, we demonstrated that high glucose increased oxidative stress in primary rat PSCs, thereby facilitating the activation of these cells, while antioxidant treatment attenuated high glucose-induced PSC activation.

High doses of glucosamine have been known to induce apoptosis of pancreatic beta cells. The mechanism for this phenomenon has not been clearly elucidated. We aimed to explore the potential mechanisms for glucosamine toxicity in the rat insulinoma cell line INS-1 and in rat native beta cells. We also investigated whether glucagon-like peptide (GLP)-1 could be protective against glucosamine. Glucosamine exhibited dose-dependent inhibition of cell survival and an increase in the cell population at the sub-G1 phase. Glucosamine was revealed to inhibit cellular glucose uptake, resulting in the activation of AMP-activated protein kinase (AMPK). Accordingly, phosphorylation of P70S6K and ribosomal protein S6 (S6RP) was decreased. Protein glycosylation appeared not to be involved in this cytotoxicity. Pretreatment with GLP-1 alleviated glucosamine-mediated inhibition of glucose uptake and lessened AMPK activation, thus allowing recovery of the phosphorylation levels of P70S6K and S6RP. The effect of GLP-1 was blocked by the adenylyl cyclase inhibitor MDL12330A but not by the protein kinase A inhibitor H89. Taken together, these data demonstrate that glucosamine may inhibit beta-cell survival by diminishing cellular glucose uptake independent of glycosylation. This glucosamine toxicity can be blocked by GLP-1, which leads to recovery of the glucose uptake through a PKA-independent, cAMP-dependent mechanism.

In islet transplantation, a substantial part of the graft becomes nonfunctional for several reasons including hypoxia. AMP-activated protein kinase (AMPK) in mammalian cells is a regulator of energy homeostasis, and is activated by metabolic stresses such as hypoxia. However, the role of AMPK in hypoxic injury to pancreatic beta cells is not clear. When a rat beta cell line, INS-1 cell, was incubated in an anoxic chamber, phosphorylation of both AMPK and its downstream protein, acetyl-CoA carboxylase 2 increased with time. Adenovirus-mediated expression of constitutively active form of AMPK under normoxic conditions increased caspase-3 activation, suggesting induction of apoptosis. Reactive oxygen species production also increased with time during hypoxia. Pretreatment with compound C, an AMPK inhibitor, or N-acetyl-l-cysteine, an antioxidant, significantly lowered hypoxia-mediated cell death. These results suggest that AMPK, in association with oxidative stress, plays an important role in acute and severe hypoxic injury to pancreatic beta cells.

Cytokines such as interleukin-1beta (IL-1beta) stimulate inducible nitric oxide synthase (iNOS) expression and nitric oxide overproduction leading to beta-cell damage. Meanwhile, glucagon-like peptide-1 (GLP-1) and its potent analog exendin-4 (EX-4) were well known for beta-cell proliferation. However, the protective mechanisms of GLP-1 in beta-cells exposed to cytokines were not fully elucidated. Therefore, the effects of EX-4 on the IL-1beta-induced iNOS gene expression were investigated employing RINm5F beta-cells. EX-4 inhibited IL-1beta-induced iNOS protein expression and nitrite production. However, northern blot and promoter analyses showed that EX-4 failed to inhibit IL-1beta-induced iNOS mRNA expression and iNOS promoter activity. By electrophoretic mobility shift assay (EMSA), EX-4 did not alter the binding activity of NF-kappaB to the iNOS promoter. Consistent with the EMSA result, EX-4 did not inhibit nuclear translocation of p65. We also tested the effect of EX-4 on iNOS mRNA stability. Actinomycin D chase experiments showed that EX-4 did not affect the decay rate of iNOS mRNA and the promoter assay using the construct containing 3'-untranslated region of iNOS showed that EX-4 did not alter the stability of iNOS mRNA. Meanwhile, forskolin significantly inhibited IL-1beta-induced iNOS protein, which was reversed by H-89, a protein kinase A (PKA) inhibitor. Moreover, EX-4 pretreatment restored IL-1beta-induced decrease in cAMP toward control level. Additionally, the cycloheximide chase study demonstrated that EX-4 significantly accelerated iNOS protein degradation. We therefore concluded that EX-4 inhibited IL-1beta-induced iNOS protein and nitrite production via cAMP/PKA system irrespective of both transcriptional and posttranscriptional mechanisms of iNOS gene, and this inhibitory effect of EX-4 appears to be regulated at posttranslational level.

Glucagon-like peptide-1 (GLP-1) induces several immediate early response genes such as c-fos, c-jun, and early growth response-1 (Egr-1), which are involved in cell proliferation and differentiation. We recently reported that exendin-4 (EX-4), a potent GLP-1 agonist, upregulated Egr-1 expression via phosphorylation of CREB, a transcription factor in INS-1 β-cells. This study was designed to investigate the role of another transcription factors, serum response factor (SRF) and Yin Yang-1 (YY1), in EX-4-induced Egr-1 expression. EX-4 significantly increased Egr-1 mRNA and subsequently its protein level. EX-4-induced Egr-1 expression was inhibited by pretreatment with a PKA inhibitor, H-89, and an MEK inhibitor, PD 98059. The siRNA-mediated inhibition of PKA and ERK1 resulted in significant reduction of EX-4-induced Egr-1 expression. Promoter analyses showed that SRE clusters were essential for Egr-1 transcription, and YY1 overexpression did not affect Egr-1 promoter activity. EMSA results demonstrated that EX-4-induced transient increase in DNA–protein complex on SRE site, and that both SRF and phospho-SRF were bound to this site. Treatment of either YY1 consensus oligonucleotide or YY1 antibody did not effect the change of density or migration of the DNA–protein complex. Collectively, EX-4-induced Egr-1 expression is largely dependent on cAMP-mediated extracellular signal-regulated kinase activation, and EX-4 induces Egr-1 transcription via the interaction of SRF and phospho-SRF to SRE sites. J. Cell. Biochem. 104: 2261–2271, 2008. © 2008 Wiley-Liss, Inc.

Objectives Pancreatic acini of diabetic rats release amylase less than normal acini on cholecystokinin (CCK) stimulation. Pancreatic enzyme secretion by CCK is closely related to the second messenger inositol 1,4,5-trisphosphate (IP3), which mobilizes intracellular calcium stores via the endoplasmic reticulum-located receptor IP3 (IP3R). Recently, we observed altered intracellular calcium response on CCK-8 stimulation in streptozotocin (STZ)- treated diabetic rat acini. Methods To determine whether IP3R is involved in altered calcium response, we measured inositol phosphate (IP) formation and the expression and phosphorylation of type III IP3R protein in diabetic acini. Also, CCK receptor mRNA expression was examined to determine whether the changes in IP formation and IP3R protein phosphorylation in diabetic acini might result from the defect at the postreceptor level. Results CCK-8-induced IP formation at all concentrations used was significantly reduced in diabetic acini, though IP formation was increased in a concentration-dependent manner. The expression of type III IP3R protein was significantly reduced in diabetic acini. Additionally, CCK-8-stimulated phosphorylation of type III IP3R protein was not observed in diabetic acini. However, the reduction of CCK receptor mRNA expression was not detected in diabetic acini. Conclusion Our results indicate that altered calcium response to CCK-8 in diabetic acini might be associated with a post-CCK receptor defect including the changes in IP formation, type III IP3R protein expression, and phosphorylation of type III IP3R protein.

Pancreatic acini of streptozotocin (STZ)-induced diabetic rats release amylase less than normal acini on cholecystokinin (CCK) stimulation. Pancreatic enzyme secretion has been closely related to the intracellular calcium concentration ([Ca2(+)](i)) of the acinar cell. In the present study, sequential changes of the intracellular calcium signal which probably underlie the altered enzyme secretion in response to CCK-8 were investigated using pancreatic acini from diabetic rats.Diabetic rats were prepared by single intravenous injection of STZ (70 mg/kg). Stimulating experiments with CCK-8 were performed 7 days later. Pancreatic acini were isolated by collagenase digestion. Amylase release and [Ca2(+)](i) were measured by colorimethod and calcium imaging, respectively. The geometry of intracellular calcium signal was analyzed.Normal acini exhibited concentration-dependent [Ca2(+)](i) increase and regular oscillatory calcium signal on CCK-8 stimulation. Amylase release was also concentration-dependent. However, diabetic acini showed significantly less [Ca2(+)](i) increase, prolonged time to peak [Ca2(+)](i), decreased calcium spikes number, and decreased amylase release compared with normal acini. The decreased [Ca2(+)](i) in diabetic acini was restored significantly by insulin treatment.Relatively decreased amylase release in diabetic pancreatic acini in response to CCK, appears to be associated with altered calcium signal due to insulin deficiency.

Background Glucose toxicity that is caused by chronic exposure to a high glucose concentration leads to islet dysfunction and induces apoptosis in pancreatic β-cells. Heme oxygenase-1 (HO-1) has been identified as an anti-apoptotic and cytoprotective gene. The purpose of this study is to investigate whether HO-1 up-regulation when using metalloprotophyrin (cobalt protoporphyrin, CoPP) could protect pancreatic β-cells from high glucose-induced apoptosis. Methods Reverse transcription-polymerase chain reaction was performed to analyze the CoPP-induced mRNA expression of HO-1. Cell viability of INS-1 cells cultured in the presence of CoPP was examined by acridine orange/propidium iodide staining. The generation of intracellular reactive oxygen species (ROS) was measured using flow cytometry. Glucose stimulated insulin secretion (GSIS) was determined following incubation with CoPP in different glucose concentrations. Results CoPP increased HO-1 mRNA expression in both a dose- and time-dependent manner. Overexpression of HO-1 inhibited caspase-3, and the number of dead cells in the presence of CoPP was significantly decreased when exposed to high glucose conditions (HG). CoPP also decreased the generation of intracellular ROS by 50% during 72 hours of culture with HG. However, decreased GSIS was not recovered even in the presence of CoPP. Conclusion Our data suggest that CoPP-induced HO-1 up-regulation results in protection from high glucose-induced apoptosis in INS-1 cells; however, glucose stimulated insulin secretion is not restored.

Background: Despite a recent breakthough in human islet transplantation for treating type 1 diabetes mellitus, the limited availability of donor pancreases remains a major obstacle. Endocrine cells within the gut epithelium (enteroendocrine cells) and pancreatic β cells share similar pathways of differentiation during embryonic development. In particular, K-cells that secrete glucose-dependent insulinotropic polypeptide (GIP) have been shown to express many of the key proteins found in β cells. Therefore, we hypothesize that K-cells can be transdifferentiated into β cells because both cells have remarkable similarities in their embryonic development and cellular phenotypes. Methods: K-cells were purified from heterogeneous STC-1 cells originati ng from an endocrine tumor of a mouse intestine. In addition, a K-cell subclone expressing stable Nkx6.1, called “Kn4-cells,” was successfully obtained. In vitro differentiation of K-cells or Kn4-cells into β cells was completed after exendin-4 treatment and serum deprivation. The expressions of insulin mRNA and prot ein were examined by RT-PCR and immunocytochemistry. The interacellular insulin content was also measured. Results: K-cells were found to express glucokinase and GIP as assessed by RT-PCR and Western blot analysis. RT-PCR showed that K-cells also expressed Pdx-1, Neur oD1/Beta2, and MafA, but not Nkx6.1. After exendin-4 treatment and serum deprivation, insulin mRNA and insulin or C-peptide were clearly detected in Kn4-cells. The intracellular insulin content was al so increased significantly in these cells. Conclusion: K-cells are an attractive potential source of insulin-producing cells for treatment of type 1 diabetes mellitus. However, more experiments are necessary to optimize a strategy for converting K-cells into β cells. (Korean Diabetes J 33:475-484, 2009)

Background: Despite a recent breakthrough in human islet transplantation for treating diabetes mellitus, the limited availability of insulin-producing tissue is still a major obstacle. This has led to a search for alternative sources of transplantable insulin-producing cells including pancreatic duct cells. We aimed to establish in vitro culture of pancreatic duct cells from a partial pancreas tissue in human, which could be harnessed to differentiate into pancreatic β cells.Methods: We isolated pancreatic duct cells from small pieces of pancreas tissue (1~3 g) derived from non-diabetic humans (n = 8) undergoing pancreatic surgery due to cancer. Pancreas tissue was finely minced after injection of collagenase P into the parenchyma. The mince was incubated in a shaking water bath at 37℃ for 25 min and passed through a 150 μm mesh. The released cells were recovered, washed, and plated in a dish containing CMRL culture medium with serum.Results: Isolated pancreatic cells grew in monolayer and became confluent in 1~2 wks showing typical epithelial cobblestone morphology. Immunochemistry demonstrated that ~90% of the cultured cells were cytokeratin7-positive duct cells. To induce β cell differentiation, the cells were incubated in DMEM/F12 culture medium without serum. In addition, treatment with Matrigel overlay, exendin-4, cholera toxin or forskolin was done. Though β cell differentiation was found by immunostaining and RT-PCR, the differentiation efficiency was very low. Over-expression of neurogenin-3 by recombinant adenovirus did not increase β cell differentiation of the cultured duct cells significantly. Conclusion: We established in vitro culture of pancreatic duct cells from a partial pancreas tissue in human, which differentiate into pancreatic cells. However, a strategy to optimize β cell differentiation in this model is needed. (J Kor Diabetes Assoc 31:236~242, 2007)

Background: The incidence of atherosclerosis is well correlated with the progression of type 2 diabetes mellitus. High plasma glucose in uncontrolled diabetic patients evokes many vascular complications such as atherosclerosis. Specifically, high glucose was reported to induce thrombospondin-1 (TSP-1), which activates matrix metalloproteinase-2 (MMP-2) and leads to the invasion of vascular smooth muscle cells (VSMCs) into the intima. Catechins with antioxidant effects are known to inhibit MMP-2 activity. Therefore, this study was aimed at revealing the effect of epicatechin, one of catechins, on high glucose-induced TSP-1 and the invasiveness of VSMCs. Methods: VSMCs were primarily isolated from Sprague-Dawley rat aorta. The VSMCs were incubated with different doses (30, 100 and 300 μM) of epicatechin under high glucose concentration (30 mM). The TSP-1 protein and mRNA expressions were analyzed by performing Western blotting and Northern blot analyses, respectively. RT-PCR was performed to observe the MMP-2 mRNA expression. Gelatin zymography was performed for the measurement of MMP-2 activity. Invasion assays were performed to evaluate the invasiveness of VSMCs. Results: Epicatechin inhibited the high glucose-induced TSP-1 expression and the MMP-2 activity in a dose-dependent manner. Also, epicatechin inhibited the high glucose-induced invasiveness of VSMCs across the matrix barrier in a dose-dependent fashion. Conclusion: Collectively, epicatechin may prevent the high glucose-induced proliferation and invasion of VSMCs by inhibiting the TSP-1 expression and the MMP-2 activity. Therefore, epicatechin appears to play a protective role in the development of atherosclerosis. (J Kor Soc Endocrinol 21:302~310, 2006)