Insulinoma INS-1 Cells Research Articles

MEN1 tumorigenesis in the pituitary and pancreatic islet requires Cdk4 but not Cdk2.

Recent studies suggest that physiological and tumorigenic proliferation of mammalian cells is controlled by multiple cyclin-dependent kinases (CDKs) largely in tissue-specific manners. We and others previously demonstrated that adult mice deficient for the D-cyclin-dependent kinase CDK4 (Cdk4−/− mice) exhibit hypoplasia in the pituitary and pancreatic islet due to primary postnatal defects in proliferation. Intriguingly, those neuroendocrine tissues affected in Cdk4−/− mice are the primary targets of tumorigenesis in the syndrome of multiple endocrine neoplasia type-1 (MEN1). Mice with heterozygous disruption of the tumor suppressor Men1 gene (Men1+/−) develop tumors in the pituitary, pancreatic islets and other neuroendocrine tissues, which is analogous to humans with MEN1 mutations. To explore the genetic interactions between loss of Men1 and activation of CDKs, we examined the impact of Cdk4 or Cdk2 disruption on tumorigenesis in Men1+/− mice. A majority of Men1+/− mice with wild-type CDKs developed pituitary and islet tumors by 15 months of age. Strikingly, Men1+/−; Cdk4−/− mice did not develop any tumors, and their islets and pituitaries remained hypoplastic with decreased proliferation. In contrast, Men1+/−; Cdk2−/− mice showed pituitary and islet tumorigenesis comparable to those in Men1+/− mice. Pituitaries of Men1+/−; Cdk4−/− mice showed no signs of loss of heterozygosity (LOH) in the Men1 locus, while tumors in Men1+/− mice and Men1+/−; Cdk2−/− mice exhibited LOH. Consistently, CDK4 knockdown in INS-1 insulinoma cells inhibited glucose-stimulated cell cycle progression with a significant decrease in phosphorylation of retinoblastoma protein (RB) at specific sites including Ser780. CDK2 knockdown had minimum effects on RB phosphorylation and cell cycle progression. These data suggest that CDK4 is a critical downstream target of MEN1-dependent tumor suppression and is required for tumorigenic proliferation in the pituitary and pancreatic islet, whereas CDK2 is dispensable for tumorigenesis in these neuroendocrine cell types.

Novel AGLP-1 albumin fusion protein as a long-lasting agent for type 2 diabetes

Glucagon like peptide-1 (GLP-1) regulates glucose mediated-insulin secretion, nutrient accumulation, and β-cell growth. Despite the potential therapeutic usage for type 2 diabetes (T2D), GLP-1 has a short half-life in vivo (t1/2 ＜2 min). In an attempt to prolong half-life, GLP-1 fusion proteins were genetically engineered: GLP-1 human serum albumin fusion (GLP-1/HSA), AGLP-1/HSA which has an additional alanine at the N-terminus of GLP-1, and AGLP-1-L/HSA, in which a peptide linker is inserted between AGLP-1 and HSA. Recombinant fusion proteins secreted from the Chinese Hamster Ovary-K1 (CHO-K1) cell line were purified with high purity (＞96%). AGLP-1 fusion protein was resistant against the dipeptidyl peptidase-IV (DPP-IV). The fusion proteins activated cAMP-mediated signaling in rat insulinoma INS-1 cells. Furthermore, a C57BL/6N mice pharmacodynamics study exhibited that AGLP-1-L/HSA effectively reduced blood glucose level compared to AGLP-1/HSA. [BMB Reports 2013; 46(12): 606-610]

Novel insights into pancreatic β-cell glucolipotoxicity from real-time functional analysis of mitochondrial energy metabolism in INS-1E insulinoma cells

High circulating glucose and non-esterified (free) fatty acid levels can cause pancreatic β-cell failure. The molecular mechanisms of this β-cell glucolipotoxicity are yet to be established conclusively. In the present paper we report on the involvement of mitochondrial dysfunction in fatty-acid-induced β-cell failure. We have used state-of-the-art extracellular flux technology to functionally probe mitochondrial energy metabolism in intact INS-1E insulinoma cells in real-time. We show that 24-h palmitate exposure at high glucose attenuates the glucose-sensitivity of mitochondrial respiration and lowers coupling efficiency of glucose-stimulated oxidative phosphorylation. These mitochondrial defects coincide with an increased level of ROS (reactive oxygen species), impaired GSIS (glucose-stimulated insulin secretion) and decreased cell viability. Palmitate lowers absolute glucose-stimulated respiration coupled to ATP synthesis, but does not affect mitochondrial proton leak. Palmitate is not toxic when administered at low glucose unless fatty acid β-oxidation is inhibited. Palmitoleate, on the other hand, does not affect mitochondrial respiration, ROS levels, GSIS or cell viability. Although palmitoleate protects against the palmitate-induced ROS increase and cell viability loss, it does not protect against respiratory and insulin secretory defects. We conclude that mitochondrial dysfunction contributes to fatty-acid-induced GSIS impairment, and that glucolipotoxic cell viability and GSIS phenotypes are mechanistically distinct.

Extracts from Leonurus sibiricus L. increase insulin secretion and proliferation of rat INS-1E insulinoma cells

Ethnopharmacological relevanceTraditional Mongolian medicine (TMM) uses preparations from herbs as one form of medication for the treatment of a diversity of diseases including diabetes mellitus (DM). We evaluated the effect of extracts from the plant Leonurus sibiricus L. (LS), used in TMM to treat typical symptoms of type 2 DM, on insulin secretion, electrophysiological properties, intracellular calcium concentration and cell proliferation of INS-1E insulinoma cells under standard cell culture conditions (SCC; 11.1mM glucose). Materials and methodsInsulin secretion was measured by ELISA, electrical properties were assessed by whole cell patch clamping, intracellular calcium concentration (Cai) by Fluo-4 time lapse imaging, insulin receptor expression was verified by RT-PCR and cell proliferation assessed by CellTiter-Glo® cell viability assay. ResultsInsulin released from INS-1E cells into the culture medium over 24h was significantly increased in presence of 500mg/L aqueous LS extract (LS OWE) as well as methanolic LS extract (LS MeOH/H2O) but not in the presence of the butanol-soluble extract (LS MeOH/BuOH). Acute application of LS OWE resulted in a depolarization of the cell membrane potential paralleled by an initial increase and subsequent decline and silencing of action potential frequency, by KATP channel inhibition, persisting depolarization and an increase in Cai. The electrophysiological effects were comparable to those of 100μM tolbutamide, which, however failed to elevate insulin secretion under SCC. Furthermore all LS extracts stimulated INS-1E cell proliferation. ConclusionsThe finding that extracts from Leonurus sibiricus L. enhance insulin secretion and/or foster cell proliferation may provide possible explanations for the underlying therapeutic principles in the empirical use of LS-containing formulations in DM and DM-related disorders as applied in TMM.

Mitochondrial phosphate transport during nutrient stimulation of INS-1E insulinoma cells

Here, we have investigated the role of inorganic phosphate (Pi) transport in mitochondria of rat clonal β-cells. In α-toxin-permeabilized INS-1E cells, succinate and glycerol-3-phosphate increased mitochondrial ATP release which depends on exogenous ADP and Pi. In the presence of substrates, addition of Pi caused mitochondrial matrix acidification and hyperpolarisation which promoted ATP export. Dissipation of the mitochondrial pH gradient or pharmacological inhibition of Pi transport blocked the effects of Pi on electrochemical gradient and ATP export. Knock-down of the phosphate transporter PiC, however, neither prevented Pi-induced mitochondrial activation nor glucose-induced insulin secretion. Using 31P NMR we observed reduction of Pi pools during nutrient stimulation of INS-1E cells. Interestingly, Pi loss was less pronounced in mitochondria than in the cytosol. We conclude that matrix alkalinisation is necessary to maintain a mitochondrial Pi pool, at levels sufficient to stimulate energy metabolism in insulin-secreting cells beyond its role as a substrate for ATP synthesis.

In vitro and mouse in vivo characterization of the potent free fatty acid 1 receptor agonist TUG-469

Activation of the G protein-coupled free fatty acid receptor 1 (FFA1; formerly known as GPR40) leads to an enhancement of glucose-stimulated insulin secretion from pancreatic β-cells. TUG-469 has previously been reported as a potent FFA1 agonist. This study was performed to confirm the higher in vitro potency of TUG-469 compared to the reference FFA1 agonist GW9508 and to prove in vivo activity in a pre-diabetic mouse model. The in vitro pharmacology of TUG-469 was studied using Ca(2+)-, cAMP-, and impedance-based assays at recombinant FFA1 and free fatty acid receptor 4, formerly known as GPR120 (FFA4) expressing 1321N1 cells and the rat insulinoma cell line INS-1. Furthermore, we investigated the systemic effect of TUG-469 on glucose tolerance in pre-diabetic New Zealand obese (NZO) mice performing a glucose tolerance test after intraperitoneal administration of 5 mg/kg TUG-469. In comparison to GW9508, TUG-469 showed a 1.7- to 3.0-times higher potency in vitro at 1321N1 cells recombinantly expressing FFA1. Both compounds increased insulin secretion from rat insulinoma INS-1 cells. TUG-469 is > 200-fold selective for FFA1 over FFA4. Finally, a single dose of 5 mg/kg TUG-469 significantly improved glucose tolerance in pre-diabetic NZO mice. TUG-469 turned out as a promising candidate for further drug development of FFA1 agonists for treatment of type 2 diabetes mellitus.

Tcf19 is a novel islet factor necessary for proliferation and survival in the INS-1 β-cell line

Recently, a novel type 1 diabetes association locus was identified at human chromosome 6p31.3, and transcription factor 19 (TCF19) is a likely causal gene. Little is known about Tcf19, and we now show that it plays a role in both proliferation and apoptosis in insulinoma cells. Tcf19 is expressed in mouse and human islets, with increasing mRNA expression in nondiabetic obesity. The expression of Tcf19 is correlated with β-cell mass expansion, suggesting that it may be a transcriptional regulator of β-cell mass. Increasing proliferation and decreasing apoptotic cell death are two strategies to increase pancreatic β-cell mass and prevent or delay diabetes. siRNA-mediated knockdown of Tcf19 in the INS-1 insulinoma cell line, a β-cell model, results in a decrease in proliferation and an increase in apoptosis. There was a significant reduction in the expression of numerous cell cycle genes from the late G1 phase through the M phase, and cells were arrested at the G1/S checkpoint. We also observed increased apoptosis and susceptibility to endoplasmic reticulum (ER) stress after Tcf19 knockdown. There was a reduction in expression of genes important for the maintenance of ER homeostasis (Bip, p58IPK, Edem1, and calreticulin) and an increase in proapoptotic genes (Bim, Bid, Nix, Gadd34, and Pdia2). Therefore, Tcf19 is necessary for both proliferation and survival and is a novel regulator of these pathways.

Melatonin‐mediated insulin synthesis during endoplasmic reticulum stress involves HuD expression in rat insulinoma INS‐1E cells

In this study, we investigated how melatonin mediates insulin synthesis through endoplasmic reticulum (ER) via HuD expression in rat insulinoma INS-1E cells. Under ER stress condition (thapsigargin with/without melatonin, tunicamycin with/without melatonin), phosphorylation of AMP-activated protein kinase (p-AMPK) was significantly increased when compared with only with/without melatonin (control/melatonin). Insulin receptor substrate (IRS) two protein was significantly reduced under conditions of ER stress when compared with control/melatonin, but no expression of IRS1 protein was observed. In thapsigargin treatment, melatonin (10, 50μm) increased IRS2 protein expression in a dose-dependent manner. p-Akt (Ser473) expression significantly decreased under ER stress condition prior to control/melatonin. Melatonin (10, 50μm) significantly reduced nuclear and cellular p85α expressions in a dose-dependent manner when compared with only thapsigargin or tunicamycin. These results indicate the activation of the aforementioned expressions under regulation of the pathway, AMPK → IRS2 → Akt/PKB → PI3K (p85α). However, mammalian target of rapamycin and raptor protein, mTORC1, was found to be independent of the ER stress response. In thapsigargin treatment, melatonin increased nuclear mammalian RNA-binding protein (HuD) expression and reduced cellular HuD expression and subsequently resulted in a decrease in cellular insulin level and rise in insulin secretion in a dose-dependent manner. In tunicamycin treatment, HuD and insulin proteins showed similar expression tendencies. These results indicate that ER stress/melatonin, especially thapsigargin/melatonin, increased nuclear HuD expression and subsequently resulted in a decrease in intracellular biosynthesis; it is hypothesized that extracellular secretion of insulin may be regulated by melatonin.

Identification of circulating microRNAs in HNF1A-MODY carriers

HNF1A-MODY is a monogenic form of diabetes caused by mutations in the HNF1A gene. Here we identify, for the first time, HNF1A-MODY-associated microRNAs (miRNAs) that can be detected in the serum of HNF1A-MODY carriers. An miRNA array was carried out in rat INS-1 insulinoma cells inducibly expressing the common human Pro291fsinsC-HNF1A frame shift mutation. Differentially expressed miRNAs were validated by quantitative real-time PCR. Expression of miRNAs in the serum of HNF1A-MODY carriers (n = 31), MODY-negative family members (n = 10) and individuals with type 2 diabetes mellitus (n = 17) was quantified by absolute real-time PCR analysis. Inducible expression of Pro291fsinsC-HNF1A in INS-1 cells caused a significant upregulation of three miRNAs (miR-103, miR-224, miR-292-3p). The differential expression of two miRNAs (miR-103 and miR-224) was validated in vitro. Strongly elevated levels of miR-103 and miR-224 could be detected in the serum of HNF1A-MODY carriers compared with MODY-negative family controls. Serum levels of miR-103 distinguished HNF1A-MODY carriers from HbA1c-matched individuals with type 2 diabetes mellitus. Our study demonstrates that the pathophysiology of HNF1A-MODY is associated with the overexpression of miR-103 and miR-224. Furthermore, our study demonstrates that these miRNAs can be readily detected in the serum of HNF1A-MODY carriers.

Ras-related C3 botulinum toxin substrate 1 (RAC1) regulates glucose-stimulated insulin secretion via modulation of F-actin

Aims/hypothesisThe small G-protein ras-related C3 botulinum toxin substrate 1 (RAC1) plays various roles in mammalian cells, such as in the regulation of cytoskeletal organisation, cell adhesion, migration and morphological changes. The present study examines the effects of RAC1 ablation on pancreatic beta cell function.MethodsIsolated islets from pancreatic beta cell-specific Rac1-knockout (betaRac1 −/−) mice and RAC1 knockdown INS-1 insulinoma cells treated with small interfering RNA were used to investigate insulin secretion and cytoskeletal organisation in pancreatic beta cells.ResultsBetaRac1 −/− mice showed decreased glucose-stimulated insulin secretion, while there were no apparent differences in islet morphology. Isolated islets from the mice had blunted insulin secretion in response to high glucose levels. In RAC1 knockdown INS-1 cells, insulin secretion was also decreased in response to high glucose levels, consistent with the phenotype of betaRac1 −/− mice. Even under high glucose levels, RAC1 knockdown INS-1 cells remained intact with F-actin, which inhibits the recruitment of the insulin granules, resulting in an inhibition of insulin secretion.Conclusions/interpretationIn RAC1-deficient pancreatic beta cells, F-actin acts as a barrier for insulin granules and reduces glucose-stimulated insulin secretion.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-013-2849-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

PPAR-γ Activation Increases Insulin Secretion through the Up-regulation of the Free Fatty Acid Receptor GPR40 in Pancreatic β-Cells

BackgroundIt has been reported that peroxisome proliferator-activated receptor (PPAR)-γ and their synthetic ligands have direct effects on pancreatic β-cells. We investigated whether PPAR-γ activation stimulates insulin secretion through the up-regulation of GPR40 in pancreatic β-cells.MethodsRat insulinoma INS-1 cells and primary rat islets were treated with rosiglitazone (RGZ) and/or adenoviral PPAR-γ overexpression. OLETF rats were treated with RGZ.ResultsPPAR-γ activation with RGZ and/or adenoviral PPAR-γ overexpression increased free fatty acid (FFA) receptor GPR40 expression, and increased insulin secretion and intracellular calcium mobilization, and was blocked by the PLC inhibitors, GPR40 RNA interference, and GLUT2 RNA interference. As a downstream signaling pathway of intracellular calcium mobilization, the phosphorylated levels of CaMKII and CREB, and the downstream IRS-2 and phospho-Akt were significantly increased. Despite of insulin receptor RNA interference, the levels of IRS-2 and phospho-Akt was still maintained with PPAR-γ activation. In addition, the β-cell specific gene expression, including Pdx-1 and FoxA2, increased in a GPR40- and GLUT2-dependent manner. The levels of GPR40, phosphorylated CaMKII and CREB, and β-cell specific genes induced by RGZ were blocked by GW9662, a PPAR-γ antagonist. Finally, PPAR-γ activation up-regulated β-cell gene expressions through FoxO1 nuclear exclusion, independent of the insulin signaling pathway. Based on immunohistochemical staining, the GLUT2, IRS-2, Pdx-1, and GPR40 were more strongly expressed in islets from RGZ-treated OLETF rats compared to control islets.ConclusionThese observations suggest that PPAR-γ activation with RGZ and/or adenoviral overexpression increased intracellular calcium mobilization, insulin secretion, and β-cell gene expression through GPR40 and GLUT2 gene up-regulation.

CDK5 Regulatory Subunit-associated Protein 1-Like 1 (CDKAL1) Is a Tail-anchored Protein in the Endoplasmic Reticulum (ER) of Insulinoma Cells

Genome-wide association studies have led to the identification of numerous susceptibility genes for type 2 diabetes. Among them is Cdkal1, which is associated with reduced β-cell function and insulin release. Recently, CDKAL1 has been shown to be a methylthiotransferase that modifies tRNA(Lys) to enhance translational fidelity of transcripts, including the one encoding proinsulin. Here, we report that out of several CDKAL1 isoforms deposited in public databases, only isoform 1, which migrates as a 61-kDa protein by SDS-PAGE, is expressed in human islets and pancreatic insulinoma INS-1 and MIN6 cells. We show that CDKAL1 is a novel member of the tail-anchored protein family and exploits the TCR40/Get3-assisted pathway for insertion of its C-terminal transmembrane domain into the endoplasmic reticulum. Using endo-β-N-acetylglucosaminidase H and peptide:N-glycosidase F sensitivity assays on CDKAL1 constructs carrying an N-glycosylation site within the luminal domain, we further established that CDKAL1 is an endoplasmic reticulum-resident protein. Moreover, we observed that silencing CDKAL1 in INS-1 cells reduces the expression of secretory granule proteins prochromogranin A and proICA512/ICA512-TMF, in addition to proinsulin and insulin. This correlated with reduced glucose-stimulated insulin secretion. Taken together, our findings provide new insight into the role of CDKAL1 in insulin-producing cells and help to understand its involvement in the pathogenesis of diabetes.

Endoplasmic Reticulum Redox State Is Not Perturbed by Pharmacological or Pathological Endoplasmic Reticulum Stress in Live Pancreatic β-Cells

Accumulation of unfolded, misfolded and aggregated proteins in the endoplasmic reticulum (ER) causes ER stress. ER stress can result from physiological situations such as acute increases in secretory protein biosynthesis or pathological conditions that perturb ER homeostasis such as alterations in the ER redox state. Here we monitored ER redox together with transcriptional output of the Unfolded Protein Response (UPR) in INS-1 insulinoma cells stably expressing eroGFP (ER-redox-sensor) and mCherry protein driven by a GRP78 promoter (UPR-sensor). Live cell imaging, flow cytometry and biochemical characterization were used to examine these parameters in response to various conditions known to induce ER stress. As expected, treatment of the cells with the reducing agent dithiothreitol caused a decrease in the oxidation state of the ER accompanied by an increase in XBP-1 splicing. Unexpectedly however, other treatments including tunicamycin, thapsigargin, DL-homocysteine, elevated free fatty acids or high glucose had essentially no influence on the ER redox state, despite inducing ER stress. Comparable results were obtained with dispersed rat islet cells expressing eroGFP. Thus, unlike in yeast cells, ER stress in pancreatic β-cells is not associated with a more reducing ER environment.

Localization and regulation of pancreatic selenoprotein P

Progressive loss of pancreatic β-cell mass is a crucial feature of type 2 diabetes mellitus. As β-cells express very low amounts of the antioxidant enzymes catalase and glutathione peroxidase (GPx), they appear to be particularly vulnerable to oxidative damage in the pathogenesis of diabetes. Here, we investigated the pancreatic expression pattern and regulation of selenoprotein P (Sepp1), which may serve as an additional antioxidant enzyme inside and outside of cells. Sepp1 was detected in rodent pancreas by immunofluorescence and real-time RT-PCR. Regulation of Sepp1 biosynthesis in INS-1 rat insulinoma cells was investigated by real-time RT-PCR, luciferase gene reporter assay, and immunoblotting. Sepp1 and Gpx1 gene expressions in rat pancreas were 58 and 22% respectively of the liver values. Pancreatic Sepp1 expression was restricted to the endocrine tissue, with Sepp1 being present in the α- and β-cells of mouse islets. In INS-1 insulinoma cells, Sepp1 expression was stimulated by the selenium compound sodium selenate and diminished in the presence of high glucose (16.7 vs 5 mM) concentrations. Sepp1 mRNA stability was also lowered at 16.7 mM glucose. Moreover, Sepp1 mRNA levels were decreased in isolated murine islets cultured in high-glucose (22 mM) medium compared with normal glucose (5.5 mM) medium. Pancreatic Sepp1 expression was elevated upon treatment of mice with the β-cell toxin streptozotocin. This study shows that pancreatic islets express relatively high levels of Sepp1 that may fulfill a function in antioxidant protection of β-cells. Downregulation of Sepp1 expression by high glucose might thus contribute to glucotoxicity in β-cells.

Forkhead Box Protein A2 (FOXA2) Protein Stability and Activity Are Regulated by Sumoylation

The forkhead box protein A2 (FOXA2) is an important regulator of glucose and lipid metabolism and organismal energy balance. Little is known about how FOXA2 protein expression and activity are regulated by post-translational modifications. We have identified that FOXA2 is post-translationally modified by covalent attachment of a small ubiquitin related modifier-1 (SUMO-1) and mapped the sumoylation site to the amino acid lysine 6 (K6). Preventing sumoylation by mutating the SUMO acceptor K6 to arginine resulted in downregulation of FOXA2 protein but not RNA expression in INS-1E insulinoma cells. K6R mutation also downregulated FOXA2 protein levels in HepG2 hepatocellular carcinoma cells, HCT116 colon cancer cells and LNCaP and DU145 prostate cancer cells. Further, interfering with FOXA2 sumoylation through siRNA mediated knockdown of UBC9, an essential SUMO E2 conjugase, resulted in downregulation of FOXA2 protein levels. Stability of sumoylation deficient FOXA2K6R mutant protein was restored when SUMO-1 was fused in-frame. FOXA2 sumoylation and FOXA2 protein levels were increased by PIAS1 SUMO ligase but not a SUMO ligase activity deficient PIAS1 mutant. Although expressed at lower levels, sumoylation deficient FOXA2K6R mutant protein was detectable in the nucleus indicating that FOXA2 nuclear localization is independent of sumoylation. Sumoylation increased the transcriptional activity of FOXA2 on Pdx-1 area I enhancer. Together, our results show that sumoylation regulates FOXA2 protein expression and activity.

Phospholipase iPLA2γ–dependent regulation of uncoupling protein UPC2 in insulinoma INS-1E cells

Phospholipase iPLA2γ–dependent regulation of uncoupling protein UPC2 in insulinoma INS-1E cells

Determining the retinoic acid response element in the liver glucokinase gene promoter using deletion and linker‐scan analysis

Glucokinase is a key enzyme of hepatic glucose utilization when plasma glucose level is high. We have reported that retinoids in a liver derived lipophilic extract synergized with insulin to induce the expression of the glucokinase gene (Gck) in primary rat hepatocytes. Experiments were designed to determine the retinoic acid response element (RARE) in Gck promoter. We have shown that retinoic acid (RA) activated the minimal luciferase reporter construct containing 234‐bp (−237/−3, Gck‐234‐WT‐LUC) of the Gck promoter sequence both in primary rat hepatocytes and INS‐1 insulinoma cells. A series of reporter constructs containing overlapping 10‐ to 12‐bp scramble mutations covering the nucleotide sequence from −237 to −3 were made to narrow down the RARE to a 20‐bp region. Subsequently, a series of 5‐ to 6‐bp linker‐scan scramble mutations covering the entire 20‐bp region were made to narrow down the RARE to a 5‐bp region. RA induced Gck‐234‐WT‐LUC activity by 2.74±0.25‐fold in INS‐1 cells. The scramble mutation covering the sequence −177/−173 destroyed the RA‐induced luciferase activity. Moreover, single nucleotide mutations at −177, −176, and −175 significantly impaired the response of the reporter constructs to RA treatment. We conclude that the RA‐induced Gck transcription is mediated by a transcription activator associated with the RARE sequence covering −177/−175 of the liver Gck promoter.Grant Funding Source: American Heart Association

Abstract P218: Identification of One Retinoic Acid Responsive Element in Glucokinase Gene Promoter

Introduction: Studies have shown that vitamin A (VA) status affected hepatic glycogen content and body lipid levels in rats. The active metabolite of VA, retinoic acid (RA), regulates gene expression via activation of both retinoic acid receptor (RAR) and retinoid X receptor (RXR) bound to retinoic acid responsive element (RARE). We have reported that retinoids synergized with insulin to induce glucokinase gene ( Gck ) in primary rat hepatocyes. Hypothesis: We hypothesize that the rat promoter of Gck contains RARE(s). Methods: To identify the RARE(s) in the Gck promoter, we have made (1) a series of luciferase reporter gene constructs containing shortened DNA fragments of Gck promoter; (2) expression vectors containing cDNAs of rat RARα, RXRα and RXRγ; (3) linker-scan mutant reporter constructs covering 0.2 kb of the proximal Gck promoter region and a 20 bp region which disrupt the RA response. The responses of these reporter constructs to RA and the effects of the co-transfection of RAR and RXR on their activation were analyzed in primary hepatocytes and INS-1 insulinoma cells using dual luciferase assay. Results: (1) RA and RA + insulin, not insulin, activated the reporter gene constructs containing 1, 0.8, 0.6, 0.4, and 0.2 kb of Gck promoter in primary hepatocytes (from 1.7- to 2.6-fold). (2) RA alone did not activate Gck promoter constructs in INS-1 cells. However, co-transfection of RAR with reporter gene constructs restored and improved RA response of them in INS-1 cells and primary hepatocytes, respectively. (3) Two consecutive linker-scan mutant reporter constructs covering about 20 bp of the proximal region of Gck promoter, but not the adjacent ones, completely lost the response to RA. (4) One of the five linker-scan mutant reporter constructs covering this 20 bp region completed disrupted the RA response, demonstrating the identification of a RARE. Conclusions: RA induces the activation of Gck promoter constructs in primary hepatocytes. INS-1 cells are a useful tool for analyzing liver Gck promoter activation by RA with co-transfection of RAR/RXR. For the first time, one RARE is identified in the proximal region of rat Gck promoter. Further studies are needed to determine the roles of this identified RARE in mediating Gck expression in responses to hormonal and nutritional stimuli. Funding(This research has received full or partial funding support from the American Heart Association, National Center)

Activation of the unfolded protein response pathway causes ceramide accumulation in yeast and INS-1E insulinoma cells

Sphingolipids are not only important components of membranes but also have functions in protein trafficking and intracellular signaling. The LCB1 gene encodes a subunit of the serine palmitoyltransferase, which is responsible for the first step of sphingolipid synthesis. Here, we show that activation of the unfolded protein response (UPR) can restore normal ceramide levels and viability in yeast cells with a conditional defect in LCB1. Dependence on UPR was demonstrated by showing the HAC1-dependence of the suppression. A similar induction of ceramides by UPR seems to take place in mammalian cells. In rat pancreatic INS-1E cells, UPR activation induces the transcription of the CerS6 gene, which encodes a ceramide synthase. This correlates with the specific accumulation of ceramide with a C16 fatty acyl chain upon UPR activation. Therefore, our study reveals a novel connection between UPR induction and ceramide synthesis that seems to be conserved between yeast and mammalian cells.

The Transcription Factor C/EBP delta Has Anti-Apoptotic and Anti-Inflammatory Roles in Pancreatic Beta Cells

In the course of Type 1 diabetes pro-inflammatory cytokines (e.g., IL-1β, IFN-γ and TNF-α) produced by islet-infiltrating immune cells modify expression of key gene networks in β-cells, leading to local inflammation and β-cell apoptosis. Most known cytokine-induced transcription factors have pro-apoptotic effects, and little is known regarding “protective” transcription factors. To this end, we presently evaluated the role of the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) on β-cell apoptosis and production of inflammatory mediators in the rat insulinoma INS-1E cells, in purified primary rat β-cells and in human islets. C/EBPδ is expressed and up-regulated in response to the cytokines IL-1β and IFN-γ in rat β-cells and human islets. Small interfering RNA-mediated C/EBPδ silencing exacerbated IL-1β+IFN-γ-induced caspase 9 and 3 cleavage and apoptosis in these cells. C/EBPδ deficiency increased the up-regulation of the transcription factor CHOP in response to cytokines, enhancing expression of the pro-apoptotic Bcl-2 family member BIM. Interfering with C/EBPδ and CHOP or C/EBPδ and BIM in double knockdown approaches abrogated the exacerbating effects of C/EBPδ deficiency on cytokine-induced β-cell apoptosis, while C/EBPδ overexpression inhibited BIM expression and partially protected β-cells against IL-1β+IFN-γ-induced apoptosis. Furthermore, C/EBPδ silencing boosted cytokine-induced production of the chemokines CXCL1, 9, 10 and CCL20 in β-cells by hampering IRF-1 up-regulation and increasing STAT1 activation in response to cytokines. These observations identify a novel function of C/EBPδ as a modulatory transcription factor that inhibits the pro-apoptotic and pro-inflammatory gene networks activated by cytokines in pancreatic β-cells.