Mouse Pancreatic Beta-cell Line Research Articles

The L-type amino acid transporter 1 enhances drug delivery to the mouse pancreatic beta cell line (MIN6)

l-type amino acid transporter 1 (LAT1) is a membrane transporter responsible for carrying large, neutral l-configured amino acids as well as appropriate (pro)drugs into a cell. It has shown a great potential to improve drug delivery across the blood-brain barrier and to increase cell uptake into several brain and cancer cell types. However, besides the brain, the LAT1-utilizing compounds are also delivered more efficiently into the pancreas in vivo. In this study, we quantified the expression of LAT1 along several other membrane transporters in mouse pancreatic β-cell line (MIN6). Furthermore, we studied the function of LAT1 in MIN6 cells, and its ability to deliver non-steroidal anti-inflammatory drug (NSAID)-derived prodrugs there. The results showed that LAT1 was highly abundant in MIN6 cells, with an even expression on cell pseudoislets. The l-leucine uptake as a probe substrate was efficient, with comparable affinity and capacity to previously studied immortalized mouse microglia (BV2). The NSAID-derived prodrugs utilized LAT1 for their delivery and were uptaken into MIN6 cells 2–300 times more efficiently when compared to their parent drugs. A similar increase in pancreatic delivery was observed also in vivo, where the pancreatic exposure was 2–10 times higher with selected prodrugs, indicating an excellent correlation between in vitro uptake and in vivo pancreatic delivery. Finally, the LAT1-utilizing prodrugs were able to reverse the effects of cytokines on insulin secretion in MIN6 cells, showing that improved delivery via LAT1 can enhance drug effects in the mouse pancreatic β-cell line.

Structural and quantitative characterization of membrane N-glycans from MIN6 mouse pancreatic beta cells using liquid chromatography-quadrupole-Orbitrap tandem mass spectrometry

MIN6, a mouse pancreatic beta cell line, is used in diabetes research, and the cellular N-glycoproteins in membrane are important in regulating the metabolism of insulin secretion. However, the identities of N-glycans in MIN6 cells are yet to be fully elucidated. In this study, the structures of N-glycans were analyzed using liquid chromatography-electrospray ionization-higher energy collisional dissociation-tandem mass spectrometry. The abundances (%) of each N-glycan relative to the total N-glycans (100 %) were also obtained. Fifty N-glycans (with relative abundance of each > 0.5 %) were obtained, revealing 22 bisecting N-acetylglucosamine (GlcNAc; associated with cell adhesion and growth; sum of relative abundance of each: 27.1 %), 21 core-fucosylated (associated with glucose sensing and insulin secretion regulation; 28.3 %), and 16 sialylated (N-acetylneuraminic acid; related to the expression of glucose transporters and diabetes;15.5 %) N-glycans. Membranes contained higher bisecting GlcNAc and core-fucosylation, similar sialylation, but less high-mannosylation than the lysate (the cellular contents). Notably, all bisecting GlcNAc N-glycans were categorized into structures with (16.6 %) or without (10.5 %) core-fucosylation and with (6.9 %) or without (20.2 %) sialylation. The bisecting GlcNAc structures were not found in human islets; moreover, sialylation levels were 6.9 times higher than for human islets. These structural characteristics of N-glycans affect their cell adhesion and distribution through homologous interactions between beta cells, leading to increased insulin secretion efficiency. This study is the first to identify the structures and quantities of 50 N-glycans in MIN6 cell membranes that may play an important role in regulating the functions of pancreatic beta cells.

Benzophenones alter autophagy and ER stress gene expression in pancreatic beta cells in vitro

Benzophenones (BPs) are endocrine disruptors frequently used in sunscreens and food packaging as UV blockers. Our goal was to assess the effect of benzophenone 2 (BP2) and 3 (BP3) on gene expression related to autophagy process and ER stress response in pancreatic beta cells. To that end, the mouse pancreatic beta cell line MIN6B1 was treated with 10µM BP2 or BP3 in the presence or absence of the autophagy-inhibitor chloroquine (CQ, 10µM) or the autophagy-inducer rapamycin (RAPA, 50nM) during 24h. BP3 inhibited the expression of the autophagic gene Ulk1, and additional effects were uncovered when autophagy was modified by CQ and RAPA. BP3 counteracted CQ-induced Lamp2 expression but did not compensate CQ-induced Sqstm1/p62 gene transcription, neither BP2. Nevertheless, the BPs did not alter the autophagic flux. In relation to ER stress, BP3 inhibited unspliced and spliced Xbp1 mRNA levels in the presence or absence of CQ, totally counteracted CQ-induced Chop gene expression, and partially reverted CQ-induced Grp78/Bip mRNA levels, while BP2 also partially inhibited Grp78/Bip mRNA induction by CQ. In conclusion, BPs, principally BP3, affect cellular adaptive responses related to autophagy, lysosomal biogenesis, and ER stress in pancreatic beta cells, indicating that BP exposure could lead to beta cell dysfunction.

Antiproliferative and Pro-Oxidant Effect of Polyphenols in Aqueous Leaf Extract of Passiflora alata Curtis on Activated T Lymphocytes from Non-Obese Diabetic (NOD SHILT/J) Mice.

The antioxidant, anti-inflammatory and antiproliferative properties of Passiflora alata Curtis are due to the presence of polyphenols in its composition. Our previous work showed that non-obese diabetic (NOD) mice undergoing treatment with aqueous leaf extract of P. alata present reduced insulitis in the pancreas, possibly due to its anti-inflammatory properties. However, depending on the concentration and their ability to interact with other molecules, these phenolic compounds may promote oxidation reactions in some cellular components, such as proteins and lipids, thus presenting a pro-oxidant effect. The present work aimed to evaluate the in vitro effects of aqueous leaf extract of P. alata and its polyphenols (vitexin, isoorientin, rutin and catechin) on lymphocyte proliferation and viability, the cell cycle and oxidative stress. Our results showed that T lymphocytes stimulated with concanavalin A mitogen (ConA) and in the presence of IC50 concentrations of P. alata extract and polyphenols undergo cell injury via inhibition of proliferation, with these effects being more pronounced concerning CD4+ T cells (P. alata, 3.54 ± 0.34%; isoorientin, 57.07 ± 6.4%; vitexin, 16.95 ± 1.11%; catechin, 37.9 ± 4.2% and rutin, 40.14 ± 4.5%), compared to the non-treated group (77.17 ± 6.29) (p < 0.0001 for all comparisons). This process includes late apoptosis/necrosis induction (P. alata, 77.5 ± 0.7%; vitexin, 83 ± 3.3%; isoorientin, 83.8 ± 1.4%; catechin, 83 ± 1.9% and rutin, 74.9 ± 3.2, while the control presented 53.6% ± 3.1 (p < 0.0001 for all comparisons)) and mitochondrial depolarization leading to cell-death induction. Furthermore, an in vitro model of a mixed culture of NOD mice T cells with a mouse pancreatic beta-cell line (MIN6) showed increased intracellular nitric oxide and lipid peroxidation in NOD T cells submitted to P. alata extract (46.41 ± 3.08) compared to the untreated control group (33.57 ± 1.99, p = 0.01315). These results suggest that aqueous leaf extract of P. alata and the polyphenols in these leaves represent a target for translational research showing the plant’s benefits for developing new drugs with immunomodulatory properties against inflammatory diseases such as diabetes mellitus.

Generation of a Beta-Cell Transplant Animal Model of Diabetes Using CRISPR Technology.

Since insulin deficiency results from pancreatic beta-cell destruction, all type 1 and most type 2 diabetes patients eventually require life-long insulin injections. Insulin gene synthesis could also be impaired due to insulin gene mutations as observed in diabetic patients with MODY 10. At this point, insulin gene therapy could be very effective to recompense insulin deficiency under these circumstances. For this reason, an HIV-based lentiviral vector carrying the insulin gene under the control of insulin promoter (LentiINS) was generated, and its therapeutic efficacy was tested in a beta-cell transplant model lacking insulin produced by CRISPR/Cas9-mediated genetically engineered pancreatic beta cells. To generate an insulin knockout beta-cell transplant animal model of diabetes, a dual gene knockout plasmid system involving CRISPR/Cas9 was transfected into a mouse pancreatic beta cell line (Min6). Fluorescence microscopy and antibiotic selection were utilized to select the insulin gene knockout clones. Transplantation of the genetically engineered pancreatic beta cells under the kidney capsule of STZ-induced diabetic rats revealed LentiINS- but not LentiLacZ-infected Ins2KO cells transiently reduced hyperglycemia similar to that of MIN6 in diabetic animals. These results suggest LentiINS has the potential to functionally restore insulin production in an insulin knockout beta-cell transplant animal model of diabetes.

G Protein‐Coupled Receptor Kinase 6 (GRK6) Regulation of Insulin Processing and Secretion

We performed exome sequencing in a Type 2 Diabetes (T2D) patient belonging to a T2D family containing two affected and five unaffected individuals revealing an autosomal dominant inheritance pattern. A mutation in the GRK6 gene, coding for G protein-coupled receptor kinase 6, was identified that segregates with the phenotype. GRK6 belongs to a family of serine/threonine kinases that phosphorylate agonist occupied G protein-coupled receptors (GPCRs) effectively modulating downstream signaling pathways including those involved in metabolism. To understand the potential role of GRK6 in T2D, we utilized pharmacological inhibition and genetic deletion of GRK6 by both shRNA and CRISPR in the mouse pancreatic beta-cell line, MIN6. We then assayed the ability of these cells to process and secrete insulin in response to glucose. Small molecule inhibition of GRK5/GRK6 caused an increase in insulin secretion whereas insulin processing was reduced. Insulin processing was also diminished in GRK6 knockdown cells. Furthermore, GRK6 knockdown attenuated insulin secretion but enhanced proinsulin secretion consistent with a processing defect and the aforementioned patient phenotype. The insulin processing defect in GRK6 knockdown cells was also seen in CRISPR mediated GRK6 knockout cells. The altered insulin profile in MIN6 cells lacking GRK6 function appears to be caused by changes in the expression and activity of the proprotein convertases, the enzymes responsible for proinsulin to insulin conversion, as pharmacological inhibition, knockdown, and knockout of GRK6 all resulted in less expression and activity of these enzymes. To support this, we performed rescue experiments where GRK6 re-expression in knockdown cells partially recovered insulin and proprotein convertase levels in cells. Taken together, our data show that GRK6 regulates insulin processing and provides novel insight into how GRK6 might contribute to T2D.

Berberine Alleviates Oxidative Stress in Islets of Diabetic Mice by Inhibiting miR-106b Expression and Up-Regulating SIRT1.

Mounting studies have indicated the role of berberine, SIRT1, and oxidative stress in diabetes, respectively. However, few studies have demonstrated their correlation and regulation function in diabetes. Therefore, the protective effect of berberine in diabetic and the underlying core mechanism were investigated in the current study. Diabetic mice model in vivo were established. Mouse pancreatic beta-cell line NIT-1 cells were treated with 30 mM high glucose to induce diabetic condition in vitro. Serum biochemical parameters (glucose, total cholesterol, and triglycerides) were detected. Oxidative stress indicators (MDA, SOD1), along with miR-106b and SIRT1 expression in islets and cells were also assessed. Direct targeting relationship between miR-106b and SIRT1 was discussed by dual luciferase reporter gene assay. Diabetic model in vivo and in vitro were both established successfully. The expression of serum biochemical parameters was increased, and oxidative stress parameters, and miR-106b, SIRT1 were abnormally expressed in diabetic mice and NIT-1 cells. Meanwhile, berberine could alleviate oxidative stress injury in diabetic progression. Through dual luciferase reporter gene assay, we found that SIRT1 was a target gene of miR-106b. In addition, miR-106b over-expression could reverse the protection of berberine in NIT-1 cells against from oxidative stress induced by high glucose. Berberine could attenuate oxidative stress of diabetic mice at least partly through miR-106b/SIRT1 pathway and affecting the function of islets, which might be beneficial in reducing the cardiovascular risk factors in diabetes. J. Cell. Biochem. 118: 4349-4357, 2017. © 2017 Wiley Periodicals, Inc.

Proteomics analysis of rough endoplasmic reticulum in pancreatic beta cells

Pancreatic beta cells have well-developed ER to accommodate for the massive production and secretion of insulin. ER homeostasis is vital for normal beta cell function. Perturbation of ER homeostasis contributes to beta cell dysfunction in both type 1 and type 2 diabetes. To systematically identify the molecular machinery responsible for proinsulin biogenesis and maintenance of beta cell ER homeostasis, a widely used mouse pancreatic beta cell line, MIN6 cell was used to purify rough ER. Two different purification schemes were utilized. In each experiment, the ER pellets were solubilized and analyzed by 1D SDS-PAGE coupled with HPLC-MS/MS. A total of 1467 proteins were identified in three experiments with ≥95% confidence, among which 1117 proteins were found in at least two separate experiments and 737 proteins found in all three experiments. GO analysis revealed a comprehensive profile of known and novel players responsible for proinsulin biogenesis and ER homeostasis. Further bioinformatics analysis also identified potential beta cell specific ER proteins as well as ER proteins present in the risk genetic loci of type 2 diabetes. This dataset defines a molecular environment in the ER for proinsulin synthesis, folding and export and laid a solid foundation for further characterizations of altered ER homeostasis under diabetes-causing conditions. All MS data have been deposited in the ProteomeXchange with identifier PXD001081 (http://proteomecentral.proteomexchange.org/dataset/PXD001081).

Nephrin, a transmembrane protein, is involved in pancreatic beta-cell survival signaling

Nephrin, a cell surface signaling receptor, regulates podocyte function in health and disease. We study the role of nephrin in β-cell survival signaling. We report that in mouse islet β-cells and the mouse pancreatic beta-cell line (βTC-6 cells) nephrin is associated and partly co-localized with PI3-kinase. Incubation of cells with functional anti-nephrin antibodies induced nephrin clustering at the plasma membrane, nephrin phosphorylation and recruitment of PI3-kinase to nephrin thus resulting in increased PI3K-dependent Akt phosphorylation and augmented phosphorylation/inhibition of pro-apoptotic Bad and FoxO. Nephrin silencing abolished Akt activation and increased susceptibility of cells to apoptosis. High glucose impaired nephrin signaling, increased nephrin internalization and up-regulated PKCα expression. Interestingly, a marked decrease in nephrin expression and phosphorylated Akt was observed in pancreatic islets of db/db lepr−/− diabetic mice. Our findings revealed that nephrin is involved in β-cell survival and suggest that glucose-induced changes in nephrin signaling may contribute to gradual pancreatic β-cell loss in type 2 diabetes.

The expression and function of histamine H3 receptors in pancreatic beta cells

Histamine and its receptors in the CNS play important roles in energy homeostasis. Here, we have investigated the expression and role of histamine receptors in pancreatic beta cells, which secrete insulin. The expression of histamine receptors in pancreatic beta cells was examined by RT-PCR, Western blotting and immunostaining. Insulin secretion assay, ATP measurement and calcium imaging studies were performed to determine the function and signalling pathway of histamine H₃ receptors in glucose-induced insulin secretion (GIIS) from MIN6 cells, a mouse pancreatic beta cell line. The function and signalling pathway of H₃ receptors in MIN6 cell proliferation were examined using pharmacological assay and Western blotting. Histamine H₃ receptors were expressed in pancreatic beta cells. A selective H₃ receptor agonist, imetit, and a selective inverse H₃ receptor agonist, JNJ-5207852, had inhibitory and facilitatory effects, respectively, on GIIS in MIN6 cells. Neither imetit nor JNJ-5207852 altered intracellular ATP concentration, or intracellular calcium concentration stimulated by glucose and KCl, indicating that GIIS signalling was affected by H3 receptor signalling downstream of the increase in intracellular calcium concentration. Moreover, imetit attenuated bromodeoxyuridine incorporation in MIN6 cells. The phosphorylation of cAMP response element-binding protein (CREB), which facilitated beta cell proliferation, was inhibited, though not significantly, by imetit, indicating that activated H₃ receptors inhibited MIN6 cell proliferation, possibly by decreasing CREB phosphorylation. Histamine H₃ receptors were expressed in mouse beta cells and could play a role in insulin secretion and, possibly, beta cell proliferation.

PERK phosphorylation on tyrosine 561 delays ER stress‐induced PERK activation

The PKR‐like endoplasmic reticulum (ER) kinase (PERK) is an ER transmembrane ser/thr protein kinase that plays an essential role in mediating the unfolded protein response (UPR) activated during stress conditions in the ER lumen. The importance of PERK in pancreatic beta‐cell homeostasis is demonstrated in Wolcott‐ Rallison syndrome, a neonatal or early infancy form of insulin‐dependent diabetes in humans with inactivating mutations in the PERK gene. In this study, we provide strong evidence that PERK is phosphorylated on tyrosine 561 (Y561) in pervanadate‐treated MIN6 cells, a mouse pancreatic beta cell line in culture. In addition, we demonstrate that increased phosphorylation of PERK on Y561 delays PERK activation in response to pharmacological agents that induce ER stress. Interestingly, we discovered that the adaptor protein Nck1 directly interacts with PERK phosphorylated on Y561 through its SH2 domain. Finally, using recombinant wild‐type and kinase dead PERK proteins, our data strongly suggest that PERK phosphorylation on Y561 is dependent on PERK kinase activity. All together, our results indicate that PERK phosphorylation on Y561 and binding to Nck1 play an important role in regulating PERK activity. Supported by the Canadian Diabetes Association.

An siRNA Screen in Pancreatic Beta Cells Reveals a Role for Gpr27 in Insulin Production

The prevalence of type 2 diabetes in the United States is projected to double or triple by 2050. We reasoned that the genes that modulate insulin production might be new targets for diabetes therapeutics. Therefore, we developed an siRNA screening system to identify genes important for the activity of the insulin promoter in beta cells. We created a subclone of the MIN6 mouse pancreatic beta cell line that expresses destabilized GFP under the control of a 362 base pair fragment of the human insulin promoter and the mCherry red fluorescent protein under the control of the constitutively active rous sarcoma virus promoter. The ratio of the GFP to mCherry fluorescence of a cell indicates its insulin promoter activity. As G protein coupled receptors (GPCRs) have emerged as novel targets for diabetes therapies, we used this cell line to screen an siRNA library targeting all known mouse GPCRs. We identified several known GPCR regulators of insulin secretion as regulators of the insulin promoter. One of the top positive regulators was Gpr27, an orphan GPCR with no known role in beta cell function. We show that knockdown of Gpr27 reduces endogenous mouse insulin promoter activity and glucose stimulated insulin secretion. Furthermore, we show that Pdx1 is important for Gpr27's effect on the insulin promoter and insulin secretion. Finally, the over-expression of Gpr27 in 293T cells increases inositol phosphate levels, while knockdown of Gpr27 in MIN6 cells reduces inositol phosphate levels, suggesting this orphan GPCR might couple to Gq/11. In summary, we demonstrate a MIN6-based siRNA screening system that allows rapid identification of novel positive and negative regulators of the insulin promoter. Using this system, we identify Gpr27 as a positive regulator of insulin production.

Metformin prevents endoplasmic reticulum stress-induced apoptosis through AMPK-PI3K-c-Jun NH2 pathway

Type 2 diabetes mellitus is thought to be partially associated with endoplasmic reticulum (ER) stress toxicity on pancreatic beta cells and the result of decreased insulin synthesis and secretion. In this study, we showed that a well-known insulin sensitizer, metformin, directly protects against dysfunction and death of ER stress-induced NIT-1 cells (a mouse pancreatic beta cell line) via AMP-activated protein kinase (AMPK) and phosphatidylinositol-3 (PI3) kinase activation. We also showed that exposure of NIT-1 cells to metformin (5mM) increases cellular resistance against ER stress-induced NIT-1 cell dysfunction and death. AMPK and PI3 kinase inhibitors abolished the effect of metformin on cell function and death. Metformin-mediated protective effects on ER stress-induced apoptosis were not a result of an unfolded protein response or the induced inhibitors of apoptotic proteins. In addition, we showed that exposure of ER stressed-induced NIT-1 cells to metformin decreases the phosphorylation of c-Jun NH2 terminal kinase (JNK). These data suggest that metformin is an important determinant of ER stress-induced apoptosis in NIT-1 cells and may have implications for ER stress-mediated pancreatic beta cell destruction via regulation of the AMPK–PI3 kinase–JNK pathway.

Cholesterol induces mitochondrial dysfunction and apoptosis in mouse pancreatic beta-cell line MIN6 cells

Reduction of pancreatic β-cell mass is a key element leading to type 2 diabetes. Obesity and overweight with high levels of lipids including cholesterol are tightly linked to type 2 diabetes. The direct impact of cholesterol on pancreatic β-cells, however, has not been extensively studied. In this study, MIN6 mouse β-cell line was used to test the effect of cholesterol on pancreatic β-cell apoptosis over different doses and durations. It was found that cholesterol dose- and time-dependently induced cell death of MIN6 cells above 160 μM after 6 h treatment in vitro. Annexin-V staining revealed that cholesterol treatment significantly induced apoptosis in MIN6 cells. Cholesterol treatment resulted in the loss of the ability to retain Rhodamine 123, indicating mitochondrial damage in MIN6 cells. Cholesterol-induced cell apoptosis and mitochondrial damage were blocked by low-temperature condition. In addition, glutathione also protected MIN6 cells from cholesterol-induced cell death. It is concluded that high level of cholesterol induces cell apoptosis in MIN6 cells, which is in part due to mitochondrial dysfunction. We suggest that excessive uptake of cholesterol in β-cells may contribute to β-cell apoptosis and dysfunction and the deterioration of type 2 diabetes.

Neurotensin receptor-2 and -3 are crucial for the anti-apoptotic effect of neurotensin on pancreatic β-TC3 cells

The neuropeptide neurotensin (NT) has been recently shown to protect pancreatic beta cells from toxic agents-induced apoptosis through interaction with the NT receptor-2 (NTSR2) and activation of the phosphatidyl inositol-3 kinase pathway. However, expression of the NT receptor-3/sortilin (NTSR3) in the mouse pancreatic beta cell line β-TC3 led us to investigate its possible functional role in these cells. By using siRNA, immunoprecipitation, co-localization and caspase-3 assays, we provide evidence for a functional endogenous interaction between NTSR2 and NTSR3. Expression of both receptors is necessary for the protective action of NT on staurosporine-induced caspase-3 activity in β-TC3 cells. Moreover, NTSR2 and NTSR3 co-immunoprecipitate and are co-localized at the plasma membrane. Thus, the NT response in beta cells is controlled by the formation of a functional complex between NTSR2 and NTSR3.

Calcium elevation in mouse pancreatic beta cells evoked by extracellular human islet amyloid polypeptide involves activation of the mechanosensitive ion channel TRPV4.

To investigate the mechanism by which human islet amyloid polypeptide (hIAPP) fibril formation results in calcium influx across the plasma membrane of pancreatic beta cells, and its association with apoptosis. Cytoplasmic intracellular calcium concentrations ([Ca(2+)](i)) were monitored for 2 h as the 340/380 nm fluorescence ratio in fura-2 loaded cells of the MIN6 mouse pancreatic beta cell line. Cell morphology was evaluated by transmission electron microscopy, and viability by FACS. hIAPP (10 micromol/l) increased [Ca(2+)](i) in 21% of MIN6 cells in standard buffer, and in 8% of cells in Na(+)-free buffer. Transient receptor potential (TRP) channel inhibitors (gadolinium and ruthenium red) prevented the [Ca(2+)](i) rise under both conditions, whilst nifedipine was only effective in the presence of Na(+). hIAPP increased apoptosis in both insulinoma cells and islets in primary culture, and cell viability was partially rescued by ruthenium red (p < 0.001). By RT-PCR, we detected expression of the mechanosensitive TRP cation channel subfamily V member 4 (Trpv4) in MIN6 cells and mouse pancreas. Small interference RNA against Trpv4 prevented hIAPP-induced [Ca(2+)](i) rises, decreased hIAPP-triggered expression of the endoplasmic reticulum (ER) stress response, and reduced hIAPP-triggered cell death by 50% (p < 0.05). Alterations in [Ca(2+)](i) play a key role in hIAPP-induced beta cell cytotoxicity. By electron microscopy, we detected extracellular hIAPP aggregates adjacent to irregular invaginated regions of the plasma membrane. We propose that TRPV4 channels may sense physical changes in the plasma membrane induced by hIAPP aggregation, enabling Ca(2+) entry, membrane depolarisation and activation of L-type Ca(2+) channels. Decreasing the activity of TRPV4 prevented hIAPP-induced [Ca(2+)](i) changes, reduced hIAPP-triggered ER stress and improved cell viability.

Pharmacological regulation of insulin secretion in MIN6 cells through the fatty acid receptor GPR40: identification of agonist and antagonist small molecules

1. Long chain fatty acids have recently been identified as agonists for the G protein-coupled receptors GPR40 and GPR120. Here, we present the first description of GW9508, a small-molecule agonist of the fatty acid receptors GPR40 and GPR120. In addition, we also describe the pharmacology of GW1100, a selective GPR40 antagonist. These molecules were used to further investigate the role of GPR40 in glucose-stimulated insulin secretion in the MIN6 mouse pancreatic beta-cell line. 2. GW9508 and linoleic acid both stimulated intracellular Ca2+ mobilization in human embryonic kidney (HEK)293 cells expressing GPR40 (pEC50 values of 7.32+/-0.03 and 5.65+/-0.06, respectively) or GPR120 (pEC50 values of 5.46+/-0.09 and 5.89+/-0.04, respectively), but not in the parent HEK-293 cell line. 3. GW1100 dose dependently inhibited GPR40-mediated Ca2+ elevations stimulated by GW9508 and linoleic acid (pIC50 values of 5.99+/-0.03 and 5.99+/-0.06, respectively). GW1100 had no effect on the GPR120-mediated stimulation of intracellular Ca2+ release produced by either GW9508 or linoleic acid. 4. GW9508 dose dependently potentiated glucose-stimulated insulin secretion in MIN6 cells, but not in primary rat or mouse islets. Furthermore, GW9508 was able to potentiate the KCl-mediated increase in insulin secretion in MIN6 cells. The effects of GW9508 on insulin secretion were reversed by GW1100, while linoleic acid-stimulated insulin secretion was partially attenuated by GW1100. 5. These results add further evidence to a link between GPR40 and the ability of fatty acids to acutely potentiate insulin secretion and demonstrate that small-molecule GPR40 agonists are glucose-sensitive insulin secretagogues.

Genetically modified rice seeds accumulating GLP-1 analogue stimulate insulin secretion from a mouse pancreatic beta-cell line

Glucagon-like peptide-1 (7-36) amide (GLP-1) is the most potent physiological insulinotropic hormone in humans. We produced large amounts of a GLP-1 analogue, [Ser8, Gln26, Asp34]-GLP-1, which is resistant to trypsin-digestion, as part of a chimeric rice seed storage protein, a 26kDa globulin, in genetically modified rice seeds. Junction sites between GLP-1 analogue and globulin were replaced by tryptic cleavage sites. The highest level of GLP-1 analogue accumulation was ≈20–50μg per seed. We found that GLP-1 analogue derived from trypsin-digested genetically modified rice seeds stimulated insulin secretion from a mouse pancreatic beta-cell line, MIN6.

Cytokine-mediated Down-regulation of the Transcription Factor cAMP-response Element-binding Protein in Pancreatic β-Cells

Cytokines are known to induce apoptosis of pancreatic beta-cells. Impaired expression of the anti-apoptotic gene bcl-2 is one of the mechanisms involved. In this study, we identified a defect involving transcription factor cAMP-response element-binding protein (CREB) in the expression of bcl-2. Exposure of mouse pancreatic beta-cell line, MIN6 cells, to cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma) led to a significant (p < 0.01) decrease in Bcl-2 protein and mRNA levels. Cytokines decreased (56%) the activity of the bcl-2 promoter that contains a cAMP-response element (CRE) site. Similar decreases were seen with a luciferase reporter gene driven by tandem repeats of CRE and a CREB-specific Gal4-luciferase reporter, suggesting a defect at the level of CREB. The active phospho form (serine 133) of CREB diminished significantly (p < 0.01) in cells exposed to cytokines. Examination of signaling pathways upstream of CREB revealed a reduction in the active form of Akt. Cytokine-induced decrease of bcl-2 promoter activity was partially restored when cells were cotransfected with a constitutively active form of Akt. Several end points of cytokine action including decreases in phospho-CREB, phospho-Akt, and BCl-2 levels and activation of caspase-9 were observed in isolated mouse islets. Overexpression of wild-type CREB in MIN6 cells by plasmid transfection and adenoviral infection led to protection against cytokine-induced apoptosis. Adenoviral transfer of dominant-negative forms of CREB, on the other hand, resulted in activation of caspase-9 and exaggeration of cytokine-induced beta-cell apoptosis. Together, these results point to CREB as a novel target for strategies aimed at improving the survival of beta-cells.

Differential gene expression in well-regulated and dysregulated pancreatic beta-cell (MIN6) sublines.

To identify genes involved in regulated insulin secretion, we have established and characterized two sublines derived from the mouse pancreatic beta-cell line MIN6, designated B1 and C3. They have a similar insulin content, but differ in their secretory properties. B1 responded to glucose in a concentration- and cell confluence-dependent manner, whereas C3 did not. B1 cells were stimulated by phorbol 12-myristate 13-acetate, leucine, arginine, glibenclamide, isobutylmethylxanthine, and KCl, whereas C3 did not respond (leucine, arginine, and glibenclamide) or responded to a lesser extent (isobutylmethylxanthine, phorbol 12-myristate 13-acetate, and KCl). Although intracellular Ca(2+) rose in response to glucose in B1 but not C3 cells, KCl increased intracellular Ca(2+) in a similar manner in both sublines. GLUT-1, GLUT-2, Kir6.2, and SUR1 expression was not significantly different between B1 and C3 cells, whereas E-cadherin was more abundantly expressed in B1 cells. A more complete list of differentially expressed genes was established by suppression subtractive hybridization and high density (Affymetrix) oligonucleotide microarrays. Genes were clustered according to known or putative function. Those involved in metabolism, intracellular signaling, cytoarchitecture, and cell adhesion are of potential interest. These two sublines should be useful for identification of the genes and mechanisms involved in regulated insulin secretion of the pancreatic beta-cell.