Thapsigargin down-regulates protein levels of GRP78/BiP in INS-1E cells

Pancreatic β-cells have a well-developed endoplasmic reticulum (ER) and express large amounts of chaperones and protein disulfide isomerases (PDI) to meet the high demand for synthesis of proteins. We have observed an unexpected decrease in chaperone protein level in the β-cell model INS-1E after exposure to the ER stress inducing agent thapsigargin. As these cells are a commonly used model for primary β-cells and has been shown to be vulnerable to ER stress, we hypothesize these cells are incapable of mounting a chaperone defense upon activation of ER stress. To investigate the chaperone expression during an ER stress response, induced by thapsigargin in INS-1E cells, we used quantitative mass spectrometry based proteomics. The results displayed a decrease of GRP78/BiP, PDIA3 and PDIA6. Decrease of GRP78/BiP was verified by Western blot and occurred in parallel with enhanced levels of p-eIF2α and CHOP. In contrast to INS-1E cells, GRP78/BiP was not decreased in MIN6 cell or rat and mouse islets after thapsigargin exposure. Investigation of the decreased protein levels of GRP78/BiP indicates that this is not a consequence of reduced mRNA expression. Rather the reduction results from the combined effect of reduced protein synthesis and enhanced proteosomal degradation and possibly also degradation via autophagy. Induction of ER stress with thapsigargin leads to lower protein levels of GRP78/BiP, PDIA3 and PDIA6 in INS-1E cells which may contribute to the susceptibility of ER stress in this β-cell model.     

Mitochondrial protein patterns correlating with impaired insulin secretion from INS-1E cells exposed to elevated glucose concentrations

Extended hyperglycaemia leads to impaired glucose-stimulated insulin secretion (GSIS) and eventually beta-cell apoptosis in individuals with type 2 diabetes mellitus. In an attempt to dissect mechanisms behind the detrimental effects of glucose, we focused on measuring changes in expression patterns of mitochondrial proteins. Impaired GSIS was observed from INS-1E cells cultured for 5 days at 20 or 27 mM glucose compared to cells cultured at 5.5 or 11 mM glucose. After culture, mitochondria were isolated from the INS-1E cells by differential centrifugation. Proteins of the mitochondrial fraction were bound to a strong anionic surface (SAX2) protein array and mass spectra generated by SELDI-TOF-MS. Analysis of the spectra revealed proteins with expression levels that correlated with the glucose concentration of the culture medium. Indeed, such differentially expressed proteins created patterns of protein changes, which correlated with impairment of GSIS. In conclusion, the study reveals the first glucose-induced differentially expressed patterns of beta-cell mitochondrial proteins obtained by SELDI-TOF-MS.     

Inhibition of cyclic nucleotide phosphodiesterase during exposure to WI-38 cells to prostaglandin E1.

Short term incubation of WI-38 cultures with 5.7 micron prostaglandin E1 (PGE1) caused cyclic AMP phosphodiesterase activity in fibroblast homogenates to fall by 25 to 35% as compared to controls. The PGE1-induced decline in phosphodiesterase activity coincided with a rapid increase in intracellular cyclic AMP levels in response to the hormone and was rapidly reversed by washing the cultures free of the prostaglandin before homogenizing the cells. The effect of PGE1 on WI-38 phosphodiesterase activity was localized to the enzyme form(s) present in 27,000 times g supernatant fractions of cell homogenates. These data suggest that the pattern of cyclic AMP accumulation in WI-38 fibroblasts exposed to PGE1 may be related, at least in part, to decreased phosphodiesterase activity during hormone stimulation.     

Two-dimensional gel proteome reference map of INS-1E cells

The insulin-producing INS-1E rat cell line is widely used as a model for studying β-cells. It is a well-characterized cell line, mainly used in diabetes research. We established a 2-DE reference map for INS-1E cells. Using MALDI-TOF/TOF-MS/MS, we identified 546 spots. These included various proteins with an important role in β-cell physiology and with known roles as crucial proteins for diabetes development. We believe that the availability of this reference map will enhance our knowledge of β-cell physiology.     

Palmitate-induced changes in protein expression of insulin secreting INS-1E cells

Elevated blood levels of glucose and lipids in individuals with type 2 diabetes mellitus have been observed to cause impairment of insulin secretion from pancreatic β-cells. Chronic exposure to either of the circulating fatty acid oleate or palmitate has different effects on the β-cell. Whereas palmitate causes functional impairment of the β-cell and apoptosis, oleate has only minor negative effects on β-cell function and mass. The aim of the present study was to delineate mechanisms by which the fatty acids affect the β-cell differently. In particular, the aim was to identify β-cell proteins exclusively regulated by palmitate. INS-1E cells were cultured for 24 h in medium supplemented with palmitate or oleate. After culture, cells were lysed and subjected to two-dimensional gel electrophoresis. Proteins specifically regulated by palmitate were excised from the gel and identified by peptide mass fingerprinting using MALDI-TOF MS. Proteins exclusively regulated by palmitate were classified into proteins of carbohydrate or protein metabolism and Ca²⁺ or mRNA binding proteins. The specific palmitate-induced down-regulation of enzymes of glycolysis, proteins of protein turnover and anti-apoptotic protein may contribute to explain the different effects exerted by palmitate and oleate on β-cell function and mass.     

Different secretory response of pancreatic islets and insulin secreting cell lines INS-1 and INS-1E to osmotic stimuli

Objective of this study was to characterize osmotically-induced insulin secretion in two tumor cell lines. We compared response of freshly isolated rat pancreatic islets and INS-1 and INS-1E tumor cell lines to high glucose, 30 % hypotonic medium and 20 % hypertonic medium. In Ca(2+)-containing medium glucose induced insulin release in all three cell types. Hypotonicity induced insulin secretion from islets and INS-1 cells but not from INS-1E cells, in which secretion was inhibited despite similar increase in cell volume in both cell types. GdCl(3) (100 micromol/l) did not affect insulin response from INS-1E cells to hypotonic challenge. Hypertonic medium inhibited glucose-induced insulin secretion from islets but not from tumor cells. Noradrenaline (1 micromol/l) inhibited glucose-induced but not swelling-induced insulin secretion from INS-1 cells. Surprisingly, perifusion with Ca(2+)-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity while that of INS-1 cells was partially inhibited. Functioning glucose-induced insulin secretion is not sufficient prerequisite for hypotonicity-induced response in INS-1E cells suggesting that swelling-induced exocytosis is not essential step in the mechanism mediating glucose-induced insulin secretion. Both cell lines are resistant to inhibitory effect of hyperosmolarity on glucose-induced insulin secretion. Response of INS-1E cells to hypotonicity is inhibited by the presence of Ca(2+) in medium.     

Glucose induces anion conductance and cytosol-to-membrane transposition of ICln in INS-1E rat insulinoma cells.

The metabolic coupling of insulin secretion by pancreatic beta cells is mediated by membrane depolarization due to increased glucose-driven ATP production and closure of K(ATP) channels. Alternative pathways may involve the activation of anion channels by cell swelling upon glucose uptake. In INS-1E insulinoma cells superfusion with an isotonic solution containing 20 mM glucose or a 30% hypotonic solution leads to the activation of a chloride conductance with biophysical and pharmacological properties of anion currents activated in many other cell types during regulatory volume decrease (RVD), i.e. outward rectification, inactivation at positive membrane potentials and block by anion channel inhibitors like NPPB, DIDS, 4-hydroxytamoxifen and extracellular ATP. The current is not inhibited by tolbutamide and remains activated for at least 10 min when reducing the extracellular glucose concentration from 20 mM to 5 mM, but inactivates back to control levels when cells are exposed to a 20% hypertonic extracellular solution containing 20 mM glucose. This chloride current can likewise be induced by 20 mM 3-Omethylglucose, which is taken up but not metabolized by the cells, suggesting that cellular sugar uptake is involved in current activation. Fluorescence resonance energy transfer (FRET) experiments show that chloride current activation by 20 mM glucose and glucose-induced cell swelling are accompanied by a significant, transient redistribution of the membrane associated fraction of ICln, a multifunctional 'connector hub' protein involved in cell volume regulation and generation of RVD currents.     

Dynamic regulation of uncoupling protein 2 content in INS-1E insulinoma cells

Uncoupling protein 2 (UCP2) regulates glucose-stimulated insulin secretion in pancreatic beta-cells. UCP2 content, measured by calibrated immunoblot in INS-1E insulinoma cells (a pancreatic beta-cell model) grown in RPMI medium, and INS-1E mitochondria, was 2.0 ng/million cells (7.9 ng/mg mitochondrial protein). UCP2 content was lower in cells incubated without glutamine and higher in cells incubated with 20 mM glucose, and varied from 1.0-4.4 ng/million cells (2.7-14.5 ng/mg mitochondrial protein). This dynamic response to nutrients was achieved by varied expression rates against a background of a very short UCP2 protein half-life of about 1 h.     

Secreted PDZD2 exerts concentration-dependent effects on the proliferation of INS-1E cells

PDZD2 (PDZ domain containing 2) is a multi-PDZ protein expressed in pancreas and many other tissues. PDZD2 shows extensive homology to pro-interleukin-16 (pro-IL-16) and is localized mainly to the endoplasmic reticulum. We have recently demonstrated that PDZD2, like pro-IL-16, is proteolytically cleaved at its C-terminus to generate a secreted protein, sPDZD2 (for secreted PDZD2). To understand the possible functional role of PDZD2 in pancreas, we investigated the cellular distribution of PDZD2 in adult pancreas using an antiserum that recognizes both the full-length and secreted forms of PDZD2. Immunohistochemical analysis revealed a strong expression of PDZD2 in pancreatic islet beta cells but not alpha cells. Consistent with the beta-cell-enriched expression of PDZD2, immunoblot analysis indicated expression of both full-length PDZD2 and sPDZD2 in the insulinoma cell line INS-1E. A recombinant sPDZD2 protein was synthesized for study of its functional effect on INS-1E cells. In culture media with limiting serum, co-incubation with sPDZD2 stimulated the proliferation of INS-1E cells. The mitogenic effect of sPDZD2 was concentration-dependent, and was associated with a slight inhibition of the insulin promoter activity at high sPDZD2 concentrations. As a potential mitogen of beta-like cells, sPDZD2 may be useful for the optimization of beta-cell growth and differentiation in vitro.     

Glucose and GLP-1 stimulate cAMP production via distinct adenylyl cyclases in INS-1E insulinoma cells

In β cells, both glucose and hormones, such as GLP-1, stimulate production of the second messenger cAMP, but glucose and GLP-1 elicit distinct cellular responses. We now show in INS-1E insulinoma cells that glucose and GLP-1 produce cAMP with distinct kinetics via different adenylyl cyclases. GLP-1 induces a rapid cAMP signal mediated by G protein–responsive transmembrane adenylyl cyclases (tmAC). In contrast, glucose elicits a delayed cAMP rise mediated by bicarbonate, calcium, and ATP-sensitive soluble adenylyl cyclase (sAC). This glucose-induced, sAC-dependent cAMP rise is dependent upon calcium influx and is responsible for the glucose-induced activation of the mitogen-activated protein kinase (ERK1/2) pathway. These results demonstrate that sAC-generated and tmAC-generated cAMP define distinct signaling cascades.     

The impact of ANG II and IV on INS-1 cells and on blood glucose and plasma insulin

The impact of angiotensin (ANG) for peripheral, global effects is well known. Local ANG systems including that of the insulin-releasing β cell are not well investigated. In insulin-secreting cell line (INS-1), AT₁ and AT₄ receptors for ANG II and IV were demonstrated by Western blots. Only small amounts of ANG II-binding sites of low affinity were observed. ANG II and SARILE displaced binding of ¹²⁵I-ANG II. ANG II and IV as well as their non-degradable analogs SARILE and Nle-ANG IV increased the glucose-induced insulin release in a bell-shaped way; the maximum effect was at ～1?nM. The increase was antagonized by 1 µM losartan or 10 µM divalinal (AT₁ and AT₄ receptor antagonists, respectively). The insulin release was accompanied by a ⁴⁵Ca²⁺ uptake in the case of ANG II and ANG IV. Divalinal abolished the effect of ANG IV and Nle-ANG IV on this parameter. ANG IV reduced the increase in blood glucose during a glucose tolerance test with corresponding, albeit smaller effects on plasma insulin. Using confocal laser scanning microscopy, transfected insulin-regulated aminopeptidase (IRAP) with AT₄ receptors was shown to be accumulated close to the nucleus and the cytosolic membrane, whereas GLUT4 was not detectable. IRAP was inhibited by ANG IV. In conclusion, AT₁ and AT₄ receptors may be involved in diabetic homeostasis. Effects are mediated by insulin release, which is accompanied by an influx of extracellular Ca²⁺. The impact of ANG IV/IRAP agonists may be worth being used as antidiabetics.     

Connexin36 contributes to INS-1E cells survival through modulation of cytokine-induced oxidative stress,ER stress and AMPK activity

Cell-to-cell communication mediated by gap junctions made of Connexin36 (Cx36) contributes to pancreatic β-cell function. We have recently demonstrated that Cx36 also supports β-cell survival by a still unclear mechanism. Using specific Cx36 siRNAs or adenoviral vectors, we now show that Cx36 downregulation promotes apoptosis in INS-1E cells exposed to the pro-inflammatory cytokines (IL-1β, TNF-α and IFN-γ) involved at the onset of type 1 diabetes, whereas Cx36 overexpression protects against this effect. Cx36 overexpression also protects INS-1E cells against endoplasmic reticulum (ER) stress-mediated apoptosis, and alleviates the cytokine-induced production of reactive oxygen species, the depletion of the ER Ca²⁺ stores, the CHOP overexpression and the degradation of the anti-apoptotic protein Bcl-2 and Mcl-1. We further show that cytokines activate the AMP-dependent protein kinase (AMPK) in a NO-dependent and ER-stress-dependent manner and that AMPK inhibits Cx36 expression. Altogether, the data suggest that Cx36 is involved in Ca²⁺ homeostasis within the ER and that Cx36 expression is downregulated following ER stress and subsequent AMPK activation. As a result, cytokine-induced Cx36 downregulation elicits a positive feedback loop that amplifies ER stress and AMPK activation, leading to further Cx36 downregulation. The data reveal that Cx36 plays a central role in the oxidative stress and ER stress induced by cytokines and the subsequent regulation of AMPK activity, which in turn controls Cx36 expression and mitochondria-dependent apoptosis of insulin-producing cells.     

Identification of glucose response proteins in two biological models of beta-cell adaptation to chronic high glucose exposure.

High resolution 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with computerized analysis of gel images was used to search for proteins whose biosynthesis was induced or repressed in pancreatic islet cells chronically exposed to high glucose in an in situ and a tissue culture model of islet cell adaptation to excessive fuel load. The in situ model involved a 4-day intravenous infusion of either 50% glucose or 0.45% saline solution, followed by islet isolation, [35S]methionine labeling at 3 and 18 mM glucose for both groups, and protein analysis by 2-dimensional SDS-PAGE. The tissue culture model involved a 7-day culture of isolated rat islets in RPMI 1640 with 10% fetal calf serum containing either 3 or 30 mM glucose, followed by radiolabeling and 2-dimensional PAGE of proteins as in the in situ model. A small fraction of about 1.5% of the approximately 2000 identifiable proteins can be characterized as adaptive proteins. Of these altogether 58 proteins in the two models, 5 proteins were demonstrable in both models and two of these (proteins 1526 and 7622) are particularly noteworthy. Protein 1526 (Mr 57,000; pI 5.09) showed the same response pattern in both models and its expression was most enhanced when islets from chronically glucose-infused animals or those cultured for 7 days at 30 mM were radiolabeled at 18 mM glucose. Protein 7622 (Mr 68,000; pI 6.50) (also known as GSP-65; Collins, H.W., Buettger, C., and Matschinsky, F.M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5494-5498) showed a different labeling pattern in the two models: stimulation of [35S]methionine incorporation by 18 mM glucose both in control and experimental islets from the infusion study, but lack of such stimulation of radiolabeling in islets cultured for 7 days at 30 mM glucose in contrast to islets cultured at 3 mM. The experimental strategy and the methodology are evaluated and the significance of the results is discussed. Potentials of the approach and plans for future experiments are considered.     

IGFs, basic FGF, and glucose modulate proliferation and apoptosis induced by IFNgamma but not by IL-1beta in rat INS-1E beta-cells.

We investigated the effects of glucose and beta-cell growth factors (IGF-I, IGF-II, bFGF) on growth and apoptosis in the presence and absence of apoptosis inducing cytokines (IFNgamma, Il-1beta, TNFalpha). Rat INS-1E beta-cell viability was measured by WST-1 viability assay and cell counting, apoptosis by FACS analysis of annexin-V-FITC and fluorescein-dUTP (TUNEL-staining)-positive cells. Glucose alone maintained INS-1E beta-cell viability at high physiological concentrations (6.2-12.5 mmol/l), addition of IGF-II alone or in combination with bFGF further increased these glucose effects. The cytokines IFNg and IL-1beta, but not TNFalpha strongly induced INS-1E beta-cell apoptosis. Interestingly, glucose alone induced apoptosis at extremely low or very high concentrations. In combination with IFNg, low glucose (1.6 mmol/l) increased apoptosis by 25.6% (1SD 5.0%) and high glucose (50 mmol/l) by 22.8% (1SD 2.8%) compared to 12.5 mmol/l glucose. In contrast, glucose failed to modulate IL-1beta-induced apoptosis. Most importantly, IGF-II and bFGF inhibited apoptosis induced by IFNg, but not by IL-1beta. Therefore, IGF signaling, supported by bFGF and optimal glucose levels, maintains beta-cell viability in vitro. Cytokines IFNg and IL-1beta differentially interfere with intracellular signaling cascades stimulated by IGFs and bFGF or glucose, respectively.