Proprotein-processing endoprotease furin controls growth of pancreatic beta-cells

We have previously reported that in the well-differentiated beta-cell line MIN6 cells, the beta-cell-specific differentiated characteristics, such as insulin content, expression of prohormone convertases PC2 and PC3, and glucose-regulated insulin secretion, diminished when the proprotein-processing endoprotease furin was highly expressed. Since furin converts many growth-related protein precursors to their bioactive forms, we compared the four pancreatic islet cell lines RINm5F, betaTC3, betaHC9, and MIN6 with respect to cell growth rate, furin expression, endoprotease activity, and insulin content. RINm5F cells exhibited the strongest furin expression, higher furin-type endoprotease activity, and the fastest cell growth, but had the least insulin content. In contrast, MIN6 cells exhibited only a weak furin expression, little furin-type endoprotease activity, and the slowest cell growth, but had the highest insulin content. To test whether furin-expressing cells secrete growth-promoting factors cleaved by furin, we prepared conditioned media from RINm5F and furin cDNA-introduced MIN6 (MIN6-F) cells. The conditioned media from RINm5F and MIN6-F induced increased DNA synthesis and promoted the growth of normal MIN6 cells, compared with the medium from the empty vector-introduced MIN6-0 cells. We then examined the effect of the protease inhibitors alpha1-antitrypsin and its variants by infecting their vaccinia recombinants to the four cell lines. All conditioned media from each cell line expressing the furin-specific alpha1-antitrypsin variant exhibited the least DNA synthetic capacity on normal MIN6 cells. Furthermore, all three sublines of MIN6-F grew faster than MIN6-0 and MIN6. Thus, we suggest that the islet cells with higher furin expression may induce increased production of growth factors, which result in an increase in cell growth, through an autocrine/paracrine mechanism.     

Nutrient stimulation results in a rapid Ca2+-dependent threonine phosphorylation of myosin heavy chain in rat pancreatic islets and RINm5F cells

Activation of protein kinases plays an important role in the Ca2+-dependent stimulation of insulin secretion by nutrients. The aim of the present study was to identify kinase substrates with the potential to regulate secretion because these have been poorly defined. Nutrient stimulation of the rat insulinoma RINm5F cell line and rat pancreatic islets resulted in an increase in the threonine phosphorylation of a 200-kDa protein. This was secondary to the gating of voltage-dependent Ca2+ channels because it was reproduced by depolarizing KCl concentrations and blocked by the Ca2+ channel antagonist, verapamil. The peak rises in [Ca2+]i preceded or were coincident with the maximal threonine phosphorylation in response to both glyceraldehyde and KCl. In digitonin-permeabilized RINm5F cells a rise in Ca2+ from 0.1 to 0.15 microM was sufficient to increase phosphorylation. Protein kinase C, protein kinase A, and Ca2+/calmodulin-dependent kinase II did not appear to be responsible for the phosphorylation, yet the Ca2+ dependence of the response suggests possible involvement of other members of the Ca2+/calmodulin-dependent kinase family. The 200-kDa protein was identified as myosin heavy chain by immunoprecipitation with a polyclonal nonmuscle myosin antibody. Phosphopeptide mapping indicated that the site of phosphorylation on myosin heavy chain was the same for both KCl- and glyceraldehyde-stimulated cells. Phosphoamino acid analysis confirmed a low basal phosphothreonine content of myosin heavy chain, which increased 6-fold in response to KCl. A lesser (2-fold) increase in serine phosphorylation was also detected using this technique. Although myosin IIA and IIB were shown to be present in RINm5F cells and rat islets, myosin IIA was the predominant threonine-phosphorylated species, suggesting that the two myosin species might be independently regulated. Our results identify myosin heavy chain as a novel kinase substrate in pancreatic beta-cells and suggest that it might play an important role in the regulation of insulin secretion.     

Altered bcl-2 and bax expression and intracellular Ca2+ signaling in apoptosis of pancreatic cells and the impairment of glucose-induced insulin secretion

Apoptosis is the process of cellular self-destruction, and genes such as bcl-2 and bax are known to inhibit and promote apoptosis, respectively. In this study, we show that apoptosis can be induced in pancreatic beta-cell lines, and we investigate the apoptotic pathways through the bcl-2 and bax genes and intracellular Ca2+. Serum deprivation induces apoptosis in the MIN6 and RINm5F pancreatic beta-cell lines, and alters the bcl-2 messenger RNA (mRNA) and protein. KCl, BayK, A23187, and ionomycin elicit an elevation of cytosolic/nuclear Ca2+, which, however, is insufficient to evoke apoptosis or to alter bcl-2 or bax mRNA expression in MIN6 cells. The extracellular Ca2+ chelators, EGTA and 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetrapotassium salt, hydrate, evoke apoptosis and also alter the ratio of bcl-2 to bax mRNA and protein concomitantly with the depletion of cytosolic/nuclear Ca2+. This indicates that there are at least two apoptotic pathways in pancreatic beta-cells: through serum deprivation and through a decrease in cytosolic/nuclear Ca2+. MIN6 cells exhibit reduced insulin secretion induced by glucose regardless of the molecular pathway of apoptosis. Apoptosis in pancreatic beta-cells, therefore, may be closely related to the impairment of insulin secretion in certain pathological conditions such as diabetes mellitus.     

Thrombin-dependent inhibition of fibrinolysis

AIM: To study the mechanisms underlying oxytocin (Oxy)-induced insulin release. METHODS: In a clonal pancreatic beta-cell line, RINm5F cells. RESULTS: Oxy increased insulin release and [Ca2+]i in a concentration-dependent manner. Oxy-induced insulin release was not altered by pretreatment with pertussis toxin (PT). U-73122 (2-8 mumol.L-1), a phospholipase C (PLC) inhibitor, concentration-dependently inhibited Oxy-induced increases in [Ca2+]i with IC50 value of 2.8 +/- 0.2 mumol.L-1. In addition, U-73122 diminished the Oxy-induced increase in intracellular concentration of inositol 1, 4, 5-triphosphate (IP3). U-73122 at 8 mumol.L-1 totally abolished the Oxy-induced increases in [Ca2+]i and IP3; however it reduced the Oxy-induced increase in insulin release only by 36% and 63% in the monolayer and suspended cell preparations, respectively. CONCLUSION: Oxy increases insulin release through both PLC and non-PLC mediated signal transduction mechanisms.     

Effects of veratridine on the action potentials and contractility of right and left ventricles from normo- and hypertensive rats

Induction of nitric oxide synthase and generation of nitric oxide in pancreatic islet beta-cells may mediate cytokine-induced dysfunction leading to insulin-dependent diabetes mellitus. Nitric oxide generation can be regulated by availability of arginine substrate which, in turn, may be affected by substrate utilization in competing pathways such as the arginase-catalysed formation of ornithine and urea. In this study we have investigated the activity of arginase in the rat insulinoma-derived cell line RINm5F and the effect on this of interleukin 1beta, the nitric oxide synthase reaction intermediate NG-hydroxy-l-arginine and the nitric oxide-generating compounds 3-morpholinosydnonimine and S-nitrosoglutathione. Cytosols from RINm5F cells treated with or without interleukin 1beta (0.1nM, 18h) were incubated (45min, 37 degrees C) with [U-14C]arginine. Radiolabelled products ([14C]citrulline from nitric oxide synthase, [14C]ornithine and [14C]urea from arginase) were separated by high-performance liquid chromatography or ion-exchange chromatography. Interleukin 1beta increased citrulline production (from 0.01+/-0.002 to 0.58+/-0.03 pmol/microg cell protein), indicating induction of nitric oxide synthase, and significantly decreased production of both ornithine (from 4.60+/-0.20 to 3.40+/-0.20 pmol/microg) and urea (0.93+/-0.05 to 0.69+/-0.04 pmol/microg) (P<0.001), indicating decreased activity of arginase. Arginase was significantly inhibited by NG-hydroxy-l-arginine (IC50=50 microM), S-nitrosoglutathione (500 microM: 69+/-7% of control) and 3-morpholinosydnonimine (1 mM: 57+/-7% of control) (P<0.05). We conclude that during cytokine-directed beta-cell assault nitric oxide synthase-catalysed production of NG-hydroxy-l-arginine and nitric oxide may inhibit arginase thereby increasing the availability of arginine for nitric oxide production.     

Endoproteolysis of glucagon-like peptide (GLP)-1 (7-36) amide by ectopeptidases in RINm5F cells

This study concerns whether the pancreatic beta cell expresses cell-surface ectopeptidases that are capable of proteolysis of peptide hormones and neuropeptides that modify glucose-dependent insulin release. These biochemical investigations of the RINm5F cell line found that these cells express ectopeptidases. We have characterized the limited endoproteolysis of GLP-1 (7-36) amide that occurs in the presence of RINm5F plasma membranes. The products and the sensitivity to specific peptidase inhibitors of the proteolysis is characteristic of neutral endopeptidase (NEP) 24.11. Vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), amylin, glucagon, glucose-dependent insulinotropic polypeptide (GIP), and exendin-4 also undergo proteolysis in the presence of RIN cell membranes. NEP 24.11-activity in RIN cell membranes was confirmed using a specific fluorogenic assay, by histochemistry, and by comparison with the recombinant enzyme with respect to the kinetics of proteolysis of GLP-1 (7-36) amide and of a fluorogenic substrate. Specific fluorogenic assays revealed the presence of aminopeptidase N and the absence of aminopeptidase A and of dipeptidylpeptidase IV.     

Identification of a quantitative trait locus influencing plasma insulin levels after 70% pancreatectomy using the OLETF rat

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese-type non-insulin-dependent diabetes mellitus (NIDDM) in humans. The OLETF rat has poor capacity for proliferation of pancreatic beta-cells after partial pancreatectomy, which may be the critical pathogenetic event in NIDDM development. The poor pancreatic beta-cell proliferation in this model is characterized by reduction in beta-cell mass and decrease in insulin content in the remnant pancreas. Our investigation was designed to identify quantitative trait loci (QTLs) responsible for beta-cell mass and plasma insulin levels after partial pancreatectomy by performing a genome-wide scan in an F2 intercross obtained by mating the OLETF and the Fischer-344 (F344) rats. We have identified a suggestive QTL for the plasma insulin levels, near D20Mgh5 on rat chromosome 20, with a maximum lod score of 3.75 which accounts for 20% of the total variance, while no QTLs were detected for beta-cell mass. This chromosome 20 QTL, whose OLETF allele is associated with low plasma insulin levels through acting in an incompletely recessive manner, may affect insulin secretion itself rather than beta-cell proliferation.     

In utero undernutrition impairs rat beta-cell development

The role of nutrition on the development of the endocrine pancreas was studied in a rat model obtained by maternal food restriction. A 50% food restriction was applied to female rats from day 15 of pregnancy and resulted in intrauterine growth-retardation (IUGR) in the offspring. At day 1 postnatal, beta-cell mass was significantly decreased in IUGR pups as compared to controls (0.70 +/- 0.06 vs 1.07 +/- 0.06 mg, p < 0.0001), as well as insulin content. This change in beta-cell mass can be attributed to a reduced number of islets, since the density of insulin-positive aggregates in pancreatic sections of IUGR rats was 20% lower than in controls. Proliferative capacity of beta cells, as measured by 5-bromo-2-deoxyuridine (BrdU) labelling index, was not altered in growth-retarded animals. Body as well as pancreatic weight were fully recovered in IUGR pups after 21 days of normal feeding by control mothers. However, these animals retained a 25% decrease in insulin content, 40% decrease in beta-cell mass (1.58 +/- 0.18 vs 2.78 +/- 0.42 mg, p < 0.001) and a strong reduction in the density of insulin positive aggregates per cm2, as compared to controls, suggesting that the total islet number was likely to be reduced. Beta-cell proliferative capacity remained normal. In conclusion, in utero undernutrition in rats does not impede postnatal growth but durably impairs beta-cell development. Impairment of beta-cell differentiation might be suggested.     

Glucose stimulation of pancreatic beta-cell lines induces expression and secretion of dynorphin

To investigate adaptive responses of pancreatic beta-cells to hyperglycemia, genes induced by glucose stimulation were identified by subtraction cloning. Among 53 clones representing differentially expressed genes, 20 encoded the endogenous opioid precursor, prodynorphin. The amino acid sequence of murine prodynorphin is identical to the rat protein in sequences comprising the opioid peptides and 86% identical in the remainder of the molecule. Stimulation of MIN6 cells increased prodynorphin RNA levels to more than 20-fold in proportion to physiological glucose concentrations. Similar induction levels were observed in murine betaTC3 and rat Rinm5F beta-cell lines. Prodynorphin RNA expression increased within 1 h of glucose stimulation, achieved maximal levels by 4 h, and remained elevated for at least 24 h. By using RIA, MIN6 cells were shown to contain and secrete increased amounts of dynorphin-A following glucose stimulation. Treatment of MIN6 cells with KCl, forskolin, or isobutyl-methyl-xanthine strongly induced prodynorphin RNA expression, suggesting that induction may be related to secretion-coupled signaling pathways. The induction of prodynorphin in several beta-cell lines is consistent with previous demonstrations of beta-cell synthesis of other endogenous opioids, including beta-endorphin, and suggests that opioids may have a potentially significant role in regulating beta-cell secretion.     

Regulation of insulinoma cell proliferation and insulin accumulation by peptides and second messengers

The regulation of clonal rat insulinoma (RINm5F) cell proliferation and hormone accumulation was investigated with the aim of identifying putative compounds capable of inducing differentiation, i.e. decreased growth and increased insulin accumulation, by the tumor cells. In particular, interest was focused on the role of a number of peptides as well as pharmacological probes modulating various signal transduction systems and which have been shown to regulate normal beta-cell proliferation and insulin accumulation. Growth hormone stimulated insulin accumulation and inhibited DNA synthesis, whereas galanin and insulin-like growth factor I caused a moderate suppression of insulin accumulation but did not affect proliferation, while epidermal growth factor, transforming growth factor beta, platelet-derived growth factor, acidic and basic fibroblast growth factor, bradykinin and somatostatin were virtually inactive on all parameters tested. Exogenous prostaglandins E2 and F1 alpha were inactive, while the cycloxygenase inhibitor indomethacin slightly suppressed insulin accumulation. The cytokine IL-1 beta caused a significant decrease in both beta-cell mitogenesis and insulin accumulation, effects that were mediated through nitric oxide generation. The vitamin A derivative retinyl acetate slightly inhibited serum-stimulated DNA synthesis, but did not affect insulin accumulation. The vitamin E alpha-tocopherol significantly enhanced insulin release but did not affect mitogenesis. By contrast, gamma-tocopherol was inactive on both these parameters. The alpha-adrenergic agonist clonidine evoked a slight inhibition of serum-stimulated DNA synthesis, without influencing insulin accumulation, whereas phenylephrine did not affect any of these parameters. Carbamylcholine increased insulin accumulation, but not cell proliferation, whereas the adenylyl cyclase activator forskolin suppressed mitogenesis but did not affect insulin accumulation. Inhibition of protein kinase C with staurosporine or prolonged treatment with phorbol ester suppressed DNA synthesis, as did the tyrosine kinase inhibitor genistein. Stimulating Ca2+ influx by closing ATP-dependent K+ channels with glibenclamide enhanced DNA synthesis, while opening of these channels with diazoxide suppressed cell growth. Conversely, preventing Ca2+ influx by the Ca2+ channel antagonist D-600, chelating intracellular Ca2+ by fura-2 AM or inhibiting the Ca2+/calmodulin-dependent protein kinase by calmidazol resulted in a decreased DNA synthesis. On the other hand, uncontrolled influx or mobilization of Ca2+ by ionomycin or thapsigargin resulted in an arrested DNA synthesis. The present paper shows that RINm5F insulinoma cell proliferation and insulin accumulation can be modulated by various peptidergic and pharmacological agents regulating certain signal transduction pathways. However, mitogenesis in the insulinoma cells seemingly is controlled in a vastly different manner in comparison to that in normal beta-cells. The most spectacular finding in this screening study, i.e. that growth hormone, contrarily to its effect on normal beta-cells, suppresses insulinoma cell growth, merits further elucidation of the underlying mechanisms. Possibly the hormone might become of utility in a clinical setting in the treatment of patients with insulin-producing tumors.     

Inhibition of insulin release by a formamidine pesticide amitraz and its metabolites in a rat beta-cell line: an action mediated by alpha-2 adrenoceptors, a GTP-binding protein and a decrease in cyclic AMP

We tested the hypothesis that the formamidine pesticide amitraz and its metabolites inhibit insulin release from a rat beta-cell line (RINm5F) by decreasing intracellular cyclic AMP levels and examined whether GTP-binding proteins were involved in the process. Amitraz and its active metabolite BTS27271 (0.1-10 microM) inhibited insulin release that was induced by 100 microM of IBMX in a dose-dependent manner. Other metabolites of amitraz tested failed to inhibit insulin release. A potent and specific alpha-2 adrenoceptor agonist, medetomidine (0.1 microM), abolished the IBMX-induced insulin release. Amitraz, BTS27271 and medetomidine also decreased intracellular cyclic AMP concentrations that were elevated by IBMX administration. Moreover, all three drugs inhibited the insulin release stimulated by forskolin (1 microM), an adenylyl cyclase activator. Yohimbine (0.01, 0.1 and 1 microM), an alpha-2 adrenoceptor antagonist, prevented the inhibitory effect of amitraz and BTS27271 on insulin release, whereas prazosin (1 microM), an alpha-1 adrenoceptor antagonist, did not. Yohimbine, but not prazosin, also prevented the effect of amitraz, BTS27271 and medetomidine on IBMX-stimulated accumulation of cyclic AMP. Pretreatment of cells with PTX (0.1 micrograms/ml) for 16 h, antagonized the effects of amitraz and BTS27271 on IBMX-induced increase in insulin release. Thus, one mechanism by which amitraz and its metabolite BTS27271 decrease insulin release is by inhibiting adenylyl cyclase, an action mediated through a PTX-sensitive G-protein.     

Insulinoma cells contain an isoform of Ca2+/calmodulin-dependent protein kinase II delta associated with insulin secretion vesicles

The Ca2+/calmodulin dependent protein kinase II (CaM kinase II) is thought to play an important part in glucose-stimulated insulin secretion. To determine which of the known subtypes (alpha, beta, gamma, delta) occur in insulin-secreting cells, we amplified all types of CaM kinase II by RT-PCR and found the beta3-, gamma-, delta2- and delta6-subtypes in RINm5F insulinoma cells. None of the other 8 delta-subtypes was present. Antibodies generated against the bacterially expressed association domain of the delta2-subtype recognized the recombinant gamma and delta-subtypes. In INS-1 and RINm5F cells, as well as freshly isolated rat islets, only a 55-kDa protein corresponding in size to the delta2-subtype expressed in NIH3T3 fibroblasts was detected. The delta2-subtype therefore appears to represent the predominant subtype of CaM kinase II present in insulin secreting cells. The enzyme was primarily associated with cytoskeletal structures, and very little was present in the soluble compartment or detergent soluble fraction in INS-1- or RINm5F-cells. An analysis of its subcellular distribution was performed by sucrose and Nycodenz density gradient fractionation of INS-1 cells and detection of CaM kinase II delta by immune blots. The enzyme codistributed with insulin used as a marker for secretory granules but not with the lighter synaptic-like microvesicles detected with an antibody against synaptophysin, plasma membranes (syntaxin 1), lysosomes (arylsulfatase), or mitochondria (cytochrome c oxidase). CaM kinase II delta2 thus is identified as the subtype associated with insulin secretory granules and is likely to be involved in insulin secretion.