Distribution of somatostatin receptors in RINm5F insulinoma cells

Previous studies with heterogeneous populations of pancreatic cells have provided evidence for the presence of somatostatin (SRIF) receptors in cytosol and secretion vesicles, as well as the plasma membrane. To examine the distribution of SRIF receptors between soluble and membrane fractions in a homogeneous pancreatic islet cell population, we have used the clonal RINm5F insulinoma cell line. These cells contain specific, high affinity binding sites for [125I-Try11]SRIF on the cell surface, and occupancy of these sites by SRIF and SRIF analogs correlates with inhibition of insulin secretion. Stable, steady state binding was achieved using both intact cells and membranes by performing binding incubations with [25I-Tyr11]SRIF at 22 C. Half-maximal inhibition of [125I-Tyr11]SRIF binding occurred with 0.21 +/- 0.11 nM SRIF in membranes and 0.35 +/- 0.30 nM SRIF in cells. In contrast, the binding of [125I-Tyr11]SRIF to cytosolic macromolecules was not reduced by concentrations of SRIF as high as 100 nM, demonstrating that this binding was of much lower affinity. RINm5F membranes were further purified using a Percoll gradient to prepare a microsomal fraction, which was enriched in adenylate cyclase activity, and a secretory granule fraction, which was enriched in insulin. [125I-Tyr11]SRIF binding to the microsomal fraction (3.8 +/- 0.3 fmol/mg) was 3 times higher than to secretion granules (1.2 +/- 0.2 fmol/mg). Thus, high affinity SRIF binding sites were most abundant in microsomal membranes and were low or undetectable in secretory granules and cytosol. To determine whether translocation of SRIF receptors to the plasma membrane accompanied insulin secretion, we examined the effects of various insulin secretagogues on [125I-Tyr11]SRIF binding to intact cells. Leucine (20 mM), glyceraldehyde (15 mM), forskolin (1 microM), and glucagon (1 microM) stimulated insulin release 1.5- to 4.0-fold in different experiments. However, these secretagogues did not increase [125I-Tyr11]SRIF binding. In summary, our results indicate that high affinity SRIF receptors in RINm5F cells are located primarily on the plasma membrane and that the concentration of SRIF receptors at the cell surface is independent of the secretory activity of the cells.     

Tumour formation and insulin secretion by clonal RINm5F cells following repeated subcutaneous transplantation in NEDH rats

The effects of repeated s.c. transplantation of clonal insulin-secreting RINm5F cells in NEDH rats on tumour morphology, insulin-glucose homeostasis and the function of RINm5F cells re-established in culture were examined after maintenance in vivo for periods of up to 308 days. Transplantation of RINm5F cells for ten consecutive passages consistently produced a single encapsulated vascularized tumour associated with the development in recipient rats of hyperinsulinaemia, hypoglycaemia and eventual neuroglycopenic coma. At the tenth passage, tumour-bearing rats exhibited a markedly enhanced rate of insulin-stimulated glucose uptake by 19 days, with evidence of a large and variable insulin response to i.p. glucose. Survival was 22 +/- 2 days, and resected RINm5F cell tumours exhibited weak immunostaining for insulin in scattered cells, with strands of fibrous tissue separating clusters of tumour cells many of which had distinct polarity. There was no detectable immunostaining for glucagon, somatostatin or pancreatic polypeptide. The insulin content and insulin secretory output of RINm5F cells re-established in culture after 20, 146, 259 or 308 days propagation in vivo were generally enhanced compared with non-passaged RINm5F cells. The magnitude of the effect was not appreciably affected by the duration of maintenance in vivo, but it was critically dependent upon the subsequent period of culture in vitro. Thus, whereas 2-day cultured RINm5F cells from the eighth tumour passage exhibited a greater than 100-fold increment of insulin content and release, with enhanced secretory responsiveness to leucine, arginine, theophylline, K+ (25 mmol/l) or Ca2+ (7.6 mmol/l), RINm5F cells cultured for a further 19 days had almost completely lost the attributes resulting from 259 days of maintenance in vivo. The results indicate substantial enhancement of the functional capabilities of RINm5F cells in vivo, and suggest that the resulting tumours afford a novel model in NEDH rats of responsive trabecular-type insulinomas.     

Stimulation of insulin release is uncoupled from insulin biosynthesis in tumoral RINm5F cells.

RINm5F cells, an insulin-secreting subclone of a rat insulinoma cell line, were incubated in serum-free medium up to 24 hours in the presence or absence of glucagonlike peptide-1(7-36)amide in various concentrations, 3-isobutyl-1 methylxanthine (1 mM), choleratoxin (10 nM), carbachol (1 mM), and potassium (40 mM). Insulin release and biosynthesis were measured by the immunoreactive insulin content of the cells and the medium. Steady-state levels of insulin-specific mRNA were determined by Northern and slot blot analysis. Short-term insulin release is significantly stimulated by all secretagogues tested. A significant increase of insulin biosynthesis by any of the various secretagogues was not detectable on the peptide and mRNA level. Sodium butyrate (1 mM), a differentiating agent, increased insulin-specific mRNA levels in RINm5F cells after 72 hours. In conclusion, substances known to stimulate short-term insulin release in RINm5F cells do not stimulate insulin biosynthesis, indicating an uncoupling of insulin secretion and biosynthesis in these transformed beta cells.     

Pancreastatin increases cytosolic Ca2+ in insulin secreting RINm5F cells.

We have investigated the effect of pancreastatin on cytosolic Ca2+ concentration in the insulin secreting cell line RINm5F. Changes in [Ca2+]i induced by pancreastatin were detected by Fluo-3 fluorescence using both flow cytometry and batch analysis measurements, and turned out to be from 90 to 315 nM equivalent to 80% of that caused by ATP, which increased [Ca2+]i from 90 nM to 400 nM. This effect of pancreastatin did not depend on extracellular calcium and was not mediated by alpha-adrenergic receptors since it was not prevented by the alpha-blocker yohimbine. It is concluded that pancreastatin has a role in the homeostasis of free cytosolic calcium in the insulin secreting cell line Rinm5F.     

Magnesium requirement for somatostatin inhibition of insulin secretion

Rat pancreas perfusions were performed using a perfusate with a fixed calcium concentration of 5 mEq/l and magnesium varying from 0 to 0.6 mEq/dl. Insulin secretion was stimulated by a constant glucose infusion of 300 mg/dl. This glucose concentration produces the typical biphasic insulin secretory response. We observed that in the absence of magnesium, somatostatin concentrations of 0.5 and 2.0 ng/ml were without effect on first phase insulin secretion. However, these same somatostatin levels produced 50% or more inhibition of insulin secretion in the presence of magnesium at 0.3 or 0.6 mEq/l. Similarly, in the absence of magnesium, somatostatin at 50 ng/ml failed to inhibit second phase insulin secretion, whereas this same somatostatin level produced about 50% inhibition of insulin secretion in the presence of magnesium at 0.3 mEq/l. Thus, altering perfusate magnesium concentrations without changing calcium is an important determinant of the degree of inhibition of secretion produced by somatostatin. In particular, in the absence of magnesium ion, somatostatin concentrations which would 'normally' produce 50% inhibition of secretion (ID50) are without effect. Therefore, magnesium ion is necessary for the full inhibitory effect of somatostatin to occur. These results suggest that inhibitors, as well as potentiators, of the insulin secretory process may act by altering intracellular/membrane calcium-magnesium ratios, but in opposite directions.     

Improved metabolic stimulus for glucose-induced insulin secretion through GK and PFK-2/FBPase-2 coexpression in insulin-producing RINm5F cells

The glucose sensor enzyme glucokinase plays a pivotal role in the regulation of glucose-induced insulin secretion in pancreatic beta-cells. Activation of glucokinase represents a promising concept for the treatment of type 2 diabetes. Therefore, we analyzed the glucokinase activation through its physiological interaction partner, the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) and the resulting effect on glucose metabolism in insulin-producing cells. In RINm5F-GK-PFK-2/FBPase-2 cells stably overexpressing glucokinase plus islet PFK-2/FBPase-2, colocalization between both enzymes as well as elevation of glucokinase activity were significantly increased at a stimulatory glucose concentration of 10 mmol/liter. RINm5F-GK-PFK-2/FBPase-2 cells showed under this culture condition a significant increase in glucose utilization and in the ATP/ADP ratio compared with RINm5F-GK cells, which only overexpress glucokinase. Also glucose-induced insulin secretion was elevated in RINm5F-GK-PFK-2/FBPase-2 cells in comparison to RINm5F-GK cells. Furthermore, pyruvate accumulation and lactate production in RINm5F-GK-PFK-2/FBPase-2 cells were significantly lower at both 10 and 30 mmol/liter glucose than in RINm5F-GK and RINm5F cells. The significant improvement of glucose metabolism after PFK-2/FBPase-2 overexpression is apparently not exclusively the result of high glucokinase enzyme activity. Stabilization of the closed glucokinase conformation by PFK-2/FBPase-2 may not only activate the enzyme but also improve metabolic channeling in beta-cells.     

Effects of islet hormones on insulin secretion from cloned B cell lines, HIT-T15 and RINm5F

The effects of the islet cell hormones glucagon, somatostatin-28 and pancreatic polypeptide on insulin secretion from cultured cloned pancreatic B cells (HIT-T15 and RINm5F) have been investigated. Glucagon stimulates the secretion of insulin from HIT-T15 cells in the absence and presence of glucose and from RINm5F cells in the absence and presence of glyceraldehyde. HIT-T15 cells were more sensitive to the stimulatory effect of glucagon than RINm5F cells. Somatostatin-28 and pancreatic polypeptide, both alone and in combination, reduced glucose- and glucagon-stimulated insulin release from HIT-T15 cells and glyceraldehyde- and glucagon-stimulated insulin release from RINm5F cells. HIT-T15 cells were more sensitive to the inhibitory actions of somatostatin-28 and pancreatic polypeptide than RINm5F cells. This study supports the hypothesis that insulin release from normal B cells may be modified by the paracrine activity of islet hormones, glucagon, somatostatin and pancreatic polypeptide and probably occurs before any fine tuning imposed by subsequently released insulin.     

Does somatostatin inhibition of insulin secretion involve two mechanisms of action?

Somatostatin, the hypothalamic growth hormone release inhibitory factor (GHRIF), directly inhibits both the first and second phases of insulin secretion. The sensitivities of these two phases of insulin secretion to somatostatin differ remarkably. The first phase of secretion is approximately 25 to 50 times more sensitive to somatostatin inhibition than is the second phase. In addition, somatostatin inhibition of insulin secretion during the second phase is "reversed" by supplemental calcium, whereas the somatostatin effect on the first phase is unaffected by additional calcium. These findings suggest that the cellular events which produce the two phases of insulin secretion are separate processes, and that somatostatin has a dual mechanism of action in inhibiting insulin secretion.     

Cytokines activate genes of the endocytotic pathway in insulin-producing RINm5F cells

Aims/hypothesis Cytokines are important humoral mediators of beta cell destruction in autoimmune diabetes. The aim of this study was to identify novel cytokine-induced genes in insulin-producing RINm5F cells, which may contribute to beta cell death or survival.Methods A global gene expression profile in cytokine-exposed insulin-producing RINm5F cells was achieved by automated restriction fragment differential display PCR. The expression of selected candidate genes was confirmed by real-time RT-PCR analysis.Results Exposure of RINm5F cells to IL-1 or to a cytokine mixture (IL-1, TNF-, IFN-) for 6 h resulted in the differential expression of a functional gene cluster. Apart from the well-known up-regulation of the cytokine-responsive genes iNOS, NF-B, MnSOD and Hsp70, several genes that belong to the functional cluster of the endocytotic pathway were identified. These endocytotic genes comprised: clathrin, megalin, synaptotagmin and calcineurin, which were up-regulated by IL-1 or the cytokine mixture. In contrast, the expression of the calcineurin inhibitor CAIN and of the GDP/GTP exchange protein Rab3 was down-regulated by cytokines. Other up-regulated cytokine-responsive genes were: agrin, murine adherent macrophage protein mRNA (MAMA) and transport-associated protein (TAP1/MTP), whereas the plasma membrane calcium ATPase (PMCA) 2 and PMCA 3 genes were down-regulated by cytokines.Conclusions/interpretation Our results indicate that genes of the endocytotic pathway are regulated by pro-inflammatory cytokines. This might affect the density of cytokine receptors at the beta cell surface and concomitantly the sensitivity of the cells to cytokine toxicity. A better understanding of the functional cross-talk between endocytotic and cytokine signalling pathways could further the development of novel strategies to protect pancreatic beta cells against toxic effects of pro-inflammatory cytokines.Electronic Supplementary Material Electronic supplementary material is available in the online version of this article at Abbreviations CAIN calcineurin inhibitor - EP extension-protected adaptor - Hsp70 heat shock protein 70 - iNOS inducible nitric oxide synthase - MAMA murine adherent macrophage protein - MnSOD manganese superoxide dismutase - NF-B nuclear factor kappa B - NO nitric oxide - PMCA plasma membrane calcium ATPase - Rab3 rab3 GDP/GTP exchange protein - RFDD-PCR restriction fragment differential display PCR - SD standard adaptor - Syt synaptotagmin V - TAP1 transport-associated protein 1     

Cholinergic stimulation of insulin release from cloned B-cell lines HIT-T15 and RINm5F

Summary Acetylcholine stimulated the release of insulin from cultured HIT-T15 and RINm5F cells in the absence of glucose confirming that the insulin response to cholinergic stimulation is preserved in these two cloned B-cell lines. In addition acetylcholine potentiated glucose stimulated insulin release from HIT-T15 but not from RINm5F cells. Perifused HIT-T15 cells responded to square wave acetylcholine challenge with a monophasic release of insulin while the release profile for RINm5F cells was irregular and reduced. Acetylcholine stimulated insulin release from both cell lines was reduced by atropine but unaffected by hexamethonium confirming a mechanism of action involving muscarinic receptor activation.     

Suppression of endogenous insulin secretion by exogenous insulin in patients with insulinoma

OBJECTIVE: Previous studies have demonstrated that endogenous insulin secretion is not suppressed by exogenous insulin in patients with insulinoma. In this study we examined whether insulin secretion in insulinoma patients is suppressed by exogenous insulin during hypoglycaemia. SUBJECTS AND METHODS: Sixteen insulinoma patients (5 men and 11 women) and 10 normal subjects were studied. Hyperinsulinaemic glucose clamp studies were performed at both euglycaemia (4.5 mmol/l glucose) and hypoglycaemia (2.5 mmol/l glucose). RESULTS: In normal subjects, plasma C-peptide levels were suppressed by 66% during the euglycaemic hyperinsulinaemic clamps (P < 0.01). In contrast, in insulinoma patients, plasma C-peptide levels increased by 25% during the clamps (P < 0.05). In the hypoglycaemic hyperinsulinaemic clamps, plasma C-peptide levels were nearly completely (91%) suppressed in normal subjects and partially (39%) suppressed in patients with insulinoma (P < 0.01). The decrease in C-peptide levels during the hypoglycaemic clamps was > 30% in 12 (75%) of 16 insulinoma patients and > 50% in 8 (50%) patients. CONCLUSIONS: This study demonstrated that in patients with insulinoma, insulin secretion was not suppressed by exogenous insulin during euglycaemia but was suppressed during hypoglycaemia, although the degree of suppression was less than that in normal subjects. Our results suggest that the feedback regulation of insulin secretion by exogenous insulin is partially retained in patients with insulinoma.     

Characteristics of the receptors which mediate the stimulation of ACTH secretion by vasopressin in conscious dogs

In order to characterize the receptors which mediate the adrenocorticotropic hormone (ACTH) response to vasopressin in conscious animals, plasma 11-hydroxycorticosteroid concentration was measured in conscious dogs during the infusion of vasopressin or structural analogs of vasopressin which exhibit selective antidiuretic or vasoconstrictor activity. Vasopressin (1.0 ng/kg/min for 60 min) increased mean arterial pressure, decreased heart rate and increased plasma corticosteroid concentration from 1.0 +/- 0.2 to 2.2 +/- 0.2 micrograms/dl (p less than 0.001). A specific antagonist of the vasoconstrictor activity of vasopressin, d(CH2)5MeTyrAVP (10 micrograms/kg), completely blocked the cardiovascular and corticosteroid responses to vasopressin. A selective vasoconstrictor (V1) agonist, PheOrnOT (1.0 ng/kg/min), which produced the same cardiovascular responses as vasopressin, increased plasma corticosteroid concentration from 1.1 +/- 0.1 to 2.9 +/- 0.9 micrograms/dl (p less than 0.005). In marked contrast, a selective antidiuretic (V2) agonist, dDAVP (1.0 ng/kg/min) had no effect on blood pressure, heart rate or plasma corticosteroid concentration. These results indicate that the stimulation of ACTH release by vasopressin in conscious dogs is mediated by receptors which resemble vasoconstrictor-type (V1) receptors rather than antidiuretic-type (V2) receptors.     

Inhibition of glucose stimulated insulin secretion by neuropeptide Y is mediated via the Y1 receptor and inhibition of adenylyl cyclase in RIN 5AH rat insulinoma cells

Summary Neuropeptide Y (NPY) has been shown to inhibit insulin secretion from the islets of Langerhans. We show that insulin secretion in the insulinoma cell line RIN 5AH is inhibited by NPY. ¹²⁵I-Peptide YY (PYY) saturation and competition-binding studies using NPY fragments and analogues on membranes prepared from this cell line show the presence of a single class of NPY receptor with a Y1 receptor subtype-like profile. Inhibition of insulin secretion in this cell line by NPY fragments and analogues also shows a Y1 receptor-like profile. Both receptor binding and inhibition of insulin secretion showed the same orders of potency with NPY > [Pro³⁴]-NPY > NPY 3–36 > > NPY 13–36. The Y1 receptor antagonist, BIBP 3226, blocks NPY inhibition of insulin secretion from, and inhibits ¹²⁵I-PYY binding to, RIN 5AH cells. Northern blot analysis using a Y1-receptor specific probe shows that NPY Y1 receptors are expressed by RIN 5AH cells. Y5 receptors are not expressed in this cell line. Neuropeptide Y inhibition of insulin secretion is blocked by incubation with pertussis toxin, implying that the effect is via a G-protein (G_i or G_o) coupled receptor. Neuropeptide Y inhibits the activation of adenylyl cyclase by isoprenaline in RIN 5AH cell lysates, and the stimulation of cAMP by glucagon-like peptide-1 (7–36) amide (GLP-1). It also blocks insulin secretion stimulated by GLP-1, but not by dibutyryl cyclic AMP. Hence, we suggest that NPY inhibits insulin secretion from RIN 5AH cells via a Y1 receptor linked through G_i to the inhibition of adenylyl cyclase. [Diabetologia (1998) 41: 1482–1491] Received: 10 November 1997 and in final revised form: 16 June 1998     

Gamma-aminobutyric acidA receptors mediate 3 alpha-hydroxy-5 alpha-pregnan-20-one-induced gonadotropin secretion

3 alpha-Hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-THP), can selectively release LH in estrogen-primed ovariectomized rats. This progesterone metabolite does not bind to the progesterone receptor. Recently, 3 alpha,5 alpha-THP has been reported to be a potent modulator of the gamma-aminobutyric acidA (GABAA) receptor in the brain. Therefore, the purpose of this study was to determine whether 3 alpha,5 alpha-THP's effect on gonadotropin secretion is GABAA receptor mediated. Ovariectomized immature rats were primed for 2 days with estradiol (2 micrograms/rat.day). On the morning of the third day, 3 alpha,5 alpha-THP was administered either with or without prior treatment with progesterone receptor antagonist (RU486) or the GABAA receptor antagonist picrotoxin. When 3 alpha,5 alpha-THP was administered alone, a dose-related effect on LH and FSH release was observed. The 0.8 mg/kg BW dose of 3 alpha,5 alpha-THP stimulated both LH and FSH release, whereas the 1.6 mg/kg BW dose released only LH. The GABAA receptor antagonist picrotoxin had no significant effect on LH or FSH secretion. However, administration of picrotoxin 30 min before 0.8 mg/kg BW 3 alpha,5 alpha-THP resulted in antagonism of 3 alpha,5 alpha-THP's ability to release LH and FSH. The effects of 1.6 mg/kg BW 3 alpha,5 alpha-THP on serum LH were also blocked by picrotoxin. Picrotoxin was ineffective in altering the gonadotropin-stimulating response of progesterone and deoxycorticosterone. These steroids do not bind to the GABAA receptor. The progesterone receptor antagonist RU486 administered alone had no effect on serum LH or FSH levels. When RU486 was administered before 3 alpha,5 alpha-THP treatment, it was ineffective in blocking 3 alpha,5 alpha-THP's ability to release LH. These studies indicate that the GABAA receptor is responsible for mediating 3 alpha,5 alpha-THP-induced gonadotropin secretion.     

L-arginine stimulates cyclic guanosine 3',5'-monophosphate formation in rat islets of Langerhans and RINm5F insulinoma cells: evidence for L-arginine:nitric oxide synthase.

L-Arginine (L-Arg) is metabolized by nitric oxide synthase to the reactive intermediate nitric oxide. Since nitric oxide stimulates guanylyl cyclase and cGMP synthesis, L-Arg effects on cGMP accumulation in isolated pancreatic islets of the rat and RINm5F insulinoma cells were determined. Both L-Arg and glucose stimulation increased islet cGMP levels, and glucose potentiated the response to L-Arg alone. A competitive inhibitor of L-Arg metabolism to nitric oxide, NG-monomethyl-L-arginine, reduced glucose- and L-Arg-stimulated insulin release and glucose-induced increases in cGMP; however, basal insulin release was slightly increased. D-Arg and L-ornithine did not affect islet cGMP levels, although insulin release was stimulated. RINm5F cell cGMP levels and insulin release increased in response to L-Arg in a concentration- and time-related manner, whereas glucose and L-histidine were without effect. 8-Bromo-cGMP also slightly increased RINm5F cell insulin release. Sodium nitroprusside as a source of nitric oxide increased RINm5F cell cGMP production. Methylene blue and LY83583, inhibitors of soluble guanylyl cyclase activation, reduced RINm5F cell cGMP levels in the presence and absence of L-Arg; LY83583 also reduced glucose-stimulated cGMP levels in islets. Insulin release by glucose and L-Arg was also inhibited by methylene blue and LY83583 in islets. We conclude that glucose and L-Arg stimulate guanylyl cyclase activity and cGMP formation in beta-cells at least in part through metabolism to the reactive intermediate nitric oxide. However, neither nitric oxide nor cGMP synthesis is obligatory for insulin secretion.     

HIV-1蛋白酶抑制剂Nelfinavir降低大鼠胰岛素瘤INS-1细胞胰岛素释放

HIV 1蛋白酶抑制剂Nelfinavir处理 48h ,显著降低大鼠INS 1细胞基础胰岛素分泌和葡萄糖刺激的胰岛素释放 ,Nelfinavir对后者的抑制作用更强 ,提示Nelfinavir长期治疗可能导致胰岛 β细胞功能损害。     

Binding sites for peptide-histidine-isoleucine (PHI) on rat insulinoma-derived RINm5F cells

Specific binding sites for ¹²⁵I-labelled rat peptide-histidine-isoleucine (PHI) were identified on rat insulinoma-derived RINm5F cells. The concentrations of peptides producing half-maximal displacement of label were rat PHI, 0.36 ± 0.14 nM, vasoactive intestinal polypeptide (VIP), 0.38 ± 0.13 nM and secretin, approximately 0.2 μM. Glucagon and glucagon-like peptide-1(7–36)amide were without effect on binding. PHI and VIP produced dose-dependent increases in cAMP production in the cells that were significantly (P < 0.05) above unstimulated rates for ligand concentrations between 10⁻⁸ and 10 ⁻⁶ M. Both PHI and VIP produced a small but significant (P < 0.05) enhancement in the rate of release of immunoreactive insulin from the cells but the effect was not dose dependent.