Dexamethasone-induced insulin resistance is associated with increased connexin 36 mRNA and protein expression in pancreatic rat islets

Augmented glucose-stimulated insulin secretion (GSIS) is an adaptive mechanism exhibited by pancreatic islets from insulin-resistant animal models. Gap junction proteins have been proposed to contribute to islet function. As such, we investigated the expression of connexin 36 (Cx36), connexin 43 (Cx43), and the glucose transporter Glut2 at mRNA and protein levels in pancreatic islets of dexamethasone (DEX)-induced insulin-resistant rats. Study rats received daily injections of DEX (1 mg/kg body mass, i.p.) for 5 days, whereas control rats (CTL) received saline solution. DEX rats exhibited peripheral insulin resistance, as indicated by the significant postabsorptive insulin levels and by the constant rate for glucose disappearance (K-ITT). GSIS was significantly higher in DEX islets (1.8-fold in 16.7 mmol/L glucose vs. CTL, p < 0.05). A significant increase of 2.25-fold in islet area was observed in DEX vs. CTL islets (p < 0.05). Cx36 mRNA expression was significantly augmented, Cx43 diminished, and Glut2 mRNA was unaltered in islets of DEX vs. CTL (p < 0.05). Cx36 protein expression was 1.6-fold higher than that of CTL islets (p < 0.05). Glut2 protein expression was unaltered and Cx43 was not detected at the protein level. We conclude that DEX-induced insulin resistance is accompanied by increased GSIS and this may be associated with increase of Cx36 protein expression.

LOW-PROTEIN DIET IMPAIRS GLUCOSE-INDUCED INSULIN-SECRETION FROM AND CA-45 UPTAKE BY PANCREATIC RAT ISLETS

Glucose-induced insulin secretion rom and Ca-45 uptake by isolated pancreatic islets, derived from rats fed with normal (NPD) or low protein diet (LPD), were studied. Insulin secretion from both types of islets in response to increasing concentrations of glucose followed an S-shaped pattern. However, basal secretion observed at substimulatory concentrations of glucose (0-5.6 mM), as well as maximal release, obtained at 16.7 mM or higher glucose concentrations were significantly reduced in islets from LPD. Furthermore, in LPD rat islets, the dose-response curve to glucose was clearly shifted to the right compared with NPD islets, with the half-maximal response occurring at 8.5 and 14.4 mM glucose for NPD and LPD islets, respectively. In islets from NPD rats, the Ca-45 content, after 5 or 90 min in the presence of 8.3 mM glucose, was higher than that observed for islets kept at 2.8 mM glucose and increased further at 16.7 mM glucose. After 5 min of incubation, the Ca-45 uptake by LPD islets in the presence of 8.3 mM glucose was slightly higher than basal values (2.8 mM glucose); however, no further increase in the Ca-45 uptake was noticed at 16.7 mM glucose. In LPD islets a significant increase in Ca-45 uptake over basal values was registered only at 16.7 mM glucose, after 90 min of incubation. These data indicate that the poor secretary response to glucose observed in islets from LPD rats may be related to a defect in the ability of glucose to increase Ca2+ uptake and/or to reduce Ca2+ efflux from beta-cells.

AMINO-ACID-SEQUENCE OF TSTX-V, AN ALPHA-TOXIN FROM TITYUS-SERRULATUS SCORPION-VENOM, AND ITS EFFECT ON K+ PERMEABILITY OF BETA-CELLS FROM ISOLATED RAT ISLETS OF LANGERHANS

Highly purified Tityustoxin V (TsTX-V), an alpha-toxin isolated from the venom of the Brazilian scorpion Tityus serrulatus, was obtained by ion exchange chromatography on carboxymethylcellulose-52. It was shown to be homogeneous by reverse phase high performance liquid chromatography, N-terminal sequencing (first 39 residues) of the reduced and alkylated protein and by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and tricine. Following enzymatic digestion, the complete amino acid sequence (64 residues) was determined. The sequence showed higher homology with the toxins from the venoms of the North African than with those of the North and South American scorpions. Using the rate of Rb-86(+) release from depolarized rat pancreatic beta-cells as a measure of K+ permeability changes, TsTX-V (5.6 mu g/ml) was found to increase by 2.0-2.4-fold the rate of marker outflow in the presence of 8.3 mM glucose. This effect was persistent and slowly reversible, showing similarity to that induced by 100 mu-M veratridine, an agent that increases the open period of Na+ channels, delaying their inactivation. It is suggested that, by extending the depolarized period, TsTX-V indirectly affects beta-cell voltage-dependent K+ channels, thus increasing K+ permeability.

PROLACTIN INDUCES MATURATION OF GLUCOSE SENSING MECHANISMS IN CULTURED NEONATAL RAT ISLETS

The effects of PRL treatment on insulin content and secretion, and Rb-86 and Ca-45 fluxes from neonatal rat islets maintained in culture for 7-9 days were studied. PRL treatment enhanced islet insulin content by 40% and enhanced early insulin secretion evoked by 16.7 mm glucose. Insulin release stimulated by oxotremorine-M, a muscarinic agonist, in the presence of glucose (8.3 or 16.7 mm) was unchanged by PRL treatment. However, PRL treatment potentiated phorbol 12,13-dibutyrate-stimulated insulin secretion in the presence of the above glucose concentrations. PRL treatment potentiated the reduction in Rb-86 efflux induced by glucose or tolbutamide and enhanced the increase in Rb-86 efflux evoked by diazoxide. PRL treatment slightly potentiated the increment in Ca-45 uptake induced by high concentrations of K+, but failed to affect the increment evoked by 16.7 mm glucose. Since glucose-induced Ca-45 uptake was not affected by PRL, we suggest that the enhancement in first phase insulin secretion evoked by glucose in the PRL-treated islets occurs at a step in the secretory process that may involve protein kinase-C. These data further support observations that PRL treatment increases islet sensitivity to glucose.

High doses of dexamethasone induce increased beta-cell proliferation in pancreatic rat islets

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

INGAP-PP up-regulates the expression of genes and proteins related to K-ATP(+) channels and ameliorates Ca2+ handling in cultured adult rat islets

Islet Neogenesis Associated Protein (INGAP) increases pancreatic beta-cell mass and potentiates glucose-induced insulin secretion. Here, we investigated the effects of the pentadecapeptide INGAP-PP in adult cultured rat islets upon the expression of proteins constitutive of the K-ATP(+) channel, Ca2+ handling, and insulin secretion. The islets were cultured in RPMI medium with or without INGAP-PP for four days. Thereafter, gene (RT-PCR) and protein expression (Western blotting) of Foxa2, SUR1 and Kir6.2, cytoplasmic Ca2+ ([Ca2+](i)), static and dynamic insulin secretion, and Rb-86 efflux were measured. INGAP-PP increased the expression levels of Kir6.2, SUR1 and Foxa2 genes, and SUR1 and Foxa2 proteins. INGAP-PP cultured islets released significantly more insulin in response to 40 mM KCl and 100 mu M tolbutamide. INGAP-PP shifted to the left the dose-response curve of insulin secretion to increasing concentrations of glucose (EC50 of 10.0 +/- 0.4 vs. 13.7 +/- 1.5 mM glucose of the controls). It also increased the first phase of insulin secretion elicited by either 22.2 mM glucose or 100 mu M tolbutamide and accelerated the velocity of glucose-induced reduction of Rb-86 efflux in perifused islets. These effects were accompanied by a significant increase in [Ca2+](i) and the maintenance of a considerable degree of [Ca2+](i) oscillations. These results confirm that the enhancing effect of INGAP-PP upon insulin release, elicited by different secretagogues, is due to an improvement of the secretory function in cultured islets. Such improvement is due, at least partly, to an increased K-ATP(+) channel protein expression and/or changing in the kinetic properties of these channels and augmented [Ca2+](i) response. Accordingly, INGAP-PP could potentially be used to maintain the functional integrity of cultured islets and eventually, for the prevention and treatment of diabetes. (C) 2008 Elsevier B.V. All rights reserved.

Synergistic effect of glucose and prolactin on GLUT2 expression in cultured neonatal rat islets

We studied the synergistic effect of glucose and prolactin (PRL) on insulin secretion and GLUT2 expression in cultured neonatal rat islets. After 7 days in culture, basal insulin secretion (2.8 mM glucose) was similar in control and PRL-treated islets (1.84 ± 0.06% and 2.08 ± 0.07% of the islet insulin content, respectively). At 5.6 and 22 mM glucose, insulin secretion was significantly higher in PRL-treated than in control islets, achieving 1.38 ± 0.15% and 3.09 ± 0.21 % of the islet insulin content in control and 2.43 ± 0.16% and 4.31 ± 0.24% of the islet insulin content in PRL-treated islets, respectively. The expression of the glucose transporter GLUT2 in B-cell membranes was dose-dependently increased by exposure of the islet to increasing glucose concentrations. This effect was potentiated in islets cultured for 7 days in the presence of 2 μg/ml PRL. At 5.6 and 10 mM glucose, the increase in GLUT2 expression in PRL-treated islets was 75% and 150% higher than that registered in the respective control. The data presented here indicate that insulin secretion, induced by different concentrations of glucose, correlates well with the expression of the B-cell-specific glucose transporter GLUT2 in pancreatic islets.

Long-term effect of prolactin treatment on glucose-induced insulin secretion in cultured neonatal rat islets

Insulin secretion and 45SCa2+ uptake and efflux were studied in neonatal rat islets maintained in culture for 7 or 19 days in the absence or presence of prolactin (PRL). Insulin secretion in response to glucose (G), leucine (Leu), arginine (Arg) and carbachol (Cch) was augmented after 7 and 19 days in culture, compared to basal secretion (G 2.8 mM), in both PRL- treated and control islets. However, the increase in insulin secretion induced by the above secretagogues was higher in islets cultured in the presence of PRL for 19 days. In PRL-treated islets, the 45Ca2+ content after a 5 min incubation in the presence of G, Leu, Arg and Cch was significantly higher than the control only in islets cultured for 19 days. Except with Arg, the 45Ca2+ uptake in PRL-treated islets after a 90 min incubation was also significantly higher than the control only in islets cultured for 19 days. Finally, Leu-induced alterations in the 45Ca2+ efflux were higher in PRL-treated than in control islets cultured for 7 or 19 days. In the absence of external Ca2+, the reduction in 45Ca2+ efflux induced by glucose was also significantly higher in PRL-treated than in control islets. This effect was slightly potentiated after 19 days in culture. These data further support the hypothesis that PRL treatment enhances maturation of the secretory mechanism in neonatal islets. This effect can be potentiated even more if the treatment is prolonged.

Glucocorticoids in vivo induce both insulin hypersecretion and enhanced glucose sensitivity of stimulus-secretion coupling in isolated rat islets

Although glucocorticoids are widely used as antiinflammatory agents in clinical therapies, they may cause serious side effects that include insulin resistance and hyperinsulinemia. To study the potential functional adaptations of the islet of Langerhans to in vivo glucocorticoid treatment, adult Wistar rats received dexamethasone (DEX) for 5 consecutive days, whereas controls (CTL) received only saline. The analysis of insulin release in freshly isolated islets showed an enhanced secretion in response to glucose in DEX-treated rats. The study of Ca2 2+ signals by fluorescence microscopy also demonstrated a higher response to glucose in islets from DEX-treated animals. However, no differences in Ca2 2+signals were found between both groups with tolbutamide or KCl, indicating that the alterations were probably related to metabolism. Thus, mitochondrial function was explored by monitoring oxidation of nicotinamide dinucleotide phosphate autofluorescence and mitochondrial membrane potential. Both parameters revealed a higher response to glucose in islets from DEX-treated rats. The mRNA and protein content of glucose transporter-2, glucokinase, and pyruvate kinase was similar in both groups, indicating that changes in these proteins were probably not involved in the increased mitochondrial function. Additionally,weexplored the status of Ca2 2+-dependent signaling kinases. Unlike calmodulin kinase II, we found an augmented phosphorylation level of protein kinase Cα as well as an increased response of the phospholipase C/inositol 1,4,5-triphosphate pathway in DEX-treated rats. Finally, an increased number of docked secretory granules were observed in the β-cells of DEX animals using transmission electron microscopy. Thus, these results demonstrate that islets from glucocorticoid-treated rats develop several adaptations that lead to an enhanced stimulus-secretion coupling and secretory capacity. Copyright © 2010 by The Endocrine Society.

Long-term dexamethasone treatment alters the histomorphology of acinar cells in rat parotid and submandibular glands

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ebselen and cytokine-induced nitric oxide synthase expression in insulin-producing cells

Interleukin-1 (IL-1) may be a mediator of β-cell damage in insulin-dependent diabetes mellitus (IDDM). The IL-1 mechanism of action on insulin-producing cells probably includes activation of the transcription nuclear factor κB (NF-κB), increased transcription of the inducible form of nitric oxide synthase (iNOS) and the subsequent production of nitric oxide (NO). Reactive oxygen intermediates, particularly H2O2, have been proposed as second messengers for NF-κB activation. In the present study, we tested whether ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a glutathione peroxidase mimicking compound, could counteract the effects of IL-1β, H2O2 and alloxan in rat pancreatic islets and in the rat insulinoma cell line RINm5F (RIN cells). Some of these experiments were also reproduced in human pancreatic islets. Ebselen (20 μM) prevented the increase in nitrite production by rat islets exposed to IL-1β for 6 hr and induced significant protection against the acute inhibitory effects of alloxan or H2O2 exposure, as judged by the preserved glucose oxidation rates. However, ebselen failed to prevent the increase in nitrite production and the decrease in glucose oxidation and insulin release by rat islets exposed to IL-1β for 24 hr. Ebselen prevented the increase in nitrite production by human islets exposed for 14 hr to a combination of cytokines (IL-1β, tumor necrosis factor-α and interferon-γ). In RIN cells, ebselen counteracted both the expression of iNOS mRNA and the increase in nitrite production induced by 6 hr exposure to IL-β but failed to block IL-1β-induced iNOS expression following 24 hr exposure to the cytokine. Moreover, ebselen did not prevent IL-1β-induced NF-κB activation. As a whole, these data indicate that ebselen partially counteracts cytokine-induced NOS activation in pancreatic β-cells, an effect not associated with inhibition of NF-κB activation.

Endurance training stimulates growth and survival pathways and the redox balance in rat pancreatic islets

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Glucose- and insulin-induced phosphorylation of the insulin receptor and its primary substrates IRS-1 and IRS-2 in rat pancreatic islets

The presence of tyrosine-phosphorylated proteins was studied in cultured rat pancreatic islets, Immunoblotting performed with total extracts of islets cultured in the presence of 1.8 or 5.6 mM glucose revealed at least three distinct tyrosine-phosphorylated bands (25 kDa, 95 kDa and 165-185 kDa). After 12 h incubation in medium containing 1.8 mM glucose, a pulse exposition to 11 or 22 mM glucose or to 10(-7) M insulin led to a substantial increase in the phosphorylation of all three bands, with no appearance of novel bands. Immunoprecipitation with specific antibodies demonstrated that the signal detected at 95 kDa corresponds to the beta subunit of the insulin receptor (IR) while the band at 165-185 kDa corresponds to the early substrates of the insulin receptor, IRS-1 and IRS-2. Immunoprecipitation with IRS-I or IRS-2 antisera detected their association with the lipid metabolizing enzyme phosphatidylinositol 3-kinase (PI 3-kinase), Thus, this is the first demonstration that elements involved in the insulin-signalling pathway of traditional target tissues are also present in pancreatic islets and are potentially involved in auto- and paracrine-signalling in this organ.

Insulin signaling proteins in pancreatic islets of insulin-resistant rats induced by glucocorticoid

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Repair of critical-size defects with autogenous periosteum-derived cells combined with bovine anorganic apatite/collagen: an experimental study in rat calvaria

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)