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 confers resistance to the inhibitory effects of interleukin1 beta on insulin secretion in pancreatic islets

High protein content in the diet during childhood and adolescence has been associated to the onset insulin-dependent diabetes mellitus. We investigated the effect of interleukin-1 beta (IL-I beta) on insulin secretion, glucose metabolism, and nitrite formation by islets isolated from rats fed with normal protein (NP, 17%) or low protein (LP, 6%) after weaning. Pretreatment of islets with IL-1 beta for 1 h or 34 h inhibited the insulin secretion induced by glucose in both groups, but it was less marked in LP than in NP group. Islets from LP rats exhibited a decreased IL-1 beta -induced nitric oxide (NO) production, lower inhibition of D-[(UC)-C-14]-glucose oxidation to (CO2)-C-14, and less pronounced effect of IL-1 beta on alpha -ketoisocaproic acid-induced insulin secretion than NP islets. However, when the islets were stimulated by high concentrations of K+ the inhibitory effect of IL-1 beta on insulin secretion was not different between groups. In conclusion, protein restriction protects beta -cells of the deleterious effect of IL-1 beta, apparently, by decreasing NO production. The lower NO generation in islets from protein deprived rats may be due to increased free fatty acids oxidation and consequent alteration in Ca2+ homeostasis. (C) 2001 Elsevier B.V. All rights reserved.

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

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

Pancreatic islets from dexamethasone-treated rats show alterations in global gene expression and mitochondrial pathways

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

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)

Reduced insulin secretion in response to nutrients in islets from malnourished young rats is associated with a diminished calcium uptake

Changes in Ca-45 uptake and insulin secretion in response to glucose, leucine, and arginine were measured in isolated islets derived from 4-week-old rats born of mothers maintained with normal protein (NP, 17%) or low protein (LP, 6%) diet during pregnancy and lactation. Glucose provoked a dose-dependent stimulation of insulin secretion in both groups of islets, with basal (2.8 mmol/L glucose) and maximal release (27.7 mmol/L glucose) significantly reduced in LP compared with NP islets. In the LP group the concentration-response curve to glucose was shifted to the right compared with the NP group, with the half-maximal response occurring at 16.9 and 13.3 mmol/L glucose, respectively. In LP islets, glucose-induced first and second phases of insulin secretions were drastically reduced. In addition, insulin response to individual amino acids, or in association with glucose, was also significantly reduced in the LP group compared with NP islets. Finally, in LP islets the Ca-45 uptake after 5 minutes or 90 minutes of incubation (which reflect mainly the entry and retention, respectively, of Ca2+), was lower than in NP islets. These data indicate that in malnourished rats both initial and sustained phases of insulin secretion in response to glucose were reduced. This poor secretory response to nutrients seems to be the consequence of an altered Ca2+ handling by malnourished islet cells. (J. Nutr. Biochem. 10:37-43, 1999) (C) Elsevier B.V. 1999. All rights reserved.

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.

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.

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.

Leucine Supplementation Augments Insulin Secretion in Pancreatic Islets of Malnourished Mice

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

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.