Morphofunctional Alterations in Endocrine Pancreas of Short- and Long-term Dexamethasone-treated Rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Long-term dexamethasone therapy may induce peripheral insulin resistance (IR), which in turn elicits increased beta-cell function and proliferation. However, whether such adaptive compensations occur during short-term treatment with dexamethasone is unclear. Here, we compared morphofunctional parameters in endocrine pancreas after short- and long-term dexamethasone administration. Groups of rats received daily i.p. injection of 1 mg/kg b.w. dexamethasone for 1 (DEX-1), 3 (DEX-3), or 5 consecutive days (DEX-5), whilst control rats were saline-treated (CTL). Despite the absence of apparent IR in DEX-1 rats, this group exhibited increased circulating insulin levels and glucose-stimulated insulin secretion (GSIS), compared to the CTL group (p < 0.05). Evident IR as well as marked hyperinsulinemia and GSIS, as judged by the static and dynamic insulin secretion values, were observed in DEX-3 and DEX-5 rats (p < 0.05). GSIS in islets cultured with 1 mu M dexamethasone was lower compared to the control (p < 0.05). Marked increases in beta-cell proliferation were observed in DEX-3 and DEX-5 rats, compared to CTL and DEX-1 rats (p < 0.05). The alterations observed in DEX-3 rats were more pronounced in DEX-5 rats, which also exhibited a higher content of islet Cdk4 and Cd2 proteins, compared to the CTL group (p < 0.05). We conclude that short-term dexamethasone treatment (DEX-1) induces an increase in beta-cell function that does not require the presence of discernible IR. As the treatment continues, the IR develops rapidly, and increased insulin secretion as well as beta-cell hyperplasia is demanded for the appropriate maintenance of glucose homeostasis.434275281Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)INOD (Instituto Nacional de Obesidade e Diabetes)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Reduced pancreatic beta-cell mass is associated with decreased FoxO1 and Erk1/2 protein phosphorylation in low-protein malnourished rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)A low-protein diet leads to functional and structural pancreatic islet alterations, including islet hypotrophy. Insulin-signaling pathways are involved in several adaptive responses by pancreatic islets. We determined the levels of some insulin-signaling proteins related to pancreatic islet function and growth in malnourished rats. Adult male Wistar rats (N = 20 per group) were fed a 17% protein (normal-protein diet; NP) or 6% protein (low-protein diet; LP), for 8 weeks. At the end of this period, blood glucose and serum insulin and albumin levels were measured. The morphometric parameters of the endocrine pancreas and the content of some proteins in islet lysates were determined. The beta-cell mass was significantly reduced (congruent to 65%) in normoglycemic but hypoinsulinemic LP rats compared to NP rats. Associated with these alterations, a significant 30% reduction in insulin receptor substrate-1 and a 70% increase in insulin receptor substrate-2 protein content were observed in LP islets compared to NP islets. The phosphorylated serine-threonine protein kinase (pAkt)/Akt protein ratio was similar in LP and NP islets. The phosphorylated forkhead-O1 (pFoxO1)/FoxO1 protein ratio was decreased by 43% in LP islets compared to NP islets (P < 0.05). Finally, the ratio of phosphorylated-extracellular signal-related kinase 1/2 (pErk1/2) to total Erk1/2 protein levels was decreased by 71% in LP islets compared to NP islets (P < 0.05). Therefore, the reduced beta-cell mass observed in LP rats is associated with the reduction of phosphorylation in mitogenic-related signals, FoxO1 and Erk proteins. The cause/effect basis of this association remains to be determined.4210935941Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [02/04310-4, 04/11684-9, 03/10829-0

Involvement of the cholinergic pathway in glucocorticoid-induced hyperinsulinemia in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Aims: We investigated the contribution of the cholinergic nervous system to dexamethasone-induced insulin resistance and hyperinsulinemia in rats. Methods: Seventy-day-old Wistar male rats were distributed in groups: control (CTL), vagotomized (VAG), and sham operated (SHAM). On the 90th day of life, half of the rats were treated daily with 1 mg/kg of dexamethasone for 5 days (CTL DEX, VAG DEX, and SHAM DEX). Results: In the presence of 8.3 mM glucose plus 100 mu M carbachol (Cch), isolated islets from CTL DEX secreted significantly more insulin than CTL. Cch-enhancement of secretion was further increased in islets from VAG CTL and VAG DEX than SHAM CTL and SHAM DEX, respectively. In CTL DEX islets, M3R and PLC beta 1 and phosphorylated PKC alpha, but not PKC alpha, protein content was significantly higher compared with each respective control. In islets from VAG DEX, the expression of M3R protein increased significantly compared to VAG CTL and SHAM DEX. Vagotomy per se did not affect insulin resistance, but attenuated fasted and fed insulinemia in VAG DEX, compared with SHAM DEX rats. Conclusion: These data indicate an important participation of the cholinergic nervous system through muscaric receptors in dexamethasone-induced hyperinsulinemia in rats. (C) 2009 Elsevier Ireland Ltd. All rights reserved.872184191Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Functional alterations in endocrine pancreas of rats with different degrees of dexamethasone-induced insulin resistance

Objectives: We have analyzed the peripheral insulin and glucose sensitivity in vivo, and islet function ex vivo in rats with different degrees of insulin resistance induced by dexamethasone (DEX). Methods: Dexamethasone, in the concentrations of 0.1 (DEX 0.1), 0.5 (DEX 0.5), and 1.0 mg/kg body weight (DEX 1.0) was administered daily, intraperitoneally, to adult Wistar rats for 5 days, whereas controls received saline. Results: Dexamethasone treatment induced peripheral insulin resistance in a dose-dependent manner. At the end of the treatment, only DEX 1.0 rats showed significant increase of postabsorptive blood glucose and serum triglycerides, and nonesterified fatty acids levels. Incubation of pancreatic islets in increasing glucose concentrations (2.8-22 mM) led to an augmented insulin secretion in all DEX-treated rats. Leucine, carbachol, and high KCl concentrations induced the insulin release in DEX 0.5 and DEX 1.0, whereas arginine augmented secretion in all DEX-treated groups. Conclusions: We demonstrate that in DEX 0.5 and, especially in DEX 0.1 groups, but not in DEX 1.0, the adaptations that occurred in the endocrine pancreas are able to counteract metabolic disorders (glucose intolerance and dyslipidemia). These animal models seem to be interesting approaches for the study of degrees of subjacent effects that may mediate type 2 diabetes (DEX 1.0) and islet function alterations, without collateral effects (DEX 0.1 and DEX 0.5).36328429

Tetracaine stimulates extracellular Ca2+-independent insulin release

The effect of the local anesthetic, tetracaine, on Ca-45 efflux, cytoplasmic Ca2+ concentration [Ca2+](i) and insulin secretion in pancreatic B-cells was studied. At a physiological level of [Ca2+](o), tetracaine (0.1-5 mM) dose-dependently inhibited insulin secretion induced by 22 mM glucose. Paradoxically, at the same glucose concentration but in the absence of external Ca2+, tetracaine dose-dependently increased insulin secretion. At a low glucose level (2.8 mM) tetracaine failed to affect secretion, either in the presence or absence of external Ca2+. At high (12 mM) or low (2.8 mM) glucose, [Ca2+](i) was increased by tetracaine in a dose-dependent manner. Tetracaine (2 mM) also increased the Ca-45 efflux from isolated islets. This effect was of the same magnitude at both low and high glucose concentrations, and was independent of the presence of extracellular Ca2+. Finally, tetracaine increased Ca-45 efflux from islets perifused in the presence of thapsigargin. In conclusion, our data indicate that tetracaine releases Ca2+ from a thapsigargin-insensitive store in pancreatic B-cells. Under suitable experimental conditions, insulin release can be elicited by a [Ca2+](o)-independent pathway. The existence of a ryanodine-like Ca2+ channel in pancreatic B-cells is proposed.3274170025726

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 (2.3 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 mu g/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.30335936

Cellular changes in the prostatic stroma of glucocorticoid-treated rats

Glucocorticoid hormones (GCs) have been widely used for the treatment of prostate cancer because of their inhibitory property against tumour growth. However, their mechanism of action in the prostate has received little attention. Excess GCs can lead to peripheral insulin resistance resulting in hyperglycaemia and hyperinsulinaemia. Insulin plays an important role as a cellular stimulant and high levels are related to low levels of androgens. Our objective has been to describe the effects of insulin resistance induced by dexamethasone treatment on the morphology of rat ventral prostate. Mate adult Wistar rats received daily intraperitoneal injections of dexamethasone or saline for five consecutive days after which the rats were killed and the ventral prostate was removed, weighed and prepared for conventional and transmission electron microscopy (TEM). Dexamethasone treatment resulted in atrophy and decreased proliferative activity of prostatic epithelial cells. TEM analysis revealed changes in the epithelium-stroma interface, with some interruptions in the basement membrane. Fibroblasts showed a secretory phenotype with dilated endoplasmic reticulum. Smooth muscle cells exhibited a contractile pattern with 50% atrophy, an irregular membrane and twisted nuclei. Mitochondrial alterations, such as enlarged size and high electron density in the mitochondrial matrix, were also detected in smooth muscle cells. Insulin resistance induced by dexamethasone is thus associated with epithelial atrophy similar to that described for diabetic rats. However, GCs are responsible for morphological changes in the stromal cell population suggesting the activation of fibroblasts and atrophy of the smooth muscle cells.332349950

Dexamethasone treatment in vivo counteracts the functional pancreatic islet alterations caused by malnourishment in rats

The effects of dexamethasone (Dex) on the metabolic parameters, peripheral insulin, and glucose sensitivity in vivo as well as on islet function ex vivo of rats submitted to low-protein diet were analyzed. Dexamethasone (1.0 mg/kg body weight) was administered intraperitoneally daily to adult Wistar rats fed on a normal-protein diet or low-protein diet (LPD) for 5 days, whereas control rats fed on a normal-protein diet or low-protein diet (LP) received saline alone. At the end of the experimental period, LP rats showed a significant reduction in serum insulin, total serum protein, and serum albumin levels compared with rats fed on a normal-protein diet (P < .05). All these parameters tended to be normalized in LPD rats (P < .05); furthermore, these rats exhibited increased serum glucose and nonesterified fatty acid levels compared with LP rats (P < .05). Rats submitted to the low-protein diet demonstrated normal peripheral glucose sensitivity and improved peripheral insulin sensitivity, which was reversed by Dex treatment. A reduced area of islets from LP rats was partially recovered in LPD rats (P < .05). At 16.7 mmol/L glucose, insulin secretion from LPD islets was also partially recovered and was significantly higher than that from LP islets (P < .05). In conclusion, induction of insulin resistance by Dex treatment reverses most of the metabolic alterations in rats submitted to a low-protein diet. In addition, several islet functions were also improved by Dex, confirming the plasticity of pancreatic islets in adverse conditions. (C) 2008 Elsevier Inc. All rights reserved.57561762

Increased pancreatic islet mass is accompanied by activation of the insulin receptor substrate-2/serine-threonine kinase pathway and augmented cyclin D(2) protein levels in insulin-resistant rats

It is well known that glucocorticoids induce peripheral insulin resistance in rodents and humans. Here, we investigated the structural and ultrastructural modifications, as well as the proteins involved in beta-cell function and proliferation, in islets from insulin-resistant rats. Adult male Wistar rats were made insulin resistant by daily administration of dexamethasone (DEX; 1mg/kg, i.p.) for five consecutive days, whilst control (CTL) rats received saline alone. Structure analyses showed a marked hypertrophy of DEX islets with an increase of 1.7-fold in islet mass and of 1.6-fold in islet density compared with CTL islets (P < 0.05). Ultrastructural evaluation of islets revealed an increased amount of secreting organelles, such as endoplasmic reticulum and Golgi apparatus in DEX islets. Mitotic figures were observed in DEX islets at structural and ultrastructural levels. Beta-cell proliferation, evaluated at the immunohistochemical level using anti-PCNA (proliferating cell nuclear antigen), showed an increase in pancreatic beta-cell proliferation of 6.4-fold in DEX islets compared with CTL islets (P < 0.0001). Increases in insulin receptor substrate-2 (IRS-2), phosphorylated-serine-threonine kinase AKT (p-AKT), cyclin D(2) and a decrease in retinoblastoma protein (pRb) levels were observed in DEX islets compared with CTL islets (P < 0.05). Therefore, during the development of insulin resistance, the endocrine pancreas adapts itself increasing beta-cell mass and proliferation, resulting in an amelioration of the functions. The potential mechanisms that underlie these events involve the activation of the IRS-2/AKT pathway and activation of the cell cycle, mediated by cyclin D(2). These adaptations permit the maintenance of glycaemia at near-physiological ranges.89426427