Junia Carolina Santos-Silva

Pancreatic Alpha-Cell Dysfunction Contributes to the Disruption of Glucose Homeostasis and Compensatory Insulin Hypersecretion in Glucocorticoid-Treated Rats

Glucocorticoid (GC)-based therapies can cause insulin resistance (IR), glucose intolerance, hyperglycemia and, occasionally, overt diabetes. Understanding the mechanisms behind these metabolic disorders could improve the management of glucose homeostasis in patients undergoing GC treatment. For this purpose, adult rats were treated with a daily injection of dexamethasone (1 mg/kg b.w., i.p.) (DEX) or saline as a control for 5 consecutive days. The DEX rats developed IR, augmented glycemia, hyperinsulinemia and hyperglucagonemia. Treatment of the DEX rats with a glucagon receptor antagonist normalized their blood glucose level. The characteristic inhibitory effect of glucose on glucagon secretion was impaired in the islets of the DEX rats, while no direct effects were found on α-cells in islets that were incubated with DEX in vitro. A higher proportion of docked secretory granules was found in the DEX α-cells as well as a trend towards increased α-cell mass. Additionally, insulin secretion in the presence of glucagon was augmented in the islets of the DEX rats, which was most likely due to their higher glucagon receptor content. We also found that the enzyme 11βHSD-1, which participates in GC metabolism, contributed to the insulin hypersecretion in the DEX rats under basal glucose conditions. Altogether, we showed that GC treatment induces hyperglucagonemia, which contributes to an imbalance in glucose homeostasis and compensatory β-cell hypersecretion. This hyperglucagonemia may result from altered α-cell function and, likely, α-cell mass. Additionally, blockage of the glucagon receptor seems to be effective in preventing the elevation in blood glucose levels induced by GC administration.

Diacerhein Improves Glucose Tolerance and Insulin Sensitivity in Mice on a High-Fat Diet

Obesity and type 2 diabetes are characterized by insulin resistance, and the common basis of these events is a chronic and systemic inflammatory process marked by the activation of the c-Jun N-terminal kinase (JNK) and inhibitor-κB kinase (IKKβ)/nuclear factor-κB (NFκB) pathways, up-regulated cytokine synthesis, and endoplasmic reticulum dysfunction. The aim of this study was to evaluate the effects of diacerhein administration, an antiinflammatory drug that reduces the levels of inflammatory cytokines, on insulin sensitivity and signaling in diet-induced obese (DIO) mice. Swiss mice were fed with conventional chow (control group) or a high-fat diet (DIO group). Later, DIO mice were randomly subdivided into a new subgroup (DAR) that received 20 mg/kg diacerhein for 10 d. Western blotting was used to quantify the expression and phosphorylation of insulin receptor, insulin receptor substrate 1, and Akt and of inflammatory mediators that modulate insulin signaling in a negative manner (IKKβ, JNK, and inducible nitric oxide synthase). We show here, for the first time, that the administration of diacerhein in DIO mice improved endoplasmic reticulum stress, reduced JNK and IKKβ phosphorylation, and resulted in a marked improvement in fasting glucose, a decrease in macrophage infiltration in adipose tissue, and a reduced expression and activity of proinflammatory mediators accompanied by an improvement in the insulin signaling mainly in the liver and adipose tissue. Taken together, these results indicate that diacerhein treatment improves insulin sensitivity in obesity, mediated by the reversal of subclinical inflammation, and that this drug may be an alternative therapy for insulin resistance.

Enhanced glucose-induced intracellular signaling promotes insulin hypersecretion: pancreatic beta-cell functional adaptations in a model of genetic obesity and prediabetes.

Obesity is associated with insulin resistance and is known to be a risk factor for type-2 diabetes. In obese individuals, pancreatic beta-cells try to compensate for the increased insulin demand in order to maintain euglycemia. Most studies have reported that this adaptation is due to morphological changes. However, the involvement of beta-cell functional adaptations in this process needs to be clarified. For this purpose, we evaluated different key steps in the glucose-stimulated insulin secretion (GSIS) in intact islets from female ob/ob obese mice and lean controls. Obese mice showed increased body weight, insulin resistance, hyperinsulinemia, glucose intolerance and fed hyperglycemia. Islets from ob/ob mice exhibited increased glucose-induced mitochondrial activity, reflected by enhanced NAD(P)H production and mitochondrial membrane potential hyperpolarization. Perforated patch-clamp examination of beta-cells within intact islets revealed several alterations in the electrical activity such as increased firing frequency and higher sensitivity to low glucose concentrations. A higher intracellular Ca(2+) mobilization in response to glucose was also found in ob/ob islets. Additionally, they displayed a change in the oscillatory pattern and Ca(2+) signals at low glucose levels. Capacitance experiments in intact islets revealed increased exocytosis in individual ob/ob beta-cells. All these up-regulated processes led to increased GSIS. In contrast, we found a lack of beta-cell Ca(2+) signal coupling, which could be a manifestation of early defects that lead to beta-cell malfunction in the progression to diabetes. These findings indicate that beta-cell functional adaptations are an important process in the compensatory response to obesity.

Cell-to-cell contact dependence and junctional protein content are correlated with in vivo maturation of pancreatic beta cells.

In this study, we investigated the cellular distribution of junctional proteins and the dependence on cell-cell contacts of pancreatic beta cells during animal development. Fetus and newborn rat islets, which display a relatively poor insulin secretory response to glucose, present an immature morphology and cytoarchitecture when compared with young and adult islets that are responsive to glucose. At the perinatal stage, beta cells display a low junctional content of neural cell adhesion molecule (N-CAM), α- and β-catenins, ZO-1, and F-actin, while a differential distribution of N-CAM and Pan-cadherin was seen in beta cells and nonbeta cells only from young and adult islets. In the absence of intercellular contacts, the glucose-stimulated insulin secretion was completely blocked in adult beta cells, but after reaggregation they partially reestablished the secretory response to glucose. By contrast, neonatal beta cells were poorly responsive to sugar, regardless of whether they were arranged as intact islets or as isolated cells. Interestingly, after 10 days of culturing, neonatal beta cells, known to display increased junctional protein content in vitro, became responsive to glucose and concomitantly dependent on cell-cell contacts. Therefore, our data suggest that the developmental acquisition of an adult-like insulin secretory pattern is paralleled by a dependence on direct cell-cell interactions.

Vagotomy ameliorates islet morphofunction and body metabolic homeostasis in MSG-obese rats

The parasympathetic nervous system is important for β-cell secretion and mass regulation. Here, we characterized involvement of the vagus nerve in pancreatic β-cell morphofunctional regulation and body nutrient homeostasis in 90-day-old monosodium glutamate (MSG)-obese rats. Male newborn Wistar rats received MSG (4 g/kg body weight) or saline [control (CTL) group] during the first 5 days of life. At 30 days of age, both groups of rats were submitted to sham-surgery (CTL and MSG groups) or subdiaphragmatic vagotomy (Cvag and Mvag groups). The 90-day-old MSG rats presented obesity, hyperinsulinemia, insulin resistance, and hypertriglyceridemia. Their pancreatic islets hypersecreted insulin in response to glucose but did not increase insulin release upon carbachol (Cch) stimulus, despite a higher intracellular Ca2+ mobilization. Furthermore, while the pancreas weight was 34% lower in MSG rats, no alteration in islet and β-cell mass was observed. However, in the MSG pancreas, increases of 51% and 55% were observed in the total islet and β-cell area/pancreas section, respectively. Also, the β-cell number per β-cell area was 19% higher in MSG rat pancreas than in CTL pancreas. Vagotomy prevented obesity, reducing 25% of body fat stores and ameliorated glucose homeostasis in Mvag rats. Mvag islets demonstrated partially reduced insulin secretion in response to 11.1 mM glucose and presented normalization of Cch-induced Ca2+ mobilization and insulin release. All morphometric parameters were similar among Mvag and CTL rat pancreases. Therefore, the higher insulin release in MSG rats was associated with greater β-cell/islet numbers and not due to hypertrophy. Vagotomy improved whole body nutrient homeostasis and endocrine pancreatic morphofunction in Mvag rats.

Propranolol treatment lowers blood pressure, reduces vascular inflammatory markers and improves endothelial function in obese mice

&NA; Obesity‐associated hypertension is accompanied by a number of cardiovascular risk factors including vascular insulin resistance (IR) and higher sympathetic nervous activity. Therefore, autonomic blockade was demonstrated to reverse hypertension, endothelial dysfunction and IR in obese individuals. We hypothesized that &bgr;‐AR blockade with propranolol would restore endothelial function and vascular insulin signaling in obesity, associated with an anti‐inflammatory effect. Body weight, systolic blood pressure (SBP), plasma biochemical parameters and aortic endothelial function were analyzed in mice fed standard diet (control group) or a high fat diet (HFD) that were treated with vehicle (water) or propranolol (10 mg/kg/day) for 8 weeks. Propranolol treatment did not modify obesogenic effect of HFD feeding. However, propranolol was effective in preventing the rise in SBP, the hyperinsulinemia and the impaired endothelium‐dependent relaxation to acetylcholine and to insulin in obese mice. Protective effect of propranolol administration in endothelial function was associated with increased nitric oxide (NO) production and phosphorylation of Akt (Ser473) and eNOS (Ser1177), but with reduced phospho‐IRS‐1(Ser307) and phospho‐ERK1/2 (Thr202/Tyr204). In addition, &bgr;‐blocker propranolol prevented the NF‐kB nuclear translocation and the increase in phospho‐I&kgr;B‐&agr; (Ser32) and in interleukin(IL)‐6 expression in aorta of obese mice, without significant changes in either aortic reactive oxygen species production or in circulating IL‐6 and TNF‐&agr; levels. In &bgr;2‐AR knockout mice, despite increasing body weight and visceral fat, HFD did not increase SBP and showed a partial improvement of endothelial function, revealing a role of &bgr;2‐AR in cardiovascular effects of obesity. In conclusion, our results suggest that &bgr;‐AR blockade with propranolol is effective to prevent the endothelial dysfunction, vascular IR and pro‐inflammatory state displayed in HFD‐induced obesity, independent of changes in body weight. Graphical abstract Figure. No caption available.

Taurine Supplementation Enhances Insulin Secretion Without Altering Islet Morphology in Non-obese Diabetic Mice

Taurine (TAU) is a sulfated amino acid that improves pancreatic islet function and regulates β-cell mass in pre- and diabetic states. We herein analyzed glucose homeostasis and islet morphofunction in non-obese diabetic (NOD) mice supplemented with 2 % TAU in their drinking water from birth until 90-days of age. TAU-supplemented female NOD mice (TAU group) showed a better glucose tolerance without modification in insulinemia, when compared to non-supplemented NOD mice (CTL). Glucose-induced insulin secretion was higher in islets isolated from female and male TAU groups. In addition, a better insulin release was observed at 30 mM K+ in islets from female TAU mice. These effects were accompanied by a higher total intracellular Ca2+ concentration in islets from female and male TAU mice. TAU-treated mice did not show any alteration in β-cell and islet areas, compared with CTL mice. Islets from TAU female mice presented a higher ratio of phosphorylated Akt and ERK (extracellular signal-regulated kinase) related to Akt and ERK protein content, respectively, in comparison with CTL islets. Additional experiments using isolated islets from Swiss mice showed that 3 mM TAU prevented the reduction in insulin secretion induced by 12 h incubation with IL1-β or IL1-β + IFN-γ. In conclusion, TAU supplementation improved NOD islet function without altering endocrine pancreatic morphometry, an effect that may be associated with a protective TAU effect upon cytokine-induced islet dysfunction, together with an improved protein expression of Akt and ERK.

Protein malnutrition blunts the increment of taurine transporter expression by a high-fat diet and impairs taurine reestablishment of insulin secretion

Taurine (Tau) restores β‐cell function in obesity; however, its action is lost in malnourished obese rodents. Here, we investigated the mechanisms involved in the lack of effects of Tau in this model. C57BL/6 mice were fed a control diet (CD)(14% protein) oraprotein‐restricted diet (RD) (6% protein) for 6 wk. Afterward, mice received a high‐fat diet (HFD) for 8 wk [CD + HFD (CH) and RD + HFD (RH)] with or without 5% Tau supplementation after weaning on their drinking water [CH + Tau (CHT) and RH + Tau (RHT)]. The HFD increased insulin secretion through mitochondrial metabolism in CH and RH. Tau prevented all those alterations in CHT only. The expression of the taurine transporter (Tau‐T), as well as Tau content in pancreatic islets, was increased in CH but had no effect on RH. Protein malnutrition programs β cells and impairs Tau‐induced restoration of mitochondrial metabolism and biogenesis. This may be associated with modulation of the expression of Tau‐T in pancreatic islets, which may be responsible for the absence of effect of Tau in protein‐malnourished obese mice.—Branco, R.C. S., Camargo, R. L., Batista, T. M., Vettorazzi, J. F., Borck, P. C., dos Santos‐Silva, J. C. R., Boschero, A. C., Zoppi, C. C., Carneiro, E. M. Protein malnutrition blunts the increment of taurine transporter expression by a high‐fat diet and impairs taurine reestablishment of insulin secretion. FASEB J. 31, 4078–4087 (2017). www.fasebj.org—Branco, Renato Chaves Souto, Camargo, Rafael Ludemann, Batista, Thiago Martins, Vettorazzi, Jean Franciesco, Borck, Patrícia Cristine, dos Santos‐Silva, Junia Carolina Rebelo, Boschero, Antonio Carlos, Zoppi, Cláudio Cesar, Carneiro, Everardo Magalhães Protein malnutrition blunts the increment of taurine transporter expression by a high‐fat diet and impairs taurine reestablishment of insulin secretion. FASEB J. 31, 4078–4087 (2017)

ARHGAP21 prevents abnormal insulin release through actin rearrangement in pancreatic islets from neonatal mice.

AIMS ARHGAP21 is a Rho GTPase-activating protein (RhoGAP) that associates with many proteins and modulates several cellular functions, including actin cytoskeleton rearrangement in different tissues. However, it is unknown whether ARHGAP21 is expressed in pancreatic beta cells and its function in these cells. Herein, we assess the participation of ARHGAP21 in insulin secretion. MAIN METHODS Neonatal mice were treated with anti-sense oligonucleotide against ARHG AP21 (AS) for 2 days, resulting in a reduction of the proteins expression of about 60% in the islets. F-actin depolimerization, insulin secretion,mRNA level of genes involved in insulin secretion, maturation and proliferation were evaluated in islets from both control and AS-treated mice. KEY FINDINGS ARHGAP21 co-localized with actin inMIN6 beta cells and with insulin in neonatal pancreatic islets. F-actin was reduced in AS-islets, as judged by lower phalloidin intensity. Insulin secretion was increased in islets from AS-treated mice, however no differences were observed in the GSIS (glucose-stimulated insulin secretion). In these islets, the pERK1/2 was increased, as well as the gene expressions of VAMP2 and SNAP25, proteins that are present in the secretory machinery. Maturation and cell proliferation were not affected in islets from AS-treated mice. SIGNIFICANCE In conclusion, our data show, for the first time, that ARHGAP21 is expressed and participates in the secretory process of pancreatic beta cells. Its effect is probably via pERK1/2, which modulates the rearrangement of the cytoskeleton. ARHGAP21 also controls the expression of genes that encodes proteins of the secretory machinery.

Prolonged fasting elicits increased hepatic triglyceride accumulation in rats born to dexamethasone-treated mothers

We investigated the effect of dexamethasone during the last week of pregnancy on glucose and lipid metabolism in male offspring. Twelve-week old offspring were evaluated after fasting for 12-hours (physiological) and 60-hours (prolonged). Physiological fasting resulted in glucose intolerance, decreased glucose clearance after pyruvate load and increased PEPCK expression in rats born to dexamethasone-treated mothers (DEX). Prolonged fasting resulted in increased glucose tolerance and increased glucose clearance after pyruvate load in DEX. These modulations were accompanied by accumulation of hepatic triglycerides (TG). Sixty-hour fasted DEX also showed increased citrate synthase (CS) activity, ATP citrate lyase (ACLY) content, and pyruvate kinase 2 (pkm2), glucose transporter 1 (slc2a1) and lactate dehydrogenase-a (ldha) expressions. Hepatic AKT2 was increased in 60-hour fasted DEX, in parallel with reduced miRNAs targeting the AKT2 gene. Altogether, we show that metabolic programming by prenatal dexamethasone is characterized by an unexpected hepatic TG accumulation during prolonged fasting. The underlying mechanism may depend on increased hepatic glycolytic flux due to increased pkm2 expression and consequent conversion of pyruvate to non-esterified fatty acid synthesis due to increased CS activity and ACLY levels. Upregulation of AKT2 due to reduced miRNAs may serve as a permanent mechanism leading to increased pkm2 expression.