Gustavo J. Santos

Cafeteria diet inhibits insulin clearance by reduced insulin-degrading enzyme expression and mRNA splicing.

Insulin clearance plays a major role in glucose homeostasis and insulin sensitivity in physiological and/or pathological conditions, such as obesity-induced type 2 diabetes as well as diet-induced obesity. The aim of the present work was to evaluate cafeteria diet-induced obesity-induced changes in insulin clearance and to explain the mechanisms underlying these possible changes. Female Swiss mice were fed either a standard chow diet (CTL) or a cafeteria diet (CAF) for 8 weeks, after which we performed glucose tolerance tests, insulin tolerance tests, insulin dynamics, and insulin clearance tests. We then isolated pancreatic islets for ex vivo glucose-stimulated insulin secretion as well as liver, gastrocnemius, visceral adipose tissue, and hypothalamus for subsequent protein analysis by western blot and determination of mRNA levels by real-time RT-PCR. The cafeteria diet induced insulin resistance, glucose intolerance, and increased insulin secretion and total insulin content. More importantly, mice that were fed a cafeteria diet demonstrated reduced insulin clearance and decay rate as well as reduced insulin-degrading enzyme (IDE) protein and mRNA levels in liver and skeletal muscle compared with the control animals. Furthermore, the cafeteria diet reduced IDE expression and alternative splicing in the liver and skeletal muscle of mice. In conclusion, a cafeteria diet impairs glucose homeostasis by reducing insulin sensitivity, but it also reduces insulin clearance by reducing IDE expression and alternative splicing in mouse liver; however, whether this mechanism contributes to the glucose intolerance or helps to ameliorate it remains unclear.

CNTF protects MIN6 cells against apoptosis induced by Alloxan and IL-1β through downregulation of the AMPK pathway.

Our group previously demonstrated that CNTF protects pancreatic islets against apoptosis induced by IL1β. In addition, it is known that AMPK knockout protects beta cells from IL1β-mediated apoptosis, however how AMPK activation leads to apoptosis remains unknown. The present study was designed to investigate the possible role of AMPK pathway modulation in CNTF protective effects against apoptosis induced by IL1β or Alloxan and how AMPK activation leads to beta cells apoptosis. First, we observed that apoptosis of MIN6 cells, induced by Alloxan as well as IL-1β, requires activation of the AMPK pathway, and also that CNTF protective effects are dependent on downregulation of AMPK. In addition, we found that Alloxan induces AMPK differently from IL1β, as Alloxan acts mainly through CaMKII while IL1β acts through LKB1 phosphorylation. Meanwhile, CNTF by itself inhibited the AMPK pathway and protected against AMPK activation induced by Alloxan or IL1β via downregulation of CaMKII. Finally, AMPK-dependent MIN6 cell apoptosis, induced by IL1β or Alloxan, required increased iNOS expression, an effect that was reversed by CNTF downregulation of AMPK pathway and iNOS expression. In conclusion, IL1β upregulates the LKB1-AMPK-INOS pathway, while Alloxan acts through CaMKII-AMPK-INOS, both ultimately leading to beta cell death. In this context, CNTF protects beta cells against apoptosis, induced by either IL1β or Alloxan, through downregulation of the CaMKII-AMPK-INOS pathway.

Augmented β-Cell Function and Mass in Glucocorticoid-Treated Rodents Are Associated with Increased Islet Ir-β/AKT/mTOR and Decreased AMPK/ACC and AS160 Signaling

Glucocorticoid (GC) therapies may adversely cause insulin resistance (IR) that lead to a compensatory hyperinsulinemia due to insulin hypersecretion. The increased β-cell function is associated with increased insulin signaling that has the protein kinase B (AKT) substrate with 160 kDa (AS160) as an important downstream AKT effector. In muscle, both insulin and AMP-activated protein kinase (AMPK) signaling phosphorylate and inactivate AS160, which favors the glucose transporter (GLUT)-4 translocation to plasma membrane. Whether AS160 phosphorylation is modulated in islets from GC-treated subjects is unknown. For this, two animal models, Swiss mice and Wistar rats, were treated with dexamethasone (DEX) (1 mg/kg body weight) for 5 consecutive days. DEX treatment induced IR, hyperinsulinemia, and dyslipidemia in both species, but glucose intolerance and hyperglycemia only in rats. DEX treatment caused increased insulin secretion in response to glucose and augmented β-cell mass in both species that were associated with increased islet content and increased phosphorylation of the AS160 protein. Protein AKT phosphorylation, but not AMPK phosphorylation, was found significantly enhanced in islets from DEX-treated animals. We conclude that the augmented β-cell function developed in response to the GC-induced IR involves inhibition of the islet AS160 protein activity.

Ciliary neurotrophic factor protects mice against streptozotocin-induced type 1 diabetes through SOCS3: the role of STAT1/STAT3 ratio in β-cell death.

Background: CNTF promotes islet survival, possibly protecting mice against type 1 diabetes. Results: CNTF inhibits STZ- and IL1β-induced apoptosis of islets and increases SOCS3 expression. Conclusion: CNTF protects against STZ-induced diabetes, which depends on increased SOCS3 expression and reduced STAT1/STAT3 ratio. Significance: Understanding the mechanisms that determine pancreatic islet fate is crucial for the prevention and treatment of diabetes. Type 1 diabetes is characterized by a loss of islet β-cells. Ciliary neurotrophic factor (CNTF) protects pancreatic islets against cytokine-induced apoptosis. For this reason, we assessed whether CNTF protects mice against streptozotocin-induced diabetes (a model of type 1 diabetes) and the mechanism for this protection. WT and SOCS3 knockdown C57BL6 mice were treated for 5 days with citrate buffer or 0.1 mg/kg CNTF before receiving 80 mg/kg streptozotocin. Glycemia in non-fasted mice was measured weekly from days 0–28 after streptozotocin administration. Diabetes was defined as a blood glucose > 11.2 mmol/liter. Wild-type (WT) and SOCS3 knockdown MIN6 cells were cultured with CNTF, IL1β, or both. CNTF reduced diabetes incidence and islet apoptosis in WT but not in SOCS3kd mice. Likewise, CNTF inhibited apoptosis in WT but not in SOCS3kd MIN6 cells. CNTF increased STAT3 phosphorylation in WT and SOCS3kd mice and MIN6 cells but reduced STAT1 phosphorylation only in WT mice, in contrast to streptozotocin and IL1β. Moreover, CNTF reduced NFκB activation and required down-regulation of inducible NO synthase expression to exert its protective effects. In conclusion, CNTF protects mice against streptozotocin-induced diabetes by increasing pancreatic islet survival, and this protection depends on SOCS3. In addition, SOCS3 expression and β-cell fate are dependent on STAT1/STAT3 ratio.

Acute exercise restores insulin clearance in diet-induced obese mice

The aim of this study was to investigate the insulin clearance in diet-induced obese (DIO) mice submitted to acute endurance exercise (3h of treadmill exercise at 60-70% VO2max). Glucose-stimulated insulin secretion in isolated islets; ipGTT; ipITT; ipPTT; in vivo insulin clearance; protein expression in liver, skeletal muscle, and adipose tissue (insulin degrading enzyme (IDE), insulin receptor subunitβ(IRβ), phospho-Akt (p-Akt) and phospho-AMPK (p-AMPK)), and the activity of IDE in the liver and skeletal muscle were accessed. In DIO mice, acute exercise reduced fasting glycemia and insulinemia, improved glucose and insulin tolerance, reduced hepatic glucose production, and increased p-Akt protein levels in liver and skeletal muscle and p-AMPK protein levels in skeletal muscle. In addition, insulin secretion was reduced, whereas insulin clearance and the expression of IDE and IRβ were increased in liver and skeletal muscle. Finally, IDE activity was increased only in skeletal muscle. In conclusion, we propose that the increased insulin clearance and IDE expression and activity, primarily, in skeletal muscle, constitute an additional mechanism, whereby physical exercise reduces insulinemia in DIO mice.

Endurance training inhibits insulin clearance and IDE expression in Swiss mice.

Introduction Endurance training improves peripheral insulin sensitivity in the liver and the skeletal muscle, but the mechanism for this effect is poorly understood. Recently, it was proposed that insulin clearance plays a major role in both glucose homeostasis and insulin sensitivity. Therefore, our goal was to determine the mechanism by which endurance training improves insulin sensitivity and how it regulates insulin clearance in mice. Methods Mice were treadmill-trained for 4 weeks at 70–80% of maximal oxygen consumption (VO2 max) for 60 min, 5 days a week. The glucose tolerance and the insulin resistance were determined using an IPGTT and an IPITT, respectively, and the insulin decay rate was calculated from the insulin clearance. Protein expression and phosphorylation in the liver and the skeletal muscle were ascertained by Western blot. Results Trained mice exhibited an increased VO2 max, time to exhaustion, glucose tolerance and insulin sensitivity. They had smaller fat pads and lower plasma concentrations of insulin and glucose. Endurance training inhibited insulin clearance and reduced expression of IDE in the liver, while also inhibiting insulin secretion by pancreatic islets. There was increased phosphorylation of both the canonical (IR-AKT) and the non-canonical (CaMKII-AMPK-ACC) insulin pathways in the liver of trained mice, whereas only the CaMKII-AMPK pathway was increased in the skeletal muscle. Conclusion Endurance training improved glucose homeostasis not only by increasing peripheral insulin sensitivity but also by decreasing insulin clearance and reducing IDE expression in the liver.

Interleukin-6 increases the expression and activity of insulin-degrading enzyme

Impairment of the insulin-degrading enzyme (IDE) is associated with obesity and type 2 diabetes mellitus (T2DM). Here, we used 4-mo-old male C57BL/6 interleukin-6 (IL-6) knockout mice (KO) to investigate the role of this cytokine on IDE expression and activity. IL-6 KO mice displayed lower insulin clearance in the liver and skeletal muscle, compared with wild type (WT), due to reduced IDE expression and activity. We also observed that after 3-h incubation, IL-6, 50 and 100 ng ml−1, increased the expression of IDE in HEPG2 and C2C12 cells, respectively. In addition, during acute exercise, the inhibition of IL-6 prevented an increase in insulin clearance and IDE expression and activity, mainly in the skeletal muscle. Finally, IL-6 and IDE concentrations were significantly increased in plasma from humans, after an acute exercise, compared to pre-exercise values. Although the increase in plasma IDE activity was only marginal, a positive correlation between IL-6 and IDE activity, and between IL-6 and IDE protein expression, was observed. Our outcomes indicate a novel function of IL-6 on the insulin metabolism expanding the possibilities for new potential therapeutic strategies, focused on insulin degradation, for the treatment and/or prevention of diseases related to hyperinsulinemia, such as obesity and T2DM.

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.