Sandra Mara Ferreira

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.

Modulation of the peroxiredoxin system by cytokines in insulin-producing RINm5F cells: Down-regulation of PRDX6 increases susceptibility of beta cells to oxidative stress

Peroxiredoxins are a family of six antioxidant enzymes (PRDX1-6), and may be an alternative system for the pancreatic beta cells to cope with oxidative stress. This study investigated whether the main diabetogenic pro-inflammatory cytokines or the anti-inflammatory cytokine IL-4 modulate PRDXs levels and putative intracellular pathways important for this process in the insulin-producing RINm5F cells. RINm5F cells expressed significant amounts of PRDX1, PRDX3 and PRDX6 enzymes. Only PRDX6 was modulated by cytokines, showing both mRNA and protein down-regulation following incubation of RINm5F cells with TNF-alpha and IFN-gamma but not with IL-1beta. Separately IFN-gamma or TNF-alpha decreased PRDX6 protein but not mRNA levels. The blockage of the JNK signalling and of the calpains and proteasome proteolysis systems restored PRDX6 protein levels. IL-4 alone did not modulate PRDXs levels. However, pre/co-incubation with IL-4 substantially prevented the decrease in PRDX6 induced by pro-inflammatory cytokines. Knockdown of PRDX6 increased susceptibility of RINm5F cells to the deleterious effects of pro-inflammatory cytokines and to oxidative stress. These results show that, from the PRDXs significantly expressed in RINm5F cells, only PRDX6 is modulated by the diabetogenic cytokines IFN-gamma and TNF-alpha. This PRDX6 down-regulation depends on the calpain and proteasome systems and JNK signalling. PRDX6 is an important enzyme for protection against oxidative stress and the interaction between pro- and anti-inflammatory cytokines might be important to determine the antioxidant capacity of the cells.

Hyperinsulinemia caused by dexamethasone treatment is associated with reduced insulin clearance and lower hepatic activity of insulin-degrading enzyme.

OBJECTIVES Glucocorticoid treatment induces insulin resistance (IR), which is counteracted by a compensatory hyperinsulinemia, due to increased pancreatic β-cell function. There is evidence for also reduced hepatic insulin clearance, but whether this correlates with altered activity of insulin-degrading enzyme (IDE) in the liver, is not fully understood. Here, we investigated whether hyperinsulinemia, in glucocorticoid-treated rodents, is associated with any alteration in the insulin clearance and activity of the IDE in the liver. MATERIALS/METHODS Adult male Swiss mice and Wistar rats were treated with the synthetic glucocorticoid dexamethasone intraperitoneally [1mg/kg body weight (b.w.)] for 5 consecutive days. RESULTS Glucocorticoid treatment induced IR and hyperinsulinemia in both species, but was more impactful in rats that also displayed glucose intolerance and hyperglycemia. Insulin clearance was reduced in glucocorticoid-treated rats and mice, as judged by the reduction of insulin decay rate and increased insulin area-under-the-curve (47% and 87%, respectively). These results were associated with reduced activity (35%) of hepatic IDE in rats and a tendency to reduction (p=0.068) in mice, without alteration in hepatic IDE mRNA content, in both species. CONCLUSION In conclusion, the reduced insulin clearance in glucocorticoid-treated rodents was due to the reduction of hepatic IDE activity, at least in rats, which may contributes to the compensatory hyperinsulinemia. These findings corroborate the idea that short-term and/or partial inhibition of IDE activity in the liver could be beneficial for the glycemic control.

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.

Metabolic memory of ß-cells controls insulin secretion and is mediated by CaMKII.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) functions both in regulation of insulin secretion and neurotransmitter release through common downstream mediators. Therefore, we hypothesized that pancreatic ß-cells acquire and store the information contained in calcium pulses as a form of “metabolic memory”, just as neurons store cognitive information. To test this hypothesis, we developed a novel paradigm of pulsed exposure of ß-cells to intervals of high glucose, followed by a 24-h consolidation period to eliminate any acute metabolic effects. Strikingly, ß-cells exposed to this high-glucose pulse paradigm exhibited significantly stronger insulin secretion. This metabolic memory was entirely dependent on CaMKII. Metabolic memory was reflected on the protein level by increased expression of proteins involved in glucose sensing and Ca2+-dependent vesicle secretion, and by elevated levels of the key ß-cell transcription factor MAFA. In summary, like neurons, human and mouse ß-cells are able to acquire and retrieve information.

Acute Exercise Improves Insulin Clearance and Increases the Expression of Insulin-Degrading Enzyme in the Liver and Skeletal Muscle of Swiss Mice.

The effects of exercise on insulin clearance and IDE expression are not yet fully elucidated. Here, we have explored the effect of acute exercise on insulin clearance and IDE expression in lean mice. Male Swiss mice were subjected to a single bout of exercise on a speed/angle controlled treadmill for 3-h at approximately 60–70% of maximum oxygen consumption. As expected, acute exercise reduced glycemia and insulinemia, and increased insulin tolerance. The activity of AMPK-ACC, but not of IR-Akt, pathway was increased in the liver and skeletal muscle of trained mice. In an apparent contrast to the reduced insulinemia, glucose-stimulated insulin secretion was increased in isolated islets of these mice. However, insulin clearance was increased after acute exercise and was accompanied by increased expression of the insulin-degrading enzyme (IDE), in the liver and skeletal muscle. Finally, C2C12, but not HEPG2 cells, incubated at different concentrations of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) for 3-h, showed increased expression of IDE. In conclusion, acute exercise increases insulin clearance, probably due to an augmentation of IDE expression in the liver and skeletal muscle. The elevated IDE expression, in the skeletal muscle, seems to be mediated by activation of AMPK-ACC pathway, in response to exercise. We believe that the increase in the IDE expression, comprise a safety measure to maintain glycemia at or close to physiological levels, turning physical exercise more effective and safe.

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.

Effects of Displacement and Trajectory Length on the Variability Pattern of Reaching Movements

The design of the present study enabled the authors to distinguish between the possible effects of movement displacement and trajectory length on the pattern of final positions of planar reaching movements. With their eyes closed, 9 subjects performed series of fast and accurate movements from different initial positions to the same target. For some series, the movements were unconstrained and were therefore performed along an approximately straight vertical line. For other series, an obstacle was positioned so that trajectory length was increased because of an increase in movement curvature. Ellipses of variability obtained by means of principal component analysis applied to the scatter of movement final positions enabled the authors to assess the pattern of movement variable errors. The results showed that the orientation of the ellipses was not affected by movement displacement or by trajectory length, whereas variable errors increased with movement displacement. An increase in trajectory length as a consequence of increased curvature caused no change in variable error. From the perspective of current motor control theory, that finding was quite unexpected. Further studies are required so that one can distinguish among the possible effects of various kinematics, kinetics, and other variables that could affect the pattern of variable errors of reaching movements.