COVID-19 lockdown effects on the anthropometrics, aerobic capacity, muscle function and metabolic control in children and adolescents with overweight and obesity

Abstract Objective: The lockdown due to a novel Coronavirus (COVID-19) pandemic negatively impacted the daily physical activity levels and sedentary behavior of children and adolescents. The purpose of this study was to determine the effects of lockdown on the anthropometric measurements, aerobic capacity, muscle function, lipid profile and glycemic control in overweight and obese children and adolescents. Methods: 104 children and adolescents with overweight and obesity were divided in a non-lock-down group (NL) (n = 48) and a lockdown group (L) (n = 56). Both NL and L groups were evaluated on three consecutive days, day one: anthropometric measurements; day two: aerobic capacity and muscle function and day three: lipid profile and glycemic control. Data are presented as mean ± standard deviation (SD) and median plus interquartile range (IQR) according to their assumption of normality. Results: The L group increased the body weight (81.62 ± 22.04 kg vs 74.04 ± 24.46 kg; p = 0.05), body mass index (32.54 ± 5,49 kg/m² vs 30.48 ± 6.88 kg/m²; p = 0.04), body mass index by z-score (3.10 ± 0.60 SD vs 2.67 ± 0.85 SD; p = 0.0015), triglycerides [141.00 mg/dl IQR (106.00-190.00 mg/dl) vs 103.00 mg/dl IQR (78.50- 141.50 mg/dl); p = 0.001], fasting insulin [31.00 mU/L IQR (25.01 - 47-17 mU/L vs 21.82 mU/L IQR (16.88 - 33.10 mU/L; p = 0.001)] and Conclusions: The lockdown due COVID-19 pandemic had a negative impact on the anthropometric measurements, lipid profile, and glycemic control of overweight and obese children and adolescents.

NADPH oxidase contributes to oxidative damage and mitochondrial impairment induced by acute ethanol treatment in rat hippocampal neurons.

Acute ethanol treatment induces neurodegeneration in cultured neurons and can lead to brain damage in animal models. Neuronal cells exposed to ethanol showed an increase in reactive oxygen species (ROS), oxidative damage and mitochondrial impairment contributing to synaptic failure. However, the underlying mechanisms of these events are not well understood. Here, we studied the contribution of NADPH oxidase, as a relevant source of ROS production in the brain, to mitochondrial impairment and oxidative stress induced by ethanol. We used primary hippocampal neurons subjected to an acute treatment of ethanol at increasing concentrations (25, 50, and 75 mM, 24 h), and we evaluated ROS production, mitochondrial function, and synaptic vesicle activity. Our studies showed that after ethanol administration, hippocampal neurons presented an increase in ROS levels, mitochondrial dysfunction, calcium handling defects, and synaptic impairment. Interestingly, treatment with the NADPH inhibitor, apocynin, significantly prevented oxidative stress, mitochondrial dysfunction, and the impairment of synaptic vesicle activity induced by ethanol treatment. These results indicate that NADPH oxidase could be a key participant in the molecular mechanism by which alcohol affects the brain.

Mifepristone enhances insulin-stimulated Akt phosphorylation and glucose uptake in skeletal muscle cells.

Mifepristone is the only FDA-approved drug for glycaemia control in patients with Cushing's syndrome and type 2 diabetes. Mifepristone also has beneficial effects in animal models of diabetes and patients with antipsychotic treatment-induced obesity. However, the mechanisms through which Mifepristone produces its beneficial effects are not completely elucidated. PURPOSE: To determine the effects of mifepristone on insulin-stimulated glucose uptake on a model of L6 rat-derived skeletal muscle cells. RESULTS: Mifepristone enhanced insulin-dependent glucose uptake, GLUT4 translocation to the plasma membrane and Akt Ser473 phosphorylation in L6 myotubes. In addition, mifepristone reduced oxygen consumption and ATP levels and increased AMPK Thr172 phosphorylation. The knockdown of AMPK prevented the effects of mifepristone on insulin response. CONCLUSIONS: Mifepristone enhanced insulin-stimulated glucose uptake through a mechanism that involves a decrease in mitochondrial function and AMPK activation in skeletal muscle cells.

New Implications for the Melanocortin System in Alcohol Drinking Behavior in Adolescents: The Glial Dysfunction Hypothesis.

Alcohol dependence causes physical, social, and moral harms and currently represents an important public health concern. According to the World Health Organization (WHO), alcoholism is the third leading cause of death worldwide, after tobacco consumption and hypertension. Recent epidemiologic studies have shown a growing trend in alcohol abuse among adolescents, characterized by the consumption of large doses of alcohol over a short time period. Since brain development is an ongoing process during adolescence, short- and long-term brain damage associated with drinking behavior could lead to serious consequences for health and wellbeing. Accumulating evidence indicates that alcohol impairs the function of different components of the melanocortin system, a major player involved in the consolidation of addictive behaviors during adolescence and adulthood. Here, we hypothesize the possible implications of melanocortins and glial cells in the onset and progression of alcohol addiction. In particular, we propose that alcohol-induced decrease in α-MSH levels may trigger a cascade of glial inflammatory pathways that culminate in altered gliotransmission in the ventral tegmental area and nucleus accumbens (NAc). The latter might potentiate dopaminergic drive in the NAc, contributing to increase the vulnerability to alcohol dependence and addiction in the adolescence and adulthood.

Dissecting signalling by individual Akt/PKB isoforms, three steps at once.

The serine/threonine kinase Akt/PKB (protein kinase B) is key for mammalian cell growth, survival, metabolism and oncogenic transformation. The diverse level and tissue expression of its three isoforms, Akt1/PKBα, Akt2/PKBß and Akt3/PKBγ, make it daunting to identify isoform-specific actions in vivo and even in isolated tissues/cells. To date, isoform-specific knockout and knockdown have been the best strategies to dissect their individual overall functions. In a recent article in the Biochemical Journal, Kajno et al. reported a new strategy to study isoform selectivity in cell lines. Individual Akt/PKB isoforms in 3T3-L1 pre-adipocytes are first silenced via shRNA and stable cellular clones lacking one or the other isoform are selected. The stably silenced isoform is then replaced by a mutant engineered to be refractory to inhibition by MK-2206 (Akt1(W80A) or Akt2(W80A)). Akt1(W80A) or Akt2(W80A) are functional and effectively recruited to the plasma membrane in response to insulin. The system affords the opportunity to acutely control the activity of the endogenous non-silenced isoform through timely addition of MK-2206. Using this approach, it is confirmed that Akt1/PKBα is the preferred isoform sustaining adipocyte differentiation, but both Akt1/PKBα and Akt2/PKBß can indistinctly support insulin-dependent FoxO1 (forkhead box O1) nuclear exclusion. Surprisingly, either isoform can also support insulin-dependent glucose transporter (GLUT) 4 translocation to the membrane, in contrast with the preferential role of Akt2/PKBß assessed by knockdown studies. The new strategy should allow analysis of the plurality of Akt/PKB functions in other cells and in response to other stimuli. It should also be amenable to high-throughput studies to speed up advances in signal transmission by this pivotal kinase.

The cholesterol-lowering agent methyl-ß-cyclodextrin promotes glucose uptake via GLUT4 in adult muscle fibers and reduces insulin resistance in obese mice.

Insulin stimulates glucose uptake in adult skeletal muscle by promoting the translocation of GLUT4 glucose transporters to the transverse tubule (T-tubule) membranes, which have particularly high cholesterol levels. We investigated whether T-tubule cholesterol content affects insulin-induced glucose transport. Feeding mice a high-fat diet (HFD) for 8 wk increased by 30% the T-tubule cholesterol content of triad-enriched vesicular fractions from muscle tissue compared with triads from control mice. Additionally, isolated muscle fibers (flexor digitorum brevis) from HFD-fed mice showed a 40% decrease in insulin-stimulated glucose uptake rates compared with fibers from control mice. In HFD-fed mice, four subcutaneous injections of MßCD, an agent reported to extract membrane cholesterol, improved their defective glucose tolerance test and normalized their high fasting glucose levels. The preincubation of isolated muscle fibers with relatively low concentrations of MßCD increased both basal and insulin-induced glucose uptake in fibers from controls or HFD-fed mice and decreased Akt phosphorylation without altering AMPK-mediated signaling. In fibers from HFD-fed mice, MßCD improved insulin sensitivity even after Akt or CaMK II inhibition and increased membrane GLUT4 content. Indinavir, a GLUT4 antagonist, prevented the stimulatory effects of MßCD on glucose uptake. Addition of MßCD elicited ryanodine receptor-mediated calcium signals in isolated fibers, which were essential for glucose uptake. Our findings suggest that T-tubule cholesterol content exerts a critical regulatory role on insulin-stimulated GLUT4 translocation and glucose transport and that partial cholesterol removal from muscle fibers may represent a useful strategy to counteract insulin resistance.

Insulin elicits a ROS-activated and an IP3-dependent Ca²âº release, which both impinge on GLUT4 translocation.

Insulin signaling includes generation of low levels of H2O2; however, its origin and contribution to insulin-stimulated glucose transport are unknown. We tested the impact of H2O2 on insulin-dependent glucose transport and GLUT4 translocation in skeletal muscle cells. H2O2 increased the translocation of GLUT4 with an exofacial Myc-epitope tag between the first and second transmembrane domains (GLUT4myc), an effect additive to that of insulin. The anti-oxidants N-acetyl L-cysteine and Trolox, the p47(phox)-NOX2 NADPH oxidase inhibitory peptide gp91-ds-tat or p47(phox) knockdown each reduced insulin-dependent GLUT4myc translocation. Importantly, gp91-ds-tat suppressed insulin-dependent H2O2 production. A ryanodine receptor (RyR) channel agonist stimulated GLUT4myc translocation and insulin stimulated RyR1-mediated Ca(2+) release by promoting RyR1 S-glutathionylation. This pathway acts in parallel to insulin-mediated stimulation of inositol-1,4,5-trisphosphate (IP3)-activated Ca(2+) channels, in response to activation of phosphatidylinositol 3-kinase and its downstream target phospholipase C, resulting in Ca(2+) transfer to the mitochondria. An inhibitor of IP3 receptors, Xestospongin B, reduced both insulin-dependent IP3 production and GLUT4myc translocation. We propose that, in addition to the canonical α,ß phosphatidylinositol 3-kinase to Akt pathway, insulin engages both RyR-mediated Ca(2+) release and IP3-receptor-mediated mitochondrial Ca(2+) uptake, and that these signals jointly stimulate glucose uptake.

Insulin-dependent H2O2 production is higher in muscle fibers of mice fed with a high-fat diet.

Insulin resistance is defined as a reduced ability of insulin to stimulate glucose utilization. C57BL/6 mice fed with a high-fat diet (HFD) are a model of insulin resistance. In skeletal muscle, hydrogen peroxide (H2O2) produced by NADPH oxidase 2 (NOX2) is involved in signaling pathways triggered by insulin. We evaluated oxidative status in skeletal muscle fibers from insulin-resistant and control mice by determining H2O2 generation (HyPer probe), reduced-to-oxidized glutathione ratio and NOX2 expression. After eight weeks of HFD, insulin-dependent glucose uptake was impaired in skeletal muscle fibers when compared with control muscle fibers. Insulin-resistant mice showed increased insulin-stimulated H2O2 release and decreased reduced-to-oxidized glutathione ratio (GSH/GSSG). In addition, p47phox and gp91phox (NOX2 subunits) mRNA levels were also high (~3-fold in HFD mice compared to controls), while protein levels were 6.8- and 1.6-fold higher, respectively. Using apocynin (NOX2 inhibitor) during the HFD feeding period, the oxidative intracellular environment was diminished and skeletal muscle insulin-dependent glucose uptake restored. Our results indicate that insulin-resistant mice have increased H2O2 release upon insulin stimulation when compared with control animals, which appears to be mediated by an increase in NOX2 expression.

Testosterone increases GLUT4-dependent glucose uptake in cardiomyocytes.

Testosterone exerts important effects in the heart. Cardiomyocytes are target cells for androgens, and testosterone induces rapid effects via Ca(2+) release and protein kinase activation and long-term effects via cardiomyocyte differentiation and hypertrophy. Furthermore, it stimulates metabolic effects such as increasing glucose uptake in different tissues. Cardiomyocytes preferentially consume fatty acids for ATP production, but under particular circumstances, glucose uptake is increased to optimize energy production. We studied the effects of testosterone on glucose uptake in cardiomyocytes. We found that testosterone increased uptake of the fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-2-deoxyglucose and [(3) H]2-deoxyglucose, which was blocked by the glucose transporter 4 (GLUT4) inhibitor indinavir. Testosterone stimulation in the presence of cyproterone or albumin-bound testosterone-induced glucose uptake, which suggests an effect that is independent of the intracellular androgen receptor. To determine the degree of GLUT4 cell surface exposure, cardiomyocytes were transfected with the plasmid GLUT4myc-eGFP. Subsequently, testosterone increased GLUT4myc-GFP exposure at the plasma membrane. Inhibition of Akt by the Akt-inhibitor-VIII had no effect. However, inhibition of Ca(2+) /calmodulin protein kinase (CaMKII) (KN-93 and autocamtide-2 related inhibitory peptide II) and AMP-activated protein kinase (AMPK) (compound C and siRNA for AMPK) prevented glucose uptake induced by testosterone. Moreover, GLUT4myc-eGFP exposure at the cell surface caused by testosterone was also abolished after CaMKII and AMPK inhibition. These results suggest that testosterone increases GLUT4-dependent glucose uptake, which is mediated by CaMKII and AMPK in cultured cardiomyocytes. Glucose uptake could represent a mechanism by which testosterone increases energy production and protein synthesis in cardiomyocytes.

Electrical stimuli release ATP to increase GLUT4 translocation and glucose uptake via PI3Kγ-Akt-AS160 in skeletal muscle cells.

Skeletal muscle glucose uptake in response to exercise is preserved in insulin-resistant conditions, but the signals involved are debated. ATP is released from skeletal muscle by contractile activity and can autocrinely signal through purinergic receptors, and we hypothesized it may influence glucose uptake. Electrical stimulation, ATP, and insulin each increased fluorescent 2-NBD-Glucose (2-NBDG) uptake in primary myotubes, but only electrical stimulation and ATP-dependent 2-NBDG uptake were inhibited by adenosine-phosphate phosphatase and by purinergic receptor blockade (suramin). Electrical stimulation transiently elevated extracellular ATP and caused Akt phosphorylation that was additive to insulin and inhibited by suramin. Exogenous ATP transiently activated Akt and, inhibiting phosphatidylinositol 3-kinase (PI3K) or Akt as well as dominant-negative Akt mutant, reduced ATP-dependent 2-NBDG uptake and Akt phosphorylation. ATP-dependent 2-NBDG uptake was also inhibited by the G protein ßγ subunit-interacting peptide ßark-ct and by the phosphatidylinositol 3-kinase-γ (PI3Kγ) inhibitor AS605240. ATP caused translocation of GLUT4myc-eGFP to the cell surface, mechanistically mediated by increased exocytosis involving AS160/Rab8A reduced by dominant-negative Akt or PI3Kγ kinase-dead mutants, and potentiated by myristoylated PI3Kγ. ATP stimulated 2-NBDG uptake in normal and insulin-resistant adult muscle fibers, resembling the reported effect of exercise. Hence, the ATP-induced pathway may be tapped to bypass insulin resistance.