Metabolic Consequences of Anabolic Steroids, Insulin, and Growth Hormone Abuse in Recreational Bodybuilders: Implications for the World Anti-Doping Agency Passport.

BACKGROUND: Hormonal doping in recreational sports is a public-health concern. The World Anti-Doping Agency (WADA) promoted the creation of the Athlete Biological Passport, aiming to monitor athlete's biological variables over time to facilitate indirect detection of doping. Detection tests for anabolic androgenic steroids (AAS) and growth hormone (GH) are available while insulin abuse cannot be revealed. We have determined in recreational bodybuilders the metabolic effects associated with different patterns of hormone abuse. All analyses were conducted using Statistical Package for Social Sciences (SPSS) 21.0 software (SPSS Chicago, IL). RESULTS: We have assessed plasma concentrations of selected metabolic markers and fatty acid content in erythrocyte membranes of 92 male bodybuilders and in 45 healthy controls. Hormonal abuse was identified by anonymous questionnaires. 43% (%) of recruited bodybuilders regularly abused hormones, i.e., anabolic androgenic steroids (95%) often associated with GH (30%) and/or insulin (38%). HDL-cholesterol was lower in insulin and/or GH abusers. Alanine (ALT) and aspartic (AST) transaminases were greater in hormone abusing bodybuilders than in non-doping bodybuilders and controls. Insulin doping was selectively associated with increased plasma ALT-to-AST ratio. In erythrocyte membranes, elongase activity (i.e., stearic-to-palmitic ratio) was lower in insulin and/or growth hormone doping, whereas increased Δ-9 desaturase activity (i.e., palmitoleic-to-palmitic ratio) was selectively associated with insulin doping. CONCLUSIONS: In conclusion, our study demonstrates that insulin and GH abuse are characterized by multiple alterations of specific metabolic markers. Although further studies are needed to test whether longitudinal monitoring of selected metabolic marker such as muscle contraction time, HDL levels, ALT-AST ratio as well as the activities of selected enzymes (e.g. Δ-9 desaturase and elongase), could contribute to the detection of insulin and GH abuse in sport.

High-protein diet with excess leucine prevents inactivity-induced insulin resistance in women.

BACKGROUND AND AIMS: Muscle inactivity leads to muscle atrophy and insulin resistance. The branched-chain amino acid (BCAA) leucine interacts with the insulin signaling pathway to modulate glucose metabolism. We have tested the ability of a high-protein BCAA-enriched diet to prevent insulin resistance during long-term bed rest (BR). METHODS: Stable isotopes were infused to determine glucose and protein kinetics in the postabsorptive state and during a hyperinsulinemic-euglycemic clamp in combination with amino acid infusion (Clamp + AA) before and at the end of 60 days of BR in two groups of healthy, young women receiving eucaloric diets containing 1 g of protein/kg per day (n = 8) or 1.45 g of protein/kg per day enriched with 0.15 g/kg per day of BCAAs (leucine/valine/isoleucine = 2/1/1) (n = 8). Body composition was determined by Dual X-ray Absorptiometry. RESULTS: BR decreased lean body mass by 7.6 ± 0.3 % and 7.2 ± 0.8 % in the groups receiving conventional or high protein-BCAA diets, respectively. Fat mass was unchanged in both groups. At the end of BR, percent changes of insulin-mediated glucose uptake significantly (p = 0.01) decreased in the conventional diet group from 155 ± 23 % to 84 ± 10 % while did not change significantly in the high protein-BCAA diet group from 126 ± 20 % to 141 ± 27 % (BR effect, p = 0.32; BR/diet interaction, p = 0.01; Repeated Measures ANCOVA). In contrast, there were no BR/diet interactions on proteolysis and protein synthesis Clamp + AA changes in the conventional diet and the high protein-BCAA diet groups. CONCLUSION: A high protein-BCAA enriched diet prevented inactivity-induced insulin resistance in healthy women.

Lipoprotein(a) as a Risk Factor for Cardiovascular Diseases: Pathophysiology and Treatment Perspectives.

Cardiovascular disease (CVD) is still a leading cause of morbidity and mortality, despite all the progress achieved as regards to both prevention and treatment. Having high levels of lipoprotein(a) [Lp(a)] is a risk factor for cardiovascular disease that operates independently. It can increase the risk of developing cardiovascular disease even when LDL cholesterol (LDL-C) levels are within the recommended range, which is referred to as residual cardiovascular risk. Lp(a) is an LDL-like particle present in human plasma, in which a large plasminogen-like glycoprotein, apolipoprotein(a) [Apo(a)], is covalently bound to Apo B100 via one disulfide bridge. Apo(a) contains one plasminogen-like kringle V structure, a variable number of plasminogen-like kringle IV structures (types 1-10), and one inactive protease region. There is a large inter-individual variation of plasma concentrations of Lp(a), mainly ascribable to genetic variants in the Lp(a) gene: in the general po-pulation, Lp(a) levels can range from <1 mg/dL to >1000 mg/dL. Concentrations also vary between different ethnicities. Lp(a) has been established as one of the risk factors that play an important role in the development of atherosclerotic plaque. Indeed, high concentrations of Lp(a) have been related to a greater risk of ischemic CVD, aortic valve stenosis, and heart failure. The threshold value has been set at 50 mg/dL, but the risk may increase already at levels above 30 mg/dL. Although there is a well-established and strong link between high Lp(a) levels and coronary as well as cerebrovascular disease, the evidence regarding incident peripheral arterial disease and carotid atherosclerosis is not as conclusive. Because lifestyle changes and standard lipid-lowering treatments, such as statins, niacin, and cholesteryl ester transfer protein inhibitors, are not highly effective in reducing Lp(a) levels, there is increased interest in developing new drugs that can address this issue. PCSK9 inhibitors seem to be capable of reducing Lp(a) levels by 25-30%. Mipomersen decreases Lp(a) levels by 25-40%, but its use is burdened with important side effects. At the current time, the most effective and tolerated treatment for patients with a high Lp(a) plasma level is apheresis, while antisense oligonucleotides, small interfering RNAs, and microRNAs, which reduce Lp(a) levels by targeting RNA molecules and regulating gene expression as well as protein production levels, are the most widely explored and promising perspectives. The aim of this review is to provide an update on the current state of the art with regard to Lp(a) pathophysiological mechanisms, focusing on the most effective strategies for lowering Lp(a), including new emerging alternative therapies. The purpose of this manuscript is to improve the management of hyperlipoproteinemia(a) in order to achieve better control of the residual cardiovascular risk, which remains unacceptably high.

Sarcopenic obesity in free-living older adults detected by the ESPEN-EASO consensus diagnostic algorithm: Validation in an Italian cohort and predictive value of insulin resistance and altered plasma ghrelin profile.

Aging and obesity are synergistic sarcopenia risk factors (RF). Their association in sarcopenic obesity (SO) enhances morbidity and mortality, but consensus on SO diagnostic criteria is limited. ESPEN and EASO issued a consensus algorithm for SO screening (obesity and clinical SO suspicion) and diagnosis [low muscle strength by hand-grip (HGS) and low muscle mass by BIA], and we investigated its implementation in older adults (>65-years), as well as SO-associated metabolic RF [insulin resistance (IR: HOMA) and plasma acylated (AG) and unacylated (UnAG) ghrelin, with predictive value also assessed from 5-year-prior observations]. Older adults with obesity from the Italian MoMa study on metabolic syndrome in primary care (n = 76) were studied. 7 of 61 individuals with positive screening had SO (SO+; 9 % of cohort). No individuals with negative screening had SO. SO+ had higher IR, AG and plasma AG/UnAG ratio (p < 0.05 vs negative screening and SO-), and both IR and ghrelin profile predicted 5-year SO risk independent of age, sex and BMI. The current results provide the first ESPEN-EASO algorithm-based investigation of SO in free-living older adults, with 9 % prevalence in those with obesity and 100 % algorithm sensitivity, and they support IR and plasma ghrelin profile as SO risk factors in this setting.

[Identifying possible homozygous familial hypercholesterolemia patients: an Italian experts' opinion]. / Inquadramento diagnostico del paziente con possibile ipercolesterolemia familiare omozigote: il parere di un gruppo italiano di esperti.

Homozygous familial hypercholesterolemia (HoFH) is a rare genetic disease characterized by high plasma levels of low-density lipoprotein cholesterol (LDL-C) and massive risk of premature atheromasia and cardiovascular events. HoFH is caused by mutations in several genes, such as LDLR, APOB, PCSK9 and LDLRAP1. If untreated, the average age of death is 18 years old, but fatalities within the first 5 years of age have been recorded. Therefore, early diagnosis and treatment are crucial, in order to prevent and/or delay the cardiovascular complications of LDL-C exposure. Because HoFH is a rare disorder, it is not frequently encountered in daily clinical practice at the primary/secondary unspecialized cardiological centers. Then the availability of practical indications helping to identify HoFH patients or to arise a suspect of HoFH is particularly strategic to promptly start the appropriate lipid-lowering therapy. For such a purpose, a group of Italian experts suggests three useful algorithms to identify cases requiring accurate and specialized clinical evaluation as potential HoFH patients. These cases with suspected HoFH should be addressed to specialized centres for the optimal management of these patients.

Epidemiology of atherosclerotic cardiovascular disease in polygenic hypercholesterolemia with or without high lipoprotein(a) levels.

Background and aims: Epidemiology of atherosclerotic cardiovascular disease might be different in patients with polygenic hypercholesterolemia plus high levels (≥30 mg/dl) of Lp(a) (H-Lpa) than in those with polygenic hypercholesterolemia alone (H-LDL). We compared the incidence of peripheral artery disease (PAD), coronary artery disease (CAD), and cerebrovascular disease (CVD) in patients with H-Lpa and in those with H-LDL. Methods: Retrospective analysis of demographics, risk factors, vascular events, therapy, and lipid profile in outpatient clinical data. Inclusion criteria was adult age, diagnosis of polygenic hypercholesterolemia, and both indication and availability for Lp(a) measurement. Results: Medical records of 258 patients with H-Lpa and 290 H-LDL were reviewed for occurrence of vascular events. The median duration of follow-up was 10 years (IQR 3-16). In spite of a similar reduction of LDL cholesterol, vascular events occurred more frequently, and approximately 7 years earlier (P = 0.024) in patients with H-Lpa than in H-LDL (HR 1.96 1.21-3.17, P = 0.006). The difference was around 10 years for acute events (TIA, Stroke, acute coronary events) and one year for chronic ones (P = 0.023 and 0.525, respectively). Occurrence of acute CAD was higher in H-Lpa men (HR 3.1, 95% CI 1.2-7.9, P = 0.007) while, among women, PAD was observed exclusively in H-Lpa subjects with smoking habits (P = 0.009). Conclusions: Patients with high Lp(a) levels suffer from a larger and earlier burden of the disease compared to those with polygenic hypercholesterolemia alone. These patients are at higher risk of CAD if they are men, and of PAD if they are women.

Early lean mass sparing effect of high-protein diet with excess leucine during long-term bed rest in women.

Muscle inactivity leads to muscle atrophy. Leucine is known to inhibit protein degradation and to promote protein synthesis in skeletal muscle. We tested the ability of a high-protein diet enriched with branched-chain amino acids (BCAAs) to prevent muscle atrophy during long-term bed rest (BR). We determined body composition (using dual energy x-ray absorptiometry) at baseline and every 2-weeks during 60 days of BR in 16 healthy young women. Nitrogen (N) balance was assessed daily as the difference between N intake and N urinary excretion. The subjects were randomized into two groups: one received a conventional diet (1.1 ± 0.03 g protein/kg, 4.9 ± 0.3 g leucine per day) and the other a high protein, BCAA-enriched regimen (1.6 ± 0.03 g protein-amino acid/kg, 11.4 ± 0.6 g leucine per day). There were significant BR and BR × diet interaction effects on changes in lean body mass (LBM) and N balance throughout the experimental period (repeated measures ANCOVA). During the first 15 days of BR, lean mass decreased by 4.1 ± 0.9 and 2.4 ± 2.1% (p < 0.05) in the conventional and high protein-BCAA diet groups, respectively, while at the end of the 60-day BR, LBM decreased similarly in the two groups by 7.4 ± 0.7 and 6.8 ± 2.4%. During the first 15 days of BR, mean N balance was 2.5 times greater (p < 0.05) in subjects on the high protein-BCAA diet than in those on the conventional diet, while we did not find significant differences during the following time intervals. In conclusion, during 60 days of BR in females, a high protein-BCAA diet was associated with an early protein-LBM sparing effect, which ceased in the medium and long term.

Mechanism of action and therapeutic use of bempedoic acid in atherosclerosis and metabolic syndrome.

Bempedoic acid is a new cholesterol-lowering drug, which has recently received US FDA and EMA approval. This drug targets lipid and glucose metabolism as well as inflammation via downregulation of ATP-citrate lyase and upregulation of AMP-activated protein kinase (AMPK). The primary effect is the reduction of cholesterol synthesis in the liver and its administration is generally not associated to unwanted muscle effects. Suppression of hepatic fatty acid synthesis leads to decreased triglycerides and, possibly, improved non-alcoholic fatty liver disease. Bempedoic acid may decrease gluconeogenesis leading to improved insulin sensitivity, glucose metabolism, and metabolic syndrome. The anti-inflammatory action of bempedoic acid is mainly achieved via activation of AMPK pathway in the immune cells, leading to decreased plasma levels of C-reactive protein. Effects of bempedoic acid on atherosclerotic cardiovascular disease, type 2 diabetes and chronic liver disease have been assessed in randomized clinical trials but require further confirmation. Safety clinical trials in phase III indicate that bempedoic acid administration is generally well-tolerated in combination with statins, ezetimibe, or proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors to achieve low-density lipoprotein cholesterol targets. The aim of this narrative review on bempedoic acid is to explore the underlying mechanisms of action and potential clinical targets, present existing evidence from clinical trials, and describe practical management of patients.

n-3 PUFA dietary lipid replacement normalizes muscle mitochondrial function and oxidative stress through enhanced tissue mitophagy and protects from muscle wasting in experimental kidney disease.

INTRODUCTION AND METHODS: Skeletal muscle mitochondrial dysfunction may cause tissue oxidative stress and consequent catabolism in chronic kidney disease (CKD), contributing to patient mortality. We investigated in 5/6-nephrectomized (Nx) rats the impact of n3-polyunsaturated fatty-acids (n3-PUFA) isocaloric partial dietary replacement on gastrocnemius muscle (Gm) mitochondrial master-regulators, ATP production, ROS generation and related muscle-catabolic derangements. RESULTS: Nx had low Gm mitochondrial nuclear respiratory factor-2 and peroxisome proliferator-activated receptor gamma coactivator-1alpha, low ATP production and higher mitochondrial fission-fusion protein ratio with ROS overproduction. n3-PUFA normalized all mitochondrial derangements and pro-oxidative tissue redox state (oxydized to total glutathione ratio). n3-PUFA also normalized Nx-induced muscle-catabolic proinflammatory cytokines, insulin resistance and low muscle weight. Human uremic serum reproduced mitochondrial derangements in C2C12 myotubes, while n3-PUFA coincubation prevented all effects. n3-PUFA also enhanced muscle mitophagy in-vivo and siRNA-mediated autophagy inhibition selectively blocked n3-PUFA-induced normalization of C2C12 mitochondrial ROS production. CONCLUSIONS: In conclusion, dietary n3-PUFA normalize mitochondrial master-regulators, ATP production and dynamics in experimental CKD. These effects occur directly in muscle cells and they normalize ROS production through enhanced mitophagy. Dietary n3-PUFA mitochondrial effects result in normalized catabolic derangements and protection from muscle wasting, with potential positive impact on patient survival.

Skeletal Muscle in Hypoxia and Inflammation: Insights on the COVID-19 Pandemic.

SARS-CoV-2 infection is often associated with severe inflammation, oxidative stress, hypoxia and impaired physical activity. These factors all together contribute to muscle wasting and fatigue. In addition, there is evidence of a direct SARS-CoV-2 viral infiltration into skeletal muscle. Aging is often characterized by sarcopenia or sarcopenic obesity These conditions are risk factors for severe acute COVID-19 and long-COVID-19 syndrome. From these observations we may predict a strong association between COVID-19 and decreased muscle mass and functions. While the relationship between physical inactivity, chronic inflammation, oxidative stress and muscle dysfunction is well-known, the effects on muscle mass of COVID-19-related hypoxemia are inadequately investigated. The aim of this review is to highlight metabolic, immunity-related and redox biomarkers potentially affected by reduced oxygen availability and/or muscle fatigue in order to shed light on the negative impact of COVID-19 on muscle mass and function. Possible countermeasures are also reviewed.

Statin-Associated Myopathy: Emphasis on Mechanisms and Targeted Therapy.

Hyperlipidemia is a major risk factor for cardiovascular morbidity and mortality. Statins are the first-choice therapy for dyslipidemias and are considered the cornerstone of atherosclerotic cardiovascular disease (ASCVD) in both primary and secondary prevention. Despite the statin-therapy-mediated positive effects on cardiovascular events, patient compliance is often poor. Statin-associated muscle symptoms (SAMS) are the most common side effect associated with treatment discontinuation. SAMS, which range from mild-to-moderate muscle pain, weakness, or fatigue to potentially life-threatening rhabdomyolysis, are reported by 10% to 25% of patients receiving statin therapy. There are many risk factors associated with patient features and hypolipidemic agents that seem to increase the risk of developing SAMS. Due to the lack of a "gold standard", the diagnostic test for SAMS is based on a clinical criteria score, which is independent of creatine kinase (CK) elevation. Mechanisms that underlie the pathogenesis of SAMS remain almost unclear, though a high number of risk factors may increase the probability of myotoxicity induced by statin therapy. Some of these, related to pharmacokinetic properties of statins and to concomitant therapies or patient characteristics, may affect statin bioavailability and increase vulnerability to high-dose statins.

Ruling Out Coronavirus Disease 2019 in Patients with Pneumonia: The Role of Blood Cell Count and Lung Ultrasound.

Coronavirus disease 2019 (COVID-19) is characterized by a distinctive blood leucocyte pattern and B-lines on lung ultrasound (LUS) as marker of alveolar-interstitial syndrome. We aimed to evaluate the accuracy of blood leucocyte count alone or in combination with LUS for COVID-19 diagnosis. We retrospectively enrolled consecutive patients diagnosed with community acquired pneumonia (CAP) at hospital admission to derive and validate cutoff values for blood cell count that could be predictive of COVID-19 before confirmation by the nucleic acid amplification test (NAAT). Cutoff values, generated and confirmed in inception (41/115, positive/negative patients) and validation (100/180, positive/negative patients) cohorts, were ≤17 and ≤10 cells/mm3 for basophils and eosinophils, respectively. Basophils and/or eosinophils below cutoff were associated with sensitivity of 98% (95%CI, 94-100) and negative likelihood ratio of 0.04 (95%CI, 0.01-0.11). In a subgroup of 265 subjects, the sensitivity of B-line on LUS was 15% lower (p < 0.001) than that of basophils and/or eosinophils below cutoff. The combination of B-lines with basophils and eosinophils below cutoff was associated with a moderate increase of the positive likelihood ratio: 5.0 (95%CI, 3.2-7.7). In conclusion, basophil and eosinophil counts above the generated cutoff virtually rule out COVID-19 in patients with CAP. Our findings can help optimize patient triage pending the NAAT results.

The Aging Muscle in Experimental Bed Rest: A Systematic Review and Meta-Analysis.

Background: Maintaining skeletal muscle mass and function in aging is crucial for preserving the quality of life and health. An experimental bed rest (BR) protocol is a suitable model to explore muscle decline on aging during inactivity. Objective: The purpose of this systematic review and meta-analysis was, therefore, to carry out an up-to-date evaluation of bed rest, with a specific focus on the magnitude of effects on muscle mass, strength, power, and functional capacity changes as well as the mechanisms, molecules, and pathways involved in muscle decay. Design: This was a systematic review and meta-analysis study. Data sources: We used PubMed, Medline; Web of Science, Google Scholar, and the Cochrane library, all of which were searched prior to April 23, 2020. A manual search was performed to cover bed rest experimental protocols using the following key terms, either singly or in combination: "Elderly Bed rest," "Older Bed rest," "Old Bed rest," "Aging Bed rest," "Aging Bed rest," "Bed-rest," and "Bedrest". Eligibility criteria for selecting studies: The inclusion criteria were divided into four sections: type of study, participants, interventions, and outcome measures. The primary outcome measures were: body mass index, fat mass, fat-free mass, leg lean mass, cross-sectional area, knee extension power, cytokine pattern, IGF signaling biomarkers, FOXO signaling biomarkers, mitochondrial modulation biomarkers, and muscle protein kinetics biomarkers. Results: A total of 25 studies were included in the qualitative synthesis, while 17 of them were included in the meta-analysis. In total, 118 healthy elderly volunteers underwent 5-, 7-, 10-, or 14-days of BR and provided a brief sketch on the possible mechanisms involved. In the very early phase of BR, important changes occurred in the skeletal muscle, with significant loss of performance associated with a lesser grade reduction of the total body and muscle mass. Meta-analysis of the effect of bed rest on total body mass was determined to be small but statistically significant (ES = -0.45, 95% CI: -0.72 to -0.19, P < 0.001). Moderate, statistically significant effects were observed for total lean body mass (ES = -0.67, 95% CI: -0.95 to -0.40, P < 0.001) after bed rest intervention. Overall, total lean body mass was decreased by 1.5 kg, while there was no relationship between bed rest duration and outcomes (Z = 0.423, p = 672). The meta-analyzed effect showed that bed rest produced large, statistically significant, effects (ES = -1.06, 95% CI: -1.37 to -0.75, P < 0.001) in terms of the knee extension power. Knee extension power was decreased by 14.65 N/s. In contrast, to other measures, meta-regression showed a significant relationship between bed rest duration and knee extension power (Z = 4.219, p < 0.001). Moderate, statistically significant, effects were observed after bed rest intervention for leg muscle mass in both old (ES = -0.68, 95% CI: -0.96 to -0.40, P < 0.001) and young (ES = -0.51, 95% CI: -0.80 to -0.22, P < 0.001) adults. However, the magnitude of change was higher in older (MD = -0.86 kg) compared to younger (MD = -0.24 kg) adults. Conclusion: Experimental BR is a suitable model to explore the detrimental effects of inactivity in young adults, old adults, and hospitalized people. Changes in muscle mass and function are the two most investigated variables, and they allow for a consistent trend in the BR-induced changes. Mechanisms underlying the greater loss of muscle mass and function in aging, following inactivity, need to be thoroughly investigated.

Central adiposity markers, plasma lipid profile and cardiometabolic risk prediction in overweight-obese individuals.

BACKGROUND: Waist circumference (WC) is the currently recommended marker of central fat for cardiometabolic risk screening. Alternative surrogate markers have been recently proposed to better reflect the metabolic impact of central fat accumulation per se, based on WC normalization by height (Weight-to-Height Ratio - WtoH; Body Roundness Index - BRI) or body mass index (BMI) without (A Body Shape Index - ABSI) or with inclusion of plasma triglyceride and HDL-cholesterol concentrations (Visceral Adiposity Index - VAI). METHODS: We investigated associations between WtoH, BRI, ABSI or VAI and insulin resistance (HOMA-index) or metabolic syndrome (MetS) in a general population cohort from the North-East Italy Mo.Ma. study (n = 1965, age = 49 ± 13 years, BMI = 26.7 ± 5.2 kg/m2). Baseline values were also evaluated as predictors of future insulin resistance and MetS in overweight-obese individuals undergoing 5-year follow-up (Ow-Ob) (n = 263; age = 54 ± 9, BMI = 30,7 ± 4,1). RESULTS: Compared to WC or BMI, basal WtoH and BRI were similarly associated with baseline HOMA and MetS prevalence after multiple adjustments (P < 0.001) and all markers similarly predicted 5-year HOMA and MetS (P < 0.001). Under basal conditions, superimposable results were observed for VAI whereas ABSI was less accurate or unable to identify baseline HOMA and MetS (p < 0.05 vs WtoH-BRI-VAI-WC-BMI). VAI had highest 5-year risk predictive value in Ow-Ob [ROC Area Under the Curve (AUC) VAI > WtoH-BRI-WC-BMI; p < 0.05] while no predictive value was in contrast observed for ABSI (ROC AUC ABSI < WtoH-BRI-WC-BMI; p < 0.05). Using alternate formulae with plasma lipid inclusion in ABSI and removal from VAI calculations completely reversed their 5-year predictive value and AUC. CONCLUSIONS: The current findings do not support replacement of WC with height-normalized anthropometric central fat surrogate markers to predict cardiometabolic risk in the general and overweight-obese population. BMI-normalization impairs risk assessment unless plasma lipid concentrations are available and included in calculations.

Alkalinization with potassium bicarbonate improves glutathione status and protein kinetics in young volunteers during 21-day bed rest.

BACKGROUND & AIMS: Physical inactivity is associated with lean body mass wasting, oxidative stress and pro-inflammatory changes of cell membrane lipids. Alkalinization may potentially counteract these alterations. We evaluated the effects of potassium bicarbonate supplementation on protein kinetics, glutathione status and pro- and anti-inflammatory polyunsaturated fatty acids (PUFA) in erythrocyte membranes in humans, during experimental bed rest. METHODS: Healthy, young, male volunteers were investigated at the end of two 21-day bed rest periods, one with, and the other without, daily potassium bicarbonate supplementation (90 mmol × d-1), according to a cross-over design. Oxidative stress in erythrocytes was evaluated by determining the ratio between reduced (GSH) and oxidized glutathione (GSSG). Glutathione turnover and phenylalanine kinetics, a marker of whole body protein metabolism, were determined by stable isotope infusions. Erythrocyte membranes PUFA composition was analyzed by gas-chromatography. RESULTS: At the end of the two study periods, urinary pH was 10 ± 3% greater in subjects receiving potassium bicarbonate supplementation (7.23 ± 0.15 vs. 6.68 ± 0.11, p < 0.001). Alkalinization increased total glutathione concentrations by 5 ± 2% (p < 0.05) and decreased its rate of clearance by 38 ± 13% (p < 0.05), without significantly changing GSH-to-GSSG ratio. After alkalinization, net protein balance in the postabsorptive state improved significantly by 17 ± 5% (p < 0.05) as well as the sum of n-3 PUFA and the n-3-to-n-6 PUFA ratio in erythrocyte membranes (p < 0.05). CONCLUSIONS: Alkalinization during long-term inactivity is associated with improved glutathione status, anti-inflammatory lipid pattern in cell membranes and reduction in protein catabolism at whole body level. This study suggests that, in clinical conditions characterized by inactivity, oxidative stress and inflammation, alkalinization could be a useful adjuvant therapeutic strategy.

Acylated ghrelin treatment normalizes skeletal muscle mitochondrial oxidative capacity and AKT phosphorylation in rat chronic heart failure.

BACKGROUND: Chronic heart failure (CHF) is associated with skeletal muscle abnormalities contributing to exercise intolerance, muscle loss, and negative impact on patient prognosis. A primary role has been proposed for mitochondrial dysfunction, which may be induced by systemic and tissue inflammation and further contribute to low insulin signalling. The acylated form of the gastric hormone ghrelin (AG) may improve mitochondrial oxidative capacity and insulin signalling in both healthy and diseased rodent models. METHODS: We investigated the impact of AG continuous subcutaneous administration (AG) by osmotic minipump (50 nmol/kg/day for 28 days) compared with placebo (P) on skeletal muscle mitochondrial enzyme activities, mitochondrial biogenesis regulators transcriptional expression and insulin signalling in a rodent post-myocardial infarction CHF model. RESULTS: No statistically significant differences (NS) were observed among the three group in cumulative food intake. Compared with sham-operated, P had low mitochondrial enzyme activities, mitochondrial biogenesis regulators transcripts, and insulin signalling activation at AKT level (P < 0.05), associated with activating nuclear translocation of pro-inflammatory transcription factor nuclear factor-κB. AG completely normalized all alterations (P < 0.05 vs P, P = NS vs sham-operated). Direct AG activities were strongly supported by in vitro C2C12 myotubes experiments showing AG-dependent stimulation of mitochondrial enzyme activities. No changes in mitochondrial parameters and insulin signalling were observed in the liver in any group. CONCLUSIONS: Sustained peripheral AG treatment with preserved food intake normalizes a CHF-induced tissue-specific cluster of skeletal muscle mitochondrial dysfunction, pro-inflammatory changes, and reduced insulin signalling. AG is therefore a potential treatment for CHF-associated muscle catabolic alterations, with potential positive impact on patient outcome.

Unacylated Ghrelin: A Novel Regulator of Muscle Intermediate Metabolism With Potential Beneficial Effects in Chronic Kidney Disease.

In patients with chronic kidney disease (CKD), malnutrition with loss of skeletal muscle mass has a negative impact on morbidity and mortality. Emerging evidence indicates that a cluster of oxidative stress, inflammation, and insulin resistance directly contributes to skeletal muscle catabolism by favoring protein breakdown over synthesis. Ghrelin is a gastric hormone discovered and initially studied in its acylated orexigenic form. More recently, a role of unacylated ghrelin (UnAG) has been described to reduce skeletal muscle mitochondrial reactive oxygen species generation, inflammation, and insulin resistance both in experimental models and in clinical studies. UnAG administration could therefore represent a potential comprehensive therapeutic approach for CKD-related metabolic and nutritional complications. Studies of UnAG administration in experimental and clinical CKD are needed to test the hypothesis that UnAG may chronically improve nutritional status and outcome in CKD patients.

Unacylated ghrelin normalizes skeletal muscle oxidative stress and prevents muscle catabolism by enhancing tissue mitophagy in experimental chronic kidney disease.

Unacylated ghrelin (UnAG) may lower skeletal muscle oxidative stress, inflammation, and insulin resistance in lean and obese rodents. UnAG-induced autophagy activation may contribute to these effects, likely involving removal of dysfunctional mitochondria (mitophagy) and redox state maintenance. In chronic kidney disease (CKD) oxidative stress, inflammation and insulin resistance may negatively influence patient outcome by worsening nutritional state through muscle mass loss. Here we show in a 5/6 nephrectomy (Nx) CKD rat model that 4 d s.c. UnAG administration (200 µg twice a day) normalizes CKD-induced loss of gastrocnemius muscle mass and a cluster of high tissue mitochondrial reactive oxygen species generation, high proinflammatory cytokines, and low insulin signaling activation. Consistent with these results, human uremic serum enhanced mitochondrial reactive oxygen species generation and lowered insulin signaling activation in C2C12 myotubes while concomitant UnAG incubation completely prevented these effects. Importantly, UnAG enhanced muscle mitophagy in vivo and silencing RNA-mediated autophagy protein 5 silencing blocked UnAG activities in myotubes. UnAG therefore normalizes CKD-induced skeletal muscle oxidative stress, inflammation, and low insulin signaling as well as muscle loss. UnAG effects are mediated by autophagy activation at the mitochondrial level. UnAG administration and mitophagy activation are novel potential therapeutic strategies for skeletal muscle metabolic abnormalities and their negative clinical impact in CKD.-Gortan Cappellari, G., Semolic, A., Ruozi, G., Vinci, P., Guarnieri, G., Bortolotti, F., Barbetta, D., Zanetti, M., Giacca, M., Barazzoni, R. Unacylated ghrelin normalizes skeletal muscle oxidative stress and prevents muscle catabolism by enhancing tissue mitophagy in experimental chronic kidney disease.

Unacylated Ghrelin Reduces Skeletal Muscle Reactive Oxygen Species Generation and Inflammation and Prevents High-Fat Diet-Induced Hyperglycemia and Whole-Body Insulin Resistance in Rodents.

Excess reactive oxygen species (ROS) generation and inflammation may contribute to obesity-associated skeletal muscle insulin resistance. Ghrelin is a gastric hormone whose unacylated form (UnAG) is associated with whole-body insulin sensitivity in humans and may reduce oxidative stress in nonmuscle cells in vitro. We hypothesized that UnAG 1) lowers muscle ROS production and inflammation and enhances tissue insulin action in lean rats and 2) prevents muscle metabolic alterations and normalizes insulin resistance and hyperglycemia in high-fat diet (HFD)-induced obesity. In 12-week-old lean rats, UnAG (4-day, twice-daily subcutaneous 200-µg injections) reduced gastrocnemius mitochondrial ROS generation and inflammatory cytokines while enhancing AKT-dependent signaling and insulin-stimulated glucose uptake. In HFD-treated mice, chronic UnAG overexpression prevented obesity-associated hyperglycemia and whole-body insulin resistance (insulin tolerance test) as well as muscle oxidative stress, inflammation, and altered insulin signaling. In myotubes, UnAG consistently lowered mitochondrial ROS production and enhanced insulin signaling, whereas UnAG effects were prevented by small interfering RNA-mediated silencing of the autophagy mediator ATG5. Thus, UnAG lowers mitochondrial ROS production and inflammation while enhancing insulin action in rodent skeletal muscle. In HFD-induced obesity, these effects prevent hyperglycemia and insulin resistance. Stimulated muscle autophagy could contribute to UnAG activities. These findings support UnAG as a therapeutic strategy for obesity-associated metabolic alterations.

Gastric bypass-induced weight loss alters obesity-associated patterns of plasma pentraxin-3 and systemic inflammatory markers.

BACKGROUND: Systemic inflammation contributes to obesity-associated complications. The short pentraxin C-reactive protein (CRP) is a validated inflammatory marker, whereas long pentraxin-3 (PTX3) limits inflammation and is adaptively stimulated by proinflammatory cytokines in vitro. Severely obese (SO) patients (body mass index [BMI]>40] have the highest obesity-associated complications and increasingly undergo surgical treatment. SO-associated changes in plasma PTX3 and their interactions with systemic inflammation are, however, unknown. OBJECTIVE: We sought to determine potential alterations in plasma PTX3 and their associations with changes in inflammatory markers before and after weight loss induced by laparoscopic Roux-en-Y gastric bypass (LRYGB). SETTING: University hospital in Trieste, Italy. METHODS: Plasma PTX3, CRP, and cytokines, including tumor necrosis factor α and interleukin 6 were measured in (1) 24 individuals with severe, class III obesity (SO; age = 42 ± 1 yr, female/male = 18/6, BMI = 45 ± 1 kg/m(2)) before and 3, 6, and 12 months after LRYGB; and (2) age- and sex-matched normal-weight (N; n = 56, BMI = 22 ± .2 kg/m(2)) or class I obese individuals (O; n = 44, BMI = 31.2 ± .3 kg/m(2)). RESULTS: SO, but not O, had higher plasma PTX3 compared with N, associated with highest proinflammatory cytokines and CRP (P<.05 versus N-O). In all patients, plasma interleukin 6 and tumor necrosis factor α were associated positively with PTX3 (P<.05). Plasma CRP and proinflammatory cytokines declined during LRYGB-induced weight loss. In contrast, high PTX3 further increased and remained elevated (P<.05 versus basal). CONCLUSIONS: Obesity level and energy balance modulate interactions between PTX3 and systemic inflammation. Elevated PTX3 is a novel, potentially adaptive alteration associated with proinflammatory cytokines in SO. Their differential changes conversely suggest circulating PTX3 as a novel negative inflammatory marker in SO undergoing LRYGB-induced weight loss.