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Ghrelin is a gastric hormone circulating in acylated (AG) and unacylated (UnAG) forms. AG represents ~10% of total plasma ghrelin, has an appetite-stimulating effect and is the only form for which a receptor has been identified. UnAG has no orexigenic effects, and its circulating levels are positively associated with insulin sensitivity in metabolic syndrome patients. Skeletal muscle oxidative stress and inflammation are key negative modulators of tissue and whole-body insulin action, and UnAG was recently reported to reduce oxidative stress in non-muscle cells in-vitro. Its potential direct involvement in the regulation of muscle intermediate metabolism in vivo and its clinical impact remain however largely unknown. In the current studies, we first investigated potential associations between plasma AG, UnAG and HOMA insulin resistance index in the general population. In 719 individuals from the North-East-Italy MoMa epidemiological study, TG and UnAG but not of AG were negatively associated with HOMA after adjusting for gender and body mass both at baseline and at 5-year follow-up, and changes in TG and UnAG but not AG were negatively associated with changes in HOMA independently of potential confounders. We next tested the hypothesis that UnAG increases insulin sensitivity by modulating oxidative stress, inflammation and insulin action in skeletal muscle tissue. In healthy male rats, UnAG administration consistently reduced muscle mitochondrial ROS production, reduced inflammation and activated insulin-anabolic signalling. Analogous findings were observed in transgenic mice with systemic UnAG overexpression, and in UnAG-treated myotubes in vitro, thereby supporting a potential direct hormone effect. Importantly, these findings were not observed with AG administration nor in liver samples, thus further indicating independent and tissue specific UnAG actions. Autophagy, the removal of dysfunctional organelles, is an emerging protective mechanism in both cardiac and skeletal muscle. We therefore also hypothyzed that UnAG could reduce ROS production by inducing muscle autophagy. In cardiomiocytes UnAG reduced ROS production in association with increased dysfunctional mitochondria removal and in UnAG but not AG treated myotubes silencing of autophagy promoter ATG5 restored ROS generation. We next investigated the potential clinical relevance of UnAG actions in disease models characterized by muscle oxidative stress, inflammation and insulin resistance. In high-fat diet (HFD)-induced obese and diabetic mice, chronic UnAG overexpression prevented hyperglycemia and whole-body insulin resistance, as well as muscle oxidative stress, inflammation and altered insulin signalling. In rodent chronic kidney disease induced by 5/6 nephrectomy, protein-energy wasting, enhanced skeletal muscle ROS production, increased tissue inflammation and impaired insulin signalling were also completely normalized by UnAG treatment. Importantly, these findings were associated with a recovery of gastrocnemious muscle mass. While a specific receptor for UnAG needs to be identified, our combined findings consistently point towards a novel independent role of UnAG as a regulator of muscle metabolic pathways maintaining energy status and tissue anabolism. Underlying mechanism appear to involve the modulation of mitochondrial function with reduced ROS generation, which could be mediated at least in part by autophagy induction. Data from disease models also suggests that modulation of ghrelin acylation to enhance UnAG availability is a potential novel target in the treatment of metabolic derangements in disease states characterized by metabolic and nutritional complications.

Functional screening of expression libraries in vivo would offer the possibility of identifying novel biotherapeutics without a priori knowledge of their biochemical function. Here we describe a procedure for the functional selection of tissue-protective factors based on the in vivo delivery of arrayed cDNA libraries from the mouse secretome using adeno-associated virus (AAV) vectors. Application of this technique, which we call FunSel, in the context of acute ischaemia, revealed that the peptide ghrelin protects skeletal muscle and heart from ischaemic damage. When delivered to the heart using an AAV9 vector, ghrelin markedly reduces infarct size and preserves cardiac function over time. This protective activity associates with the capacity of ghrelin to sustain autophagy and remove dysfunctional mitochondria after myocardial infarction. Our findings describe an innovative tool to identify biological therapeutics and reveal a novel role of ghrelin as an inducer of myoprotective autophagy., Cell-based screening assays allow functional testing of chemicals but do not mimic the in vivo situation well. Here, the authors report a method for the discovery of secreted cytoprotective factors in mice and use it to demonstrate that the hormone ghrelin protects cardiac muscle from ischaemic damage.

BACKGROUND: Obesity [Body Mass Index (BMI) > 30 kg/m2] is a risk factor for disease conditions enhancing hospitalization and mortality risks, but higher BMI was paradoxically reported to reduce mortality in several acute and chronic diseases. Unintentional weight loss (WL) is conversely associated with disease development and may worsen patient outcome, but the impact of weight loss and its interaction with obesity in modulating risk of death in hospitalized patients remain undefined. METHODS: We investigated the ESPEN nutritionDay database of non-critically ill hospitalized patients to assess the impact of self-reported 3-month WL (WL1:2.5-6.6%; WL2: 6.6-12.6%, WL3: >12.6%) and its interaction with BMI in modulating 30-day in-hospital mortality. Multivariate Cox regression was used to estimate hazard ratios (HR), with stable weight (WL0) as reference category. RESULTS: In 110835 nDay patients, 30-day mortality increased with increasing WL. Male gender, increasing disease severity index PANDORA score (age, nutrient intake, mobility, fluid status, cancer and main patient group) and not having had surgery also predicted 30-day mortality. HR for 30-day mortality remained significantly higher compared to WL0 for WL2 and WL3 after multiple adjustment. Adjusted HR and its increments through increasing weight loss categories were comparable in lean (BMI30 kg/m2). Impact of gender, PANDORA score and surgery on 30-day mortality were conversely comparable in the three BMI groups. CONCLUSIONS: These results indicate that self-reported WL could represent a relevant prognostic factor in every hospitalized patient. Overweight and obesity per se have no protective impact against WL-associated mortality.

With prolonged pandemic conditions, and emerging evidence but persisting low awareness of the importance of nutritional derangements, ESPEN has promoted in close collaboration with World Health Organization-Europe a call for papers on all aspects relating COVID-19 and nutrition as well as nutritional care, in the Society Journals Clinical Nutrition and Clinical Nutrition ESPEN. Although more COVID-related papers are being submitted and continue to be evaluated, ESPEN and WHO present the current editorial to summarize the many published findings supporting major interactions between nutritional status and COVID-19. These include 1) high risk of developing the disease and high risk of severe disease in the presence of pre-existing undernutrition (malnutrition) including micronutrient deficiencies; 2) high risk of developing malnutrition during the course of COVID-19, with substantial impact on long-term sequelae and risk of long COVID; 3) persons with obesity are also prone to develop or worsen malnutrition and its negative consequences during the course of COVID-19; 4) malnutrition screening and implementation of nutritional care may improve disease outcomes; 5) social and public health determinants contribute to the interaction between nutritional status and COVID-19, including negative impact of lockdown and social limitations on nutrition quality and nutritional status. We believe the evidence supports the need to consider COVID-19 as (also) a case of malnutrition-enhanced disease and disease-related malnutrition, with added risk for persons both with and without obesity. Similarities with many other disease conditions further support recommendations to implement standard nutritional screening and care in COVID-19 patients, and they underscore the relevance of appropriate nutritional and lifestyle prevention policies to limit infection risk and mitigate the negative health impact of acute pandemic bouts.

Ghrelin is a gastric hormone circulating in acylated (AG) and unacylated (UnAG) forms. This narrative review aims at presenting current emerging knowledge on the impact of ghrelin forms on energy balance and metabolism. AG represents ~ 10% of total plasma ghrelin, has an appetite-stimulating effect and is the only form for which a receptor has been identified. Moreover, other metabolic AG-induced effects have been reported, including the modulation of glucose homeostasis with stimulation of liver gluconeogenesis, the increase of fat mass and the improvement of skeletal muscle mitochondrial function. On the other hand, UnAG has no orexigenic effects, however recent reports have shown that it is directly involved in the modulation of skeletal muscle energy metabolism by improving a cluster of interlinked functions including mitochondrial redox activities, tissue inflammation and insulin signalling and action. These findings are in agreement with human studies which show that UnAG circulating levels are positively associated with insulin sensitivity both in metabolic syndrome patients and in a large cohort from the general population. Moreover, ghrelin acylation is regulated by a nutrient sensor mechanism, specifically set on fatty acids availability. These recent findings consistently point towards a novel independent role of UnAG as a regulator of muscle metabolic pathways maintaining energy status and tissue anabolism. While a specific receptor for UnAG still needs to be identified, recent evidence strongly supports the hypothesis that the modulation of ghrelin-related molecular pathways, including those involved in its acylation, may be a potential novel target in the treatment of metabolic derangements in disease states characterized by metabolic and nutritional complications. Level of evidence Level V, narrative review.

Obesity is a major health risk factor, and obesity-induced morbidity and complications account for huge costs for affected individuals, families, healthcare systems, and society at large. In particular, obesity is strongly associated with the development of insulin resistance, which in turn plays a key role in the pathogenesis of obesity-associated cardiometabolic complications, including metabolic syndrome components, type 2 diabetes, and cardiovascular diseases. Insulin sensitive tissues, including adipose tissue, skeletal muscle, and liver, are profoundly affected by obesity both at biomolecular and functional levels. Altered adipose organ function may play a fundamental pathogenetic role once fat accumulation has ensued. Modulation of insulin sensitivity appears to be, at least in part, related to changes in redox balance and oxidative stress as well as inflammation, with a relevant underlying role for mitochondrial dysfunction that may exacerbate these alterations. Nutrients and substrates as well as systems involved in host-nutrient interactions, including gut microbiota, have been also identified as modulators of metabolic pathways controlling insulin action. This review aims at providing an overview of these concepts and their potential inter-relationships in the development of insulin resistance, with particular regard to changes in adipose organ and skeletal muscle.

Deterioration of muscle strength during cancer results in functional limitation, poor quality of life and reduced survival. The indirect effects on muscle strength of nutritional interventions based on protein and amino acid derivatives targeted at improving muscle mass are poorly documented. A scoping review was performed to examine the available evidence on the effects of proteins, amino acids and their derivatives on muscle strength in adult cancer patients. Pubmed and Scopus databases were searched to identify research articles published in the last 10 years. Fourteen studies met the inclusion criteria, showing that changes in muscle strength following protein or amino acid supplementation are generally concordant with those in muscle mass in cancer patients. Administration of both energy and proteins in the presence of reduced oral intakes results in more robust effects on both muscle strength and mass. It is not clear whether this is due to the correction of the energy deficit or to an interaction between proteins and other macronutrients. The optimal mixture, type, and dose of amino acid/protein supplementation alone or in combination with other anabolic strategies should be determined to provide the best nutritional approach in cancer.

Background: Ghrelin may exert positive effects on cardiac structure and function in heart failure (HF) patients. Methods: We assessed ghrelin levels in 266 dilated cardiomyopathy (DCM) patients and in 200 age, gender and body mass index (BMI) matched controls. Further, we evaluated the expression of ghrelin and growth hormone secretagogue-receptor (GHSR) in the myocardium of 41 DCM patients and in 11 controls. Results: DCM patients had significantly lower levels of total, acylated and unacylated ghrelin when compared to controls (p < 0.05 for all). In controls, we observed a negative correlation of ghrelin with age, male gender and BMI. These correlations were lost in the DCM group, except for male gender. Total ghrelin was higher in patients with more recent diagnosis when compared to patients with longer duration of the DCM (p = 0.033). Further, total ghrelin was higher in patients with lower left ventricular systolic function (p = 0.05). Ghrelin prepropeptide was expressed more in DCM patients than in controls (p = 0.0293) while GHSR was expressed less in DCM patients (p < 0.001). Furthermore, ghrelin showed an inverse correlation with its receptor (ρ = −0.406, p = 0.009), and this receptor showed a significant inverse correlation with Interleukin-1β (ρ = −0.422, p = 0.0103). Conclusion: DCM duration and severity are accompanied by alterations in the ghrelin−GHSR system.

Unacylated ghrelin (UnGhr) exerts several beneficial actions on vascular function. The aim of this study was to assess the effects of UnGhr on high-fat induced endothelial dysfunction and its underlying mechanisms. Thoracic aortas from transgenic mice, which were overexpressing UnGhr and being control fed either a standard control diet (CD) or a high-fat diet (HFD) for 16 weeks, were harvested and used for the assessment of vascular reactivity, endothelial nitric oxide synthase (eNOS) expression and activity, thiobarbituric acid reactive substances (TBARS) and glutathione levels, and aortic lipid accumulation by Oil Red O staining. Relaxations due to acetylcholine and to DEA-NONOate were reduced (p < 0.05) in the HFD control aortas compared to vessels from the CD animals. Overexpression of UnGhr prevented HFD-induced vascular dysfunction, while eNOS expression and activity were similar in all vessels. HFD-induced vascular oxidative stress was demonstrated by increased (p < 0.05) aortic TBARS and glutathione in wild type (Wt) mice; however, this was not seen in UnGhr mice. Moreover, increased (p < 0.05) HFD-induced lipid accumulation in vessels from Wt mice was prevented by UnGhr overexpression. In conclusion, chronic UnGhr overexpression results in improved vascular function and reduced plaque formation through decreased vascular oxidative stress, without affecting the eNOS pathway. This research may provide new insight into the mechanisms underlying the beneficial effects of UnGhr on the vascular dysfunction associated with obesity and the metabolic syndrome.

Hip fractures are strongly associated with mortality in the elderly. Studies investigating predisposing factors have suggested a negative impact of poor nutritional, cognitive and functional status on patient survival, however their independent prognostic impact as well as their interactions remain undefined. This study aimed to determine whether poor nutritional status independently predicts 1 year post-fracture mortality after adjusting for cognitive and functional status and for other clinically relevant covariates. METHODS: 1211 surgically treated hip fracture elderly (age ≥ 65) patients consecutively admitted to the Orthopaedic Surgery Unit of the "Azienda Sanitaria Universitaria Integrata Trieste" (ASUITs), Cattinara Hospital, Trieste, Italy and managed by a dedicated orthogeriatric team. Pre-admission nutritional status was evaluated by Mini Nutritional Assessment (MNA) questionnaire, cognitive status by Short Portable Mental Status Questionnaire (SPMSQ) and functional status by Activity of Daily Living (ADL) questionnaire. All other clinical data, including comorbidities, type of surgery, post-operative complications (delirium, deep vein thrombosis, cardiovascular complications, infections, need for blood transfusions) were obtained by hospital clinical records and by mortality registry. RESULTS: Poor nutritional status (defined as MNA ≤23.5), increased cognitive and functional impairment were all associated with 3-, 6- and 12 month mortality (p < 0.001). Both cognitive and functional impairment were associated with poor nutritional status (p < 0.001). Logistic regression analysis demonstrated that the association between nutritional status and 3-, 6- and 12- month mortality was independent of age, gender, comorbidities, type of surgery and post-operative complications as well as of cognitive and functional impairment (p < 0.001). In contrast, the associations between mortality and cognitive and functional impairment were independent (p < 0.001) of demographic (age, gender) and clinical covariates but not of malnutrition. Kaplan-Meier analysis showed a lower mean survival time (p < 0.001) in patients with poor nutritional status compared with those well-nourished. CONCLUSIONS: In hip fracture elderly patients, poor nutritional status strongly predicts 1 year mortality, independently of demographic, functional, cognitive and clinical risk factors. The negative prognostic impact of functional and cognitive impairment on mortality is mediated by their association with poor nutritional status

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.

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.

Elderly and patients affected by chronic diseases face a high risk of muscle loss and impaired physical function. Omega 3 fatty acids (FA) attenuate inflammation and age-associated muscle loss, prevent systemic insulin resistance and improve plasma lipids, potentially impacting on sarcopenia. This paper aims to review recent randomized clinical studies assessing the effects a chronic omega 3 FA supplementation on inflammatory and metabolic profile during conditions characterized by sarcopenia (aging, insulin resistance, type 2 diabetes, chronic renal failure). A comprehensive search of three online databases was performed to identify eligible trials published between 2012 and 2017. A total of 36 studies met inclusion criteria. Omega 3 FA yielded mixed results on plasma triglycerides in the elderly and no effects in renal patients. No changes in systemic insulin resistance were observed. Inflammation markers did not benefit from omega 3 FA in insulin resistant and in renal subjects while decreasing in obese and elderly. Muscle related parameters improved in elderly and in renal patients. In conclusion, in aging- and in chronic disease-associated sarcopenia omega 3 FA are promising independently of associated anabolic stimuli or of anti-inflammatory effects. The evidence for improved glucose metabolism in insulin resistant and in chronic inflammatory states is less solid.

Background: Endothelial dysfunction is a key vascular alteration in chronic kidney disease (CKD). Omega 3 (n-3) polyunsaturated fatty acids (PUFA) reduce vascular oxidative stress and inflammation. We investigated whether n-3 PUFA could reverse endothelial dysfunction in CKD by improving endothelial nitric oxide synthase (eNOS) function and oxidative stress. Methods: 5/6 nephrectomized male Wistar rats (CKD; n = 10) and sham operated animals (SHAM; n = 10) were treated for 6 weeks with standard diet. An additional group of CKD rats were fed an n-3 PUFA enriched diet (CKD + PUFA; n = 10). We then measured endothelium-dependent (EDD) and -independent vasodilation, markers of endothelial function and of oxidative stress in thoracic aortas. Results: Compared to SHAM, in CKD aortas EDD and eNOS expression were reduced (p < 0.05) and 3-nitrotyrosine levels were increased, while expression of NADPH oxidase subunits NOX4 and p22phox was similar. In-vitro incubation with Tiron failed to reverse endothelial dysfunction in CKD. In CKD + PUFA, EDD improved (p < 0.05) compared with CKD rats, while blockade of eNOS by L-NAME worsened EDD. These effects were accompanied by increased (p < 0.05) eNOS and reduced (p < 0.05) expression of NOX4 and 3-nitrotyrosine levels. Conclusion: Collectively, these findings indicate that n-3 PUFA improve endothelial dysfunction by restoring NO bioavailability in CKD.

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.

AIMS/HYPOTHESIS: Insulin effects reportedly involve reactive oxygen species (ROS) and oxidative stress in vitro, but skeletal muscle oxidative stress is an emerging negative regulator of insulin action following high-fat feeding. NEFA may enhance oxidative stress and insulin resistance. We investigated the acute impact of insulin with or without NEFA elevation on muscle ROS generation and insulin signalling, and the potential association with altered muscle mitochondrial function. METHODS: We used hyperinsulinaemic-euglycaemic clamping, 150 min, without or with lipid infusion to modulate plasma NEFA concentration in lean rats. RESULTS: Insulin and glucose (Ins) infusion selectively enhanced xanthine oxidase-dependent muscle ROS generation. Ins with lipid infusion (Ins+NEFA) lowered whole-body glucose disposal and muscle insulin signalling, and these effects were associated with high muscle mitochondrial ROS generation and activation of the proinflammatory nuclear factor-κB inhibitor (IκB)-nuclear factor-κB (NFκB) pathway. Antioxidant infusion prevented NEFA-induced systemic insulin resistance and changes in muscle mitochondrial ROS generation, IκB-NFκB pathway and insulin signalling. Changes in insulin sensitivity and signalling were independent of changes in mitochondrial enzyme activity and ATP production, which, in turn, were not impaired by changes in ROS generation under any condition. CONCLUSIONS/INTERPRETATION: Acute muscle insulin effects include enhanced ROS generation through xanthine oxidase. Additional NEFA elevation enhances mitochondrial ROS generation, activates IκB-NFκB and reduces insulin signalling. These alterations are not associated with acute reductions in mitochondrial enzyme activity and ATP production, and are reversed by antioxidant infusion. Thus, NEFA acutely cause systemic and muscle insulin resistance by enhancing muscle oxidative stress through mitochondrial ROS generation and IκB-NFκB activation.

Glucose-induced changes of artery anatomy and function account for diabetic vascular complications, which heavily impact disease morbidity and mortality. Since fibronectin containing extra domain A (EDA + FN) is increased in diabetic vessels and participates to vascular remodeling, we wanted to elucidate whether and how EDA + FN is implicated in diabetes-induced endothelial dysfunction using isometric-tension recording in a murine model of diabetes. In thoracic aortas of EDA−/−, EDA+/+ (constitutively lacking and expressing EDA + FN respectively), and of wild-type mice (EDAwt/wt), streptozotocin (STZ)-induced diabetes impaired endothelial vasodilation to acetylcholine, irrespective of genotype. However STZ + EDA−/− mice exhibited increased endothelial dysfunction compared with STZ + EDA+/+ and with STZ + EDAwt/wt. Analysis of the underlying mechanisms revealed that STZ + EDA−/− mice show increased oxidative stress as demonstrated by enhanced aortic superoxide anion, nitrotyrosine levels and expression of NADPH oxidase NOX4 and TGF-β1, the last two being reverted by treatment with the antioxidant n-acetylcysteine. In contrast, NOX1 expression and antioxidant potential were similar in aortas from the three genotypes. Interestingly, reduced eNOS expression in STZ + EDA+/+ vessels is counteracted by increased eNOS coupling and function. Although EDA + FN participates to vascular remodelling, these findings show that it plays a crucial role in limiting diabetic endothelial dysfunction by preventing vascular oxidative stress.

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.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).University hospital in Trieste, Italy.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)).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).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.

Summary Changes in liver mitochondrial function with more oxidized redox state and enhanced inflammation may contribute to the onset of obesity- and insulin resistance-associated hepatic complications, including non-alcoholic fatty liver disease and steato-hepatitis. Unacylated ghrelin (UnAG) is a gastric hormone reported to be associated with lower oxidative stress in different cell types, but its potential effects on liver mitochondrial function, redox state and inflammation in vivo remains undetermined. We investigated the impact of chronic UnAG overexpression (Tg Myh6/Ghrl) leading to systemic upregulation of circulating hormone on mitochondrial ATP production, redox state (oxidized-to-total glutathione) and inflammation markers in lean mice. Compared to wild-type animals (wt), Tg Myh6/Ghrl had superimposable liver weight, triglyceride content and plasma lipid profile. Liver mitochondrial enzyme activities and ATP production as well as oxidized-to-total glutathione were also similar in the two groups. In addition, no differences were observed in tissue inflammation marker TNF-alpha between wild-type and Tg Myh6/Ghrl animals. Thus, chronic systemic UnAG upregulation does not alter liver triglyceride content, mitochondrial function, redox state and inflammation markers in lean mice. These findings do not support a major role of UnAG as a physiological modulator of in vivo liver oxidative-lipid metabolism and inflammation.

Background: Menopause is associated with increased arterial stiffness, an independent marker of cardiovascular risk. Omega-3 polyunsaturated fatty acids (N3-PUFAs) are thought to have multiple cardiovascular benefits, including prevention of arterial stiffness. We investigated whether treatment with N3-PUFA prevents increase in arterial stiffness in ovariectomized rats, an animal model of experimental menopause. Methods: A total of 43 Wistar rats, 2 months old, were divided into 3 groups, control, sham surgery, normal diet (CTRL, n = 15); ovariectomy, normal diet (OVX, n = 14); and ovariectomy with N3-PUFA supplementation (0.8 g/kg/d in daily gavages administration; OVX + O3, n = 14). Two months after surgery, carotid–femoral pulse wave velocity (PWV) and arterial blood pressure (BP) were measured by carotid and femoral cannulation. Aortic morphometric measurements were performed after dissection. Results: Ovariectomy caused a significant increase in BP ( P < .05), PWV ( P < .0001), and elastic modulus ( P = .001) compared to CTRL. After ovariectomy, N3-PUFA supplementation completely prevented increase in arterial stiffness ( P < .0001 vs OVX) and BP ( P < .05 vs OVX) and resulted in a significant increase in body weight and aortic thickness. Conclusions: In an experimental model of menopause, N3-PUFA supplementation prevents arterial stiffening and other vascular changes induced by ovariectomy. These results represent a therapeutic benefit of N3-PUFAs in prevention of postmenopausal cardiovascular disease.

Objective Increments in red blood cell count (RBC), hemoglobin (Hb) and hematocrit (Ht) levels are reportedly associated with higher insulin resistance (IR). Obesity may cause IR, but underlying factors remain incompletely defined, and interactions between obesity, hematological parameters and IR are incompletely understood. We therefore determined whether: 1) BMI and obesity per se are independently associated with higher RBC, hemoglobin and hematocrit; 2) hematological parameters independently predict insulin resistance in obese individuals. Design and Methods We investigated the associations between BMI, hematological parameters and insulin resistance as reflected by homeostasis model assessment (HOMA) in a general population cohort from the North-East Italy MoMa epidemiological study (M/F = 865/971, age = 49±1). Results In all subjects, age-, sex- and smoking-adjusted hematological parameters were positively associated with BMI in linear regression (P

Menopause is associated with endothelial dysfunction and oxidative stress. In this condition, reduced n-3 polyunsaturated fatty acids (n-3 PUFAs) contribute to cardiovascular disease. We investigated whether treatment with n-3 PUFA reverses endothelial dysfunction and oxidative stress in experimental menopause. Thirty female rats underwent either sham-surgery or bilateral ovariectomy or bilateral ovariectomy+oral n-3 PUFA (0.8 g kg(-1) day(-1) for 2 months). Ovariectomy caused endothelial dysfunction to acetylcholine, which was reversed by superoxide scavenger Tiron. Erythrocyte membrane lipid composition was characterized by reduced n-3 PUFA total content and omega-3 index, and by concomitant increase in n-6:n-3 PUFA ratio. Ovariectomy-related oxidative stress, demonstrated by both enhanced superoxide production and 3-nitrotyrosine expression in aorta, was associated with increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit NOX-4 protein expression. Endothelial nitric oxide synthase (eNOS) functional inhibition by l-NG-nitroarginine methyl ester, protein expression and activity did not change. In ovariectomized rats, treatment with n-3 PUFA increased n-3 PUFA total content and omega-3 index and decreased n-6:n-3 PUFA ratio in erythrocyte membrane, reversed vascular oxidative stress, endothelial dysfunction, aortic 3-nitrotyrosine and markedly lowered NOX-4 protein expression; eNOS protein expression also increased, paralleled by reversal of inhibitory binding to Caveolin-1, while ex-vivo functional inhibition and NOS synthesis were unchanged. These findings demonstrate in vivo a therapeutic benefit of n-3 PUFA on menopause-associated endothelial dysfunction by reversal of alterations in membrane lipid composition induced by ovariectomy and by reduction of vascular oxidative stress. In this setting they also identify NOX-4 as a potential target to reduce oxidative stress-mediated vascular complications.

OBJECTIVE: Oxidative stress and inflammation characterize hemodialysis (HD) and are associated with malnutrition, cardiovascular disease, and poor clinical outcome. p66(shc) stimulates oxidative stress and atherogenesis. The objective of the present study was to assess p66(shc) expression levels in HD and their associations with inflammatory and oxidative stress markers. DESIGN: p66(shc) messenger ribonucleic acid (mRNA) was compared with systemic oxidative stress and inflammation markers in control subjects and patients on HD before and after a single HD session in a cross-sectional analysis. SETTING: Outpatient hemodialysis unit. PATIENTS: The study included stable HD patients (n = 21, men/women: 18/3) who were on HD 3 times per week for a minimum of 8 weeks; age-matched control subjects (n = 22, men/women:17/5). MAIN OUTCOME MEASURE: mRNA levels of p66(shc), tumor necrosis factor α (TNF-α), and pentraxin 3 (PTX3), p66(shc) protein levels in white blood cells, lipid peroxidation (in the form of plasma thiobarbituric acid-reactive substance [TBARS]) and serum C-reactive protein. RESULTS: In patients on dialysis, of the p66(shc), TNF-α, and PTX3 mRNAs, p66(shc) protein levels were higher (P < .05) than in control subjects, as well as plasma TBARS and C-reactive protein (P < .05). p66(shc) mRNA directly correlated with TBARS (r = 0.69, P = .0005) and with TNF-α mRNA (r = 0.63, P = .003). These associations were confirmed in the whole study population (TBARS: r = 0.541, P = .0003; TNF-α: r = 0.581, P < .0001), whereas in the control group only the positive association between p66(shc) and TNF-α was detected. TNF-α was directly correlated with PTX3 both in HD patients (r = 0.72, P = .0005) and in the whole study group (r = 0.678, P < .0001). The dialysis session affected neither p66(shc) and TNF-α mRNA nor p66(shc) protein expression, whereas it further increased (P = .002) PTX3 mRNA. As compared with predialysis levels, TBARS were reduced (P < .05) after dialysis. In these conditions, p66(shc) remained directly correlated with TNF-α (r = 0.901, P < .0001). CONCLUSIONS: Increased p66(shc) gene expression correlates with TNF-α mRNA and with levels of markers of oxidative stress in HD. We suggest a novel link between HD-associated inflammation and p66(shc) gene expression contributing to systemic oxidative stress.

Aging is characterized by activation of inducible over endothelial nitric oxide synthase (iNOS and eNOS), impaired antioxidant activity and increased oxidative stress, which reduces nitric oxide bioavailability and causes endothelial dysfunction. Caloric restriction (CR) blunts oxidative stress. We investigated whether CR impacts endothelial dysfunction in aging and the underlying mechanisms. Aortas from young (YC, 6 months of age) and old (OC, 24 months of age) rats ad-libitum fed and from old rats caloric-restricted for 3-weeks (OR, 26%) were investigated. Endothelium-dependent vasorelaxation was impaired in OC, associated with reduced eNOS and increased iNOS expression (P