Lack of Fibronectin Extra Domain A Alternative Splicing Exacerbates Endothelial Dysfunction in Diabetes

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\u2009+\u2009FN) is increased in diabetic vessels and participates to vascular remodeling, we wanted to elucidate whether and how EDA\u2009+\u2009FN 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\u2009+\u2009FN respectively), and of wild-type mice (EDA(wt/wt)), streptozotocin (STZ)-induced diabetes impaired endothelial vasodilation to acetylcholine, irrespective of genotype. However STZ\u2009+\u2009EDA(-/-) mice exhibited increased endothelial dysfunction compared with STZ\u2009+\u2009EDA(+/+) and with STZ\u2009+\u2009EDA(wt/wt). Analysis of the underlying mechanisms revealed that STZ\u2009+\u2009EDA(-/-) mice show increased oxidative stress as demonstrated by enhanced aortic superoxide anion, nitrotyrosine levels and expression of NADPH oxidase NOX4 and TGF-\u3b21, 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\u2009+\u2009EDA(+/+) vessels is counteracted by increased eNOS coupling and function. Although EDA\u2009+\u2009FN participates to vascular remodelling, these findings show that it plays a crucial role in limiting diabetic endothelial dysfunction by preventing vascular oxidative stress

A negative impact of recent weight loss on in-hospital mortality is not modified by overweight and obesity

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

Poor nutritional status but not cognitive or functional impairment per se independently predict 1 year mortality in elderly patients with hip-fracture

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 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 6423.5), 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 statu

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 \ub1 13 years, BMI = 26.7 \ub1 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 \ub1 9, BMI = 30,7 \ub1 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 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

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 \ub5g 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

n-3 PUFA-Enriched Diet Preserves Skeletal Muscle Mitochondrial Function and Redox State and Prevents Muscle Mass Loss in Mice with Chronic Heart Failure

Rationale and Methods: Skeletal muscle derangements, potentially including mitochondrial dysfunction with altered mitochondrial dynamics and high reactive oxygen species (ROS) generation, may lead to protein catabolism and muscle wasting, resulting in low exercise capacity and reduced survival in chronic heart failure (CHF). We hypothesized that 8-week n-3-PUFA isocaloric partial dietary replacement (Fat = 5.5% total cal; EPA + DHA = 27% total fat) normalizes gastrocnemius muscle (GM) mitochondrial dynamics regulators, mitochondrial and tissue pro-oxidative changes, and catabolic derangements, resulting in preserved GM mass in rodent CHF [Myocardial infarction (MI)-induced CHF by coronary artery ligation, left-ventricular ejection fraction <50%]. Results: Compared to control animals (Sham), CHF had a higher GM mitochondrial fission-fusion protein ratio, with low ATP and high ROS production, pro-inflammatory changes, and low insulin signalling. n-3-PUFA normalized all mitochondrial derangements and the pro-oxidative state (oxidized to total glutathione ratio), associated with normalized GM cytokine profile, and enhanced muscle-anabolic insulin signalling and prevention of CHF-induced GM weight loss (all p < 0.05 vs. CHF and p = NS vs. S). Conclusions:n-3-PUFA isocaloric partial dietary replacement for 8 weeks normalizes CHF-induced derangements of muscle mitochondrial dynamics regulators, ROS production and function. n-3-PUFA mitochondrial effects result in preserved skeletal muscle mass, with potential to improve major patient outcomes in clinical settings

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 hyper-glycemia in high-fat diet (HFD)-induced obesity. In 12-week-old lean rats, UnAG (4-day, twice-daily subcutaneous 200-mg injections) reduced gastrocnemius mitochondrial ROS generation and inflammatory cytokines while enhancing AKT-dependent signaling and insulinstimulated 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

Unacylated ghrelin does not alter mitochondrial function, redox state and triglyceride content in rat liver in vivo

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

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

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

NOVEL METABOLIC ROLES OF UNACYLATED GHRELIN: FROM PATHOPHYSIOLOGY TO DISEASE MODELS TREATMENT

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