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Purpose Aims of the present study were to investigate a wide array of psychological symptoms through validated psychometric tests, before and after 5 weeks of deep Transcranial Magnetic Stimulation (dTMS) in individuals with obesity, and to identify possible relationships with neuroendocrine parameters. Methods Forty-five patients with obesity (33 F, 12 M; age 48.8 ± 9.9 years; body wt 97.6 ± 14.2 Kg; BMI 36.2 ± 4.2) were randomized into two groups: 26 received high frequency (HF) dTMS and 19 Sham stimulation for 5 weeks. At baseline and after the 5-week treatment, all patients underwent the following psychometric evaluations: Food Cravings Questionnaire-Trait (FCQ-T) and its subscales, Barratt Impulsiveness Scale-11 (BIS-11), State and Trait Anxiety Inventory (STAI-y1 and STAI-y2), and Beck Depression Inventory (BDI). Hormonal and neuroendocrine markers were assessed at the first and last dTMS session. Results By adjusting for baseline variables and treatment arms, a significant decrease in body wt and BMI was found in HF group, both with univariate (p = 0.019) and multivariate analyses (p = 0.012). Impulsivity significantly decreased in HF group, both with univariate (p = 0.031) and multivariate analyses (p = 0.011). A positive association between the impulsivity score change and the leptin level variation (p = 0.031) was found. Conclusion The decrease of impulsivity together with the BMI reduction in individuals with obesity, treated with real stimulation, suggests that impulsivity may be a risk factor for obesity. Treatment with dTMS revealed to be effective in reducing both BMI and impulsivity by enhancing inhibitory capacity of Pre-Frontal Cortex (PFC), and modulating neuroendocrine system, especially leptin.

Cardiovascular complications are known as the main cause of mortality in subjects with diabetes mellitus (DM). In particular, ischemic heart disease is more common in patients with DM than in nondiabetics and promising therapeutically strategies to repair heart damages, including progenitor cell therapy or reprogramming non-cardiac cells, are inadequate in patients with DM. Emerging evidence indicates that hyperglycemia and oxidative stress are the primary causes of cardiac regenerative therapies’ failure in DM. Thus, it is essential to develop new therapeutic approaches aimed at contrasting the cardiac hyperglycemic microenvironment. Several researchers have proposed pulsed electromagnetic field (PEMF), an innovative medical device, to regenerate bone tissue, but very few works have studied PEMF possible use in other pathological condition and, in particularly, in cardiac damages. For the first time, we investigated the effects of PEMF preconditioning treatment on ischemic damages in H9c2 rat cardiomyocytes grown in hyperglycemic condition (25 mmol/L glucose). H9c2 cardiomyocytes were directly exposed to PEMF for 30 minutes (PEMF were generated by a commercially device having these features: 1,33 ms pulses at 75 Hz-1,5 mT) before ischemic injury. Ischemia was simulated by treating H9c2 cells with 5μM rotenone for 3 hours. Control cells were not pretreated with PEMF. Protein kinase B (Akt) and extracellular signal-regulated kinases (ERKs) activation were increased by PEMF stimuli: these kinases are closely related to attenuated cell apoptosis and oxidative stress. Indeed, our results indicated that PEMF treatment could significantly reduce reactive oxygen species (ROS) levels in hyperglycemic H9c2. Taken together, our preliminary study indicated that PEMF could be a potential strategy to improve the efficacy of cardiac regenerative therapies in diabetic patients. Disclosure I. Terruzzi: None. P. Senesi: None. F. Vacante: None. L. Luzi: Consultant; Self; McKinsey & Company. Research Support; Self; Allergan, Gelesis, SunStar Inc. Speaker's Bureau; Self; A. Menarini Diagnostics, AstraZeneca, Eli Lilly and Company, GlaxoSmithKline plc., Menarini Group, Novartis AG, Novo Nordisk Inc., Sunstar Foundation.

In a few months, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has become the main health problem worldwide. Epidemiologic studies revealed that populations have different vulnerabilities to SARS-CoV-2. Severe outcomes of the coronavirus disease 2019 (COVID-19) with an increased risk of death are observed in patients with metabolic syndrome, as well as diabetic and heart conditions (frail population). Excessive proinflammatory cytokine storm could be the main cause of increased vulnerability in this frail population. In patients with diabetes and/or heart disease, a low inflammatory state is often associated with gut dysbiosis. The increase amount of microbial metabolites (i.e., trimethylamine N-oxide and lipopolysaccharide), which generate an inflammatory microenvironment, is probably associated with an improved risk of severe illness from COVID-19. Nutritional interventions aimed at restoring the gut microbial balance could represent preventive strategies to protect the frail population from COVID-19. This narrative review presents the possible molecular mechanisms by which intestinal dysbiosis that enhances the inflammatory state could promote the spread of SARS-CoV-2 infection. Some nutritional strategies to counteract inflammation in frail patients are also analyzed., Highlights • Severe outcome of coronavirus disease 2019, with an increased risk of death, is observed in patients with metabolic syndrome, as well as diabetic and heart conditions • Intestinal dysbiosis could contribute to the aberrant inflammatory response triggered by severe acute respiratory syndrome coronavirus 2 • Nutraceutical strategies aimed at restoring eubiosis condition could represent a valid adjuvant intervention for coronavirus disease 2019 management

Background and Aims. Hepatocellular carcinoma (HCC) is the common tumor of the liver. Unfortunately, most HCC seem to be resistant to conventional chemotherapy and radiotherapy. The poor efficacy of antitumor agents is also due, at least in part, to the inefficient drug delivery and metabolism exerted by the steatotic/cirrhotic liver that hosts the tumor. Thus, novel approaches in chemotherapy may be needed to improve the survival rate in patients with HCC. Metformin (METF) has been found to lower HCC risk; however, the mechanisms by which METF performs its anticancer activity are not completely elucidated. Previous studies have showed METF action on growth inhibition in the liver in a dose/time-dependent manner and its antitumor role by targeting multiple pathways. We investigated molecular effects of METF in an in vitro human hepatoma model (HepG2), studying cell cycle regulators, tumorigenesis markers, and insulin-like growth factor (IGF) axis regulation. Materials and Methods. HepG2 cells were treated with METF (400 μM) for 24, 48, and 72 hours. METF action on cell cycle progression and cellular pathways involved in metabolism regulation was evaluated by gene expression analysis, immunofluorescence, and Western blot assay. Results. By assessing HepG2 cell viability, METF significantly decreased growth cell capacity raising KLF6/p21 protein content. Moreover, METF ameliorated the cancer microenvironment reducing cellular lipid drop accumulation and promoting AMPK activity. The overexpression of IGF-II molecule and the IGF-I receptor that plays a main role in HCC progression was counteracted by METF. Furthermore, the protein content of HCC principal tumor markers, CK19 and OPN, linked to the metastasis process was significantly reduced by METF stimulus. Conclusion. Our data show that METF could suppress HepG2 proliferation, through induction of cell cycle arrest at the G0/G1 phase. In addition, METF effect on the cancer microenvironment and on the IGF axis leads to the development of new METF therapeutic use in HCC treatment.

Diabetic heart disease (DHD) results in structural and functional deterioration due to progressive loss of cardiomyocytes and cardiac stem cells, leading to a premature aging of the diabetic heart. The causes of DHD development are not completely clarified, but it is evident that higher oxidative stress of diabetes induces cardiac structural alterations. Recently, the use of stem cells or cardiac progenitors have inspired the most promising therapeutic strategies but these approaches are limited in diabetic patients. Herein we investigate L-Carnitine (LC) and Resveratrol (RSV) actions on oxidative stress and differentiation process in H9c2 rat cardiomyocytes. H9c2, during proliferation or differentiation condition, were treated with LC (0.5 or 5mM) or RSV (0.1 or 25μM). LC significantly enhanced anti-oxidative response by increasing (48 and 72h) mitochondrial SOD2 protein expression and STAT3 activation. LC did not induce p21 translocation at the nuclear level, demonstrating not to have an inhibitory effect on the cell cycle. LC decreased protein synthesis of CaMKII, whose activation is associated with hypertrophic process. In addition, the protective LC effect on H9c2 cardiomyocytes in stress condition was evaluated treating cells with LC 48h before exposure to H2O2: LC significantly reduced intracellular ROS concentration in a dose-dependent manner. RSV 0.1μM while increasing CaMKII content, did not show any effect on the proliferative potential of H9c2. RSV 25μM induced a significant proliferation decrease in favor of H9c2 differentiation, as shown by ERKs pathway activation and α-actinin protein synthesis promotion. RSV 25μM significantly reduced ROS generation induced by rotenone in neo cardiomyotubes (72h). The ability to improve the microenvironment damaged by oxidative stress and to stimulate cardiac progenitor differentiation could recognize LC/RSV as valid adjuvant therapies to preserve cardiomyocytes survival and ameliorate heart regeneration. Disclosure I. Terruzzi: None. P. Senesi: None. A. Montesano: None. F. Vacante: None. L. Luzi: Speaker's Bureau; Self; A. Menarini Diagnostics, AstraZeneca, Eli Lilly and Company. Research Support; Self; Gelesis. Consultant; Self; McKinsey & Company. Speaker's Bureau; Self; Menarini Group, Merck Sharp & Dohme Corp.. Research Support; Self; Novartis AG. Speaker's Bureau; Self; Novo Nordisk A/S. Research Support; Self; Sunstar Foundation. Speaker's Bureau; Self; Smith & Nephew.

As the rate of obesity and type-2 diabetes is constantly increasing, there is a need to develop new therapeutic approaches with better effects and lower side effects at lower prices. The use of traditional foods with beneficial health properties could be a solution. Lupin, a legume rich in proteins, exerts a hypoglycemic effects and positively modifies proteins involved in glucose homeostasis, in both animals and humans. The aim of this study was to investigate the beneficial effects of lupin flour supplementation on glucose homeostasis using an in vivo model. The condition of insulin resistance (IR) was induced by diet containing corn oil (4%). Ten-week-old male C57BL/6 mice were given ad libitum access to a corn bean meal diet for 9 weeks (CD) or a cornbean meal diet enriched with 2% lupine flour (LC). Food intake was evaluated daily and body weight was measured weekly. Intra-peritoneal glucose tolerance test (IPGTT) was performed to assess the body’s ability to metabolize glucose. Food was removed 15 h before the start of IPGTT, and mice body weights after food deprivation were used to calculate glucose doses. Blood glucose and plasma insulin concentrations were measured. The quantity of food ingested was similar between groups. At the 9th week of treatment, the time-course of glucose clearance in both groups remained elevated for 120 min after glucose administration, indicating a glucose intolerance state. However, HOMA-IR was significantly lower (p=0.02) in LC than in CD group: the LC group showed significantly lower plasma insulin (2.09 ± 0.53 ng/mL) (p = 0.002) than the CD group (3.34 ± 0.56 ng/mL) while plasma glucose did not differ between groups (140 ± 24.9 mg/dL vs. 138 ± 35.5 mg/dL). These biological effects of lupine flour strengthen its promising potential as a new therapeutic agent of treatment for human insulin resistance conditions. Disclosure I. Terruzzi: None. F. Vacante: None. P. Senesi: None. A. Montesano: None. R. Codella: None. L. Luzi: Speaker's Bureau; Self; A. Menarini Diagnostics, AstraZeneca, Eli Lilly and Company. Research Support; Self; Gelesis. Consultant; Self; McKinsey & Company. Speaker's Bureau; Self; Menarini Group, Merck Sharp & Dohme Corp.. Research Support; Self; Novartis AG. Speaker's Bureau; Self; Novo Nordisk A/S. Research Support; Self; Sunstar Foundation. Speaker's Bureau; Self; Smith & Nephew.

Background. Metabolic alterations as hyperglycemia and inflammation induce myocardial molecular events enhancing oxidative stress and mitochondrial dysfunction. Those alterations are responsible for a progressive loss of cardiomyocytes, cardiac stem cells, and consequent cardiovascular complications. Currently, there are no effective pharmacological measures to protect the heart from these metabolic modifications, and the development of new therapeutic approaches, focused on improvement of the oxidative stress condition, is pivotal. The protective effects of levocarnitine (LC) in patients with ischemic heart disease are related to the attenuation of oxidative stress, but LC mechanisms have yet to be fully understood.Objective. The aim of this work was to investigate LC’s role in oxidative stress condition, on ROS production and mitochondrial detoxifying function in H9c2 rat cardiomyocytes during hyperglycemia.Methods. H9c2 cells in the hyperglycemic state (25 mmol/L glucose) were exposed to 0.5 or 5 mM LC for 48 and 72 h: LC effects on signaling pathways involved in oxidative stress condition were studied by Western blot and immunofluorescence analysis. To evaluate ROS production, H9c2 cells were exposed to H2O2after LC pretreatment.Results. Ourin vitrostudy indicates how LC supplementation might protect cardiomyocytes from oxidative stress-related damage, preventing ROS formation and activating antioxidant signaling pathways in hyperglycemic conditions. In particular, LC promotes STAT3 activation and significantly increases the expression of antioxidant protein SOD2. Hyperglycemic cardiac cells are characterized by impairment in mitochondrial dysfunction and the CaMKII signal: LC promotes CaMKII expression and activation and enhancement of AMPK protein synthesis. Our results suggest that LC might ameliorate metabolic aspects of hyperglycemic cardiac cells. Finally, LC doses herein used did not modify H9c2 growth rate and viability.Conclusions. Our novel study demonstrates that LC improves the microenvironment damaged by oxidative stress (induced by hyperglycemia), thus proposing this nutraceutical compound as an adjuvant in diabetic cardiac regenerative medicine.

The purpose of this study is to investigate Ranolazine action on skeletal muscle differentiation and mitochondrial oxidative phenomena. Ranolazine, an antianginal drug, which acts blocking the late INaL current, was shown to lower hemoglobin A1c in patients with diabetes. In the present study, we hypothesized an action of Ranolazine on skeletal muscle cells regeneration and oxidative process, leading to a reduction of insulin resistance. 10 μM Ranolazine was added to C2C12 murine myoblastic cells during proliferation, differentiation and newly formed myotubes. Ranolazine promoted the development of a specific myogenic phenotype: increasing the expression of myogenic regulator factors and inhibiting cell cycle progression factor (p21). Ranolazine stimulated calcium signaling (calmodulin-dependent kinases) and reduced reactive oxygen species levels. Furthermore, Ranolazine maintained mitochondrial homeostasis. During the differentiation phase, Ranolazine promoted myotubes formation. Ranolazine did not modify kinases involved in skeletal muscle differentiation and glucose uptake (extracellular signal-regulated kinases 1/2 and AKT pathways), but activated calcium signaling pathways. During proliferation, Ranolazine did not modify the number of mitochondria while decreasing osteopontin protein levels. Lastly, neo-formed myotubes treated with Ranolazine showed typical hypertrophic phenotype. In conclusion, our results indicate that Ranolazine stimulates myogenesis and reduces a pro-oxidant inflammation/oxidative condition, activating a calcium signaling pathway. These newly described mechanisms may partially explain the glucose lowering effect of the drug.

Background and aims Lupin seed is referred to as an antidiabetic product in traditional medicine. Conglutin-γ, a lupin seed glycoprotein, was found to cause a significant plasma glucose reduction when orally administered to rats in glucose overload trials. Conglutin-γ was identified as being responsible for the claimed biological activity, and the aim of this work was to envisage its hypothetical insulin-mimetic cellular mechanism of action. Insulin is responsible for proteosynthesis control through IRS/AKT/P70S6k/PHAS1 pathways modulation, glucose homeostasis through PKC/Flotillin-2/caveolin-3/Cbl activation and muscle differentiation/hypertrophy via muscle-specific MHC gene transcription control. Methods and results To assess whether conglutin-γ modulates the same insulin-activated kinases, myoblastic C2C12 cells were incubated after 72 h of differentiation with 100 nM insulin or 0.5 mg/mL (∼10 μM) conglutin-γ. Metformin-stimulated cells were used as a positive control. The effect on the above mentioned pathways was evaluated after 5, 10, 20 and 30 min. In the control cells medium insulin, conglutin-γ and metformin were not added. We demonstrated that insulin or conglutin-γ cell stimulation resulted in the persistent activation of protein synthetic pathway kinases and increased glucose transport, glut4 translocation and muscle-specific gene transcription regulation. Conclusions Our results indicate that conglutin-γ may regulate muscle energy metabolism, protein synthesis and MHC gene transcription through the modulation of the same insulin signalling pathway, suggesting the potential therapeutic use of this natural legume protein in the treatment of diabetes and other insulin-resistant conditions, as well as the potential conglutin-γ influence on muscle cells differentiation and regulation of muscle growth.

Metformin (METF), historical antihyperglycemic drug, is a likely candidate for lifespan extension, treatment and prevention of sedentariness damages, insulin resistance, and obesity. Skeletal muscle is a highly adaptable tissue, capable of hypertrophy response to resistance training and of regeneration after damage. Aims of this work were to investigate METF ability to prevent sedentariness damage and to enhance skeletal muscle function. Sedentary 12-week-old C57BL/6 mice were treated with METF (250 mg/kg per day, in drinking water) for 60 days. METF role on skeletal muscle differentiation was studiedin vitrousing murine C2C12 myoblasts. Muscular performance evaluation revealed that METF enhanced mice physical performance (EstimatedVO2max). Biochemical analyses of hepatic and muscular tissues indicated that in liver METF increased AMPK and CAMKII signaling. In contrast, METF inactivated ERKs, the principal kinases involved in hepatic stress. In skeletal muscle, METF activated AKT, key kinase in skeletal muscle mass maintenance. Inin vitrostudies, METF did not modify the C2C12 proliferation capacity, while it positively influenced the differentiation process and myotube maturation. In conclusion, our novel results suggest that METF has a positive action not only on the promotion of healthy aging but also on the prevention of sedentariness damages.

Obesity, due to the combination of inherited genes and environmental factors, is continually increasing. We evaluated the relationship between polymorphisms of methylene-tetrahydrofolate reductase (MTHFR C677T and A1298C), methionine synthase (MTR A2756G), methionine synthase reductase (MTRR A66G), betaine:homocysteine methyltransferase (BHMT G742A) and cystathionine beta-synthase (CBS 68-bp ins) genes and the risk of obesity. We studied these polymorphic variants in 54 normal and 82 obese subjects [body mass index (BMI)=22.4+/-1.8, 34.1+/-7.1; ages 35.2+/-10.7, 43.3+/-10.6 respectively]. Levels of total plasma homocysteine (t-Hcy), folates, and vitamins B6 and B12 were not significantly different, while leptin concentration was significantly higher (p=0.005) in the obese patients compared to the lean controls. The frequency of only (a) MTHFR (AC), (b) MTR (AG), and (c) MTRR (AG) heterozygous genotypes was statistically different in the obese compared to the control group (p=0.03, p=0.007, and p=0.01). Single (a), (b), and (c) heterozygous genotypes had a significant risk of developing obesity [p=0.02, 0.01, and 0.03; odds ratio (OR)=2.5, 3.0, and 2.4; 95% confidence interval (CI)=1.2-5.3, 1.3-7.1, and 1.2-5.1 respectively] and the risk remarkably increased for combined genotypes a+b, a+c, b+c, and a+b+c (p=0.002, 0.002, 0.016, 0.006; OR=7.7, 5.4, 5.8, 15.4; 95% CI=1.9-30.4, 1.7-16.8, 1.4-23.2, 1.6- 152.3). These findings suggest that in obese subjects, Hcy cycle efficiency is impaired by MTHFR, MTR, and MTRR inability to supply methyl-group donors, providing evidence that MTHFR, MTR, and MTRR gene polymorphisms are genetic risk factors for obesity.

Skeletal muscle regeneration and hypertrophy are important adaptive responses to both physical activity and pathological stimuli. This research was performed to investigate DNA demethylation action on the late phase of muscle differentiation and early stage of hypertrophy. The epigenetic process involved in myogenesis was studied with the DNA-demethylating agent 5-azacytidine (AZA). We induced muscle differentiation in C2C12 mouse myoblasts in the presence of 5 μM AZA and growth or differentiation medium for 48, 72, and 96 h. To study a potential AZA hypertrophic effect, we stimulated 72 h differentiated myotubes with AZA for 24 h. Unstimulated cells were used as control. By western blot and immunofluorescence analysis, we examined AZA action on myogenic regulatory factors expression, hypertrophic signaling pathway and myotube morphology. During differentiation, protein levels of myogenic markers, Myf6 and Myosin Heavy Chain (MyHC), were higher in AZA stimulated cells compared to control. Myostatin and p21 analysis revealed morphological changes which reflect a tendency to hypertrophy in myotubes. In AZA stimulated neo formed myotubes, we observed that IGF-I pathway, kinases p70 S6, 4E-BP1, and ERK1/2 were activated. Furthermore, AZA treatment increased MyHC protein content in stimulated neo myotubes. Our work demonstrates that DNA demethylation could plays an important role in promoting the late phase of myogenesis, activating endocellular pathways involved in protein increment and stimulating the hypertrophic process.