L-Carnitine counteracts in vitro fructose-induced hepatic steatosis through targeting oxidative stress markers

Purpose: Nonalcoholic fatty liver disease (NAFLD) is defined by excessive lipid accumulation in the liver and involves an ample spectrum of liver diseases, ranging from simple uncomplicated steatosis to cirrhosis and hepatocellular carcinoma. Accumulating evidence demonstrates that high fructose intake enhances NAFLD development and progression promoting inhibition of mitochondrial \u3b2-oxidation of long-chain fatty acids and oxidative damages. l-Carnitine (LC), involved in \u3b2-oxidation, has been used to reduce obesity caused by high-fat diet, which is beneficial to ameliorating fatty liver diseases. Moreover, in the recent years, various studies have established LC anti-oxidative proprieties. The objective of this study was to elucidate primarily the underlying anti-oxidative mechanisms of LC in an in vitro model of fructose-induced liver steatosis. Methods: Human hepatoma HepG2 cells were maintained in medium supplemented with LC (5 mM LC) with or without 5 mM fructose (F) for 48 h and 72 h. In control cells, LC or F was not added to medium. Fat deposition, anti-oxidative, and mitochondrial homeostasis were investigated. Results: LC supplementation decreased the intracellular lipid deposition enhancing AMPK activation. However, compound C (AMPK inhibitor-10 \u3bcM), significantly abolished LC benefits in F condition. Moreover, LC, increasing PGC1 \u3b1 expression, ameliorates mitochondrial damage-F induced. Above all, LC reduced ROS production and simultaneously increased protein content of antioxidant factors, SOD2 and Nrf2. Conclusion: Our data seemed to show that LC attenuate fructose-mediated lipid accumulation through AMPK activation. Moreover, LC counteracts mitochondrial damages and reactive oxygen species production restoring antioxidant cellular machine. These findings provide new insights into LC role as an AMPK activator and anti-oxidative molecule in NAFLD

Modulation of cell cycle progression by 5-azacytidine is associated with early myogenesis induction in murine myoblasts

Myogenesis is a multistep process, in which myoblasts withdraw from the cell cycle, cease to divide, elongate and fuse to form multinucleated myotubes. Cell cycle transition is controlled by a family of cyclin-dependent protein kinases (CDKs) regulated by association with cyclins, negative regulatory subunits and phosphorylation. Muscle differentiation is orchestrated by myogenic regulatory factors (MRFs), such as MyoD and Myf-5. DNA methylation is crucial in transcriptional control of genes involved in myogenesis. Previous work has indicated that treatment of fibroblasts with the DNA-demethylating agent 5-azacytidine (AZA) promotes MyoD expression. We studied the effects of AZA on cell cycle regulation and MRFs synthesis during myoblast proliferation and early myogenesis phases in C2C12 cells. During the proliferation phase, cells were incubated in growth medium with 5\u3bcM AZA (GMAZA) or without AZA (GM) for 24 hours. At 70% confluence, cells were kept in growth medium in order to spontaneously achieve differentiation or transferred to differentiation medium with 5\u3bcM AZA (DMAZA) or without AZA (DM) for 12 and 24 hours. Cells used as control were unstimulated. In the proliferation phase, AZA-treated cells seemed to lose their characteristic circular shape and become elongated. The presence of AZA resulted in significant increases in the protein contents of Cyclin-D (FC:1.23 GMAZA vs GM p 640.05), p21 (FC: 1.23 GMAZA vs GM p 640.05), Myf-5 (FC: 1.21 GMAZA vs GM p 640.05) and MyoD (FC: 1.20 GMAZA vs GM p 640.05). These results propose that AZA could inhibit cell proliferation. During 12 hours of differentiation, AZA decreased the downregulation of genes involved in cell cycle arrest and in restriction point (G1 and G1/S phase) and the expression of several cyclins, E2F Transcription Factors, cyclin-dependent kinase inhibitors, specific genes responsible of cell cycle negative regulation. During 24 hours of differentiation, AZA induced an increment in the protein expression of Myf-5 (FC: 1.57 GMAZA vs GM p 640.05), MyoD (FC: 1.14 DM vs GM p 640.05; FC: 1.47 DMAZA vs GM p 640.05), p21 (FC: 1.36 GMAZA vs GM p 640.01; FC: 1.49 DM vs GM p 640.05; FC: 1.82 DMAZA vs GM p 640.01) and MyHC (FC: 1.40 GMAZA vs GM p 640.01; FC: 2.39 DM vs GM p 640.05; FC: 3.51 DMAZA vs GM p 640.01). Our results suggest that AZA-induced DNA demethylation can modulate cell cycle progression and enhance myogenesis. The effects of AZA may open novel clinical uses in the field of muscle injury research and treatment

Potential therapeutic role of L-carnitine in skeletal muscle oxidative stress and atrophy conditions

The targeting of nutraceutical treatment to skeletal muscle damage is an emerging area of research, driven by the need for new therapies for a range of muscle-associated diseases. L-Carnitine (CARN) is an essential nutrient and plays a key role in mitochondrial \u3b2-oxidation and in the ubiquitin-proteasome system regulation. As a dietary supplement to improve athletic performance, CARN has been studied for its potential to enhance \u3b2-oxidation. However, CARN effects on myogenesis, mitochondrial activity, and hypertrophy process are not completely elucidated. This in vitro study aims to investigate CARN role on skeletal muscle remodeling, differentiation process, and myotubes formation. We analyzed muscle differentiation and morphological features in C2C12 myoblasts exposed to 5 mM CARN. Our results showed that CARN was able to accelerate C2C12 myotubes formation and induce morphological changes, characterizing the start of hypertrophy process. In addition, CARN improved AKT activation and downstream cellular signaling pathways involved in skeletal muscle atrophy process prevention. Also, CARN positively regulated the pathways involved in oxidative stress defense. In this work, we provide an interesting novel mechanism of the potential therapeutic use of CARN to treat pathological conditions characterized by skeletal muscle morphological and functional impairment, oxidative stress production, and atrophy process in aging

Resveratrol promotes myogenesis and hypertrophy in murine myoblasts

Background: Nutrigenomics elucidate the ability of bioactive food components to influence gene expression, protein synthesis, degradation and post-translational modifications.Resveratrol (RSV), natural polyphenol found in grapes and in other fruits, has a plethora of health benefits in a variety of human diseases: cardio- and neuroprotection, immune regulation, cancer chemoprevention, DNA repair, prevention of mitochondrial disorder, avoidance of obesity-related diseases. In skeletal muscle, RSV acts on protein catabolism and muscle function, conferring resistance against oxidative stress, injury and cell death, but its action mechanisms and protein targets in myogenesis process are not completely known. Myogenesis is a dynamic multistep process regulated by Myogenic Regulator Factors (MRFs), responsible of the commitment of myogenic cell into skeletal muscle: mononucleated undifferentiated myoblasts break free from cell cycle, elongate and fuse to form multinucleated myotubes. Skeletal muscle hypertrophy can be defined as a result of an increase in the size of pre-existing skeletal muscle fibers accompanied by increased protein synthesis, mainly regulated by Insulin Like Growth Factor 1 (IGF-1), PI3-K/AKT signaling pathways.Aim of this work was the study of RSV effects on proliferation, differentiation process and hypertrophy in C2C12 murine cells.Methods: To study proliferative phase, cells were incubated in growth medium with/without RSV (0.1 or 25 \u3bcM) until reaching sub confluence condition (24, 48, 72 h). To examine differentiation, at 70% confluence, cells were transferred in differentiation medium both with/without RSV (0.1 or 25 \u3bcM) for 24, 48, 72, 96 hours. After 72 hours of differentiation, the genesis of hypertrophy in neo-formed myotubes was analyzed.Results: Data showed that RSV regulates cell cycle exit and induces C2C12 muscle differentiation. Furthermore, RSV might control MRFs and muscle-specific proteins synthesis. In late differentiation, RSV has positive effects on hypertrophy: RSV stimulates IGF-1 signaling pathway, in particular AKT and ERK 1/2 protein activation, AMPK protein level and induces hypertrophic morphological changes in neo-formed myotubes modulating cytoskeletal proteins expression.Conclusions: RSV might control cell cycle promoting myogenesis and hypertrophy in vitro, opening a novel field of application of RSV in clinical conditions characterized by chronic functional and morphological muscle impairment

Metformin Counteracts HCC Progression and Metastasis Enhancing KLF6/p21 Expression and Downregulating the IGF Axis

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 (400M) for 24, 48, and 72hours. 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

Semiquantitative analysis of mercury in landfill leachates using double-pulse laser-induced breakdown spectroscopy.

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L-carnitine supplementation attenuates NAFLD progression and complications in a methionine and choline deficient mouse model

Background and Aims Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease in developed countries. Lipid droplets accumulation, subsequent generation of reactive oxygen species (ROS) and fibrosis deposition cause the progression to non-alcoholic steatohepatitis (NASH). L-Carnitine (LC) has been proposed to prevent liver damage of various etiologies. The aim of the study was to investigate the effects of LC supplementation on liver fat deposition, oxidative stress and fibrosis development mechanism in a mouse model of steatohepatitis induced by a methionine-choline deficient diet (MCDD). In the same model we also analyzed the role of LC on cardiac tissue, considering the highest rates of mortality in NAFLD due to cardiovascular events. Methods The MCD diet model, best approximates the histological features of human NASH. Ten-week-old male C57BL/6 mice were divided into 3 experimental groups: one group (n=10) were fed with 120g/week of normal diet (CNT) and two groups (n=10 each group) with 120g/week of MCDD for nr. 6 weeks. After the first 3 weeks, one of the MCDD food group was enriched with 200mg/kg/die oral LC (MCDD+LC) until the end of the experiments. Results The LC supplementation delayed liver lipid accumulation (-28%, p=0.0005) and increased pCAMKII protein level (p=0.0308) in respect of MCDD group. LC supplementation also showed a role in controlling liver ROS generation (p=0.0386), restored pERK protein (p=0.005) and consequently delayed hepatotoxicity by the action of PPAR\u3b3 (p=0.0088) on Nf-\u199B expression (p=0.0009) compared with the MCDD group. Finally, LC supplementation caused a sharp decrease of \u3b1SMA (p=0.04) compared to the MCDD group. In parallel, we investigated the LC role to control cardiac stress and ROS production. LC decreased ROS production (p=0.01). To control fibrosis progression, LC significantly decreases pSTAT3/STAT3 (p=0.01) and pERK2/ERK2 expression (p=0.02) compared with MCDD group. As in liver, LC significantly decreased \u3b1SMA protein level (p=0.04) in relation to MCDD group. Conclusions We have demonstrated that LC decreased the severity of experimental NAFLD progression controlling lipid accumulation, oxidative stress imbalance and fibrosis progression in both liver and cardiac tissues analyzed. LC affected the shared oxidative stress mechanism pathway. Further studies are required to determine whether a long-term LC supplementation is able to control the pathophysiologic evolution of this disease and its related complications

L-Carnitine Reduces Oxidative Stress and Promotes Cells Differentiation and Bone Matrix Proteins Expression in Human Osteoblast-Like Cells

Bone fragility and associated fracture risk are major problems in aging. Oxidative stress and mitochondrial dysfunction play a key role in the development of bone fragility. Mitochondrial dysfunction is closely associated with excessive production of reactive oxygen species (ROS). L-Carnitine (L-C), a fundamental cofactor in lipid metabolism, has an important antioxidant property. Several studies have shown how L-C enhances osteoblastic proliferation and activity. In the current study, we investigated the potential effects of L-C on mitochondrial activity, ROS production, and gene expression involved in osteoblastic differentiation using osteoblast-like cells (hOBs) derived from elderly patients. The effect of 5mM L-C treatment on mitochondrial activity and L-C antioxidant activity was studied by ROS production evaluation and cell-based antioxidant activity assay. The possible effects of L-C on hOBs differentiation were assessed by analyzing gene and protein expression by Real Time PCR and western blotting, respectively. L-C enhanced mitochondrial activity and improved antioxidant defense of hOBs. Furthermore, L-C increased the phosphorylation of Ca2+/calmodulin-dependent protein kinase II. Additionally, L-C induced the phosphorylation of ERK1/2 and AKT and the main kinases involved in osteoblastic differentiation and upregulated the expression of osteogenic related genes, RUNX2, osterix (OSX), bone sialoprotein (BSP), and osteopontin (OPN) as well as OPN protein synthesis, suggesting that L-C exerts a positive modulation of key osteogenic factors. In conclusion, L-C supplementation could represent a possible adjuvant in the treatment of bone fragility, counteracting oxidative phenomena and promoting bone quality maintenance

Betaine promotes cell differentiation of human osteoblasts in primary culture

BACKGROUND: Betaine (BET), a component of many foods, is an essential osmolyte and a source of methyl groups; it also shows an antioxidant activity. Moreover, BET stimulates muscle differentiation via insulin like growth factor I (IGF-I). The processes of myogenesis and osteogenesis involve common mechanisms with skeletal muscle cells and osteoblasts sharing the same precursor. Therefore, we have hypothesized that BET might be effective on osteoblast cell differentiation. METHODS: The effect of BET was tested in human osteoblasts (hObs) derived from trabecular bone samples obtained from waste material of orthopedic surgery. Cells were treated with 10 mM BET at 5, 15, 60 min and 3, 6 and 24 h. The possible effects of BET on hObs differentiation were evaluated by real time PCR, western blot and immunofluorescence analysis. Calcium imaging was used to monitor intracellular calcium changes. RESULTS: Real time PCR results showed that BET stimulated significantly the expression of RUNX2, osterix, bone sialoprotein and osteopontin. Western blot and immunofluorescence confirmed BET stimulation of osteopontin protein synthesis. BET stimulated ERK signaling, key pathway involved in osteoblastogenesis and calcium signaling. BET induced a rise of intracellular calcium by means of the calcium ions influx from the extracellular milieu through the L-type calcium channels and CaMKII signaling activation. A significant rise in IGF-I mRNA at 3 and 6 h and a significant increase of IGF-I protein at 6 and 24 h after BET stimulus was detected. Furthermore, BET was able to increase significantly both SOD2 gene expression and protein content. CONCLUSIONS: Our study showed that three signaling pathways, i.e. cytosolic calcium influx, ERK activation and IGF-I production, are enhanced by BET in human osteoblasts. These pathways could have synergistic effects on osteogenic gene expression and protein synthesis, thus potentially leading to enhanced bone formation. Taken together, these results suggest that BET could be a promising nutraceutical therapeutic agent in the strategy to counteract the concomitant and interacting impact of sarcopenia and osteoporosis, i.e. the major determinants of senile frailty and related mortality