Mitochondrial dysfunction and epigenetics underlying the link between early-life nutrition and non-alcoholic fatty liver disease.

Early-life malnutrition plays a critical role in foetal development and predisposes to metabolic diseases later in life, according to the concept of 'developmental programming'. Different types of early nutritional imbalance, including undernutrition, overnutrition and micronutrient deficiency, have been related to long-term metabolic disorders. Accumulating evidence has demonstrated that disturbances in nutrition during the period of preconception, pregnancy and primary infancy can affect mitochondrial function and epigenetic mechanisms. Moreover, even though multiple mechanisms underlying non-alcoholic fatty liver disease (NAFLD) have been described, in the past years, special attention has been given to mitochondrial dysfunction and epigenetic alterations. Mitochondria play a key role in cellular metabolic functions. Dysfunctional mitochondria contribute to oxidative stress, insulin resistance and inflammation. Epigenetic mechanisms have been related to alterations in genes involved in lipid metabolism, fibrogenesis, inflammation and tumorigenesis. In accordance, studies have reported that mitochondrial dysfunction and epigenetics linked to early-life nutrition can be important contributing factors in the pathogenesis of NAFLD. In this review, we summarise the current understanding of the interplay between mitochondrial dysfunction, epigenetics and nutrition during early life, which is relevant to developmental programming of NAFLD.

A Novel Splice Variant of Human TGF-ß Type II Receptor Encodes a Soluble Protein and Its Fc-Tagged Version Prevents Liver Fibrosis in vivo.

We describe, for the first time, a new splice variant of the human TGF-ß type II receptor (TßRII). The new transcript lacks 149 nucleotides, resulting in a frameshift and the emergence of an early stop codon, rendering a truncated mature protein of 57 amino acids. The predicted protein, lacking the transmembrane domain and with a distinctive 13-amino-acid stretch at its C-terminus, was named TßRII-Soluble Endogenous (TßRII-SE). Binding predictions indicate that the novel 13-amino-acid stretch interacts with all three TGF-ß cognate ligands and generates a more extensive protein-protein interface than TßRII. TßRII-SE and human IgG1 Fc domain were fused in frame in a lentiviral vector (Lv) for further characterization. With this vector, we transduced 293T cells and purified TßRII-SE/Fc by A/G protein chromatography from conditioned medium. Immunoblotting revealed homogeneous bands of approximately 37 kDa (reduced) and 75 kDa (non-reduced), indicating that TßRII-SE/Fc is secreted as a disulfide-linked homodimer. Moreover, high-affinity binding of TßRII-SE to the three TGF-ß isoforms was confirmed by surface plasmon resonance (SPR) analysis. Also, intrahepatic delivery of Lv.TßRII-SE/Fc in a carbon tetrachloride-induced liver fibrosis model revealed amelioration of liver injury and fibrosis. Our results indicate that TßRII-SE is a novel member of the TGF-ß signaling pathway with distinctive characteristics. This novel protein offers an alternative for the prevention and treatment of pathologies caused by the overproduction of TGF-ß ligands.

Interplay between early-life malnutrition, epigenetic modulation of the immune function and liver diseases.

Early-life nutrition plays a critical role in fetal growth and development. Food intake absence and excess are the two main types of energy malnutrition that predispose to the appearance of diseases in adulthood, according to the hypothesis of 'developmental origins of health and disease'. Epidemiological data have shown an association between early-life malnutrition and the metabolic syndrome in later life. Evidence has also demonstrated that nutrition during this period of life can affect the development of the immune system through epigenetic mechanisms. Thus, epigenetics has an essential role in the complex interplay between environmental factors and genetics. Altogether, this leads to the inflammatory response that is commonly seen in non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome. In conjunction, DNA methylation, covalent modification of histones and the expression of non-coding RNA are the epigenetic phenomena that affect inflammatory processes in the context of NAFLD. Here, we highlight current understanding of the mechanisms underlying developmental programming of NAFLD linked to epigenetic modulation of the immune system and environmental factors, such as malnutrition.

Paradoxical role of the NADPH oxidase NOX4 in early preneoplastic stages of hepatocytes induced by amino acid deprivation.

BACKGROUND: The NADPH oxidase (NOX) 4 is an important source of ROS in signal transduction that acts as a liver tumor suppressor. Transforming Growth Factor ß (TGF-ß) and Epidermal Growth Factor Receptor (EGFR) pathways are involved in the modulation of NOX4 expression. Data showed that recurrent protein deprivation induces changes distinctive of a preneoplastic profile. However, the mechanisms underneath these changes have not been completely understood. METHODS: Hepatocytes that survived to the lack of amino acids (Aa) (Sel line) were cultured in complete or Aa free medium. We elucidated the molecular mechanisms that support such preneoplastic alterations employing biochemical and molecular biology assays. RESULTS: Sel line showed increased phospho-AKT and phospho-ERKs levels, diminished caspase-3 activity, augmented cell proliferation and overactivation of EGFR pathway, reminiscent of a preneoplastic phenotype. NOX4 was upregulated in these cells by TGF-ß canonical pathway, however ROS levels were not found increased as a result of an increment of antioxidant enzymes. Inhibition of TGF-ß receptor diminished NOX4 and strikingly, after EGFR inhibition, NOX4 levels also decreased. Therefore, both TGF-ß and EGFR pathways are shown to be involved in the upregulation of NOX4 in Sel line. CONCLUSIONS: This work provides novel results regarding to the regulation of NOX4 in the preneoplastic transformation of hepatocytes in the absence of Aa, in the context of TGF-ß and EGFR pathways. GENERAL SIGNIFICANCE: The advances in the understanding of the molecular mechanisms whose deregulation ultimately causes Hepatocellular Carcinoma (HCC) are essential to prevent it and to design diagnostic biomarkers and therapeutic tools.

Testosterone modulates FoxO3a and p53-related genes to protect C2C12 skeletal muscle cells against apoptosis.

The loss of muscle mass and strength with aging, sarcopenia, is a prevalent condition among the elderly, associated with skeletal muscle dysfunction and enhanced muscle cell apoptosis. We have previously demonstrated that testosterone protects against H2O2-induced apoptosis in C2C12 muscle cells, at different levels: morphological, biochemical and molecular. Since we have observed that testosterone reduces p-p53 and maintains the inactive state of FoxO3a transcription factor, induced by H2O2, we analyzed if the hormone was exerting its antiapoptotic effect at transcriptional level, by modulating pro and antiapoptotic genes associated to them. We detected the upregulation of the proapoptotic genes Puma, PERP and Bim, and MDM2 in response to H2O2 at different periods of the apoptotic process, and the downregulation of the antiapoptotic gene Bcl-2, whereas testosterone was able to modulate and counteract H2O2 effects. Furthermore, ERK and JNK kinases have been demonstrated to be linked to FoxO3a phosphorylation and thus its subcellular distribution. This work show some transcription level components, upstream of the classical apoptotic pathway, that are activated during oxidative stress and that are points where testosterone exerts its protective action against apoptosis, exposing some of the puzzle pieces of the intricate network that aged skeletal muscle apoptosis represents.

17ß-Estradiol Protects Skeletal Myoblasts From Apoptosis Through p53, Bcl-2, and FoxO Families.

17ß-Estradiol (E2 ) protects several nonreproductive tissues from apoptosis, including skeletal muscle. Previously, we showed that E2 at physiological concentrations prevented apoptosis induced by H2 O2 in skeletal myoblasts, reverting PKCδ, JNK, and p66Shc activation and exerting a beneficial action over mitochondria. Since genomic actions underlying the regulation of nuclear gene transcription are a common property of this steroid, the present work characterizes the transcriptional activity modulated by E2 to exert its antiapoptotic effect. We report that E2 protects skeletal myoblasts against apoptosis induced by H2 O2 modulating p53 and FoxO transcription factors and then their target genes Bcl-2, Bim, Puma, PERP, and MDM2, without affecting Noxa gene. The results presented in this work support the notion that the transcription factors FoxO and p53 coordinate apoptosis in C2C12 cells, and deepens our knowledge about a putative molecular mechanism by which E2 exerts beneficial effects against oxidative stress in skeletal myoblasts. J. Cell. Biochem. 118: 104-115, 2017. © 2016 Wiley Periodicals, Inc.

17ß-Estradiol and testosterone in sarcopenia: Role of satellite cells.

The loss of muscle mass and strength with aging, referred to as sarcopenia, is a prevalent condition among the elderly. Although the molecular mechanisms underlying sarcopenia are unclear, evidence suggests that an age-related acceleration of myocyte loss via apoptosis might be responsible for muscle perfomance decline. Interestingly, sarcopenia has been associated to a deficit of sex hormones which decrease upon aging. The skeletal muscle ability to repair and regenerate itself would not be possible without satellite cells, a subpopulation of cells that remain quiescent throughout life. They are activated in response to stress, enabling them to guide skeletal muscle regeneration. Thus, these cells could be a key factor to overcome sarcopenia. Of importance, satellite cells are 17ß-estradiol (E2) and testosterone (T) targets. In this review, we summarize potential mechanisms through which these hormones regulate satellite cells activation during skeletal muscle regeneration in the elderly. The advance in its understanding will help to the development of potential therapeutic agents to alleviate and treat sarcopenia and other related myophaties.

17ß-Estradiol Abrogates Apoptosis Inhibiting PKCδ, JNK, and p66Shc Activation in C2C12 Cells.

17ß-Estradiol (E2) protects several non-reproductive tissues from apoptosis, including skeletal muscle. Previously, we showed that E2 at physiological concentrations prevented apoptosis induced by H2O2 in skeletal myoblasts. As we have also demonstrated a clear beneficial action of this hormone on skeletal muscle mitochondria, the present work further characterizes the signaling mechanisms modulated by E2 that are involved in mitochondria protection, which ultimately result in antiapoptosis. Here, we report that E2 through estrogen receptors (ERs) inhibited the H2O2-induced PKCδ and JNK activation, which results in the inhibition of phosphorylation and translocation to mitochondria of the adaptor protein p66Shc. In conjunction, the inhibition by the hormone of this H2O2-triggered signaling pathway results in protection of mitochondrial potential membrane. Our results provide basis for a putative mechanism by which E2 exerts beneficial effects on mitochondria, against oxidative stress, in skeletal muscle cells.

High passage numbers induce resistance to apoptosis in C2C12 muscle cells

Cell lines with high passage numbers exhibit alterations in cell morphology and functions. In the present work, C2C12 skeletal muscle cells with either low (<20) or high (>60) passage numbers (identified as l-C2C12 or h-C2C12, respectively) were used to investigate the apoptotic response to H2O2 as a function of culture age h-C2C12. We found that older cultures (h-C2C12 group) were depleted of mitochondrial DNA (mtDNA). When we analyzed the behavior of Bad, Bax, caspase-3 and mitochondrial transmembrane potential, we observed that cells in the h-C2C12 group were resistant to H2O2 induction of apoptosis. We propose serially cultured C2C12 cells as a refractory model to H2O2-induced apoptosis. In addition, the data obtained in this work suggest that mtDNA is required for apoptotic cell death in skeletal muscle C2C12 cells.(AU)

High passage numbers induce resistance to apoptosis in C2C12 muscle cells

Cell lines with high passage numbers exhibit alterations in cell morphology and functions. In the present work, C2C12 skeletal muscle cells with either low (<20) or high (>60) passage numbers (identified as l-C2C12 or h-C2C12, respectively) were used to investigate the apoptotic response to H2O2 as a function of culture age h-C2C12. We found that older cultures (h-C2C12 group) were depleted of mitochondrial DNA (mtDNA). When we analyzed the behavior of Bad, Bax, caspase-3 and mitochondrial transmembrane potential, we observed that cells in the h-C2C12 group were resistant to H2O2 induction of apoptosis. We propose serially cultured C2C12 cells as a refractory model to H2O2-induced apoptosis. In addition, the data obtained in this work suggest that mtDNA is required for apoptotic cell death in skeletal muscle C2C12 cells.

Estradiol exerts antiapoptotic effects in skeletal myoblasts via mitochondrial PTP and MnSOD.

17ß-Estradiol (E(2)) protects several non-reproductive tissues from apoptosis, including skeletal muscle. We have shown that E(2) at physiological concentrations prevented apoptosis induced by H(2)O(2) in C2C12 skeletal myoblasts. As we also demonstrated the presence of estrogen receptors in mitochondria, the present work was focused on the effects of E(2) on this organelle. Specifically, we evaluated the actions of E(2) on the mitochondrial permeability transition pore (MPTP) by the calcein-acetoxymethylester/cobalt method using fluorescence microscopy and flow cytometry. Pretreatment with E(2) prevented MPTP opening induced by H(2)O(2), which preceded loss of mitochondrial membrane potential. In addition, it was observed that H(2)O(2) induced translocation of Bax to mitochondria; however, in the presence of the steroid this effect was abrogated suggesting that members of the Bcl-2 family may be regulated by E(2) to exert an antiapoptotic effect. Moreover, E(2) increased mitochondrial manganese superoxide dismutase protein expression and activity, as part of a mechanism activated by E(2) that improved mitochondrial performance. Our results suggest a role of E(2) in the regulation of apoptosis with a clear action at the mitochondrial level in C2C12 skeletal myoblast cells.

High passage numbers induce resistance to apoptosis in C2C12 muscle cells.

Cell lines with high passage numbers exhibit alterations in cell morphology and functions. In the present work, C2C12 skeletal muscle cells with either low (< 20) or high (> 60) passage numbers (identified as 1-C2C12 or h-C2C12, respectively) were used to investigate the apoptotic response to H2O2 as a function of culture age h-C2C12. We found that older cultures (h-C2C12 group) were depleted of mitochondrial DNA (mtDNA). When we analyzed the behavior of Bad, Bax, caspase-3 and mitochondrial transmembrane potential, we observed that cells in the h-C2C12 group were resistant to H2O2 induction of apoptosis. We propose serially cultured C2Cl2 cells as a refractory model to H2O2-induced apoptosis. In addition, the data obtained in this work suggest that mtDNA is required for apoptotic cell death in skeletal muscle C2C12 cells.

High passage numbers induce resistance to apoptosis in C2C12 muscle cells. / High passage numbers induce resistance to apoptosis in C2C12 muscle cells.

Cell lines with high passage numbers exhibit alterations in cell morphology and functions. In the present work, C2C12 skeletal muscle cells with either low (< 20) or high (> 60) passage numbers (identified as 1-C2C12 or h-C2C12, respectively) were used to investigate the apoptotic response to H2O2 as a function of culture age h-C2C12. We found that older cultures (h-C2C12 group) were depleted of mitochondrial DNA (mtDNA). When we analyzed the behavior of Bad, Bax, caspase-3 and mitochondrial transmembrane potential, we observed that cells in the h-C2C12 group were resistant to H2O2 induction of apoptosis. We propose serially cultured C2Cl2 cells as a refractory model to H2O2-induced apoptosis. In addition, the data obtained in this work suggest that mtDNA is required for apoptotic cell death in skeletal muscle C2C12 cells.