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.

Glucose and Amino Acid Metabolic Dependencies Linked to Stemness and Metastasis in Different Aggressive Cancer Types.

Malignant cells are commonly characterised by being capable of invading tissue, growing self-sufficiently and uncontrollably, being insensitive to apoptosis induction and controlling their environment, for example inducing angiogenesis. Amongst them, a subpopulation of cancer cells, called cancer stem cells (CSCs) shows sustained replicative potential, tumor-initiating properties and chemoresistance. These characteristics make CSCs responsible for therapy resistance, tumor relapse and growth in distant organs, causing metastatic dissemination. For these reasons, eliminating CSCs is necessary in order to achieve long-term survival of cancer patients. New insights in cancer metabolism have revealed that cellular metabolism in tumors is highly heterogeneous and that CSCs show specific metabolic traits supporting their unique functionality. Indeed, CSCs adapt differently to the deprivation of specific nutrients that represent potentially targetable vulnerabilities. This review focuses on three of the most aggressive tumor types: pancreatic ductal adenocarcinoma (PDAC), hepatocellular carcinoma (HCC) and glioblastoma (GBM). The aim is to prove whether CSCs from different tumour types share common metabolic requirements and responses to nutrient starvation, by outlining the diverse roles of glucose and amino acids within tumour cells and in the tumour microenvironment, as well as the consequences of their deprivation. Beyond their role in biosynthesis, they serve as energy sources and help maintain redox balance. In addition, glucose and amino acid derivatives contribute to immune responses linked to tumourigenesis and metastasis. Furthermore, potential metabolic liabilities are identified and discussed as targets for therapeutic intervention.

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.

Protein malnutrition during fetal programming induces fatty liver in adult male offspring rats.

We evaluated the effects of protein malnutrition on liver morphology and physiology in rats subjected to different malnutrition schemes. Pregnant rats were fed with a control diet or a low protein diet (LPD). Male offspring rats received a LPD during gestation, lactation, and until they were 60 days old (MM group), a late LPD that began after weaning (CM), or a LPD administrated only during the gestation-lactation period followed by a control diet (MC). On day 60, blood was collected and the liver was dissected out. We found a decrease in MM rats' total body (p < 0.001) and liver (p < 0.05) weight. These and CM rats showed obvious liver dysfunction reflected by the increase in serum glutamic pyruvic transaminase (SGOT) (MM p < 0.001) and serum glutamic pyruvic transaminase (SGPT) (MM and CM p < 0.001) enzymes, and liver content of cholesterol (MM and CM p < 0.001) and triglycerides (MM p < 0.01; CM p < 0.001), in addition to what we saw by histology. Liver dysfunction was also shown by the increase in gamma glutamyl transferase (GGT) (MM, MC, and CM p < 0.001) and GST-pi1 (MM and CM p < 0.001, MC p < 0.05) expression levels. MC rats showed the lowest increment in GST-pi1 expression (MC vs. MM; p < 0.001, MC vs. CM; p < 0.01). ROS production (MM, CM, and MC: p < 0.001), lipid peroxidation (MM, CM, and MC p < 0.001), content of carbonyl groups in liver proteins (MM and CM p < 0.001, MC p < 0.01), and total antioxidant capacity (MM, CM, and MC p < 0.001) were increased in the liver of all groups of malnourished animals. However, MM rats showed the highest increment. We found higher TNF-α (MM and CM p < 0.001), and IL-6 (MM and CM p < 0.001) serum levels and TGF-ß liver content (MM p < 0.01; CM p < 0.05), in MM and CM groups, while MC rats reverted the values to normal levels. Pro-survival signaling pathways mediated by tyrosine or serine/threonine kinases (pAKT) (MM and CM p < 0.001; MC p < 0.01) and extrasellular signal-regulated kinase (pERKs) (MM p < 0.01; CM p < 0.05) appeared to be activated in the liver of all groups of malnourished rats, suggesting the presence of cells resistant to apoptosis which would become cancerous. In conclusion, a LPD induced liver damage whose magnitude was related to the developmental stage at which malnutrition occurs and to its length.

Lack of amino acids in mouse hepatocytes in culture induces the selection of preneoplastic cells.

Protein malnutrition occurs when there is insufficient protein to meet metabolic demands. Previous works have indicated that cycles of protein fasting/refeeding enhance the incidence of early lesions during chemical carcinogenesis in rat liver. The general objective of this work was to study the effect of aminoacids (Aa) deprivation on the proliferation and survival of hepatocytes, to understand its possible involvement in the generation of pre-neoplastic stages in the liver. Lack of Aa in the culture medium of an immortalized mice hepatocyte cell line induced loss in cell viability, correlating with apoptosis. However, a subpopulation of cells was able to survive, which showed a more proliferative phenotype and resistance to apoptotic stimuli. Escaping to Aa deprivation-induced death is coincident with an activated mTOR signaling and higher levels of phospho-AKT and phospho-ERKs, which correlated with increased activation of EGFR/SRC pathway and overexpression of EGFR ligands, such as TGF-α and HB-EGF. Lack of Aa induced a rapid increase in reactive oxygen species (ROS) production. However, cells that survived showed an enhancement in the levels of reduced glutathione and a higher expression of γ-GCS, the regulatory enzyme of glutathione synthesis, which can be interpreted as an adaptation of the cells to counteract the oxidative stress. In conclusion, results presented in this paper indicate that it is possible to isolate a subpopulation of hepatocytes that are able to grow in the absence of Aa, showing higher capacity to proliferate and survive, reminiscent of a preneoplastic phenotype.

Alternation between dietary protein depletion and normal feeding cause liver damage in mouse.

The effect of frequent protein malnutrition on liver function has not been intensively examined. Thus, the effects of alternating 5 days of a protein and amino acid-free diet followed by 5 days of a complete diet repeated three times (3 PFD-CD) on female mouse liver were examined. The expression of carbonic anhydrase III (CAIII), fatty acid synthase (FAS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutathione S-transferase P1 (GSTP1) in liver were assessed by proteomics, reverse transcriptase-polymerase chain reaction and Northern blotting. The activities of liver GSTs, glutathione reductase (GR) and catalase (CAT), as well as serum glutamic-oxaloacetic transaminase (SGOT) and glutamic-pyruvic transaminase (SGPT) were also tested. Additionally, oxidative damage was examined by measuring of protein carbonylation and lipid peroxidation. Liver histology was examined by light and electron microscopy. Compared with control mice, 3 PFD-CD increased the content of FAS protein (+90%) and FAS mRNA (+30%), while the levels of CAIII and CAIII mRNAs were decreased (-48% and -64%, respectively). In addition, 3 PFD-CD did not significantly change the content of GSTP1 but produced an increase in its mRNA level (+20%), while it decreased the activities of both CAT (-66%) and GSTs (-26%). After 3 PFD-CD, liver protein carbonylation and lipid peroxidation were increased by +55% and +95%, respectively. In serum, 3 PFD-CD increased the activities of both SGOT (+30%) and SGPT (+61%). In addition, 3 PFD-CD showed a histological pattern characteristic of hepatic damage. All together, these data suggest that frequent dietary amino acid deprivation causes hepatic metabolic and ultrastructural changes in a fashion similar to precancerous or cancerous conditions.

Oxidative stress in mouse liver caused by dietary amino acid deprivation: protective effect of methionine.

The aim of this work was to evaluate the effects of a diet depleted of amino acids (protein-free diet, or PFD), as well as the supplementation with methionine (PFD+Met), on the antioxidant status of the female mouse liver. With this purpose, cytosolic protein spots from two-dimensional non-equilibrium pH gel electrophoresis were identified by several procedures, such as mass spectrometry, Western blot, gel matching and enzymatic activity. PFD decreased the contents of catalase (CAT), peroxiredoxin I (Prx-I), and glutathione peroxidase (GPx) by 67%, 37% and 45%, respectively. Gene expression analyses showed that PFD caused a decrease in CAT (-20%) and GPx (-30%) mRNA levels but did not change that of Prx-I. It was also found that, when compared to a normal diet, PFD increased the liver contents of both reactive oxygen species (+50%) and oxidized protein (+88%) and decreased that of glutathione (-45%). Supplementation of PFD with Met prevented these latter effects to varying degrees, whereas CAT, Prx-I and GPx mRNA levels resulted unmodified. Present results suggest that dietary amino acid deprivation deranges the liver antioxidant defences, and this can be, in part, overcome by supplementation with Met.

Primary cell culture of Echinococcus granulosus developed from the cystic germinal layer: biological and functional characterization.

Cell cultures of parasitic helminths are an invaluable tool for investigations of basic biological processes, as well as for development of improved chemotherapeutic agents and molecular interactions between host and parasite. We carried out a simple and efficient methodology to isolate Echinococcus granulosus germinal cells which were maintained for at least 4 months while cultivated in the presence of reducing agents and hormones. Microscopic analysis of the primary cell culture revealed the presence of cells with similar Echinococcus germinal cell morphology and behaviour. Population doubling time was estimated at 48 h, showing a rapid division rate. To discard possible host contamination, the specificity of the primary culture was tested by nested PCR, analyzing mdh gene expression and obtaining only one product with the expected size. We also studied the expression of specific E. granulosus proteins in primary cell culture. The novel and systematized method described here constitutes a powerful tool for investigations in cystic echinococcosis on biochemical and biological aspects related to the life cycle of the parasite and mechanisms of host-parasite interactions. This method also constitutes a powerful tool for the design of more efficient therapeutic alternatives.

Appendicular tuberculosis: the resurgence of an old disease with difficult diagnosis.

Gastrointestinal tuberculosis (TB) is quite rare, representing only 3% of all extra-pulmonary cases. Blind gut and ileum are the most common gastrointestinal localizations, while appendix involvement is infrequent. Appendix involvement is usually related to symptoms of acute appendicitis since the caseous necrosis may lead to adhesions and surgical complications such as perforation. For this reason patients with suspected appendicular TB usually undergo surgery even without a secure diagnosis. In these cases, due to the absence of specific symptoms and signs, the diagnosis is delayed after surgery, thus resulting in a high percentage of important, and sometimes lethal, complications. Histopathological examination is often the only way to reach a diagnosis and to establish specific antibiotic therapy, while an early diagnosis could avoid surgical treatment. We report a case of appendicular TB not only for its rarity but also to discuss the difficulty in its diagnosis.

Laparo-endoscopic "Rendezvous" to treat cholecysto-choledocolithiasis: Effective, safe and simplifies the endoscopist's work.

AIM: To investigate our clinical experience with combined laparo-endoscopic Rendezvous (RV) for the treatment of patients affected by gallstones and common bile duct (CBD) stones and especially to study the never evaluated opinion of the endoscopist concerning the difficulty of the intraoperative endoscopic procedure during the RV in comparison with standard endoscopic retrograde cholangio-pancreatography (ERCP). METHODS: Eighty consecutive patients affected by cholecystolithiasis and diagnosed or suspected CBD stones were treated with a standardized "tailored" RV. The relevant technical features, the feasibility, the effectiveness in stone clearance, the safety but also the simple evaluation of difficulty and agreement of the endoscopist were analyzed with a questionnaire. RESULTS: The feasibility was 97.5% and the effectiveness 100% concerning CBD clearance and solution of coexisting problems at the papilla. Minor morbidity was 3.3%, the operating time was prolonged by a mean of 14 min, the mean hospital stay was 3.8 d and only one stone's recurrence occurred. The endoscopist evaluated the procedure to be simpler than standard ERCP-ES in 81.2% of the cases. CONCLUSION: Simultaneous RV carries high effectiveness and safety at least comparable to those reported for other options. The endoscopist is very often satisfied with this approach because of the minimization of some steps of the endoscopic procedure and avoidance of relevant iatrogenic risk factors. If the mandatory collaboration between surgeons and endoscopists is guaranteed, this approach can often be preferable for the patient, the surgeon, the endoscopist and the hospital.

[Perineo-ano-rectal injuries: clinical experience]. / I traunmi perineo-ano-rettali: esperienza clinica.

Traumatic lesions involving the rectum, perineum and anus are infrequent but difficult to treat, requiring experience with trauma and colo-proctological surgery. The aim of the treatment is to repair the lesions and to minimise the early complications which are the main cause of failure and of late complications and disability. The most complicated lesions present problems concerning either the surgical strategy or the surgical timing, both of which are essential for a successful outcome. The Authors analyse their recent clinical experience with 7 patients with complex traumatic lesions involving the rectum, perineum and anus, excluding those of gynaecological/obstetric origin and those not involving the sphincter. They evaluated the clinical history, causes and types of lesions, as well as treatment, complications and outcomes. Five of the lesions were caused by impalement, one by an explosion and one by a motorboat propeller blade. Six of the patients (85.7%) were treated by direct primary repair and one (14.3%) by secondary repair after a previous colostomy. All 7 patients achieved complete recovery of the lesions. Only two cases (28.6%) of early complications and one case (14.3%) of persistent minimal sphincter dysfunction occurred. On the basis of these good results, the clinical experience and the literature, the Authors suggest that these perineo-ano-rectal lesions, though often complex, may often be cured by early surgery, confining colostomy only to particular cases. In addition to experience with trauma and the timing of colo-proctological surgery, a knowledge of all the available surgical options is mandatory to achieve the best results.

Laparoscopic resection of a large GIST of the stomach: is it preferable in elderly patients? A case report.

Gastrointestinal stromal tumour (GIST) of the stomach is extremely rare in the elderly. Surgical resection of the stomach by partial gastrectomy or wedge resection is the standard treatment. Today the resection can also be performed laparoscopically, especially in the case of small tumours as well as for larger GIST though there are unclearly defined oncological limits. The authors report the successful treatment of a large 7.5 cm GIST of the stomach by laparoscopic wedge resection in a 78-year-old patient. The GIST was almost entirely located intraperitoneally between the stomach and the spleen and could be radically resected with a minimal touch technique. The patient recovered promptly and manifested no recurrence at a 2-year follow-up. The authors focus on the main factors supporting the indication for laparoscopic resection of large gastric GIST, especially in the elderly. The surgical risk/benefit ratios of the different approaches, the surgeon's skills in laparoscopically respecting the rules of oncological surgery, and informed consent of the patient in relation to the limited scientific evidence concerning the main risk factors of recurrence are all important considerations.

Adrenal enucleation in MSG-damaged hyperleptinemic male rats transiently restores adrenal sensitivity to leptin.

It is known that the neonatal treatment of rats with monosodium L-glutamate (MSG) induces several metabolic abnormalities, resulting in enhanced adiposity and hyperleptinemia. Our study was designed to explore the consequences of MSG-induced chronic hyperleptinemia on adrenal sensitivity to the inhibitory effect of exogenous leptin. Neonatal male rats treated with MSG or vehicle (controls, CTR) were followed during 150 days in order to study changes observed over development in body weight, food consumption as well as in vivo hypothalamo-pituitary-adrenal (HPA) axis and adipocyte functions. During adulthood, adrenal response to adrenocorticotropin (ACTH) was evaluated both in vitro and in vivo in order to determine the adrenal sensitivity to the inhibitory effect of leptin. For this purpose, sham-operated as well as CTR and MSG rats with bilateral adrenal enucleation (AE) were used. Our results indicate that: (1) between 30 and 150 days of age, MSG animals developed hypophagia, accompanied by arrest in body weight gain, and concomitant enhanced basal levels of all HPA axis components and of leptin; (2) adrenals from of 150-day- old MSG rats displayed an in vitro adrenocortical hyperresponse to ACTH stimulation as well as an adrenal refractoriness to the physiological inhibitory effect of leptin on ACTH-stimulated glucocorticoid output, and (3) bilateral AE in adult MSG-treated rats transiently reversed the MSG-induced hyperleptinemia, restoring normal leptin levels as well as a normal adrenal sensitivity to the inhibitory effect of leptin. Our data indicate that adrenal exposure to the chronically high plasma leptin levels observed in MSG rats is involved in the loss of the inhibitory regulatory effect of leptin at the adrenal level, being therefore, at least in part, responsible for the increased total and free glucocorticoid production measured in MSG adult rats. Furthermore, this study strongly suggests that the adrenal overfunction, frequently associated with different phenotypes of obesity, could be due to an adrenal resistance to the leptin-negative regulation.

Participation of the central serotonergic pathway in the endotoxin-stimulated hypothalamo-pituitary-adrenal axis function

The aim of the present study was to determine whether the central serotoninergic pathway (5HT) plays any stimulatory/inhibitory role in endotoxin-stimulated hypothalamo-pituitary-adrenal (HPA) axis function in the male rat. For this purpose, animals inhibited or not of central 5HT synthesis were injected with vehicle alone or containing endotoxin (LPS) and killed 2 h later. The results indicate that the inhibition of the 5HT pathway did not modify the LPS-induced hypoglycemia but that significantly reduced ACTH release in plasma. Although the inhibition of 5HT did not modify the CRH-ergic system, it diminished hypothalamic vasopressin (AVP) content in basal condition. After LPS injection, rats inhibited of 5HT synthesis showed a significant activation of the hypothalamic AVP-ergic system whereas control rats did not. These data clearly indicate that decreased HPA axis function after 5HT inhibition could be partially compensated by the facilitation of the AVP neuronal activity.