Arabidopsis thaliana sirtuins control proliferation and glutamate dehydrogenase activity.

Sirtuins are part of a gene family of NAD-dependent deacylases that act on histone and non-histone proteins and control a variety of activities in all living organisms. Their roles are mainly related to energy metabolism and include lifetime regulation, DNA repair, stress resistance, and proliferation. A large amount of knowledge concerning animal sirtuins is available, but data about their plant counterparts are scarce. Plants possess few sirtuins that have, like in animals, a recognized role in stress defense and metabolism regulation. However, engagement in proliferation control, which has been demonstrated for mammalian sirtuins, has not been reported for plant sirtuins so far. In this work, srt1 and srt2 Arabidopsis mutant seedlings have been used to evaluate in vivo the role of sirtuins in cell proliferation and regulation of glutamate dehydrogenase, an enzyme demonstrated to be involved in the control of cell cycle in SIRT4-defective human cells. Moreover, bioinformatic analyses have been performed to elucidate sequence, structure, and function relationships between Arabidopsis sirtuins and between each of them and the closest mammalian homolog. We found that cell proliferation and GDH activity are higher in mutant seedlings, suggesting that both sirtuins exert a physiological inhibitory role in these processes. In addition, mutant seedlings show plant growth and root system improvement, in line with metabolic data. Our data also indicate that utilization of an easy to manipulate organism, such as Arabidopsis plant, can help to shed light on the molecular mechanisms underlying the function of genes present in interkingdom species.

Diiodothyronines regulate metabolic homeostasis in primary human hepatocytes by modulating mTORC1 and mTORC2 activity.

Until three decades, ago 3,5-diiodothyronine (3,5-T2) and 3,3'-diiodothyronine (3,3'-T2) were considered products of thyroid hormone catabolism without biological activity. Some metabolic effects have been described in rodents, but the physiological relevance in humans and the mechanisms of action are unknown. Aim of this work was to investigate the role and the mechanisms of action of 3,5-T2 and 3,3'-T2 in the regulation of metabolic homeostasis in human liver. We used primary human hepatocytes freshly isolated from donors and grown on Matrigel as the golden standard in vitro model to study human hepatic metabolism. Results show that diiodothyronines in the range of plasma physiological concentrations reduced hepatic lipid accumulation, by modulating the activity of the mTORC1/Raptor complex through an AMPK-mediated mechanism, and stimulated the mTORC2/Rictor complex-activated pathway, leading to the down regulation of the expression of key gluconeogenic genes. Hence, we propose that diiodothyronines act as key regulators of hepatic metabolic homeostasis in humans.

Circadian Rhythms and Hormonal Homeostasis: Pathophysiological Implications.

Over recent years, a deeper comprehension of the molecular mechanisms that control biological clocks and circadian rhythms has been achieved. In fact, many studies have contributed to unravelling the importance of the molecular clock for the regulation of our physiology, including hormonal and metabolic homeostasis. Here we will review the structure, organisation and molecular machinery that make our circadian clock work, and its relevance for the proper functioning of physiological processes. We will also describe the interconnections between circadian rhythms and endocrine homeostasis, as well as the underlying consequences that circadian dysregulations might have in the development of several pathologic affections. Finally, we will discuss how a better knowledge of such relationships might prove helpful in designing new therapeutic approaches for endocrine and metabolic diseases.

LPS-induced TNF-α factor mediates pro-inflammatory and pro-fibrogenic pattern in non-alcoholic fatty liver disease.

Lipopolysaccharide (LPS) is currently considered one of the major players in non-alcoholic fatty liver disease (NAFLD) pathogenesis and progression. Here, we aim to investigate the possible role of LPS-induced TNF-α factor (LITAF) in inducing a pro-inflammatory and pro-fibrogenic phenotype of non-alcoholic steatohepatitis (NASH).We found that children with NAFLD displayed, in different liver-resident cells, an increased expression of LITAF which correlated with histological traits of hepatic inflammation and fibrosis. Total and nuclear LITAF expression increased in mouse and human hepatic stellate cells (HSCs). Moreover, LPS induced LITAF-dependent transcription of IL-1ß, IL-6 and TNF-α in the clonal myofibroblastic HSC LX-2 cell line, and this effect was hampered by LITAF silencing. We showed, for the first time in HSCs, that LITAF recruitment to these cytokine promoters is LPS dependent. However, preventing LITAF nuclear translocation by p38MAPK inhibitor, the expression of IL-6 and TNF-α was significantly reduced with the aid of p65NF-ĸB, while IL-1ß transcription exclusively required LITAF expression/activity. Finally, IL-1ß levels in plasma mirrored those in the liver and correlated with LPS levels and LITAF-positive HSCs in children with NASH.In conclusion, a more severe histological profile in paediatric NAFLD is associated with LITAF over-expression in HSCs, which in turn correlates with hepatic and circulating IL-1ß levels outlining a panel of potential biomarkers of NASH-related liver damage. The in vitro study highlights the role of LITAF as a key regulator of the LPS-induced pro-inflammatory pattern in HSCs and suggests p38MAPK inhibitors as a possible therapeutic approach against hepatic inflammation in NASH.

Emodin prevents intrahepatic fat accumulation, inflammation and redox status imbalance during diet-induced hepatosteatosis in rats.

High-fat and/or high-carbohydrate diets may predispose to several metabolic disturbances including liver fatty infiltration (hepatosteatosis) or be associated with necro-inflammation and fibrosis (steatohepatitis). Several studies have emphasized the hepatoprotective effect of some natural agents. In this study, we investigated the potential therapeutic effects of the treatment with emodin, an anthraquinone derivative with anti-oxidant and anti-cancer abilities, in rats developing diet-induced hepatosteatosis and steatohepatitis. Sprague-Dawley rats were fed a standard diet (SD) for 15 weeks, or a high-fat/high-fructose diet (HFD/HF). After 5 weeks, emodin was added to the drinking water of some of the SD and HFD/HF rats. The experiment ended after an additional 10 weeks. Emodin-treated HFD/HF rats were protected from hepatosteatosis and metabolic derangements usually observed in HFD/HF animals. Furthermore, emodin exerted anti-inflammatory activity by inhibiting the HFD/HF-induced increase of tumor necrosis factor (TNF)-α. Emodin also affected the hepatocytes glutathione homeostasis and levels of the HFD/HF-induced increase of glutathionylated/phosphorylated phosphatase and tensin homolog (PTEN). In conclusion, we demonstrated that a natural agent such as emodin can prevent hepatosteatosis, preserving liver from pro-inflammatory and pro-oxidant damage caused by HFD/HF diet. These findings are promising, proposing emodin as a possible hindrance to progression of hepatosteatosis into steatohepatitis.

3,5,3'-triiodothyronine (T3) stimulates cell proliferation through the activation of the PI3K/Akt pathway and reactive oxygen species (ROS) production in chick embryo hepatocytes.

Thyroid hormones (THs) have a wide variety of essential roles in vertebrates, ranging from the regulation of key metabolic processes to cell proliferation and apoptosis. The classical mechanism of action of THs is genomic; 3,5,3'-triiodothyronine (T3) binds to specific nuclear receptors (TRs) and modifies the expression of specific genes. Recently, a new category of mechanisms, termed nongenomic, has been discovered for T3. These mechanisms include, among others, the rapid activation of signal transduction pathways, such as PI3K/Akt and MAPK, which eventually lead to cell proliferation. These effects are mediated in some cell types by a plasma membrane receptor, identified as integrin αvß3, and in other cell types by cytoplasmic TRß1. The aim of this work was to analyze the effect of T3 on the cell growth of chick embryo hepatocytes at two different stages of development, 14 and 19 days, and to determine the activation of the signal transduction pathways, focusing on the potential involvement of a plasma membrane receptor and the possible participation of PI3K/Akt and reactive oxygen species (ROS). Our results clearly show that T3 stimulates cell proliferation at both stages of development through the activation of the PI3K/Akt pathway and the production of small amounts of ROS, which operate as effective second messengers. Moreover, we prove that these effects are not initiated at the plasma membrane receptor for T3.

Redox homeostasis and posttranslational modifications/activity of phosphatase and tensin homolog in hepatocytes from rats with diet-induced hepatosteatosis.

High-fat and high-carbohydrate diets may predispose to simple steatosis, alone or associated with necroinflammation and fibrosis (steatohepatitis). However, there are few reports about the real effect of these nutrients on hepatocyte redox homeostasis and consequent molecular derangement. Here, we investigated whether different diets would induce oxidative damage in primary rat hepatocytes and thereby affect the activity of phosphatase and tensin homolog (PTEN). We used Sprague-Dawley rats fed, for 14 weeks, a standard diet (SD), a high-fat/low-carbohydrate diet (HFD-LC), a normal-fat/high-fructose diet (NFD-HF), or a high-fat/high-fructose diet (HFD-HF). Metabolic and histological parameters were analyzed in blood and liver samples, while oxidative stress markers and related posttranscriptional modification of PTEN were analyzed in isolated hepatocytes. Our results indicate that different dietetic hypercaloric regimens caused liver damage and a significant increase of body and liver weight, as well as elevated plasma levels of alanine aminotransferase, triglycerides and insulin. Hepatocytes from NFD-HF and HFD-HF rats displayed a decrement of cell viability and proliferation rate. Hepatocytes from animals treated with hypercaloric regimens also exhibited oxidative stress greater than SD hepatocytes. Finally, NFD-HF and HFD-HF hepatocytes showed an increased PTEN phosphorylation and decreased PTEN activity, which seem strongly correlated to an increased glutathionylation of the protein. In conclusion, we demonstrate that fructose-enriched diets cause a tissue and hepatocyte damage that might exacerbate those observed in the presence of high-fat alone and might render, via redox homeostasis imbalance, the hepatocytes more prone to posttranslational modifications and activity alteration of PTEN.

Hypertonic stress regulates amino acid transport and cell cycle proteins in chick embryo hepatocytes.

Hyperosmotic stress affects cell growth, decreasing cell volume and increasing the uptake of organic osmolytes. However, the sensitivity of embryonic cells to osmotic treatment remains to be established. We have analysed some aspects of cell-cycle control and amino-acid transport in hypertonic conditions during prenatal life. The effects of hyperosmotic stress on amino-acid uptake mediated by system A, (3)H-thymidine incorporation, and regulation of cell-cycle proteins were analysed in chick embryo hepatocytes. Hypertonic stress increased system A activity and caused cell-cycle delay. Effects on amino-acid transport involved p38 kinase activation and new carrier synthesis. Cyclin D1, cdk4 (cyclin-dependent kinase 4) and PCNA (proliferating-cell nuclear antigen) levels decreased, whereas cyclin E, p21 and p53 levels were unchanged. Incorporation of (3)H-leucine indicated decreased synthesis of cyclin D1. In contrast, analysis of mRNA by qRT-PCR (quantitative real-time PCR) showed a net increase of cyclin D1 transcripts, suggesting post-transcriptional regulation. The data show that chick embryo hepatocytes respond to hyperosmotic conditions by arresting cell growth to prevent DNA damage and increasing osmolyte uptake to regulate cell volume, indicating that the adaptive response to environmental stress exists during prenatal life.

Mirnome analysis reveals novel molecular determinants in the pathogenesis of diet-induced nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is an emerging disease with a broad spectrum of liver conditions. The complex molecular pathogenesis of NAFLD is still unclear. In this study, we conducted an analysis of microRNA (miRNA) expression profiles in liver of rats made NAFLD by different diets. To this aim, Sprague-Dawley rats were fed ad libitum for 3 months with different diets: standard diet (SD), diet enriched in fats and low in carbohydrates (HFD), SD with high fructose (SD-HF) and diet with high levels of fats and fructose (HFD-HF). Our results demonstrated that the treatment with different dietetic regimens caused a significant increase of the body weight and the alteration of some metabolic parameters compared with control animals, as well as various liver injuries. The miRNAs analysis showed the significant downregulation of three miRNAs (miR-122, miR-451 and miR-27) and the upregulation of miR-200a, miR-200b and miR-429 in HFD, SD-HF and HFD-HF rats. Besides, miR-21 expression was significantly decreased only in fructose-enriched diets. These miRNAs target molecules involved in the control of lipid and carbohydrate metabolism, signal transduction, cytokine and chemokine-mediated signaling pathway and apoptosis. Western blot analysis of PKCδ, LITAF, ALDOLASE-A, p38MAPK, PTEN, LIPIN1, EPHRIN-A1, EPHA2 and FLT1 showed a diet-induced deregulation of all these proteins. Interestingly, the expression pattern of LITAF, PTEN, LIPIN1, EPHRIN-A1, EPHA2 and FLT1 might be well explained by the trend of their specific mRNAs, by potentially regulatory miRNAs, or both. In conclusion, we highlight for the first time the potential involvement of novel determinants (miRNAs and proteins) in the molecular pathogenesis of diet-induced NAFLD.

Multiple parameters are involved in the effects of cadmium on prenatal hepatocytes.

Cadmium, a toxic heavy metal, expresses its toxicity by affecting several cellular functions, such as enzyme activities, DNA repair systems, redox state of the cell and signal transduction. Although the liver is a known target organ, the mechanisms involved in cadmium toxicity are not yet clarified, especially during prenatal development. Here we consider the effects of cadmium on viability, proliferation, adhesion and defence mechanisms in primary adult and fetal rat hepatocytes. Fetal hepatocytes are less sensitive to cadmium toxicity, they appear to be unaffected or even stimulated by treatments that strongly inhibit DNA synthesis in adult cells. The behaviour of proteins involved in cell cycle regulation also differs from adult cells, according to the proliferative state. In addition, following Cd exposure, E-cadherin/beta-catenin complex disassembles in both cell types, with fetal cells being influenced at higher doses. The beta-catenin is not found in the nucleus, ruling out a direct role on DNA synthesis stimulation. Finally, metallothionein is more easily inducible in fetal hepatocytes, while Cd intracellular concentrations and HSP protein levels are not differentially affected. In conclusion, multiple cellular targets are affected by Cd in primary hepatocytes and the adverse effects of the metal are always better counteracted by fetal cells.

Cell-cycle protein modifications in the kidney of Mus spretus from Doñana National Park.

On April 1998 a tailing dam of the Aznalcollar pyrite mine partially collapsed and released to the Guadiamar river acidic water and mud containing toxic metals threatening the Doñana National Park, a Spanish wildlife reserve located near the estuary of Guadalquivir river. To assess the possible biological effects on terrestrial ecosystems, biochemical markers were assayed in the kidneys of Algerian mice (Mus spretus) collected in several areas of Doñana and Guadiamar river. Biomarkers assayed are proteins involved in cell cycle regulation, in particular cyclins and their associated kinases, and some cell cycle inhibitors. Moreover Mitogen Activated Protein Kinases (MAPK), a signal transduction system involved in cell division, p53, a protein involved in growth arrest after DNA damage, and HSP70, an early stress-induced protein, were assayed. The kidneys of animals collected one year after the ecological disaster had increased levels of PCNA (proliferating cell nuclear antigen), indicating an increased number of cells in the S phase of cell cycle. This shift of cells from G0 to S phase is due to increased levels of cyclins D1, E, and A, to decreased levels of p21 and p27 cdk inhibitors, and to activation of MAPK cascade. On the other hand, p53 and HSP70 levels are not changed. These data demonstrate that the presence of toxic metals after ecological disaster provoked the induction of kidney cell proliferation interpretable as a compensatory cell growth after tissue damage and apoptosis, and that could lead to the genomic instability characteristic of cancer cell.