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Thyme-like plants including Thymbra spicata L. are widely used as food and folk medicinal remedies in the Mediterranean area. This study aimed to explore the in vitro antitumor potential of polyphe...

Background and aims Several chronic multifactorial diseases originate from energy unbalance between food intake and body energy expenditure, including non-alcoholic fatty liver disease (NAFLD), diabetes, and cardiovascular disorders. Vascular endothelium plays a central role in body homeostasis, and NAFLD is often associated with endothelial dysfunction (ED), the first step in atherosclerosis. Both sugars and fatty acids (FAs) are fuel sources for energy production, but their excess leads to liver steatosis which may trigger ED through a network of mechanisms which need to be clarified. Here, we investigated the crosstalk pathways between in vitro cultured steatotic hepatocytes (FaO) and endothelial cells (HECV) being mediated by soluble factors. Methods and results We employed the conditioned medium approach to test how different extent and features of hepatic steatosis distinctively affect endothelium leading to ED. The steatogenic media collected from steatotic hepatocytes were characterized by high triglyceride content and led to lipid accumulation and fat-dependent dysfunction in HECV cells. We found a parallelism between (i) extent of hepatocyte steatosis and level of lipid accumulation in HECV cells; (ii) type of hepatocyte steatosis (with macro- or microvesicular LDs) and extent of oxidative stress, lipid peroxidation, nitric oxide release and expression of ED markers in HECV cells. Conclusions The present findings seem to suggest that, in addition to triglycerides, other soluble mediators should be released by steatotic hepatocytes and may influence lipid accumulation and function of HECV cells. Further studies need to depict the exact profile of soluble factors involved in steatotic hepatocyte-endothelium crosstalk.

Aims Adipocyte hypertrophy is the main cause of obesity. A deeper understanding of the molecular mechanisms regulating adipocyte dysfunction may help to plan strategies to treat/prevent obesity and its metabolic complications. Here, we investigated in vitro the molecular alterations associated with early adipocyte hypertrophy, focusing on mitochondrial dysfunction. Main methods As model of adipocyte hypertrophy, we employed 3T3-L1 preadipocytes firstly differentiated into mature adipocytes, then cultured with long-chain fatty acids. As a function of differentiation and hypertrophy, we assessed triglyceride content, lipid droplet size, radical homeostasis by spectrophotometry and microscopy, as well as the expression of PPARγ, adiponectin and metallothioneins. Mitochondrial status was investigated by electron microscopy, oxygraph 2 k (O2K) high-resolution respirometry, fluorimetry and western blot. Key findings Compared to mature adipocytes, hypertrophic adipocytes showed increased triglyceride accumulation and lipid peroxidation, larger or unique lipid droplet, up-regulated expression of PPARγ, adiponectin and metallothioneins. At mitochondrial level, early-hypertrophic adipocytes exhibited: (i) impaired mitochondrial oxygen consumption with parallel reduction in the mitochondrial complexes; (ii) no changes in citrate synthase and HSP60 expression, and in the inner mitochondrial membrane polarization; (iii) no stimulation of mitochondrial fatty acid oxidation. Our findings indicate that the content, integrity, and catabolic activity of mitochondria were rather unchanged in early hypertrophic adipocytes, while oxygen consumption and oxidant production were altered. Significance In the model of early adipocyte hypertrophy exacerbated oxidative stress and impaired mitochondrial respiration were observed, likely depending on reduction in the mitochondrial complexes, without changes in mitochondrial mass and integrity.

Thyme-like plants including

Phenolic compounds (PC) of virgin olive oil exert several biochemical and pharmacological beneficial effects. Some dietary PC seem to prevent/improve obesity and metabolic-related disorders such as non-alcoholic fatty liver disease (NAFLD). We investigated the possible effects of PC extracted from olive pomace (PEOP) and of the main single molecules present in the extract (tyrosol, apigenin, oleuropein, p-coumaric and caffeic acid) in protecting hepatocytes and endothelial cells against triglyceride accumulation and oxidative stress.Rat hepatoma and human endothelial cells were exposed to a mixture of oleate/palmitate to mimic the condition of NAFLD and atherosclerosis, respectively. Then, cells were incubated for 24 h in the absence or in the presence of PC or PEOP. Different parameters were evaluated, such as lipid accumulation and oxidative stress-related markers.In hepatic cells, expression of peroxisome proliferator-activated receptors (PPARs) and of stearoyl-CoA desaturase 1 (SCD-1) were assessed as index of lipid metabolism. In endothelial cells, expression of intercellular adhesion molecule-1 (ICAM-1), activation of nuclear factor kappa-B (NF-kB), release of nitric oxide (NO), and wound-healing rate were assessed as index of inflammation.PEOP extract ameliorated hepatic lipid accumulation and lipid-dependent oxidative imbalance thus showing potential applications as therapeutic agent tuning down hepatosteatosis and atherosclerosis.

Hepatic steatosis is the hallmark of non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome and insulin resistance with potential evolution towards non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma. Key roles of autophagy and oxidative stress in hepatic lipid accumulation and NAFLD progression are recognized. Here, we employed a rat hepatoma cell model of NAFLD progression made of FaO cells exposed to oleate/palmitate followed or not by TNFα treatment to investigate the molecular mechanisms through which silybin, a lipid-lowering nutraceutical, may improve hepatic lipid dyshomeostasis. The beneficial effect of silybin was found to involve amelioration of the fatty acids profile of lipid droplets, stimulation of the mitochondrial oxidation and upregulation of a microRNA of pivotal relevance in hepatic fat metabolism, miR-122. Silybin was also found to restore the levels of Aquaporin-9 (AQP9) and glycerol permeability while reducing the activation of the oxidative stress-dependent transcription factor NF-κB, and autophagy turnover. In conclusion, silybin was shown to have molecular effects on signaling pathways that were previously unknown and potentially protect the hepatocyte. These actions intersect TG metabolism, fat-induced autophagy and AQP9-mediated glycerol transport in hepatocytes.

S-adenosylmethionine (SAMe) is an endogenous methyl donor derived from ATP and methionine that has pleiotropic functions. Most SAMe is synthetized and consumed in the liver, where it acts as the main methylating agent and in protection against the free radical toxicity. Previous studies have shown that the administration of SAMe as a supernutrient exerted many beneficial effects in various tissues, mainly in the liver. In the present study, we aimed to clarify the direct effects of SAMe on fatty acid-induced steatosis and oxidative stress in hepatic and endothelial cells. Hepatoma FaO cells and endothelial HECV cells exposed to a mixture of oleate/palmitate are reliable models for hepatic steatosis and endothelium dysfunction, respectively. Our findings indicate that SAMe was able to significantly ameliorate lipid accumulation and oxidative stress in hepatic cells, mainly through promoting mitochondrial fatty acid entry for &beta, oxidation and external triglyceride release. SAMe also reverted both lipid accumulation and oxidant production (i.e., ROS and NO) in endothelial cells. In conclusion, these outcomes suggest promising beneficial applications of SAMe as a nutraceutical for metabolic disorders occurring in fatty liver and endothelium dysfunction.

Overconsumption of fats and sugars is a major cause of development of non‑alcoholic fatty liver disease (NAFLD). The main objectives of the present study were to explore the pathways sustaining the interfering metabolic effects of excess fructose and fatty acids in hepatocytes, and to clarify the mechanisms through which the nutraceutical silybin rescues the functional and metabolic alterations that are associated with the NALFD progression. Cultured hepatocytes were exposed to fructose and fatty acids, alone or in combination, to induce different grades of steatosis in vitro. Cell viability, apoptosis, free radical production, lipid content, lipid peroxidation and activity of lipogenic enzymes were assessed by spectrophotometric assays. Oxygen consumption and mitochondrial respiration parameters were measured using a Seahorse analyzer. Expression of markers for liver steatosis and dysfunction were also evaluated by reverse transcription‑quantitative polymerase chain reaction. The data revealed that fructose and fatty acid combination in vitro had a positive interference on lipogenic pathways, leading to more severe steatosis and liver dysfunction, reduced cell viability, increased apoptosis, oxidative stress and mitochondrial respiration. Hepatic cell abnormalities were almost completely alleviated by silybin treatment. These findings suggest that silybin may serve as a novel and cost‑effective dietary supplement for treatment and/or prevention of hepatosteatosis associated with NAFLD progression.

Quartz is a well-known occupational fibrogenic agent able to cause fibrosis and other severe pulmonary diseases such as silicosis and lung cancer. The silicotic pathology owes its severity to the structural and chemo-physical properties of the particles such as shape, size and abundance of surface radicals. In earlier studies, we reported that significant amounts of surface radicals can be generated on crystalline silica by chemical aggression with ascorbic acid (AA), a vitamin naturally abundant in the lung surfactant, and this reaction led to enhanced cytotoxicity and production of inflammatory mediators in a macrophage cell line. However in the lung, other cells acting in the development of silicosis, like fibroblasts and endothelial cells, can come to direct contact with inhaled quartz. We investigated the cytotoxic/pro-inflammatory effects of AA-treated quartz microcrystals (QA) in human primary fibroblasts and endothelial cells as compared to unmodified microcrystals (Q). Our results show that, in fibroblasts, the abundance of surface radicals on quartz microcrystals (Q vs QA) significantly enhanced cell proliferation (with or without co-culture with macrophages), reactive oxygen species (ROS) production, NF-κB nuclear translocation, smooth muscle actin, fibronectin, Bcl-2 and tissue inhibitor of metalloproteinase-1 expression and collagen production. Contrariwise, endothelial cells reacted to the presence of quartz microcrystals independently from the abundance of surface radicals showing similar levels of cytotoxicity, ROS production, cell migration, MCP-1, ICAM-I and fibronectin gene expression when challenged with Q or QA. In conclusion, our in vitro experimental model demonstrates an important and quite unexplored direct contribute of silica surface radicals to fibroblast proliferation and fibrogenic responses.

Ethnopharmacological relevance Thymbra spicata, a member of the Lamiaceae family, is native to eastern Mediterranean area. Leaves of this plant are rich in phenolic compounds and are a popular remedy of traditional medicine in Lebanon to prevent and/or counteract hyperlipidemia and hyperglycemia. Aim of the study To evaluate the antisteatotic and antioxidant activities of extracts from leaves of Thymbra spicata L. using in vitro models of non-alcoholic fatty liver disease (NAFLD), a leading cause of liver-related morbidity and mortality worldwide, for whom no effective treatments are still available. Materials and methods Two different extracts from Thymbra spicata L. aerial parts were prepared using water (TW) or ethanol (TE) as solvent. Their chemical composition was characterized by gas and liquid chromatography coupled with mass spectrometry. Both extracts were tested on cultured hepatic and endothelial cells treated to mimic in vitro a multisistemic pathology such as NAFLD. We assayed the effects on lipid accumulation, free radical production, lipid peroxidation, cell migration. Results Both the total phenolic and the total flavonoid contents were higher in the ethanolic extract. Rosmarinic acid was the most abundant polyphenol in TW, while TE was richer in carvacrol. Our findings demonstrated that both extracts ameliorated lipid accumulation, oxidative stress and inflammation in the NAFLD cellular models. However, the aqueous extract was more effective to reduce hepatic steatosis, and the ethanolic extract had higheranti-oxidant potential and wound healing activity. Conclusions T. spicata extracts could be promising bioactive products to develop natural therapeutic agents or dietary supplements to treat NAFLD and obesity-related metabolic disease. Our findings suggest that while the ethanolic extract might be used in preventing endothelium dysfunction, the aqueous extract would act better as lipid-lowering agent.

It is a common notion that thyroid status is linked to energy expenditure and body weight, and thyroid hormones (THs) have always deserved attention for their potential use as pharmacological agents to counteract obesity and lipid metabolism-related disorders. Besides 3,5,3′,5′-tetraiodo- l -thyronine (thyroxine or T4) and 3,3′,5,-triiodo- l -thyronine (T3), other iodothyronines can be produced in the thyroid or in peripheral tissues through the process of stepwise deiodination. Among these TH metabolites, 3,5-diodo- l -thyronine (T2) is of particular interest for its effects on energy metabolism. After a brief overview of TH production and actions at the cellular level, this chapter will focus on the mechanisms underlying the lipid-lowering effects of the iodothyronines T3 and T2, with particular regard to the regulation of lipid metabolism in the liver.

Non Alcoholic Fatty Liver Disease (NAFLD) is characterised by fat accumulation in hepatocytes in the form of triacyglycerols (TAGs) within cytosolic lipid droplets. Fucoidans (FUs) are biologically active polysaccharides usually isolated from brown marine algae, but recently identified also in terrestrial plants. In this study, we aimed to investigate the anti-oxidant and anti-steatotic effects of FUs purified from C. compressa, F. hermonis, and E. globulus. To this aim, we used a validated NAFLD in vitro model consisting of rat hepatoma FaO cells exposed to an oleate/palmitate mixture. Such a model is suitable for rapid investigation of direct effects of natural and artificial compounds, together with satisfying the strategy of 3Rs for laboratory use of animals. Our results indicated that all FUs display anti-oxidant and anti-steatotic activities. Steatotic FaO cells may be employed to further study the biological effects of FUs.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease often associated with overnutrition. Number and morphometry of lipid droplets (LDs) define micro vs macrovesicular steatosis, influence the morphology and function of hepatocytes and possibly their stiffness. The link between grade and features of steatosis and biomechanical properties of single hepatocytes requires deeper investigations. In vitro NAFLD models with distinct steatosis conditions were set by exposing FaO hepatoma cells to single or combined fructose (Fru), fatty acids (FA), and tumor necrosis factor (TNF)α. Single Cell Force Spectroscopy and Quantitative Phase Microscopy quantified the single cell stiffness and a series of morphometric parameters; the mRNA expression of genes involved in lipid metabolism was quantified by real-time PCR. In our models, LD size and number increased with Fru and FA as single agents, and more with combined Fru/FA (macrovesicular steatosis), while FA/TNFα combination increased LD number with a reduction in their size (microvesicular steatosis). We found that the changes in LD size and number influenced cell stiffness and morphometry as follows: (i) single cell elasticity increased in macrovesicular steatosis (maximally with combined Fru/FA); (ii) FA-induced steatosis resulted in cells thinner and larger, whereas combined FA/TNFα shrunk the hepatocytes. Taken together the data on hepatocyte biomechanics show that, in addition to extent of lipid accumulation, cell stiffness is mainly influenced by LD size, while cell morphometry directly relates to LD number. Our findings suggest that a novel mechanobiology perspective might provide future contributions in NAFLD research.

Transgenic mice overexpressing spermine oxidase (SMO) in the cerebral cortex (Dach-SMO mice) showed increased vulnerability to excitotoxic brain injury and kainate-induced epileptic seizures. To investigate the mechanisms by which SMO overexpression leads to increased susceptibility to kainate excitotoxicity and seizure, in the cerebral cortex of Dach-SMO and control mice we assessed markers for astrocyte proliferation and neuron loss, and the ability of kainate to evoke glutamate release from nerve terminals and astrocyte processes. Moreover, we assessed a possible role of astrocytes in an in vitro model of epileptic-like activity in combined cortico-hippocampal slices recorded with a multi-electrode array device. In parallel, as the brain is a major metabolizer of oxygen and yet has relatively feeble protective antioxidant mechanisms, we analyzed the oxidative status of the cerebral cortex of both SMO-overexpressing and control mice by evaluating enzymatic and non-enzymatic scavengers such as metallothioneins. The main findings in the cerebral cortex of Dach-SMO mice as compared to controls are the following: astrocyte activation and neuron loss; increased oxidative stress and activation of defense mechanisms involving both neurons and astrocytes; increased susceptibility to kainate-evoked cortical epileptogenic activity, dependent on astrocyte function; appearance of a glutamate-releasing response to kainate from astrocyte processes due to activation of Ca(2+)-permeable AMPA receptors in Dach-SMO mice. We conclude that reactive astrocytosis and activation of glutamate release from astrocyte processes might contribute, together with increased reactive oxygen species production, to the vulnerability to kainate excitotoxicity in Dach-SMO mice. This mouse model with a deregulated polyamine metabolism would shed light on roles for astrocytes in increasing vulnerability to excitotoxic neuron injury.

Objectives: Oxidative stress seems to be involved in Rett syndrome (RTT). The aim of this study was to assess the antioxidant status in RTT children with MECP2 gene mutations with respect to healthy controls, and to explore novel blood antioxidant markers for RTT severity. Methods: In erythrocytes from RTT females aged 2–14 years (n = 27) and age-matched controls (n = 27), we measured the levels of malonaldehyde and the activity of two antioxidant enzymes, Cu/Zn-superoxide dismutase and catalase, by spectrophotometric assays. In leukocytes, the expression of metallothioneins, the main non-enzymatic antioxidants, was assessed by real-time RT-PCR. In nine selected RTT children, methylome analysis was also performed. Results: Blood of RTT patients showed increased lipid peroxidation and a dysregulated pattern of MT expression, while enzymatic activities did not change significantly with respect to controls. Moreover, we observed no epigenetic dysregulation in CpG-enriched promoter regions of the analysed genes but significant hypomethylation in the random loci. Conclusions: As the haematic level of MT-1A directly correlates with the phenotype severity, this metallothionein can represent a marker for RTT severity. Moreover, the attempt to link the level of blood oxidative stress with MECP2 mutation and specific clinical features led us to draw some interesting conclusions.

Background Oxidative stress is implicated in pathogenesis of alcoholic liver disease (ALD). This study investigated the possible correlation among the erythrocyte indices of oxidative stress, the leukocyte panels of antioxidant proteins (metallothioneins), the serum biochemical parameters and the liver steatosis grade. Methods A total of 118 cases including 60 alcoholic subjects and 58 controls were enrolled. All the alcoholic subjects were screened for body mass index (BMI), liver steatosis, and blood chemistry and serology. The level of oxidative stress and oxidative stress-related parameters were measured in the blood and correlated with clinical findings. Results Alcoholic subjects showed higher BMI, moderate/severe hepatic steatosis, increase in the levels of triglycerides, cholesterol, glucose, γ-glutamyl-transpeptidase (GGT), alanine aminotransferase (ALT), bilirubin, alpha 1 and beta 2 globulins, iron and a decrease in the levels of aspartate aminotransferase (AST) and beta 1 globulin with respect to the reference values. Moreover, alcoholic subjects showed: (i) an increase in Thiobarbituric Acid Reactive Substance (TBARS) content representing a good estimation of global oxidative stress; (ii) a stimulation of the activities of the antioxidant enzymes catalase and SOD; (iii) a modulation of expression of metallothioneins, with a down-regulation of MT-1A and an up-regulation of MT-1E isoforms. Conclusions Our data suggest that alcoholism is strongly associated with altered pattern of blood metallothioneins; this parameter combined with the score calculated by an ad hoc implemented algorithm (HePaTest) could offer a non-invasive alternative approach for evaluating alcohol-related damages and could be used in follow-up of alcoholic patients.

In vertebrate systems, many endocrine disruptors (EDs) can also interfere with energy and lipid metabolism, thus acting as metabolic disruptors. At the cellular level, these effects are mainly mediated by interactions with nuclear receptors/transcription factors, leading to the modulation of genes involved in lipid homeostasis, as well as by rapid, receptor-independent pathways. Several potential metabolic disruptors are found in aquatic environments. In fish, different EDs have been shown to affect hepatic lipid homeostasis both in vivo and in vitro. However, little information is available in aquatic invertebrates due to our poor knowledge of the regulatory pathways of lipid metabolism. In this work, primary cell cultures from the digestive gland of the bivalve Mytilus galloprovincialis were utilized to investigate the effects of model EDs (bisphenol A (BPA) and perfluorooctane sulphonate (PFOS)) on lipid homeostasis. Both compounds (at 24 and 3h of exposure) increased intracellular lipid and tryglyceride-TAG content, with strongest effects of PFOS at 10-7M. Acyl-CoA oxidase activity was unaffected, whereas some changes in the activity of glycolytic, antioxidant/biotransformation enzymes were observed; however, no clear relationship was found with lipid accumulation. Evaluation of mitochondrial membrane potential Δψm and determination of extracellular TAG content indicate that PFOS interferes with mitochondrial function and lipid secretion, whereas BPA mainly affects lipid secretion. Experiments with specific inhibitors showed that activation of PI-3 kinase and extracellularly regulated mitogen-activated protein kinase (ERK MAPK) plays a key role in mediating lipid accumulation. Mussel digestive gland cells represent a simple in vitro model for screening the metabolic effects of EDs in marine invertebrates.

AIM: To investigate in vitro the therapeutic effect and mechanisms of silybin in a cellular model of hepatic steatosis. METHODS: Rat hepatoma FaO cells were loaded with lipids by exposure to 0.75 mmol/L oleate/palmitate for 3 h to mimic liver steatosis. Then, the steatotic cells were incubated for 24 h with different concentrations (25 to 100 μmol/L) of silybin as phytosome complex with vitamin E. The effects of silybin on lipid accumulation and metabolism, and on indices of oxidative stress were evaluated by absorption and fluorescence microscopy, quantitative real-time PCR, Western blot, spectrophotometric and fluorimetric assays. RESULTS: Lipid-loading resulted in intracellular triglyceride (TG) accumulation inside lipid droplets, whose number and size increased. TG accumulation was mediated by increased levels of peroxisome proliferator-activated receptors (PPARs) and sterol regulatory element-binding protein-1c (SREBP-1c). The lipid imbalance was associated with higher production of reactive oxygen species (ROS) resulting in increased lipid peroxidation, stimulation of catalase activity and activation of nuclear factor kappa-B (NF-κB). Incubation of steatotic cells with silybin 50 μmol/L significantly reduced TG accumulation likely by promoting lipid catabolism and by inhibiting lipogenic pathways, as suggested by the changes in carnitine palmitoyltransferase 1 (CPT-1), PPAR and SREBP-1c levels. The reduction in fat accumulation exerted by silybin in the steatotic cells was associated with the improvement of the oxidative imbalance caused by lipid excess as demonstrated by the reduction in ROS content, lipid peroxidation, catalase activity and NF-κB activation. CONCLUSION: We demonstrated the direct anti-steatotic and anti-oxidant effects of silybin in steatotic cells, thus elucidating at a cellular level the encouraging results demonstrated in clinical and animal studies.

Despite some advances in the understanding of amyotrophic lateral sclerosis (ALS) pathogenesis, significant achievements in treating this disease are still lacking. Mesenchymal stromal (stem) cells (MSCs) have been shown to be effective in several models of neurological disease. To determine the effects of the intravenous injection of MSCs in an ALS mouse model during the symptomatic stage of disease, MSCs (1 × 10⁶) were intravenously injected in mice expressing human superoxide dismutase 1 (SOD1) carrying the G93A mutation (SOD1/G93A) presenting with experimental ALS. Survival, motor abilities, histology, oxidative stress markers and [³H]D-aspartate release in the spinal cord were investigated. MSC injection in SOD1/G93A mice improved survival and motor functions compared with saline-injected controls. Injected MSCs scantly home to the central nervous system and poorly engraft. We observed a reduced accumulation of ubiquitin agglomerates and of activated astrocytes and microglia in the spinal cord of MSC-treated SOD1/G93A mice, with no changes in the number of choline acetyltransferase- and glutamate transporter type 1-positive cells. MSC administration turned around the upregulation of metallothionein mRNA expression and of the activity of the antioxidant enzyme glutathione S-transferase, both associated with disease progression. Last, we observed that MSCs reverted both spontaneous and stimulus-evoked neuronal release of [³H]D-aspartate, a marker of endogenous glutamate, which is upregulated in SOD1/G93A mice. These findings suggest that intravenous administration of MSCs significantly improves the clinical outcome and pathological scores of mutant SOD1/G93A mice, thus providing the rationale for their exploitation for the treatment of ALS.

Experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis, is characterized by demyelination, inflammation and neurodegeneration of CNS in which free radicals play a role. Recently, the efficacy of murine mesenchimal stem cells (MSCs) as treatment of EAE induced in mice by the encephalitogenic peptide MOG(35-55) was demonstrated. The present study analyzed some markers of oxidative stress, inflammation/degeneration and apoptosis such as metallothioneins (MTs), antioxidant enzymes (superoxide dismutase, catalase and glutathione-S-transferase), poly(ADP-ribose) polymerase-1 and p53 during EAE progression and following MSC treatment. Expression of the three brain MT isoforms increased significantly in EAE mice compared with healthy controls, but while expression of MT-1 and MT-3 increased along EAE course, MT-2 was up-regulated at the onset, but returned to levels similar to those of controls in chronic phase. The changes in the transcription and activity of the antioxidant enzymes and in expression of poly(ADP-ribose) polymerase-1 and p53 showed the same kinetics observed for MT-1 and MT-3 during EAE. Interestingly, i.v. administration of MSCs reduced the EAE-induced increases in levels/activities of all these proteins. These results support an antioxidant and neuroprotective activity for MSCs that was also confirmed in vitro on neuroblastoma cells exposed to an oxidative insult.

This study is focused on the role of UCP-2 in hepatic oxidative metabolism following acute CCl(4) administration to rats. UCP-2 mRNA, almost undetectable in the liver of controls, was significantly increased 24 h after CCl(4) administration, peaked at 72 h and then tended to disappear. UCP-2 protein, undetectable in controls, increased 48-72 h after CCl(4) treatment. Experiments with isolated liver cells indicated that in control rats UCP-2 was expressed in non-parenchymal cells and not in hepatocytes, whereas in CCl(4)-treated rats UCP-2 expression was induced in hepatocytes and was not affected in non-parenchymal cells. Addition of CCl(4) to the culture medium of hepatocytes from control rats failed to induce UCP-2 expression. Liver mitochondria from CCl(4)-treated rats showed an increase of H(2)O(2) release at 12-24 h, followed by a rise of TBARS. Vitamin E protected liver from CCl(4) injury and reduced the expression of UCP-2. Treatment with GdCl(3) prior to CCl(4), in order to inhibit Kupffer cells, reduced TBARS and UCP-2 mRNA increase in hepatic mitochondria. Our data indicate that CCl(4) induces the expression of UCP-2 in hepatocytes with a redox-dependent mechanism involving Kupffer cells. A role of UCP-2 in moderating CCl(4)-induced oxidative stress during tissue regeneration after injury is suggested.

Excess ethanol consumption and fatty acid intake lead to a cumulative effect on liver steatosis through still unclear mechanisms. This study aimed to characterize the lipid homoeostasis alterations under the exposure of hepatocytes to ethanol alone or combined with fatty acids. FaO hepatoma cells were incubated in the absence (C) or in the presence of 100 mM ethanol (EtOH) or 0.35 mM oleate/palmitate (FFA) alone or in the combination (FFA/EtOH). Content of intra- and extra-cellular triglycerides (TAGs) and of lipid droplets (LDs), expression of lipogenic and lipolytic genes, and oxidative stress-related parameters were evaluated. Exposure to either FFAs or EtOH given separately led to steatosis which was augmented when they were combined. Our results show that FFA/EtOH: (i) increased the LD number, but reduced their size compared to separate treatments; (ii) up-regulated PPARγ and SREBP-1c and down-regulated sirtuin-1 (SIRT1); (iii) impaired FFA oxidation; (iv) did not change lipid secretion and oxidative stress. Our findings indicate that one of the major mechanisms of the metabolic interference between ethanol and fat excess is the impairment of FFA oxidation, in addition to lipogenic pathway stimulation. Interestingly, ethanol combined with FFAs led to a shift from macrovesicular to microvesicular steatosis that represents a more dangerous condition.

Introduction Systemic delivery of bone marrow-derived mesenchymal stem cells (MSC) seems to be of benefit in the treatment of multiple sclerosis (MS), an autoimmune disease of the central nervous system (CNS) sustained by migration of T cells across the brain blood barrier (BBB) and subsequent induction of inflammatory lesions into CNS. MSC have been found to modulate several effector functions of T cells. In this study, we investigated the effects of MSC on adhesion molecules and receptors on T cell surface that sustain their transendothelial migration. Methods We used different co-culture methods combined with real-time PCR and flow cytometry to evaluate the expression both at the mRNA and at the plasma-membrane level of α4 integrin, β2 integrin, ICAM-1 and CXCR3. In parallel, we assessed if MSC are able to modulate expression of adhesion molecules on the endothelial cells that interact with T cells during their transendothelial migration. Results Our in vitro analyses revealed that MSC: (i) inhibit proliferation and activation of both peripheral blood mononuclear cells (PBMC) and CD3+-selected lymphocytes through the release of soluble factors; (ii) exert suppressive effects on those surface molecules highly expressed by activated lymphocytes and involved in transendothelial migration; (iii) inhibit CXCL10-driven chemotaxis of CD3+ cells; (iv) down-regulated expression of adhesion molecules on endothelial cells. Conclusions Taken together, these data demonstrate that the immunosuppressive effect of MSC does not exclusively depends on their anti-proliferative activity on T cells, but also on the impairment of leukocyte migratory potential through the inhibition of the adhesion molecules and receptors that are responsible for T cell trafficking across BBB. This could suggest a new mechanism through which MSC modulate T cell responses.

Thyroid hormones play a crucial role in new neuron production and maturation during brain development. However, the knowledge about the involvement of these hormones on adult neurogenesis is still incomplete. Hippocampus is an anatomical region where neurogenesis occurs throughout adulthood and where high levels of thyroid hormone receptors have been found. In this work the possible involvement of thyroid hormones in the regulation of adult neurogenesis in the granule cell layer of rat hippocampus dentate gyrus was investigated using an experimental model of adult-onset pharmacologically-induced hypothyroidism. Neurogenesis was assessed by means of the thymidine analogue 5-bromo-2′-deoxyuridine 24 h and 30 days after its last administration in order to study neural precursor proliferation and newborn cell survival, respectively. Mitotic activity of the neural precursors was not affected by thyroid hormone deficiency; on the contrary, newborn cell survival dramatically decreased under these conditions when compared with controls, leading to a lower number of immature neurons being added to the granule cell layer. Moreover, in conditions of hypothyroidism, new neurons exhibit a delay in neuronal differentiation showing a prolonged expression of the neuritogenesis-associated immature neuron marker TUC-4 and a very immature morphology. Finally, the total number and size of granule cells, and granule cell layer volume decreased in hypothyroid rats. These results suggest that thyroid hormones play a role in regulating new neuron production during adult life in dentate gyrus of rat hippocampus.

Tetrabromobisphenol A (2,2-bis(3,5-dibromo-4-hydroxyphenyl propane-TBBPA) is the most produced brominated flame retardant, detected in the environment and in biological samples. TBBPA shares structural similarities with thyroid hormones (THs), and it has been shown to interfere with different aspects of TH physiology, this raising concern on its possible effects as an endocrine disruptor in humans and wildlife. THs play a major role in lipid metabolism, with the liver representing one of their main target tissues. At the cellular level, THs act through interactions with TH receptors (TRs), as well as through TR-independent mechanisms. Rat hepatoma FaO cells (a liver cell line defective for functional TRs) overloaded with lipids have been utilized as a model to investigate the anti-steatotic effects of THs in the hepatocyte. In this work, the possible effects of TBBPA in steatotic FaO cells were investigated. Exposure to TBBPA for 24 h reduced triglyceride (TAG) content and the size of lipid droplets (LDs); similar effects were obtained with equimolar doses (10(-6) M) of T3 (3,3',5-L-triiodothyronine). TBBPA and T3 showed common effects on transcription of genes involved in lipid homeostasis. In particular, TBBPA mainly up-regulated mRNA levels for LD-associated oxidative tissue-enriched PAT protein (OXPAT), peroxisome proliferator-activated receptor (PPAR) isoform β/δ, and the mitochondrial uncoupling protein 2 (UCP2). The results demonstrate that TBBPA can decrease lipid accumulation in steatotic cells through stimulation of oxidative pathways. These data identify novel thyromimetic actions of TBBPA at the cellular level.

OBJECTIVE: The levels of uncoupling protein 2 (UCP2) mRNA and determinants of respiration (ATP synthesis, proton leak and non-mitochondrial respiration) were evaluated in Kupffer cells isolated from the livers of normal euthyroid, acute hyperthyroid and chronic hyperthyroid rats. METHODS: After liver perfusion, Kupffer cells were purified by density-gradient centrifugation followed by counterflow centrifugal elutriation. UCP2 mRNA levels were measured by Northern blot and respiratory parameters by polarographic method. RESULTS: In cells isolated from hyperthyroid (tri-iodothyronine (T(3))-treated) rats, the effect of T(3) treatment on the UCP2 mRNA level varied: it was more than doubled (P

The insulin-like growth factors (IGFs) and insulin-like growth factor binding proteins (IGFBPs), which regulate IGF activity, play a fundamental role in renal cell proliferation and differentiation. The thyroid hormone is considered to be required for kidney development; excess induces local hypertrophy and hyperplasia. The aim of the present study was to investigate the possible involvement of the IGF/IGFBP system in thyroid hormone-induced renal growth during the development of the rat. Our results show that thyroid hormone withdrawal by 6-propyl-2-thiouracil (PTU)-treatment of rats at all ages had no effect on renal IGFBP-4 mRNA levels, whereas the abundance of the serum protein was decreased compared to controls. Intraperitoneal triiodothyronine (T3) administration to hypothyroid rats resulted in renal hypertrophy associated with a significant upregulation of IGFBP-4 expression with increased levels of renal IGFBP-4 mRNA and serum protein. T3-induced upregulation of IGFBP-4 expression suggests the involvement of the local IGF/IGFBP system in T3-induced renal hypertrophy.

Previously, we have observed that epidermal growth factor (EGF), a potent mitogen for cultured hepatocytes, stimulates the production of IGF-I and IGF-binding proteins (IGFBPs) by cultured hepatocytes from adult rats. This study was undertaken to investigate the possibility that other growth factors of hepatic origin could specifically be involved in the regulation of IGF-I and IGFBP expression. The effects of transforming growth factor-alpha (TGF-alpha), through EGF receptors to induce a mitogenic response, and transforming growth factor-beta1 (TGF-beta1), produced by non-parenchymal liver cells and able to inhibit hepatocyte proliferation in vivo and in culture, have been studied in cultured adult rat hepatocytes. Our results demonstrate that TGF-alpha and TGF-beta1 significantly stimulate IGF-I and IGFBP secretion by cultured hepatocytes but no change in the abundance of IGF-I and IGFBP mRNAs was observed with respect to controls. Cycloheximide is able to inhibit both basal and TGF-stimulated release of IGF-I and a similar effect was elicited by octreotide, the somatostatin analog, known to directly affect hepatic IGF-I gene expression. Our findings show the role of the liver in the secretion of IGF-I and IGFBPs, not only under endocrine and nutritional control but also under autocrine and paracrine control.

Excess fat accumulation renders the liver more vulnerable to ethanol, but it is still unclear how alcohol enhances lipid dysmetabolism and oxidative stress in a pre-existing steatosis condition. The effects produced by binge ethanol consumption in the liver of male Wistar rats fed a standard (Ctrl) or a high-fat diet HFD were compared. The liver status was checked through tissue histology and standard serum parameters. Alteration of hepatic lipid homeostasis and consequent oxidative unbalance were assessed by quantifying the mRNA expression of the lipid-regulated peroxisome proliferator-activated receptors (PPARs), of the cytochromes CYP2E1 and CYP4A1, and of some antioxidant molecules such as the metallothionein isoforms MT1 and MT2 and the enzymes catalase and superoxide dismutase. The number of adipose differentiation-related protein (ADRP)-positive lipid droplets (LDs) was evaluated by immunohistochemical staining. As a response to the double insult of diet and ethanol the rat liver showed: (1) a larger increase in fat accumulation within ADRP-positive LDs; (2) stimulation of lipid oxidation in the attempt to limit excess fat accumulation; (3) induction of antioxidant proteins (MT2, in particular) to protect the liver from the ethanol-induced overproduction of oxygen radicals. The data indicate an increased susceptibility of fatty liver to ethanol and suggest that the synergistic effect of diet and ethanol on lipid dysmetabolism might be mediated, at least in part, by PPARs and cytochromes CYP4A1 and CYP2E1.

Amyotrophic lateral sclerosis (ALS) is a late-onset fatal neurodegenerative disease reflecting degeneration of upper and lower motoneurons (MNs). The cause of ALS and the mechanisms of neuronal death are still largely obscure, thus impairing the establishment of efficacious therapies. Glutamate (Glu)-mediated excitotoxicity plays a major role in MN degeneration in ALS. We recently demonstrated that the activation of Group I metabotropic Glu autoreceptors, belonging to both type 1 and type 5 receptors (mGluR1 and mGluR5), at glutamatergic spinal cord nerve terminals, produces excessive Glu release in mice over-expressing human superoxide-dismutase carrying the G93A point mutation (SOD1(G93A)), a widely used animal model of human ALS. To establish whether these receptors are implicated in ALS, we generated mice expressing half dosage of mGluR1 in the SOD1(G93A) background (SOD1(G93A)Grm1(crv4/+)), by crossing the SOD1(G93A) mutant mouse with the Grm1(crv4/+) mouse, lacking mGluR1 because of a spontaneous recessive mutation. SOD1(G93A)Grm1(crv4/+) mice showed prolonged survival probability, delayed pathology onset, slower disease progression and improved motor performances compared to SOD1(G93A) mice. These effects were associated to reduction of mGluR5 expression, enhanced number of MNs, decreased astrocyte and microglia activation, normalization of metallothionein and catalase mRNA expression, reduced mitochondrial damage, and decrease of abnormal Glu release in spinal cord of SOD1(G93A)Grm1(crv4/+)compared to SOD1(G93A) mice. These results demonstrate that a lower constitutive level of mGluR1 has a significant positive impact on mice with experimental ALS, thus providing the rationale for future pharmacological approaches to ALS by selectively blocking Group I metabotropic Glu receptors.

Background/Aims: Fatty acids are the main energy stores and the major membrane components of the cells. In the hepatocyte, fatty acids are esterified to triacylglycerols (TAGs) and stored in lipid droplets (LDs). The lipid lowering action of 3,5-diiodo-L-thyronine (T 2 ) on an in vitro model of hepatosteatosis was investigated in terms of fatty acid and protein content of LDs, lipid oxidation and secretion. Methods: FaO cells were exposed to oleate/ palmitate, then treated with T 2 . Results: T 2 reduced number and size of LDs, and modified their acyl composition by decreasing the content of saturated (SFA) vs monounsaturated (MUFA) fatty acids thus reversing the SFA/MUFA ratio. The expression of the LD-associated proteins adipose differentiation-related protein (ADRP), oxidative tissue-enriched PAT protein (OXPAT), and adipose triglyceride lipase (ATGL) was increased in ‘steatotic’ cells and further up-regulated by T 2 . Moreover, T 2 stimulated the mitochondrial oxidation by up-regulating carnitine-palmitoyl-transferase (CPT1), uncoupling protein 2 (UCP2) and very long-chain acylcoenzyme A dehydrogenase (VLCAD). Conclusions: T 2 leads to mobilization of TAGs from LDs and stimulates mitochondrial oxidative metabolism of fatty acids, in particular of SFAs, and thus enriches of MUFAs the LDs. This action may protect the hepatocyte from excess of SFAs that are more toxic than MUFAs.

OBJECTIVE: In normal subjects, the major form of circulating IGF is the GH-dependent 150 kDa complex. The liver appears to be the main source of the three components of the 150 kDa complex and, in particular, hepatocytes synthesize the insulin-like growth factor (IGF) peptide and the acid-labile subunit (ALS), whereas Kupffer and sinusoidal endothelial cells produce IGF-binding protein-3 (IGFBG-3). We have studied the effects of the somatostatin analog octreotide, IGF-II des(1-3)IGF-I, transforming growth factor (TGF)-beta 1 and tri-iodothyronine (T3) on ALS secretion into the medium conditioned by rat hepatocytes in primary culture. METHODS: The regulation of ALS release was evaluated in the conditioned medium of adult rat hepatocytes exposed to increasing concentrations of test substances or to vehicle alone (control), after gel filtration in basic conditions, by immunoblot using an antiserum generated against the N-terminal 34 amino acids of human ALS. RESULTS: The results demonstrate that: 1) octreotide in vitro produces a dose-dependent inhibition of both basal and GH-stimulated ALS secretion into the hepatocyte conditioned medium; 2) the release of ALS by adult rat hepatocytes is not affected by the presence during the incubation of des(1-3)IGF-I or IGF-II; 3) an inhibitory effect, although only with very high doses, can be observed after treatment with TGF-beta 1; and 4) a small but significant increase of ALS released into the medium can be seen when hepatocytes are treated with T3. CONCLUSIONS: Evaluation of the effect of substances known to affect the production of IGF peptides, the IGFBPs, or both, on adult rat hepatocytes in primary culture revealed no powerful stimulator, but instead a potent inhibitor of ALS release/synthesis. Our data suggest that the effect of somatostatin on the 150 kDa complex is mediated not only by the reduction in GH concentration, but also by a direct inhibition of ALS release or synthesis.

In normal subjects, the major form of circulating IGF is the GH-dependent 150 K complex. As demonstrated by gel-permeation chromatography, the acid-labile subunit (ALS) purified from human serum, incubated for 2 h at 20°C with [125I]IGF-I and rIGFBP-3, is able to increase not only the molecular weight (mol. wt.) of the IGF-IGFBP-3 complex, but also the amount of IGF-I bound. In both charcoal and polyethylene glycol ligand binding assays, competitive binding curves for the displacement of [125I]IGF-I from rIGFBP-3 by increasing concentrations of unlabeled IGF-I showed an increased binding activity of rIGFBP-3 in the presence of ALS. The effect of ALS on rIGFBP-3 binding activity was dose dependent. In addition, ligand and immunoblot revealed that ALS and rIGFBP-3 are able to form a high mol. wt. complex in the absence of IGF peptide. On the basis of these data, ALS seems to have a more complex function than that of simply increasing the mol. wt of the IGF-IGFBP-3 complex. The regulation of ALS synthesis by rat hepatocytes in primary culture has also been evaluated by immunoblot. In agreement with the in vivo finding of an inhibitory effect of octreotide (a somatostatin analog) on the formation of the 150 K complex in acromegalic subjects, we could observed in vitro that octreotide produces a dose-dependent inhibition of ALS secretion into the hepatocyte conditioned medium. TGF-β1 was also inhibitory at high doses. On the contrary, we could not evidence any effect of IGF-I or IGF-II, while a small increase has been noted after incubation with T3.

studies. It has been demonstrated that hepatocytes cultured in serum-free medium are in quiescent state but can enter S phase when cultured in the presence of growth factors such as epidermal growth factor, the powerful mitogen produced mainly in salivary glands. EGF acts via a 170 Kd membrane receptor glycoprotein wich has intrinsic tyrosine kinase activity. Tyrosine kinase-mediated autophosphorylat ion is considered a critical event in EGF signal transduction. However, the mechanism of EGF action as well as the events associated with EGF-stimulated DNA synthesis in cultured hepatocytes have not been completely elucidated. We have emp]oyed hepatocytes isolated from adult rats and cultured in the presence of EGF to study the time-course of events possibly associated with DNA synthesis such as the expression of the protooncogene c-myc, the expression of mRNA for IGFI, the growth factor produced by adult rat liver, the activity of DNA topoisomerase I (Topo I) and poly ADP-ribose polymerase (pADPRP), the enzymes involved in the regulation of DNA replication and chromatin functional activity, respectively.

Bisphenol A (BPA), used in the manufacture of polycarbonate plastic and epoxy resin, is one of the most abundant endocrine disruptors in the environment, considered as a xenoestrogen. BPA has recently become of additional public health concern because of increasing evidence of deleterious effects on metabolism. Dietary intake seems the most important route for BPA exposure, followed by rapid biotransformation in the gut and liver and elimination in the urine. Although hepatocytes can represent a significant target for this compound, little is known on the direct effects and mechanisms of action of BPA on lipid homeostasis at the cellular level. In this work, the effects of BPA (0.3–3–30–300 ng mL −1 , 24 h) were investigated in rat FaO hepatoma, a well differentiated liver cell line. At both 30 and 300 ng mL −1 , BPA significantly increased intracellular triglyceride (TAG) content and lipid accumulation in lipid droplets (LDs), without affecting cell viability. The effects of BPA were associated with decreased mRNA levels of the transcription factors Peroxisome Proliferator-Activated Receptor (PPAR) isoforms α and βδ, as well as of their downstream genes acyl-CoA oxidase (AOX) and carnitine palmitoyl transferase (CPT1) involved in lipid oxidation. No increase in transcription of lipogenic genes was observed. BPA also decreased mRNA levels of ApolipoproteinB (apoB) and the extracellular TAG content, indicating alterations in lipid secretion. FaO cells did not express Estrogen Receptor α (ERα and showed a very low expression of ERβ compared to rat liver. All the effects of BPA were prevented by cell pretreatment with Wortmannin, indicating the involvement of phosphatidyl inositol-3 kinase activation. The results demonstrate a direct action of BPA on lipid homeostasis in FaO cells through interference with lipid oxidation and secretion, and add further information on the cellular pathways that can be perturbed by this compound.

Iodothyronines influence lipid metabolism and energy homeostasis. Previous studies demonstrated that 3,5-l-diiodothyronine (T2), as well as 3,3′,5-l-triiodothyronine (T3), was able to both prevent and reverse hepatic steatosis in rats fed a high-fat diet, and this effect depends on a direct action of iodothyronines on the hepatocyte. However, the involvement of thyroid hormone receptors (TRs) in mediating the lipid-lowering effect of iodothyronines was not elucidated. In this study, we investigated the ability of T2 and T3 to reduce the lipid overloading using the rat hepatoma FaO cells defective for functional TRs. The absence of constitutive mRNA expression of both TRα1 and TRβ1 in FaO cells was verified by RT-qPCR. To mimic the fatty liver condition, FaO cells were treated with a fatty acid mixture and then exposed to pharmacological doses of T2 or T3 for 24 h. Lipid accumulation, mRNA expression of the peroxisome proliferator-activated receptors (PPAR-α, -γ, -δ) the acyl-CoA oxidase (AOX), and the stearoyl CoA desaturase (SCD1), as well as fuel-stimulated O2 consumption in intact cells, were evaluated. Lipid accumulation was associated with an increase in triacylglycerol content, PPARγ mRNA expression, and a decrease in PPARδ and SCD1 mRNA expression. The addition of T2 or T3 to lipid-overloaded cells resulted in i) reduction in lipid content; ii) downregulation of PPARα, PPARγ, and AOX expression; iii) increase in PPARδ expression; and iv) stimulation of mitochondrial uncoupling. These data demonstrate, for the first time, that in the hepatocyte, the lipid-lowering actions of both T2 and T3 are not mediated by TRs.

Background & Aims Previous studies have demonstrated that 3,5-l-diiodothyronine (T 2 ) is able to prevent lipid accumulation in the liver of rats fed a high-fat diet. Whether this effect is due to a direct action of T 2 on the liver has not been elucidated. In this study, we investigated the ability of T 2 to reduce the excess lipids in isolated hepatocytes treated with fatty acids (FFAs). The effects of T 2 were compared with those elicited by 3,3′,5-l-triiodothyronine (T 3 ). Methods To mimic the fatty liver condition, primary cultures of rat hepatocytes were overloaded with lipids, by exposure to FFAs ("fatty hepatocytes"), and then treated with T 2 or T 3 . Lipid content, morphometry of lipid droplets (LDs), and expression of the adipocyte differentiation-related protein (ADRP) and the peroxisome proliferator-activated receptors (PPAR-α, -γ, -δ) were evaluated. Activities of the lipolytic enzyme acyl CoA oxidase – AOX and the antioxidant enzymes superoxide dismutase – SOD and catalase – CAT were also determined. Results FFA-induced lipid accumulation was associated with an increase in both number/size of LDs and expression of ADRP , PPAR-γ, and PPAR-δ/β mRNAs, as well as in the activities of AOX, SOD, and CAT. The addition of T 2 or T 3 to "fatty hepatocytes" resulted in a reduction in: (i) lipid content and LD diameter; (ii) PPAR-γ and PPAR-δ expression; (iii) activities of AOX and antioxidant enzymes. Conclusions These data demonstrate, for the first time, a direct action of both T 2 and T 3 in reducing the excess fat in cultured hepatocytes.

Excess energy is stored as neutral lipids in lipid droplets (LDs) whose surface is coated by PAT proteins, each playing a distinct cellular function. The adipocyte differentiation-related protein (ADRP) and tail-interacting protein (TIP47) are expressed almost ubiquitously, whereas the oxidative tissue-enriched PAT protein (OXPAT) is expressed in specific tissues, such as the liver. In rat liver, only ADRP expression has been documented. This study was aimed at identifying OXPAT and TIP47 transcripts in rat hepatocytes, and investigating how their expression is modulated by excess lipids, using fat-enriched hepatocytes to mimic different degrees of steatosis. Primary rat hepatocytes were exposed to fatty acids (FFAs) for 12, 24 and 36 h. Lipid accumulation was estimated by spectrophotometric quantification of triacylglycerol. Expression of PAT proteins as well as of PPARgamma was evaluated by real-time RT-PCR. Hepatocytes exposed to FFAs showed progressive lipid accumulation. The increase in lipid content was associated with the induction of PAT protein expression. At 12 h, OXPAT and TIP47 mRNA expression was up-regulated. At longer times, the level of OXPAT transcripts remained high, whereas that of TIP47 slowly declined. Conversely, ADRP expression showed a time-dependent increase with exposure to FFAs. This study demonstrates, for the first time, the presence of OXPAT and TIP47 transcripts in rat hepatocytes, as well as their up-regulation with lipid accumulation. The distinct time courses observed for the three PAT proteins during FFA exposure might reflect the different roles played by each protein in lipid metabolism in the hepatocyte. Up-regulation of TIP47 and OXPAT might represent an early response to excess lipids, while, in correspondence with a lipid overload, up-regulation of ADRP could address lipids towards storage.

Growth hormone has been established as a primary regulator of IGF-I gene expression in adults, not only in liver but also in many extrahepatic tissues. We considered the possibility that IGF-I production by adult rat liver could also be stimulated by epidermal growth factor (EGF), a peptide known to be involved in liver regeneration. Chromatographic analysis performed after acid treatment of conditioned media revealed the presence of both immunoreactive (IR) IGF-I and IGF binding protein (IGFBP). Both IR IGF-I and IGFBP were present in the conditioned medium of adult rat hepatocytes in basal conditions. The stimulation of IGF-I and IGFBP secretion by EGF appears to be dose-dependent with a significant increment already evident at 5 nM. That EGF stimulates secretion is supported by the finding that IGF-I and IGFBP-1 mRNA levels are increased after EGF supplementation. We conclude that adult rat hepatocytes spontaneously produce IGF-I and IGFBP, and that EGF is able to increase their synthesis and secretion. This non-growth hormone-dependent regulation of IGF-I and IGFBP-1 production by adult rat hepatocytes in culture indicates an important autocrine/paracrine role for IGF-I, particularly during liver regeneration after extensive organ mass loss.

In rats fed a high fat diet (HFD), long-term administration of 3,5-diiodo-L-thyronine (T2), a naturally occurring iodothyronine, was shown to reduce body-weight gain, fat mass, and hepatic lipid accumulation. This work was aimed at investigating the mechanisms of T2 action in the liver of HFD rats. The results show that HFD induces liver lipid peroxidation and stimulates the activity of enzymes involved in hydrogen peroxide (H2O2) metabolism, catalase in particular. Moreover, quantitative RT-PCR revealed HFD-induced upregulation of the transcription factor PPARα, as well as of metallothionein isoforms (MT-1 and MT-2). T2 administration prevented the HDF-induced lipid peroxidation, as well as the increase in H2O2 metabolism, and reduced the upregulation of both PPARα and MT-2. These data demonstrate that in the liver of HFD rats, T2 prevents both lipid accumulation and oxidative stress associated with increased fat metabolism.

We have previously demonstrated that triiodothyronine (T 3 ) stimulates hepatic IGFBP-4 expression in rats. Since there is evidence that some of the genes whose expression is regulated by T 3 are also sensitive to 3,5-diiodothyronine (T 2 ), we used the adult rat hepatocyte model in primary cultures directly exposed to T 2 to evaluate insulin-like growth factor binding protein-4 (IGFBP-4) expression by Northern and Ligand blot analyses in this study. Our results demonstrate that T 2 , like T 3 , is able to enhance IGFBP-4 mRNA and protein after 12-24 h of incubation. The potency of the two iodothyronines is comparable as judged by dose-dependence experiments. The T 2 -induced IGFBP-4 increase is independent from ongoing protein synthesis but dependent on active transcription. Since T 3 and T 2 do not affect IGF-I production, it appears that the iodothyronines affect the hepatic IGF system at the IGFBP level. Our data, demonstrating that T 2 mimics the stimulatory effect of T 3 on IGFBP-4 expression by rat hepatocytes, allow us to include IGFBP-4 gene among the genes regulated by the two iodothyronines.

Hepatic insulin-like growth factor binding protein (IGFBP) expression is controlled by diverse factors including thyroid hormone, which enhances IGFBP-4 production in hepatocytes. In the present work, we have investigated whether hepatic IGFBP-4 expression is regulated by retinoic acid (RA), which acts via nuclear receptors belonging to the steroid/thyroid hormone receptor superfamily. Primary cultures of adult rat hepatocytes were incubated with two natural stereoisomers of RA, all-trans RA and 9-cis RA (atRA and 9cRA), and with the synthetic RA receptor (RAR)-selective agonist TTNPB. IGFBP-4 mRNA abundance was measured by Northern blot and protein production was evaluated by Ligand blot on hepatocyte-conditioned culture media. Our results indicate that atRA, 9cRA, and TTNPB increase IGFBP-4 expression by cultured hepatocytes, both at the mRNA and protein level. The RARs play a definite role in this regulation, which is independent from ongoing protein synthesis but dependent on active transcription. AtRA and thyroid hormone act synergistically in increasing hepatic IGFBP-4 expression. Our data establish a role for hormonal factors such as thyronines and retinoids in regulating the hepatic IGF system directly at the IGFBP-4 level.