Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells.

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive fat accumulation leads to non-alcoholic fatty liver disease (NAFLD),  which is potentially reversible and may evolve into non-alcoholic steatohepatitis (NASH) and eventually cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms linking lipid accumulation in hepatocytes with the progression to NASH, irreversible liver damage, fibrosis, cirrhosis, and even HCC still remains unclear. To this end, several in vitro and in vivo models have been developed to elucidate the pathological processes that cause NAFLD. In the present study, we describe a cellular model for the induction of liver vesicular steatosis that consists of DMSO-differentiated human hepatic HepaRG cells treated with the fatty acid salt sodium oleate. Indeed, sodium oleate-treated HepaRG cells accumulate lipid droplets in the cytoplasm and show typical features of steatosis. This in vitro human model represents a valuable alternative to in vivo mice models as well as to the primary human hepatocytes. We also present a comparison of several methods for the quantification and evaluation of fat accumulation in HepaRG cells, including Oil Red O staining, cytofluorimetric Bodipy measurement, metabolic gene expression analysis by qPCR, and coherent anti-Stokes Raman scattering (CARS) microscopy. CARS imaging combines the chemical specificity of Raman spectroscopy, a chemical analysis technique well-known in materials science applications, with the benefits of high-speed, high-resolution non-linear optical microscopies to allow precise quantification of lipid accumulation and lipid droplet dynamics. The establishment of an efficient in vitro model for the induction of vesicular steatosis, alongside an accurate method for the quantification and characterization of lipid accumulation, could lead to the development of early stage diagnosis of NAFLD via the identification of molecular markers, and to the generation of new treatment strategies.

Targeting a phospho-STAT3-miRNAs pathway improves vesicular hepatic steatosis in an in vitro and in vivo model.

Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease. Although genetic predisposition and epigenetic factors contribute to the development of NAFLD, our understanding of the molecular mechanism involved in the pathogenesis of the disease is still emerging. Here we investigated a possible role of a microRNAs-STAT3 pathway in the induction of hepatic steatosis. Differentiated HepaRG cells treated with the fatty acid sodium oleate (fatty dHepaRG) recapitulated features of liver vesicular steatosis and activated a cell-autonomous inflammatory response, inducing STAT3-Tyrosine-phosphorylation. With a genome-wide approach (Chromatin Immunoprecipitation Sequencing), many phospho-STAT3 binding sites were identified in fatty dHepaRG cells and several STAT3 and/or NAFLD-regulated microRNAs showed increased expression levels, including miR-21. Innovative CARS (Coherent Anti-Stokes Raman Scattering) microscopy revealed that chemical inhibition of STAT3 activity decreased lipid accumulation and deregulated STAT3-responsive microRNAs, including miR-21, in lipid overloaded dHepaRG cells. We were able to show in vivo that reducing phospho-STAT3-miR-21 levels in C57/BL6 mice liver, by long-term treatment with metformin, protected mice from aging-dependent hepatic vesicular steatosis. Our results identified a microRNAs-phosphoSTAT3 pathway involved in the development of hepatic steatosis, which may represent a molecular marker for both diagnosis and therapeutic targeting.

Metabolic Changes Associated With Muscle Expression of SOD1G93A.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder, classified into sporadic or familial forms and characterized by motor neurons death, muscle atrophy, weakness, and paralysis. Among the familial cases of ALS, approximately 20% are caused by dominant mutations in the gene coding for superoxide dismutase (SOD1) protein. Of note, mutant SOD1 toxicity is not necessarily limited to the central nervous system. ALS is indeed a multi-systemic and multifactorial disease that affects whole body physiology and induces severe metabolic changes in several tissues, including skeletal muscle. Nevertheless, whether alterations in the plasticity, heterogeneity, and metabolism of muscle fibers are the result of motor neuron degeneration or alternatively occur independently of it remain to be elucidated. To address this issue, we made use of a mouse model (MLC/SOD1G93A) that overexpresses the SOD1 mutant gene selectively in skeletal muscle. We found an alteration in the metabolic properties of skeletal muscle characterized by alteration in fiber type composition and metabolism. Indeed, we observed an alteration of muscle glucose metabolism associated with the induction of Phosphofructokinases and Pyruvate dehydrogenase kinase 4 expression. The upregulation of Pyruvate dehydrogenase kinase 4 led to the inhibition of Pyruvate conversion into Acetyl-CoA. Moreover, we demonstrated that the MLC/SOD1G93A transgene was associated with an increase of lipid catabolism and with the inhibition of fat deposition inside muscle fibers. All together these data demonstrate that muscle expression of the SOD1G93A gene induces metabolic changes, along with a preferential use of lipid energy fuel by muscle fibers. We provided evidences that muscle metabolic alterations occurred before disease symptoms and independently of motor neuron degeneration, indicating that skeletal muscle is likely an important therapeutic target in ALS.

Subacute loss of vision in one eye · rash on hands and feet · plaques with scaling on genitals · Dx?

A 67-year-old man presented to the hospital with subacute loss of vision in his left eye. The visual changes began 2 weeks earlier, with a central area of visual loss that had since progressed to near complete vision loss in the left eye. Physical examination revealed patchy alopecia, a scaling and hyperkeratotic rash of his hands and feet, and blanching, erythematous plaques with associated scaling on the scrotum and glans penis. Ophthalmologic examination revealed 1/200 vision in his left eye with a large plaque occupying a substantial portion of the superior quadrant, smaller perifoveal plaques in both of his eyes, and a small infiltrate above the left optic nerve head. The patient also described fatigue, loss of taste, and an unintentional weight loss of 7 to 10 kg over the previous 6 months. He had seen his primary care provider 3 months prior for a burning sensation and scaling rash on his feet and hands, and was prescribed a topical steroid.

The histone deacetylase inhibiting drug Entinostat induces lipid accumulation in differentiated HepaRG cells.

Dietary overload of toxic, free metabolic intermediates leads to disrupted insulin signalling and fatty liver disease. However, it was recently reported that this pathway might not be universal: depletion of histone deacetylase (HDAC) enhances insulin sensitivity alongside hepatic lipid accumulation in mice, but the mechanistic role of microscopic lipid structure in this effect remains unclear. Here we study the effect of Entinostat, a synthetic HDAC inhibitor undergoing clinical trials, on hepatic lipid metabolism in the paradigmatic HepaRG liver cell line. Specifically, we statistically quantify lipid droplet morphology at single cell level utilizing label-free microscopy, coherent anti-Stokes Raman scattering, supported by gene expression. We observe Entinostat efficiently rerouting carbohydrates and free-fatty acids into lipid droplets, upregulating lipid coat protein gene Plin4, and relocating droplets nearer to the nucleus. Our results demonstrate the power of Entinostat to promote lipid synthesis and storage, allowing reduced systemic sugar levels and sequestration of toxic metabolites within protected protein-coated droplets, suggesting a potential therapeutic strategy for diseases such as diabetes and metabolic syndrome.

Impaired consciousness in epilepsy investigated by a prospective responsiveness in epilepsy scale (RES).

PURPOSE: Impaired consciousness in epileptic seizures has a major negative impact on patient quality of life. Prior work on epileptic unconsciousness has mainly used retrospective and nonstandardized methods. Our goal was to validate and to obtain initial data using a standardized prospective testing battery. METHODS: The responsiveness in epilepsy scale (RES) was used on 52 patients during continuous video-electroencephalography (EEG) monitoring. RES begins with higher-level questions and commands, and switches adaptively to more basic sensorimotor responses depending on patient performance. RES continues after seizures and includes postictal memory testing. Scoring was conducted based on video review. KEY FINDINGS: Testing on standardized seizure simulations yielded good intrarater and interrater reliability. We captured 59 seizures from 18 patients (35% of participants) during 1,420 h of RES monitoring. RES impairment was greatest during and after tonic-clonic seizures, less in partial seizures, and minimal in auras and subclinical seizures. In partial seizures, ictal RES impairment was significantly greater if EEG changes were present. Maximum RES impairment (lowest ictal score) was also significantly correlated with long postictal recovery time, and poor postictal memory. SIGNIFICANCE: We found that prospective testing of responsiveness during seizures is feasible and reliable. RES impairment was related to EEG changes during seizures, as well as to postictal memory deficits and recovery time. With a larger patient sample it is hoped that this approach can identify brain networks underlying specific components of impaired consciousness in seizures. This may allow the development of improved treatments targeted at preventing dysfunction in these networks.