The ability to store fat in the form of cytoplasmic triglyceride droplets is conserved from Saccharomyces cerevisiae to humans. Although much is known regarding the composition and catabolism of lipid droplets, the molecular components necessary for the biogenesis of lipid droplets have remained obscure. Here we report the characterization of a conserved gene family important for lipid droplet formation named fat-inducing transcript (FIT). FIT1 and FIT2 are endoplasmic reticulum resident membrane proteins that induce lipid droplet accumulation in cell culture and when expressed in mouse liver. shRNA silencing of FIT2 in 3T3-LI adipocytes prevents accumulation of lipid droplets, and depletion of FIT2 in zebrafish blocks diet-induced accumulation of lipid droplets in the intestine and liver, highlighting an important role for FIT2 in lipid droplet formation in vivo. Together these studies identify and characterize a conserved gene family that is important in the fundamental process of storing fat.adipocytes ͉ diabetes ͉ FIT ͉ obesity ͉ triglyceride T he ability to store energy in the form of triglyceride (TG) is conserved from Saccharomyces cerevisiae to humans. TGs are stored in the cytoplasm surrounded by a monolayer of phospholipid in distinct structures or organelles given numerous names, such as lipid particles, oil bodies, adiposomes, eicosasomes, and, more commonly, lipid droplets (1). Under normal physiological conditions, lipid droplets are involved in maintaining energy balance at the cellular and whole-organism levels. Yet under conditions of extreme lipid droplet acquisition, as in obesity, the risk for acquiring common debilitating diseases such as type 2 diabetes and cardiovascular diseases is increased (2).Despite their central role in energy homeostasis, only recently have the composition and functions of many of the components of lipid droplets from S. cerevisiae, Drosophila, and mammalian cells been revealed. In general, lipid droplets are composed of a core of neutral lipids, primarily TGs, surrounded by a monolayer of phospholipids and lipid droplet-associated proteins (3-7). In mammalian cells, the catabolism of lipid droplets is a highly regulated process involving hormonal signals, droplet-associated proteins, and lipases (8-10). Although much has been learned about the components and catabolism of lipid droplets, the molecular mechanism of lipid droplet biogenesis has remained unknown. The prevailing view is that lipid droplets are formed at the endoplasmic reticulum (ER) because the ER is the site of TG biosynthesis, and lipid droplets are often observed in close association with the cytoplasmic face of the ER (11-13). A widely accepted model of lipid droplet biogenesis involves the formation of a core or lens of newly synthesized TG between the leaflets of the ER membrane that buds off with the cytoplasmic leaflet of the ER surrounding the neutral lipid core and acquires exchangeable cytosolic lipid droplet-associated proteins (14). However, this view was recently challenged by observatio...

Background: Animal models of non-alcoholic steatohepatitis (NASH) are important tools in preclinical research and drug discovery. Gubra-Amylin NASH (GAN) diet-induced obese (DIO) mice represent a model of fibrosing NASH. The present study directly assessed the clinical translatability of the model by head-to-head comparison of liver biopsy histological and transcriptome changes in GAN DIO-NASH mouse and human NASH patients. Methods: C57Bl/6 J mice were fed chow or the GAN diet rich in saturated fat (40%), fructose (22%) and cholesterol (2%) for ≥38 weeks. Metabolic parameters as well as plasma and liver biomarkers were assessed. Liver biopsy histology and transcriptome signatures were compared to samples from human lean individuals and patients diagnosed with NASH. Results: Liver lesions in GAN DIO-NASH mice showed similar morphological characteristics compared to the NASH patient validation set, including macrosteatosis, lobular inflammation, hepatocyte ballooning degeneration and periportal/perisinusoidal fibrosis. Histomorphometric analysis indicated comparable increases in markers of hepatic lipid accumulation, inflammation and collagen deposition in GAN DIO-NASH mice and NASH patient samples. Liver biopsies from GAN DIO-NASH mice and NASH patients showed comparable dynamics in several gene expression pathways involved in NASH pathogenesis. Consistent with the clinical features of NASH, GAN DIO-NASH mice demonstrated key components of the metabolic syndrome, including obesity and impaired glucose tolerance. Conclusions: The GAN DIO-NASH mouse model demonstrates good clinical translatability with respect to the histopathological, transcriptional and metabolic aspects of the human disease, highlighting the suitability of the GAN DIO-NASH mouse model for identifying therapeutic targets and characterizing novel drug therapies for NASH.

Background: FIT2 is an ER protein purported to be important for triglyceride lipid droplet formation. Results: FIT2 deficiency in adipose tissue results in lipodystrophy and metabolic dysfunction. Conclusion: FIT2 is required for normal triglyceride storage in adipose tissue. Significance: This study provides the first proof-of-principle mouse models indicating that FIT2 is essential for normal triglyceride storage in adipose tissue.

SUMO-modification of nuclear proteins has profound effects on gene expression. However, non-toxic chemical tools that modulate sumoylation in cells are lacking. Here, to identify small molecule sumoylation inhibitors we developed a cell-based screen that focused on the well-sumoylated substrate, human Liver Receptor Homolog-1 (hLRH-1, NR5A2). Our primary gene-expression screen assayed two SUMO-sensitive transcripts, APOC3 and MUC1, that are upregulated by SUMO-less hLRH-1 or by siUBC9 knockdown, respectively. A polyphenol, tannic acid (TA) emerged as a potent sumoylation inhibitor in vitro (IC50 = 12.8 µM) and in cells. TA also increased hLRH-1 occupancy on SUMO-sensitive transcripts. Most significantly, when tested in humanized mouse primary hepatocytes, TA inhibits hLRH-1 sumoylation and induces SUMO-sensitive genes, thereby recapitulating the effects of expressing SUMO-less hLRH-1 in mouse liver. Our findings underscore the benefits of phenotypic screening for targeting post-translational modifications, and illustrate the potential utility of TA for probing the cellular consequences of sumoylation.DOI: http://dx.doi.org/10.7554/eLife.09003.001

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Background: Fat storage-inducing transmembrane protein 2 (FIT2) is implicated to be important in the formation of triacylglyceride lipid droplets. Results: Mice with skeletal muscle-specific overexpression of FIT2 had increased muscle triacylglycerides, were completely protected from diet-induced weight gain, and had altered muscle energy metabolism. Conclusion: FIT2 plays an unexpected function in regulating muscle energy metabolism. Significance: This is the first study describing a function for FIT2 in energy metabolism.

Cardiovascular disease and malignancy are the most common cause of death in Non-alcoholic Steatohepatitis (NASH) patients. Aside from lifestyle modification, there is currently no treatment for NASH. Activation of Liver Receptor Homolog-1 (Lrh-1), known to bind phospholipid ligands, has been shown to effectively reduce liver triglyceride (TG) in DIO mice, raising Lrh-1 as a possible target for treating NASH. Despite this finding, hepatic TGs are equivalent in controls and liver-specific Lrh-1 knockout (LKO or Lrh1 AlbCre ) mice, regardless of diet. Given this discrepancy, we sought to characterize Lrh-1’s role in hepatic lipid metabolism by acutely deleting Lrh-1 in the adult liver, thus eliminating potential compensatory developmental effects associated with LKO. To acutely eliminate Lrh-1 in hepatocytes, 6-week old Lrh-1 fl/f l male mice were infected with AVV8-TBG-eGFP (Control) or AAV8-TBG-Cre (LKO AAVCre ) via retro-orbital injection and fed chow or high fat diet. LKO AAVCre mice developed hepatic steatosis after six weeks on standard chow or high fat diet. Furthermore, LKO AAVCre hepatocytes exhibited large lipid droplets, which were visible as early as 2 wks post-infection, thus suggesting that lipid handling is significantly altered in LKO AAVCre hepatocytes, independent of fatty acid transport or oxidation. LKO AAVCre exhibited lower Pcsk9 expression, which correlated with decreased fasting plasma LDL-C. Consistent with other studies showing that perturbations in phospholipid pools affect lipid storage, lipidomic analyses revealed a significant reduction in phospholipid species containing arachidonic acid (AA), thus reducing the overall diversity of key membrane phospholipids. RNA-Seq analyses from LKO AAVCre livers confirmed that factors promoting lipid droplet size ( Cidec , Plin4 ) were greatly increased while key enzymes in biosynthesis of unsaturated fatty acids were reduced ( Fads1, Fads2 and Elovl5 ). In addition, expression of human LRH-1 in LKO AAVCre decreased hepatic TG and improved glucose tolerance in DIO mice, in a ligand dependent manner. Collectively our data establish a novel role for Lrh-1 as a key regulator of lipid storage, thereby providing the first in vivo evidence as to why phospholipid serve as Lrh-1 ligands.

The global production of fossil-based plastics has reached critical levels, and their substitution with bio-based polymers is an urgent requirement. Poly(3-hydroxybutyrate) (PHB) is a biopolymer that can be produced via microbial cultivation, but efficient microorganisms and low-cost substrates are required. Halomonas boliviensis LC1, a moderately halophilic bacterium, is an effective PHB producer, and hydrolysates of the residual stalks of quinoa (Chenopodium quinoa Willd.) can be considered a cheap source of sugars for microbial fermentation processes in quinoa-producing countries. In this study, H. boliviensis LC1 was adapted to a cellulosic hydrolysate of quinoa stalks obtained via acid-catalyzed hydrothermal pretreatment and enzymatic saccharification. The adapted strain was cultivated in hydrolysates and synthetic media, each of them with two different initial concentrations of glucose. Cell growth, glucose consumption, and PHB formation during cultivation were assessed. The cultivation results showed an initial lag in microbial growth and glucose consumption in the quinoa hydrolysates compared to cultivation in synthetic medium, but after 33 h, the values were comparable for all media. Cultivation in hydrolysates with an initial glucose concentration of 15 g/L resulted in a higher glucose consumption rate (0.15 g/(L h) vs. 0.14 g/(L h)) and volumetric productivity of PHB (14.02 mg/(L h) vs. 10.89 mg/(L h)) than cultivation in hydrolysates with 20 g/L as the initial glucose concentration. During most of the cultivation time, the PHB yield on initial glucose was higher for cultivation in synthetic medium than in hydrolysates. The produced PHBs were characterized using advanced analytical techniques, such as high-performance size-exclusion chromatography (HPSEC), Fourier transform infrared (FTIR) spectroscopy, 1H nuclear magnetic resonance (NMR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). HPSEC revealed that the molecular weight of PHB produced in the cellulosic hydrolysate was lower than that of PHB produced in synthetic medium. TGA showed higher thermal stability for PHB produced in synthetic medium than for that produced in the hydrolysate. The results of the other characterization techniques displayed comparable features for both PHB samples. The presented results show the feasibility of producing PHB from quinoa stalks with H. boliviensis.