BackgroundNon-alcoholic fatty liver disease is the most common form of chronic liver disease in industrialized countries. Recent studies have highlighted the association between peroxisomal dysfunction and hepatic steatosis. Peroxisomes are intracellular organelles that contribute to several crucial metabolic processes, such as facilitation of mitochondrial fatty acid oxidation (FAO) and removal of reactive oxygen species through catalase or plasmalogen synthesis. Statins are known to prevent hepatic steatosis and non-alcoholic steatohepatitis (NASH), but underlying mechanisms of this prevention are largely unknown.MethodsSeven-week-old C57BL/6J mice were given normal chow or a methionine- and choline-deficient diet (MCDD) with or without various statins, fluvastatin, pravastatin, simvastatin, atorvastatin, and rosuvastatin (15 mg/kg/day), for 6 weeks. Histological lesions were analyzed by grading and staging systems of NASH. We also measured mitochondrial and peroxisomal FAO in the liver.ResultsStatin treatment prevented the development of MCDD-induced NASH. Both steatosis and inflammation or fibrosis grades were significantly improved by statins compared with MCDD-fed mice. Gene expression levels of peroxisomal proliferator-activated receptor α (PPARα) were decreased by MCDD and recovered by statin treatment. MCDD-induced suppression of mitochondrial and peroxisomal FAO was restored by statins. Each statin's effect on increasing FAO and improving NASH was independent on its effect of decreasing cholesterol levels.ConclusionStatins prevented NASH and increased mitochondrial and peroxisomal FAO via induction of PPARα. The ability to increase hepatic FAO is likely the major determinant of NASH prevention by statins. Improvement of peroxisomal function by statins may contribute to the prevention of NASH.

Iridovirus is a causative agent of epizootics among cultured rock bream (Oplegnathus fasciatus) in Korea. Here, we report the complete genomic sequence of rock bream iridovirus (RBIV). The genome of RBIV was 112080 bp long and contained at least 118 putative open reading frames (ORFs), and its genome organization was similar to that of infectious spleen and kidney necrosis virus (ISKNV). Of the RBIV's 118 ORFs, 85 ORFs showed 60-99% amino acid identity to those of ISKNV. Phylogenetic analysis of major capsid protein (MCP), DNA repair protein RAD2, and DNA polymerase type-B family indicated that RBIV is closely related to red sea bream iridovirus (RSIV), Grouper sleepy disease iridovirus (GSDIV), Dwarf gourami iridovirus (DGIV), and ISKNV. The genome sequence provides useful information concerning the evolution and divergence of iridoviruses in cultured fish.

Fibrosis of adipose tissue induces ectopic fat accumulation and insulin resistance by inhibiting adipose tissue expandability. Mechanisms responsible for the induction of adipose tissue fibrosis may provide therapeutic targets but are poorly understood. In this study, high-fat diet (HFD)–fed wild-type (WT) and iNOS−/− mice were used to examine the relationship between nitric oxide (NO) produced by macrophages and adipose tissue fibrosis. In contrast to WT mice, iNOS−/− mice fed an HFD were protected from infiltration of proinflammatory macrophages and adipose tissue fibrosis. Hypoxia-inducible factor 1α (HIF-1α) protein level was increased in adipose tissue of HFD-fed WT mice, but not iNOS−/− mice. In contrast, the expression of mitochondrial biogenesis factors was decreased in HFD-fed WT mice, but not iNOS−/− mice. In studies with cultured cells, macrophage-derived NO decreased the expression of mitochondrial biogenesis factors, and increased HIF-1α protein level, DNA damage, and phosphorylated p53 in preadipocytes. By activating p53 signaling, NO suppressed peroxisome proliferator–activated receptor γ coactivator 1α expression, which induced mitochondrial dysfunction and inhibited preadipocyte differentiation in adipocytes. The effects of NO were blocked by rosiglitazone. The findings suggest that NO produced by macrophages induces mitochondrial dysfunction in preadipocytes by activating p53 signaling, which in turn increases HIF-1α protein level and promotes a profibrogenic response in preadipocytes that results in adipose tissue fibrosis.

ObjectiveLipotoxic hepatocyte injury is a primary event in non-alcoholic steatohepatitis (NASH), but the mechanisms of lipotoxicity are not fully defined. Sphingolipids and free cholesterol (FC) mediate hepatocyte injury, but their link in NASH has not been explored. We examined the role of free cholesterol and sphingomyelin synthases (SMSs) that generate sphingomyelin (SM) and diacylglycerol (DAG) in hepatocyte pyroptosis, a specific form of programmed cell death associated with inflammasome activation, and NASH.DesignWild-type C57BL/6J mice were fed a high fat and high cholesterol diet (HFHCD) to induce NASH. Hepatic SMS1 and SMS2 expressions were examined in various mouse models including HFHCD-fed mice and patients with NASH. Pyroptosis was estimated by the generation of the gasdermin-D N-terminal fragment. NASH susceptibility and pyroptosis were examined following knockdown of SMS1, protein kinase Cδ (PKCδ), or the NLR family CARD domain-containing protein 4 (NLRC4).ResultsHFHCD increased the hepatic levels of SM and DAG while decreasing the level of phosphatidylcholine. Hepatic expression of Sms1 but not Sms2 was higher in mouse models and patients with NASH. FC in hepatocytes induced Sms1 expression, and Sms1 knockdown prevented HFHCD-induced NASH. DAG produced by SMS1 activated PKCδ and NLRC4 inflammasome to induce hepatocyte pyroptosis. Depletion of Nlrc4 prevented hepatocyte pyroptosis and the development of NASH. Conditioned media from pyroptotic hepatocytes activated the NOD-like receptor family pyrin domain containing 3 inflammasome (NLRP3) in Kupffer cells, but Nlrp3 knockout mice were not protected against HFHCD-induced hepatocyte pyroptosis.ConclusionSMS1 mediates hepatocyte pyroptosis through a novel DAG-PKCδ-NLRC4 axis and holds promise as a therapeutic target for NASH.

The cell-surface markers used routinely to define the undifferentiated state and pluripotency of human embryonic stem cells (hESCs) are those used in mouse embryonic stem cells (mESCs) because of a lack of markers directly originated from hESC itself. To identify more hESC-specific cell-surface markers, we generated a panel of monoclonal antibodies (MAbs) by immunizing the irradiated cell clumps of hESC line Miz-hES1, and selected 26 MAbs that were able to bind to Miz-hES1 cells but not to mESCs, mouse embryonic fibroblast cells, and STO cells. Most antibodies did not bind to human neural progenitor cells derived from the Miz-hES1 cells, either. Of these, MAb 20-202S (IgG1, κ) immunoprecipitated a cell-surface protein of 72-kDa from the lysate of biotin-labeled Miz-hES1 cells, which was identified to be heat shock 70-kDa protein 8 isoform 1 (HSPA8) by quadrupole time-of-flight tandem mass spectrometry.Immunocytochemical analyses proved that the HSPA8 protein was also present on the surface of hESC lines Miz-hES4, Miz-hES6, and HSF6. Two-color flow cytometric analysis of Miz-hES1 and HSF6 showed the coexpression of the HSPA8 protein with other hESC markers such as stage-specific embryonic antigen 3 (SSEA3), SSEA4, TRA-1-60, and TRA-1-81. Flow cytometric and Western blot analyses using various cells showed that MAb 20-202S specifically bound to the HSPA8 protein on the surface of Miz-hES1, contrary to other anti-HSP70 antibodies examined. Furthermore, the surface expression of the HSPA8 protein on Miz-hES1 was markedly downregulated upon differentiation. These data indicate that a novel MAb 20-202S recognizes the HSPA8 protein on the surface of hESCs and suggest that the HSPA8 protein is a putative cell-surface marker for undifferentiated hESCs. Stem Cells 2005;23:1502-1513

Free cholesterol (FC) accumulation in the liver is an important pathogenic mechanism of nonalcoholic steatohepatitis (NASH). Plasmalogens, key structural components of the cell membrane, act as endogenous antioxidants and are primarily synthesized in the liver. However, the role of hepatic plasmalogens in metabolic liver disease is unclear. In this study, we found that hepatic levels of docosahexaenoic acid (DHA)-containing plasmalogens, expression of glyceronephosphate O-acyltransferase (Gnpat, the rate-limiting enzyme in plasmalogen biosynthesis), and expression of Pparα were lower in mice with NASH caused by accumulation of free cholesterol (FC) in the liver. Cyclodextrin-induced depletion of FC transactivated Δ-6 desaturase by increasing Srebp2 expression in cultured hepatocytes. DHA, the major product of Δ-6 desaturase activation, activated GNPAT, thereby explaining the association between high hepatic FC and decreased Gnpat expression. Gnpat siRNA treatment significantly decreased Pparα expression in cultured hepatocytes. In addition to GNPAT, DHA activated PPARα and increased expression of Pparα and its target genes, suggesting that DHA in the DHA-containing plasmalogens contributed to activation of PPARα. Accordingly, administration of the plasmalogen precursor alkyl glycerol (AG) prevented hepatic steatosis and NASH through a PPARα-dependent increase in fatty acid oxidation. Gnpat+/− mice were more susceptible to hepatic lipid accumulation and less responsive to the preventive effect of fluvastatin on NASH development, suggesting that endogenous plasmalogens prevent hepatic steatosis and NASH. Conclusions Increased hepatic FC in animals with NASH decreased plasmalogens, thereby sensitizing animals to hepatocyte injury and NASH. Our findings uncover a novel link between hepatic FC and plasmalogen homeostasis via GNPAT regulation. Further study of AG or other agents that increase hepatic plasmalogen levels may identify novel therapeutic strategies against NASH.

The administration of mesenchymal stem cells (MSCs) was shown to attenuate overt as well as early diabetic nephropathy in rodents, but the underlying mechanism of this beneficial effect is largely unknown. Inflammation and mitochondrial dysfunction are major pathogenic factors in diabetic nephropathy. In this study, we found that the repeated administration of MSCs prevents albuminuria and injury to tubular epithelial cells (TECs), an important element in the progression of diabetic nephropathy, by improving mitochondrial function. The expression of M1 macrophage markers was significantly increased in diabetic kidneys compared with that in control kidneys. Interestingly, the expression of arginase-1 (Arg1), an important M2 macrophage marker, was reduced in diabetic kidneys and increased by MSC treatment. In cultured TECs, conditioned media from lipopolysaccharide-activated macrophages reduced peroxisomal proliferator-activated receptor gamma coactivator 1α (Pgc1a) expression and impaired mitochondrial function. The coculture of macrophages with MSCs increased and decreased the expression of Arg1 and M1 markers, respectively. Treatment with conditioned media from cocultured macrophages prevented activated macrophage-induced mitochondrial dysfunction in TECs. In the absence of MSC coculture, Arg1 overexpression in macrophages reversed Pgc1a suppression in TECs. These observations suggest that MSCs prevent the progression of diabetic nephropathy by reversing mitochondrial dysfunction in TECs via the induction of Arg1 in macrophages.

LATS2 is a tumor suppressor gene implicated in the control of cell growth and the cell cycle. Here, we investigated the posttranscriptional regulation of LATS2 expression by tristetraprolin (TTP). Our results show that the expression level of LATS2 is inversely correlated with TTP expression in human cancer cell lines. Overexpression of TTP reduced the expression level of LATS2. Conversely, treatment with small interfering RNA against TTP increased the expression level of LATS2 through stabilization of LATS2 mRNA and suppressed the proliferation of A549 human lung cancer cells. LATS2 mRNA contains AUrich elements (AREs) within the 3-untranslated region, and TTP destabilized a luciferase mRNA containing LATS2 ARE. In addition, RNA electrophoretic mobility shift assay revealed that TTP directly bound to the ARE of LATS2 mRNA. These results establish LATS2 mRNA as a physiological target of TTP and suggest the possibility that TTP controls cell growth through regulation of LATS2 mRNA stability.LATS2 is a new member of the LATS tumor suppressor family (1-3). The LATS2 gene is located at chromosome 13q11-12 and encodes a 1046-amino acid protein that contains a C-terminal serine/threonine kinase domain (4). According to recent publications, LATS2 appears to coordinate the cell cycle, cell proliferation, and cell death. LATS2 promotes the stabilization of p53 by inactivating MDM2 (5), plays a role in maintenance of mitotic fidelity (6), and regulates spindle organization through recruitment of ␥-tubulin to the centrosome (7). Overexpression of LATS2 results in G 2 /M arrest via inhibition of Cdc2 kinase activity (8), inhibition of G 1 /S transition via down-regulation of cyclin E/Cdk2 kinase activity (9), and induction of apoptosis via down-regulation of apoptotic inhibitors such as Bcl-2 and Bcl-x L (10). Deletion of LATS2 in flies (11, 12) and mice (13) accelerates cell proliferation and tumor development, indicating that the reduced function of the LATS2 gene leads to accelerated cell proliferation.Expression of LATS2 is down-regulated in several human cancer cells (14 -18). The mechanisms for down-regulation have been the subject of substantial interest. It has been reported that the expression of LATS2 in breast cancer (14), astrocytoma (17), and acute lymphoblastic leukemia (18, 19) is down-regulated by hypermethylation of the promoter. In addition, LATS2 mRNA is down-regulated at the post-transcriptional level by microRNA-372 and microRNA-373 in testicular germ cell tumors (20), gastric cancer cells (21), and esophageal cancer cells (22), suggesting that the expression of the LATS2 gene is controlled at the post-transcriptional level as well as the transcriptional level.Post-transcriptional regulation of gene expression is also mediated by AU-rich elements (AREs) 4 located in the 3Ј-untranslated region (3Ј-UTR) of a variety of short-lived mRNAs such as cytokines and proto-oncogenes (23). The destabilizing function of AREs is believed to be regulated by ARE-binding proteins (24). Numerous ARE-binding proteins ha...