Ezetimibe ameliorates steatohepatitis via AMP activated protein kinase-TFEB-mediated activation of autophagy and NLRP3 inflammasome inhibition.

Impairment in macroautophagy/autophagy flux and inflammasome activation are common characteristics of nonalcoholic steatohepatitis (NASH). Considering the lack of approved agents for treating NASH, drugs that can enhance autophagy and modulate inflammasome pathways may be beneficial. Here, we investigated the novel mechanism of ezetimibe, a widely prescribed drug for hypercholesterolemia, as a therapeutic option for ameliorating NASH. Human liver samples with steatosis and NASH were analyzed. For in vitro studies of autophagy and inflammasomes, primary mouse hepatocytes, human hepatoma cells, mouse embryonic fibroblasts with Ampk or Tsc2 knockout, and human or primary mouse macrophages were treated with ezetimibe and palmitate. Steatohepatitis and fibrosis were induced by feeding Atg7 wild-type, haploinsufficient, and knockout mice a methionine- and choline-deficient diet with ezetimibe (10 mg/kg) for 4 wk. Human livers with steatosis or NASH presented impaired autophagy with decreased nuclear TFEB and increased SQSTM1, MAP1LC3-II, and NLRP3 expression. Ezetimibe increased autophagy flux and concomitantly ameliorated lipid accumulation and apoptosis in palmitate-exposed hepatocytes. Ezetimibe induced AMPK phosphorylation and subsequent TFEB nuclear translocation, related to MAPK/ERK. In macrophages, ezetimibe blocked the NLRP3 inflammasome-IL1B pathway in an autophagy-dependent manner and modulated hepatocyte-macrophage interaction via extracellular vesicles. Ezetimibe attenuated lipid accumulation, inflammation, and fibrosis in liver-specific Atg7 wild-type and haploinsufficient mice, but not in knockout mice. Ezetimibe ameliorates steatohepatitis by autophagy induction through AMPK activation and TFEB nuclear translocation, related to an independent MTOR ameliorative effect and the MAPK/ERK pathway. Ezetimibe dampens NLRP3 inflammasome activation in macrophages by modulating autophagy and a hepatocyte-driven exosome pathway.

Association between betatrophin/ANGPTL8 and non-alcoholic fatty liver disease: animal and human studies.

Betatrophin/angiopoietin-like protein 8 (ANGPTL8) is a liver-secreted protein recently identified as a potent stimulator of beta cell proliferation in mice. However, it is unclear how betatrophin is regulated in humans with non-alcoholic fatty liver disease (NAFLD). We investigated the role of betatrophin in mice and in humans with and without NAFLD. Serum betatrophin levels were examined by ELISA in 164 subjects, including 96 patients with NAFLD. Levels were significantly elevated in subjects with NAFLD compared with controls (1.301 ± 0.617 vs. 0.900 ± 0.574 µg/L, P < 0.001), even after stratification by diabetic or obesity status. Circulating betatrophin positively correlated with obesity or glycemic indices, liver enzyme profiles, and NAFLD status, and was confirmed by multivariate regression analyses (ß = 0.195, P = 0.040). However, when including insulin resistance index in the model, the significant association between betatrophin level and NAFLD was diminished due to a mediation effect of insulin resistance on this relationship. Palmitate or tunicamycin increased betatrophin expression in HepG2 cells, while a chemical chaperone blocked its induction. Hepatic expression of betatrophin was elevated in mice with NAFLD including db/db or ob/ob mice and mice with a high-fat or methionine-choline deficient diet. In conclusion, circulating betatrophin was increased in mice and humans with NAFLD and its expression was induced by endoplasmic reticulum stress in hepatocytes (Clinical trial no. NCT02285218).

Metformin Restores Parkin-Mediated Mitophagy, Suppressed by Cytosolic p53.

Metformin is known to alleviate hepatosteatosis by inducing 5' adenosine monophosphate (AMP)-kinase-independent, sirtuin 1 (SIRT1)-mediated autophagy. Dysfunctional mitophagy in response to glucolipotoxicities might play an important role in hepatosteatosis. Here, we investigated the mechanism by which metformin induces mitophagy through restoration of the suppressed Parkin-mediated mitophagy. To this end, our ob/ob mice were divided into three groups: (1) ad libitum feeding of a standard chow diet; (2) intraperitoneal injections of metformin 300 mg/kg; and (3) 3 g/day caloric restriction (CR). HepG2 cells were treated with palmitate (PA) plus high glucose in the absence or presence of metformin. We detected enhanced mitophagy in ob/ob mice treated with metformin or CR, whereas mitochondrial spheroids were observed in mice fed ad libitum. Metabolically stressed ob/ob mice and PA-treated HepG2 cells showed an increase in expression of endoplasmic reticulum (ER) stress markers and cytosolic p53. Cytosolic p53 inhibited mitophagy by disturbing the mitochondrial translocation of Parkin, as demonstrated by immunoprecipitation. However, metformin decreased ER stress and p53 expression, resulting in induction of Parkin-mediated mitophagy. Furthermore, pifithrin-α, a specific inhibitor of p53, increased mitochondrial incorporation into autophagosomes. Taken together, these results indicate that metformin treatment facilitates Parkin-mediated mitophagy rather than mitochondrial spheroid formation by decreasing the inhibitory interaction with cytosolic p53 and increasing degradation of mitofusins.

Metformin alleviates hepatosteatosis by restoring SIRT1-mediated autophagy induction via an AMP-activated protein kinase-independent pathway.

Metformin activates both PRKA and SIRT1. Furthermore, autophagy is induced by either the PRKA-MTOR-ULK1 or SIRT1-FOXO signaling pathways. We aimed to elucidate the mechanism by which metformin alleviates hepatosteatosis by examining the molecular interplay between SIRT1, PRKA, and autophagy. ob/ob mice were divided into 3 groups: one with ad libitum feeding of a standard chow diet, one with 300 mg/kg intraperitoneal metformin injections, and one with 3 g/d caloric restriction (CR) for a period of 4 wk. Primary hepatocytes or HepG2 cells were treated with oleic acid (OA) plus high glucose in the absence or presence of metformin. Both CR and metformin significantly improved body weight and glucose homeostasis, along with hepatic steatosis, in ob/ob mice. Furthermore, CR and metformin both upregulated SIRT1 expression and also stimulated autophagy induction and flux in vivo. Metformin also prevented OA with high glucose-induced suppression of both SIRT1 expression and SIRT1-dependent activation of autophagy machinery, thereby alleviating intracellular lipid accumulation in vitro. Interestingly, metformin treatment upregulated SIRT1 expression and activated PRKA even after siRNA-mediated knockdown of PRKAA1/2 and SIRT1, respectively. Taken together, these results suggest that metformin alleviates hepatic steatosis through PRKA-independent, SIRT1-mediated effects on the autophagy machinery.

Glycated albumin causes pancreatic ß-cells dysfunction through autophagy dysfunction.

Growing evidence suggests that advanced glycation end-products (AGEs) are cytotoxic to pancreatic ß-cells. The aims of this study were to investigate whether glycated albumin (GA), an early precursor of AGEs, would induce dysfunction in pancreatic ß-cells and to determine which kinds of cellular mechanisms are activated in GA-induced ß-cell apoptosis. Decreased viability and increased apoptosis were induced in INS-1 cells treated with 2.5 mg/mL GA under 16.7mM high-glucose conditions. Insulin content and glucose-stimulated secretion from isolated rat islets were reduced in 2.5 mg/mL GA-treated cells. In response to 2.5 mg/mL GA in INS-1 cells, autophagy induction and flux decreased as assessed by green fluorescent protein-microtubule-associated protein 1 light chain 3 dots, microtubule-associated protein 1 light chain 3-II conversion, and SQSTM1/p62 in the presence and absence of bafilomycin A1. Accumulated SQSTM1/p62 through deficient autophagy activated the nuclear factor-κB (p65)-inducible nitric oxide synthase-caspase-3 cascade, which was restored by treatment with small interfering RNA against p62. Small interfering RNA treatment against autophagy-related protein 5 significantly inhibited the autophagy machinery resulting in a significant increase in iNOS-cleaved caspase-3 expression. Treatment with 500µM 4-phenyl butyric acid significantly alleviated the expression of endoplasmic reticulum stress markers and iNOS in parallel with upregulated autophagy induction. However, in the presence of bafilomycin A1, the decreased viability of INS-1 cells was not recovered. Glycated albumin, an early precursor of AGE, caused pancreatic ß-cell death by inhibiting autophagy induction and flux, resulting in nuclear factor-κB (p65)-iNOS-caspase-3 cascade activation as well as by increasing susceptibility to endoplasmic reticulum stress and oxidative stress.

Potential association between coronary artery disease and the inflammatory biomarker YKL-40 in asymptomatic patients with type 2 diabetes mellitus.

BACKGROUND: Inflammation plays an important role in coronary artery disease from the initiation of endothelial dysfunction to plaque formation to final rupture of the plaque. In this study, we investigated the potential pathophysiological and clinical relevance of novel cytokines secreted from various cells including adipocytes, endothelial cells, and inflammatory cells, in predicting coronary artery disease (CAD) in asymptomatic subjects with type 2 diabetes mellitus. METHODS: We enrolled a total of 70 asymptomatic type 2 diabetic patients without a documented history of cardiovascular disease, and determined serum levels of chemerin, omentin-1, YKL-40, and sCD26. We performed coronary computed tomographic angiography (cCTA) in all subjects, and defined coronary artery stenosis ≥ 50 % as significant CAD in this study. RESULTS: Subjects were classified into two groups: patients with suspected coronary artery stenosis on cCTA (group I, n = 41) and patients without any evidence of stenosis on cCTA (group II, n = 29). Group I showed significantly higher YLK-40 levels and lower HDL-C levels than group II (p = 0.038, 0.036, respectively). Levels of chemerin, omentin-1, and sCD26 were not significantly different between the two groups. Serum YKL-40 levels were positively correlated with systolic/diastolic BP, fasting/postprandial triglyceride levels, and Framingham risk score. Furthermore, YKL-40 levels showed moderate correlation with the degree of coronary artery stenosis and the coronary artery calcium score determined from cCTA. In multivariate logistic analysis, after adjusting for age, gender, smoking history, hypertension, and LDL-cholesterol, YLK-40 levels showed only borderline significance. CONCLUSIONS: YKL-40, which is secreted primarily from inflammatory cells, was associated with several CVD risk factors and was elevated in type 2 diabetic patients with suspected coronary artery stensosis on cCTA. These results suggest the possibility that the inflammatory biomarker YKL-40 might be associated with coronary artery disease in asymptomatic patients with type 2 diabetes mellitus.

Dimethyl sulfoxide reduces hepatocellular lipid accumulation through autophagy induction.

Induction of autophagy is known not only to regulate cellular homeostasis but also to decrease triglyceride accumulation in hepatocytes. The aim of this study is to investigate whether DMSO (dimethyl sulfoxide) has a beneficial role in free fatty acid-induced hepatic fat accumulation. In HepG2 cells, treatment with 0.5 mM palmitate for six hours significantly increased lipid and triglyceride (TG) accumulation, assessed by Oil-red O staining and TG quantification assay. Treatment with 0.01% DMSO for 16 h statistically reduced palmitate-induced TG contents. Pretreatment of 10 mM 3-methyladenine (3MA) for 2 h restored hepatocellular lipid contents, which were attenuated by treatment with DMSO. DMSO increased LC3-II conversion and decreased SQSTM1/p62 expression in a time and dose-dependent manner. In addition, the number of autophagosomes and autolysosomes, as seen under an electron microscopy, as well as the percentage of RFP-LAMP1 colocalized with GFP-LC3 dots in cells transfected with both GFP-LC3 and RFP-LAMP1, as seen under a fluorescent microscopy, also increased in DMSO-treated HepG2 cells. DMSO also suppressed p-eIF2α/p-EIF2S1, ATF4, p-AKT1, p-MTOR and p-p70s6k/p-RPS6KB2 expression as assessed by western blotting. Knockdown of ATF4 expression using siRNA suppressed ATF4 expression and phosphorylation of AKT1, MTOR and RPS6KB2, but increased LC3-II conversion. DMSO reduced not only soluble but also insoluble mtHTT (mutant huntingtin aggregates) expressions, which were masked in the presence of autophagy inhibitor. DMSO, a kind of chemical chaperone, activated autophagy by suppressing ATF4 expression and might play a protective role in the development of fatty acid-induced hepatosteatosis.

Direct generation of neurosphere-like cells from human dermal fibroblasts.

Neural stem cell (NSC) transplantation replaces damaged brain cells and provides disease-modifying effects in many neurological disorders. However, there has been no efficient way to obtain autologous NSCs in patients. Given that ectopic factors can reprogram somatic cells to be pluripotent, we attempted to generate human NSC-like cells by reprograming human fibroblasts. Fibroblasts were transfected with NSC line-derived cellular extracts and grown in neurosphere culture conditions. The cells were then analyzed for NSC characteristics, including neurosphere formation, gene expression patterns, and ability to differentiate. The obtained induced neurosphere-like cells (iNS), which formed daughter neurospheres after serial passaging, expressed neural stem cell markers, and had demethylated SOX2 regulatory regions, all characteristics of human NSCs. The iNS had gene expression patterns that were a combination of the patterns of NSCs and fibroblasts, but they could be differentiated to express neuroglial markers and neuronal sodium channels. These results show for the first time that iNS can be directly generated from human fibroblasts. Further studies on their application in neurological diseases are warranted.

Terpestacin inhibits tumor angiogenesis by targeting UQCRB of mitochondrial complex III and suppressing hypoxia-induced reactive oxygen species production and cellular oxygen sensing.

Cellular oxygen sensing is required for hypoxia-inducible factor-1alpha stabilization, which is important for tumor cell survival, proliferation, and angiogenesis. Here we find that terpestacin, a small molecule previously identified in a screen of microbial extracts, binds to the 13.4-kDa subunit (UQCRB) of mitochondrial Complex III, resulting in inhibition of hypoxia-induced reactive oxygen species generation. Consequently, such inhibition blocks hypoxia-inducible factor activation and tumor angiogenesis in vivo, without inhibiting mitochondrial respiration. Overexpression of UQCRB or its suppression using RNA interference demonstrates that it plays a crucial role in the oxygen sensing mechanism that regulates responses to hypoxia. These findings provide a novel molecular basis of terpestacin targeting UQCRB of Complex III in selective suppression of tumor progression.

Functional analysis of a histone deacetylase-like protein of Thermus caldophilus GK24 in mammalian cell.

The function of eukaryotic histone deacetylase (HDAC) has been extensively studied for its critical role in transcriptional regulation and carcinogenesis. However that of the prokaryotic counterpart remains largely unknown. Recently, we cloned HDAC-like protein in Thermus caldophilus GK24 (Tca HDAC) from a genomic library of the microorganism based on homology analysis with human HDAC1. To explore the function of Tca HDAC in mammalian cells, Tca HDAC gene expressing vector was transfected into a human fibrosarcoma cell line, HT1080. Tca HDAC was mainly localized in nuclei of the mammalian cells as a human HDAC1 was, due to an N-terminal HDAC association domain. We further generated histidine-substituted Tca HDAC mutants and investigated their role in biochemical and cellular activity of the enzyme. Tca HDAC mutants exhibited dramatic loss of enzymatic activity and conditioned media (CM) from HT1080 cells transfected with mutant Tca HDAC was unable to stimulate angiogenic phenotypes of endothelial cells in vitro whereas that of wild Tca HDAC did. Collectively, these results demonstrate that a prokaryotic histone deacetylase from T. caldophilus GK24 is functionally active in mammalian cells and its function in gene expression is conserved from prokaryotes to eukaryotes.

Cloning, expression, and biochemical characterization of a new histone deacetylase-like protein from Thermus caldophilus GK24.

Histone deactylases (HDACs) are members of an ancient enzyme family found in eukaryotes as well as in prokaryotes such as archaebacteria and eubacteria. We here report a new histone deacetylase (Tca HDAC) that was cloned from the genomic library of Thermus caldophilus GK24 based on homology analysis with human histone deacetylase1 (HDAC1). The gene contains an open reading frame encoding 375 amino acids with a calculated molecular mass of 42,188 Da and the deduced amino acid sequence of Tca HDAC showed a 31% homology to human HDAC1. The Tca HDAC gene was over-expressed in Escherichia coli using a Glutathione-S transferase (GST) fusion vector (pGEX-4T-1) and the purified protein showed a deacetylase activity toward the fluorogenic substrate for HDAC. Moreover, the enzyme activity was inhibited by trichostatin A, a specific HDAC inhibitor, in a dose-dependent manner. Optimum temperature and pH of the enzyme was found to be approximately 70 degrees C and 7.0, respectively. In addition, zinc ion is required for catalytic activity of the enzyme. Together, these data demonstrate that Tca HDAC is a new histone deacetylase-like enzyme from T. caldophilus GK24 and will be a useful tool for deciphering the role of HDAC in the prokaryote and development of new biochemical reactions.