Downregulation of microRNA-451 in non-alcoholic steatohepatitis inhibits fatty acid-induced proinflammatory cytokine production through the AMPK/AKT pathway

Mechanisms associated with the progression of non-alcoholic fatty liver disease (NAFLD) remain unclear. We attempted to identify the pattern of altered gene expression at different time points in a high fat diet (HFD)-induced NAFLD mouse model. The early up-regulated genes are mainly involved in the innate immune responses, while the late up-regulated genes represent the inflammation processes. Although recent studies have shown that microRNAs play important roles in hepatic metabolic functions, the pivotal role of microRNAs in the progression of NAFLD is not fully understood. We investigated the functions of miR-451, which was identified as a target gene in the inflammatory process in NAFLD. miR-451 expression was significantly decreased in the palmitate (PA)-exposed HepG2 cells and in liver tissues of HFD-induced non-alcoholic steatohepatitis (NASH) mice. Its decreased expressions were also observed in liver specimens of NASH patients. In vitro analysis of the effect of miR-451 on proinflammatory cytokine provided evidence for negative regulation of PA-induced interleukin (IL)-8 and tumor necrosis factor-alpha (TNF-α) production. Furthermore, miR-451 over-expression inhibited translocation of the PA-induced NF-κB p65 subunit into the nucleus. Our result showed that Cab39 is a direct target of miRNA-451 in steatotic cells. Further study showed that AMPK activated through Cab39 inhibits NF-κB transactivation induced in steatotic HepG2 cells. miR-451 over-expression in steatotic cells significantly suppressed PA-induced inflammatory cytokine. These results provide new insights into the negative regulation of miR-451 in fatty acid-induced inflammation via the AMPK/AKT pathway and demonstrate potential therapeutic applications for miR-451 in preventing the progression from simple steatosis to severely advanced liver disease.     

miRNA-130b-5p promotes hepatic stellate cell activation and the development of liver fibrosis by suppressing SIRT4 expression

Liver fibrosis is a progressive disease accompanied by the deposition of extracellular matrix (ECM). Numerous reports have demonstrated that alterations in the expression of microRNAs (miRNAs) are related to liver disease. However, the effect of individual miRNAs on liver fibrosis has not been studied. Hepatic stellate cells (HSCs), being responsible for producing ECM, exert an important influence on liver fibrosis. Then, microarray analysis of non-activated and activated HSCs induced by transforming growth factor β1 (TGF-β1) showed that miR-130b-5p expression was strongly up-regulated during HSC activation. Moreover, the progression of liver fibrosis had a close connection with the expression of miR-130b-5p in different liver fibrosis mouse models. Then, we identified that there were specific binding sites between miR-130b-5p and the 3′ UTR of Sirtuin 4 (SIRT4) via a luciferase reporter assay. Knockdown of miR-130b-5p increased SIRT4 expression and ameliorated liver fibrosis in mice transfected with antagomiR-130b-5p oligos. In general, our results suggested that miR-130b-5p promoted HSC activation by targeting SIRT4, which participates in the AMPK/TGF-β/Smad2/3 signalling pathway. Hence, regulating miR-130b-5p maybe serve as a crucial therapeutic treatment for hepatic fibrosis.     

MiR-22重组腺病毒的构建及对HepG2细胞葡萄糖摄取的影响

文中构建了miR-22重组腺病毒Ad-miR-22,分析了其对HepG2细胞胰岛素信号通路及葡萄糖摄取的抑制作用。通过PCR方法,扩增了miR-22的前体及侧翼序列,酶切后克隆至腺病毒穿梭载体pAdTrack-CMV中,构建穿梭质粒pAdT-22,经PCR及测序鉴定。穿梭质粒经PmeⅠ线性化后,直接转化含有腺病毒骨架载体的感受态细胞BJ5183,产生重组腺病毒质粒Ad-miR-22,最后经PacⅠ线性化后转染包装细胞系293A。重组腺病毒经过3轮扩增后感染HepG2细胞,通过荧光定量PCR检测miR-22表达水平。通过葡萄糖摄取实验观察Ad-miR-22对HepG2细胞葡萄糖摄取的影响。采用Western blotting检测Ad-miR-22对HepG2细胞SIRT1在蛋白质水平的表达及GSK-3β磷酸化水平的影响。采用荧光定量PCR检测miR-22对PEPCK及G6Pase等基因在mRNA水平表达的影响。结果表明,重组腺病毒Ad-miR-22感染显著增加HepG2细胞miR-22表达水平。此外,Ad-miR-22显著抑制胰岛素诱导的HepG2葡萄糖摄取,并通过下调GSK-3β磷酸化抑制胰岛素信号通路的激活。Ad-miR-22反转胰岛素对糖异生关键酶表达的抑制作用,并下调SIRT1基因在蛋白质水平的表达。综上所述,构建了miR-22的重组腺病毒,发现其显著增加糖异生,抑制HepG2细胞葡萄糖摄取,该作用可能与miR-22调节SIRT1在蛋白质水平的表达有关。     

Effect of Bombyx mori on the Liver Protection of Non-Alcoholic Fatty Liver Disease Based on In Vitro and In Vivo Models

Edible insects, Bombyx mori (silkworm; SW), which feed on mulberry leaves, have been consumed by humans for a long time as supplements or traditional medication. Non-alcoholic fatty liver disease (NAFLD) is a liver metabolic disorder that affects many people worldwide. We examined the hepatoprotective effects of SW using in vitro and high-fat and high-fructose (HFHF) diet-induced obese in vivo model mice by real-time PCR, immunoblot analysis, and fecal microbiota analysis. SW significantly reduced lipid accumulation and expression of the lipogenic genes in HepG2 cells and the livers of HFHF-induced mice. SW caused significant reductions in triglycerides, and total cholesterol in serum and upregulation of fatty acid oxidation markers compared to the HFHF group. Besides, SW significantly induced phosphorylation of AMPK and ACC in both models, suggesting roles in AMPK activation and the ACC signaling pathway. Furthermore, the gut microbiota analysis demonstrated that SW treatment reduced Firmicutes to Bacteroidetes ratios and the relative abundance of the Lachnospiraceae family compared to HFHF-induced obese mice. These results provide a novel therapeutic agent of hepatoprotective effects of SW for non-alcoholic hepatic steatosis that targets hepatic AMPK and ACC-mediated lipid metabolism.     

SIRT1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase

Resveratrol may protect against metabolic disease through activating SIRT1 deacetylase. Because we have recently defined AMPK activation as a key mechanism for the beneficial effects of polyphenols on hepatic lipid accumulation, hyperlipidemia, and atherosclerosis in type 1 diabetic mice, we hypothesize that polyphenol-activated SIRT1 acts upstream of AMPK signaling and hepatocellular lipid metabolism. Here we show that polyphenols, including resveratrol and the synthetic polyphenol S17834, increase SIRT1 deacetylase activity, LKB1 phosphorylation at Ser(428), and AMPK activity. Polyphenols substantially prevent the impairment in phosphorylation of AMPK and its downstream target, ACC (acetyl-CoA carboxylase), elevation in expression of FAS (fatty acid synthase), and lipid accumulation in human HepG2 hepatocytes exposed to high glucose. These effects of polyphenols are largely abolished by pharmacological and genetic inhibition of SIRT1, suggesting that the stimulation of AMPK and lipid-lowering effect of polyphenols depend on SIRT1 activity. Furthermore, adenoviral overexpression of SIRT1 stimulates the basal AMPK signaling in HepG2 cells and in the mouse liver. AMPK activation by SIRT1 also protects against FAS induction and lipid accumulation caused by high glucose. Moreover, LKB1, but not CaMKKbeta, is required for activation of AMPK by polyphenols and SIRT1. These findings suggest that SIRT1 functions as a novel upstream regulator for LKB1/AMPK signaling and plays an essential role in the regulation of hepatocyte lipid metabolism. Targeting SIRT1/LKB1/AMPK signaling by polyphenols may have potential therapeutic implications for dyslipidemia and accelerated atherosclerosis in diabetes and age-related diseases.     

Rapamycin-mediated CD36 translational suppression contributes to alleviation of hepatic steatosis

Rapamycin, a mammalian target of rapamycin (mTOR)-specific inhibitor, has the effect of anti-lipid deposition on non-alcoholic fatty liver disease (NAFLD), but the mechanisms with which rapamycin alleviates hepatic steatosis are not fully disclosed. CD36 is known to facilitate long-chain fatty acid uptake and contribute to NAFLD progression. Hepatic CD36 expression is closely associated with hepatic steatosis, while mTOR pathway is involved in CD36 translational control. This study was undertaken to investigate whether rapamycin alleviates hepatic steatosis via the inhibition of mTOR pathway-dependent CD36 translation. Human hepatoblastoma HepG2 cells were treated with palmitate and C57BL/6J mice were fed with high fat diet (HFD) to induce hepatic steatosis. Hepatic CD36 protein expression was significantly increased with lipid accumulation in palmitate-treated HepG2 cells or HFD-fed C57BL/6J mice. Rapamycin reduced hepatic steatosis and CD36 protein expression, but it had no influence on CD36 mRNA expression. Rapamycin had no effect on CD36 protein stability, but it significantly decreased CD36 translational efficiency. We further confirmed that rapamycin inhibited the phosphorylation of mTOR and its downstream translational regulators including p70 ribosomal protein S6 kinase (p70S6K), eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and eukaryotic initiation factor 4E (eIF4E). This study demonstrates that rapamycin inhibits hepatic CD36 translational efficiency through the mTOR pathway, resulting in reduction of CD36 protein expression and alleviation of hepatic steatosis.     

Interleukin-38 alleviates hepatic steatosis through AMPK/autophagy-mediated suppression of endoplasmic reticulum stress in obesity models

Interleukin-38 (IL-38), recently recognized as a cytokine with anti-inflammatory properties that mitigate type 2 diabetes, has been associated with indicators of insulin resistance and nonalcoholic fatty liver disease (NAFLD). This study investigated the impact of IL-38 on hepatic lipid metabolism and endoplasmic reticulum (ER) stress. We assessed protein expression levels using Western blot analysis, while monodansylcadaverine staining was employed to detect autophagosomes in hepatocytes. Oil red O staining was utilized to examine lipid deposition. The study revealed elevated serum IL-38 levels in high-fat diet (HFD)-fed mice and IL-38 secretion from mouse keratinocytes. IL-38 treatment attenuated lipogenic lipid accumulation and ER stress markers in hepatocytes exposed to palmitate. Furthermore, IL-38 treatment increased AMP-activated protein kinase (AMPK) phosphorylation and autophagy. The effects of IL-38 on lipogenic lipid deposition and ER stress were nullified in cultured hepatocytes by suppressing AMPK through small interfering (si) RNA or 3-methyladenine (3MA). In animal studies, IL-38 administration mitigated hepatic steatosis by suppressing the expression of lipogenic proteins and ER stress markers while reversing AMPK phosphorylation and autophagy markers in the livers of HFD-fed mice. Additionally, AMPK siRNA, but not 3MA, mitigated IL-38-enhanced fatty acid oxidation in hepatocytes. In summary, IL-38 alleviates hepatic steatosis through AMPK/autophagy signaling-dependent attenuation of ER stress and enhancement of fatty acid oxidation via the AMPK pathway, suggesting a therapeutic strategy for treating NAFLD.     

Inflammatory Stress Increases Hepatic CD36 Translational Efficiency via Activation of the mTOR Signalling Pathway

Inflammatory stress is an independent risk factor for the development of non-alcoholic fatty liver disease (NAFLD). Although CD36 is known to facilitate long-chain fatty acid uptake and contributes to NAFLD progression, the mechanisms that link inflammatory stress to hepatic CD36 expression and steatosis remain unclear. As the mammalian target of rapamycin (mTOR) signalling pathway is involved in CD36 translational activation, this study was undertaken to investigate whether inflammatory stress enhances hepatic CD36 expression via mTOR signalling pathway and the underlying mechanisms. To induce inflammatory stress, we used tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) stimulation of the human hepatoblastoma HepG2 cells in vitro and casein injection in C57BL/6J mice in vivo. The data showed that inflammatory stress increased hepatic CD36 protein levels but had no effect on mRNA expression. A protein degradation assay revealed that CD36 protein stability was not different between HepG2 cells treated with or without TNF-α or IL-6. A polysomal analysis indicated that CD36 translational efficiency was significantly increased by inflammatory stress. Additionally, inflammatory stress enhanced the phosphorylation of mTOR and its downstream translational regulators including p70S6K, 4E-BP1 and eIF4E. Rapamycin, an mTOR-specific inhibitor, reduced the phosphorylation of mTOR signalling pathway and decreased the CD36 translational efficiency and protein level even under inflammatory stress resulting in the alleviation of inflammatory stress-induced hepatic lipid accumulation. This study demonstrates that the activation of the mTOR signalling pathway increases hepatic CD36 translational efficiency, resulting in increased CD36 protein expression under inflammatory stress.     

Downregulation of miR-192 causes hepatic steatosis and lipid accumulation by inducing SREBF1: Novel mechanism for bisphenol A-triggered non-alcoholic fatty liver disease

Exposure to Bisphenol A (BPA) has been associated with the development of nonalcoholic fatty liver disease (NAFLD) but the underlying mechanism remains unclear. Given that microRNA (miRNA) is recognized as a key regulator of lipid metabolism and a potential mediator of environmental cues, this study was designed to explore whether exposure to BPA-triggered abnormal steatosis and lipid accumulation in the liver could be modulated by miR-192. We showed that male post-weaning C57BL/6 mice exposed to 50 μg/kg/day of BPA by oral gavage for 90 days displayed a NAFLD-like phenotype. In addition, we found in mouse liver and human HepG2 cells that BPA-induced hepatic steatosis and lipid accumulation were associated with decreased expression of miR-192, upregulation of SREBF1 and a series of genes involved in de novo lipogenesis. Downregulation of miR-192 in BPA-exposed hepatocytes could be due to defective pre-miR-192 processing by DROSHA. Using HepG2 cells, we further confirmed that miR-192 directly acted on the 3′UTR of SREBF1, contributing to dysregulation of lipid homeostasis in hepatocytes. MiR-192 mimic and lentivirus-mediated overexpression of miR-192 improved BPA-induced hepatic steatosis by suppressing SREBF1. Lastly, we noted that lipid accumulation was not a strict requirement for developing insulin resistance in mice after BPA treatment. In conclusion, this study demonstrated a novel mechanism in which NAFLD associated with BPA exposure arose from alterations in the miR-192-SREBF1 axis.     

Hypoxia inducible factor-1 promotes liver fibrosis in nonalcoholic fatty liver disease by activating PTEN/p65 signaling pathway

Obesity is a major contributor to the development of steatohepatitis and fibrosis from nonalcoholic fatty liver disease (NAFLD). Hypoxia aggravates progression of NAFLD. In mice on high-fat diet (HFD), hepatic steatosis leads to liver tissue hypoxia, evidenced by accumulation of hypoxia inducible factor-1-alpha (HIF-1α), which is a central regulator of the global response to hypoxia. Hepatocyte cell signaling is an important factor in hepatic fibrogenesis. We here hypothesize that HIF-1α knockout in hepatocyte may protect against liver fibrosis. We first found that HFD led to 80% more hepatic collagen deposition than Hif1a^−/−hep mice, which was confirmed by a-SMA staining of liver tissue. Body weight and liver weight were similar between groups. We then found the increasing HIF1a expression and decreasing PTEN expression in the mice on HFD and in PA-treated HepG2 cells. Finally, we found that HIF1 mediated PTEN/nfkb-p65 pathway plays an important role in the development of NAFLD to liver fibrosis. Collectively, these results identify a novel HIF1a/PTEN/NF-κ Bp65 signaling pathway in NAFLD, which could be targeted for the therapy.     

The role of AMPKα2 in the HFD-induced nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is associated with hepatic steatosis, inflammation and liver fibrosis and has become one of the leading causes of hepatocellular carcinoma and liver failure. However, the underlying molecular mechanism of hepatic steatosis and the progression to nonalcoholic steatohepatitis (NASH) are not fully understood. Herein, we discovered that AMPKα2 catalytic subunit showed reduced expression in the liver following high fat diet (HFD) feeding to mice. Importantly, knockout of AMPKα2 in mice aggravated NAFLD, hepatic steatosis, inflammation and fibrosis. On the other hand, hepatocyte-targeted overexpression of AMPKα2 prevented or reversed NAFLD indications. In vivo mechanistic studies revealed that increased phosphorylation of IKKα/β and NF-κB in HFD-fed AMPKα2^−/− mice compared to WT mice, and treatment of these mouse cohorts with an inhibitor of NF-κB signaling for 4 weeks, effectively attenuated the progression of steatohepatitis and metabolic disorder features. In summary, AMPKα2 provides a protective role in the process of hepatic steatosis to NASH progression through suppression of liver NF-κB signaling.     

Resveratrol alleviates alcoholic fatty liver in mice

Alcoholic fatty liver is associated with inhibition of sirtuin 1 (SIRT1) and AMP-activated kinase (AMPK), two critical signaling molecules regulating the pathways of hepatic lipid metabolism in animals. Resveratrol, a dietary polyphenol, has been identified as a potent activator for both SIRT1 and AMPK. In the present study, we have carried out in vivo animal experiments that test the ability of resveratrol to reverse the inhibitory effects of chronic ethanol feeding on hepatic SIRT1-AMPK signaling system and to prevent the development of alcoholic liver steatosis. Resveratrol treatment increased SIRT1 expression levels and stimulated AMPK activity in livers of ethanol-fed mice. The resveratrol-mediated increase in activities of SIRT1 and AMPK was associated with suppression of sterol regulatory element binding protein 1 (SREBP-1) and activation of peroxisome proliferator-activated receptor gamma coactivator alpha (PGC-1alpha). In parallel, in ethanol-fed mice, resveratrol administration markedly increased circulating adiponectin levels and enhanced mRNA expression of hepatic adiponectin receptors (AdipoR1/R2). In conclusion, resveratrol treatment led to reduced lipid synthesis and increased rates of fatty acid oxidation and prevented alcoholic liver steatosis. The protective action of resveratrol is in whole or in part mediated through the upregulation of a SIRT1-AMPK signaling system in the livers of ethanol-fed mice. Our study suggests that resveratrol may serve as a promising agent for preventing or treating human alcoholic fatty liver disease.     

Sirtuin 1 (SIRT1) protein degradation in response to persistent c-Jun N-terminal kinase 1 (JNK1) activation contributes to hepatic steatosis in obesity

SIRT1 is involved in the pathogenesis of obesity, diabetes, and aging. However, it is not clear how SIRT1 activity is regulated by intracellular kinases in cells. In this study, we investigated SIRT1 phosphorylation and protein degradation in response to JNK1 activation in obese mice. Mouse SIRT1 is phosphorylated by JNK1 at Ser-46 (Ser-47 in human SIRT1), which is one of the four potential residues targeted by JNK1. The phosphorylation induces a brief activation of SIRT1 function and degradation of SIRT1 thereafter by the proteasome. Ubiquitination occurs in SIRT1 protein after the phosphorylation. Mutation of Ser-46 to alanine prevents the phosphorylation, ubiquitination, and degradation. In vivo, SIRT1 undergoes an extensive degradation in hepatocytes in obesity as a consequence of persistent activation of JNK1. The degradation leads to inhibition of SIRT1 function, which contributes to development of hepatic steatosis. The degradation disappears in obesity when JNK1 is inactivated in mice. JNK2 exhibits an opposite activity in the regulation of SIRT1 degradation. The JNK1-SIRT1 pathway provides a new molecular mechanism for the pathogenesis of hepatic steatosis in obesity.     

Effects of homocysteine and its related compounds on oxygen consumption of the rat heart tissue homogenate: the role of different gasotransmitters

Accumulating evidence indicates that microRNAs are implicated in tumor initiation and progression through negatively regulating oncogenes or tumor suppressor genes. In the present study, we report that the expression of miR-200a was significantly lower in renal cell carcinoma (RCC) specimens and RCC cell lines. Restoration of miR-200a suppressed cell growth, arrested cell cycle progression, and promoted cell apoptosis in RCC cell lines. We next used qRT-PCR array technology to identify Sirtuin 1 (SIRT1) as one of the downregulated proteins during miR-200a overexpression in 786-O cells. Following a further assay by luciferase reporter system, SIRT1 was validated as a direct target of miR-200a. Moreover, siRNA-mediated knockdown of SIRT1 could partially phenocopy the effects of miR-200a overexpression. In contrast, overexpression of truncated SIRT1 (without an endogenous 3′-UTR) could rescue the effect of miR-200a overexpression on 786-O cells, which suggested that SIRT1 3′-UTR is targeted by miR-200a specifically. These observations provide further evidence for a critical tumor-suppressive role of the miR-200a in RCC in addition to identifying a novel regulatory mechanism, which may contribute to SIRT1 upregulation in RCC.     

Circ_0057558 promotes nonalcoholic fatty liver disease by regulating ROCK1/AMPK signaling through targeting miR-206

Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic liver disorders that is featured by the extensive deposition of fat in the hepatocytes. Current treatments are very limited due to its unclear pathogenesis. Here, we investigated the function of circ_0057558 and miR-206 in NAFLD. High-fat diet (HFD) feeding mouse was used as an in vivo NAFLD model and long-chain-free fatty acid (FFA)-treated liver cells were used as an in vitro NAFLD model. qRT-PCR was used to measure levels of miR-206, ROCK1 mRNA, and circ_0057558, while Western blotting was employed to determine protein levels of ROCK1, p-AMPK, AMPK, and lipogenesis-related proteins. Immunohistochemistry were performed to examine ROCK1 level. Oil-Red O staining was used to assess the lipid deposition in cells. ELISA was performed to examine secreted triglyceride (TG) level. Dual-luciferase assay was used to validate interactions of miR-206/ROCK1 and circ_0057558/miR-206. RNA immunoprecipitation was employed to confirm the binding of circ_0057558 with miR-206. Circ_0057558 was elevated while miR-206 was reduced in both in vivo and in vitro NAFLD models. miR-206 directly bound with ROCK1 3’-UTR and suppressed lipogenesis and TG secretion through targeting ROCK1/AMPK signaling. Circ_0057558 directly interacted with miR-206 to disinhibit ROCK1/AMPK signaling. Knockdown of circ_0057558 or overexpression of miR-206 inhibited lipogenesis, TG secretion and expression of lipogenesis-related proteins. ROCK1 knockdown reversed the effects of circ_0057558 overexpression. Injection of miR-206 mimics significantly ameliorated NAFLD progression in vivo. Circ_0057558 acts as a miR-206 sponge to de-repress the ROCK1/AMPK signaling and facilitates lipogenesis and TG secretion, which greatly contributes to NAFLD development and progression.Subject terms: Diseases, Gastrointestinal diseases