Effects of 2-Methoxyestradiol, a Main Metabolite of Estradiol on Hepatic ABCA1 Expression in HepG2 Cells

ATP-binding cassette transporter A1 (ABCA1) is a key regulator of lipid efflux, and the absence of ABCA1 induces hepatic lipid accumulation, which is one of the major causes of fatty liver. 2-Methoxyestradiol (2-ME2) has been demonstrated to protect against fatty liver. In this study, we investigated the effects of 2-ME2 on the hepatic lipid content and ABCA1 expression. We found that 2-ME2 dose-dependently increased ABCA1 expression, and therefore, the lipid content was significantly decreased in HepG2 cells. 2-ME2 enhanced the ABCA1 promoter activity; however, this effect was reduced after the inhibition of the PI3K pathway. The overexpression of Akt or p110 induced ABCA1 promoter activity, while dominant-negative Akt diminished the ability of 2-ME2 on ABCA1 promoter activity. Further, 2-ME2 stimulated the rapid phosphorylation of Akt and FoxO1 and reduced the nuclear accumulation of FoxO1. Chromatin immunoprecipitation confirmed that FoxO1 bonded to the ABCA1 promoter region. The binding was reduced by 2-ME2, which facilitated ABCA1 gene transcription. Furthermore, mutating FoxO1-binding sites in the ABCA1 promoter region or treatment with FoxO1-specific siRNA disrupted the effect of 2-ME2 on ABCA1 expression. All of our results demonstrated that 2-ME2 might upregulate ABCA1 expression via the PI3K/Akt/FoxO1 pathway, which thus reduces the lipid content in hepatocytes.

Traditional Chinese medicine Lingguizhugan decoction ameliorate HFD-induced hepatic-lipid deposition in mice by inhibiting STING-mediated inflammation in macrophages

Background Stimulator of IFN genes (STING) is highly expressed in the livers of non-alcoholic fatty liver disease (NAFLD) patients and high fat diet (HFD) induced NAFLD mice model. The STING signaling-mediated inflammation has been shown to play a critical role in metabolic disorders. Lingguizhugan decoction (LGZG), a Traditional Chinese herbal decoction, has been applied to treat metabolic disorders for many years. However, whether LGZG can alleviate the progression of NAFLD through inhibiting inflammation remains unclear. This study was to determine the role of STING-mediated inflammation in the HFD-induced hepatic-lipid deposition treated with LGZG. Methods The anti-inflammatory and anti-steatotic effects of LGZG in vivo were detected by H&E staining, immunofluorescence and immuno-chemistry. Mice bone-marrow-derived macrophages (BMDMs) and primary liver macrophages were treated with STING-specific agonist (DMXAA), LGZG and its critical components respectively. The treated culture supernatant of BMDMs and primary liver macrophages from each group was co-cultured with palmitic acid-treated mouse primary hepatocytes or mouse liver cell line AML-12 respectively to detect whether the activation of STING-mediated pathway is involved in the anti-steatotic effect of LGZG. The hepatocyte lipid deposition in vivo and in vitro were detected by oil red staining. Mitochondrial DNA release of mouse liver extracts were detected by real time PCR. The expression of proteins and inflammatory cytokines related to STING-TBK1-NF-κB pathway was detected by western blotting and ELISA. Results LGZG significantly ameliorated HFD induced hepatic steatosis, oxidative stress, hepatic mitochondrial damage and mitochondrial DNA release, which was correlated with reduction of the expression level of STING as well as the infiltration of STING-positive macrophages in the livers of HFD fed mice. The critical components of LGZG directly inhibited the activation of STING-TBK1-NF-κB pathway in liver macrophages induced by DMXAA, LPS, thereby reducing the release of IFNβ and TNFα. Co-incubating the culture supernatant of LGZG treated liver macrophages and PA-stimulated hepatocytes significantly inhibited the PA-induced lipid deposition. Conclusion This study demonstrates that LGZG can ameliorate HFD-induced hepatic-lipid deposition through inhibiting STING-TBK1-NF-κB pathway in liver macrophages, which provides novel insight for elucidating the molecular mechanism of LGZG alleviating HFD induced hepatic steatosis.

Hepatic MIR20B promotes nonalcoholic fatty liver disease by suppressing PPARA

Background:Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation and imbalances in lipid metabolism in the liver. Although nuclear receptors (NRs) play a crucial role in hepatic lipid metabolism, the underlying mechanisms of NR regulation in NAFLD remain largely unclear. Methods:Using network analysis and RNA-seq to determine the correlation between NRs and microRNA in human NAFLD patients, we revealed that MIR20B specifically targets PPARA. MIR20B mimic and anti-MIR20B were administered to human HepG2 and Huh-7 cells and mouse primary hepatocytes as well as high fat diet (HFD)- or methionine-deficient diet (MCD)-fed mice to verify the specific function of MIR20B in NAFLD. We tested the inhibition of the therapeutic effect of a PPARα agonist, fenofibrate, by Mir20b and the synergic effect of combination of fenofibrate with anti-Mir20b in NAFLD mouse model. Results:We revealed that MIR20B specifically targets PPARA through miRNA regulatory network analysis of nuclear receptor genes in NAFLD. The expression of MIR20B was upregulated in free fatty acid (FA)-treated hepatocytes and the livers of both obesity-induced mice and NAFLD patients. Overexpression of MIR20B significantly increased hepatic lipid accumulation and triglyceride levels. Furthermore, MIR20B significantly reduced FA oxidation and mitochondrial biogenesis by targeting PPARA. In Mir20b-introduced mice, the effect of fenofibrate to ameliorate hepatic steatosis was significantly suppressed. Finally, inhibition of Mir20b significantly increased FA oxidation and uptake, resulting in improved insulin sensitivity and a decrease in NAFLD progression. Moreover, combination of fenofibrate and anti-Mir20b exhibited the synergic effect on improvement of NAFLD in MCD-fed mice. Conclusions:Taken together, our results demonstrate that the novel MIR20B targets PPARA, plays a significant role in hepatic lipid metabolism, and present an opportunity for the development of novel therapeutics for NAFLD. Funding:This research was funded by Korea Mouse Phenotyping Project (2016M3A9D5A01952411), the National Research Foundation of Korea (NRF) grant funded by the Korea government (2020R1F1A1061267, 2018R1A5A1024340, NRF-2021R1I1A2041463, 2020R1I1A1A01074940), and the Future-leading Project Research Fund (1.210034.01) of UNIST.

MIF-2/D-DT is an atypical atherogenic chemokine that promotes advanced atherosclerosis and hepatic lipogenesis

Atherosclerosis is the underlying cause of cardiovascular diseases (CVDs) such as myocardial infarction and ischemic stroke. It is a lipid-triggered chronic inflammatory condition of the arterial vascular wall that is driven by various inflammatory pathways including atherogenic cytokines and chemokines. D-dopachrome tautomerase (D-DT), also known as macrophage migration inhibitory factor-2 (MIF-2), belongs to the MIF protein family, which is best known for its pathogenic role in a variety of inflammatory and immune conditions including CVDs. While MIF is well known as a promoter of atherogenic processes, MIF-2 has not been studied in atherosclerosis. Here, we investigated atherosclerosis in hyperlipidemic Mif-2-/-Apoe-/- mice and studied the role of MIF-2 in various atherogenic assays in vitro. We found that global Mif-2 deficiency as well as its pharmacological blockade by 4-CPPC protected against atherosclerotic lesion formation and vascular inflammation in models of early and advanced atherogenesis. On cellular level, MIF-2 promoted monocyte migration in 2D and 3D and monocyte arrest on aortic endothelial monolayers, promoted B-cell chemotaxis in vitro and B-cell homing in vivo, and increased macrophage foam cell formation. Dose curves and direct comparison in a 3D migration set-up suggest that MIF-2 may be a more potent chemokine than MIF for monocytes and B cells. We identify CXCR4 as a novel receptor for MIF-2. The evidence relies on a CXCR4 inhibitor, CXCR4 internalization experiments, MIF-2/CXCR4 binding studies by yeast-CXCR4 transformants, and fluorescence spectroscopic titrations with a soluble CXCR4 surrogate. Of note, Mif-2-/- Apoe-/- mice exhibited decreased plasma cholesterol and triglyceride levels, lower body weights, smaller livers, and profoundly reduced hepatic lipid accumulation compared to Apoe-/- mice. Mechanistic experiments in Huh-7 hepatocytes suggest that MIF-2 regulates the expression and activation of sterol-regulatory element binding protein-1 and -2 (SREBP-1, SREBP-2) to induce lipogenic downstream genes such as FASN and LDLR, while it attenuated the activation of the SREBP inhibiting AMPK pathway. Studies using receptor Inhibitors showed that SREBP activation and hepatic lipoprotein uptake by MIF-2 is mediated by both CXCR4 and CD74. Lastly and in line with a combined role of MIF-2 in vascular inflammation and hepatic lipid accumulation, MIF-2 was found to be profoundly upregulated in unstable human carotid plaques, underscoring a critical role for MIF-2 in advanced stages of atherosclerosis. Together, these data identify MIF-2 as a novel atherogenic chemokine and CXCR4 ligand that not only promotes lesion formation and vascular inflammation but also strongly affects hepatic lipogenesis in an SREBP-mediated manner, possibly linking atherosclerosis and hepatic steatosis.

The Beneficial Additive Effect of Silymarin in Metformin Therapy of Liver Steatosis in a Pre-Diabetic Model

The combination of plant-derived compounds with anti-diabetic agents to manage hepatic steatosis closely associated with diabetes mellitus may be a new therapeutic approach. Silymarin, a complex of bioactive substances extracted from Silybum marianum, evinces an antioxidative, anti-inflammatory, and hepatoprotective activity. In this study, we investigated whether metformin (300 mg/kg/day for four weeks) supplemented with micronized silymarin (600 mg/kg/day) would be effective in mitigating fatty liver disturbances in a pre-diabetic model with dyslipidemia. Compared with metformin monotherapy, the metformin–silymarin combination reduced the content of neutral lipids (TAGs) and lipotoxic intermediates (DAGs). Hepatic gene expression of enzymes and transcription factors involved in lipogenesis (Scd-1, Srebp1, Pparγ, and Nr1h) and fatty acid oxidation (Pparα) were positively affected, with hepatic lipid accumulation reducing as a result. Combination therapy also positively influenced arachidonic acid metabolism, including its metabolites (14,15-EET and 20-HETE), mitigating inflammation and oxidative stress. Changes in the gene expression of cytochrome P450 enzymes, particularly Cyp4A, can improve hepatic lipid metabolism and moderate inflammation. All these effects play a significant role in ameliorating insulin resistance, a principal background of liver steatosis closely linked to T2DM. The additive effect of silymarin in metformin therapy can mitigate fatty liver development in the pre-diabetic state and before the onset of diabetes.

Cannabis sativa L. (var. indica) Exhibits Hepatoprotective Effects by Modulating Hepatic Lipid Profile and Mitigating Gluconeogenesis and Cholinergic Dysfunction in Oxidative Hepatic Injury

Cannabis sativa L. is a crop utilized globally for recreational, therapeutic, and religious purposes. Although considered as an illicit drug in most countries, C. sativa until recently started gaining attention for its medicinal application. This study sought to investigate the hepatoprotective effect of C. sativa on iron-mediated oxidative hepatic injury. Hepatic injury was induced ex vivo by incubating hepatic tissues with Fe2+, which led to depleted levels of reduced glutathione, superoxide dismutase, catalase and ENTPDase activities, triglyceride, and high-density lipoprotein–cholesterol (HDL-C). Induction of hepatic injury also caused significant elevation of malondialdehyde, nitric oxide, cholesterol, and low-density lipoprotein–cholesterol (LDL-C) levels while concomitantly elevating the activities of ATPase, glycogen phosphorylase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, amylase, and lipase. Treatment with the hexane, dichloromethane (DCM), and ethanol extracts of C. sativa leaves significantly (p < 0.05) reversed these levels and activities to almost near normal. However, there was no significant effect on the HDL-C level. The extracts also improved the utilization of glucose in Chang liver cells. High-performance liquid chromatography (HPLC) analysis showed the presence of phenolics in all extracts, with the ethanol extract having the highest constituents. Cannabidiol (CBD) was identified in all the extracts, while Δ-9-tetrahydrocannabinol (Δ-9-THC) was identified in the hexane and DCM extracts only. Molecular docking studies revealed strong interactions between CBD and Δ-9-THC with the β2 adrenergic receptor of the adrenergic system. The results demonstrate the potential of C. sativa to protect against oxidative-mediated hepatic injury by stalling oxidative stress, gluconeogenesis, and hepatic lipid accumulation while modulating cholinergic and purinergic activities. These activities may be associated with the synergistic effect of the compounds identified and possible interactions with the adrenergic system.

Structural and Functional Modulation of Gut Microbiota by Jiangzhi Granules during the Amelioration of Nonalcoholic Fatty Liver Disease

Recently, accumulating evidence revealed that nonalcoholic fatty liver disease (NAFLD) is highly associated with the dysbiosis of gut microbiota. Jiang Zhi Granule (JZG), which is composed of five widely used Chinese herbs, has shown hypolipidemic effect, while whether such effect is mediated by gut microbiota is still unclear. Here, we found that both low and high doses of JZG (LJZ and HJZ) could improve hepatic steatosis and function, as well as insulin resistance in NAFLD mice. 16S rRNA gene sequencing revealed that JZG treatment could reverse the dysbiosis of intestinal flora in NAFLD mice, exhibiting a dose-dependent effect. Notably, HJZ could significantly reduce the relative abundance of Desulfovibrionaceae, while increasing the relative abundance of such as S24_7 and Lachnospiraceae. PICRUSt analysis showed that HJZ could significantly alter the functional profile of gut microbiota, including the reduction of the lipopolysaccharide biosynthesis and sulfur metabolism pathway, which is verified by the decreased levels of fecal hydrogen sulfide (H2S) and serum lipopolysaccharide binding protein (LBP). In addition, hepatic mRNA sequencing further indicated that the HJZ group can regulate the peroxisome proliferator-activated receptor (PPAR) pathway and inflammatory signaling pathway, as validated by RT-PCR and Western blot. We also found that different doses of JZG may regulate lipid metabolism through differentiated pathways, as LJZ mainly through the promotion of hepatic lipid hydrolysis, while HJZ mainly through the improvement of hepatic lipid oxidation. Taken together, JZG could modulate gut dysbiosis with dose-effect, alleviate inflammation level, and regulate hepatic lipid metabolism, which may subsequently contribute to the improvement of NAFLD. Our study revealed the underlying mechanisms in the improvement of NAFLD by a Chinese herbal compound, providing future guidance for clinical usage.

Hepatic Lipid Metabolism Disorder and Atherosclerosis

: Lipid metabolism disorder plays a fundamental role in the pathogenesis of atherosclerosis. As the largest metabolic organ of the human body, liver has a key role in lipid metabolism by influencing fat production, fat decomposition, and the intake and secretion of serum lipoproteins. Numerous clinical and experimental studies have indicated that the dysfunction of hepatic lipid metabolism is closely tied to the onset of atherosclerosis. However, the identity and functional role of hepatic lipid metabolism responsible for these associations remain unknown. This review presented that cholesterol synthesis, cholesterol transport, and the metabolism of triglyceride, lipoproteins, and fatty acids are all associated with hepatic lipid metabolism and atherosclerosis. Moreover, we also discussed the roles of gut microbiota, inflammatory response, and oxidative stress in the pathological association between hepatic lipid metabolism and atherosclerosis. These significant evidences support strongly that hepatic lipid metabolism disorders may increase the risk of atherosclerosis.