Simultaneous Dual-Channel Tracking Lipid Droplets Dynamic Behaviors and Revealing the Protective Role in Nonalcoholic Fatty Liver in mice.

Objective Our recent study demonstrated that growth differentiation factor 5 (GDF5) could promote white adipose tissue thermogenesis and alleviate high-fat diet- (HFD-) induced obesity in fatty acid-binding protein 4- (Fabp4-) GDF5 transgenic mice (TG). Here, we further investigated the effects of systemic overexpression of the GDF5 gene in adipocytes HFD-induced nonalcoholic fatty liver disease (NAFLD). Methods Fabp4-GDF5 TG mice were administered an HFD feeding. NAFLD-related indicators associated with lipid metabolism and inflammation were measured. A GDF5 lentiviral vector was constructed, and the LO2 NAFLD cell model was induced by FFA solution (oleic acid and palmitic acid). The alterations in liver function, liver lipid metabolism, and related inflammatory indicators were analyzed. Results The liver weight was significantly reduced in the TG group, which was in accordance with the significantly downregulated expression of TNFα, MCP1, Aim2, and SREBP-1c and significantly upregulated expression of CPT-1α and ACOX2 in TG mouse livers. Compared to that of cells in the FAA-free control group, LO2 cells with in situ overexpression of GDF5 developed lipid droplets after FFA treatment; the levels of triglycerides, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were significantly increased in both the GDF5 lentivirus and control lentivirus groups compared with those of the FAA-free group. Additionally, the levels of FAS, SREBP-1, CPT-1α, and inflammation-associated genes, such as ASC and NLRC4, were unaltered despite GDF5 treatment. Conclusion Systemic overexpression of GDF5 in adipose tissue in vivo significantly reduced HFD-induced NAFLD liver damage in mice. The overexpression of GDF5 in hepatocytes failed to improve lipid accumulation and inflammation-related reactions induced by mixed fatty acids, suggesting that the protective effect of GDF5 in NAFLD was mainly due to the reduction in adipose tissue and improvements in metabolism. Hence, our study suggests that the management of NAFLD should be targeted to reduce the overall amount of body fat and improve metabolic status before the progression to nonalcoholic steatohepatitis occurs.

Type 2 diabetes mellitus (T2DM) is a common chronic metabolic disease. Accumulating evidence has demonstrated that nonalcoholic fatty liver disease (NAFLD) shares common typical features with T2DM, and they affect each other extensively. Thus, NAFLD has emerged as a novel target for T2DM prevention and care. Although Corni Fructus (CF) and its extracts have a therapeutic effect on T2DM, its effects and mechanisms on T2DM with NAFLD are far from elucidated. In this study, a mouse model of T2DM with NAFLD complication was established in ICR mice by feeding a high-fat, high-sugar (HFHS) diet and intraperitoneally injecting with a low dose of streptozotocin (STZ). Then, the effects of iridoid glycosides (IG) extracted from CF on this mouse model were investigated. We found that 4-week IG administration remarkably alleviated hyperglycemia and insulin resistance and significantly reduced inflammation, oxidative stress, and fat accumulation in the liver of T2DM with NAFLD mice. Further studies showed that IG inhibited the NF-κB but enhanced the PI3K-AKT signaling pathway. In summary, these results indicated that the IG from CF has potential therapeutic effects on T2DM with NAFLD.

Background As the most common chronic liver disease, non-alcoholic fatty liver disease (NAFLD) affects 10-30% of the general population. Characterized by excessive lipid droplet formation in hepatocytes, fatty liver may progress to nonalcoholic steatohepatitis (NASH), eventually leading to cirrhosis and liver failure. Currently, there have been no effective therapeutic or preventive measures for NAFLD. Thus, there is an urgent need to identify hepatic mediators that promote lipid droplet accumulation as well as the underlying molecular mechanisms in the liver during the pathogenesis of NAFLD. We previously reported that E4 promoter-binding protein 4 (E4BP4) is an insulin-induced stabilizer of the lipogenic factor SREBP-1c and promotes the SREBP-1c-mediated lipogenesis in hepatocytes. Our most recent mass spectrometry data revealed that E4BP4 may undergo SUMOylation, a reversible post-translational modification that was shown to regulate E4BP4-dependent NK cell development. In this study, we set out to determine how hepatic E4BP4 regulates lipid droplet formation and liver steatosis a high-fat diet-induced NAFLD mouse model and investigate how E4BP4 SUMOylation impacts the process. Results Compared with E4bp4flox/flox mice, E4bp4 liver-specific KO (E4bp4-LKO) mice exhibit decreased lipid accumulation in the liver despite similar body weight after 12 weeks of high-fat diet feeding. Our microarray analysis showed a reduction in the expression of lipid droplet biding genes such as Cidea, Cidec (Fsp27), Plin4 in the liver of E4bp4-LKO mice. Restoring E4bp4 expression in E4bp4-LKO primary hepatocytes was sufficient to induce lipid droplet formation and Fsp27β expression, while overexpression of Fsp27β increased lipid droplets and triglycerides in E4bp4-LKO primary mouse hepatocytes and promoted lipid accumulation HFD-fed E4bp4-LKO mice. Meanwhile, we generated an E4BP4 mutant, namely E4BP4-5KR, which contains five lysine to arginine (5KR) substitutions in the five SUMOylation motifs. Our in vitro SUMOylation assay confirmed the SUMOylation of E4BP4-WT but not E4BP4-5KR mutant in the presence of E1/2, SUMO2/3, and ATP. We detected more lipid droplet formation in hepa1 cells transfected with E4bp4-5KR vs. E4bp4-WT. Lastly, the abundance of SUMOylated E4BP4 was reduced in the liver of high-fat diet-fed mice vs. regular chow-fed mice. Conclusion All together, our data demonstrated that E4bp4 drives lipid droplet formation and live steatosis in high-fat diet-fed mice likely through its regulation of lipid-droplet binding genes. Our study also highlights the critical role of deSUMOylation of hepatic E4BP4 in promoting NAFLD and suggests that E4BP4 SUMOylation in the liver could be targeted for treating NAFLD.

Helicobacter pylori infection aggravates diet-induced nonalcoholic fatty liver in mice

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation, which is the most common form of chronic liver disease. Multiple clinical studies using natural compounds such as flavonoids have been conducted to treat NAFLD. In the present study, the pharmacological effect of Citrus aurantium L. (Rutaceae) peel extract (CAE), which contains over 27% of polymethoxyflavone nobiletin, on NAFLD was evaluated using a high-fat diet (HFD) animal model susceptible to developing NAFLD. C57BL/6 mice were fed an HFD (60% kcal of energy derived from fat) for 8 weeks to induce obesity. Obese mice were randomly allocated to four groups of eight mice each (HFD alone, HFD with silymarin, HFD with 50 mg/kg CAE, and HFD with 100 mg/kg CAE). After 8 weeks of treatment, all mice were euthanized, and plasma and liver tissues were analyzed biochemically and histopathologically. The results indicate that CAE treatment significantly reduced HFD-induced NAFLD, as shown by decreased serum lipid index and prevented liver histopathology. The expression of genes involved in lipid synthesis including free fatty acid (FFA), peroxisome-proliferator-activated receptor γ (PPAR-γ), sterol receptor element binding protein 1c (SREBP-1c), and fatty acid synthesis enzyme was suppressed by CAE treatment. Moreover, compared to untreated mice, CAE-treated HFD mice showed decreased pro-inflammatory cytokine expression. These results demonstrated that CAE prevented HFD-induced NAFLD by reducing plasma levels of triglyceride and cholesterol and de novo lipid synthesis.

The accumulation of hepatic lipid droplets (LDs) is a hallmark of non-alcoholic fatty liver disease (NAFLD). Appropriate degradation of hepatic LDs and oxidation of complete free fatty acids (FFAs) are important for preventing the development of NAFLD. Histone deacetylase (HDAC) is involved in the impaired lipid metabolism seen in high-fat diet (HFD)-induced obese mice. Here, we evaluated the effect of MS-275, an inhibitor of HDAC1/3, on the degradation of hepatic LDs and FFA oxidation in HFD-induced NAFLD mice. To assess the dynamic degradation of hepatic LDs and FFA oxidation in fatty livers of MS-275-treated HFD C57BL/6J mice, an intravital two-photon imaging system was used and biochemical analysis was performed. The MS-275 improved hepatic metabolic alterations in HFD-induced fatty liver by increasing the dynamic degradation of hepatic LDs and the interaction between LDs and lysozyme in the fatty liver. Numerous peri-droplet mitochondria, lipolysis, and lipophagy were observed in the MS-275-treated mouse fatty liver. Biochemical analysis revealed that the lipolysis and autophagy pathways were activated in MS-275 treated mouse liver. In addition, MS-275 reduced the de novo lipogenesis, but increased the mitochondrial oxidation and the expression levels of oxidation-related genes, such as PPARa, MCAD, CPT1b, and FGF21. Taken together, these results suggest that MS-275 stimulates the degradation of hepatic LDs and mitochondrial free fatty acid oxidation, thus protecting against HFD-induced NAFLD.

The protective effects of the total glycosides from Ligustri Lucidi Fructus against nonalcoholic fatty liver (NAFL) in mice were investigated. Liver injury was induced by the administration of high fat diet for 60 days. During this period, the model group received high fat diet only; the treatment groups received various drugs plus high fat diet. Compared with the model group, the total glycosides significantly decreased the contents of triglyceride (TG) and cholesterol (TC), as well as the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum. Moreover, the contents of TG and TC in liver tissue and the liver index were reduced. Histological findings also confirmed antisteatosis. Compared with the model group, total glycosides significantly reduced the levels of the sterol regulatory element binding protein-1c (SREBP-1c) and liver X receptor-a (LXR-α) protein, and down-regulated the expression of SREBP-1c, LXR-α and interleukin-6 (IL-6) mRNA in the liver. These results suggest that the total glycosides are effective in the treatment of NAFL of mice. Their mode of action is associated with inhibiting SREBP-1c, LXR-α and IL-6 mRNA, reducing lipid synthesis factor SREBP-1c and LXR-α protein and gene expression, suppressing inflammatory responses, then decreasing serum lipid and hepatic lipid.

Gypenosides improve the intestinal microbiota of non-alcoholic fatty liver in mice and alleviate its progression

The objective of this study was to investigate the protective effect of Crataegus pinnatifida polysaccharide (CPP) on non-alcoholic fatty liver disease (NAFLD) induced by a high-fat diet (HFD) in mice. The findings demonstrated that CPP improved free fatty acid (FFA)-induced lipid accumulation in HepG2 cells and effectively reduced liver steatosis and epididymal fat weight in NAFLD mice, as well as decreased serum levels of TG, TC, AST, ALT, and LDL-C. Furthermore, CPP exhibited inhibitory effects on the expression of fatty acid synthesis genes FASN and ACC while activating the expression of fatty acid oxidation genes CPT1A and PPARα. Additionally, CPP reversed disturbances in intestinal microbiota composition caused by HFD consumption. CPP decreased the firmicutes/Bacteroidetes ratio, increased Akkermansia abundance, and elevated levels of total short-chain fatty acid (SCFA) content specifically butyric acid and acetic acid. Our results concluded that CPP may intervene in the development of NAFLD by regulating of intes-tinal microbiota imbalance and SCFAs production. Our study highlights that CPP has a potential to modulate lipid-related pathways via alterations to gut microbiome composition thereby ex-erting inhibitory effects on obesity and NAFLD development.

Reduction of Hepatosteatosis and Lipid Levels by an Adipose Differentiation-Related Protein Antisense Oligonucleotide

Lipophagy as a form of autophagy, can degrade lipid droplets, thereby acting as a critical regulator of cellular lipid metabolism, helping maintain the intracellular lipid homeostasis. In this study, we developed LP-SCUN, a far-red fluorescent probe for pinpointing lipid droplets with high sensitivity to solvent polarity. This probe allows for in situ imaging of lipophagy, tracking how lipid droplets move from non-polar to polar environments within lysosomes, leading to noticeable changes in fluorescence signals. The triphenylamine and triethylene glycol monomethyl ether groups of LP-SCUN facilitated its selective binding toward lipid droplets. Significantly, lipophagy and subsequent formation of autolysosomes decreased the environmental polarity, leading to a significant decrease in red fluorescence intensity (λex = 500 nm and λem = 643 nm). As such LP-SCUN was suitable for the in situ and real-time tracking of the cellular lipophagic processes. With this research we used LP-SCUN to elucidate the mechanism of lipid metabolism in liver cells of palmitate-induced hyperlipidemia cell models and high-fat diet-induced hyperlipidemia animal models. The results indicated that non-alcoholic fatty liver disease (NAFLD) can trigger an increase in cell lipophagy levels, while the inhibition of cell lipophagy can effectively alleviate the damage caused by NAFLD to cells and liver tissue in mice.

Insulin plays important roles in apoptosis and lipid droplet (LD) formation, and it is one of the determinants involved in increasing fat mass. However, the mechanisms underlying insulin-induced enlargement of fat mass remain unclear. Our previous study suggested that insulin-induced increases in LDs are related to c-Jun N-terminal kinase (JNK)2-mediated upregulation of cell death-inducing DNA fragmentation factor-α-like effector (CIDE)C in human adipocytes. However, other genes involved in insulin/JNK2-induced LD formation are unknown. Here, we explored insulin/JNK2-regulated genes to clarify the mechanism of enlargement of LDs. Microarray analysis revealed that an insulin/JNK2 pathway mostly regulates expression of genes involved in lipid metabolism, including sterol regulatory element binding protein (SREBP)-1, a key transcription factor of lipogenesis. The JNK inhibitor SP600125 blocked insulin-induced upregulation of SREBP-1c expression. Small interfering RNA-mediated depletion of JNK2 suppressed insulin-induced nuclear accumulation of the active form of SREBP-1 protein and upregulation of SREBP-1c. Furthermore, depletion of JNK2 attenuated insulin-induced upregulation of SREBP-1c target lipogenic enzymes, leading to reduced de novo fatty acid synthesis. In addition, JNK2 coimmunoprecipitated with SREBP-1, reinforcing the correlation between JNK2 and SREBP-1. These results suggest that SREBP-1c is a novel insulin/JNK2-regulated gene and that the JNK2/SREBP-1c pathway mediates insulin-induced fatty acid synthesis, which may lead to enlargement of LDs in human adipocytes.

Several clinical studies suggested that light-to-moderate alcohol intake could alleviate nonalcoholic fatty liver disease (NAFLD), but the underlying mechanism is still poorly understood. Mice fed a high-fat diet (HFD) were submitted or not to moderate ethanol intake for 3months (ca. 10g/kg/day) via drinking water. Biochemical, analytical and transcriptomic analyses were performed in serum and liver. Serum ethanol concentrations in ethanol-treated HFD mice comprised between 0.5 and 0.7g/l throughout the experiment. NAFLD improvement was observed in ethanol-treated HFD mice as assessed by reduced serum transaminase activity. This was associated with less microvesicular and more macrovacuolar steatosis, the absence of apoptotic hepatocytes and a trend towards less fibrosis. Liver lipid analysis showed increased amounts of fatty acids incorporated in triglycerides and phospholipids, reduced proportion of palmitic acid in total lipids and higher desaturation index, thus suggesting enhanced stearoyl-coenzyme A desaturase activity. mRNA expression of several glycolytic and lipogenic enzymes was upregulated. Genome-wide expression profiling and gene set enrichment analysis revealed an overall downregulation of the expression of genes involved in collagen fibril organization and leukocyte chemotaxis and an overall upregulation of the expression of genes involved in oxidative phosphorylation and mitochondrial respiratory chain complex assembly. In addition, mRNA expression of several proteasome subunits was upregulated in ethanol-treated HFD mice. Moderate chronic ethanol consumption may alleviate NAFLD by several mechanisms including the generation of non-toxic lipid species, reduced expression of profibrotic and proinflammatory genes, restoration of mitochondrial function and possible stimulation of proteasome activity.

PARP1-mediated PPARα poly(ADP-ribosyl)ation suppresses fatty acid oxidation in non-alcoholic fatty liver disease

Activation of oval cells (OCs) has been related to hepatocyte injury during chronic liver diseases including non-alcoholic fatty liver disease (NAFLD). However, OCs plasticity can be affected under pathological environments. We previously found protection against hepatocyte cell death by inhibiting protein tyrosine phosphatase 1B (PTP1B). Herein, we investigated the molecular and cellular processes involved in the lipotoxic susceptibility in OCs expressing or not PTP1B. Palmitic acid (PA) induced apoptotic cell death in wild-type (Ptpn1+/+) OCs in parallel to oxidative stress and impaired autophagy. This lipotoxic effect was attenuated in OCs lacking Ptpn1 that showed upregulated antioxidant defences, increased unfolded protein response (UPR) signaling, higher endoplasmic reticulum (ER) content and elevated stearoyl CoA desaturase (Scd1) expression and activity. These effects in Ptpn1-/- OCs concurred with an active autophagy, higher mitochondrial efficiency and a molecular signature of starvation, favoring lipid droplet (LD) formation and dynamics. Autophagy blockade in Ptpn1-/- OCs reduced Scd1 expression, mitochondrial fitness, LD formation and restored lipoapoptosis, an effect also recapitulated by Scd1 silencing. PTP1B immunostaining was detected in OCs from mouse liver and, importantly, LDs were found in OCs from Ptpn1-/- mice with NAFLD. In conclusion, we demonstrated that Ptpn1 deficiency restrains lipoapoptosis in OCs through a metabolic rewiring towards a "starvation-like" fate, favoring autophagy, mitochondrial fitness and LD formation. Dynamic LD-lysosomal interations likely ensure lipid recycling and, overall, these adaptations protect against lipotoxicity. The identification of LDs in OCs from Ptpn1-/- mice with NAFLD opens therapeutic perspectives to ensure OC viability and plasticity under lipotoxic liver damage.