Steatotic HepG2 Cells Research Articles

Engineered Lipids for Intracellular Reactive Oxygen Species Scavenging in Steatotic Hepatocytes.

Intracellular reactive oxygen species (ROS) in steatotic cells pose a problem due to their potential to cause oxidative stress and cellular damage. Delivering engineered phospholipids to intracellular lipid droplets in steatotic hepatic cells, using the cell's inherent intracellular lipid transport mechanisms are investigated. Initially, it is shown that tail-labeled fluorescent lipids assembled into liposomes are able to be transported to intracellular lipid droplets in steatotic HepG2 cells and HHL-5 cells. Further, an antioxidant, an EUK salen-manganese derivative, which has superoxide dismutase-like and catalase-like activity, is covalently conjugated to the tail of a phospholipid and formulated as liposomes for administration. Steatotic HepG2 cells and HHL-5 cells incubated with these antioxidant liposomes have lower intracellular ROS levels compared to untreated controls and non-covalently formulated antioxidants. This first proof-of-concept study illustrates an alternative strategy to equip native organelles in mammalian cells with engineered enzyme activity.

Silibinin ameliorates deoxycholic acid-induced pyroptosis in steatotic HepG2 cells by inhibiting NLRP3 inflammasome activation

Nonalcoholic steatohepatitis (NASH) represents an inflammatory subtype of nonalcoholic fatty liver disease (NAFLD). The activation of the NOD-like receptor protein 3 (NLRP3) inflammasome triggers pyroptosis, thus propelling the progression from simple steatosis to NASH. Silibinin, a hepatoprotective compound derived from milk thistle, exerts diverse hepatoprotective effects. However, the direct impact of silibinin on NLRP3 inflammasome activation and its ability to mitigate pyroptosis remain uncertain. To address this, we utilized an in vitro model of NASH, employing HepG2 cells treated with deoxycholic acid (DCA) and free fatty acids. Subsequently, we treated these model cells with silibinin for 24 h. Our findings demonstrated that, although there were no significant changes in cellular lipid content, silibinin effectively ameliorated hepatocyte injuries. Silibinin treatment inhibited the activation of the NLRP3 inflammasome and suppressed DCA-induced pyroptosis. Additionally, molecular docking analysis revealed that silibinin exhibited a binding affinity to components of the NLRP3 inflammasome similar to that of MCC950, a selective NLRP3 inhibitor. These results suggest that silibinin may alleviate inflammation in DCA-exposed HepG2 cells by mitigating pyroptosis, possibly through its binding affinity and inhibition of the NLRP3 inflammasome. Overall, our study indicates that silibinin holds promise as a therapeutic agent for NASH by modulating pyroptosis and inhibiting NLRP3 inflammasome activation.

Dietary cholesterol drives the development of nonalcoholic steatohepatitis by altering gut microbiota mediated bile acid metabolism in high-fat diet fed mice

Nonalcoholic fatty liver disease (NAFLD) is the most widespread chronic liver disorder globally. Unraveling the pathogenesis of simple fatty liver to nonalcoholic steatohepatitis (NASH) has important clinical significance for improving the prognosis of NAFLD. Here, we explored the role of a high-fat diet alone or combined with high cholesterol in causing NASH progression. Our results demonstrated that high dietary cholesterol intakes accelerate the progression of spontaneous NAFLD and induces liver inflammation in mice. An elevation of hydrophobic unconjugated bile acids cholic acid (CA), deoxycholic acid (DCA), muricholic acid and chenodeoxycholic acid, was observed in high-fat and high-cholesterol diet fed mice. Full-length sequencing of the 16S rDNA gene of gut microbiota revealed a significant increase in the abundance of Bacteroides, Clostridium, and Lactobacillus that possess bile salt hydrolase activity. Furthermore, the relative abundance of these bacterial species was positively correlated with content of unconjugated bile acids in liver. Moreover, the expression of genes related to bile acid reabsorption (organic anion-transporting polypeptides, Na+-taurocholic acid cotransporting polypeptide, apical sodium dependent bile acid transporter and organic solute transporter β) was found to be increased in mice with a high-cholesterol diet. Lastly, we observed that hydrophobic bile acids CA and DCA induce an inflammatory response in free fatty acids-induced steatotic HepG2 cells. In conclusion, high dietary cholesterol promotes the development of NASH by altering gut microbiota composition and abundance and thereby influencing with bile acid metabolism.

Hepatic-Targeted Nano-enzyme with Resveratrol Loading for Precise Relief of Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is characterized by massive lipid deposition in hepatocytes and is often associated with hepatic inflammation and other severe metabolic syndromes. The intervention of NASH can prevent its further progression into hepatocarcinoma. In this study we have successfully constructed liver-targeted Ce-based hollow mesoporous nanocarriers loaded with bioactive drugs. This may provide an effective approach for eliminating NASH. Liver-section-specific targeting was realized by covalently linked galactose (Gal), which can be specifically recognized by receptors in the membranes of hepatocytes. Meanwhile, resveratrol (Res), a drug used to treat NASH, was efficiently loaded into the pores and cavity of CeO2 (Res@H-CeO2 -Gal). In steatotic HepG2 cells (free fatty acid induction), this nanosystem was found to enhance cellular Res internalization for improved anti-lipogenesis activity. In mice with NASH, Res@H-CeO2 -Gal increased Res delivery to liver sections for a reduction in lipid accumulation and enhanced anti-inflammatory activity from the antioxidant capacity of Ce-based nanocarriers. This effectively recovered NASH mice to the normal state. These findings show that the hepatic targeting and Res delivery nanoplatform could act as a safe and promising strategy for the elimination of NASH and other liver diseases.

Comparative Transcriptome Analysis Reveals That Exendin-4 Improves Steatosis in HepG2 Cells by Modulating Signaling Pathways Related to Lipid Metabolism.

No therapy exists for non-alcoholic fatty liver disease (NAFLD). However, glucagon-like peptide receptor agonists (GLP-1RAs) showed a beneficial effect on NAFLD, although the underpinning mechanisms remain unclear due to their pleiotropic effects. We examined the implicated signaling pathways using comparative transcriptomics in a cell model of steatosis to overcome pleiotropy. We treated steatotic HepG2 cells with the GLP-1RA Exendin-4 (Ex-4). We compared the transcriptome profiles of untreated steatotic, and Ex-4-treated steatotic cells, and used Ingenuity Pathway Analysis (IPA) to identify the signaling pathways and associated genes involved in the protective effect of Ex-4. Ex-4 treatment significantly reduces steatosis. RNA-seq analysis revealed 209 differentially expressed genes (DEGs) between steatotic and untreated cells, with farnesoid X receptor/retinoid X receptor (FXR/RXR) (p = 8.9 × 10−7) activation being the top regulated canonical pathway identified by IPA. Furthermore, 1644 DEGs were identified between steatotic cells and Ex-4-treated cells, with liver X receptor/retinoid X receptor (LXR/RXR) (p = 2.02 × 10−7) and FXR/RXR (p = 3.28 × 10−7) activation being the two top canonical pathways. The top molecular and cellular functions between untreated and steatotic cells were lipid metabolism, molecular transport, and small molecular biochemistry, while organismal injury and abnormalities, endocrine system disorders, and gastrointestinal disease were the top three molecular and cellular functions between Ex-4-treated and steatotic cells. Genes overlapping steatotic cells and Ex-4-treated cells were associated with several lipid metabolism processes. Unique transcriptomic differences exist between steatotic cells and Ex-4-treated steatotic cells, providing an important resource for understanding the mechanisms that underpin the protective effect of GLP-1RAs on NAFLD and for the identification of novel therapeutic targets for NAFLD.

Exendin-4 alleviates steatosis in an in vitro cell model by lowering FABP1 and FOXA1 expression via the Wnt/-catenin signaling pathway

Non-alcoholic fatty liver disease (NAFLD) is the leading chronic liver disease worldwide. Agonists of the glucagon-like peptide-1 receptor (GLP-1R), currently approved to treat type 2 diabetes, hold promise to improve steatosis and even steatohepatitis. However, due to their pleiotropic effects, the mechanisms underlying their protective effect on NAFLD remain elusive. We aimed to investigate these mechanisms using an in vitro model of steatosis treated with the GLP-1R agonist Exendin-4 (Ex-4). We established steatotic HepG2 cells by incubating the cells with 400 µM oleic acid (OA) overnight. Further treatment with 200 nM Ex-4 for 3 h significantly reduced the OA-induced lipid accumulation (p < 0.05). Concomitantly, Ex-4 substantially reduced the expression levels of Fatty Acid-Binding Protein 1 (FABP1) and its primary activator, Forkhead box protein A1 (FOXA1). Interestingly, the silencing of β-catenin with siRNA abolished the effect of Ex-4 on these genes, suggesting dependency on the Wnt/β-catenin pathway. Additionally, after β-catenin silencing, OA treatment significantly increased the expression of nuclear transcription factors SREBP-1 and TCF4, whereas Ex-4 significantly decreased this upregulation. Our findings suggest that direct activation of GLP-1R by Ex-4 reduces OA-induced steatosis in HepG2 cells by reducing fatty acid uptake and transport via FABP1 downregulation.

Hepatoprotective Constituents of Macrocybe gigantea (Agaricomycetes) from India.

Non-alcoholic fatty liver disease (NAFLD) is one of the most frequent, chronic liver diseases worldwide and currently has no specific therapy. Our previous study indicated the anti-NAFLD effect of Macrocybe gigantea (Massee) Pegler & Lodge in high-fat diet-fed animals. This study aimed to isolate and identify the active hepatoprotective constituents from M. gigantea using fatty acid induced steatotic HepG2 cells as in vitro model. The effect of the test materials on the viability of HepG2 cells was analyzed using MTT assay. The HepG2 cells were treated with a mixture of palmitate-oleate to induce steatosis; after 24 h of treatment with the test materials, the intracellular lipid content was estimated using Oil Red O staining. The levels of transaminases were also estimated in the spent media. Bioassay-guided isolation of hepatoprotective constituents from M. gigantea yielded two compounds viz., ergosterol and linoleic acid; their structures were confirmed using spectroscopic data. Among these two compounds, ergosterol significantly lowered the levels of intracellular triglyceride content of fatty acid induced HepG2 cells; it also lowered the leakage of transaminases. The reductions caused by linoleic acid were not statistically significant at the tested concentrations. Detailed investigations on efficacy and safety of these compounds and M. gigantea might yield some useful leads for the management of NAFLD.

Corosolic acid ameliorates non-alcoholic steatohepatitis induced by high-fat diet and carbon tetrachloride by regulating TGF-β1/Smad2, NF-κB, and AMPK signaling pathways.

Hawthorn (Crataegus pinnatifida Bunge. var. major) is an edible and medicinal fruit that is very common in food and traditional Chinese medicine. Corosolic acid (CA), a pentacyclic triterpenoid, which is an active component of hawthorn (Crataegus pinnatifida Bunge. var. major), has been exhibiting various pharmacological activities such as antidiabetic, antibacterial, anticancer, antiinflammatory, and antioxidant effects. The study aimed to evaluate the effect of CA on non-alcoholic steatohepatitis (NASH) in mice induced by 60 kcal% high-fat diet (HFD) and carbon tetrachloride (CCl4 ). CA lowered liver index and serum AST, ALT, TG, and TC levels compared to those in the model group. Histological analyses of the liver tissues of mice treated with CA revealed significantly decreased number of lipid droplets and alleviated inflammation and fibrosis. CA inhibited the transcripts of pro-fibrogenic markers (including α-SMA, collagen I, and TIMP-1) and the levels of pro-inflammatory cytokines (including TNF-α, IL-1β, caspase-1, and IL-6) associated with hepatic fibrosis, and NF-κB translocation and TGF-β1/Smad2 and AMPK pathways. In addition, CA reduced lipid accumulation via the regulation of AMPK and NF-κB activation in FFA-induced steatotic HepG2 cells. CA also decreased α-SMA, collagen I expressions, and Smad2 phosphorylation, which were reduced by TGF-β1 treatment in LX2 cells. Our results suggested that CA ameliorated NASH through regulating TGF-β1/Smad2, NF-κB, and AMPK signaling pathways, and CA could be developed as a potential health functional food or therapeutic agent for NASH patients.

Hepatoprotective effect of selected isoandrographolide derivatives on steatotic HepG2 cells and High Fat Diet fed rats

Non-alcoholic Fatty Liver Disease (NAFLD) is one of the growing epidemics of the globe. This study was aimed to evaluate the anti-NAFLD effect of selected IAN derivatives using in silico, in vitro and in vivo models. In silico tools viz., DataWarrior, SwissADME and Gaussian 09 were used to predict the pharmacokinetic properties and electronic distribution patterns of the derivatives; docking analysis was done with Autodock against PPARα. Toxicities of the derivatives were assessed in HepG2 cells using MTT assay. Anti-NAFLD efficacies of the derivatives were assessed in free fatty acid induced steatotic HepG2 cells. In vivo anti-NAFLD effect of active isoandrographolide (IAN) derivative, 19-propionyl isoandrographolide (IAN-19P) was assessed in High Fat Diet fed rats. In silico and in vitro studies indicated that IAN-19P showed improved drug-likeness and drug score. The toxicity of IAN-19P to HepG2 cells was comparatively less than IAN and other derivatives. In free fatty acid induced steatotic HepG2 cells, treatment with IAN-19P significantly lowered intracellular triglyceride content and leakage of LDH and transaminases. Treating High Fat Diet fed animals with IAN-19P significantly lowered plasma lipids, transaminases, LDH and GGT levels. The treatment with IAN-19P upregulated the expressions of PPARα and CPT-1. IAN-19P did not produce any noticeable adverse effect till 2 g/kg concentration in acute and 250 mg/kg concentration in subacute toxicity studies. This study indicated the beneficial effect of IAN-19P for the treatment of NAFLD; however robust investigations are needed to establish the potential of IAN-19P to treat NAFLD.

The alleviation of lipid deposition in steatosis hepatocytes by capsaicin-loaded α-lactalbumin nanomicelles via promoted endocytosis and synergetic multiple signaling pathways

A capsaicin delivery system was developed by self-assembling of α-lactalbumin polypeptides into nanomicelles. The capsaicin was loaded into the α-lactalbumin micelles via hydrophobic interaction with an embedding amount of 126.58 mg/g. Steatotic HepG2 cells were treated with the capsaicin-loaded nanomicelles and the anti-lipogenesis effect of the cells was significantly improved. The endocytosis rate of capsaicin was improved by nanomicelles. Moreover, the nanomicelles could escape from lysosomes and overcome P-glycoprotein efflux pump effect, which prevented the capsaicin being pumped out of cells. Furthermore, the capsaicin-loaded nanomicelles showed a deep penetration and anti-lipogenesis effect on steatotic HepG2 spheroid model. Western blotting results suggested that the capsaicin-loaded nanomicelles remarkably inhibited lipogenesis, promoted lipid hydrolysis and macro-autophagy compared with free capsaicin. The present study suggested that the α-lactalbumin nanocarriers exhibited a considerable potential in delivery of hydrophobic bioactive ingredients into cells effectively and maximized their bioactivities in functional foods.

Optimized preparation of eugenol microcapsules and its effect on hepatic steatosis in HepG2 cells

This study was aimed at evaluating the potential of peach gum (PG) and gelatin in the microencapsulation of eugenol and the intervention of eugenol microcapsules on hepatic steatosis in vitro. Response surface method (RSM) was used to optimize the encapsulation conditions of eugenol microcapsules. The microcapsules were characterized by scanning electron microscopy (SEM), dynamic Light Scattering (DLS), Fourier transform infrared spectroscopy (FT-IR) and release behavior in vitro was determined. The effect of eugenol microcapsules on free fatty acids (FFA) treated hepatocellular cells (HepG2) cells was evaluated by oil red O staining and intracellular total cholesterol (TC) and triglyceride (TG) determination. The results showed that the optimal encapsulation conditions were as follows: the PG-gelatin ratio was 1.6:1.4, the core-wall ratio was 1.6:1.4, the pH was 4 and the emulsification speed was 9000 r/min. The optimized microcapsules were smooth spherical with a size of about 3.09 ± 0.58 μm and the encapsulation was confirmed by FT-IR. In vitro release behavior showed that eugenol microcapsules could be released stably in a neutral environment for 72 h. Oil red O staining showed that 50 and 100 μM eugenol microcapsules could significantly inhibit the lipid accumulation and reduce the TC and TG in steatotic HepG2 cells induced by FFA. Therefore, PG and gelatin can be used as excellent carriers for the microencapsulation of volatile compounds in the field of biomedical industry, and eugenol microcapsules is a promising preparation for the treatment of nonalcoholic fatty liver disease (NAFLD).

9-PAHSA Prevents Mitochondrial Dysfunction and Increases the Viability of Steatotic Hepatocytes.

Nonalcoholic fatty liver disease (NAFLD) is quickly becoming the most common liver disease worldwide. Within the NAFLD spectrum, patients with nonalcoholic steatohepatitis (NASH) are at the highest risk of developing cirrhosis and disease progression to hepatocellular carcinoma. To date, therapeutic options for NASH patients have been ineffective, and therefore, new options are urgently needed. Hence, a model system to develop new therapeutic interventions is needed. Here, we introduce two new in vitro models of steatosis induction in HepG2 cells and primary murine hepatocytes. We used a recently discovered novel class of bioactive anti-inflammatory lipids called branched fatty acid esters of hydroxyl fatty acids. Among these bioactive lipids, palmitic-acid-9-hydroxy-stearic-acid (9-PAHSA) is the most promising as a representative nondrug therapy based on dietary supplements or nutritional modifications. In this study, we show a therapeutic effect of 9-PAHSA on lipotoxicity in steatotic primary hepatocytes and HepG2 cells. This could be shown be increased viability and decreased steatosis. Furthermore, we could demonstrate a preventive effect in HepG2 cells. The outcome of 9-PAHSA administration is both preventative and therapeutically effective for hepatocytes with limited damage. In conclusion, bioactive lipids like 9-PAHSA offer new hope for prevention or treatment in patients with fatty liver and steatosis.

CKIP-1 augments autophagy in steatotic hepatocytes by inhibiting Akt/mTOR signal pathway

Nonalcoholic fatty liver disease (NAFLD), which is characterized by aberrant accumulation of intrahepatic triglycerides and lipid droplets (LDs) in the liver cells, is becoming increasingly prevalent at an alarming rate worldwide. LDs can be consumed by either hydrolysis or autophagy, which is shown to be of importance in the regulation of hepatic lipid metabolism. We have shown that deficiency of pleckstrin homology domain-containing casein kinase 2 interacting protein-1 (CKIP-1), a scaffold protein that interacts with various proteins in multiple signal pathways, in mice aggravates high-fat diet induced fatty liver. However, its underlying mechanisms remain largely unknown. In this study, we found that the mRNA and protein levels of CKIP-1 decreased dramatically in steatotic HepG2 cells induced by oleic acid (OA) treatment. Coincidently, hepatic autophagy was also dynamically regulated in steatotic HepG2 cells. In addition, overexpression of CKIP-1 activated autophagy by suppression of Akt/mTOR signaling, which in turn reduced lipid accumulation. Moreover, these phenomena were reversed in CKIP-1-shRNA transfected steatotic hepatocytes. To further evaluate the potential role of CKIP-1 in autophagy, we determined the level of autophagy related proteins in CKIP-1 knockout mice. These results supported our findings in vitro. In summary, we found CKIP-1 to be a positive regulator of hepatic autophagy and a promising therapeutic target for treatment of NAFLD.

Mitochondrial Energy-Regulating Effect of Atractyloside Inhibits Hepatocellular Steatosis Through the Activation of Autophagy.

Background and AimAtractyloside (ATR), a mitochondrial uncoupler, is known for its specific inhibition of mitochondrial oxidative phosphorylation. Previous studies have reported that moderate mitochondrial uncoupling effect is beneficial to increase the decomposition and clearance of hepatic lipid, prevent the occurrence of fatty liver diseases. Moreover, the beneficial effects of mitochondrial uncouplers on type 2 diabetes and metabolic syndromes have been consistently observed. The present study investigated the effect of ATR on steatosis level of HepG2 cells treated with free fatty acid (FFA).MethodsIntracellular triglyceride level and Oil Red O staining were assessed, the mitochondrial adaptation and ADP/ATP ratio were analyzed, the protein level of AMPK, mTOR and LC3B, autophagic flux, and the co-localization of LC3B with lipid droplets was performed.ResultsATR treatment inhibited the activity of mitochondrial respiratory chain complexes I and IV, decreased the mitochondrial membrane potential, and increased the ADP/ATP ratio in the FFA-treated cells. Furthermore, ATR increased the gene expression and protein level of LC3B and promoted the autophagic flux processing from early autophagosome to late autolysosome by increasing the protein level of AMPKα and decreasing the protein level of mTOR. An increased number of autophagosomes (LC3B) was also observed in the lipid droplets. ATR treatment accelerated lipid degradation in the FFA-treated cells, and the lowest lipid content was observed in the cell group with 7.5 μM ATR.ConclusionLow concentrations (2.5, 5, and 7.5 μM) of ATR treatment could activate autophagy to accelerate the degradation of TGs in steatosis HepG2 cells; the mechanism may be related to the activation of the AMPK/mTOR pathway induced by the increased ADP/ATP ratio. In addition, the ideal concentration of ATR for improving steatotic HepG2 cells was 7.5 μM.

Combination of luteolin and lycopene effectively protect against the “two-hit” in NAFLD through Sirt1/AMPK signal pathway

AimLuteolin and lycopene are common natural products, widely existing in nature, and both of which were reported to have various biological functions including anti-inflammatory, anti-obesity and anti-NAFLD. In the present study, we aimed to evaluate the therapeutic efficacy of luteolin and lycopene in combination and its latent molecular mechanisms in vitro and in vivo models of NAFLD. Main methodsSodium palmitate (PA)-induced steatotic HepG2 cells and primary hepatocytes, and high-fat diet-induced C57BL/6J obese mice were treated with luteolin, lycopene and their combination. Metabolic parameters were measured. Key findingsWe found that luteolin (20 μM) + lycopene (10 μM) was the best therapeutic combination in PA-induced HepG2 cells, and significantly improve cell viability and lipid accumulation in PA-induced HepG2 cells and primary hepatocytes. In addition, luteolin (20 mg/kg) + lycopene (20 mg/kg) could ameliorate increased body weight and hepatocyte steatosis; regulate serum triglycerides, serum total cholesterol, hepatic triglycerides and hepatic total cholesterol; decrease serum alanine transaminase and aspartate transaminase. Furthermore, in vivo and in vitro, luteolin, lycopene and their combination had no effect on Sirt1 expression, but all of them could upregulate the expression of NAMPT, which could increase the level of NAD+, the co-substrate of Sirt1, indirectly activating Sirt1/AMPK pathway, and then inhibited lipogenesis and increased β-oxidation, defensing the “first hit”; they also inactivated nuclear factor-κB (NF-κB) and decreased the levels of IL-6, IL-1β and TNF-α, defensing the “second hit”. SignificanceThus, luteolin and lycopene in combination can effectively ameliorate “two-hit” in NAFLD through activation of the Sirt1/AMPK pathway.

Berbamine induced AMPK activation regulates mTOR/SREBP-1c axis and Nrf2/ARE pathway to allay lipid accumulation and oxidative stress in steatotic HepG2 cells

Non-alcoholic fatty liver disease is emanating as a global cataclysm. This study was designed to investigate the antioxidative, anti-inflammatory and fat metabolism-regulating potential of berbamine (BBM), a natural bis-benzylisoquinoline alkaloid. BBM attenuated intracellular lipid accumulation in oleic-acid exposed HepG2 cells (0.5 mM) by inhibiting fatty acid uptake, lipogenesis, and promoting fatty acid β-oxidation by activating AMP-activated kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR)-α. Berbamine (5 μM) induced AMPK activation (P < 0.001) via LKB1 (Ser-428) and elevated AMP:ATP ratio (P < 0.001). AMPK activation negatively regulated mTOR and also constrained the nuclear translocation of SREBP-1c and inhibited the lipogenic proteins, stearoyl-CoA desaturase-1 (SCD-1) and fatty acid synthase (FAS) (P < 0.001). BBM stimulated nuclear translocation of redox-sensitive nuclear factor erythroid-2-related factor-2 (Nrf2) and increased hepatic expression of Nrf2 responsive enzymes, HO-1 and Nqo-1. BBM treatment reduced the oxidative burst and pro-inflammatory responses by significantly enhancing hepatic antioxidant defenses [SOD (P < 0.001), catalase (P < 0.001) and cellular glutathione (P < 0.01)] and diminishing NF-κB regulated pro-inflammatory cytokines (TNF-α, and IL-6) levels respectively. TEM analysis confirmed the disruption of mitochondrial structure and reduction in mitochondrial size (50.97%, P < 0.001) in steatotic HepG2 cells which was significantly prevented by 5 μM BBM treatment (71.84% as compared to control, P < 0.01). Pre-treatment of Compound C (AMPK inhibitor, 25 μM) greatly repressed the anti-steatotic properties exhibited by BBM confirming the involvement of AMPK signaling pathway. In summary, the results manifest that BBM reduces intracellular lipid accumulation via AMPK/mTOR/SREBP-1c axis mediated regulation of lipid metabolism and upsurged nuclear stability of Nrf2 by promoting AMPK/Nrf2 association to ameliorate oxidative stress/proinflammatory response.

In Steatotic Cells, ATP-Citrate Lyase mRNA Is Efficiently Translated through a Cap-Independent Mechanism, Contributing to the Stimulation of De Novo Lipogenesis.

Non-alcoholic fatty liver disease (NAFLD) is a chronic disease in which excessive amount of lipids is accumulated as droplets in hepatocytes. Recently, cumulative evidences suggested that a sustained de novo lipogenesis can play an important role in NAFLD. Dysregulated expression of lipogenic genes, including ATP-citrate lyase (ACLY), has been found in liver diseases associated with lipid accumulation. ACLY is a ubiquitous cytosolic enzyme positioned at the intersection of nutrients catabolism and cholesterol and fatty acid biosyntheses. In the present study, the molecular mechanism of ACLY expression in a cell model of steatosis has been reported. We identified an internal ribosome entry site (IRES) in the 5′ untranslated region of the ACLY mRNA, that can support an efficient mRNA translation through a Cap-independent mechanism. In steatotic HepG2 cells, ACLY expression was up-regulated through IRES-mediated translation. Since it has been demonstrated that lipid accumulation in cells induces endoplasmic reticulum (ER) stress, the involvement of this cellular pathway in the translational regulation of ACLY has been also evaluated. Our results showed that ACLY expression was increased in ER-stressed cells, through IRES-mediated translation of ACLY mRNA. A potential role of the Cap-independent translation of ACLY in NAFLD has been discussed.

Resveratrol Treatment Enhances the Cellular Response to Leptin by Increasing OBRb Content in Palmitate-Induced Steatotic HepG2 Cells.

The interaction of leptin with its hepatic longest receptor (OBRb) promotes the phosphorylation of signal transducer and activator of transcription-3 (STAT3), protecting the liver from lipid accumulation. However, leptin signalling is disrupted in hepatic steatosis, causing leptin resistance. One promising strategy to combat this problem is the use of bioactive compounds such as polyphenols. Since resveratrol (RSV) is a modulator of lipid homeostasis in the liver, we investigated whether treatment with different doses of RSV restores appropriate leptin action and fat accumulation in palmitate-induced steatotic human hepatoma (HepG2) cells. Both RSV metabolism and the expression of molecules implicated in leptin signalling were analysed. RSV at a 10 μM concentration was entirely metabolized to resveratrol-3-sulfate after 24 and counteracted leptin resistance by increasing the protein levels of OBRb. In addition, RSV downregulated the expression of lipogenic genes including fatty acid synthase (Fas) and stearoyl-CoA desaturase-1 (Scd1) without any significant change in Sirtuin-1 (SIRT1) enzymatic activity. These results demonstrate that RSV restored leptin sensitivity in a cellular model of hepatic steatosis in a SIRT1-independent manner.

Multiple extraction conditions to produce phytochemical- and antioxidant-rich Alternanthera sessilis (red) extracts that attenuate lipid accumulation in steatotic HepG2 cells

Alternanthera sessilis (red) is an edible herb used by Malaysians to alleviate hyperlipidaemia. This study aimed to produce phytochemical- and antioxidant-rich A. sessilis (red) whole plant extracts using multiple extraction conditions. The lipid-lowering effects of the extracts against steatosis in HepG2 cells were assessed. Ethanolic extracts (100%, 50 °C, 24 h) showed the highest total phenolic content (83.5 mg GAE/g extract) while water extracts (50 °C, 24 h) had the highest extraction yield (20.5%). Ethyl acetate extracts (50 °C, 24 h) showed the highest flavonoids (641 mg RE/g), triterpenoids (46.1 mg UAE/g) and betalains (amaranthin: 72.7 mg/g; betaxanthin: 74.5 mg/g; betanin: 73.1 mg/g). Despite this, the ethanolic extracts showed the highest antioxidant activities (DPPH IC50: 82.6 μg/ml; TEAC: 0.51 mmol TE/g; FRAP: 1.95 mmol Fe2+/g). UHPLC analyses showed the presence of 4 polyphenolic compounds from the extracts; trans-ferulic acid, rutin, quercetin and apigenin. Different concentrations (5–40 μg/ml) of ethanolic, ethyl acetate and water extracts produced at 50 °C, 24 h were selected for cell-based assays. Two assays were used to determine their preventive and ameliorative effects against palmitate-induced lipid accumulation in HepG2 cells. Results indicated that the ethanolic extracts were the most effective at preventing steatosis (intracellular lipid content reduced by 28.3%) while water extracts showed the highest decrease of steatosis (intracellular lipid content reduced by 30%). The results were comparable with the common drug fenofibrate (0.1 mM) for hyperlipidaemia, indicating the potential of this plant's extract to reduce hepatic lipid accumulation.

Antihyperlipidemic effect of iridoid glycoside deacetylasperulosidic acid isolated from the seeds of Spermacoce hispida L. - A traditional antiobesity herb

Ethnobotanical relevanceThe interest on herbal health supplements for obesity is increasing globally. Our previous ethnobotanical survey in Tiruvallur district, Tamil Nadu, India indicated the use of Spermacoce hispida L. seeds for the treatment of obesity. Aim of the studyThis study was aimed to validate the traditional claim and to identify the antihyperlipidemic principle in the seeds of Spermacoce hispida using bioassay guided fractionation method. MethodsBioassay monitored fractionation of the aqueous extract from Spermacoce hispida seeds was carried out using triton WR 1339 induced hyperlipidemic animals. It yielded deacetylasperulosidic acid (DAA) as the active ingredient. Pharmacokinetic properties of DAA were predicted using DataWarrior and SwissADME tools. In vitro antiobesity and antihyperlipidemic effects of DAA were evaluated in 3T3L1 preadipocytes and HepG2 cells, respectively. The chronic antihyperlipidemic efficacy of DAA was evaluated in high fat diet fed rats. ResultsDAA did not show any mutagenic and tumorigenic properties. It bound with PPARα with comparable ligand efficiency as fenofibrate. The treatment with DAA significantly lowered the proliferation of matured adipocytes, but not preadipocytes. The treatment of steatotic HepG2 cells with DAA significantly decreased the LDH leakage by 43.03% (P < 0.05) at 50 μM concentration. In triton WR 1339 induced hyperlipidemic animals, the treatment with 50 mg/kg dose significantly lowered the TC, TG and LDL-c levels by 40.27, 46.00 and 63.65% respectively. In HFD fed animals, the treatment at 10 mg/kg decreased BMI and AC/TC ratio without altering SRBG. It also improved serum lipid, transaminases and phosphatases levels of HFD fed animals. The treatment lowered adipocyte hypertrophy and steatosis of hepatocytes. ConclusionThis preliminary report supported the traditional use of Spermacoce hispida for the treatment of obesity. Further detailed investigations on the long term safety, efficacy and molecular mode of action of Spermacoce hispida and DAA will throw more light on their usefulness for the management of obesity.