Zehong Yang,Yuanyuan Yu,Nannan Sun,Limian Zhou,Dong Zhang,HaiXin Chen,Wei Miao,Weihang Gao,Canyang Zhang,Changhui Liu,Xiaoying Yang,Xiaojie Wu,Yong Gao
Ginsenosides Rc, as a novel SIRT6 activator, protects mice against high fat diet induced NAFLD
Ginsenosides Rc,NAFLD,PPAR-α,SIRT6
Background Hepatic lipid disorder impaired mitochondrial homeostasis and intracellular redox balance, triggering development of non-alcohol fatty liver disease (NAFLD), while effective therapeutic approach remains inadequate. Ginsenosides Rc has been reported to maintain glucose balance in adipose tissue, while its role in regulating lipid metabolism remain vacant. Thus, we investigated the function and mechanism of ginsenosides Rc in defending high fat diet (HFD)-induced NAFLD. Methods Mice primary hepatocytes (MPHs) challenged with oleic acid & palmitic acid were used to test the effects of ginsenosides Rc on intracellular lipid metabolism. RNAseq and molecular docking study were performed to explore potential targets of ginsenosides Rc in defending lipid deposition. Wild type and liver specific sirtuin 6 (SIRT6, 50721) deficient mice on HFD for 12 weeks were subjected to different dose of ginsenosides Rc to determine the function and detailed mechanism in vivo. Results We identified ginsenosides Rc as a novel SIRT6 activator via increasing its expression and deacetylase activity. Ginsenosides Rc defends OA&PA-induced lipid deposition in MPHs and protects mice against HFD-induced metabolic disorder in dosage dependent manner. Ginsenosides Rc (20mg/kg) injection improved glucose intolerance, insulin resistance, oxidative stress and inflammation response in HFD mice. Ginsenosides Rc treatment accelerates peroxisome proliferator activated receptor alpha (PPAR-α, 19013)-mediated fatty acid oxidation in vivo and in vitro. Hepatic specific SIRT6 deletion abolished ginsenoside Rc-derived protective effects against HFD-induced NAFLD. Conclusion Ginsenosides Rc protects mice against HFD-induced hepatosteatosis by improving PPAR-α-mediated fatty acid oxidation and antioxidant capacity in a SIRT6 dependent manner, and providing a promising strategy for NAFLD.
Ginsenosides Rc, as a novel SIRT6 activator, protects mice against high fat diet induced NAFLD

Hepatic lipid disorder impaired mitochondrial homeostasis and intracellular redox balance, triggering development of non-alcohol fatty liver disease (NAFLD), while effective therapeutic approach remains inadequate. Ginsenosides Rc has been reported to maintain glucose balance in adipose tissue, while its role in regulating lipid metabolism remain vacant. Thus, we investigated the function and mechanism of ginsenosides Rc in defending high fat diet (HFD)-induced NAFLD.

Mice primary hepatocytes (MPHs) challenged with oleic acid & palmitic acid were used to test the effects of ginsenosides Rc on intracellular lipid metabolism. RNAseq and molecular docking study were performed to explore potential targets of ginsenosides Rc in defending lipid deposition. Wild type and liver specific sirtuin 6 (SIRT6, 50721) deficient mice on HFD for 12 weeks were subjected to different dose of ginsenosides Rc to determine the function and detailed mechanism in vivo.

We identified ginsenosides Rc as a novel SIRT6 activator via increasing its expression and deacetylase activity. Ginsenosides Rc defends OA&PA-induced lipid deposition in MPHs and protects mice against HFD-induced metabolic disorder in dosage dependent manner. Ginsenosides Rc (20mg/kg) injection improved glucose intolerance, insulin resistance, oxidative stress and inflammation response in HFD mice. Ginsenosides Rc treatment accelerates peroxisome proliferator activated receptor alpha (PPAR-α, 19013)-mediated fatty acid oxidation in vivo and in vitro. Hepatic specific SIRT6 deletion abolished ginsenoside Rc-derived protective effects against HFD-induced NAFLD.

Ginsenosides Rc protects mice against HFD-induced hepatosteatosis by improving PPAR-α-mediated fatty acid oxidation and antioxidant capacity in a SIRT6 dependent manner, and providing a promising strategy for NAFLD.

Excessive lipid deposition in the liver leads to nonalcoholic fatty liver disease (NAFLD), which accounts for 25% of the global population and represents a spectrum of hepatic pathologies, including simple steatosis, nonalcoholic steatohepatitis (NASH) and cirrhosis [1]. Although a close link to obesity has been approved, the specific pathogenesis of NAFLD remains partly obscure [2]. Among the numerous aetiological mechanisms, a “two-hit model” theory is well established [3]. Diet or genetic factors-induced insulin resistance derives lipid accumulation in hepatocytes and starts the first hit. Chronic lipid exposure, especially high free fatty acids levels destroy lysosomal-mitochondrial axis and erupt intracellular ROS overproduction, stimulating lipid peroxidation and cytokine activity, the second hit occurs [4]. Thus, the ease of hepatic lipid disorder may effectively defend the occur of NAFLD phenotype [5]. Collective studies confirmed that de novo increased lipogenesis and impaired fatty acid oxidation leads to lipid accumulation in the liver and are obvious in NAFLD subjects [6,7]. Transcription factors peroxisome proliferator activated receptor alpha (PPAR-α, 19013) and sterol regulatory element binding protein-1 (SREBP-1, 20787) have been proposed to play a particularly important role in maintaining lipid homeostasis [[8], [9], [10]]. Especially PPAR-α, it is transcriptionally controlled by multiple nutritional and hormonal factors, including insulin and leptin [11]. PPAR-α deficient mice spontaneously appeared hyperketonemia, hypoglycemic and NAFLD symptoms, accompanying with a pronounced reactive oxygen species (ROS) overproduction [12]. Moreover, PPAR-α could serves as a potential target for the treatment of NAFLD. Pharmacological or genetic activation of the nuclear receptor PPAR facilitates gluconeogenesis and acetyle-CoA to provide ATP and NADP during physiological state via increasing the genes involved in mitochondrial and microsomal fatty acid oxidation, and could be potentially used for the treatment of NAFLD clinically [13]. Sirtuin 6 (SIRT6, 50721) belongs to the NAD + -dependent class III deacetylases and is characterized by the unique chromatically location [14]. Previous studies suggested that SIRT6 is involved in the regulation of genomic stability, DNA repair, glycolipid metabolism, aging, cardiovascular disease and metabolism [15]. SIRT6 transgenic (TG) mice effectively reduced high fat diet-induced physiological damage, while SIRT6 deficiency in adipose tissue impairs thermogenic functions of brown adipose tissue and predispose mice to obese [16]. In liver, SIRT6 potentially interact with PPAR-α to maintain the fatty acid oxidation rate and improve mitochondrial stress during high fat diet feeding [17]. Hepatocyte-specific SIRT6 deletion predisposes mice to nonalcoholic steatohepatitis via up-regulation of basic leu cine zipper transcription factor 1 (Bach1, 12013), a nuclear factor, erythroid derived 2, like 2 (Nfe2l2, 18024) repressor [18]. Although multiple genes have been proved to alter hepatic lipid metabolism via targeting SIRT6/PPAR axis, pharmacological molecular, which targets SIRT6 to therapy NAFLD has never been reported. Ginsenoside Rc has been previous reported to prevent inflammation, oxidation response and protect the central nervous system [19]. In 3T3-L1 adipocytes, ginsenoside Rc stimulates anti-adipogenic activity via down-regulating CCAAT/enhancer binding protein (C/EBP), alpha (Cebpa, 12606) and peroxisome proliferator activated receptor gamma (PPARγ, 19016) [20]. Ginsenoside Rc administration suppressed hormones (corticotropin, dibutyryl cyclic AMP and epinephrine)-induced lipolysis in rat epididymal fat cells, suggesting a potential role of ginsenoside Rc in reducing hepatic lipid burden [21]. However, the effects and the detailed molecular mechanisms by which ginsenoside Rc regulates hepatic lipid metabolism remain to be fully understood. Our study identified ginsenoside Rc as a novel SIRT6 activator and protects mice against high fat diet induced NAFLD in a SIRT6-PPAR-α work manner for the first time. Ginsenoside Rc administration effectively alleviates HFD-induced oxidative, inflammation response and mitochondrial stress dependent on SRIT6-PPAR-α axis. Hepatic specific SIRT6 deletion abrogated ginsenoside Rc derived protective effects against lipid and glucose disorder during HFD exposure, suggesting a potential role of ginsenoside Rc-SIRT6 interaction in the treatment of NAFLD and metabolic syndrome.

Wild-type C57BL/6J (WT) mice(6-8weeks) were purchased from the Animal Laboratory Center of Guangdong Province (Guangzhou, China). The ginsenosides Rc (CAS.No11021-14-0) was purchased from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai,China) with a purity of 98%. Mice with floxed alleles of Sirt6 were crossed with mice that express Cre recombinase driven by albumin promoter in the liver to generate liver specific Sirt6-deficient (LKO) mice. All mice were housed in individually ventilated cages and maintained on a 12 h light-dark cycle. The mice were fed a HFD (60% fat; Research Diets) ad libitum with free access to water. All animal experiments were conducted under protocols approved by and in accordance with the guidelines of the Animal Ethics Committee of Guangzhou University of Chinese Medicine. All surgeries were performed under sodium pentobarbital anaesthesia, and all efforts were made to minimize suffering. Based on previous papers [[22], [23], [24]], WT and LKO mice on HFD for 12 weeks were injected intraperitoneally with ginsenoside Rc (5 mg/kg, 10 mg/kg, 20 mg/kg) or saline (Control) once a day for another 4 weeks. Serum and hepatic ALT, AST, TG and TC were determined using an automated Monarch device (Science and Technology Park, Guangzhou, China). Serum TNFα, IL-6 levels were measured using an ELISA assay (eBioscience, San Diego, CA, USA). Serum ketone body were determined by ELISA (R&D Systems, Minneapolis, USA).

For GTT and PTT, mice fasted for 16 h received an intraperitoneal injection of glucose (1g/kg) or pyruvate sodium (1g/kg). For ITT, mice fasted for 6 h received an intraperitoneal injection of human insulin (0.75 IU/kg). Then we calculated homeostasis model assessment of insulin resistance (HOMA-IR) according to the following formula: (HOMA-IR index = fasting serum insulin (μUI/mL) × fasting blood glucose (mM)/22.5.). Blood glucose concentrations were measured from tail blood at the indicated times using a One-Touch Ultra" glucometer (Aikang Inc., Hangzhou, China) and serum insulin was measured using an ultrasensitive ELISA kit (CUSABIO BIOTECH CO., LTD., Wuhan, China; Cat.CSB-E05071m).

Mouse primary hepatocytes (MPHs) were isolated and cultured as previously reported [25]. MPHs were seeded in 96 well plates, and 5 wells were repeated. After culturing in cell incubator for 12h, change to a new medium containing Rc (drug concentration gradient: 0, 20, 40, 80, 200, 500μM), and set blank wells and control wells were incubated for 24 hours after administration. Aspirate the medium, add and incubated with the pre-mixed medium containing CCK-8 (100μL 1640 medium, 10μL CCK-8 solution), and then measure the OD value at 450nm with a microplate reader. MPHs were harvested after incubation for 48 h with 500 μM oleic acid (OA) & 300 μM palmitic acid (PA) (OA&PA model group) and OA&PA plus 10μM/20μM/40μM Rc (OA&PA + Rc group). All experiments were repeated four times.

MPHs were incubated with 0.4μci/ml [9,10-3H] oleic acid (PerkinElmer Life Sciences) and 100 μM unlabeled oleic acid (conjugated with BSA) in krebs-Ringer buffer(119 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2.6 mM MgSO4,24.6 mM NaHCO3, 2.6 mM KH2PO4, 10 mM HEPES, pH 7.4) for 1 h at 37°C. The supernatant was collected and incubated with 1.3 M perchloric acid. All solutions were centrifuged at 16,000 g for 10 minutes, and the supernatant was neutralized with 2 M KOH and 0.6 M MOPS. 3ml scintillation was added into solution and measure [3H] radioactivity.

The mtDNA copy number was used as a marker for mitochondrial density using qPCR as previously reported. Briefly, total DNA was isolated from liver using a Universal Genomic DNA Extraction kit (Tiangen, Beijing, China) according to the manufacturer's instructions. The mitochondrial DNA copy number was calculated from the ratio of mitochondrial-encoded gene COXII/nuclear-encoded gene ribosomal protein S18 (Rps18, 20084). The primer sequences of COXII and Rps18 were presented in Supplemental Table 1.

MPHs were seeded in 6-well plates and treated as above, followed by incubation with DCFH-DA (5 uM) at 37°C for 0.5h, avoiding light. Then, the cells were rinsed with PBS for three times, and the fluorescence emission of the cells was detected by fluorescence microscopy. For, in vivo ROS analysis, mice were injected with DCFH-DA (10mM) via tail vein for one hour before sacrificed and livers were collected. The luciferase reporter for the liver ROS levels were detected using berthold technologies LB983 NC100.

Normal distribution of all data was first tested. If the data fit a normal distribution, the statistical analysis was performed using the two-tailed unpaired t-test (SPSS 17.0); if not, the statistical analysis was carried out using the nonparametric test (SPSS 17.0). All data were presented as mean ± SEM. p < 0.05 was considered statistically significant.

To investigate the function of ginsenoside Rc on abnormal lipid metabolism, OA&PA-incubated MPHs were used. As our hypothesis, ginesenoside Rc displayed a lower cytotoxicity (Fig. 1A). And, ginesenoside Rc treatment effectively reduced the intracellular triglycerides (TG) levels in OA&PA-incubated MPHs in a dosage dependent manner, while the total cholesterol (TC) levels was slightly affected (Fig. 1B). Subsequent Oil red O staining analysis also reveals a decreased lipid deposition in OA&PA-incubated MPHs treated with ginsenoside Rc (20μM, 40μM) (Fig. 1C). Our further mechanism analysis revealed that ginsenoside Rc administration significantly increased fatty acid oxidation rate in OA&PA-incubated MPHs, while the lipogenesis was slightly affected (Fig. 1D). Ginsenoside Rc treatment obviously upregulated the genes involved in fatty acid oxidation, along with a slight suppression of lipogenic genes, which leads to a decreased lipid deposition in OA&PA-incubated MPHs (Fig. 1E). Collectively, these data suggested a potential role of ginsenoside Rc in promoting lipid catabolism.

Considering the protective effects ginsenoside Rc against lipid accumulation in vitro, we assumed that ginsenoside Rc may defend mice against high fat diet induced hepatic steatosis. Thus, different dose of ginsenoside Rc were daily injected (ip) into HFD mice for 4 weeks. Our results demonstrated that ginsenoside Rc treatment for 4 weeks decreased the body weight 12.3% of wild type (WT) mice feed on HFD in a dosage dependent manner (Fig. 2A). The ginsenoside Rc-induced reduction of body weight was manifested by a decreased in fat mass, while the lean mass was unaffected (Fig. 2B). Ginsenoside Rc treatment also significantly decreased the liver weight of WT mice fed on HFD (Fig. 2C). Our subsequent histological analysis (H&E) and Oil Red O staining displayed a reversed apparent hepatosteatosis in WT mice fed on HFD after ginsenoside Rc treatment (20mg/kg) (Fig. 2D). Ginsenoside Rc treatment effectively reduced the intracellular hepatic and serum TG levels. In addition, ginsenoside Rc treatment reduced the HOMA-IR and AST/ALT levels in HFD mice, implying an improvement of NAFLD severity. (Fig. 2E & Supplemental Table 2 & Fig.S1). Furthermore, our mechanism analysis revealed that ginsenoside Rc administration stimulates hepatic fatty acid rates, as revealed by the circulating ketone body levels and genes involved in hepatic lipoglysis and fatty acid oxidation in WT mice fed on HFD, increasing the hepatic lipid consumption (Fig.2F & Fig.S2A). Of note, ginsenoside Rc treatment for 4 weeks significantly reduced the fasting blood glucose levels and insulin levels, suggesting an improvement of glucose metabolism disorder in WT mice on HFD (Fig. 2G and H). The high fat diet exposure induced glucose intolerance and insulin resistance in HFD mice were effectively ameliorated by ginsenoside Rc treatment (Fig. 2I and J). Moreover, the pyruvate tolerance was also increased due to ginsenoside Rc administration, indicating an attenuation of hepatic glucogenesis process in WT mice fed on HFD (Fig. 2K). Consistently, the glucogenic genes including peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (PGC-1, 19017), phosphoenolpyruvate carboxykinase (PEPCK,18534) and glucose-6-46 phosphatase (G6pase, 14377) were repressed by ginsenoside Rc (Fig. 2L). Overall, these data suggested that ginsenoside Rc functions as a potent and positive regulator of insulin resistance and hepatic steatosis during HFD exposure.

To systemically determine the mechanism in which ginsenoside Rc promotes lipid catabolism, RNAseq data of liver samples from HFD mice with ginsenoside Rc or saline treatment was analyzed and 3627 genes were differently regulated by ginsenoside Rc treatment with 1590 genes upregulated and 2037 gene inhibited (Fig. 3A). Followed KEGG pathway analysis demonstrated that ginsenoside Rc treatment effectively upregulated hepatic fatty acid metabolism related pathways and PPAR-α signaling pathways in WT mice fed on HFD (Fig. 3B). As shown in the heatmap, the expression of SIRT6 and its target genes, as well as PPAR-α, along with other genes involved in hepatic lipid and glucose metabolism were increased due to ginsenoside Rc treatment, suggesting a potential role of SIRT6-PPAR-α axis in mediating ginsenoside Rc-induced protective effects (Fig. 3C & S3A). Our western blot analysis also confirmed the ginsenoside Rc-increased expression of SIRT6 in vivo (up) and in vitro (down) (Fig. 3D). We further performed docking analysis between ginsenoside Rc and SIRT6, and our data indicated a good binding affinity via hydrophobic interaction (Fig.3E & Fig.S4). Ginsenoside Rc increased the deacetylase activity of SIRT6 via this good binding affinity (Fig. 3F). Overall, these data indicated that ginsenoside Rc potentially acts as a novel SIRT6 activator through changing its expression and enzyme activity. And this interaction may increase the PPAR-α-mediated fatty acid oxidation rate and increased lipid catabolism in HFD mice.

To further validate the function of SIRT6 in mediating ginsenoside Rc-induced protective effects, we generated liver specific SIRT6-deficient (LKO) mice and subjected LKO mice to HFD exposure for 12 weeks followed by ginsenoside Rc administration. As previously reported, LKO mice bear a conditional deletion of SIRT6 at exon 2 and displayed a miss of SIRT6 protein levels in the liver, rather than other tissues (Fig. S5A). In MPHs derived from LKO mice, ginsenoside Rc at high dosage failed to reduce the OA&PA-derived lipid deposition due to SIRT6 deficiency (Fig. S5B). Compared with WT mice, ginsenoside Rc treatment has no effects of the body weight of LKO mice fed on HFD, even at high dosage (Fig. 4A). Ginsenoside Rc-induced reduction of fat mass and liver weight was abolished in LKO mice fed on HFD (Fig. 4B and C). Consistent with previous reports, LKO mice developed severer NAFLD easily, and ginsenoside Rc treatment did not alleviate the lipid droplet formation in these mice (Fig. 4D). Moreover, ginsenoside Rc failed to change the intracellular hepatic and serum TG levels, as well as the serum ALT and AST level in LKO mice fed on HFD (Fig. 4E & Supplemental Table 2). The further mechanism analysis revealed that the activation of hepatic lipoglysis and fatty acid oxidation by ginsenoside Rc was diminished due to SIRT6 knockout (Fig.4F & Fig.S2B). SIRT6 deficiency completely abrogated the protective effects of ginsenoside Rc on glucose metabolism disorder during HFD exposure, illustrated by the unchanged fasting blood glucose and insulin levels in LKO mice fed on a HFD (Fig. 4G and H). The GTT and ITT results showed that ginsenoside Rc-induced glucose tolerance and insulin sensitivity were also abolished in LKO mice fed on HFD (Fig. 4I and J). Also, ginsenoside Rc treatment failed to alter the pyruvate tolerance and hepatic genes involved in glucogenesis process in LKO mice fed on HFD (Fig. 4K and L). Collectively, these data indicated the important role of SIRT6 in mediating ginsenoside Rc-induced protective effective on hepatic glucose and lipid metabolism during HFD exposure.

Previous studies indicated that long-time high fat diet exposure impaired hepatic mitochondrial function and erupted ROS overproduction, promoting the NAFLD development. Thus, we investigate the effects of ginsenoside Rc on high-fat diet-induced mitochondrial stress and intracellular redox disorder. Interestingly, ginsenoside Rc treatment significantly increased biogenesis and morphogy of hepatic mitochondrial, suggesting an improvement of mitochondrial stress in WT mice fed on HFD (Fig. 5A and B). Moreover, the improvement of mitochondrial function contributes to ginsenoside Rc-induced intracellular redox disorder in WT mice fed on HFD, as revealed by the decreased ROS levels in the liver (left) and MPHs (right) (Fig. 5C). Of note, SIRT6 plays a crucial role in this process. Ginsenoside Rc failed to change hepatic mitochondrial biogenesis and morphogy in LKO mice fed on HFD (Fig. 5D and E). Moreover, ginsenoside Rc did not alter the ROS levels in the liver (left) and MPHs (right) derived from LKO mice (Fig. 5F). Overall, these data revealed a ginsenoside Rc-SIRT6 work manner in maintaining intracellular redox balance during HFD exposure.

Collective studies indicated that excessive intracellular fatty acids and ROS act as potent stimulators of inflammatory genes, and long-time high fat diet exposure stimulated the release of DAMP molecules, triggering severe inflammation response. Interestingly, ginsenoside Rc treatment effectively decreased serum inflammatory cytokines, including tumor necrosis factor (TNFα, 21926) and interleukin 6 (IL-6, 16193) levels (Fig. 6A and B). In contrast, SIRT6 deletion completely abolished ginsenoside Rc-induced change of serum inflammatory cytokines, including TNFα and IL-6 levels in LKO mice fed on HFD (Fig. 6C and D). These data suggested a potential SIRT6-mediated role of ginsenoside Rc in defending inflammatory response during HFD exposure.

Hepatic steatosis, contributed by impaired fatty acid oxidation and abnormal lipogenesis, acts as the first step of NAFLD and stimulators for the second hit to liver [26,27]. Accumulating studies have indicated that special oxidation process, including propionic acid oxidation, α-oxidation, ω-oxidation and unsaturated fatty acid oxidation serve as the most important pathway of lipid metabolism in mammals, especially PPAR-α-mediated mitochondrial fatty acid β-oxidation, is widely accepted as the main physiological manner to maintain hepatic lipid homeostasis [28]. Genetic or non-genetic activation of PPAR-α effectively facilitates the β-oxidation of fatty acids to produce acetyl-CoA, transforming and providing ATP and NADP during physiological state via increasing the genes involved in mitochondrial and microsomal fatty acid oxidation [29]. PPAR-α activation in hepatocytes coordinates with inhibition of lipogenesis significantly defends the progression of NAFLD in mice and in humans and thus might be of therapeutic interest. In contrast, PPAR-α deficiency mice developed hepatic steatosis in a fast and serve manner, even on chow diet. Previous studies reported that several members of ginsenosides exert multiple protective effects via targeting PPAR signaling pathways. Ginsenosides Rb1 exerted anticalcific properties through PPAR-γ/Wnt/β-catenin axis. Ginsenoside Rg1 defends hydrogen peroxide induced oxidative damage via activating PPAR pathway. Moreover, ginsenosides Rc, as one of the most important components of ginsenosides, has previously reported to inhibit adipogenesis via targeting PPARγ in adipose tissue [20]. However, the effects of ginsenosides on PPAR-α-mediated mitochondrial fatty acid β-oxidation during the development of NAFLD are rarely reported. In this study, our results demonstrated that ginsenosides Rc treatment effectively increased the hepatic fatty acid oxidation rate and the genes involved in this process, especially PPAR-α, which alleviates the hepatic steatosis during HFD exposure. Increasing evidence confirmed that NAFLD subjects are prone to glucose and lipid disorders and accompany with a series of metabolic syndromes, characterized by insulin resistance. SIRT6, one of the most important nuclear deacetylates, mainly deacetylates the histones H3 lysine 9 (H3K9) and H3 lysine 56 (H3K56), and plays a significant role in regulating hepatic lipid and glucose metabolism via regulating gluconeogenesis and sugar degradation [30,31]. Hepatic specific SIRT6 deficiency mice spontaneously displayed hypoglycemia and increased long-chain fatty acid uptake, triglyceride synthesis and fatty liver formation. SIRT6 transgenic mice effectively reduced HFD-induced insulin resistance and NAFLD phenotype via deacetylation of H3K9 in indicated gene promoter and regulating its expression [32]. Hepatic SIRT6 was reported to bind PPAR and its response element within promoter regions, activating gene transcription and β-oxidation. SIRT6 also binds to nuclear receptor coactivator 2 (NCoA-2,17978) and reduces NCOA2k780 acetylation in the liver, thereby stimulating it to activate PPAR in a SIRT6-dependent manner [17]. Genes, which targeting and increasing SIRT6, potential protects mice against HFD-induced hepatic glucose and lipid dysfunction. Although multiple pharmacological molecules have been approved to alter hepatic glucose and lipid metabolism through a sirtuin dependent manner, effective molecular, which targets SIRT6 to therapy NAFLD was has never been reported. Our results for the first time identified ginsenosides Rc as a novel and potential SIRT6 activator. Ginsenosides Rc administration significantly increased hepatic expression of SIRT6. Of note, ginsenosides Rc also showed good binding affinity with SIRT6, and ginsenosides Rc treatment markedly reduced the acetylation of H3K9 and H3K56, suggesting an elevation of SIRT6 enzyme activity. Ginsenosides Rc treatment increased the SIRT6-PPAR-α axis activity and defends HFD-induced lipid deposition in the liver. Mitochondria are the main organelles of glucose and lipid metabolism in cells, and the primary targets for excessive intracellular fatty acids, causing DNA damage and abnormal ultrastructure of mitochondrial membrane [33]. Long-time HFD exposure decreased the mitochondrial capacity to storage calcium and destroyed oxidative phosphorylation (OXPHOS) capacity, illustrated by impaired mitochondrial potential, morphogy, oxygen consumption and cellular ATP levels [34]. Dysfunction of mitochondrial respiratory chain erupts intracellular ROS overproduction, which leads to lipid peroxidation and slows down fatty acid oxidation rate, forming a vicious cycle. Moreover, ROS inhibits the secretion of very low-density lipoprotein (VLDL) by liver cells and aggravates the toxicity of lipid peroxidation products, including malondialdehyde (MDA) and nonene [35]. This will promote hepatic lipid deposition and collagen fiber synthesis, and further form fibrosis. Previous studies have indicated that SIRT6 overexpression could suppresses glucose-induced mitochondrial dysfunction and apoptosis in podocytes and retinal ganglion cells. Yet, the function of SIRT6 on hepatic ROS, especially HFD-induced ROS overproduction is never been reported. In our study, we for the first time reported that ginsenosides Rc supplement could effectively improve HFD-induced mitochondrial stress and ease hepatic excessive ROS in a SIRT6 dependent manner, suggesting a link between SIRT6 dysfunction to hepatic oxidative stress during NAFLD progress. In summary, we reported ginsenoside Rc as a novel SIRT6 activator via upregulating its expression and enzyme activity for the first time. We found ginsenoside Rc administration stimulates hepatic genes involved in PPAR-α-mediated fatty acid oxidation and reduced hepatic steatosis in HFD mice dependent on the activation of hepatic SIRT6-PPAR-α axis. Moreover, ginsenoside Rc treatment improved high fat diet-induced mitochondrial stress, oxidative stress and inflammatory damage in a SIRT6 dependent manner (Fig. 6E).

No interest is declared.