Fibronectin type III domain-containing 5 (FNDC5) protein induces browning of subcutaneous fat and mediates the beneficial effects of exercise on metabolism. However, whether FNDC5 is associated with hepatic steatosis, autophagy, fatty acid oxidation (FAO), and lipogenesis remains unknown. Herein, we show the roles and mechanisms of FNDC5 in hepatic steatosis, autophagy, and lipid metabolism. Fasted FNDC5−/− mice exhibited severe steatosis, reduced autophagy, and FAO, and enhanced lipogenesis in the liver compared with wild-type mice. Energy deprivation–induced autophagy, FAO, and AMPK activity were attenuated in FNDC5−/− hepatocytes, which were restored by activating AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Inhibition of mammalian target of rapamycin (mTOR) complex 1 with rapamycin enhanced autophagy and FAO and attenuated lipogenesis and steatosis in FNDC5−/− livers. FNDC5 deficiency exacerbated hyperlipemia, hepatic FAO and autophagy impairment, hepatic lipogenesis, and lipid accumulation in obese mice. Exogenous FNDC5 stimulated autophagy and FAO gene expression in hepatocytes and repaired the attenuated autophagy and palmitate-induced steatosis in FNDC5−/− hepatocytes. FNDC5 overexpression prevented hyperlipemia, hepatic FAO and autophagy impairment, hepatic lipogenesis, and lipid accumulation in obese mice. These results indicate that FNDC5 deficiency impairs autophagy and FAO and enhances lipogenesis via the AMPK/mTOR pathway. FNDC5 deficiency aggravates whereas FNDC5 overexpression prevents the HFD-induced hyperlipemia, hepatic lipid accumulation, and impaired FAO and autophagy in the liver.
Introduction
Nonalcoholic fatty liver disease (NAFLD) is characterized by triacylglycerol (TG) accumulation within hepatocytes (1). Hepatosteatosis is strongly associated with obesity and may progress to steatohepatitis and even to end-stage liver disease, including liver cirrhosis and hepatocellular carcinoma (2,3). Fatty acid β-oxidation (FAO) in mitochondria is a process to shorten the fatty acids into acetyl-CoA, which can be converted into ketone bodies or incorporated into the tricarboxylic acid cycle for full oxidation (4). Accumulation of lipid in the liver can be traced by the impaired FAO and increased de novo lipogenesis (5).
Autophagy is a mechanism involved in cellular homeostasis delivering cytoplasmic content to the lysosomes for degradation to macronutrients (6). Defects in autophagy play a major role in metabolic dysregulation (7). Although some studies showed the lipogenic role of autophagy, most experiments supported autophagy as a lipolytic mechanism (6). Reduced autophagic function promotes the initial development of hepatic steatosis and progression of steatosis to liver injury, and agents to augment hepatic autophagy may have therapeutic potential in nonalcoholic steatohepatitis (8–10).
Fibronectin type III domain containing 5 (FNDC5) is a type I membrane protein that has 209 amino acid residues. FNDC5 induces browning of subcutaneous adipocytes and mediates the beneficial effect of exercise on metabolism (11). Irisin, a cleaved and secreted fragment of FNDC5, acts on white adipose cells to induce a broad program of brown fat–like development (11). Our recent studies have shown that FNDC5 overexpression ameliorates hyperlipemia and enhances lipolysis in adipose tissues in obese mice (12) and that irisin inhibits hepatic gluconeogenesis and increases glycogen synthesis in type 2 diabetic mice and hepatocytes (13). However, whether FNDC5 could improve hepatosteatosis, autophagy, and FAO remains unknown. Nutrient deprivation is known to activate AMPK, resulting in the inhibition of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which regulates lipid metabolism, cellular proliferation, and autophagy (14,15). The mTORC1 inhibits peroxisome proliferator–activated receptor (PPAR)-α activity, which regulates mitochondrial functions and FAO (16). Interestingly, PPAR-α acts downstream of FNDC5 (11). The current study is designed to investigate the roles and underlying mechanisms of FNDC5 in hepatic steatosis, autophagy, FAO, and lipogenesis in FNDC5−/− mice, high-fat diet (HFD)–induced obese mice, and primary hepatocytes. Moreover, the therapeutic effects of FNDC5 were investigated.
Research Design and Methods
FNDC5−/− Mice and HFD-Induced Obese Mice
Male C57BL/6 wild-type (WT) and FNDC5−/− mice on a C57BL/6 background (Nanjing BioMedical Research Institute, Nanjing University, Nanjing, China) were used in the experiments. In HFD-induced obesity models, mice at the age of 4 weeks began to receive the HFD (21.8 kJ/g, 60% of energy as fat) for 12 weeks. Normal chow diet (14.7 kJ/g, 13% of energy as fat) was used as the control (12,17). Procedures were approved by the Nanjing Medical University Experimental Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals (NIH publication, 8th edition, 2011). Mice were caged in an environment with controlled temperature and humidity with free access to water and food under a 12-h light/dark cycle. At the end of experiments, mice were fasted overnight and then euthanized with an overdose of pentobarbital sodium (150 mg/kg, i.v.).
FNDC5 Overexpression in Mice
Mice at the age of 4 weeks began to receive the control diet or the HFD for 12 weeks. One intravenous injection of recombinant lentivirus (1 × 108 transuding units/mL, 100 μL) expressing FNDC5 or EGFP vector was administered at the end week 6 after the diet application (12). Acute experiments were performed 6 weeks after the lentivirus introduction.
Knockdown of AMPK or Atg5 by Small Interfering RNA in Hepatocytes
Primary hepatocytes were transfected with small interfering RNA (siRNA) for knockdown of AMPK or autophagy protein 5 (Atg5). Scramble siRNA was used as the control. The sequences of siRNA were as follows: AMPK: sense CGGGAUCCAUCAGCAACUATT, antisense UAGUUGCUGAUGGAUCCCGAT (18). Atg5: CCGGCCTTGGAACATCACAGTACATCTCGAGATGTACTGTGATGTTCCAAGGTTTTTG.
Primary Hepatocyte Isolation and Cell Culture
Primary hepatocytes were isolated and cultured as previously described (13,19). Briefly, mice were anesthetized with pentobarbital (50 mg/kg, i.p.). HEPES buffer containing collagenase II (0.66 mg/mL) was perfused via the portal vein. Livers were removed and excised aseptically. Cells were dispersed and filtrated. Hepatocyte suspensions were purified by centrifugation in Percoll adjusted to a density of 1.065 g/mL for 10 min at 50g to reduce the amount of nonparenchymal cells. With this method, the nonparenchymal cells are less than 1% (20). Cell viability was determined with trypan blue dye. Plates with cell viability greater than 95% were used for experiments. The hepatocytes were maintained in low glucose DMEM containing 10% FBS with penicillin (100 units/mL) and streptomycin (100 μg/mL) at 37°C in a 5% CO2 atmosphere.
Autophagy Monitoring
Cells were transfected with tandem GFP-red fluorescent protein (RFP)-LC3 adenovirus (Hanbio, Shanghai, China) for 24 h according to the instructions. Cells were treated with amino acid starvation, rapamycin, or chloroquine for 2 h to observe the autophagy flux. When autophagy inducts, GFP and RFP are both expressed as yellow dots representing autophagosomes after the images emerge. When autophagosomes fuse with lysosomes and form autolysosomes, the GFP degrades in an acid environment, but RFP-LC3 maintains, showing as red dots (21).
Oil Red O Staining and Immunohistochemistry
Livers were fixed in 4% neutral-buffered formalin phosphate and were embedded in paraffin or optimal cutting temperature compound, respectively. The tissues were subsequently sliced into 5-μm sections. Oil Red O staining was used to detect the lipid content in the livers. For immunohistochemistry evaluation, liver sections were incubated with anti-p62 antibody (Abcam Ltd., Cambridge, U.K.) or anti-LC3B antibody (Cell Signaling Technology, Danvers, MA). The anti-LC3B antibody showed stronger reactivity with LC3BII, according to the manufacturer's instructions.
Western Blot
Protein extracts were electrophoresed, blotted, and then incubated with antibodies against FNDC5, S6, phosphorylated (P)-S6, AMPKα, P-AMPKα, LC3B, Raptor, P-Raptor, ULK1, P-ULK1, and GAPDH (Cell Signaling Technology) and P62 (Abcam Ltd.) with appropriate secondary horseradish peroxidase–conjugated antibodies, and then developed.
Quantitative Real-Time PCR Analysis
RNA extracted from livers or hepatocytes was subjected to reverse transcription, and quantitative real-time PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA). Expression levels of all genes were normalized by GAPDH levels. The sequences of primers are listed Supplementary Table 1.
Measurement of Lipids and Markers of Hepatocyte Injury
Enzymatic methods were used to evaluate the levels of nonestesterified fatty acid (NEFA), TG, and cholesterol and the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum or the liver. The kits for serum NEFA serum were bought from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); for serum TG, AST, ALT, and hepatic NEFA from Jiancheng Bioengineering Institute (Nanjing, China); and for hepatic TG and cholesterol from Applygen Technologies Inc. (Beijing, China).
Assessment of FAO Rate In Vitro
Hepatocytes isolated from WT and FNDC5−/− mice were incubated with 0.05 mmol/L carnitine and 0.25 μCi [14C]palmitate (GE Healthcare Life Sciences, Pittsburgh, PA) for 24 h. A total of 100 μL of the medium was used for measuring acid-soluble metabolites with scintillation counter, and 800 μL of medium was harvested on ice and mixed with ice-cold perchloric acid (70%, 200 μL) to precipitate BSA-fatty acid complexes. The samples were centrifuged for 10 min at 14,000g, and the radioactivity of the supernatant was evaluated by liquid scintillation as captured 14CO2 (18).
Chemicals
FNDC5, lipopolysaccharide (LPS), palmitate, WY14643, carnitine, and rapamycin were bought from Sigma-Aldrich Inc. (St. Louis, MO), and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) was bought from Beyotime Biotechnology Inc. (Shanghai, China).
Statistics
Data are presented as mean ± SEM. A value of P < 0.05 was considered statistically significant. Two-tailed unpaired Student t tests were used to compare two treatment groups. One-way and two-way ANOVA were used for data analysis of more than two groups, followed by Bonferroni post hoc analysis.
Results
Fasting Causes Severe Lipid Accumulation in FNDC5-Deleted Mice
Fasting causes lipid mobilization from peripheral depots into the liver (22). To determine the role of FNDC5 in hepatic lipid accumulation, the responses of lipids to fasting were compared between FNDC5−/− mice and WT mice. Lipid accumulation in livers was increased in FNDC5−/− mice and became severe after fasting for 16 h compared with WT mice (Fig. 1A and B). Fasting caused more increases in serum NEFA and TG levels in FNDC5−/− mice than in WT mice (Fig. 1C). The efficiency of FNDC5 gene knockout was confirmed by serum FNDC5 levels and liver FNDC5 expressions (Fig. 1D–F). These results indicate that FNDC5 prevents excessive lipid accumulation in livers.
FNDC5 Deficiency Causes Defects in AMPK/PPAR-α–Mediated FAO
Reduced FAO increases hepatic lipid accumulation, and chronic starvation increases FAO gene expressions via transcriptional mechanisms (5). Activation of AMPK stimulates FAO via PPAR-α signaling (18). Thus, the roles of FNDC5 in regulating FAO and its downstream pathway were investigated. Basal and fasting-induced FAO gene expressions (Pparα, Hmgcs2, Cpt1, Acox1, Sirt3, and Cyp4a10) (Fig. 2A) and AMPK phosphorylation (Supplementary Fig. 1) were reduced in FNDC5−/− mice livers. Activation of AMPK with AICAR increased FAO gene expressions (Fig. 2B) and reduced TG levels in FNDC5−/− mice livers (Fig. 2D). Furthermore, AICAR stimulated liver AMPK activation and the subsequent mTORC1 inhibition in WT and FNDC5−/− mice (Supplementary Fig. 2). Knockdown of AMPK with siRNA increased TG contents in WT and FNDC5−/− hepatocytes (Fig. 2E). WY14643, a PPAR-α agonist, caused a greater increase in the expressions of PPAR-α target genes (Hmgcs2, Cpt1, Acox1, Ehhadh, Acsl1, Peci, Cyp4a10, and Cyp4a12) in WT hepatocytes than in FNDC5−/− hepatocytes (Fig. 2C). WY14643 inhibited palmitate-induced lipid accumulation in WT and FNDC5−/− hepatocytes (Fig. 2F and Supplementary Fig. 3). It induced a greater increase in FAO rate in WT hepatocytes than in FNDC5−/− hepatocytes (Fig. 2F). These findings indicate that FAO is reduced in FNDC5−/− mice livers and that FNDC5 is important for fasting-induced FAO gene expressions, which are mediated by the AMPK pathway. FNDC5 deficiency causes impairment in AMPK/PPAR-α–mediated FAO.
Rapamycin Prevents the Impaired FAO and Increased Lipogenesis in FNDC5 Deficiency
Because nutrient deprivation–induced AMPK represses mTORC1 activity (16), we further investigated whether mTORC1 is involved in the effects of FNDC5 on FAO. FNDC5 deficiency caused an increase in liver ribosomal S6 protein phosphorylation, a marker of mTORC1 activity (23), which was suppressed by rapamycin, an mTORC1 inhibitor (Fig. 3A). It reduced the increased liver TG contents in both fed and fasted states in FNDC5−/− mice (Fig. 3D). Rapamycin restored the reduced FAO gene expressions (Pparα, Hmgcs2, Cpt1, and Sirt3) in FNDC5−/− mice under fed and fasting conditions (Fig. 3B). The results indicate that increased mTORC1 activity contributes to the reduced FAO in mice with FNDC5 deficiency. Lipogenesis is a factor contributing to hepatic lipid accumulation (24). The mRNA levels of lipogenic genes (Srebp1c, Dgat1, Fasn, and Scd1) were raised in FNDC5−/− livers under fed and fasted conditions, which were attenuated by rapamycin (Fig. 3C). These results indicate that FNDC5 deficiency potentiates lipogenesis via increased mTORC1 activity.
FNDC5 Deficiency Causes Attenuation in Autophagy and AMPK Activity
Reduced p62 expression and increased LC3BII generation from LC3BI are markers of autophagy (25). The mRNA levels of autophagy genes, including UNC51-like kinase 1 (Ulk1), Ulk2, Atg5, Atg8, and Atg10 were downregulated in FNDC5−/− mouse livers (Fig. 4A). Fasting reduced p62 expression and increased LC3BII/LC3BI in WT livers but not in FNDC5−/− livers (Fig. 4B), which was confirmed by the liver immunohistochemistry (Supplementary Fig. 4). Moreover, amino acid deprivation caused a greater enhancement in autophagy flux in WT hepatocytes than in FNDC5−/− hepatocytes (Fig. 4C). These results indicate that autophagy in the liver is reduced in FNDC5−/− mice and that FNDC5 is required for the fasting-induced autophagy response.
Nutrient deprivation activates AMPK, which phosphorylates Raptor (an essential component of mTORC1), resulting in net repression of mTORC1 signaling (26,27). AMPK stimulates autophagy through direct ULK1 phosphorylation (28). Metformin, a biguanide antihyperglycemic agent, causes carbohydrate starvation and AMPK activation (29). We found that either amino acid starvation (Fig. 4D) or metformin (Supplementary Fig. 5) stimulated AMPK, Raptor, and ULK1 phosphorylation in WT hepatocytes but not in FNDC5−/− hepatocytes. Activation of AMPK with AICAR restored the reduced autophagy in FNDC5−/− hepatocytes (Fig. 4E) and FNDC5−/− livers (Supplementary Fig. 6). Knockdown of AMPK with siRNA attenuated autophagy in WT and FNDC5−/− hepatocytes (Fig. 4F). These findings indicate that AMPK is an essential downstream effector of FNDC5 in mediating its effect on autophagy.
Rapamycin Repairs the Attenuated Autophagy in FNDC5 Deficiency
Activation of mTORC1 is required for hepatic lipid accumulation (30). Reduced Raptor phosphorylation in FNDC5−/− livers (Fig. 4D) suggests a possibility that the increased mTORC1 might be involved in the signaling pathway of impaired autophagy. Immunohistochemistry analysis showed that inhibition of mTORC1 with rapamycin prevented the increased p62 expression and the reduced LC3B expression in FNDC5−/− livers (Fig. 5A). Consistently, Western blot analysis showed that rapamycin promoted autophagy in WT and FNDC5−/− livers (Fig. 5B) and in FNDC5−/− hepatocytes (Fig. 5D), and increased autophagy flux in WT and FNDC5−/− hepatocytes. Chloroquine, an autophagy inhibitor, blocked autophagosomes fusing with lysosome to form autolysosomes, as showed by increased autophagosome accumulation and decreased autolysosomes in both WT and FNDC5−/− hepatocytes (Fig. 5E). These results indicate that increased mTORC1 activity contributes to the attenuated autophagy in FNDC5−/− livers. Moreover, the liver function markers serum ALT and AST levels were increased in FNDC5−/− mice, which were restored by rapamycin (Fig. 5C). Palmitate-induced lipid accumulation is generally used as a cellular steatosis model (31). To ascertain whether reduced autophagy in FNDC5−/− mice is involved in lipid accumulation, the effects of rapamycin and Atg5 siRNA on palmitate-induced lipid accumulation in primary hepatocytes were investigated. Palmitate caused more lipid accumulation in FNDC5−/− hepatocytes than in WT hepatocytes, which was inhibited by rapamycin. Inhibition of autophagy by knockdown of Atg5, an essential autophagy gene, with Atg5 siRNA attenuated the role of rapamycin in reducing lipid accumulation in FNDC5−/− hepatocytes (Fig. 5F). Moreover, rapamycin reduces TG content in hepatocytes in FNDC5−/− mice (Supplementary Fig. 7). These results indicate that the ability of rapamycin to reduce lipid accumulation in FNDC5−/− hepatocytes is largely dependent on autophagy.
FNDC5 Deficiency Aggravates Hepatosteatosis, Lipid Metabolic Disturbance, and Impairment of Autophagy in HFD-Induced Obese Mice
An HFD is generally used to induce obesity in rodents (12,32). We investigated whether FNDC5 deficiency causes more severe hepatosteatosis, lipid metabolic disturbance, and impairment of autophagy in mice with obesity induced by an HFD for 12 weeks. Liver weight and the liver weight–to–body weight ratio were greater in FNDC5−/−/HFD mice than in WT/HFD mice, but the differences in body weight and food intake between WT/HFD and FNDC5−/−/HFD mice were not significant (Fig. 6A). FNDC5 deficiency aggravated the HFD-induced increases in NEFA, TG, and cholesterol levels in serum and livers (Fig. 6B). FAO gene expressions (Pparα, Hmgcs2, Cpt1, Acox1, and Sirt3) in livers were downregulated (Fig. 6E), whereas lipogenic gene expressions (Srebp1c, Dgat1, Fasn, and Scd1) were upregulated (Supplementary Fig. 8) in FNDC5−/−/HFD mice compared with WT/HFD mice. Hepatosteatosis was more severe in FNDC5−/−/HFD mice than in WT/HFD mice (Fig. 6D). In HFD mice, p62 protein expression was increased in FNDC5−/− livers compared with WT livers (Fig. 6F), and serum ALT and AST levels were higher in FNDC5−/− mice than in WT mice (Fig. 6C). Moreover, deletion of the FNDC5 gene exacerbated AMPK inhibition and enhanced mTOR activation in livers from HFD mice (Fig. 6G). These findings indicate that FNDC5 deficiency causes more severe hepatosteatosis, lipid metabolic disturbance, and impairment of autophagy in obese mice.
Exogenous FNDC5 Enhances FAO and Autophagy In Vitro
Primary WT hepatocytes incubated with FNDC5 (100 nmol/L) for 12 h or 24 h increased FAO gene expressions (Pparα, Hmgcs2, Cpt1, and Acox1) in vitro (Fig. 7A). The effects of FNDC5 for 24 h on these FAO gene expressions almost reached their maximal at the concentration of 100 nmol/L (Fig. 7B). Exogenous FNDC5 attenuated TG accumulation in FNDC5−/− hepatocytes (Supplementary Fig. 9). LPS is known to stimulate autophagy in hepatocytes (33); thus, we compared the role of FNDC5 with LPS in stimulating autophagy. FNDC5 reduced p62 and increased LC3BII levels, similar to the effects of LPS (Fig. 7C). Importantly, palmitate-induced lipid accumulation in primary FNDC5−/− hepatocytes was prevented by FNDC5 (Fig. 7D), and FNDC5 potentiated autophagy in FNDC5−/− hepatocytes (Fig. 7E). In addition, exogenous FNDC5 attenuated the AMPK inhibition and mTOR activation in FNDC5−/− hepatocytes (Fig. 7F). These results indicate that exogenous FNDC5 promotes FAO and autophagy and prevents the FNDC5 deficiency–induced lipid accumulation and autophagy impairment in vitro.
FNDC5 Overexpression Attenuates Hepatosteatosis and the FAO and Autophagy Impairment in HFD-Induced Obese Mice
The effects of lentiviral vector–mediated FNDC5 overexpression in HFD-induced obese mice were investigated to determine whether FNDC5 could be used as a therapeutic strategy for hepatosteatosis and impairment of FAO and autophagy in obesity. FNDC5 overexpression attenuated the HFD-induced increases in NEFA, TG, and cholesterol levels in serum and the liver (Fig. 8A) and reduced the HFD-induced lipid accumulation in the liver (Fig. 8B). It restored the reduced FAO gene expression (Fig. 8C) and the increased lipogenic gene expression (Fig. 8D) in HFD-induced obese mouse livers. Immunohistochemistry showed that the increased p62 expression and reduced LC3B expression in the liver were prevented by FNDC5 overexpression (Fig. 8E), which were further confirmed by Western blot analysis (Fig. 8F). FNDC5 overexpression attenuated the AMPK inhibition and mTOR activation in livers from HFD mice (Fig. 8G). The effectiveness of FNDC5 overexpression in the experiments was confirmed by the changes of serum FNDC5 levels (Supplementary Fig. 10). These findings indicate that long-term increased FNDC5 effectively prevents hepatosteatosis and attenuates the FAO and autophagy impairment in HFD-induced obese mice.
Discussion
Hepatic steatosis is generally regarded as the hepatic manifestation of the metabolic syndrome in diabetes and obesity and is thought to be the initial stage in NAFLD. NAFLD is characterized by the accumulation of TG in lipid droplets within hepatocytes (1,2,34). The primary novel findings in the current study are that FNDC5 plays critical roles in reducing hepatic lipid accumulation by restoring AMPK/mTOR-mediated autophagy, FAO, and lipogenesis. FNDC5 deficiency deteriorated hepatosteatosis, FAO, and autophagy impairment in obesity, whereas FNDC5 overexpression alleviated hepatosteatosis and improved FAO and autophagy in obesity.
Hepatic FAO is increased in response to energy demand in the fasted state (24). Fasting upregulates TG hydrolysis to supply NFFA for oxidation to meet cellular energy needs (35). An alternative energy source with respect to energy deprivation is provided by the breakdown of cellular components by autophagy (35,36). Induction of autophagy corrects hepatic lipid over-accumulation (37). Defective autophagy is involved in NAFLD (38). We found that hepatic FAO in the fed state was reduced in FNDC5−/− mice and that the fasting-induced FAO enhancement was much weaker in FNDC5−/− mice than in WT mice. Consistently, FNDC5 deficiency caused a mild hepatic lipid accumulation in the fed state but a severe hepatic lipid accumulation in the fasted state. Palmitate induced more lipid accumulation in FNDC5−/− hepatocytes than in WT hepatocytes in vitro. Although FNDC5 deficiency had no significant effect on hepatic autophagy in the fed state, the fasting-induced autophagy enhancement response in WT mice almost disappeared in FNDC5−/− mice. Inhibition of autophagy increased lipid accumulation in WT and FNDC5−/− hepatocytes. These findings indicate that FNDC5 is strongly associated with hepatic FAO and autophagy, which at least partially contribute to the reduction of hepatic lipid accumulation, particularly in the fasting state.
Hepatic steatosis is linked to being obese or overweight in most cases (39). We found that FNDC5 deficiency deteriorated hepatosteatosis accompanying FAO and autophagy impairment in HFD-induced obesity, whereas FNDC5 overexpression alleviated hepatosteatosis and repaired the reduced FAO and autophagy in HFD-induced obese mice. Exogenous FNDC5 stimulated FAO and autophagy and attenuated the palmitate-induced hepatic lipid accumulation in primary hepatocytes. These findings indicate the importance of FNDC5 in attenuating hepatic steatosis. Administration of FNDC5 or increased FNDC5 production is expected to be a therapeutic regimen for preventing hepatosteatosis, FAO, and autophagy impairment in obesity.
Most fatty acids in the liver are metabolized by FAO (40). AMPK represses mTORC1 activity (16) and maintains energy homeostasis via suppressing cellular ATP-consuming processes and stimulating ATP-producing catabolic pathways, including FAO (41). AMPK inhibits protein synthesis and mTOR signaling (42), whereas mTORC1 inhibits PPAR-α expression and function (16). PPAR-α stimulates the expression of FAO genes and is implicated in nonalcoholic steatohepatitis (43). We found that FNDC5 deficiency reduced AMPK, Raptor, and ULK1 phosphorylation. Activating AMPK or PPAR-α partially restored the downregulation of PPAR-α and FAO gene expressions. Furthermore, AMPK activation was found in association with TG levels in FNDC5−/− hepatocytes. The PPAR-α agonist WY14643 increased the FAO rate and reduced lipid accumulation in hepatocytes with FNDC5 deficiency. Inhibition of mTORC1 attenuated the increased mTORC1 activity and TG levels and partially restored the attenuated Pparα and FAO gene expressions in FNDC5−/− livers. These data indicate that FNDC5 deficiency reduces AMPK phosphorylation, which subsequently causes mTORC1 activation, and thus, inhibits PPAR-α target gene expressions and FAO. It has been shown that ghrelin upregulates autophagy via AMPK/mTOR restoration (44). Blockage of the mTOR pathway restores endoplasmic reticulum stress–induced autophagy (45). We found that the FNDC5 deficiency–induced defect in autophagy was prevented by AMPK activation and deteriorated by AMPK suppression. Inhibition of mTORC1 restored autophagy impairment and attenuated the liver injury in FNDC5−/− mice. Furthermore, rapamycin alleviated palmitate-induced lipid accumulation in FNDC5−/− hepatocytes, which was abolished by the knockdown of the essential autophagy gene Atg5. These results reveal that FNDC5 deficiency causes AMPK inhibition, mTORC1 activation, and autophagy defects. The beneficial effect of mTORC1 inhibition was largely dependent on restoration of autophagy, further suggesting that the autophagic defect in FNDC5 deficiency is partially responsible for hepatic steatosis. It is noted that rapamycin treatment strongly suppressed the liver S6 phosphorylation but has modest roles in increasing FAO and reducing lipogenic gene expressions in fasting FNDC5-knockout mice. This discrepancy suggests a possibility that some other signal pathways may be involved in regulating FAO and lipogenesis.
Lipogenesis is another important factor contributing to lipid accumulation in the liver (24). Lipogenic gene expressions resulting from mTORC1 inhibition were decreased in FNDC5−/− mice. In HFD-induced obese mice, lipogenic gene expressions were increased in FNDC5−/− mice compared with WT mice. FNDC5 overexpression prevented the increased lipogenic gene expressions in HFD-induced obese mice. On the one hand, these results indicate that FNDC5 deficiency stimulates lipogenesis via the mTOR pathway, which is involved in lipid accumulation in the liver of HFD-induced obesity. On the other hand, serum TG and NEFA levels were increased in FNDC5−/− mice, particularly in the fasting state. FNDC5 deficiency deteriorated hyperlipemia in HFD-induced obese mice. Our previous study showed that FNDC5 overexpression prevented hyperlipemia in HFD-induced obese mice (12), which was further confirmed in the current study. These results revealed that FNDC5 plays a critical role in preventing hyperlipemia.
Previous study has showed that FNDC5 overexpression in HFD-induced obese mice increases energy expenditure, attenuates hyperglycemia and insulin resistance, and activates lipolysis in adipose tissues (12). Irisin, a cleaved and secreted fragment of FNDC5, reduces gluconeogenesis, increases glycogenesis, and improves insulin resistance in streptozotocin/HFD-induced type 2 diabetes (13). Strong irisin immunoreactivity has been found in the liver (46,47). Serum irisin concentrations were inversely associated with liver TG contents in the liver in obese adults (48). A limitation in the current study is that we did not investigate whether the effects of FNDC5 are caused by its cleaved fragment irisin.
In summary, FNDC5 reduces hepatic lipid accumulation via AMPK/mTOR-mediated autophagy and FAO enhancement and de novo lipogenesis reduction. FNDC5 deficiency aggravates whereas FNDC5 overexpression prevents hepatic steatosis, hyperlipemia, impaired FAO, and autophagy, and enhanced lipogenesis in obesity (Supplementary Fig. 11). FNDC5 can be used as a therapeutic regimen for preventing hepatosteatosis and impairment of FAO and autophagy in obesity.
Article Information
Acknowledgments. The authors thank the generous support of the Collaborative Innovation Center for Cardiovascular Disease Translational Medicine.
Funding. This study was supported by National Natural Science Foundation of China (31271213, 31571167, and 91439120).
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. T.-Y.L., X.-Q.X., X.-S.R., M.-X.Z., C.-X.S., J.-J.W., Y.-B.Z., and F.Z. performed the experiments. T.-Y.L., X.-Q.X., Y.H., X.-Y.G., and G.-Q.Z. analyzed the data. T.-Y.L., X.-Y.G., Q.C., Y.-H.L., Y.-M.K., and G.-Q.Z. were involved in study design. T.-Y.-L., X.Y-.G., Q.C., Y.-H.L., Y.-M.K., and G.-Q.Z. edited the manuscript. T.-Y.L. and G.-Q.Z. wrote the manuscript. G.-Q.Z. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.