Upregulation of flavin-containing monooxygenase 3 mimics calorie restriction to retard liver aging by inducing autophagy

CR delays age-related alterations in aging liver

CR is considered to be the most successful anti-aging intervention [18]. We initially analyzed the effects of CR on whole-body and liver aging. According to previous studies, IL-6 levels and fasting insulin contents are elevated during aging [19, 20]. We observed lower serum levels of IL-6, suggesting moderate inflammation and immune response (Figure 1A), and lower fasting insulin levels in serum, indicating the amelioration of insulin resistance, in the CR group compared with the ad libitum-fed (AL) group (Figure 1B). As some studies have suggested that oxidative stress might play a part in the process of aging [21], markers reflecting hepatic oxidative damage or changes in the antioxidant system in the liver were measured. As shown in Figure 1C, 1D, the CR intervention reduced hepatic levels of malondialdehyde (MDA) and increased superoxidase dismutase (SOD) activity in the liver. It has been reported that changes in lipidomic and metabolomic signatures in the liver resulting from CR could contribute to the age-sparing effects of CR [22]. In our study, marked reductions in triglyceride (TG) and total cholesterol (TC) levels in serum and liver were observed after the CR intervention (Figure 1E–1H). We also performed Oil Red O staining to measure hepatic lipid contents. Histological analysis of liver specimens revealed that CR substantially decreased the accumulation of lipid droplets in the aging liver (Figure 1I). Previous studies of anti-aging interventions have indicated that β-gal (β-galactosidase) and p16 levels could serve as molecular markers of senescence [23]. As shown in Figure 1J–1L, the downregulation of β-gal and p16 confirmed the significant anti-aging effects of CR on the liver.

Figure 1. 40% CR for 6 months (14 to 20 months old) delays age-related alterations in aging liver. Levels of (A) IL-6 and (B) insulin in serum were measured by ELISA. Levels of (C) MDA, (D) SOD, (E) serum TG, (F) liver TG, (G) serum TC, and (H) liver TC were measured using commercial kits. (I) Lipid droplet accumulation was assessed via Oil Red O staining. (J) Western blotting was used to determine levels of senescence markers (K) β-gal and (L) p16. Results are shown as the mean ± SD of eight animals per group. *p < 0.05 compared with the control group.

CR upregulates levels of FMO3 in liver and enhances mTOR-regulated autophagy

Previous studies have shown markedly elevated levels of hepatic FMO3 mRNA in 40% CR mice [10]. Here, we investigated protein levels of FMO3 in liver after treatment with CR. As shown in Figure 2A, 2B, protein expression of FMO3 after CR was enhanced.

Figure 2. 40% CR for 6 months (14 to 20 months old) upregulates levels of FMO3 in liver and enhances mTOR-regulated autophagy. (A) Representative western blots of liver lysates from each group of mice, probed with the indicated antibodies. GADPH was used as the internal housekeeping protein control. Image analysis of (B) FMO3, (C) LC3, (D) p62, (E) p-mTOR/mTOR, and (F) p-S6K/S6K was performed using densitometry. Results are shown as the mean ± SD of eight animals per group. *p < 0.05 compared with the control group.

It has been well established that autophagy has a central role in the process of aging, as well as in all forms and at all stages of chronic liver disease [24]. Therefore, we analyzed LC3 and p62 to confirm whether the anti-aging effects of CR were modulated by autophagy. In CR mice, the LC3-II/I ratio increased significantly, while the level of p62 decreased, suggesting that CR promotes autophagy (Figure 2A, 2C, 2D). Given the critical role of mTOR signaling in autophagy, we examined whether CR influenced the activation of mTOR signaling. Our results showed that CR drastically diminished the phosphorylation of mTOR and the downstream S6K, indicating that mTOR signaling is involved in CR-induced autophagy (Figure 2A, 2E, 2F).

Overexpression of FMO3 alleviates age-related alterations in aging liver

To study the effects of FMO3 in aging mice, we used an adenovirus vector (Adv-FMO3) to activate FMO3 genes. The adenovirus vector was non-specific. The effects of CR, FMO3 overexpression, and their co-treatment were evaluated. Our studies showed that serum levels of IL-6 and insulin were significantly reduced in both the CR and FMO3 overexpression groups compared with the AL group. Combination treatment resulted in a marked decrease in serum IL-6 and insulin levels compared with the single treatments or control (Figure 3A, 3B). Next, we investigated the effects of CR, FMO3 overexpression, and their co-treatment on oxidative stress by measuring relative hepatic MDA and SOD levels. Hepatic MDA levels dramatically decreased and hepatic SOD levels drastically increased in the CR and FMO3 overexpression groups compared with the control group. The co-treatment groups showed less MDA activity and higher SOD levels compared with the single treatments or control (Figure 3C, 3D). Serum and liver lipid profiles showed that TG and TC levels in both serum and liver were markedly reduced after treatment with CR, FMO3 overexpression, and combination treatment. The co-treatment group also showed lower serum and hepatic TG and TC levels than the single treatment groups (Figure 3E–3H). Consistent with the TG and TC measurements, Oil Red O staining of the liver specimens showed a lower level of hepatic lipid accumulation in the co-treatment group compared with the other three groups, indicating a synergistic effect between FMO3 overexpression and CR. FMO3 and CR both substantially suppressed the accumulation of lipid droplets in aging liver (Figure 3I). Western blotting showed that the adenovirus expressing FMO3 (Ad-FMO3) caused robust overexpression of FMO3 in the liver (Figure 3J, 3K). Western blot analysis of the molecular markers of senescence revealed that FMO3 overexpression diminished levels of β-gal and p16, thus mimicking CR treatment. A synergistic effect was also observed in the co-treatment group (Figure 3J, 3L, 3M). Thus, FMO3 overexpression improved or reversed key serum and hepatic parameters related to inflammation, metabolism, oxidative stress, liver function, and hepatocyte senescence.

Figure 3. Overexpression of FMO3 alleviates age-related alterations in aging liver. Levels of (A) IL-6 and (B) insulin in serum were measured by ELISA. Levels of (C) SOD, (D) MDA, (E) serum TG, (F) liver TG, (G) serum TC, and (H) liver TC were measured using commercial kits. (I) Lipid droplet accumulation was assessed via Oil Red O staining. (J) Western blotting was used to determine levels of (K) FMO3, (L) β-gal, and (M) p16. Results are shown as the mean ± SD of eight animals per group. *p < 0.05 compared with the control group; ^#p < 0.05 compared with the CR group; ^&p < 0.05 compared with the FMO3 overexpression group.

FMO3 overexpression inhibits mTOR signaling and induces autophagy

To identify the mechanism of the protective effect of FMO3, we evaluated the level of autophagy and the activation of the upstream mTOR signaling that negatively regulated autophagy. As shown in Figure 4A–4C, the LC3-II/I ratio increased considerably and p62 content was greatly reduced in the CR, FMO3 overexpression, and co-treatment groups. The co-treatment group exhibited a higher LC3-II/I ratio and lower p62 level than the single treatment groups. These results indicate that FMO3 overexpression can augment autophagy and has a synergistic effect with CR. As shown in Figure 4A, 4D, 4E, FMO3 and CR both suppressed the phosphorylation of mTOR as well as the downstream S6K, and the combination treatment diminished the phosphorylation of mTOR and S6K substantially compared with single treatments or control. These results indicate that the upregulation of FMO3 acts synergistically with CR, suppressing the mTOR pathway and enhancing autophagy.

Figure 4. FMO3 overexpression inhibits mTOR signaling and induces autophagy. (A) Representative western blots of liver lysates from each group of mice, probed with the indicated antibodies. GADPH was used as the internal housekeeping protein control. Image analysis of (B) LC3, (C) p62, (D) p-mTOR/mTOR, and (E) p-S6K/S6K was performed using densitometry. Results are shown as the mean ± SD of eight animals per group. *p < 0.05 compared with the control group; ^#p < 0.05 compared with the CR group; ^&p < 0.05 compared with the FMO3 overexpression group.

Bafilomycin A1 treatment suppresses mTOR-regulated autophagy

To determine the role of autophagy specifically in the progress of FMO3-induced anti-aging effects on the liver, mice in the FMO3 overexpression group were intraperitoneally injected with a late-stage autophagy inhibitor, bafilomycin A1. The results were consistent with those obtained previously. As shown in Figure 5A–5C, compared with the control group, the FMO3 overexpression group exhibited an increased LC3II/LC3I ratio and decreased p62 content. Bafilomycin A1 is a late-stage autophagy inhibitor that prevents fusion between autophagosomes and lysosomes. The accumulation of LC3II and p62 indicated that autophagy degradation was inhibited successfully. Consistent with this, the FMO3 overexpression group also showed an immense increase in the number of autophagosomes, whereas the group receiving FMO3 overexpression combined with bafilomycin A1 treatment showed more accumulated autophagosomes than the single treatment group, resulting from the inhibition of autophagy in the late stage, as shown in the transmission electron microscopy images in Figure 5F. Notably, compared with the control group, reduced phosphorylation of mTOR and S6K was observed in the FMO3 overexpression group. Treatment with bafilomycin A1, however, abolished the FMO3-induced effects on mTOR/S6K signaling (Figure 5A, 5D, 5E).

Figure 5. Bafilomycin A1 treatment suppresses mTOR-regulated autophagy. (A) Representative western blots of liver lysates from each group of mice, probed with the indicated antibodies. GADPH was used as the internal housekeeping protein control. Image analysis of (B) LC3, (C) p62, (D) p-mTOR/ mTOR, and (E) p-S6K/S6K was performed using densitometry. (F) Autophagosomes were observed through an electron microscope (black arrows). Results are shown as the mean ± SD of eight animals per group. *p < 0.05 compared with the control group; ^#p < 0.05 compared with the FMO3 overexpression group.

Autophagy inhibition blocks FMO3-induced anti-aging effects on liver

Next, we investigated whether autophagy inhibition by bafilomycin A1 treatment could reverse the FMO3-induced beneficial effects on the liver. As shown in Figure 6A, there was a conspicuous increase in serum levels of IL-6 in mice treated with both Ad-FMO3 and bafilomycin A1 compared with those treated with Ad-FMO3 alone. Serum levels of insulin, however, showed no significant changes after autophagy inhibition (Figure 6B). Mouse livers in the autophagy inhibition group exhibited markedly increased MDA and reduced SOD levels compared with the FMO3 overexpression group, indicating that bafilomycin A1 reversed the protective effect of FMO3 overexpression on oxidative stress in the liver (Figure 6C, 6D). As shown in Figure 6E–6H, bafilomycin A1 treatment greatly increased TG and TC levels in both serum and liver. Histologically, a notable reversion of the FMO3-induced reduction of lipid accumulation in the liver was also observed (Figure 6I) through Oil Red O staining of liver tissue. According to the western blot analysis, there were no significant differences in FMO3 protein expression levels between the groups with and without bafilomycin A1 treatment (Figure 6J, 6K). Levels of β-gal and p16 greatly increased following inhibition of autophagy by bafilomycin A1, compared with the FMO3 overexpression group without autophagy inhibition (Figure 6J, 6L–6N). Thus, the inhibition of autophagy abrogated the beneficial effects of the FMO3 overexpression intervention.

Figure 6. Bafilomycin A1 treatment blocks FMO3-induced anti-aging effects on liver. Levels of (A) IL-6 and (B) insulin in serum were measured by ELISA. Levels of (C) SOD, (D) MDA, (E) serum TG, (F) liver TG, (G) serum TC, and (H) liver TC were measured using commercial kits. (I) Lipid droplet accumulation was assessed via Oil Red O staining. (J) Western blotting was used to determine the levels of (K) FMO3, (L) β-gal, and (M) p16. Results are shown as the mean ± SD of eight animals per group. *p < 0.05 compared with the control group; ^#p < 0.05 compared with the FMO3 overexpression group.

Animal models

Twelve-month-old male inbred C57BL/6 mice (weighing 30–40 g) were purchased from the Experimental Animal Center of the Chinese People’s Liberation Army no. 4 Military Medical University. Animals were maintained on a 12:12 light:dark cycle with lights on at 8:00 am and were fed a 20.5% protein rodent diet (Puluteng Ltd., Shanghai, China). Before the experiment, food consumption and body weights were monitored weekly for all mice for at least 2 weeks. Once food consumption had stabilized, food weights were calculated for the CR cohort based on the average daily food consumption of AL controls. The mice were 14 months old when we began conducting CR. During the following 6 months, all mice in the AL group had unrestricted access to food. All mice in the CR group received a 20% reduction of food supply for the first week compared with their AL intake, followed by a 30% reduction for the second week; the 40% reduction was started in the third week and continued until the end of the experiments. The CR animals were fed at the beginning of the light cycle. All groups had unrestricted access to water. Mice in the FMO+ groups were injected with Ad-FMO3 (1×109 particles/mouse), and the other groups with Ad-GFP, via the tail vein 2 weeks before sacrifice. Mice in the autophagy groups received an intraperitoneal injection of bafilomycin A1 (2 μM/kg) 24 hours after the tail intravenous injection, while the others received the same volume of 0.9% NaCl. The mice were 20 months old when they were sacrificed. The animal studies were approved by the Committee on the Ethics of Animal Experiments of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital.

Construction of adenovirus vectors

Ad-FMO3 was constructed by Yazai Biotech Ltd. (Shanghai, China). Reverse transcription polymerase chain reaction was used to amplify the cDNA fragment coding for the mouse FMO3 gene. The recombinant adenoviral vector expressing FMO3 was prepared in HepG2 cells. The titer of adenoviral vectors was determined using the gradient dilution method. The prepared adenovirus was stored at −80 °C for subsequent experiments. 1×109 particles/mouse of adenovirus [42] were delivered via the tail vein two weeks before sacrifice.

Biochemical examinations and enzyme linked immunosorbent assay (ELISA)

Levels of TC and TG in serum and liver tissue were measured using commercially available kits provided by Nanjing Jiancheng Institute of Biotechnology (Nanjing, China) according to the manufacturer’s instructions. Serum insulin and IL-6 were examined respectively using mouse insulin ELISA kits from AIS (Hong Kong, China) and mouse IL-6 ELISA kits from Biovendor (Czech Republic) following the manufacturer’s protocols.

Assay of antioxidant markers

Liver specimens were homogenized in phosphate-buffered saline to prepare 10% liver homogenate; this was then centrifuged at 4 °C and 3000 rpm for 10 min, and the supernatant was collected for the assays. Hepatic MDA activity and SOD contents were detected by a spectrophotometric method using commercially available kits provided by Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). All assays were performed according to the manufacturer’s instructions.

Western blot analysis

Western blotting was performed using a standard protocol. The liver sample was homogenized and lysed with a lysis buffer (RIPA with protease and phosphatase inhibitor). Total protein concentrations were determined using bicinchoninic acid protein assay kits (Beyotime Biotechnology, China). Proteins were separated with equal amounts in the same location from each specimen by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Thereafter, the membranes were blocked with 5% skim milk for 1 hour at room temperature and incubated overnight with the appropriate antibodies at 4 °C, followed by incubation with horseradish peroxidase-labeled secondary antibody (1:5000, Bio-Rad, USA) for 1 hour at room temperature. The primary antibodies included anti-FMO3 rabbit mAb (1:1000, Abcam, USA), mTOR rabbit mAb (1:1000, Cell Signaling Technology, USA), phospho-mTOR rabbit mAb (1:1000, Cell Signaling Technology), S6K rabbit mAb (1:1000, Cell Signaling Technology, USA), phospho-S6K rabbit mAb (1:1000, Cell Signaling Technology), p16 rabbit mAb (1:1000, Cell Signaling Technology), β-gal rabbit mAb (1:1000, Cell Signaling Technology), LC3II/I rabbit mAb (1:1000, Cell Signaling Technology), and P62 rabbit mAb (1:1000, Cell Signaling Technology). Finally, the immunoreacting bands were visualized by an enhanced chemiluminescence method using an electrochemiluminescent solution (Millipore, USA) and the ImageJ (NIH, USA) analysis system.

Oil Red O assay

Frozen liver tissue was cut into 3-μm-thick sections, then stained with filtered Oil Red O (EMD Millipore) dissolved in 60% isopropanol for 15 min at room temperature. Afterwards, the slides were incubated in hematoxylin to counterstain the nuclei and transferred to an aqueous mounting medium (Thermo Fisher Scientific). Images were captured under a light microscope (magnification x200 and x400).

Transmission electron microscopy

Liver samples were fixed in 3% glutaraldehyde at 4 °C for 24 hours, post-fixed in 1% osmium tetroxide in sodium phosphate buffer at room temperature, and cut into ultrathin sections (50–70 nm thick) with an ultramicrotome. Sections were stained with 2% uranyl acetate and lead citrate, then viewed and photographed using a Hitachi H-7650 transmission electron microscope at 120 kV. Ultrastructural analysis was performed using ImageJ (NIH).

Statistical analysis

Sample sizes, as described in the figure legends, were selected based on effect size and availability, according to the usual standard using the “resource equation” method [43]. Statistical analysis was performed using the GraphPad Prism 7.0 software. Data are presented as mean ± standard deviation (SD). One-way analysis of variance was used to assess statistical significance using Tukey post hoc analysis; p-values less than 0.05 were considered to be statistically significant.