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¹Lab for Aging Research, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China

²Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China

³Department of Clinical Medicine, Medical School of Nankai University, Tianjin, China

Corresponding author.

^* Correspondence
Hengyi Xiao, Lab for Aging Research, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, 1 Keyuan 4 Road, Gaopeng Ave, Chengdu, China. Tel.: 86 28 8516 4023; fax: 86 28 8516 4005; e‐mail: nc.ude.ucs@xiygneh,

AMPK activation is beneficial for cellular homeostasis and senescence prevention. However, the molecular events involved in AMPK activation are not well defined. In this study, we addressed the mechanism underlying the protective effect of AMPK on oxidative stress‐induced senescence. The results showed that AMPK was inactivated in senescent cells. However, pharmacological activation of AMPK by metformin and berberine significantly prevented the development of senescence and, accordingly, inhibition of AMPK by Compound C was accelerated. Importantly, AMPK activation prevented hydrogen peroxide‐induced impairment of the autophagic flux in senescent cells, evidenced by the decreased p62 degradation, GFP‐RFP‐LC3 cancellation, and activity of lysosomal hydrolases. We also found that AMPK activation restored the NAD⁺ levels in the senescent cells via a mechanism involving mostly the salvage pathway for NAD⁺ synthesis. In addition, the mechanistic relationship of autophagic flux and NAD⁺ synthesis and the involvement of mTOR and Sirt1 activities were assessed. In summary, our results suggest that AMPK prevents oxidative stress‐induced senescence by improving autophagic flux and NAD⁺ homeostasis. This study provides a new insight for exploring the mechanisms of aging, autophagy and NAD⁺ homeostasis, and it is also valuable in the development of innovative strategies to combat aging.

The AMPK pathway was inactivated in cells with H₂O₂‐induced senescence

H₂O₂ treatment‐induced fibroblast senescence has been widely used as a model of SIPS (Chen & Amos, 1994; Toussaint et al., 2000). With a modified procedure, we obtained H₂O₂‐induced senescence in NIH3T3 cells with good homogeneity. In brief, suspended cells were treated with H₂O₂ for 45 min, and then, they were incubated in complete medium for adhesion culture for five days. At three to five days post‐H₂O₂ exposure, the cells became enlarged and flattened morphologically. Strong positive staining for senescence‐associated galactosidase (SA‐β‐Gal) was observed in these cells, with the percentage of SA‐β‐Gal‐positive cells increased by 30 to 50‐fold (Fig. 1A). Another marker of senescence, senescence‐associated heterochromatic foci (SAHFs), was also positive in our H₂O₂‐treated cells (Fig. 1B). In addition, the expression levels of four other senescence‐associated genes (p53, p21, IL6, and IL8) were increased in these cells (Fig. 1C,D). The induction of senescence was obtained in the experiments using human MRC‐5 embryonic lung fibroblasts and human umbilical vein endothelial cells (HUVECs) (Fig. S1A,B, Supporting information).

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Figure 1

H₂O₂ induced senescence and AMPK pathway inhibition in NIH3T3 Cells. Cells were treated with H₂O₂ and incubated in complete medium without H₂O₂ for 3‐5 days. CTL means untreated cells. (A) Representative images of SA‐β‐Gal staining of the cells (left) and percentages of SA‐β‐Gal‐positive cells in a total of 1000 cells (right). (B) Representative images of SAHFs in cells (left) and percentages of SAHFs‐positive cells in 1000 cells (right). (C) Representative images from immunoblot assays against p53 and β‐actin. (D) Relative fold‐changes in the mRNA levels of the genes encoding p21, IL6 and IL8, as determined by qRT‐PCR. (E) Representative images from immunoblot assays against phosphorylated AMPKα (pAMPK, Thr172), AMPKα1, phosphorylated ACC (pACC, Ser79), and β‐actin. (F) The ratio of pAMPK to total AMPK was quantified by densitometry based on immunoblot images from three independent experiments. (G) Relative fold‐changes in the mRNA levels of two AMPK target genes (CPT‐1 and FAS) were monitored by qRT‐PCR assays. *P < 0.05 compared to the control (CTL). The bar represents 100 μm.

Given that the decline in AMPK activity has been reported to be associated with aging (Salminen & Kaarniranta, 2012), we tested this association in our senescence system. Beginning from the first day after H₂O₂ treatment, the levels of phosphorylated AMPKα (Thr172) and phosphorylated ACC (Ser79) markedly decreased, while the protein levels of AMPKα1 remained unchanged (Fig. 1E,F). Similarly, the expression levels of two AMPK target genes, carnitine palmitoyl transferase (CPT‐1), and fatty acid synthase (FAS), also decreased in the senescent cells (Fig. 1G). These data suggest that the AMPK pathway was downregulated in H₂O₂‐induced senescent cells.

AMPK activation prevented H₂O₂‐induced senescence

To evaluate the effects of AMPK activation on H₂O₂‐induced senescence, two known AMPK activators, metformin (Met) and berberine (BBR), were included in the culture medium after H₂O₂ treatment. As shown in Fig. 2A, both Met and BBR significantly enhanced the protein level of pAMPKα and the pACC in senescent cells. In addition, the mRNA levels of CPT‐1 gene and FAS gene increased (Fig. 2B).

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Figure 2

Activation of AMPK prevented H₂O₂‐induced senescence. A to D: H₂O₂‐treated NIH3T3 cells were incubated in complete medium with metformin (Met, 5 to 10 mM) or berberine (BBR, 5 to 10 μM) for 3 days. (A) Representative images from immunoblot assays against pAMPKα (Thr172), AMPKα1, pACC (Ser79), and β‐actin. (B) Relative fold‐changes in mRNA levels of CPT‐1 and FAS as determined by qRT‐PCR. (C) Representative images of SA‐β‐Gal staining of cells (left), and percentages of SA‐β‐Gal‐positive cells. D‐F: NIH3T3 cells were treated with H₂O₂ and incubated with Met (10 mM), BBR (10 μM) and an AMPK inhibitor, Compound C (CC, 10 μM), alone or in combination for 3 days. (D) Representative images from immunoblot assays. (E) Relative mRNA levels of CPT‐1 and FAS as determined by qRT‐PCR. (F) Representative images of SA‐β‐Gal staining of cells (left) and percentages of SA‐β‐Gal‐positive cells (right). G‐I: NIH3T3 cells without H₂O₂ treatment were used. (G) The decrease in AMPK activity in DN‐AMPK‐expressing NIH3T3 cells was shown by the decrease in AMPKα phosphorylation. (H) Representative images of SA‐β‐Gal staining of the nontransfected cells with or without CC (upper) and cells transfection with DN‐AMPK (lower). (I) The percentages of SA‐β‐Gal‐positive cells were calculated based on the images represented in H. (J) H₂O₂‐treated cells incubated with or without Met (10 mM) or BBR (10 μM) for 3 days, representative images of SA‐β‐Gal staining of cells transfected with empty vector (upper) or DN‐AMPK (lower). (K) The percentages of SA‐β‐Gal‐positive cells were calculated based on the images presented in J. *P < 0.05 and **P < 0.01 compared to the vehicle control or indicated sample, ^##P < 0.01 compared to the indicated sample. The bar represents 100 μm.

Next, we observed that when the H₂O₂‐treated cells were incubated with medium containing Met and BBR, there was a dose‐dependent decrease in the percentage of SA‐β‐Gal‐positive cells, which was similar to that caused by rapamycin treatment (Fig. 2C). The effects of AMPK activation on senescence were also evaluated in MRC‐5 cells and HUVECs (Fig. S2A,B). In addition, the preventive effects of AMPK activators were further confirmed using an AMPK inhibitor, Compound C (CC). The efficiency of CC for AMPK inactivation was first confirmed (Fig. 2D,E). As expected, when combined with CC, the effects of Met or BBR on senescence prevention were largely blunted when CC was coexisted, as indicated by the remarkable increase in SA‐β‐Gal‐positive cells (Fig. 2F). These results demonstrate that the activation of AMPK by Met and BBR can prevent H₂O₂‐induced senescence, and this prevention could be prevented by CC.

To clarify the role of AMPK in senescence protection_, the effects of chronic AMPK inhibition by CC were evaluated in normal cells. As found, many cells incubated with CC for seven days were SA‐β‐Gal‐positive and larger in size compared with the control (Fig. 2H,I), and the cells expressing dominant‐negative AMPKa1 (pDN‐AMPK) also became SA‐β‐Gal positive (Fig. 2G–I). Unsurprisingly, pDN‐AMPK overexpression also increased the SA‐β‐Gal‐positive rate in the H₂O_2‐treated cells compared with the cells transfected with the empty vector. Moreover, the antisenescence effects of Met and BBR were weakened in these cells (Fig. 2J,K). These results are consistent with the above findings, confirming the preventive effects of AMPK on senescence.

AMPK activation restored the H₂O₂‐impaired autophagic flux in senescent cells

Redressing the autophagic activity is an emerging concept for aging prevention (Rubinsztein et al., 2011). With this in mind, we investigated the status of autophagy in senescent cells, paying particular attention to the autophagic flux. The results showed that the p62 protein, dramatically accumulated in H₂O₂‐induced senescent cells without an accompanying increase in p62 mRNA (Figs 3A; S3A). Importantly, different from proliferating cells, the p62 protein did not accumulate when an autolysosomal inhibitor, HCQ, was applied to the senescent cells (Fig. 3B). This implies that almost no autolysosomal degradation capacity remained in the H₂O₂‐treated cells, so the inhibitory effects of HCQ in autolysosomes were abrogated. Next, by detecting the protein abundance of Cathepsin B, an important lysosomal protease, we found that the abundance of activated forms of the protein was significantly decreased in H₂O₂‐treated cells (Fig. 3C). To examine the status of autophagic flux, a NIH3T3 cell population stably expressing a tandem RFP‐GFP‐LC3 fusion protein was established and employed to visualize and distinguish GFP+RFP+ (yellow) and GFP‐GFP+ (red) LC3 puncta (Klionsky et al., 2012). As shown in Fig. S3B, although the formation of LC3 puncta increased in both H₂O₂ ‐treated cells and serum‐starved cells, the puncta in H₂O₂‐treated cells tended to become GFP+/RFP+ (yellow), while those in starved cells tended to be GFP‐/RFP+ (red). These results reveal that H₂O₂‐induced cellular senescence is accompanied by impaired autophagic flux.

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Figure 3

Activation of AMPK improved autophagic flux impaired by H₂O₂ treatment. (A) Cells were treated as Fig. 1, representative images from immunoblot assays against p62 and β‐actin. (B) Control and H₂O₂‐treated cells were incubated with solvent or hydroxychloroquine (HCQ, 2 μM) for 3 days; representative images from immunoblot assays against p62 and β‐actin. (C) Representative images from immunoblot assays against Cathepsin B protein. D to G: GFP‐RFP‐LC3‐expressing cells were treated with H₂O₂ then incubated for 3 days with different reagents including: Met (10 mM), BBR (10 μM), CC (10 μM), Rapa (50 nM), and HCQ (2 μM). (D) Representative confocal fluorescent images of RFP‐GFP‐LC3‐expressing cells, and the right panel shows the merged fluorescence. The bar represents 20 μm. (E) Percentages of cells with puncta like LC3 were figured up based on the images represented in D, dividing into GFP+/RFP+ group (yellow column) and GFP‐/RFP+ group (red column). (F) Representative images from immunoblot assays against LC3 and p62 proteins. (G) GFP‐RFP‐LC3‐expressing cells were incubated with indicated reagents alone or in combination for 3 days; representative confocal fluorescent images are shown as described in D. The bar represents 20 μm. (H) Percentages of cells with punctalike LC3 were figured up and grouped as described in E. (I) Representative images from immunoblot assays against LC3 and p62 proteins. (J) Representative images of SA‐β‐Gal staining of cells (left) and the percentages of SA‐β‐Gal‐positive cells (right). The bar represents 100 μm.*P < 0.05 compared to the vehicle control, ^#P < 0.05 compared to the indicated sample.

Then, the influence of AMPK activity on autophagic flux in senescent cells was investigated via several approaches. First, using RFP‐GFP‐LC3 cells, we found, similar to autophagy activator rapamycin, that Met and BBR weakened the GFP fluorescence in cells. On the contrary, similar to lysosomal inhibitor HCQ, CC enhanced GFP fluorescence and increased yellow LC3 puncta in cells (Fig. 3D,E). Second, with Western blot assay, we found that both Met and BBR alleviated the H₂O₂‐induced accumulation of the LC3 and p62 proteins, and this alleviation was consistent with the effect of rapamycin (Fig. 3F). Third, we observed that HCQ markedly blocked the effects of Met and BBR on the promotion of red LC3 puncta (Fig. 3G,H); likewise, HCQ blocked the effects of Met and BBR on the decreases in the LC3 and p62 proteins (Fig. 3I). In fact, blocking autophagic flux by HCQ aggravated H₂O₂‐induced senescence and blunted the protective effect of AMPK (Fig. 3J). Fourth, using Atg5‐silenced cells, we found that the influence of autophagic flux blockage the effect of BBR on the protection against senescence (Fig. S4A,B). Finally, we confirmed with HUVECs that BBR can restore autophagic flux and prevent cellular senescence, and this effect can be blunted by HCQ (Fig. S5A,B). Taken together, these findings indicate that AMPK activation by Met and BBR can improve the impaired autophagic flux in H₂O₂‐induced senescent cells.

AMPK restored autophagic flux associated with the amelioration of lysosomal function, mTOR inactivation, but not the nuclear translocation of TFEB

Additional evidence linking AMPK activation to autolysosome restoration was obtained by monitoring the lysosomal functions and the status of the mTOR‐TFEB signaling. The results showed that treatment with Met or BBR increased the abundance of both the proenzyme and activated forms of Cathepsin B (Fig. 4A), as well as the activity of lysosomal acid phosphatase (Fig. 4B). Moreover, we found that BBR treatment significantly suppressed mTOR phosphorylation in senescent cells, similar and even stronger than that induced by rapamycin and insulin as an mTOR activation control (Fig. 4C).

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Figure 4

AMPK restored the autophagic flux associated with the amelioration of lysosomal function, mTOR inactivation, but not the nuclear translocation of TFEB. A‐C: H₂O₂‐treated NIH3T3 cells were incubated in complete medium with different reagents for 3 days as indicated. (A) Representative images from immunoblot assays against Cathepsin B protein. (B) The acid phosphatase activity in cells. (C) Representative images from immunoblot assays against phosphorylated mTOR (pmTOR, Ser2448), phosphorylated 70S6K (P‐p70S6K, Thr389), pAMPK (Thr172), and β‐actin. (D) Immunofluorescent images of TFEB after treatment with indicated reagents. The bar represents 20 μm. (E) Immunoblot assays against TFEB protein of total cytoplasmic and nuclear subcellular fractions obtained from NIH3T3 cells with the indicated treatment. (F) Relative fold‐changes in mRNA levels of two TFEB target genes (GNS and LAMP‐1) were monitored by qRT‐PCR assays.*P < 0.05 and **P < 0.01 compared to the vehicle control.

It has reported that mTOR inactivation could result in the release TFEB from the mTORC1 complex following the nuclear translocation of TFEB and elevated transcription of multiple genes related to autophagic activity (Roczniak‐Ferguson et al., 2012). However, BBR‐induced mTOR inactivation in H₂O₂‐treated cells accompanied without increased nuclear distribution of TFEB, but with the receded (Fig. 4D,E). This situation is different from that observed in rapamycin‐treated cells, where TFEB kept in nucleus (Fig. 4D,E). To know the transcriptional function of nuclear TFEB in H₂O₂‐treated cells, we measured the expression of two representative TFEB‐targeted genes, GNS and LAMP1. We found that the transcription of GNS and LAMP1 genes reduced in H₂O₂‐treated cells, but this reduce recovered when BBR applied (Fig. 4F). Above results indicate that the positive effect of AMPK activation on autophagic flux is relevant to its role in combating lysosome dysfunction, and also to mTOR inactivation and TFEB activation as a transcriptional factor. Furthermore, our results reveal that BBR can recede the nuclear accumulation of TFEB induced by H₂O₂ treatment.

AMPK restored NAD⁺ synthesis in cells with H₂O₂‐induced senescence

Reduced cellular NAD⁺ level is a feature of aging (Gomes et al., 2013), and a link between NAD⁺ synthesis and AMPK has been suggested (Brandauer et al., 2013). For these reasons, we next examined the relationships among AMPK, NAD⁺ synthesis and senescence. As shown in Fig. 5A, the NAD⁺ level in the senescent cells was significantly decreased, which was accompanied by a decrease in the NAD/NADH ratio (Fig. S6A). Correspondingly, supplementation with nicotinamide mononucleotide (NMN), a precursor of NAD⁺ synthesis (Yoshino et al., 2011), decreased the percentage of SA‐β‐Gal‐positive cells and increased cellular NAD⁺ levels following H₂O₂ treatment (Fig. 5B,C). Importantly, Met and BBR upregulated the cellular NAD⁺ level, while CC had the opposite effect (Fig. 5D). Similar results were observed using HUVECs (Fig. S7). The ratio of NAD/NADH also increased in the Met‐ and BBR‐treated cells (Fig. S6B).

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Figure 5

The preventive effects of AMPK on the senescence associated with NAD⁺ synthesis. (A) Cellular concentrations of NAD⁺ on day 3 or day 5 after H₂O₂ treatment. (B) Representative images of SA‐β‐Gal staining of cells (left) and percentage of SA‐β‐Gal‐positive cells (right). (C) Cellular concentrations of NAD⁺ in the cells incubated with nicotinamide mononucleotide (NMN) for 3 days after H₂O₂ treatment. (D) H₂O₂‐treated cells were incubated with Met (10 mM), BBR (10 μM), or CC (10 μM) for 3 days. The concentrations of NAD⁺ are shown. (E) A schematic diagram of two pathways of NAD⁺ synthesis. Red ones indicate rate‐limiting enzymes, and blue ones illustrate the enzyme inhibitors we used. (F) Relative fold‐changes in the mRNA levels of NAMPT, as monitored by qRT‐PCR (left), and representative images from immunoblot assays against NAMPT are shown (right). The ratio of NAMPT to actin was quantified by densitometry, based on the immunoblot images from three independent experiments. (G) Cells were incubated with Met, BBR, and CC for 3 days. Representative images from immunoblots against NAMPT are shown. The ratio of NAMPT to β‐actin was quantified by densitometry based on the immunoblot images from three independent experiments. (H) H₂O₂‐treated NIH3T3 cells were incubated with NAMPT inhibitor (FK866, 5 nM), QPRT inhibitor (PHTH, 1 mM), Met (10 mM), and BBR (10 μM) alone or in combination as indicated for 3 days. The percentages of SA‐β‐Gal‐positive cells are shown. (I) The mRNA levels of NMNAT1,NMNAT2, and NMNAT3 genes in the cells infected with corresponding lentivirus‐shRNA or nontargeting shRNA (shCON) as determined by qRT‐PCR. (J) Cellular concentrations of NAD⁺ in shRNA‐infected cells. (K) Cells infected with shRNAs were treated with H₂O₂ and incubated with BBR for 3 days. Representative images of SA‐β‐Gal staining of cells (left) and percentages of SA‐β‐Gal‐positive cells (right). *P < 0.05 compared to the control (A, F) or vehicle (C, D, G, K) or shCON (I, J). The bar represents 100 μm.

The involvements of NAD⁺ synthesis assessed next. The known pathways of NAD⁺ synthesis were diagrammed in Fig. 5E. Our results showed that mRNA and protein abundance of nicotinamide phosphoribosyl transferase (NAMPT), a rate‐limiting enzyme of NAD⁺ synthesis in salvage pathway, significantly decreased in senescent cells (Fig. 5F); however, the mRNA level of quinolinic acid phosphoribosyl transferase (QPRT), a rate‐limiting enzyme of NAD⁺ synthesis in de novo pathway, was increased (Fig. S8A), while no expressional alteration in nicotinamide mononucleotide adenylyltransferase (NMNAT) (Fig. S8A). In addition, the situation of NAD⁺ consumption was examined via measuring the activity of a major NAD⁺‐consuming enzyme poly‐ADP‐ribose polymerase (PARP‐1). Resultantly, PARP‐1 activity significantly increased in senescent cells (Fig. S9). These results demonstrate that the NAD⁺ decline found in senescent cells is relevant to both its synthetic decline and consumptive elevation, and for the synthesis, the involvement of salvage pathway seems dominating.

Then, the situation upon AMPK activation was investigated. As shown, the abundance of NAMPT protein and mRNA level of QPRT were regulated positively by Met and BBR (Figs 5G, S8B), while no change in the activities of PARP‐1 observed (data not shown). As the effort to know the mechanism about AMPK improved NAD⁺ synthesis, two sets of blocking experiments were performed. The pharmacological approach showed that NAMPT inhibitor FK866 and QPRT inhibitor PHTH‐prompted senescence, but their roles were significantly repressed by Met and BBR (Fig. 5G). The shRNA‐mediated knockdown approach for the NMNAT gene showed that each of the three shNMNATs effectively suppressed NMNAT expression (Fig. 5I), and the cellular concentrations of NAD⁺ (Fig. 5J). Moreover, they markedly perturbed the protective role of AMPK activation against senescence (Fig. 5K). These results demonstrate the connection of NAD⁺ synthesis with the AMPK activity in our system and particularly emphasize the involvement of the salvage NAD⁺ synthesis pathway.

Given that NAD⁺ is a coenzyme of Sirt family deacetylases that positively regulates autophagy (Lee et al., 2008), and restrain aging (Gomes et al., 2013), the activity of Sirt1 was monitored. As the findings, the activity of Sirt1 was increased by AMPK activation in senescent cells (Fig. S10A,B). Then, we noted that EX527, a chemical inhibitor of Sirt1, suppressed the effects of AMPK on senescence and p62 accumulation (Fig. S10C). However, compared with normal cells, the ability of EX527 for blocking the p62 accumulation was only moderate. These results suggest that Sirt1 is involved in H₂O₂‐induced senescence as a downstream mediator of AMPK and NAD⁺ biosynthesis.

Reagents

Metformin and berberine were purchased from MUSTBIO technology (Chengdu, China); Compound C was from CALBIOCHEM (Darmstadt, Germany). Hydroxychloroquine (HCQ), rapamycin, and phthalic acid (PHTH) were from Sigma‐Aldrich (CA, USA). Nicotinamide mononucleotide (NMN) and FK866 were from Santa Cruz Biotechnologies (TX, USA). EX527 was from selleck (CA, USA). Antibodies against LC3 (12741), phospho‐ACC (Ser79) (3661), phospho‐mTOR (Ser2448) (5536), phospho‐p70S6K (Thr389) (9206), and p53 (2524) were purchased from Cell Signaling technology (MA, USA). Those against phospho‐AMPKα (Thr172) (ab133448), Cathepsin B (ab30443), NAMPT (ab109210), AMPKα1 (ab32047), and SQSTM1/p62 (3340‐1) were from Abcam (MA, USA). Antibodies against β‐actin and Lamin B were from Proteintech (Beijing, China), anti‐TFEB (A303‐673A‐M) was from Bethyl (TX, USA), FITC‐goat anti‐rabbit IgG was from Invitrogen (CA). The reagent used for cDNA plasmids transfection was Lipofectamine 2000 (Invitrogen, CA), and that for lentivirus‐shRNA plasmids was X‐treme GENE HP (Roche, CA, USA).

Cell culture and H₂O₂ treatment

NIH3T3 cells (murine fibroblast line) and MRC‐5 cells (human fibroblast line) were purchased from Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences (Shanghai, China), cultured in complete Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS in a humidified atmosphere with 5% CO₂ at 37 °C. HUVECs (human umbilical vein endothelial cells) (ATCC) cultured in low glucose (5.6 mM) RIPA1640 supplemented with 10% FBS. For senescence induction, a modified H₂O₂ treatment protocol was used. In brief, cells seeded in 100‐mm dishes with 5 × 10⁵ cells per dish density were trypsinized and suspended in phosphate buffer solution (PBS) at 1 × 10⁶ cells mL⁻¹ density and exposed to 400 μM (NIH3T3) or 300 μM (MRC‐5) or 700 μM (HUVECs) H₂O₂ in an Eppendorf tube at 37 °C for 45 min. During H₂O₂ treatment, the tube was turned upside down gently every 5 min. H₂O₂ treatment was terminated by a 5‐min centrifugation at 800 rpm and a washing process. Then, the cells were cultured with complete medium. During the adhesion cultivation, the cells accepted different treatments that will be described later in individual figure legends.

SA‐β‐Gal staining and SAHFs staining

Intracellular senescence‐associated‐β‐galactosidase (SA‐β‐Gal) activity was assayed using an SA‐β‐Gal staining kit (Beyotime, Beijing) according to manufacturer's instructions, and senescent cells were identified as bluish green‐stained cells under a phase‐contrast microscope. The percentage of SA‐β‐Gal‐positive cells in total cells was determined by counting 1000 cells in 7 random fields, for each group. Senescence‐associated heterochromatic foci (SAHFs) was visualized by DAPI staining after cells were fixed in situ with 4% paraformaldehyde and washed by PBS. Images with DAPI stained nuclei with blue fluorescence were taken by fluorescence microscope. The percentage of SAHF‐positive cells was determined by counting more than 1000 cells in 7 random fields, for each group. The results were expressed as mean of triplicates ± SD.

Drug treatments

For AMPK activity modulation, metformin, berberine, and Compound C were applied as described in the legends. For autophagy modulation, rapamycin and hydroxychloroquine (HCQ) were added as described in the legend of Fig. 3. For NAD⁺ precursor supplementation, nicotinamide mononucleotide (NMN) was applied as described in the legend of Fig. 5. For inhibiting NAD⁺ synthesis pathways, FK866 and phthalic acid (PHTH) were applied as described in the legend of Fig. 5.

mRFP‐GFP‐LC3 expressing cells generation and fluorescent LC3 puncta analysis

Cells were transfected with mRFP‐GFP‐LC3 plasmid (tfLC3 from addgene), and G418 (Life Technology, CA, USA) was added for selecting positive cells. As intracellular distribution of LC3 protein was tagged by the fluorescence of RFP and GFP in these cells, images were collected with fluorescent confocal microscope. Quantification of LC3 puncta was performed using Red and Green Puncta Colocalization Macro with image j program, as described (Mizushima et al., 2010), and the average numbers of LC3 puncta per cell were accounted from the data collected from more than 40 cells. Here, GFP+RFP+ puncta are yellow, and GFP‐RFP+ puncta are red. Experiments were repeated three times.

Intracellular NAD⁺ level measurement

NAD⁺, NADH, and [NAD+]/[NADH] ratio were measured from whole cells extracts using an NAD⁺/NADH quantification kit from AAT Bioquest based on enzymatic cycling reaction, according to manufacturer's instructions (AAT‐15258, CA, USA). The value was normalized according to protein concentrations. Experiments were repeated three times.

Real‐time PCR analysis

Total RNA was isolated from cultured cells using TRIzol (Takara), and 2 μg of total RNA was used for reverse transcription by QuantiTect Reverse Transcription Kit (Bio‐Rad). Quantitative real‐time polymerase chain reaction (qRT‐PCR) was performed using SYBR Green Supermix kit (Bio‐Rad, CA, USA) on a Bio‐Rad IQ5 system. PCRs were performed in triplicate, and the relative amount of cDNA was calculated by the comparative CT method using the 18S ribosomal RNA sequences as control. The primer sequences used for PCR are shown in Table S1 (Supporting information). Experiments were repeated three times.

Immunoblotting

Whole cell lysates were collected using ice‐cold lysis buffer (50 mM Tris‐base, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.1% SDS, 1% TritonX‐100, 1% Sodium deoxycholate, 1 mM PMSF, 1 mM DTT, and 1 mM protease inhibitor) and lysis for 30 min following by centrifugation. Protein concentration was determined by BCA method (Cwbio, China). And 2.5 × SDS loading buffer was added to the lysates following 10 min of boiling. Thirty μg of proteins was loaded on SDS‐PAGE gel and separated by electrophoresis, followed by blotting on a PVDF membrane (Millipore, Germany). The target proteins were probed by corresponding primary antibodies with optimized conditions and then incubated with the secondary antibody. Immunological signals were surveyed via electrochemical luminescence method, using Immobile Western Chemiluminescence HRP substrate kit (Millipore) and Fusion Solo Imaging System (VIBER LOURMAT, FRANCE). The band intensities were quantified by fusion‐capt analysis Software, VILBER LOURMAT, VALLEE, FRANCE. Experiments were repeated three times.

Statistical analysis

Data were analyzed by by one way ANOVA. Statistical analysis was performed using spss 17.0 software (SPSS, Inc, NY, USA). Error bars represent standard error of the mean (± SEM).

Fig. S1 H₂O₂ induced senescence in MRC‐5 cells and HUVECs.

Fig. S2 Activation of AMPK prevented H₂O₂‐induced senescence in MRC‐5 cells and HUVECs.

Fig. S3 H₂O₂ induced decreased autophagic flux in NIH3T3 Cells.

Fig. S4 Atg5 knockdown attenuated the effects of BBR on protection against senescence.

Fig. S5 Activation of AMPK suppressed the impairment of H₂O₂‐induced autophagic flux and decreased the senescence in HUVECs.

Fig. S6 Activation of AMPK improved the redox status in senescent cells.

Fig. S7 Activation of AMPK increased the NAD⁺ level in HUVEC Cells.

Fig. S8 The mRNA level of QPRT increased during senescence.

Fig. S9 The activity of PARP‐1 increased during senescence.

Fig. S10 Sirt1 is involved in H₂O₂‐induced senescence as a downstream mediator of AMPK and NAD⁺ biosynthesis.

Fig. S11 NAD⁺ homeostasis is required for maintaining the autophagic flux in normal cells, but not in senescent cells.

Fig. S12 Unprocessed images of western‐blot.

Click here for additional data file.^{(933K, pdf)}

We thank Prof. Jae Bum Kim for providing plasmids expressing pDN‐AMPKα1. The authors thank Dr. Canhua Huang and Yuquan Wei for continuous supports and Dr. Ping Lin, Xiujie Wang, Yi Chen for all around convenience.

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Author informationArticle notesCopyright and License informationDisclaimer

¹Lab for Aging Research, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China

²Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China

³Department of Clinical Medicine, Medical School of Nankai University, Tianjin, China

Corresponding author.

^* Correspondence
Hengyi Xiao, Lab for Aging Research, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, 1 Keyuan 4 Road, Gaopeng Ave, Chengdu, China. Tel.: 86 28 8516 4023; fax: 86 28 8516 4005; e‐mail: nc.ude.ucs@xiygneh,

AMPK activation is beneficial for cellular homeostasis and senescence prevention. However, the molecular events involved in AMPK activation are not well defined. In this study, we addressed the mechanism underlying the protective effect of AMPK on oxidative stress‐induced senescence. The results showed that AMPK was inactivated in senescent cells. However, pharmacological activation of AMPK by metformin and berberine significantly prevented the development of senescence and, accordingly, inhibition of AMPK by Compound C was accelerated. Importantly, AMPK activation prevented hydrogen peroxide‐induced impairment of the autophagic flux in senescent cells, evidenced by the decreased p62 degradation, GFP‐RFP‐LC3 cancellation, and activity of lysosomal hydrolases. We also found that AMPK activation restored the NAD⁺ levels in the senescent cells via a mechanism involving mostly the salvage pathway for NAD⁺ synthesis. In addition, the mechanistic relationship of autophagic flux and NAD⁺ synthesis and the involvement of mTOR and Sirt1 activities were assessed. In summary, our results suggest that AMPK prevents oxidative stress‐induced senescence by improving autophagic flux and NAD⁺ homeostasis. This study provides a new insight for exploring the mechanisms of aging, autophagy and NAD⁺ homeostasis, and it is also valuable in the development of innovative strategies to combat aging.

The AMPK pathway was inactivated in cells with H₂O₂‐induced senescence

H₂O₂ treatment‐induced fibroblast senescence has been widely used as a model of SIPS (Chen & Amos, 1994; Toussaint et al., 2000). With a modified procedure, we obtained H₂O₂‐induced senescence in NIH3T3 cells with good homogeneity. In brief, suspended cells were treated with H₂O₂ for 45 min, and then, they were incubated in complete medium for adhesion culture for five days. At three to five days post‐H₂O₂ exposure, the cells became enlarged and flattened morphologically. Strong positive staining for senescence‐associated galactosidase (SA‐β‐Gal) was observed in these cells, with the percentage of SA‐β‐Gal‐positive cells increased by 30 to 50‐fold (Fig. 1A). Another marker of senescence, senescence‐associated heterochromatic foci (SAHFs), was also positive in our H₂O₂‐treated cells (Fig. 1B). In addition, the expression levels of four other senescence‐associated genes (p53, f.lux License Key Crack Key For U, p21, IL6, and IL8) were increased in these cells (Fig. 1C,D). The induction of senescence was obtained in the experiments using human MRC‐5 embryonic lung fibroblasts and human umbilical vein endothelial cells (HUVECs) (Fig. S1A,B, Supporting information).

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Figure 1

H₂O₂ induced senescence and AMPK pathway inhibition in NIH3T3 Cells. Cells were treated with H₂O₂ and incubated in complete medium without H₂O₂ for 3‐5 days. CTL means untreated cells. (A) Representative images of SA‐β‐Gal staining of the cells (left) and percentages of SA‐β‐Gal‐positive cells in a total of 1000 cells (right). (B) Representative images of SAHFs in cells (left) and percentages of SAHFs‐positive cells in 1000 cells (right). (C) Representative images from immunoblot assays against p53 and β‐actin. (D) Relative fold‐changes in the mRNA levels of the genes encoding p21, IL6 and IL8, as determined by qRT‐PCR. (E) Representative images from immunoblot assays against phosphorylated AMPKα (pAMPK, Thr172), AMPKα1, phosphorylated ACC (pACC, Ser79), and β‐actin. (F) The ratio of pAMPK to total AMPK was quantified by densitometry based on immunoblot images from three independent experiments. (G) Relative fold‐changes in the mRNA levels of two AMPK target genes (CPT‐1 f.lux License Key Crack Key For U FAS) f.lux License Key Crack Key For U monitored by qRT‐PCR assays. *P < 0.05 compared to the control (CTL). The bar represents 100 μm.

Given that the decline in AMPK activity has been reported to be associated with aging (Salminen & Kaarniranta, 2012), we tested this association in our senescence system. Beginning from the first day after H₂O₂ treatment, the levels of phosphorylated AMPKα (Thr172) and phosphorylated ACC (Ser79) markedly decreased, while the protein levels of AMPKα1 remained unchanged (Fig. 1E,F). Similarly, the expression levels of two AMPK target genes, carnitine palmitoyl transferase (CPT‐1), and fatty acid synthase (FAS), also decreased in the senescent cells (Fig. 1G). These data suggest that the AMPK pathway was downregulated in H₂O₂‐induced senescent cells.

AMPK activation prevented H₂O₂‐induced senescence

To evaluate the effects of AMPK activation on H₂O₂‐induced senescence, two known AMPK activators, metformin (Met) and berberine (BBR), were included in the culture medium after H₂O₂ treatment. As shown in Fig. 2A, both Met and BBR significantly enhanced the protein level of pAMPKα and the pACC in senescent cells. In addition, the mRNA levels of CPT‐1 gene and FAS gene increased (Fig. 2B).

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Figure 2

Activation of AMPK prevented H₂O₂‐induced senescence. A to D: H₂O₂‐treated NIH3T3 cells were incubated in complete medium with metformin (Met, 5 to 10 mM) or berberine (BBR, 5 to 10 μM) for 3 days. (A) Representative images from immunoblot assays against pAMPKα (Thr172), AMPKα1, pACC (Ser79), and β‐actin. (B) Relative fold‐changes in mRNA levels of CPT‐1 and FAS as determined by qRT‐PCR. (C) Representative images of SA‐β‐Gal staining f.lux License Key Crack Key For U cells (left), and percentages of SA‐β‐Gal‐positive cells. D‐F: NIH3T3 cells were treated with H₂O₂ and incubated with Met (10 mM), BBR (10 μM) and an AMPK inhibitor, Compound C (CC, 10 μM), alone or in combination for 3 days. (D) Representative images from immunoblot assays. (E) Relative mRNA levels of CPT‐1 and FAS as determined by qRT‐PCR. (F) Representative images of SA‐β‐Gal staining of cells (left) and percentages of SA‐β‐Gal‐positive cells (right). G‐I: NIH3T3 cells without H₂O₂ treatment were used. (G) The decrease in AMPK activity in DN‐AMPK‐expressing NIH3T3 cells was shown by the decrease in AMPKα phosphorylation. (H) Representative images of SA‐β‐Gal staining of the nontransfected cells with or without CC (upper) and cells transfection with DN‐AMPK (lower). (I) The percentages of SA‐β‐Gal‐positive cells were calculated based on the images represented in H. (J) H₂O₂‐treated cells incubated with or without Met (10 mM) or BBR (10 μM) for 3 days, representative images of SA‐β‐Gal staining of cells transfected with empty vector (upper) or DN‐AMPK (lower). (K) The percentages of SA‐β‐Gal‐positive cells were calculated based on the images presented in J. *P < 0.05 and **P < 0.01 compared to the vehicle control or indicated sample, ^##P < 0.01 compared to the indicated sample. The bar represents 100 μm.

Next, f.lux License Key Crack Key For U, we observed that when the H₂O₂‐treated cells were incubated with medium containing Met and BBR, there was a dose‐dependent decrease in the percentage of SA‐β‐Gal‐positive cells, which was similar to that caused by rapamycin treatment (Fig. 2C). The effects of AMPK activation on senescence were also evaluated in MRC‐5 cells and HUVECs (Fig. S2A,B). In addition, the preventive effects of AMPK activators were further confirmed using an AMPK inhibitor, Compound C (CC). The efficiency of CC for AMPK inactivation was first confirmed (Fig. 2D,E). As expected, when combined with CC, the effects of Met or BBR on senescence prevention were largely blunted when CC was coexisted, as indicated by the remarkable increase in SA‐β‐Gal‐positive cells (Fig. 2F). F.lux License Key Crack Key For U results demonstrate that the activation of AMPK by Met and BBR can prevent H₂O₂‐induced senescence, and this prevention could be prevented by CC.

To clarify the role of AMPK in senescence protection_, the effects of chronic AMPK inhibition by CC were evaluated in normal f.lux License Key Crack Key For U. As found, many cells incubated with CC for seven days were SA‐β‐Gal‐positive and larger in size compared with the control (Fig. 2H,I), and the cells expressing dominant‐negative AMPKa1 (pDN‐AMPK) also became SA‐β‐Gal positive (Fig. 2G–I). Unsurprisingly, pDN‐AMPK overexpression also increased the SA‐β‐Gal‐positive rate in the H₂O_2‐treated cells compared with the cells transfected with the empty vector. Moreover, f.lux License Key Crack Key For U, the antisenescence effects of Met and BBR were weakened in these cells (Fig. 2J,K). These results are consistent with the above findings, confirming the preventive effects of AMPK on senescence.

AMPK activation restored the H₂O₂‐impaired autophagic flux in senescent cells

Redressing the autophagic activity is an emerging concept for aging prevention (Rubinsztein et al., 2011). With this in mind, we investigated the status of autophagy in senescent cells, paying particular attention to the autophagic flux. The results showed that the p62 protein, dramatically accumulated in H₂O₂‐induced senescent cells without an accompanying increase in p62 mRNA (Figs 3A; S3A). Importantly, different from proliferating cells, the p62 protein did not accumulate when an autolysosomal inhibitor, HCQ, was applied to the senescent cells (Fig. 3B). This implies that almost no autolysosomal degradation capacity remained in the H₂O₂‐treated cells, so the inhibitory effects of HCQ in autolysosomes were abrogated. Next, by detecting the protein abundance of Cathepsin B, an important lysosomal protease, we found that the abundance of activated forms of the protein was significantly decreased in H₂O₂‐treated cells (Fig. 3C). To examine the status of autophagic flux, a NIH3T3 cell population stably expressing a tandem RFP‐GFP‐LC3 fusion protein was established and employed to visualize and distinguish GFP+RFP+ (yellow) and GFP‐GFP+ (red) LC3 puncta (Klionsky et al., 2012). As shown in Fig. S3B, although the formation of LC3 puncta increased in both H₂O₂ ‐treated cells and serum‐starved cells, the puncta in H₂O₂‐treated cells helium music manager 14 review to become GFP+/RFP+ (yellow), while those in starved cells tended to be GFP‐/RFP+ (red). These results reveal that H₂O₂‐induced cellular senescence is accompanied by impaired autophagic flux.

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Figure 3

Activation of AMPK improved autophagic flux impaired by H₂O₂ treatment. (A) Cells were treated as Fig. 1, representative images from immunoblot assays against p62 and β‐actin. (B) Control and H₂O₂‐treated cells were incubated with solvent or hydroxychloroquine (HCQ, 2 μM) for 3 days; representative images from immunoblot assays against p62 and β‐actin. (C) Representative images from immunoblot assays against Cathepsin B protein. D to G: GFP‐RFP‐LC3‐expressing cells were treated with H₂O₂ then incubated for 3 days with different reagents including: Met (10 mM), BBR (10 μM), CC (10 μM), Rapa (50 nM), f.lux License Key Crack Key For U, and HCQ (2 μM). (D) Representative confocal fluorescent images of RFP‐GFP‐LC3‐expressing cells, and the right panel shows the merged fluorescence. The bar represents 20 μm. (E) Percentages of cells with puncta like LC3 were figured up based on the images represented in D, dividing into GFP+/RFP+ group (yellow column) and GFP‐/RFP+ group (red column). (F) Representative images from immunoblot assays against LC3 and p62 proteins. (G) GFP‐RFP‐LC3‐expressing cells were incubated with indicated reagents alone or in combination for 3 days; representative confocal fluorescent images are shown as described in D. The bar represents 20 μm. (H) Percentages of cells with punctalike LC3 were figured up and grouped as described in E. (I) Representative images from immunoblot assays against LC3 and p62 proteins. (J) Representative images of SA‐β‐Gal staining of cells (left) and the percentages of SA‐β‐Gal‐positive cells (right). The bar represents 100 μm.*P < 0.05 compared to the vehicle control, ^#P < 0.05 compared to the indicated sample.

Then, the influence of AMPK activity on autophagic flux in senescent cells was investigated via several approaches. First, f.lux License Key Crack Key For U, using RFP‐GFP‐LC3 cells, we found, similar to autophagy f.lux License Key Crack Key For U rapamycin, that Met and BBR weakened the GFP fluorescence in cells. On the contrary, similar to lysosomal inhibitor HCQ, CC enhanced GFP fluorescence and increased yellow LC3 puncta in cells (Fig. 3D,E). Second, with Western blot assay, we found that both Met and BBR alleviated the H₂O₂‐induced accumulation of the LC3 and p62 proteins, and this alleviation was consistent with the effect of rapamycin (Fig. 3F). Third, f.lux License Key Crack Key For U, we observed that HCQ markedly blocked the effects of Met and BBR on the promotion of red LC3 puncta (Fig. 3G,H); likewise, HCQ blocked the effects of Met and BBR on the decreases in the LC3 and p62 proteins (Fig. 3I). In fact, blocking autophagic flux by HCQ aggravated H₂O₂‐induced senescence and blunted the protective effect of AMPK (Fig. 3J). Fourth, using Atg5‐silenced cells, we found that the influence of autophagic flux blockage the effect of BBR on the protection against senescence (Fig. S4A,B). Finally, f.lux License Key Crack Key For U, we confirmed with HUVECs that BBR can restore autophagic flux and prevent cellular senescence, and this effect can be blunted by HCQ (Fig. S5A,B). Taken together, these findings indicate that AMPK activation by Met and BBR can improve the impaired autophagic flux in H₂O₂‐induced senescent cells.

AMPK restored autophagic flux associated with the amelioration of lysosomal function, mTOR inactivation, but not the nuclear translocation of TFEB

Additional evidence linking AMPK activation to autolysosome restoration was obtained by monitoring the lysosomal functions and the status of the mTOR‐TFEB signaling. The results showed that treatment with Met or BBR increased the abundance of both the proenzyme and activated forms of Cathepsin B (Fig. 4A), as well as the activity of lysosomal acid phosphatase (Fig. 4B). Moreover, we found that BBR treatment significantly suppressed mTOR phosphorylation in senescent cells, similar and even stronger than that induced by rapamycin and insulin as an mTOR activation control (Fig. 4C).

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Figure 4

AMPK restored the autophagic flux associated with the amelioration of lysosomal function, mTOR inactivation, but not the nuclear translocation of TFEB. A‐C: H₂O₂‐treated NIH3T3 cells were incubated in complete medium with different reagents for 3 days as indicated. (A) Representative images from immunoblot assays against Cathepsin B protein. (B) The acid phosphatase activity in cells. (C) Representative images from immunoblot assays against phosphorylated mTOR (pmTOR, Ser2448), phosphorylated 70S6K (P‐p70S6K, Thr389), pAMPK (Thr172), and β‐actin. (D) Immunofluorescent images of TFEB after treatment with indicated reagents. The bar represents 20 μm. (E) Immunoblot assays against TFEB protein of total cytoplasmic and nuclear subcellular fractions obtained from NIH3T3 cells with the indicated treatment. (F) Relative fold‐changes in mRNA levels of hma pro vpn license key 2020 android Free Activators TFEB target genes (GNS and LAMP‐1) were monitored by qRT‐PCR assays.*P < 0.05 and **P < 0.01 compared to the vehicle control.

It has reported that mTOR inactivation could result in the release TFEB from the mTORC1 complex following the nuclear translocation of TFEB and elevated transcription of multiple genes related to autophagic activity (Roczniak‐Ferguson et al., 2012), f.lux License Key Crack Key For U. However, BBR‐induced mTOR inactivation in H₂O₂‐treated cells accompanied without increased nuclear distribution of TFEB, but with the receded (Fig. 4D,E). This situation is different from that observed in f.lux License Key Crack Key For U cells, where TFEB kept in nucleus (Fig. 4D,E). To know the transcriptional function of nuclear TFEB in H₂O₂‐treated cells, we measured the expression of two representative TFEB‐targeted genes, f.lux License Key Crack Key For U, GNS and LAMP1. We found that the transcription of GNS and LAMP1 genes reduced in H₂O₂‐treated cells, but this reduce recovered when BBR applied (Fig. 4F). Above results indicate that the positive effect of AMPK activation on autophagic flux is relevant to its role in combating lysosome dysfunction, and also to mTOR inactivation and TFEB activation as a transcriptional factor. Furthermore, our results reveal that BBR can recede the nuclear accumulation of TFEB induced by H₂O₂ treatment.

AMPK restored NAD⁺ synthesis in cells with H₂O₂‐induced senescence

Reduced cellular NAD⁺ level is a feature of aging (Gomes et al., 2013), and a link between NAD⁺ synthesis and AMPK has been suggested (Brandauer et al., 2013). For these reasons, we next examined the relationships among AMPK, NAD⁺ synthesis and senescence. As shown in Fig. 5A, the NAD⁺ level in the senescent cells was significantly decreased, which was accompanied by a decrease in the NAD/NADH ratio (Fig. S6A). Correspondingly, supplementation with nicotinamide mononucleotide (NMN), a precursor of NAD⁺ synthesis (Yoshino et al., 2011), decreased the percentage of SA‐β‐Gal‐positive cells and increased cellular NAD⁺ levels following H₂O₂ treatment (Fig. 5B,C). Importantly, Met and BBR upregulated the cellular NAD⁺ level, f.lux License Key Crack Key For U, while CC had the opposite effect (Fig. 5D), f.lux License Key Crack Key For U. Similar results were observed using HUVECs (Fig. S7). The ratio of NAD/NADH also increased in the Met‐ and BBR‐treated cells (Fig. S6B).

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Figure 5

The preventive effects of AMPK on the senescence associated with NAD⁺ synthesis. (A) Cellular concentrations of NAD⁺ on day 3 or day 5 after H₂O₂ treatment, f.lux License Key Crack Key For U. (B) Representative images of SA‐β‐Gal staining of cells (left) and percentage of SA‐β‐Gal‐positive cells (right). (C) Cellular concentrations of NAD⁺ in the cells incubated with nicotinamide mononucleotide (NMN) for 3 days after H₂O₂ treatment. (D) H₂O₂‐treated cells were f.lux License Key Crack Key For U with Met (10 mM), BBR (10 μM), or CC (10 μM) for 3 days. The concentrations of NAD⁺ are shown. (E) A schematic diagram of two pathways of NAD⁺ synthesis. Red ones indicate rate‐limiting enzymes, and blue ones illustrate the enzyme inhibitors we used. (F) Relative fold‐changes in the mRNA levels of NAMPT, as monitored by qRT‐PCR (left), and representative images from immunoblot assays against NAMPT are shown (right). The ratio of NAMPT to actin was quantified by densitometry, based on the immunoblot images from three independent experiments. (G) Cells were incubated with Met, BBR, and CC for 3 days. Representative images from immunoblots against NAMPT f.lux License Key Crack Key For U shown. The ratio of NAMPT to glary utilities pro 5 keygen Activators Patch was quantified by densitometry based on the immunoblot images from three independent experiments. (H) H₂O₂‐treated NIH3T3 cells were incubated with NAMPT inhibitor (FK866, 5 nM), QPRT inhibitor (PHTH, 1 mM), Met (10 mM), and BBR (10 μM) alone or in combination as indicated for 3 days. The percentages of SA‐β‐Gal‐positive cells are shown. (I) The mRNA levels of NMNAT1,NMNAT2, and NMNAT3 genes in the cells infected with corresponding lentivirus‐shRNA or nontargeting shRNA (shCON) as determined by qRT‐PCR. (J) Cellular concentrations of NAD⁺ in shRNA‐infected cells. (K) Cells infected with shRNAs were treated with H₂O₂ and incubated with BBR for 3 days. Representative images of SA‐β‐Gal staining of cells (left) and percentages of SA‐β‐Gal‐positive cells (right). *P < 0.05 compared to the control (A, F) or vehicle (C, D, G, K) or shCON (I, J). The bar represents 100 μm.

The involvements of NAD⁺ synthesis assessed next. The known pathways of NAD⁺ synthesis were diagrammed in Fig. 5E. Our results showed that mRNA and protein abundance of nicotinamide phosphoribosyl transferase (NAMPT), a rate‐limiting enzyme of F.lux License Key Crack Key For U synthesis in salvage pathway, significantly decreased in senescent cells (Fig. 5F); however, the mRNA level of quinolinic acid phosphoribosyl transferase (QPRT), a rate‐limiting enzyme of NAD⁺ synthesis in de novo pathway, was increased (Fig. S8A), while no expressional alteration in nicotinamide mononucleotide adenylyltransferase (NMNAT) (Fig. S8A). In addition, the situation of NAD⁺ consumption was examined via measuring the activity of a major NAD⁺‐consuming enzyme poly‐ADP‐ribose polymerase (PARP‐1). Resultantly, PARP‐1 activity significantly increased in senescent cells (Fig. S9). These results demonstrate that the NAD⁺ decline found in senescent cells is relevant to both its synthetic decline and consumptive elevation, and for the synthesis, the involvement of salvage pathway seems dominating.

Then, the situation upon AMPK activation was investigated. As shown, the abundance of NAMPT protein and mRNA level of QPRT were regulated positively by Met and BBR (Figs 5G, S8B), while no change in the activities of PARP‐1 observed (data not shown). As the effort to know the mechanism about AMPK improved NAD⁺ synthesis, two sets of blocking experiments were performed. The pharmacological approach showed that NAMPT inhibitor FK866 and QPRT inhibitor PHTH‐prompted senescence, but their roles were significantly repressed by Met and BBR (Fig. 5G). The shRNA‐mediated knockdown approach for the NMNAT gene showed that each of the three shNMNATs effectively suppressed NMNAT expression (Fig. 5I), and the cellular concentrations of NAD⁺ (Fig. 5J). Moreover, they markedly perturbed the protective role of AMPK activation against senescence (Fig. 5K). These results demonstrate the connection of NAD⁺ synthesis with the AMPK activity in our system and particularly emphasize the involvement of the salvage NAD⁺ synthesis pathway.

Given that NAD⁺ is a coenzyme of Sirt family deacetylases that positively regulates autophagy (Lee et al., 2008), and restrain aging (Gomes et al., 2013), the activity of Sirt1 was monitored. As the findings, the activity of Sirt1 was increased by AMPK activation in senescent cells (Fig. S10A,B). Then, we noted that EX527, a chemical inhibitor of Sirt1, suppressed the effects of AMPK on senescence and p62 accumulation (Fig. S10C). However, f.lux License Key Crack Key For U, compared with normal cells, the ability of EX527 for blocking the p62 accumulation was only moderate. These results suggest that Sirt1 is involved in H₂O₂‐induced senescence as a downstream mediator of AMPK and NAD⁺ biosynthesis.

Reagents

Metformin and berberine were purchased from MUSTBIO technology (Chengdu, China); Compound C was from CALBIOCHEM (Darmstadt, Germany). Hydroxychloroquine (HCQ), rapamycin, and phthalic acid (PHTH) were from Sigma‐Aldrich (CA, USA). Nicotinamide mononucleotide (NMN) and FK866 were from Santa Cruz Biotechnologies (TX, USA). EX527 was from selleck (CA, USA). Antibodies against LC3 (12741), phospho‐ACC (Ser79) (3661), phospho‐mTOR (Ser2448) (5536), phospho‐p70S6K (Thr389) (9206), and p53 (2524) were purchased from Cell Signaling technology (MA, USA). Those against phospho‐AMPKα (Thr172) (ab133448), Cathepsin B (ab30443), NAMPT (ab109210), AMPKα1 (ab32047), and SQSTM1/p62 (3340‐1) were from Abcam (MA, USA). Antibodies against β‐actin and Lamin B were from F.lux License Key Crack Key For U (Beijing, China), anti‐TFEB (A303‐673A‐M) was from Bethyl (TX, f.lux License Key Crack Key For U, USA), FITC‐goat anti‐rabbit IgG was from Invitrogen (CA). The reagent used for cDNA plasmids transfection was Lipofectamine 2000 (Invitrogen, CA), and that LightBurn Free Download lentivirus‐shRNA plasmids was X‐treme GENE HP (Roche, CA, USA).

Cell culture and H₂O₂ treatment

NIH3T3 cells (murine fibroblast line) and MRC‐5 cells (human fibroblast line) were purchased from Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences (Shanghai, China), cultured in complete Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS in a humidified atmosphere with 5% CO₂ at 37 °C. HUVECs (human umbilical vein endothelial cells) (ATCC) cultured in low glucose (5.6 mM) RIPA1640 supplemented with 10% FBS. For senescence induction, a modified H₂O₂ treatment protocol was used. In brief, cells seeded in 100‐mm dishes with 5 × 10⁵ cells per dish density were trypsinized and suspended in phosphate buffer solution (PBS) at 1 × 10⁶ cells mL⁻¹ density and exposed to 400 μM (NIH3T3) or 300 μM (MRC‐5) or 700 μM (HUVECs) H₂O₂ in an Eppendorf tube at 37 °C for 45 min. During H₂O₂ treatment, the tube was turned upside down gently every 5 min. H₂O₂ treatment was terminated by a 5‐min centrifugation at 800 rpm and a washing process. Then, the cells were cultured with complete medium. During the adhesion cultivation, the cells accepted different treatments that will be described later in individual figure legends.

SA‐β‐Gal staining and Drivermax con crack Crack Key For U staining

Intracellular senescence‐associated‐β‐galactosidase (SA‐β‐Gal) activity was assayed using an SA‐β‐Gal staining kit (Beyotime, Beijing) according to manufacturer's instructions, and senescent cells were identified as bluish green‐stained cells under a phase‐contrast microscope. The percentage of SA‐β‐Gal‐positive cells in total cells was determined by counting 1000 cells in 7 random fields, for each group. Senescence‐associated heterochromatic foci (SAHFs) was visualized by DAPI staining after cells were fixed in situ with 4% paraformaldehyde and washed by PBS. Images with DAPI stained nuclei with blue fluorescence were taken by fluorescence microscope. The percentage of SAHF‐positive cells was determined by counting more than 1000 cells in 7 random fields, for each group. The results were expressed as mean of triplicates ± SD.

Drug treatments

For AMPK activity modulation, metformin, berberine, and Compound C were applied as described in the legends. For autophagy modulation, rapamycin and hydroxychloroquine (HCQ) were added as described in the legend of Fig. 3. For NAD⁺ precursor supplementation, nicotinamide mononucleotide (NMN) was applied as described in the legend of Fig. 5. For inhibiting NAD⁺ synthesis pathways, FK866 and phthalic acid (PHTH) were applied as described in the maxwell 4.2 crack Crack Key For U of Fig. 5.

mRFP‐GFP‐LC3 expressing cells generation and fluorescent LC3 puncta analysis

Cells were transfected with mRFP‐GFP‐LC3 plasmid (tfLC3 from addgene), and G418 (Life Technology, CA, USA) was added for selecting positive cells. As intracellular distribution of LC3 protein was tagged by the fluorescence of RFP and GFP in these cells, images were collected with fluorescent confocal microscope. Quantification of LC3 puncta was performed using Red and Green Puncta Colocalization Macro with image j program, as described (Mizushima et al., 2010), and the average numbers of LC3 puncta per cell were accounted from the data collected from more than 40 cells. Here, GFP+RFP+ puncta are yellow, and GFP‐RFP+ puncta are red. Experiments were repeated three times.

Intracellular NAD⁺ level measurement

NAD⁺, NADH, and [NAD+]/[NADH] ratio were measured from whole cells extracts using an NAD⁺/NADH quantification kit from AAT Bioquest based on enzymatic cycling reaction, according to manufacturer's instructions (AAT‐15258, CA, USA). The value was normalized according to protein concentrations. Experiments were repeated three times.

Real‐time PCR analysis

Total RNA was isolated from cultured cells using TRIzol (Takara), and 2 μg of total RNA was used for reverse transcription by QuantiTect Reverse Transcription Kit (Bio‐Rad). Quantitative real‐time polymerase chain reaction (qRT‐PCR) was performed using SYBR Green Supermix kit (Bio‐Rad, CA, USA) on a Bio‐Rad IQ5 system. PCRs were performed in triplicate, and the relative amount of cDNA was calculated by the comparative CT method using the 18S ribosomal RNA sequences as control. The primer sequences used for PCR are shown in Table S1 (Supporting information). Experiments were repeated three times.

Immunoblotting

Whole cell lysates were collected using ice‐cold lysis buffer (50 mM Tris‐base, f.lux License Key Crack Key For U, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.1% SDS, 1% TritonX‐100, 1% Sodium deoxycholate, 1 mM PMSF, 1 mM DTT, and 1 mM protease inhibitor) and lysis for 30 min following by centrifugation. F.lux License Key Crack Key For U concentration was determined by BCA method (Cwbio, China). And 2.5 × SDS loading buffer was added to the lysates following 10 min of boiling. Thirty μg of proteins was loaded on SDS‐PAGE gel and separated by electrophoresis, followed by blotting on a PVDF membrane (Millipore, Germany). The target proteins were probed by corresponding primary antibodies with optimized conditions and then incubated with the secondary antibody. Immunological signals were surveyed via electrochemical luminescence method, using Immobile Western Chemiluminescence HRP substrate kit (Millipore) and Fusion Solo Imaging System (VIBER LOURMAT, FRANCE). The band intensities were quantified by fusion‐capt analysis Software, VILBER LOURMAT, VALLEE, f.lux License Key Crack Key For U, FRANCE. Experiments were repeated three times.

Statistical analysis

Data were analyzed by by one way ANOVA. Iolo system mechanic crack 2019 Activators Patch analysis was performed using spss 17.0 software (SPSS, Inc, NY, USA). Error bars represent standard error of the mean (± SEM).

Fig. S1 H₂O₂ induced senescence in MRC‐5 cells and HUVECs.

Fig. S2 Activation of AMPK prevented H₂O₂‐induced senescence in MRC‐5 cells and HUVECs.

Fig. S3 H₂O₂ induced decreased autophagic flux in NIH3T3 Cells.

Fig. S4 Atg5 knockdown attenuated the effects of BBR on protection against senescence.

Fig. S5 Activation of AMPK suppressed the impairment of H₂O₂‐induced autophagic flux and decreased the senescence in HUVECs.

Fig. S6 Activation of AMPK improved the redox status in senescent cells.

Fig. S7 Activation of AMPK increased the NAD⁺ level in HUVEC Cells.

Fig. S8 The mRNA level of QPRT increased during senescence.

Fig. S9 The activity of PARP‐1 increased during senescence.

Fig. S10 Sirt1 is involved in H₂O₂‐induced senescence as a downstream mediator of AMPK and NAD⁺ f.lux License Key Crack Key For U NAD⁺ homeostasis is required for maintaining the autophagic flux in normal cells, but not in senescent cells.

Fig. S12 Unprocessed images of western‐blot.

Click here for additional data file.^{(933K, pdf)}

We thank Prof. Jae Bum Kim for providing plasmids expressing pDN‐AMPKα1. The authors thank Dr. Canhua Huang and Yuquan Wei for continuous supports and Dr. Ping Lin, Xiujie Wang, Yi Chen for all around convenience.

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