Sirt1 decreased adipose inflammation by interacting with Akt2 and inhibiting mTOR/S6K1 pathway in mice[S]

Sirtuin type 1 (Sirt1) and protein kinase B (Akt2) are associated with development of obesity and inflammation, but the molecular mechanisms of Sirt1 and Akt2 interaction on adipose inflammation remain unclear. To explore these mechanisms, a mouse model was used. Mice were fed with a high-fat diet (HFD) for 8 weeks, with interventions of resveratrol (RES) or nicotinamide (NAM) during the last 15 days. The HFD reduced Sirt1 mRNA in adipose tissue and elevated interleukin-6 (IL-6) expression. RES reduced the adipose tissue weight, increased the Sirt1 mRNA level, and reduced both mRNA and protein levels of IL-6, MCP-1, inducible nitric oxide synthase, and TNF-α by inhibiting phosphorylation of Akt2 in adipose tissue. Additionally, macrophage type I marker genes were reduced while macrophage type II marker genes were elevated by RES addition. Moreover, activation of Akt2 signal by using insulin significantly blunted the inhibitory effect of RES on adipose inflammation. Immunoprecipitation assay demonstrated that RES enhances the protein-protein interaction between Sirt1 and Akt2, but NAM inhibits this interaction. Furthermore, Sirt1 significantly reduced the levels of raptor and inactivated mammalian target of rapamycin (mTOR)C1 signal by interacting with Akt2, and confirmed that RES attenuated adipose inflammation by inhibiting the mTOR/S6K1 pathway via rapamycin.

medium containing insulin (10 g/ml; Sigma-Aldrich) and 10% FBS until the day of harvest.

Materials and vectors
Differentiated preadipocytes were treated with 100 M RES or 500 M NAM for 48 h. Plasmid vectors of His-Sirt1 (overexpression vector of Sirt1 with tag-His), Sirt1 (overexpression vector of Sirt1), His-Akt2 (overexpression vector of Akt2 with tag-His), and Akt2 (overexpression vector of Akt2) were all constructed in our laboratory. Lentiviral shRNA vector for Sirt1 (sh-Sirt1) was purchased from Gene Pharma (Shanghai, China). The lentiviral vector expressing a mutant Akt2 construct (Akt2-mutant) was generated by introducing seven silent point mutations in the Akt2 cDNA coding sequence by Gene Pharma. Plasmid vectors were transfected with X-tremeGENE™ transfection reagent (Roche, Basel, Switzerland) at a ratio of 3:1 (X-tremeGENE™ reagent: DNA) for 24-48 h before measurements of mRNA and protein. In addition, differentiated preadipocytes were infected with lentiviral vectors of Sirt1 and mutant-Akt2 for 48 h at the titer of 1 × 10 9 IFU/ml for further detection.

Cell viability and Oil Red O staining
Cell viability was measured by Cell Counting Kit 8 (CCK-8; Vazyme, Nanjing, China) as described in our previous publication (27). In brief, cells were seeded in a 96-well plate at a density of 5 × 10 3 and cultured for 12 h. Then 10 l kit buffer was added into each well and incubated at 37°C for 1 h. Absorbance was quantified at 450 nm by Vector 5 (Bio-Tek, Winooski, VT). Lipid droplets were stained with Oil Red O staining as described in our previous publication (28). In brief, the fixed cells were washed three times with PBS and stained with a working solution of Oil Red O for 30 min at room temperature, and then washed with deionized water and images of the stained cells were viewed under an inverted microscope (Nikon Instruments Europe BV, UK).

Immunoprecipitation using primary antibodies
Differentiated preadipocytes were pretransfected with plasmids using 3 l X-tremeGENE™ transfection reagent (Roche) per microgram of plasmids. Whole cell lysate was harvested in lysis buffer 48 h post transfection. The supernatant of whole cell lysate was precleaned with protein A (for rabbit primary antibody; Invitrogen, Carlsbad, CA) for 2 h and incubated with 4 g primary antibody overnight at 4°C with shaking. Immune complexes were pulled down with protein A for 2 h at 4°C with shaking. Beads were washed once with lysis buffer, three times with washing buffer, followed by SDS-PAGE and Western blot analysis.

Real-time PCR analysis
Total RNA was extracted from subcutaneous adipose tissue or differentiated preadipocytes with TRIpure reagent kit (Takara, Dalian, China) and 400 ng of total RNA was reverse transcribed using the M-MLV reverse transcriptase kit (Takara). Primers were synthesized by Shanghai Sangon Ltd. (Shanghai, China). Quantitative PCR was performed in 25 l reactions containing specific primers and SYBR Premix EX Taq (Takara). The levels of mRNAs were normalized to -actin. The expression of genes was analyzed by method of 2 Ct .

Protein extraction and Western blot analysis
Differentiated preadipocytes were lysed in RIPA buffer for 40 min at 4°C. The lysate was centrifuged at 12,000 g for 15 min at 4°C, and the supernatant was used for protein assay. Proteins (50 g) in the supernatant were separated by electrophoresis on 12% and 5% SDS-PAGE gels using slab gel apparatus and then transferred to PVDF nitrocellulose membranes (Millipore, Boston, MA) blocked with 5% skim milk powder/Tween 20/TBST at room and may offer a new potential means to prevent and treat obesity and metabolic syndrome.

Animal studies
Six-week-old Kunming male mice were purchased from the Laboratory Animal Center of Fourth Military Medical University (Xi'an, China) and used in two experiments. The use of the animals and the animal handling protocols were approved by the Animal Ethics Committee of Northwest A&F University (Xi'an, China). All mice were provided with ad libitum water and fed a standard laboratory chow diet (F4031; Bio-Serv, Flemington, NJ) initially for 2 weeks. They were then randomly divided into four groups, and had different dietary treatments for 8 weeks: one group (Control) continued consuming the chow diet, and the other three groups were fed a HFD (F3282; Bio-Serv) with or without additional supplementation. Among those three groups on the HFD, one group was fed HFD only (HFD control), another group was fed HFD with the addition of RES (150 mg/kg; Sigma-Aldrich, St. Louis, MO) for the last 15 days, and the third group was fed HFD with the addition of nicotinamide (NAM) (500 mg/ kg; Sigma-Aldrich) for the last 15 days. Mice were injected with either PBS or 10 U/kg insulin (Sigma-Aldrich) for 30 min after an Perinephric fat, epididymis fat, and subcutaneous fat were harvested and weighed on the last day of treatment. Plasma TG and glucose concentrations were measured using a TG determination kit and glucose (HK) assay kit (Sigma-Aldrich), respectively. The plasma concentrations of inducible nitric oxide synthase (iNOS), leptin, and interleukin-6 (IL-6) were detected using commercial ELISA kits from R&D and Cell Signaling Technology (Boston, MA) and Abcam (Cambridge, UK), respectively.

Primary preadipocyte culture and differentiation
Primary preadipocytes from subcutaneous white adipose tissue were isolated and cultured according to the method described previously (26). Preadipocytes were plated onto 35 mm primary culture dishes. The differentiation of preadipocytes was then performed as follows: Cells grown to 100% confluence (day 0) were exposed to induction DMEM/F12 containing dexamethasone (1 M; Sigma-Aldrich), insulin (10 g/ml; Sigma-Aldrich), IBMX (0.5 mM; Sigma-Aldrich), and 10% FBS. Four days after the induction (from day 2), cells were maintained in the induction , and GAPDH were purchased from Bioworld (Nanjing, China). Antibodies against Sirt1, leptin, IL-6, MCP-1, TNF-, p-Akt2 Ser473 , Akt2, and target-His were from Abcam. Antibody against iNOS was from Cell Signaling Technology (Danvers, MA), and Akt2-specific inhibitor, CCT128930, was from Abcam and rapamycin was from Sigma-Aldrich. Membranes were first incubated with primary antibodies at 4°C overnight, followed with HRP-conjugated secondary antibodies (Boaoshen, China) for 2 h at room temperature. Proteins were visualized using chemiluminescent peroxidase substrate (Millipore) and quantified using ChemiDoc XRS system (Bio-Rad, Hercules, CA).

Statistical analysis
Statistical analyses were conducted using SAS v8.0 (SAS Institute, Cary, NC). Data were analyzed using one-way ANOVA. Comparisons among individual means were made by Fisher's least significant difference (LSD). Data are presented as mean ± SD. P < 0.05 was considered to be significant.

Sirt1 reduced adipose deposition and decreased Akt2 level in obese mice
The body weights of mice are shown in Fig. 1A. Mice on HFD for 8 weeks had significantly higher body weight than that of week 6. The Lee index and food intake were higher compared with those of mice on chow diet (Fig. 1A-C). The Sirt1 mRNA level in the adipose tissue was reduced, but Akt2 and IL-6 mRNA levels were increased (Fig. 1D). Addition of RES significantly reduced the body weight, whereas addition of NAM had an opposite effect (Fig. 1E). No difference in food intake was found among RES, NAM, and HFD control Ser473 of subcutaneous adipose tissue in mice on HFD with addition of RES or NAM over the last 15 days; insulin was injected for 30 min (n = 6). B: Relative plasma glucose concentration of mice on HFD with addition of RES or NAM over the last 15 days; insulin was injected for 30 min (n = 6). The actual glucose content in the control group, RES group, and NAM group without insulin was 9.11 mmol/l, 7.84 mmol/l, and 10.63 mmol/l; the actual glucose content in the control group, RES group, and NAM group with insulin treatment was 5.47 mmol/l, 4.64 mmol/l, and 8.72 mmol/l. C: Relative plasma TG concentration 24 h after the insulin injection in mice on HFD with addition of RES or NAM over Sirt1 (Fig. 3H). However, in differentiated preadipocytes transfected with Akt2 dead mutant vector and Sirt1 vector, adipocyte inflammation was restored (Fig. 3I). These results together reveal that RES decreases adipose inflammation by promoting the interaction between Sirt1 and Akt2.

Sirt1 reduced adipocyte inflammation by inhibiting the Akt2/mTOR pathway
Differentiated preadipocytes were induced in this experiment. RES inhibited adipose accumulation and NAM promoted adipose accumulation; PPAR and FAS levels were decreased with RES addition; in contrast, NAM increased their levels (Fig. 4A, B). RES reduced the expression of IL-6, TNF-, and MCP-1, while it enhanced the expression of leptin with or without the insulin treatment (Fig. 4C). To further illustrate the role of Sirt1 in RESreduced inflammation, the interference or overexpression vectors of Sirt1 were used. Figure 4D demonstrates that overexpression of Sirt1 almost completely inhibited IL-6 and MCP-1 with the addition of RES, while sh-Sirt1 recovered the levels of IL-6 and MCP-1 (Fig. 4D). The reduction of raptor and p-mTOR activity indicated that Sirt1 functioned via the mTOR signal pathway (Fig. 4E). Despite insulin stimulation enhancing Akt2 activity, Sirt1 decreased Akt2 activity and reduced the expression levels of raptor and p-mTOR (Fig. 4E). Thus, these results indicate that RES inhibited adipose inflammation by activating the Sirt1/ Akt2/mTOR pathway.

The mTOR/S6K1 pathway was essential for regulation of Sirt1 in adipocyte inflammation
The essentiality of mTORC1 signal for Sirt1-regulated inflammation was determined by measuring the phosphorylation levels of mTOR Ser2448 and S6K1 Thr389 with or without RES/NMA. Interestingly, RES reduced the activity of p-mTOR Ser2448 and p-S6K1 Thr389 , whereas NAM increased those activities (Fig. 5A). A further addition of rapamycin, the mTORC1-specific inhibitor, accentuated the reduction of mTOR activity, while the addition of insulin restored the levels of p-mTOR Ser2448 and p-S6K1 Thr389 (Fig.  5A). Additionally, the RES resulted in inactivation of the mTOR signal pathway and strongly reduced the expression of IL-6, iNOS, and MCP-1; further addition of insulin elevated expression of these genes (Fig. 5B). Thus, the groups (Fig. 1F). Fat pads from three locations (epididymal, perinephric, subcutaneous) all weighed less in RES-fed mice (Fig. 1G, H). Moreover, RES increased Sirt1 expression but decreased Akt2 and IL-6 expression (Fig. 1I). Thus, the data elucidate that RES reduced body weight of mice and inhibited inflammation by elevating Sirt1 and reducing Akt2.

Sirt1 inhibited adipose inflammation and insulinstimulated Akt2 activation
We next addressed whether the alternation of Sirt1 impaired Akt2, and whether Sirt1 functioned in obesity-related inflammation. The RES treatment increased the Sirt1 level and decreased Akt2 activity in the insulin-stimulated adipose tissue, whereas NAM had the opposite effects ( Fig. 2A). Plasma TG and glucose levels were both reduced in RES mice (Fig. 2B, C). In addition, RES also decreased plasma iNOS and IL-6 levels, which were elevated after the insulin stimulation (Fig. 2D). The protein level of iNOS was decreased by RES, but leptin was increased (Fig. 2E). Consistently, RES reduced macrophage type I markers, but elevated macrophage type II markers, even when treated by insulin (Fig. 2F). The mRNA levels of inflammation marker genes, such as IL-6, TNF-, MCP-1, and iNOS, were decreased, but increased by the insulin stimulation (Fig. 2G). All these results suggest that Sirt1 and Akt2 functioned inversely in the regulation of adipose inflammation.

Sirt1 physically interacted with Akt2 in reducing mouse adipocyte inflammation
The interaction between Sirt1 and Akt2 on inflammation was examined at protein level. The transfection efficiency of plasmid vectors in differentiated preadipocytes is shown in supplemental Fig. S1. Basal Akt2 protein interacted with transfected Sirt1 protein in differentiated preadipocytes (Fig. 3A). The RES enhanced this interaction significantly, but NAM blocked the interaction between Sirt1 and Akt2 (Fig. 3A, C). The insulin treatment enhanced the interaction between Sirt1 and Akt2, as shown in Fig. 3B. The Akt2 phosphorylation level was decreased in a time-dependent manner by RES treatment, while NAM treatment showed the opposite result (Fig. 3D, E), confirming that Sirt1 downregulated Akt2 activity. The Akt2-specific inhibitor, CCT128930, significantly decreased the Akt2 phosphorylation level and reduced the levels of IL-6, MCP-1, TNF-, and iNOS (Fig. 3F, G). Consistently, CCT128930 treatment had an additive effect on the reduced adipose inflammation by the last 15 days (n = 6). The absolute TG content in the control group without insulin was 1 mmol/l, in the RES group without insulin was 0.75 mmol/l, in the NAM group without insulin was 1.3 mmol/l. The absolute TG content in the control group with insulin treatment was 1.5 mmol/l, in the RES group with insulin treatment was 1.1 mmol/l, in NAM group with insulin treatment was 1.7 mmol/l. D: Relative plasma iNOS, IL-6, and leptin concentration 24 h after insulin injection in mice on HFD with addition of RES or NAM over the last 15 days (n = 6). Absolute plasma iNOS content in the control group, RES group, and NAM group without insulin was 6.2 U/ml, 4.11 U/ml, and 8.42 U/ml; and in the control group, RES group, and NAM group with insulin treatment was 9.31 U/ml, 6.25 U/ml, and 10.3 U/ml. Absolute plasma IL-6 content in the control group, RES group, and NAM group without insulin was 16.2 pg/ml, 13.31 pg/ml, and 19 pg/ml; and in the control group, RES group, and NAM group with insulin treatment was 21.3 pg/ml, 15.7 pg/ml, and 24.8 pg/ml. Absolute plasma leptin content in the control group, RES group, and NAM group without insulin was 7.3 ng/ml, 11 ng/ml, and 5.2 ng/ml; and in the control group, RES group, and NAM group with insulin treatment was 6.11 ng/ml, 8.5 ng/ml, and 4.2 ng/ml. E: Immunoblots of iNOS and leptin 24 h after the insulin injection in mice on HFD with addition of RES or NAM over the last 15 days (n = 6). F: mRNA levels of macrophage type I (M1) or macrophage type II (M2) macrophage markers 24 h after the insulin injection in mice on HFD with addition of RES or NAM over the last 15 days (n = 6). G: mRNA levels of inflammation markers 24 h after the insulin injection in mice on HFD with addition of RES or NAM over the last 15 days (n = 6). Control: fed HFD only. Values are mean ± SD. *P < 0.05 as compared with the corresponding control. 29,30). RES is a natural plant polyphenol that prevents obesity-related chronic diseases through reducing the synthesis of lipids (31). Activation of Sirt1 by RES can lead to an enhancement of glucose tolerance and insulin sensitivity (10,32). In the current study, we found that HFD reduced the expression of Sirt1 and elevated Akt2 expression. The reversal relationship between Sirt1 and Akt2 expressions was also found by RES treatment. This reversal relationship mTORC1/S6K1 signal pathway was essential for adipocyte inflammation.

DISCUSSION
Chronic low-grade tissue inflammation is an important etiologic component of obesity-related diseases like insulin resistance, metabolic syndrome, and type 2 diabetes (7, could be altered by RES and NAM. These results are consistent with previous reports (33,34). Thus, Sirt1 is an important target to combat metabolic disorders, and for potential therapeutic interventions.
Insulin is a key regulator of glucose homeostasis, and its absence is lethal in human cells (35). The downstream effectors of insulin signal, Akts, are activated and affect metabolism, proliferation, apoptosis, growth, and inflammation of cells (36). Inhibiting the Akt signal pathway increases anti-inflammatory effects and decelerates the metabolic syndrome. The Sirt1 level is increased in response to calorie restriction, the most consistent intervention to increase lifespan and protect against deteriorations in biological functions of many key metabolic tissues (37,38). Using immunoprecipitation, we found that Sirt1 directly bound to Akt2. Our finding is consistent with the result found in porcine adipogenesis (22). Interestingly, RES promoted the interaction between Sirt1 and Akt2, but NAM reduced this interaction in this study. Phosphoinositide 3-kinase (PI3K), Akt, and their downstream kinase, mTOR, are implicated in insulin resistance, metabolic dysfunction, and inflammation (15). Thus, we focused on the influence Fig. 4. Sirt1 reduced adipocyte inflammation by inhibiting the Akt2/mTOR pathway. A: Oil Red O staining of differentiated preadipocytes for 8 days with RES or NAM treatment for 48 h (n = 3). B: Expression of adipogenic genes in differentiated preadipocytes with RES or NAM treatment for 48 h (n = 3). C: Immunoblots of inflammation markers in differentiated preadipocytes treated with RES or NAM for 48 h, followed with 10 nM insulin for 30 min (n = 3). C: Immunoblots of mTORC1 signaling differentiated preadipocytes treated with RES or NAM for 48 h, followed with 10 nM insulin for 30 min (n = 3). D: Immunoblots of Sirt1, p-Akt2, IL-6, and MCP-1 in differentiated preadipocytes pretreated with pc-Sirt1 or sh-Sirt1, and then with RES for 48 h (n = 3). E: Immunoblots of p-Akt2 and mTORC1 signaling in differentiated preadipocytes pretreated with RES or NAM for 48 h, and then with 10 nM insulin for 30 min (n = 3). pc-Sirt1: same vector as Sirt1, overexpression vector of Sirt1. sh-Sirt1: lentiviral vector containing shRNA constructs against Sirt1. The treating concentrations of RES and NAM were 100 M and 500 M, respectively, in differentiated preadipocytes. Values are mean ± SD. *P < 0.05 and **P < 0.01 as compared with the control.
of Sirt1 and Akt2 interaction on the mTOR pathway, and found that Sirt1 inhibited raptor expression and the phosphorylation of mTOR. The result implies that the mTORC1 pathway is involved in the regulation of adipose inflammation by Sirt1 and Akt2 interaction. mTOR is a serine/threonine protein kinase that regulates protein synthesis (39). Studies report that activation of the mTOR pathway plays a key role in TNF--induced inflammatory cascades (40) and is also implicated in inflammation-related diseases. mTORC1 is highly activated in tissues of obese humans and HFD-fed rodents (19,41). Inhibition of mTORC1 promotes triacylglycerol lipolysis that releases free fatty acids, blocks adipogenesis, and impairs the maintenance of fat cells (42,43). Recent studies have demonstrated that the most common cancer-promoting signaling event that converges on mTORC1 is aberrant activation of Akt kinase (44). Our data showed that Sirt1 decreased the phosphorylation level of mTOR/S6K1, and this effect was attenuated by rapamycin, which specifically inhibited mTOR signal. Conversely, the insulin treatment elevated this effect. All of these results indicate that the mTOR/ S6K1 pathway is involved in the regulation of adipose inflammation by the interaction between Sirt1 and Akt2.

CONCLUSIONS
In conclusion, the findings of this study demonstrate that: RES inhibits adipose inflammation by activating Sirt1 and promoting the interaction between Sirt1 and Akt2; the mTOR/S6K1 pathway is involved in this interaction; and inhibition of this pathway alleviated adipose inflammation (Fig. 6). These findings shed a new light that intracellular pathways could be a target for developing prevention or treatment of obesity-associated endocrine and metabolic diseases.
The authors express their gratitude to Professor C. Y. Hu, Dr. Douglas L. Vincent, and Assistant Professor Rajesh Jha from the University of Hawaii at Manoa and Shimin Liu from for University of Western Australia for helping in the revision of the manuscript.   5. The mTOR/S6K1 pathway was essential for regulation of Sirt1 in adipocyte inflammation. A: Phosphorylation and protein levels of mTOR and S6K1 in differentiated preadipocytes pretreated with RES or NAM for 48 h, and then with 5 nM rapamycin for 60 min, followed with 10 nM insulin for 30 min (n = 3). B: Immunoblots of inflammation markers in differentiated preadipocytes pretreated with RES or NAM for 48 h, and then with 5 nM rapamycin for 60 min, followed with 10 nM insulin for 30 min (n = 3). The treating concentrations of RES and NAM were 100 M and 500 M, respectively, in differentiated preadipocytes. Values are mean ± SD. *P < 0.05 as being compared with the control.