Macrophage alpha1 AMP-activated protein kinase (alpha1AMPK) antagonizes fatty acid-induced inflammation through SIRT1

Abstract

In this study, we aim to determine cellular mechanisms linking nutrient metabolism to the regulation of inflammation and insulin resistance. The nutrient sensors AMP-activated protein kinase (AMPK) and SIRT1 show striking similarities in nutrient sensing and regulation of metabolic pathways. We find that the expression, activity, and signaling of the major isoform alpha1AMPK in adipose tissue and macrophages are substantially down-regulated by inflammatory stimuli and in nutrient-rich conditions, such as exposure to lipopolysaccharide (LPS), free fatty acids (FFAs), and diet-induced obesity. Activating AMPK signaling in macrophages by 5-aminoimidazole-4-carboxamide-1-beta4-ribofuranoside or constitutively active alpha1AMPK (CA-alpha1) significantly inhibits; although inhibiting alpha1AMPK by short hairpin RNA knock-down or dominant-negative alpha1AMPK (DN-alpha1) increases LPS- and FFA-induced tumor necrosis factor alpha expression. Chromatin immunoprecipitation and luciferase reporter assays show that activation of AMPK by CA-alpha1 in macrophages significantly inhibits LPS- or FFA-induced NF-kappaB signaling. More importantly, in a macrophage-adipocyte co-culture system, we find that inactivation of macrophage AMPK signaling inhibits adipocyte insulin signaling and glucose uptake. Activation of AMPK by CA-alpha1 increases the SIRT1 activator NAD(+) content and SIRT1 expression in macrophages. Furthermore, alpha1AMPK activation mimics the effect of SIRT1 on deacetylating NF-kappaB, and the full capacity of AMPK to deacetylate NF-kappaB and inhibit its signaling requires SIRT1. In conclusion, AMPK negatively regulates lipid-induced inflammation, which acts through SIRT1, thereby contributing to the protection against obesity, inflammation, and insulin resistance. Our study defines a novel role for AMPK in bridging the signaling between nutrient metabolism and inflammation.

FIGURE 1.

Tissue distribution of α1AMPK (A), α2AMPK (B), and LKB1 (C). Total RNA was isolated from individual tissues and the expression of α1AMPK, α2AMPK, and LKB1 was measured by real time RT-PCR and normalized to cyclophilin. BAT, brown adipose tissue; Macrophage, thioglycollate-elicited peritoneal macrophage. Data are expressed as mean ± S.E., n = 3. A.U., arbitrary units.

FIGURE 2.

AMPK signaling and expression in macrophages and adipose tissue are down-regulated by LPS, FFAs, HF diets, and lipid infusion. A–C, LPS and FFAs inhibit AMPK phosphorylation (A), activity (B), and ACC phosphorylation (C) in macrophages. Bone marrow macrophages were treated with LPS (100 ng/ml) or FFAs (palmitate, oleate, and stearate mixture, 500 μm) for 2 h. D, LPS and FFAs inhibit the expression of α1AMPK and LKB1 in macrophages. RAW264.7 macrophages were treated with LPS (100 ng/ml) or FFAs (250 μm) overnight. n = 6/group. E, LPS inhibits the mRNA expression of α1AMPK and LKB1 in epididymal WAT of mice. C57/BL6J mice (male, n = 4/group) were intraperitoneally injected with LPS (2 mg/kg body weight) and WAT were dissected 2 or 6 h after injection. F, increasing circulating FFAs inhibits the mRNA expression of α1AMPK and LKB1 in epididymal WAT in mice (n = 5/group). Mice were infused with lipids (5 ml/kg·h, liposyn II; coupled with 6 units/h of heparin) for 8 h. G and H, HF feeding inhibits the mRNA expression of α1AMPK and LKB1 (G) and AMPK signaling (H) in epididymal WAT of mice (n = 8/group). For A, C, and H, AMPK signaling was measured by immunoblotting. For B, α1AMPK activity was measured using an immunocomplex assay with SAMS peptide. For D–G, mRNA levels of target genes were measured by real time RT-PCR and normalized to cyclophilin. All data are expressed as mean ± S.E. *, p < 0.05. A.U., arbitrary units.

FIGURE 3.

A and B, activation of AMPK signaling by AICAR suppresses stearate (C18:0) (A)- or LPS (B)- induced TNF-α mRNA expression in macrophages. RAW264.7 macrophages were pre-treated with various concentrations of AICAR as indicated, and then stimulated with LPS (100 ng/ml) or stearate (C18:0) (500 μm) for 4 h. TNF-α mRNA was measured by real time RT-PCR. C and D, activation of AMPK by CA-α1AMPK or AICAR inhibits stearate (C)- or LPS (D)-induced TNF-α expression. RAW264.7 macrophages were transfected with pcDNA3.1, DN-α1AMPK, or CA-α1AMPK, and then treated with vehicle (BSA for stearate and H₂O for LPS), LPS (100 ng/ml), stearate (500 μm), with or without AICAR (2 mm) as indicated for 4 h. E, activation of AMPK by AICAR or CA-α1AMPK attenuates TNF-α secretion by macrophages. RAW264.7 macrophages transfected with pcDNA3.1, DN-α1AMPK, or CA-α1AMPK or treated with AICAR (2 mm) were stimulated with stearate (250 μm) for 24 h. All data are expressed as mean ± S.E., n = 6–8, *, p < 0.05. A.U., arbitrary units.

FIGURE 4.

Inactivation of α1AMPK increases basal and stearate-stimulated TNF-α mRNA. A and B, establishment of a macrophage cell line with α1AMPK knockdown (α1KD). RAW264.7 macrophages were infected with the α1AMPK shRNA lentivirus or control lentivirus, and selected with puromycin for 8 days. α1AMPK RNA (A) and protein (B) levels were evaluated by real time RT-PCR and immunoblotting, respectively. C and D, α1AMPK knockdown increases basal and stearate- or LPS-stimulated TNF-α mRNA. The α1AMPK-knockdown and control macrophages were treated with vehicle (BSA for stearate and H₂O for LPS), stearate (500 μm), or LPS (100 ng/ml) for 4 h. E and F, inactivation of α1AMPK by DN-α1AMPK overexpression increases basal and stearate- or LPS-stimulated TNF-α mRNA. RAW264.7 macrophages were transfected with pcDNA3.1 or DN-α1AMPK, and then treated with vehicle (BSA for stearate and H₂O for LPS), LPS (100 ng/ml), or stearate (500 μm) as indicated for 4 h. TNF-α mRNA was measured by real time RT-PCR. Data are expressed as mean ± S.E., n = 6. *, p < 0.05. A.U., arbitrary units.

FIGURE 5.

Activation of AMPK suppresses NF-κB signaling in macrophages. A and B, activation of AMPK by AICAR or CA-α1AMPK inhibits NF-κB luciferase reporter activity. RAW264.7 macrophages expressing pcDNA3.1, CA-α1AMPK, or DN-α1AMPK were co-transfected with pNFκB-Luc vectors. After 5 h, cells were then treated overnight with vehicle (BSA for stearate and H₂O for LPS), LPS (100 ng/ml), and stearate (250 μm) with or without AICAR (2 mm). n = 6. C, activation of AMPK blocks NF-κB (p65) binding to the IL-6 promoter in ChIP assay. D, SYBR Green quantitative PCR was used to measure the promoter DNA immunoprecipitated by the anti-p65 antibody; n = 3. All data are expressed as mean ± S.E. *, p < 0.05. A.U., arbitrary units.

FIGURE 6.

Inactivation of macrophage α1AMPK inhibits adipocyte insulin signaling in a co-culture system. A and B, 3T3-L1 adipocytes co-cultured with α1AMPK-knockdown macrophages exhibit reduced insulin (Ins)-stimulated glucose uptake (A) and signaling (B). Insulin-stimulated glucose uptake and signaling were conducted in 3T3-L1 adipocytes co-cultured with either the knockdown (KD) or control macrophages. C, inactivation of macrophage α1AMPK stimulates JNK phosphorylation in adipocytes in the co-culture system. NS, non-specific. Data are expressed as mean ± S.E., n = 4; *, p < 0.05.

FIGURE 7.

A–C, activation of AMPK by CA-α1AMPK expression increases NAD⁺ content and NAD⁺/NADH ratio in macrophages. RAW264.7 macrophages were transfected with CA-α1AMPK expression vectors. NAD⁺ and NADH nucleotides were measured with a NAD⁺/NADH quantification kit. D and E, activation of AMPK increases SIRT1 mRNA expression in macrophages. RAW264.7 macrophages were treated with 2 mm AICAR (D) or transfected with CA-α1AMPK overnight (E). F, AICAR increases SIRT1 protein expression in macrophages. Peritoneal macrophages were treated with AICAR (2 mm) at the indicated time points. SIRT1 mRNA was measured by real time RT-PCR. SIRT1 protein expression was measured by immunoblotting. All data are expressed as mean ± S.E., n = 4; *, p < 0.05.

FIGURE 8.

A, SIRT1 deacetylates p65 in macrophages. RAW264.7 macrophages were transfected with pcDNA3.1 or expression vectors for p65, p300, SIRT1, or SIRT1(H355A). Cell lysates were used to examine p65 acetylation (lysine 310) by immunoblotting. B and C, activation of AMPK by AICAR (B) or CA-α1AMPK (C) deacetylates p65 in macrophages. RAW264.7 macrophages were transfected with expression vectors for p65, p300, and CA-α1AMPK, or treated with AICAR (2 mm). D, SIRT1 is required for full capacity of AMPK to deacetylate NF-κB. The SIRT1-knockdown or control cells were transfected with expression vectors for p65, p300, and CA-α1AMPK. A representative blot was shown in the left panel. The blots were quantitated with a Li-COR Odyssey Infrared Imager system (right panel). *, p < 0.05. E and F, SIRT1 is required for full capacity of AMPK to inhibit NF-κB transcriptional activity. The control or SIRT1-knockdown macrophages transfected with pNFκB-Luc vectors were treated with 2 mm AICAR or co-transfected with CA-α1AMPK expression vectors, and then stimulated with stearate (250 μm) (E) or LPS (100 ng/ml) (F) for 24 h. All data are expressed as mean ± S.E., n = 4; *, p < 0.05.