Mitochondrial stress-activated cGAS-STING pathway inhibits thermogenic program and contributes to overnutrition-induced obesity in mice

Abstract

Obesity is a global epidemic that is caused by excessive energy intake or inefficient energy expenditure. Brown or beige fat dissipates energy as heat through non-shivering thermogenesis by their high density of mitochondria. However, how the mitochondrial stress-induced signal is coupled to the cellular thermogenic program remains elusive. Here, we show that mitochondrial DNA escape-induced activation of the cGAS-STING pathway negatively regulates thermogenesis in fat-specific DsbA-L knockout mice, a model of adipose tissue mitochondrial stress. Conversely, fat-specific overexpression of DsbA-L or knockout of STING protects mice against high-fat diet-induced obesity. Mechanistically, activation of the cGAS-STING pathway in adipocytes activated phosphodiesterase PDE3B/PDE4, leading to decreased cAMP levels and PKA signaling, thus reduced thermogenesis. Our study demonstrates that mitochondrial stress-activated cGAS-STING pathway functions as a sentinel signal that suppresses thermogenesis in adipose tissue. Targeting adipose cGAS-STING pathway may thus be a potential therapeutic strategy to counteract overnutrition-induced obesity and its associated metabolic diseases.

Fig. 1. Fat-specific knockout of DsbA-L reduced energy expenditure in mice.

a The activities of DsbA-L^fKO (n = 8) and loxp control (n = 8) mice was measured during a 48-h period, including two light/dark cycles. b The mRNA levels of DsbA-L in inguinal WAT (iWAT) (n = 5) and BAT (n = 5) were determined by qPCR and normalized to β-actin. c Immunoblot analysis of DsbA-L, UCP1, PGC1α expression in epididymal WAT (eWAT), iWAT and BAT from C57BL/6 wild-type mice (n = 3). d Oxygen consumption of DsbA-L^fKO and loxp control mice was measured during a 48-h period, including two light/dark cycles. e Core body temperature of DsbA-L^fKO (n = 5) and loxp control (n = 6) mice at room temperature (24 °C) in the feeding condition. f Core body temperature of DsbA-L^fKO and loxp control mice exposed to cold (4 °C) in the feeding condition at different time points as indicated. Data are presented as mean ± SEM of biologically independent samples, *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed t-test.

Fig. 2. DsbA-L is positively correlated with thermogenic gene expression in brown and beige fat.

Cold exposure-induced mRNA expression in a BAT and b iWAT of DsbA-L^fKO (24 °C, n = 6; 4 °C, n = 4) and loxp control (24 °C, n = 4; 4 °C, n = 4) mice. The mRNA levels were determined by qPCR and normalized to β-actin. Immunoblot analysis for cold exposure-induced protein expression of UCP1, C/EBPβ, PGC1α, and DsbA-L in c BAT and d iWAT of DsbA-L^fKO and loxp control mice (n = 4 for each group). e Representative H&E stain of BAT and iWAT of DsbA-L^fKO and loxp control mice. Scale bar: 200 µM. f Immunoblot analysis for protein expression of thermogenic genes in DsbA-L RNAi-suppressed stable brown adipocytes and control scramble cells (n = 4 for each group). The data were semiquantified by ImageJ program. Immunoblot analysis for protein expression of thermogenic genes in g BAT and h iWAT of fat-specific DsbA-L transgenic mice (DsbA-L^fTG) (n = 3) and their control littermates (n = 3). The data were semiquantified by ImageJ program. Data are presented as mean ± SEM of biologically independent samples, *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed t-test (for comparison between two groups) or one-way ANOVA (for comparison of multiple groups).

Fig. 3. DsbA-L promotes lipolysis and fatty acid oxidation in adipose tissue.

The mRNA levels of lipolytic genes in a BAT and b iWAT of DsbA-L^fKO (n = 4) and loxp control (n = 4) mice were determined by qPCR and normalized to β-actin. Glycerol release from primary-cultured adipocytes of c BAT and d iWAT of DsbA-L^fKO (n = 3) and loxp control (n = 3) mice. Free fatty acid release from primary-cultured adipocytes of e BAT and f iWAT of DsbA-L^fKO (n = 3) and loxp control (n = 3) mice. The mRNA levels of fatty acid oxidation-related genes in g BAT and h iWAT of DsbA-L^fKO (n = 4) and loxp control (n = 4) mice were determined by qPCR and normalized to β-actin. The oxidation of ¹⁴C-palmitate in i BAT and j iWAT from DsbA-L^fKO (n = 4 for BAT and n = 5 for iWAT) and loxp (n = 6 for BAT and n = 4 for iWAT) control mice. Glycerol release from primary-cultured adipocytes of k BAT and l iWAT of DsbA-L^fTG and their control mice (n = 3 for each group). Free fatty acid release from isolated m BAT and n iWAT of DsbA-L^fTG and their control mice (n = 3 for each group). The oxidation of ¹⁴C-palmitate in o BAT and p iWAT isolated from DsbA-L^fTG and their control mice (n = 3 for each group). Data are presented as mean ± SEM of biologically independent samples, *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed t-test.

Fig. 3. DsbA-L promotes lipolysis and fatty acid oxidation in adipose tissue.

The mRNA levels of lipolytic genes in a BAT and b iWAT of DsbA-L^fKO (n = 4) and loxp control (n = 4) mice were determined by qPCR and normalized to β-actin. Glycerol release from primary-cultured adipocytes of c BAT and d iWAT of DsbA-L^fKO (n = 3) and loxp control (n = 3) mice. Free fatty acid release from primary-cultured adipocytes of e BAT and f iWAT of DsbA-L^fKO (n = 3) and loxp control (n = 3) mice. The mRNA levels of fatty acid oxidation-related genes in g BAT and h iWAT of DsbA-L^fKO (n = 4) and loxp control (n = 4) mice were determined by qPCR and normalized to β-actin. The oxidation of ¹⁴C-palmitate in i BAT and j iWAT from DsbA-L^fKO (n = 4 for BAT and n = 5 for iWAT) and loxp (n = 6 for BAT and n = 4 for iWAT) control mice. Glycerol release from primary-cultured adipocytes of k BAT and l iWAT of DsbA-L^fTG and their control mice (n = 3 for each group). Free fatty acid release from isolated m BAT and n iWAT of DsbA-L^fTG and their control mice (n = 3 for each group). The oxidation of ¹⁴C-palmitate in o BAT and p iWAT isolated from DsbA-L^fTG and their control mice (n = 3 for each group). Data are presented as mean ± SEM of biologically independent samples, *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed t-test.

Fig. 4. Activation of the cGAS–STING pathway mediates DsbA-L deficiency-induced inhibition of thermogenic gene expression.

a Cytosolic mtDNA content was quantitated via qPCR using mtDNA primers (Dloop1-3) in freshly purified brown adipocytes from DsbA-L^fKO and loxp control mice (n = 5 for each group). b Immunoblot analysis of UCP1, STING, cGAS, TNFα, DsbA-L expression, and the phosphorylation of TBK1 at Ser¹⁷² in BAT from DsbA-L^fKO and loxp control mice (n = 5 for each group). The data were semiquantified by ImageJ program. c The protein levels of UCP1, DsbA-L, and the phosphorylation of TBK1 at Ser¹⁷² and IRF3 at Ser³⁹⁶ in DsbA-L-suppressed stable brown adipocytes and scramble control adipocytes (n = 3 for each group). The data were semiquantified by ImageJ program. d 2′3′-cGAMP levels in DsbA-L-suppressed brown adipocytes and scramble control cells were measured by HPLC–ESI–MS/MS analysis. e Representative immunoblot analysis of UCP1 expression, TBK1 and IRF3 phosphorylation in DsbA-L-suppressed brown adipocytes and scramble control cells stably overexpressing myc-tagged wild-type DsbA-L and its control plasmid. Representative immunoblot analysis of UCP1 expression, TBK1, and IRF3 phosphorylation in DsbA-L-suppressed brown adipocytes and scramble control cells transiently expressing f cGAS RNAi or g STING RNAi and their respective control plasmid. h Representative immunoblot analysis of UCP1 expression, TBK1, and IRF3 phosphorylation in DsbA-L-suppressed brown adipocytes and scramble control cells treated with or without 1 μM CL316243 or 50 μM amlexanox for 12 h. i Immunoblot analysis of UCP1, STING, cGAS expression, and TBK1 phosphorylation in primary brown adipocytes isolated from DsbA-L^fTG and wild-type mice treated with or without 4 μM nigericin or 10 μM ABT-737 for 12 h. j mRNA levels of thermogenic genes in primary brown adipocytes isolated from DsbA-L^fTG and wild-type control mice treated with or without 4 μM nigericin or 10 μM ABT-737 (n = 4 for each group) for 12 h. Data are presented as mean ± SEM of biologically independent samples, *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed t-test (for comparison between two groups) or one-way ANOVA (for comparison of multiple groups).

Fig. 5. The cGAS–STING pathway inhibits cAMP-PKA signaling by activating PDE.

a Representative immunoblot analysis of UCP1 expression, and phosphorylation of PKA substrates, HSL, and TBK1 in brown adipocytes treated with 10 nM 2′3′-cGAMP at different time points as indicated. Representative immunoblot analysis of UCP1 expression, and the phosphorylation of PKA substrates, HSL and TBK1 in b STING RNAi or c cGAS RNAi brown adipocytes treated with 10 nM 2′3′-cGAMP, 10 μM ABT-737 or 4 μM nigericin for 12 h. Representative immunoblot analysis of UCP1 expression, and the phosphorylation of PKA substrates, HSL, and TBK1 in d cGAS-, e STING-, or f TBK1/IKKε-suppressed brown adipocytes treated with or without 10 nM 2′3′-cGAMP or 10 μM H89 for 12 h. g cAMP levels in scramble and DsbA-L-suppressed brown adipocytes treated with or without 250 μM IBMX or 10 μM zardaverine (Zarda) for 15 min (n = 3 for each group). h cAMP levels in brown adipocytes treated with or without 10 nM 2′3′-cGAMP for 6 h followed by treatment with or without 250 μM IBMX or 10 μM zardaverine (Zarda) for 15 min (n = 3 for each group). cAMP levels in primary adipocytes isolated from iWAT of wild-type and STING-deficient mice (STING^gt) (n = 3 for each group) treated with or without 10 nM 2′3′-cGAMP for 6 h followed by treatment of 10 μM zardaverine (Zarda) for 15 min in the i absence or j presence of CL316243. PDE activities were measured in primary adipocytes isolated from iWAT of k STING^gt or l cGAS^−/− mice (n = 3 for each group) treated with or without 10 nM 2′3′-cGAMP for 6 h. Data are presented as mean ± SEM of biologically independent samples, *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed t-test (for comparison between two groups) or one-way ANOVA (for comparison of multiple groups).

Fig. 5. The cGAS–STING pathway inhibits cAMP-PKA signaling by activating PDE.

a Representative immunoblot analysis of UCP1 expression, and phosphorylation of PKA substrates, HSL, and TBK1 in brown adipocytes treated with 10 nM 2′3′-cGAMP at different time points as indicated. Representative immunoblot analysis of UCP1 expression, and the phosphorylation of PKA substrates, HSL and TBK1 in b STING RNAi or c cGAS RNAi brown adipocytes treated with 10 nM 2′3′-cGAMP, 10 μM ABT-737 or 4 μM nigericin for 12 h. Representative immunoblot analysis of UCP1 expression, and the phosphorylation of PKA substrates, HSL, and TBK1 in d cGAS-, e STING-, or f TBK1/IKKε-suppressed brown adipocytes treated with or without 10 nM 2′3′-cGAMP or 10 μM H89 for 12 h. g cAMP levels in scramble and DsbA-L-suppressed brown adipocytes treated with or without 250 μM IBMX or 10 μM zardaverine (Zarda) for 15 min (n = 3 for each group). h cAMP levels in brown adipocytes treated with or without 10 nM 2′3′-cGAMP for 6 h followed by treatment with or without 250 μM IBMX or 10 μM zardaverine (Zarda) for 15 min (n = 3 for each group). cAMP levels in primary adipocytes isolated from iWAT of wild-type and STING-deficient mice (STING^gt) (n = 3 for each group) treated with or without 10 nM 2′3′-cGAMP for 6 h followed by treatment of 10 μM zardaverine (Zarda) for 15 min in the i absence or j presence of CL316243. PDE activities were measured in primary adipocytes isolated from iWAT of k STING^gt or l cGAS^−/− mice (n = 3 for each group) treated with or without 10 nM 2′3′-cGAMP for 6 h. Data are presented as mean ± SEM of biologically independent samples, *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed t-test (for comparison between two groups) or one-way ANOVA (for comparison of multiple groups).

Fig. 6. Knockout of cGAS or STING in mice increased PKA signaling and thermogenesis.

Immunoblot analysis of UCP1 expression and the phosphorylation of PKA substrates including HSL in a iWAT and b BAT of STING^gt and their control littermates exposed to cold stress (4 °C) or housed at room temperature (24 °C). c Representative H&E stain of BAT and iWAT of wild-type and STING^gt mice. Scale bar: 200 µM. Immunoblot analysis of UCP1 expression and the phosphorylation of PKA substrates, HSL, TBK1, and IRF3 in primary-cultured inguinal adipocytes treated with 1 μM CL316243 in the presence or absence of 10 nM 2′3′-cGAMP from d STING^gt or e cGAS^−/− mice and their wild-type control mice. Glycerol release from primary-cultured inguinal adipocytes treated with 1 μM CL316243 in the presence or absence of 10 nM 2′3′-cGAMP from f STING^gt or g cGAS^−/− mice and their wild-type control mice (n = 3 for each group). h Immunoblot analysis of UCP1 expression and the phosphorylation of PKA substrates, HSL, TBK1, and IRF3 in cGAS or STING overexpressed primary inguinal adipocytes treated with 1 μM CL316243 for 12 h. i A graphic model on the negative regulation of thermogenesis by the cGAS–STING pathway in adipose tissue. Data are presented as mean ± SEM of biologically independent samples, *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed t-test (for comparison between two groups) or one-way ANOVA (for comparison of multiple groups).

Fig. 6. Knockout of cGAS or STING in mice increased PKA signaling and thermogenesis.

Immunoblot analysis of UCP1 expression and the phosphorylation of PKA substrates including HSL in a iWAT and b BAT of STING^gt and their control littermates exposed to cold stress (4 °C) or housed at room temperature (24 °C). c Representative H&E stain of BAT and iWAT of wild-type and STING^gt mice. Scale bar: 200 µM. Immunoblot analysis of UCP1 expression and the phosphorylation of PKA substrates, HSL, TBK1, and IRF3 in primary-cultured inguinal adipocytes treated with 1 μM CL316243 in the presence or absence of 10 nM 2′3′-cGAMP from d STING^gt or e cGAS^−/− mice and their wild-type control mice. Glycerol release from primary-cultured inguinal adipocytes treated with 1 μM CL316243 in the presence or absence of 10 nM 2′3′-cGAMP from f STING^gt or g cGAS^−/− mice and their wild-type control mice (n = 3 for each group). h Immunoblot analysis of UCP1 expression and the phosphorylation of PKA substrates, HSL, TBK1, and IRF3 in cGAS or STING overexpressed primary inguinal adipocytes treated with 1 μM CL316243 for 12 h. i A graphic model on the negative regulation of thermogenesis by the cGAS–STING pathway in adipose tissue. Data are presented as mean ± SEM of biologically independent samples, *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed t-test (for comparison between two groups) or one-way ANOVA (for comparison of multiple groups).