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. 2023 Sep 24;12(19):2345.
doi: 10.3390/cells12192345.

Stimulator of Interferon Genes (STING) Triggers Adipocyte Autophagy

Kornél Z Varga  1 , Katalin Gyurina  1 , Ádám Radványi  1 , Tibor Pál  1 , László Sasi-Szabó  1 , Haidong Yu  2 , Enikő Felszeghy  1 , Tamás Szabó  1 , Tamás Röszer  1   2
Affiliations

Stimulator of Interferon Genes (STING) Triggers Adipocyte Autophagy

Kornél Z Varga et al. Cells. .

Abstract

Innate immune signaling in adipocytes affects systemic metabolism. Cytosolic nucleic acid sensing has been recently shown to stimulate thermogenic adipocyte differentiation and protect from obesity; however, DNA efflux from adipocyte mitochondria is a potential proinflammatory signal that causes adipose tissue dysfunction and insulin resistance. Cytosolic DNA activates the stimulator of interferon response genes (STING), a key signal transducer which triggers type I interferon (IFN-I) expression; hence, STING activation is expected to induce IFN-I response and adipocyte dysfunction. However, we show herein that mouse adipocytes had a diminished IFN-I response to STING stimulation by 2'3'-cyclic-GMP-AMP (cGAMP). We also show that cGAMP triggered autophagy in murine and human adipocytes. In turn, STING inhibition reduced autophagosome number, compromised the mitochondrial network and caused inflammation and fat accumulation in adipocytes. STING hence stimulates a process that removes damaged mitochondria, thereby protecting adipocytes from an excessive IFN-I response to mitochondrial DNA efflux. In summary, STING appears to limit inflammation in adipocytes by promoting mitophagy under non-obesogenic conditions.

Keywords: STING; adipocyte; immunity; inflammation; interferons; mitochondria.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Scheme summarizing the role of cGAS/STING in recognition of mitochondrial DNA (mtDNA) in the cytosol. (B) Transcript level of Sting1 and Cgas in BAT, skeletal muscle, hepatocytes, iAT and eAT of male C57/BL6 mice at 8 weeks of age. Each data point represents one biological replicate. (C) FACS plot representing STING expression level of adipocytes (ACs) and adipose tissue macrophages (ATMs) of male C57/BL6 mice at 8 weeks of age. Extended analysis is presented in [24]. (D) Histograms comparing STING levels of ACs and ATMs. Iso: isotype control. Extended analysis in [24]. (E) Transcript levels of Sting1 and Cgas in iAT and eAT of normal chow-diet (NCD)-fed or high-fat diet (HFD)-fed male C57/BL6 mice. Prevalence of STING+ ATMs in iAT and eAT, expressed as the percentage of the total ATM population. Extended analysis presented in [24]. Each data point represents one biological replicate. (F) Level of Sting1 and Cgas mRNA in mouse adipocytes cultured in vitro and treated with 10 ng/mL poly(I:C) (a TLR3 ligand) or 100 ng/mL LPS (a TLR4 ligand) for 18 h.
Figure 2
Figure 2
Expression of STING and cGAS mRNA and protein in adipocytes. (A) Venn diagram summarizing the number of equally and differently expressed mRNA transcripts of young and adult mouse iAT. A gene network associated with Sting1 was equally expressed by young and adult iAT. A protein–protein interaction map, generated by STRING [30] is shown below the Venn diagram. Extended analysis presented in [24]. (B) Immunofluorescence of in vitro cultured adipocytes from young and adult mouse iAT; nc: nucleus, scale bar 20 μm. (C) Immunostaining of STING and cGAS proteins in the iAT of young mice, showing a region containing both multilocular and unilocular adipocytes. Arrowheads label nuclei; lp: lipid droplet; cyt: cytoplasm; scale bar: 50 μm. (D) Top: Expression of STING1 and CGAS mRNA in human inguinal and abdominal adipose tissue specimens. Linear regression analysis indicates a significant positive correlation between STING1 and CGAS mRNA levels. Each data point represents one tissue donor patient. Bottom: Correlation of donor age and the adipose tissue expression levels of STING1 and CGAS. (E) Immunohistochemistry of STING and cGAS proteins in human adipose tissue, collected from the inguinal-low abdominal region. Nineteen-month-old male infant; arrowheads label nuclei; lp: lipid droplet; cyt: cytoplasm; scale bar: 25 μm. Inlet shows nuclear STING labeling of an in vitro cultured human adipocyte. Scale bar: 20 μm. (F) Body mass index z-score (BMI z-score) and BMI standard deviation score (BMI-SDS) of adipose tissue donors involved in this study. Correlation of BMI z-score with adipose tissue STING1 and CGAS mRNA levels.
Figure 3
Figure 3
Response of mouse adipocytes and macrophages to a STING ligand. (A) Left: Uptake route of the STING ligand 2′3′-cyclic-AMP-GMP (cGAMP). Membrane transport of cGAMP is facilitated by the solute carrier protein SLC19a. Right: Expression of Slc19a1 mRNA in mouse iAT, BAT, ATMs, primary adipocytes and in mouse 3T3-L1 cells. Secondary NGS analysis from [29]. (B) Ifnb expression level in iAT- and BAT-derived adipocytes, treated in vitro with 10 µg/mL cGAMP for 18 h. (C) Expression of Ucp1 mRNA and the activity of mitochondrial enzymes COX-I and SDH-A in mouse iAT-derived adipocytes treated with cGAMP for 18 h. (D) Heat map summarizing the transcriptional changes of Ifnb, Il6 and Tnfa in mouse macrophages and adipocytes, following treatment with 10 µg/mL cGAMP for 18 h. * p < 0.05, ** p < 0.01, *** p < 0.001, Student’s unpaired 2-tailed t-test.
Figure 4
Figure 4
Effect of cGAMP on autophagy in adipocytes. (A) Autophagosomes (Phs) were labeled with a CellMeterTM autophagy fluorescent imaging probe in primary mouse and human adipocytes. Adipocytes were treated with vehicle or 10 µg/mL cGAMP for 6 h. (B) Phagosome number and perimeter in vehicle-, or cGAMP-treated mouse and human adipocytes. (C) A fluorescent autophagy assay was used to estimate autophagosome number in mouse 3T3-L1 preadipocytes and adipocytes following treatment with vehicle or cGAMP. +Chlq: cells were also treated with 100 μM chloroquine for 4 h. (D,E) LC3+ and ATG5+ puncta in mouse adipocytes treated with vehicle or cGAMP. nc: nucleus, scale bar: 20 μm. Corresponding staining of preadipocytes is shown in Supplementary Figure S3C. (F) Mitochondria were labeled with BacMam 0.2 transfection system. GFP-labeled mitochondrial remnants were accumulated in autophagosomes of cGAMP-treated adipocytes. nc: nucleus, scale bar: 10 μm. Adipocytes were treated with 10 µg/mL cGAMP for 6 h. Transmission electron microscopy of phagophore (Php), phagosome (Phs), phagolysosome (Phl) and mitochondria (Mt). Scale bar: 0.1 μm. (G) Lysosomes (Lyso) were labeled with Lyso Brite Orange in human adipocytes and treated with vehicle or 10 µg/mL cGAMP for 2 h. Arrows indicate clustering of lysosomes. Scale bar: 20 μm. (H) Transmission electron microscopy of phagolysosome (Phl) and fluorescent microscopy of LC3+ and ATG5+ structures in mouse adipocytes. Adipocytes were treated with vehicle or 10 µg/mL cGAMP for 6 h. nc: nucleus. Scale bar: 0.5 μm (electron microscopy) and 10 μm (fluorescent microscopy). (I) Mean fluorescence intensity (MFI) of LC3 and ATG5 immunostaining in mouse preadipocytes and adipocytes treated with vehicle or 10 µg/mL cGAMP for 6 h. (J) Western blotting of LC3 in mouse adipocytes. Cells were treated with vehicle or cGAMP for 6 h. +Chlq: cells were also treated with 100 μM chloroquine for 4 h. * p < 0.05, ** p < 0.005, *** p < 0.001, two-tailed unpaired Student t-test (B,I) or one-way ANOVA with Dunnett’s post hoc test (C,J).
Figure 5
Figure 5
Effect of STING blockage on adipocyte autophagy. (A) H151 covalently binds to STING [27]. (B) Autophagy intensity in mouse adipocytes treated with vehicle or 0.5 μM H151 for 18 h. (C) Fluorescently labeled autophagosomes (Phs) in mouse adipocytes cultured in the presence of fetal calf serum (+FCS) or serum-deprived (−FCS) for 6 h. Cells were treated with vehicle or H151 for 6 h. (D) LC3 immunostaining of mouse adipocytes treated with vehicle of H151 during 6 h serum deprivation. Scale bar: 10 μm. (E) LC3 Western blot of mouse adipocytes following 6 h serum deprivation. Cells were treated with vehicle or H151 during serum deprivation. (F) Mitochondrial network of mouse adipocytes was labeled with MitoTracker Red (MTR) and treated with vehicle or H151 for 18 h. Scale bar: 5 μm. (G) Mitochondrial network of human adipocytes was labeled with MTR and treated with vehicle or H151 for 18 h. Arrow labels mitochondria. Scale bar: 20 μm. (H,I) FACS analysis and MFI of MTR labeling of mouse adipocyte mitochondria after 18 h H151 treatment. (J) Activity of mitochondrial COX-I in mouse and human adipocytes, following treatment with vehicle or H151 for 18 h. ** p < 0.01, *** p < 0.001. Student’s unpaired 2-tailed t-test.
Figure 6
Figure 6
Effect of STING inhibition on the inflammatory state and lipid accumulation of adipocytes. (A) Scheme of cytosolic DNA sensor pathways that respond to mitochondrial DNA (mtDNA). (B) Expression levels of Tnfa and Il6 mRNA in mouse adipocytes treated with vehicle or 0.5 μM H151 for 18 h. Cytosolic DNA sensors pathways were blocked by the NFκB inhibitor BAY 11-70082, or by transfecting cells with an Irf3-siRNA. As a comparison, adipocytes were treated with vehicle or 100 μM chloroquine for 18 h. (C) Oil red O labeling of lipid droplets in mouse preadipocytes, treated with vehicle or H151 for 18 h. Scale bar: 20 μm. (D) Number, area and perimeter of lipid droplets following treatment with vehicle or H151 for 6 h. (E) Oil red O labeling of lipid droplets in human preadipocytes, treated with vehicle or H151 for 18 h. Scale bar: 20 μm. (F) Number, area and perimeter of lipid droplets in human preadipocytes following treatment with vehicle or H151 for 6 h. * p < 0.05, ** p < 0.01, Student’s unpaired 2-tailed t-test; # p < 0.05, ### p < 0.001, one-way ANOVA with Dunnett’s post hoc test. (G) Working model summarizing the dual roles of STING in adipocytes. STING activation promotes expression of interferons (IFNs) and causes inflammation. In turn, an anti-inflammatory effect of STING exists in adipocytes by increasing autophagic removal of inflammation-provoking mitochondrial contents. Also, STING appears to control lipid content in adipocytes.
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Grants and funding

This study was supported by a German Research Fund (DFG, RO 4856-1/3 to T.R.), a Hungarian Research Fund (OTKA-NKFI 142939, to T.R.), Bolyai Research Scholarship of the Hungarian Academy of Sciences. (to T.R.), intramural funding from the Foundation of the Institute of Pediatrics, University of Debrecen, Hungary. APC was supported by the University of Debrecen, Hungary.