Mitochondrial RNA stimulates beige adipocyte development in young mice

Childhood obesity is a serious public health crisis and a critical factor that determines future obesity prevalence. Signals affecting adipocyte development in early postnatal life have a strong potential to trigger childhood obesity; however, these signals are still poorly understood. We show here that mitochondrial (mt)RNA efflux stimulates transcription of nuclear-encoded genes for mitobiogenesis and thermogenesis in adipocytes of young mice and human infants. While cytosolic mtRNA is a potential trigger of the interferon (IFN) response, young adipocytes lack such a response to cytosolic mtRNA due to the suppression of IFN regulatory factor (IRF)7 expression by vitamin D receptor signalling. Adult and obese adipocytes, however, strongly express IRF7 and mount an IFN response to cytosolic mtRNA. In turn, suppressing IRF7 expression in adult adipocytes restores mtRNA-induced mitobiogenesis and thermogenesis and eventually mitigates obesity. Retrograde mitochondrion-to-nucleus signalling by mtRNA is thus a mechanism to evoke thermogenic potential during early adipocyte development and to protect against obesity.


ELISA assays
Tissue samples were weighed and homogenized in RIPA buffer using a Roche bead mill homogenizer at 6,500 rpm for 1 min. Cell culture supernatants and plasma samples were centrifuged at 0.8 g for 10 min to remove cell debris, and supernatants were used for analysis. We used commercial ELISA kits to measure the levels of IL-6, TNF (Fisher Scientific), IRF7, Vit-D3, calcitriol and VDR (MBS1605109, MBS268259, MBS288322, MBS2701844, MyBioSource). All samples were stored at -80°C until analysis.

mtRNA isolation and in vitro transfection
Adipocyte mitochondria were isolated with a commercial mitochondrial isolation kit (Thermo Fisher Scientific, Waltham, MA), using mouse adipocytes or human THP1 cells. Mitochondrial RNA (mtRNA) was isolated by lysing the mitochondrial pellet with TRI Reagent (Sigma-Aldrich), as described 4 . Adipocytes were transfected with 2 µg of mtRNA in 6-or 24-well plates with cells at 80-90% confluency. As a transfection reagent we used Lipofectamine 3000 (Invitrogen) at a 1:3 ratio. Control cells received transfection reagent only. Cells were analyzed 18 h after transfection.

mtDNA isolation and transfection
Mitochondrial DNA (mtDNA) was isolated from mitochondria pellets using TRI Reagent (Merck Sigma-Aldrich) and reconstituted in TE buffer (10 mM Tris-HCL, 1 mM EDTA, pH 8.0). Agarose gel electrophoresis was used to examine mtDNA integrity. Adipocytes were transfected for 18 h with 1-2 µg/ml mtDNA using the TurboFect Transfection Reagent. Control cells received transfection reagent only.

Cytosolic mtRNA and mtDNA isolation
Cytosol fractions of adipocytes were collected by subcellular fractionation of the cytoplasm and the cell organelles using digitonin, as described 5,6 . Digitonin buffer contained 150 mM NaCl, 50 mM HEPES (pH 7.4) and 25 µg/ml digitonin (D141, Merck Sigma-Aldrich). Treated cells were processed until the step in which cytosol was obtained as described 4 . Cytosolic fractions (250 µl) were added to 750 µl TRI Reagent (T3934, Merck Sigma-Aldrich) and total DNA and RNA extraction was performed as described 7 . Isolated RNA was reverse transcribed into cDNA, Bactin and Ppia were used as reference genes. DNA was reconstituted in TE buffer and adjusted to 10 ng/µl. We performed qPCR using HK2 as a reference nuclear genome-encoded gene, and measured the DNA copy number of mtDNAencoded genes. We calculated the copy number according to the formula: Ct=CtTarget gene-CtReference gene mtDNA copy number=2X2 (Ct) (1)

Histology, immunofluorescence and image analysis
Tissues were fixed with 4% paraformaldehyde and embedded in paraffin, as described (1). Sections were stained with hematoxylin and eosin (Carl Roth, Karlsruhe, Germany). Antibodies are listed in Supplementary Table 2. UCP1, IFI16, AIM2 immunohistochemistry was performed on paraffin-embedded tissue sections. For histomorphometry of fat cells we used Image J, with an image-processing algorithm that incorporated the Euclidean distancebased Watershed transformation to segment the images. Briefly, binarized images were generated using Otsu's method for thresholding; enhanced images were generated using contrast limited adaptive histogram equalization (CLAHE), and finally segmented images were generated using the Watershed transformation (Extended Data Figure 8). For fluorescent microscopy of STING, cGAS, AIM2, DDX41, IFI205 and ZBP1 murine or human preadipocytes were grown on optical transparent glass-bottom plates (Greiner Bio-One GmbH, Frickenhausen, Germany) or glass coverslips, and labeled with antibodies listed in Supplementary Table 2. Negative control specimens of our fluorescent imaging and immunostaining are shown in Extended Data Figure 8. Histology images were adjusted to equal white balance after acquisition. Mitochondrial content and morphology was analyzed with ImageJ, as described 4 . Beige adipose area was measured with our custom-developed image analysis software (BeAR © v1.0, 4 ).

Quantification of UCP1 staining
In vitro UCP1 immunostaining was performed in 6-well culture plates, and samples were imaged and the optical density was measured using digital image analysis. Original images are available upon request through Figshare. Mitochondria were also labeled using an SDH-A histochemistry assay (BioOptica).

Adipocyte differentiation
Mouse or human preadipocytes of the stromal vascular fraction (SVF) were isolated and maintained as described [7][8][9] . To ensure the depletion of adipose tissue macrophages (ATMs) from the harvested preadipocytes, we used magnetic bead cell purification of the SVF with an antibody against the F4/80 antigen (Miltenyi Biotec, Bergisch Gladbach, Germany) 10 . Human subcutaneous adipose tissue preadipocytes were harvested as described 7,8 . Preadipocytes were maintained in cell culture medium supplemented with 20 g/ml insulin. To induce white differentiation of preadipocytes, we treated the cells with 50 M IBMX, 1 M dexamethasone, 1 M rosiglitazone and 20 g/ml insulin (all from Merck Sigma-Aldrich), as described 4 . Lipid content was labeled with Oil red O.

Flow cytometry analysis of DNA sensors, mitochondrial biogenesis, mitochondrial content and mitochondrial uncoupling
Mitochondrial content was analyzed with MitoTracker dyes (Thermo Fisher Scientific). Mitochondrial biogenesis was detected with the MitoBiogenesis™ Flow Cytometry Kit (Abcam, Cambridge, UK). MitoThermo Yellow (MTY), a temperature-sensitive fluorescent probe 11 , was used to assess mitochondrial thermogenesis and uncoupling, as described 12,13 . Temperature difference between the control and the test groups was expressed as Mito-T, and shown in the respective figures. MTY was developed and provided by Dr. Y-T. Chang (Center for Self-Assembly and Complexity, Institute for Basic Science & Department of Chemistry, Pohang University of Science and Technology, Pohang 37673, Republic of Korea). We used MTY for FACS analysis at 0.1 ng/ml to label 10 6 /ml cells. Cells were maintained at 37°C throughout the assay. DNA sensors (STING, p204, AIM2, DDX41) were detected with unconjugated antibodies (listed in Supplementary Table 2) and labeled with an FITC-conjugated secondary antibody for FACS analysis. Nucleic acids were labeled with Sytox Green (Thermo Fisher). Flow Repository identifiers of raw FACS data are as follows: #FR-FCM-Z236, #FR-FCM-Z2R6, #FR-FCM-ZYPU, #FR-FCM-ZYUU, #FR-FCM-Z5QA.

Imaging of mitochondrial content
For fluorescent microscopy of mitochondrial content and morphology, preadipocytes were grown on optical transparent glass-bottom plates (Greiner Bio-One GmbH, Frickenhausen, Germany) or glass coverslips. Functional mitochondria were labeled with MitoTracker Red. Mitochondria were also labeled with GFP using the BacMam 2.0 transfection system (Fisher Scientific). Mitochondrial respiration was evaluated with the WST-81 assay (Carl Roth), as described 14 . SDH-A and COX-I level was measured with flow cytometry and spectrophotometry (Abcam MitoBiogenesis Kits). Inflammasome activity was measured with the Caspase-Glo 1 Inflammasome Assay (Promega Co., Madison, WI).

High-fat diet feeding and indirect calorimetry
Respiratory exchange rate (RER), oxygen consumption (VO2) and energy expenditure (EE) were measured in each individual mouse for 24 h using a small animal indirect calorimetry system (CaloBox, Phenosys, Germany). Mean RER, VO2 and EE values were determined over 7 h in the middle of both the day and the night phases. Basal glucose levels and glucose tolerance were measured as described 7 . For HFD-feeding of mice (dams with litters P6 to P9, or mice at P28 for 12 weeks) we used a rodent HFD from SSNIFF Spezialdiäten (Soest, Germany, E15725-347) 7 . Vit-D3 was supplemented in diet, mtRNA was transfected in the iAT with magnetofection for 14 days. As a normal chow diet we used rodent diet from SSNIFF Spezialdiäten (Soest, Germany, E15051-047).

Magnetofection of mtRNA
In vivo delivery of mtRNA into the cytosol of adipocytes was achieved with magnetofection, using mtRNA-magnetic nanoparticle complexes (DogtorMag, OzBiosciences, San Diego, CA). Briefly, 15 g mtRNA-nanoparticle complexes were injected into the iAT of mice, and enrichment of the magnetic nanoparticles was ensured by magnetic exposure of the fat depot, as described 15 .

Cell viability assay
We used the Presto Blue Cell Viability Assay (Thermo Fisher Scientific) and the Rotitest Vital (Carl Roth) assays.