Brown adipose tissue CoQ deficiency activates the integrated stress response and FGF21-dependent mitohormesis

Coenzyme Q (CoQ) is essential for mitochondrial respiration and required for thermogenic activity in brown adipose tissues (BAT). CoQ deficiency leads to a wide range of pathological manifestations, but mechanistic consequences of CoQ deficiency in specific tissues, such as BAT, remain poorly understood. Here, we show that pharmacological or genetic CoQ deficiency in BAT leads to stress signals causing accumulation of cytosolic mitochondrial RNAs and activation of the eIF2α kinase PKR, resulting in activation of the integrated stress response (ISR) with suppression of UCP1 but induction of FGF21 expression. Strikingly, despite diminished UCP1 levels, BAT CoQ deficiency displays increased whole-body metabolic rates at room temperature and thermoneutrality resulting in decreased weight gain on high-fat diets (HFD). In line with enhanced metabolic rates, BAT and inguinal white adipose tissue (iWAT) interorgan crosstalk caused increased browning of iWAT in BAT-specific CoQ deficient animals. This mitohormesis-like effect depends on the ATF4-FGF21 axis and BAT-secreted FGF21, revealing an unexpected role for CoQ in the modulation of whole-body energy expenditure with wide-ranging implications for primary and secondary CoQ deficiencies.


The EMBO Journal
Ching-Fang Chang et al

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The EMBO Journal © The Author(s)  The EMBO Journal Ching-Fang Chang et al

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The EMBO Journal © The Author(s)

Figure EV1 .
Figure EV1.Enriched genes and pathways in CoQ deficient murine brown adipocytes.(A) Top 50 upregulated and top 50 downregulated mitochondrial genes from RNA sequencing results with p value ≤ 0.00418062.(B, C) Relative expression (log 10 -transformed RPKM value) of enriched genes in murine brown adipocytes treated with 4CBA (24 h).(D, E) Gene Ontology (GO) analysis of RNAseq data from 24-h 4CBA treated brown adipocytes.Representative GO categories repressed or induced.(F) Validation of tissue specific knockout of PDSS2 via qPCR analysis in BAT and liver of PDSS2 floxed (PDSS2 FL ) or BAT-specific PDSS2 knockout (PDSS2 BKO ) animals, n = 4. Results were compared using an unpaired two-tailed Student's t test.Significance presented at ***P < 0.001 compared to controls.Data describes biological replicates.Data information: (A, D, E) The Wald test was used for statistical analysis.

Figure EV2 .
Figure EV2.Genetically induced CoQ deficiency in murine brown adipocytes via knockdown of COQ2.

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A) Total CoQ levels, including CoQ 9 and CoQ 10 isoforms, of murine brown adipocytes transfected with either a scramble siRNA (CTL siRNA) or COQ2 siRNA, n = 6.(B) Gene expression of CTL siRNA and COQ2 siRNA-treated cells, n = 5. (C) UCP1 protein expression of CTL siRNA and COQ2 siRNA-treated cells, n = 12.(D) ATF4 protein expression of CTL siRNA and COQ2 siRNA treated cells, n = 3. (E) Oxygen consumption rate (OCR) of CTL siRNA and COQ2 siRNA treated cells during mitochondrial stress test, n = 5. (F) Basal, maximal, protein leak and ATP production respiration of CTL siRNA and COQ2 siRNA treated cells calculated from oxygen trace shown in (E), n = 5. (G) Fatty acid oxidation assay was performed on cells treated with CTL siRNA + palmitate (PA), CTL siRNA + PA + Etomoxir (Eto), COQ2 siRNA + PA and COQ2 siRNA + PA + Eto and OCR was measured, n = 5. (H) Basal and maximal respiration of the same cells whose oxygen trace is shown in (E), n = 5. Results were compared using a one-way ANOVA test.(I) Brown adipocytes with CTL siRNA or COQ2 siRNA treatment were stained with MitoTracker deep red, and dapi and visualized using the Zeiss LSM880, scale bar = 20 µm.Data information: Data are mean ± SEM. (A-D, F) Results were compared using an unpaired two-tailed Student's t test.Significance presented at *P < 0.05, **P < 0.01, and ***P < 0.001 compared to controls.Data describes biological replicates.

Figure EV3 .
Figure EV3.Targeting stress kinases to study ISR induction.(A)UCP1 and HRI gene expression in brown adipocytes transfected with scramble or HRI siRNA under vehicle control or 4CBA treatment for 24 h, n = 12.Pooled data for three repeated experiments.Results were compared using a one-way ANOVA test.(B) Western blot analysis of PKR and phosphorylated PKR (Thr451) levels, n = 6.(C) Phos-tag gel analysis of p-GCN2 and p-PERK compared to total GCN2 and total PERK, n = 3. (D) SDHB gene expression of cytosolic or mitochondrial cell fractions from vehicle or 4CBA-treated cells, n = 4-6.Data information: Data are mean ± SEM. (B-D) Results were compared using an unpaired two-tailed Student's t test.Significance presented at *P < 0.05, **P < 0.01, and ***P < 0.001 compared to controls.Data describes biological replicates.

Figure EV4 .
Figure EV4.Targeting ISR-related components.(A)ATF4, CHOP, and ATF5 expression in brown adipocytes with 4CBA treatment after indicated siRNA knockdown of target genes (x-axis), n = 6.Results were compared using an unpaired two-tailed Student's t test.(B) UCP1 expression in 4CBA-treated brown adipocyte with siRNA-mediated knockdown of CHOP or ATF5, n = 3. Results were compared using a one-way ANOVA test.Data information: Data are mean ± SEM.Significance presented at *P < 0.05, **P < 0.01, and ***P < 0.001 compared to controls.Data describes biological replicates.

Figure EV5 .
Figure EV5.Metabolic parameters of PDSS2 knockouts.(A) Oxygen consumption rate of PDSS2 FL or PDSS2 BKO animals was monitored under 23 and 30 °C in real-time in metabolic cages using CLAMS.Data normalized to body weight.(B, C) Averaged oxygen consumption rate.Centerline represents median and box extends from 25th to 75th percentiles.Data normalized to body weight.(D) Food consumption accumulated in three weeks.(E) Real-time feeding data from metabolic cage run at 23 and 30 °C.(F) Lean mass composition of PDSS2 FL and PDSS2 BKO animals.(G) Glucose tolerance test (GTT) of PDSS2 FL and PDSS2 BKO animals.2.0 g/kg body weight glucose dose administered via intraperitoneal (IP) injection.(H) Area under curve (AUC) analysis of GTT presented in (G).(I) Insulin tolerance test (ITT) of PDSS2 FL and PDSS2 BKO animals.0.4 IU/kg body weight insulin dose administered via IP injection.(J) Area under curve (AUC) analysis of ITT presented in (I).Data information.(A-C) n = 10-11/group.(D-F) n = 8-9/group.(G-J) n = 5-6/group.Data are mean ± SEM. Results were compared using an unpaired two-tailed Student's t test.Significance presented at *P < 0.05, and ***P < 0.001 compared to controls.Data describes biological replicates.