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Sex-specific genetic regulation of adipose mitochondria and metabolic syndrome by Ndufv2

Abstract

We have previously suggested a central role for mitochondria in the observed sex differences in metabolic traits. However, the mechanisms by which sex differences affect adipose mitochondrial function and metabolic syndrome are unclear. Here we show that in both mice and humans, adipose mitochondrial functions are elevated in females and are strongly associated with adiposity, insulin resistance and plasma lipids. Using a panel of diverse inbred strains of mice, we identify a genetic locus on mouse chromosome 17 that controls mitochondrial mass and function in adipose tissue in a sex- and tissue-specific manner. This locus contains Ndufv2 and regulates the expression of at least 89 mitochondrial genes in females, including oxidative phosphorylation genes and those related to mitochondrial DNA content. Overexpression studies indicate that Ndufv2 mediates these effects by regulating supercomplex assembly and elevating mitochondrial reactive oxygen species production, which generates a signal that increases mitochondrial biogenesis.

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Fig. 1: Sex and tissue-specific expression profiles of OXPHOS genes in both mice and humans.
Fig. 2: Adipose mitochondria levels strongly predict metabolic traits in both mice and humans.
Fig. 3: Sex-specific genetic architecture of adipose mitochondrial gene expression.
Fig. 4: Chr17 trans-eQTL controls metabolic syndrome traits in HMDP.
Fig. 5: Adipose Ndufv2 is the causal regulator of chr17 trans-eQTL locus.
Fig. 6: Adipose Ndufv2 overexpression regulated adiposity in a strain-by-sex manner.
Fig. 7: Adipose Ndufv2 overexpression regulated mitochondria in a strain-by-sex manner.
Fig. 8: Adipose Ndufv2 overexpression increased mitochondrial biogenesis and protein levels via ROS generation by altering supercomplex composition in a strain-by-sex manner.

Data availability

RNA sequencing raw data can be accessed at the Gene Expression Omnibus under accession GSE64770 (HMDP expression arrays (adipose and liver)) and GSE112947 (Gonadectomized RNA-seq data). GTEx datasets can be found online at the GTEx portal website (https://gtexportal.org/home/datasets). Uncropped scans of blots are available in the source data files. HMDP data from the authors laboratories will be made available on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank Z. Zhou, Y. Meng, S. Charugundla, D. W. Jayasekera, S. Nand and J. Ure for assistance in experiments. This work was supported by NIH grants NIH-P01HL028481, NIH-R01DK117850 and NIH-R01HL144651 (A.J.L.), NIH-R01HL125863 (J.L.M.B.), NIH-R01HL147187 (C.E.R.), NIH-1R01AA026914-01A1 (M. Liesa), NIH-R00DK120875 (K.C.K) and NIH-K99DK120875 (K.C.K); UCLA/UCSD/CTSI grants P30DK41301 (M. Liesa), UL1TR001881 (M. Liesa), P30DK063491 (M. Liesa); the American Heart Association grants A14SFRN20840000 (J.L.M.B.) and 18POST33990256 (K.C.K.); the Academy of Finland 321428 (M. Laakso); the Swedish Research Council 2018-02529 (J.L.M.B); Heart Lung Foundation 20170265 (J.L.M.B.); Astra-Zeneca through ICMC, Karolinska Institutet, Sweden (J.L.M.B.) and the Foundation Leducq 12CVD04 (L.V. and K.R.) and 18CVD02 (J.L.M.B.). The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.

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K.C.K. and A.J.L. conceived the study. K.C.K., L.V., R.A.P., L.S., M.S., L.M., E.M., C.P., T.M.M., M.P., C.E.R., K.R., J.L.M.B., M. Laakso, M.Liesa and A.J.L. performed experiments or analysed the data. K.C.K. and A.J.L. drafted the manuscript. All authors read or revised the manuscript.

Corresponding authors

Correspondence to Karthickeyan Chella Krishnan or Aldons J. Lusis.

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The authors declare no competing interests.

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Peer review information Primary handling editors: George Caputa and Isabella Samuelson. Nature Metabolism thanks Lawrence Kazak and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Chr17 locus is not associated with liver mitochondria.

Related to Fig. 3. High-resolution association mapping of 911 (1312 probes) mitochondrial gene abundance levels from (a) male and (b) female liver tissues isolated from ~100 HMDP strains to identify eQTL networks. The X- and Y- axis represent SNP and gene position on the mouse genome, respectively. Each dot represents a significant association. (c) Frequency distributions of P values of association between the chr17 trans-eQTL lead eSNP (rs48062344) and mitochondria-related transcripts in the male and female liver tissues. P values of association were calculated using (A – C) FaST-LMM that uses Likelihood-Ratio test.

Extended Data Fig. 2 Chr17 trans-eQTL do not affect the expression profiles of OXPHOS genes in male adipose or both sexes of liver tissues.

Related to Fig. 4. Volcano plots showing genetic (lead SNP of chr17 locus) differences in the expression profiles of OXPHOS genes in the (a) male adipose (n = 98 male strains; CC: 56 & TT: 42); (b) female and (c) male liver (n = 97 sex-matched strains; CC: 55 & TT: 42) isolated from HMDP. Genes corresponding to individual OXPHOS complexes are color-coded. Horizontal dotted lines represent 5% FDR-corrected significance threshold. Data are presented as log2 fold change between genotype. P values were calculated using (A – C) DESeq2 Bioconductor package that uses Wald test.

Extended Data Fig. 3 Association mapping of adipose mtDNA levels in HMDP.

Related to Fig. 4. Association mapping of mtDNA levels from (a) female and (b) male adipose in the HMDP cohort (n = 216 female and 260 male mice) Red line represents significance threshold (HMDP: P = 4.1E-06). P values of association were calculated using (A – B) FaST-LMM that uses Likelihood-Ratio test.

Extended Data Fig. 4 Liver Ndufv2 is unaffected by the chr17 trans-eQTL locus.

Related to Fig. 5. Association mapping of (a) male and (b) female liver Ndufv2 expression from HMDP. Red line represents significance threshold (P = 4.1E-06). (ce) Sex and genetic (lead SNP of chr17 locus) differences in the liver Ndufv2 expression from HMDP (n = 97 sex-matched strains; CC: 55 & TT: 42). Data are presented as median and interquartile range (boxplots). P values were calculated using (A – B) FaST-LMM that uses Likelihood-Ratio test; (C – E) Unpaired two-tailed Student’s t test.

Extended Data Fig. 5 Changes in body weight and insulin sensitivity mediated by adipose Ndufv2 overexpression.

Related to Fig. 6. Eight-week old females or males of (ab) C57BL/6 J or (cj) A/J mice were injected with AAV vectors expressing either GFP or NDUFV2 in an adipose-specific manner and fed a HF/HS diet for eight additional weeks. Changes in (A - D) fat mass and total body weight measured every two-weeks (females n = 7 per group; males n = 8 per group), (E and H) ITT, (F and I) end-point HOMA-IR and (G and J) fasting insulin levels (F – A/J n = 8 per group; M – A/J GFP n = 7, NDUFV2 n = 8) are shown. Data are presented as mean ± SEM. P values were calculated using (A – E and H) Repeated measures 2-factor ANOVA corrected by post-hoc ‘Holm-Sidak’s’ multiple comparisons test; (F, G, I and J) Unpaired two-tailed Student’s t test.

Extended Data Fig. 6 Adipose Ndufv2 overexpression regulated adiposity in a sex-by-strain manner.

Related to Fig. 6. Eight-week old males of (ae) C57BL/6 J or (fj) A/J mice were injected with AAV vectors expressing either GFP or NDUFV2 in an adipose-specific manner and fed a HF/HS diet for eight additional weeks. Metabolic traits such as (A and F) total mass and food intake; (B and G) fat and lean mass were monitored over eight weeks. Comparisons of (C and H) adipose Ndufv2 expression; (D and I) plasma free glycerol; (E and J) tissue weights from C57BL/6 J and A/J males, respectively. Data are presented as mean ± SEM (n = 8 per group). P values were calculated using (A and F) Repeated measures 2-factor or (B and G) 3-factor or (E and J) 2-factor ANOVA corrected by post-hoc ‘Holm-Sidak’s’ multiple comparisons test; (C, D, H and I) Unpaired two-tailed Student’s t test.

Extended Data Fig. 7 Adipose Ndufv2 overexpression regulated adipocyte size in a sex-by-strain manner.

Related to Fig. 7. Comparisons of adipocyte size distribution in the gonadal adipose tissues from females and males of (ab) C57BL/6 J or (cd) A/J mice overexpressing GFP or NDUFV2, respectively. Representative histological sections are shown for each group (scale: 100 µm). Red and blue shades (or bars) represent female and male NDUFV2 datapoints, while brown represents respective GFP datapoints, respectively. Data are presented as frequency distribution of adipocyte sizes or mean ± SEM (F – C57BL/6 J GFP n = 5064 cells from 7 mice, NDUFV2 n = 5249 cells from 7 mice; M – C57BL/6 J GFP n = 11721 cells from 8 mice, NDUFV2 n = 7290 cells from 7 mice; F – A/J GFP n = 5423 cells from 7 mice, NDUFV2 n = 4687 cells from 7 mice; M – A/J GFP n = 4084 cells from 8 mice, NDUFV2 n = 4863 cells from 8 mice). P values were calculated using Unpaired two-tailed Student’s t test.

Extended Data Fig. 8 Adipose Ndufv2 overexpression regulated mitochondrial respiration in a sex-by-strain manner.

Related to Fig. 7. Comparisons of mitochondrial RCR and coupling efficiency in the gonadal adipose tissues from males of (ab) C57BL/6 J or (cd) A/J mice overexpressing GFP or NDUFV2, respectively. Coupling assays of isolated gonadal adipose mitochondria from females and males of (ef) C57BL/6 J or (gh) A/J mice overexpressing GFP or NDUFV2, respectively. Data are presented as mean ± SEM (n = 4 per group). P values were calculated using 2-factor ANOVA.

Extended Data Fig. 9 Adipose Ndufv2-mediated mitochondrial regulation is not a consequence of body weight.

Related to Fig. 7. Eight-week old female C57BL/6 J mice were fed a HF/HS diet for the first six weeks without any intervention, after which were injected with AAV vectors expressing either GFP or NDUFV2 in an adipose-specific manner and diet continued for six additional weeks. Metabolic traits such as (a) total mass; (b) fat and lean mass were monitored over 12 weeks. Comparisons of (c) tissue weights; (d) adipose Ndufv2 expression; (e) mitochondrial RCR; (f) coupling efficiency and (g) coupling respiration rates between GFP and NDUFV2 animals, respectively. Data are presented as mean ± SEM (n = 6 per group). P values were calculated using (A) Repeated measures 2-factor or (B) 3-factor or (C and G) 2-factor ANOVA corrected by post-hoc ‘Holm-Sidak’s’ multiple comparisons test; (D – F) Unpaired two-tailed Student’s t test.

Extended Data Fig. 10 Ndufv2 overexpression regulated mitochondrial function in both AML12 (liver) and differentiated 3T3-L1 (adipose) cells.

Related to Fig. 8. Comparisons between GFP and NDUFV2 overexpressing AML12 (liver) cells in (a) relative normalized expression values of Ndufv2 (n = 9 per group); Coupling assays and RCR with all three complex I substrates (Pyruvate, Palmitoyl carnitine and Glutamate) either added (be) together (n = 5 per group) or (f - m) separately (n = 4 per group except Glutamate, n = 8 per group); and (N) relative normalized expression values of Ndufs4 (complex I), Sdhc (complex II), Atp5a1 (complex V) and Cpt1a (FAO) (n = 9 per group). Similarly, comparisons between control and NDUFV2 overexpressing differentiated 3T3-L1 cells in (O – R) coupling assays and RCR with different substrates (Pyruvate, Palmitoyl carnitine and Succinate) added separately (n = 6 per group). Data are presented as mean ± SEM. P values were calculated using (A and J – N) Unpaired two-tailed Student’s t test; (B – I and O – R) 2-factor ANOVA corrected by post-hoc ‘Holm-Sidak’s’ multiple comparisons test.

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Chella Krishnan, K., Vergnes, L., Acín-Pérez, R. et al. Sex-specific genetic regulation of adipose mitochondria and metabolic syndrome by Ndufv2. Nat Metab 3, 1552–1568 (2021). https://doi.org/10.1038/s42255-021-00481-w

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  • DOI: https://doi.org/10.1038/s42255-021-00481-w

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