. 2023 Oct 10;14(1):6344.

doi: 10.1038/s41467-023-41988-y.

FAM210A is essential for cold-induced mitochondrial remodeling in brown adipocytes

Jiamin Qiu^#¹, Feng Yue^#^{2

3}, Peipei Zhu⁴, Jingjuan Chen¹, Fan Xu⁵, Lijia Zhang¹, Kun Ho Kim¹, Madigan M Snyder^{1

6}, Nanjian Luo¹, Hao-Wei Xu¹, Fang Huang⁵, W Andy Tao^{4

7}, Shihuan Kuang^{8

9}

Affiliations

¹ Department of Animal Sciences, Purdue University, West Lafayette, IN, 47907, USA.
² Department of Animal Sciences, Purdue University, West Lafayette, IN, 47907, USA. fengyue@ufl.edu.
³ Department of Animal Sciences, University of Florida, Gainesville, FL, 32611, USA. fengyue@ufl.edu.
⁴ Department of Biochemistry, Purdue University, West Lafayette, IN, 47907, USA.
⁵ Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, 47907, USA.
⁶ Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907, USA.
⁷ Purdue University Institute for Cancer Research, West Lafayette, IN, 47907, USA.
⁸ Department of Animal Sciences, Purdue University, West Lafayette, IN, 47907, USA. skuang@purdue.edu.
⁹ Purdue University Institute for Cancer Research, West Lafayette, IN, 47907, USA. skuang@purdue.edu.

^# Contributed equally.

PMID: 37816711
PMCID: PMC10564795
DOI: 10.1038/s41467-023-41988-y

FAM210A is essential for cold-induced mitochondrial remodeling in brown adipocytes

Jiamin Qiu et al. Nat Commun. 2023.

. 2023 Oct 10;14(1):6344.

doi: 10.1038/s41467-023-41988-y.

Authors

Affiliations

¹ Department of Animal Sciences, Purdue University, West Lafayette, IN, 47907, USA.
² Department of Animal Sciences, Purdue University, West Lafayette, IN, 47907, USA. fengyue@ufl.edu.
³ Department of Animal Sciences, University of Florida, Gainesville, FL, 32611, USA. fengyue@ufl.edu.
⁴ Department of Biochemistry, Purdue University, West Lafayette, IN, 47907, USA.
⁵ Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, 47907, USA.
⁶ Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907, USA.
⁷ Purdue University Institute for Cancer Research, West Lafayette, IN, 47907, USA.
⁸ Department of Animal Sciences, Purdue University, West Lafayette, IN, 47907, USA. skuang@purdue.edu.
⁹ Purdue University Institute for Cancer Research, West Lafayette, IN, 47907, USA. skuang@purdue.edu.

^# Contributed equally.

PMID: 37816711
PMCID: PMC10564795
DOI: 10.1038/s41467-023-41988-y

Abstract

Cold stimulation dynamically remodels mitochondria in brown adipose tissue (BAT) to facilitate non-shivering thermogenesis in mammals, but what regulates mitochondrial plasticity is poorly understood. Comparing mitochondrial proteomes in response to cold revealed FAM210A as a cold-inducible mitochondrial inner membrane protein. An adipocyte-specific constitutive knockout of Fam210a (Fam210a^AKO) disrupts mitochondrial cristae structure and diminishes the thermogenic activity of BAT, rendering the Fam210a^AKO mice vulnerable to lethal hypothermia under acute cold exposure. Induced knockout of Fam210a in adult adipocytes (Fam210a^iAKO) does not affect steady-state mitochondrial structure under thermoneutrality, but impairs cold-induced mitochondrial remodeling, leading to progressive loss of cristae and reduction of mitochondrial density. Proteomics reveals an association between FAM210A and OPA1, whose cleavage governs cristae dynamics and mitochondrial remodeling. Mechanistically, FAM210A interacts with mitochondrial protease YME1L and modulates its activity toward OMA1 and OPA1 cleavage. These data establish FAM210A as a key regulator of mitochondrial cristae remodeling in BAT and shed light on the mechanism underlying mitochondrial plasticity in response to cold.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. BAT mitochondrial cristae remodeling and associated changes in proteomes during cold-induced thermogenesis.**
a Representative transmission electron microscopy (TEM) images showing mitochondrial remodeling in the wild-type brown adipose tissue (BAT) upon cold exposure for different times (n = 3 mice per group; scale bars, 2 µm; diagram created with BioRender.com). b Quantification of mitochondrial morphological changes in (a) (n = 35 images for 30 °C; n = 47 images for 6 h on 6 °C; n = 47 images for 6 h on 6 °C; n = 65 images for for 24 h on 6 °C; n = 50 images for 3 d and 7 d on 6 °C; 3 mice were used at each time point; number of mitochondria counted in 30 °C, and 6 h, 24 h, 3 d, 7 d on 6 °C: 539, 909, 2133, 1047, 1041; mean ± s.e.m). c Experimental workflow for the study of BAT mitochondrial proteomics of mice upon cold exposure for different times (LC-MS, liquid chromatography-mass spectrometry; diagram created with BioRender.com). d Heatmap of differentially expressed mitochondrial protein expression level in BAT upon cold exposure for different times (n = 3 mice per group; one-way ANOVA; differential expression was determined using a cutoff significance level of P < 0.05). e Gene Ontology (GO) cellular component analysis of differentially expressed proteins (Fisher’s Exact test; P values were adjusted by Benjamini–Hochberg method). f GO biological process analysis of differentially expressed proteins from the subset of mitochondrial inner membrane in (e) (Fisher’s Exact test; P values were adjusted by Benjamini–Hochberg method). g Heatmap of differentially expressed proteins of cristae formation, mitochondrion organization, and mitochondrial fusion. h Immunoblotting and quantification analysis of OPA1 (n = 3 mice per group; mean ± s.e.m). Source data are provided as a Source Data file.

**Fig. 2. Identification of FAM210A as a cold-inducible mitochondrial inner membrane protein in BAT.**
a The list of differentially expressed mitochondrial proteins with domain of unknown function (DUF) (One-way ANOVA). b Quantification of FAM210A expression in mitochondrial proteomic data (n = 3 mice per group; mean ± s.e.m; two-tailed unpaired Student’s t test; P = 0.0497, 0.006, 0.0044, and 0.0011). c Immunoblotting analysis of FAM210A in BAT from mice upon cold exposure at different time points (n = 3 mice per group). d Schematic showing the predicated domain structure and RoseTTAfold prediction of FAM210A. MTS, mitochondrial targeting sequence; TM, transmembrane region; DUF1279, domain of unknown function 1279; CC, coiled-coil domain. e Immunoblotting analysis of FAM210A in brown adipocytes (BAs) during differentiation (n = 3 independent experiments). f Immunofluorescence showing the colocalization of FAM210A with mitochondria in BAs before and after differentiation (n = 3 independent experiments; scale bars, 10 µm). g Single-molecule localization image of overexpressed *Fam210a* in Cos-7 cells (n = 3 independent experiments; scale bars, 2 µm). h Protease protection assay with mitochondrial fractions purified from BAT mitochondria (PK, proteinase K; n = 3 independent experiments). *P < 0.05, **P < 0.01. Source data are provided as a Source Data file.

**Fig. 3. Loss of *Fam210a* in adipocytes causes whitening of BAT and cold intolerance in mice.**
a Schematic diagram of the strategy used to generate adipocyte-specific *Fam210a* knockout (*Fam210a*^AKO) mice (ex, exon). b Representative image showing the morphology of adipose tissues from control and *Fam210a*^AKO mice at room temperature (RT, 22 °C) (iWAT, inguinal white adipose tissue; gWAT, gonadal white adipose tissue; n = 4 mice per group). c Representative H&E staining of BAT and iWAT from control and *Fam210a*^AKO mice at RT (n = 4 mice per group; scale bar, 50 µm). d BODIPY staining of mature BAs isolated from control and *Fam210a*^AKO mice at RT (n = 4 mice per group; scale bars: 10 µm). e Rectal core body temperature of control and *Fam210a*^AKO mice following acute cold exposure with fasting (control, n = 10 mice; *Fam210a*^AKO, n = 9 mice; mean ± s.e.m; two-tailed unpaired Student’s t test; P = 0.039056, 0.00000089, and 0.000000036). f Survival curves of control and *Fam210a*^AKO mice following acute cold exposure with fasting (euthermia is higher than 34 °C) (Control, n = 10 mice; *Fam210a*^AKO, n = 9 mice). g Infrared thermography of iBAT temperature in control and *Fam210a*^AKO mice upon cold exposure for 7 d (n = 6 mice per group). h Quantification of BAT temperature in (g) (mean ± s.e.m; two-tailed unpaired Student’s t test; P = 0.0367). i iBAT temperature of control and *Fam210a*^AKO mice upon cold exposure for 7 d by using temperature microprobe (n = 3 mice per group; mean ± s.e.m; two-tailed unpaired Student’s t test; P = 0.0154). j Oxygen consumption at light and dark. The left panel in (j) shows the oxygen consumption for 2 d; the right panel in (j) highlights the oxygen consumption after CL316,243 injection (n = 5 mice per group; mean ± s.e.m). k Regression-based analysis of energy expenditure in (j) against body mass. Data were analyzed using CaIR-ANCOVA with energy expenditure as a dependent variable and body mass as a covariate. The P values (ANCOVA) are shown at the bottom (n = 5 mice per group; mean ± s.e.m). l Serum lipid profiles in control and *Fam210a*^AKO mice after cold exposure for 7 d (NEFA non-esterified fatty acid, HDL high-density lipoprotein, LDL low-density lipoprotein; n = 4 mice per group; mean ± s.e.m; two-tailed unpaired Student’s t test; P = 0.04608, 0.017339, 0.030646, and 0.028823). *P < 0.05, **P < 0.01, ****P < 0.0001. Source data are provided as a Source Data file.

**Fig. 4. Loss of *Fam210a* impairs cold-induced browning of adipocytes and mitochondrial function.**
a, b Representative images showing the morphology (a) and tissue mass (b) of adipose tissues from control and *Fam210a*^AKO mice after cold exposure for 7 d (n = 12 mice for control BAT and iWAT; n = 9 mice for control gWAT; n = 11 mice for *Fam210a*^AKO BAT and iWAT; n = 8 mice for *Fam210a*^AKO gWAT; mean ± s.e.m; two-tailed unpaired Student’s t test; P = 0.000388, and <0.000001). c Representative H&E staining of BAT and iWAT from control and *Fam210a*^AKO mice after cold exposure for 7 d (n = 4 mice per group; scale bar, 50 µm). d Representative immunohistochemical staining of UCP1 in BAT and iWAT from control and *Fam210a*^AKO mice after cold exposure for 7 d (n = 3 mice per group; Scale bar: 50 µm). e mRNA level of thermogenic genes in BAT and iWAT of control and *Fam210a*^AKO mice after cold exposure for 7 d (n = 5 mice per group; mean ± s.e.m; two-tailed unpaired Student’s t test; P = 0.000207, 0.0145, 0.000309, 0.000328, 0.00029, 0.049247, 0.001473, and 0.000464). f, g Immunoblotting analysis of UCP1 in BAT (f) and iWAT (g) from control (Ctrl) and *Fam210a*^AKO (AKO) mice (n = 3 mice per group). h, i Immunoblotting analysis of BAT (h) and iWAT (i) mitochondrial proteins in control and *Fam210a*^AKO mice after cold exposure (n = 4 mice per group). j Fatty acid oxidation (FAO) activity measured using ¹⁴C-labeled palmitic acid in BAT from control and *Fam210a*^AKO mice with cold exposure for 7 d (n = 3 mice per group; mean ± s.e.m; two-tailed unpaired Student’s t test; P = 0.0000198). k The oxygen consumption rate (OCR) of differentiated primary BAs from control and *Fam210a*^AKO with CL316,243 treatment for 6 h before the test (Oligo Oligomycin, AA Antimycin A, Rot Rotenone; n = 3 mice per group; mean ± s.e.m). l Quantification of OCR from (k) (mean ± s.e.m; two-tailed unpaired Student’s t test; P = 0.011873 and 0.019458). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.

**Fig. 5. *Fam210a* is required for cristae and mitochondrial remodeling in BAT.**
a Representative images and quantification of isolated mitochondria pellets from control (Ctrl) and *Fam210a*^AKO (AKO) BAT (n = 6 mice per group; mean ± s.e.m; two-tailed unpaired Student’s t test; P = 0.0000003729). b Mitochondria copy number in BAT, iWAT, and gWAT from control and *Fam210a*^AKO mice at room temperature (RT) (n = 4 mice for control BAT; n = 5 mice for control iWAT and gWAT; n = 4 mice for *Fam210a*^AKO; mean ± s.e.m; two-tailed unpaired Student’s t test; P = 0.010882). c Mitochondria copy number in BAT, iWAT, and gWAT from control and *Fam210a*^AKO mice upon cold exposure (CT) for 7 d (n = 5 mice per group for BAT and iWAT; n = 4 mice per group for gWAT; mean ± s.e.m; two-tailed Student’s t test; P = 0.000395 and 0.049103). d Representative TEM images of BAT and iWAT from control and *Fam210a*^AKO mice at RT and cold exposure for 7 d (n = 3 mice per group; for 6000X, scale bars, 2 µm; for 16000X, scale bars, 1 µm). e Quantification of cristae number per mitochondrion shown in (d) (n = the number of mitochondria counted in the order they appear on the graph from left to right: 156, 104, 431, 487, 459, 76; 3 mice were used for each group; mean ± s.e.m; two-tailed unpaired Student’s t test; ****P < 0.00000001). f Schematic diagram of the strategy used to generate inducible adipocyte-specific *Fam210a* knockout mice (*Fam210a*^iAKO). g Schematic showing timing of tamoxifen (TMX) induction, cold exposure, and sampling for control and *Fam210a*^iAKO mice (diagram created with BioRender.com). h Representative TEM images of control and *Fam210a*^iAKO BAT upon cold exposure for different times (n = 3 mice per group; scale bars, 1 µm). i Quantification of Class 3 mitochondria in (h) (n = 37, 42, 34, and 24 images for the control group at 0 h, 6 h, 3 d, and 7 d after cold, respectively; n = 41, 38, 49, and 34 images for *Fam210a*^iAKO group at 0 h, 6 h, 3 d, and 7 d after cold, respectively; the number of mitochondria counted in the order they appear on the graph from left to right: 510, 620, 781, 559, 685, 861, 526, 521; 3 mice were used for each group; mean ± s.e.m; two-tailed unpaired Student’s t test; ****P < 0.00000001). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.

**Fig. 6. FAM210A regulates OPA1 cleavage through modulating YME1L and OMA1 protease activity.**
a Experimental workflow for the study of BAT mitochondrial proteomics of control and *Fam210a*^iAKO mice (diagram created with BioRender.com). b Protein abundance differences in BAT mitochondrial proteome between control and *Fam210a*^iAKO mice upon cold exposure for 3 d (FC, fold change; n = 3 mice per group; two-tailed unpaired Student’s t test; differential expression was determined using a cutoff significance level of P < 0.05). c Expression levels of proteins related to “mitochondrial morphology, dynamics & organization proteins” in BAT mitochondrial proteome of control and *Fam210a*^iAKO mice on cold for 3 d (n = 3 mice per group; mean ± s.e.m; two-tailed unpaired Student’s t test; P = 0.049728, 0.001238, and 0.015885). d Immunoblotting analysis of mitochondrial structure-related proteins in BAT of control (Ctrl) and *Fam210a*^AKO (AKO) mice upon cold exposure for 7 d (n = 4 mice per group). e Quantification of total OPA1/tubulin in (d) (mean ± s.e.m; two-tailed unpaired Student’s t test; P = 0.001). f Immunoblotting analysis of mitochondrial structure-related proteins in control (Ctrl) and *Fam210a*^iAKO (iAKO) mice upon cold exposure for different times (n = 5 mice per group for 0 and 24 h; n = 3 mice per group for 3 h; n = 6 mice per group for 6 h, 3 d, and 7 d). g Quantification of L-OPA1/S-OPA1 ratio in (f) (mean ± s.e.m; two-tailed paired Student’s t test; P = 0.000306, 0.013745, 0.000536, and 0.002041). h Immunoblotting analysis of BAT OPA1 cleavage in Ctrl and iAKO mice with BAT-local delivery of *Fam210a-c-Myc* adenovirus (FamAd) followed by cold exposure for 24 h (n = 3 mice per group). i Quantification of OPA1 cleavage in (h) (mean ± s.e.m; two-tailed paired Student’s t test; P = 0.01615). j Immunoblotting analysis of BAT OPA1 cleavage in Ctrl and iAKO mice with BAT-local delivery of o-phenanthroline (o-phe) followed by cold exposure for 24 h (n = 3 mice per group). k Quantification of OPA1 cleavage in (j) (mean ± s.e.m; two-tailed paired Student’s t test; P = 0.01612). l Representative TEM images of BAT in mice with treatments in (j) (n = 3 mice per group; scale bars, 1 µm). m Diagram depicting that FAM210A is an essential regulator of YME1L and/or OMA1, and protects L-OPA1 from excessive cleavage during cold exposure (diagram created with BioRender.com). *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

**Fig. 7. FAM210A interacts with YME1L and modulates its protease activity toward OMA1 and OPA1 cleavage.**
a YME1L, OPA1ΔC, and FAM210A were synthesized by *E. coli* cell-free protein synthesis system, and YME1L and OPA1ΔC were incubated with and without the presence of FAM210A (n = 3 independent experiments). b Quantification of OPA1ΔC protein level in (a) (mean ± s.e.m; two-tailed unpaired Student’s t test; P = 0.000762, and 0.003106). c OMA1, OPA1ΔC, and FAM210A were synthesized by *E. coli* cell-free protein synthesis system, and OMA1 and OPA1ΔC were incubated with and without the presence of FAM210A (n = 3 independent experiments). d Quantification of OPA1ΔC protein level in (c) (mean ± s.e.m; two-tailed unpaired Student’s t test). e Co-immunoprecipitation (Co-IP) of FAM210A-FLAG and YME1L in *Fam210a-flag* overexpressed HEK293 cell line (n = 3 independent experiments). f Proximity ligation assay (PLA) showing the protein interaction of FAM210A-FLAG and YME1L in *Fam210a-flag* overexpressed HEK293 cell line (n = 3 independent experiments; scale bar: 10 µm). g YME1L, OMA1, and FAM210A were synthesized by *E. coli* cell-free protein synthesis system, and YME1L and OMA1 were incubated with or without the presence of FAM210A (n = 3 independent experiments). h Quantification of OMA1 protein level in (g) (mean ± s.e.m; two-tailed unpaired Student’s t test; P = 0.02497). i Immunoblotting analysis of OPA1 in BAT of control and *Fam210a*^iAKO after cold exposure for 0 h and 6 h by using the large format electrophoresis chamber (n = 3 mice per group). j Quantification of OPA1-b/e in (i) (mean ± s.e.m; two-tailed paired Student’s t test; P = 0.02418). k Diagram depicting that FAM210A enhances YME1L activity thus suppressing OMA1, which protects L-OPA1 from over-cleavage during cold exposure to facilitate mitochondrial cristae remodeling (diagram created with BioRender.com). *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

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FAM210A is essential for cold-induced mitochondrial remodeling in brown adipocytes

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FAM210A is essential for cold-induced mitochondrial remodeling in brown adipocytes

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