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Creatine kinase B controls futile creatine cycling in thermogenic fat

Abstract

Obesity increases the risk of mortality because of metabolic sequelae such as type 2 diabetes and cardiovascular disease1. Thermogenesis by adipocytes can counteract obesity and metabolic diseases2,3. In thermogenic fat, creatine liberates a molar excess of mitochondrial ADP—purportedly via a phosphorylation cycle4—to drive thermogenic respiration. However, the proteins that control this futile creatine cycle are unknown. Here we show that creatine kinase B (CKB) is indispensable for thermogenesis resulting from the futile creatine cycle, during which it traffics to mitochondria using an internal mitochondrial targeting sequence. CKB is powerfully induced by thermogenic stimuli in both mouse and human adipocytes. Adipocyte-selective inactivation of Ckb in mice diminishes thermogenic capacity, increases predisposition to obesity, and disrupts glucose homeostasis. CKB is therefore a key effector of the futile creatine cycle.

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Fig. 1: Creatine kinase activity in BAT is dependent on CKB.
Fig. 2: CKB expression is regulated by cAMP signalling.
Fig. 3: CKB is targeted to mitochondria and regulates futile creatine cycling.
Fig. 4: Fat-selective deletion of Ckb decreases energy expenditure and predisposes mice to obesity.

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All data are available in the main Article or the Supplementary Information, and from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank P. Seale for discussions; the McGill Goodman Cancer Research Centre (GCRC) Metabolomics core facility for technical assistance; the Histology Core Facility at the McGill/GCRC for assistance with embedding and processing tissue samples; and all members of the Kazak laboratory for critical reading of the manuscript. This work was supported by a Canadian Institutes of Health Research grant (PJT-159529) and the Canadian Foundation for Innovation John R. Evans Leaders Fund (37919) (to L.K.); by Canderel and Charlotte and Leo Karassik fellowships (to J.F.R.); and by a Canderel studentship (to M.F.H.). The GCRC metabolomics core facility is funded by the Dr John R. and Clara M. Fraser Memorial Trust; the Terry Fox Foundation; the Quebec Breast Cancer Foundation and McGill University. L.K. is a Canada Research Chair in Adipocyte Biology.

Author information

Authors and Affiliations

Authors

Contributions

L.K. conceived and designed the study; J.F.R., A.R., M.F.H., B.S. and L.K. performed all the experimental work except for that described below. J.F.R. developed respirometry experiments using an oxygen electrode. M.F.H. developed creatine kinase activity assays from purified proteins and cell and tissue extracts. M.F.H. conducted immunofluorescence imaging. L.V. and K.R. collected and analysed human perirenal BAT samples by RT–PCR; L.T. analysed mined transcriptome data. C.B.D. conducted oxygen bomb calorimetry experiments. M.P.J. conducted and analysed proteomics data. L.K. wrote the manuscript, with editing from J.F.R., A.R., M.F.H., B.S. and B.M.S. L.K. supervised the project and acquired funding. All authors approved the manuscript.

Corresponding author

Correspondence to Lawrence Kazak.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature thanks Alan Saltiel, Matthew R. Skelton and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 CKB is the primary creatine kinase isoenzyme in brown adipocytes.

a, Per cent labelling (M + 3) of deuterated creatine (d3-creatine) and phosphocreatine (d3-phosphocreatine) in brown adipocytes and media (n = 3). b, Quantitative proteomics (n = 10). c, Quantitative proteomics (n = 3). d, Ribosomal profiling of AdipoQ+ and Ucp1+ adipocytes14 (n = 3). e, Ribosomal profiling from BAT (n = 5) and beige fat (n = 4)15. f, RT–qPCR (n = 3). g, Genotyping and sequencing. h, LC–MS after 1 h labelling with deuterated (M + 3) creatine (n = 3). i, j, Western blot from male (i) or female (j) mice, single-housed at 30 °C or 6 °C for 48 h. k, RT–qPCR from mice treated as in i (n = 3). l, Western blot as in i. m, RT–qPCR as in k. 30 °C (n = 7) or 6 °C (n = 5). n, Western blot of BAT. o, Western blot of protease-protected mitochondria from mice treated as in n. For gel source data, see Supplementary Fig. 1. Mice used were wild-type (C57BL/6N) male (6–8 weeks old) or female (20 weeks old) mice. All mice were reared at 22 °C and housed at 30 °C for 5 days before treatment. Data are mean ± s.e.m. of biologically independent samples. b, One-way ANOVA (Fisher’s LSD); df, two-way ANOVA (Fisher’s LSD); h, k, m, two-tailed Student’s t-tests.

Source data

Extended Data Fig. 2 Ckb silencing impairs brown adipocyte respiration.

a, RT–qPCR of primary brown adipocytes (n = 3 per group). b, Oil red O staining of primary brown adipocytes. Preads, preadipocytes. c, d, Western blot examining the effect of Ckb silencing on mitochondrial abundance and adipocyte differentiation (c) and insulin signalling (d). e, Glycerol release assay (n = 3 per group). f, Western blot examining the effect of Ckb silencing on lipolytic signalling. g, Noradrenaline-dependent OCR, obtained by subtracting basal from noradrenaline-induced respiration (from Fig. 3b). Left, shlacZ, n = 17; shCkb#1, n = 16. Right, shlacZ, n = 8; shCkb#2, n = 15. h, DNP-dependent OCR (n = 33). i, OCR after Ckb silencing (shlacZ, n = 31; shCkb#1, n = 27, shCkb#2, n = 27). Oli, oligomycin; RA, rotenone and antimycin A. j, OCR of Ucp1−/− primary brown adipocytes (n = 20 per group). k, Noradrenaline-dependent OCR, obtained by subtracting basal from noradrenaline-induced respiration from j (n = 20 per group). l, OCR of Ucp1−/− primary brown adipocytes (shlacZ, n = 17; shCkb#1, n = 7; shCkb#2, n = 10). m, Western blot (left) and OCR (right) of immortalized brown adipocytes. Gfp (P), n = 21; clone 5, n = 20; clone 3, n = 19; clone 9, n = 23. For gel source data, see Supplementary Fig. 1. Data are mean ± s.e.m. of biologically independent samples. a, e, Two-way ANOVA (Dunnett’s multiple comparisons test); g, il, m (right), multiple two-tailed Student’s t-tests.

Source data

Extended Data Fig. 3 Ckb silencing selectively impairs brown adipocyte respiration.

a, Western blot of primary brown preadipocytes after Ckb silencing. b, OCR of primary brown preadipocytes after Ckb silencing. Left, shlacZ, n = 10; shCkb#1, n = 10. Right, shlacZ, n = 10; shCkb#2, n = 10. c, OCR of primary brown adipocytes with silencing of Ckmt2 (shCkmt2#1, n = 8; shCkmt2#2, n = 11), Ckm (shCkm#1, n = 11; shCkm#2, n = 11) or Ckmt1 (shCkmt1#1, n = 23; shCkmt1#2, n = 12), compared to shlacZ (n = 23). d, RT–qPCR after silencing of creatine kinase isoforms (n = 3 per group). For gel source data, see Supplementary Fig. 1. Data are mean ± s.e.m. of biologically independent samples. b, c, Multiple two-tailed Student’s t-tests; d, two-way ANOVA (Fisher’s LSD).

Source data

Extended Data Fig. 4 CKB is targeted to mitochondria by an internal mitochondrial targeting signal-like sequence.

a, Confocal images of U-2 OS cells transfected with Ckb–Flag cDNA. Mitochondria were labelled with anti-TOM20 antibody (green); CKB–Flag was labelled with anti-Flag antibody (magenta). Scale bar, 5 μm. b, c, Western blots of mitochondrial extracts from primary brown adipocytes (b) and immortalized brown adipocytes (c) with and without protease (10 μg trypsin) treatment. d, Western blot of mitochondrial extracts after protease treatment (top) and extracts from whole cells (bottom), after Ckb silencing. e, Western blot of whole tissue lysates (WTL) and protease-protected mitochondrial extracts from BAT, heart and kidney of CkbFlag mice housed at 22 °C. Wild-type (CkbWT) mice and CkbFlag mice exposed to 30 °C or 6 °C were used to confirm the cross-reactivity of the Flag antibody with CKB–Flag. f, Quantification of mitochondrial CKB–Flag from western blots in e. g, Western blot of protease-protected BAT mitochondria from wild-type (C57BL/6N, 6–8 weeks old) male mice, reared at 22 °C, housed at 30 °C for 5 days and then subjected to single-housing at 30 °C or 6 °C. h, Western blot of whole-cell extracts. i, Western blot of cytosol extracts. j, Western blot of mitochondrial extracts with and without protease treatment. Blue arrows, CKB–Flag protein; ns, non-specific bands. k, l, Creatine kinase activity from Ckbfl/fl (n = 3), Ckb−/− brown adipocytes expressing Flag-tagged GFP (n = 3), wild-type CKB (n = 4), CKB(ΔiMTS-L) (n = 3), or CKB(C283S) (n = 3) (k) or bacteria-purified CKB–Flag variants (n = 3) (l). m, Western blots of control or Tom70-silenced mitochondrial extracts with and without protease treatment. For gel source data, see Supplementary Fig. 1. Data are mean ± s.e.m. of biologically independent samples. k, One-way ANOVA (Fisher’s LSD). Schematics were created at https://biorender.com.

Source data

Extended Data Fig. 5 Mitochondrial CKB triggers futile creatine cycling selectively in thermogenic adipocytes.

ad, Effect of creatine on the rate of respiration in mitochondria from wild-type brown adipocytes (n = 10 per group) (a), Ucp1−/− brown adipocytes (n = 14 per group) (b), 3T3-F442A white adipocytes (vehicle, n = 4; creatine, n = 5) (c), or C2C12 myoblasts (vehicle, n = 4; creatine, n = 5) (d). e, Effect of creatine on the rate of respiration in mitochondria from brown adipocytes infected with shlacZ (vehicle, n = 4; creatine, n = 5) (left), shCkb#1 (vehicle, n = 4; creatine, n = 5) (middle) or shCkb#2 (vehicle, n = 5; creatine, n = 5) (right). f, Western blot of CKB abundance in Ckbfl/fl brown adipocytes compared to Ckb−/− brown adipocytes titrated with various amounts of Flag-tagged wild-type CKB. g, Super-stoichiometric action of creatine on ADP-dependent respiration in Ckb−/− brown adipocytes without rescue (same data as Fig. 4m) (n = 4) compared to re-expression of Flag-tagged wild-type CKB to endogenous levels (n = 3) or levels fivefold above endogenous (n = 2). Endogenous re-expression and overexpression data were not different, and were thus pooled together (n = 5). Futile creatine cycling data represent the moles of liberated ADP (measured as the creatine-dependent change in ADP-dependent O2 consumption) over moles of added creatine. For gel source data, see Supplementary Fig. 1. Data are presented as mean ± s.e.m. of biologically independent samples. ae, Multiple two-tailed Student’s t-tests; g, two-tailed Student’s t-test.

Source data

Extended Data Fig. 6 Selective reduction of creatine kinase protein and activity in adipose tissues of CkbUcp1-CreERT2 and CkbAdipoQ-Cre mice.

a, Schematic of breeding strategy to generate inducible Ucp1-adipocyte-selective Ckb knockout mice (CkbUcp1-CreERT2). b, RT–qPCR of Ckb mRNA levels in various tissues of Ckbfl/fl (n = 3) and CkbUcp1-CreERT2 (n = 4) female mice housed at 22 °C. cf, Western blot of BAT (c), SAT (d), perigonadal adipose tissue (PgAT) (e) and brain (f) from Ckbfl/fl and CkbUcp1-CreERT2 male mice, housed at 22 °C (n = 3 per group). g, Creatine kinase activity in BAT lysates from Ckbfl/fl (n = 3) and CkbUcp1-CreERT2 (n = 3) male mice, housed at 22 °C. hj, Creatine kinase activity in SAT (h), PgAT (i) and brain (j) lysates from Ckbfl/fl (n = 4) and CkbUcp1-CreERT2 (n = 3) male mice, housed at 22 °C. k, Schematic of breeding strategy to generate pan-adipocyte-selective Ckb knockout mice (CkbAdipoQ-Cre). l, RT–qPCR of Ckb mRNA levels in various tissues of Ckbfl/fl (n = 5) and CkbAdipoQ-Cre (n = 3) male mice housed at 22 °C. mp, Western blot from BAT (m), SAT (n), PgAT (o) and brain (p) from Ckbfl/fl and CkbAdipoQ-Cre male mice, housed at 22 °C. qt, Creatine kinase activity in BAT (q), SAT (r), PgAT (s) and brain (t) lysates from Ckbfl/fl (n = 5) and CkbAdipoQ-Cre (n = 3) male mice, housed at 22 °C. For gel source data, see Supplementary Fig. 1. Data are mean ± s.e.m. of biologically independent samples. b, gj, l, qt, Two-tailed Student’s t-tests. Schematics were created at https://biorender.com.

Source data

Extended Data Fig. 7 Oxygen consumption, food intake and nutrient absorption.

a, CL-dependent (top) and saline-dependent (bottom) oxygen consumption of male mice at 30 °C (n = 8 per group). b, CL-dependent (top) and saline-dependent (bottom) oxygen consumption of Ckbfl/fl (n = 7) and CkbAdipoQ-Cre (n = 9) male mice at 30 °C. c, d, Body mass change (c) and cumulative food intake (d) of female mice (n = 9 per group). e, f, Body mass change (e) and cumulative food intake (f) of female mice. CkbAdipoQ-Cre (ad-lib): n = 12; Ckbfl/fl (ad-lib): n = 12 and Ckbfl/fl (pair-fed): n = 10. g, h, Lean (g) and fat (h) mass change of mice from e. i, j, ANCOVA of cumulative food intake derived from data in d, for the first (i) and last (j) week of the high-fat diet. k, l, ANCOVA of cumulative food intake derived from data in f, for the first (k) and last (l) week of the high-fat diet. m, n, ANCOVA of combined data from i and k for the first (m), and from j and l for the last (n) week of the high-fat diet. o, p, Faecal energy density (o) and output (p) (n = 4 per group). q, r, Energy output (q) and metabolic efficiency (r) of data from fh. s, Fasting blood glucose levels after a fast from zeitgeber time 0 (ZT0) to zeitgeber time 6 (ZT6). CkbAdipoQ-Cre (ad-lib), n = 11; Ckbfl/fl (ad-lib), n = 9; Ckbfl/fl (pair-fed), n = 10. Data are presented as mean ± s.e.m. of biologically independent samples. P values on graphs containing 3 experimental groups are relative to CkbAdipoQ-Cre (ad-lib) mice. ac, e, g, h, Two-way ANOVA (Fisher’s LSD); in, two-sided analysis of co-variance (ANCOVA); os, one-way ANOVA (Fisher’s LSD).

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This file contains the uncropped blots. Red boxes indicate regions of blots used to generate main figures (Fig. 1c, e, 2a, 3a, d, f, g, i) and Extended Data Figures (Extended Data Fig. 1g, i, j, l, n, o, 2c, d, f, m, 3a, 4b-e, g-j, m, 5f, 6c-f, m-p).

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Rahbani, J.F., Roesler, A., Hussain, M.F. et al. Creatine kinase B controls futile creatine cycling in thermogenic fat. Nature 590, 480–485 (2021). https://doi.org/10.1038/s41586-021-03221-y

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