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. 2021 Feb;590(7846):480-485.
doi: 10.1038/s41586-021-03221-y. Epub 2021 Feb 17.

Creatine kinase B controls futile creatine cycling in thermogenic fat

Janane F Rahbani  1   2 , Anna Roesler  1   2 , Mohammed F Hussain  1   2 , Bozena Samborska  1   2 , Christien B Dykstra  1   2 , Linus Tsai  3 , Mark P Jedrychowski  4 , Laurent Vergnes  5 , Karen Reue  5 , Bruce M Spiegelman  4 , Lawrence Kazak  6   7
Affiliations

Creatine kinase B controls futile creatine cycling in thermogenic fat

Janane F Rahbani et al. Nature. 2021 Feb.

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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Figures

Extended Data Fig. 1:
Extended Data Fig. 1:. CKB is the primary creatine kinase isoenzyme in brown adipocytes.
a, percent labeling (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+ adipocytes (n = 3). e, Ribosomal profiling from BAT (n = 5) and beige fat (n = 4). f, RT-qPCR (n = 3). g, Genotyping and sequencing. h, LC-MS after 1 hour labeling 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 hours. k, RT-qPCR from mice treated as i (n = 3). l, Western blot as i. m, RT-qPCR as 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 n. For gel source data, see Supplementary Figure 1. Mice used were wild-type (C57BL6/N) 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 treatments. Data are presented as mean ± s.e.m. of biologically independent samples. b, one-way ANOVA (Fisher’s LSD); d-f, two-way ANOVA (Fisher’s LSD); h, k, m, two-tailed Student’s t-tests.
Extended Data Fig. 2:
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 signaling (d). e, Glycerol release assay (n = 3 per group). f, Western blot examining the effect of Ckb silencing on lipolytic signaling. g, NE-dependent OCR, obtained by subtracting basal from NE-induced respiration (from Fig. 3b). Left panel: shLacZ, n = 17; shCkb#1, n = 16. Right panel: shLacZ, n = 8; shCkb#2, n = 15. h, DNP-dependent OCR (n = 33). i, OCR following 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, NE-dependent OCR, obtained by subtracting basal from NE-induced respiration from j (n = 20 per group). l, OCR of Ucp1−/− primary brown adipocytes (shLacZ, n = 17 shCkb#1, n = 7 or shCkb#2, n = 10). m, Western blot (m, left) and OCR (m, 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 Figure 1. Data are presented as mean ± s.e.m. of biologically independent samples. a, e, two-way ANOVA (Dunnett’s multiple comparisons test); g, i-l, m(right), multiple two-tailed Student’s t-tests.
Extended Data Fig. 3:
Extended Data Fig. 3:. Ckb silencing selectively impairs brown adipocyte respiration.
a, Western blot of primary brown preadipocytes following Ckb silencing. b, OCR of primary brown preadipocytes following Ckb silencing. Left panel: shLacZ, n = 10; shCkb#1, n = 10. Right panel: 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 following silencing of creatine kinase isoforms (n = 3 per group). For gel source data, see Supplementary Figure 1. Data are presented as mean ± s.e.m. of biologically independent samples. b, c, multiple two-tailed Student’s t-tests; d, two-way ANOVA (Fisher’s LSD).
Extended Data Fig. 4:
Extended Data Fig. 4:. CKB is targeted to mitochondria by an internal mitochondrial targeting signal-like.
a, Confocal images of U2OS cells transfected with Ckb.Flag cDNA. Mitochondria were labeled with anti-TOM20 antibody (green); CKB.FLAG was labeled with anti-FLAG antibody (magenta). Scale bar (white line), 5 μm. b, Western blot of mitochondrial extracts with and without protease (10 μg trypsin) treatment. c, Western blot of mitochondrial extracts with and without protease treatment. d, Western blot of mitochondrial extracts following protease treatment and extracts from whole cells, following 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. CkbFLAG mice and wild type (CkbWT mice) exposed to 30°C or 6°C were used to confirm Flag antibody cross-reactivity with CKB.FLAG. f, Quantification of mitochondrial CKB from Western blots in e. g, Western blot of protease-protected BAT mitochondria from wild-type (C57BL6/N, 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), CKBWT (n = 4), CKBΔiMTS-L (n = 3), or CKBC283S (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 Figure 1. Data are presented as mean ± s.e.m. of biologically independent samples. k, one-way ANOVA (Fisher’s LSD). Cartoons were created with biorender.com.
Extended Data Fig. 5:
Extended Data Fig. 5:. Mitochondrial CKB triggers futile creatine cycling selectively in thermogenic adipocytes.
a-d, 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) (e, left), shCkb#1 (vehicle: n = 4; creatine: n = 5) (e, middle), or shCkb#2 (vehicle: n = 5; creatine: n = 5) (e, right). f, Western blot CKB abundance in Ckbfl/fl brown adipocytes compared to Ckb−/− brown adipocytes titrated with various amounts of FLAG-tagged CKBWT. 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 CKBWT to endogenous levels (n = 3) or levels 5-fold above endogenous (n = 2). Endogenous re-expression and over-expression 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 Figure 1. Data are presented as mean ± s.e.m. of biologically independent samples. a-e, multiple two-tailed Student’s t-tests; g, two-tailed Student’s t-test.
Extended Data Fig. 6:
Extended Data Fig. 6:. Selective reduction of creatine kinase protein and activity in adipose tissues of CkbUcp1-CreERT2 and CkbAduoiQ-Cre mice.
a, Cartoon 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. c-f, Western blot of BAT (c), subcutaneous adipose tissue (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. h-j, 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, Cartoon 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. m-p, Western blot from BAT (m), SAT (n), PgAT (o), and brain (p) from Ckbfl/fl and CkbAdipoQ-Cre male mice, housed at 22°C. q-t, 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 Figure 1. Data are presented as mean ± s.e.m. of biologically independent samples. b, g-j, l, q-t, two-tailed Student’s t-tests. Cartoons were created with biorender.com.
Extended Data Fig. 7:
Extended Data Fig. 7:. Oxygen consumption, food intake and nutrient absorption.
a, CL-dependent (a, top) and saline-dependent (a, bottom) oxygen consumption of male mice at 30°C (n = 8 per group). b, CL-dependent (b, top) and saline-dependent (b, 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 HFD. k-l, ANCOVA of cumulative food intake derived from data in f, for the first (k) and last (l) week of HFD. 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 HFD. o-p, Fecal energy density (o) and output (p) (n = 4 per group). q-r, Energy output (q) and metabolic efficiency (r) of data from f-h. s, fasting blood glucose levels following a fast from ZT0-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. a-c, e, g, h, two-way ANOVA (Fisher’s LSD); i-n, two-sided analysis of co-variance (ANCOVA); o-s, one-way ANOVA (Fisher’s LSD).
Fig. 1:
Fig. 1:. Creatine kinase activity in BAT is dependent on CKB.
a, Schematic of creatine methyl-D3 (D3-creatine) supplementation of brown adipocytes to test phosphocreatine methyl-d3 (D3-phosphocreatine) synthesis. CRT, creatine transporter. b, Percent-labeling (m+3) of intracellular D3-creatine and D3-phosphocreatine, using LC-MS (n = 3 per time point). c, Cartoon of limiting dilution. d, Western blot of immortalized brown adipocyte clones (parental cell genotype: Ckbfl/fl) following infection with Gfp (P) or with Cre recombinase. Limiting dilution allowed generation of clones (3 and 9) that were devoid of CKB expression (Ckb−/−). e, LC-MS analysis of labeled (m+3) PCr (e, left) and creatine (e, right) levels after labeling for 24 hours (n = 3 per group). f, Cartoon of cold exposure. g, Western blot of BAT from wild-type (C57BL6/N, 6-8 weeks old) male mice, reared at 22°C, housed at 30°C for 7 days and compared to single-housing at 6°C. Primary brown adipocytes (cells) infected with shRNA targeting LacZ (shLZ) or Ckb (shCkb) was used to demonstrate specificity of CKB antibody. h, Cartoon of creatine kinase activity from BAT lysates (h, left). Creatine kinase activity (PCr synthesis) from BAT extracts of wild-type (C57BL6/N, 6-8 weeks old) female mice, reared at 22°C, housed at 30°C for 5 days and then subjected to single-housing at 30°C or 6°C for 48 hours (n = 3 per group) (h, right). For gel source data, see Supplementary Figure 1. Data are presented as mean ± s.e.m. of biologically independent samples. e, one-way ANOVA (Fisher’s LSD); h, two-tailed Student’s t-tests. Cartoons were created with biorender.com.
Fig. 2:
Fig. 2:. CKB expression is regulated by cAMP signaling.
a, Western blot from male mice injected i.p. with CL 316,243 (1 mg/kg) or vehicle once daily for 48 hours at 30°C. b, RT-qPCR from human perirenal BAT (pheo: n = 11; controls: n = 12). c, Creatine kinase isoform expression from publicly available RNA-seq of human cultured thermogenic adipocytes. Statistical comparisons are between undifferentiated and vehicle-treated adipocytes, and between vehicle- and forskolin-treated adipocytes (n = 5 per group). Box plots were generated in Graphpad Prism 8. Boxes stretch from the 25th to the 75th percentile; black horizontal line: median. For gel source data, see Supplementary Figure 1. Mice used were wild-type (C57BL6/N) male (6-8 weeks old). All mice were reared at 22°C and housed at 30°C for 5 days before treatments. Data are presented as mean ± s.e.m. of biologically independent samples. b, c, two-tailed Student’s t-tests.
Fig. 3:
Fig. 3:. CKB is targeted to mitochondria and regulates futile creatine cycling.
a, Western blot of primary brown adipocytes. b, Oxygen consumption rate (OCR). Left panel: shLacZ, n = 17; shCkb#1, n = 16. Right panel: shLacZ, n = 8; shCkb#2, n = 15. NE (Norepinephrine); DNP (2,4-dinitrophenol); RA (Rotenone and antimycin A). c, Quantitative proteomics of primary brown adipocyte mitochondria (n = 4). d, Cartoon of protease protection assay and Western blot of mitochondrial extracts with and without protease treatment. e, Cartoon of Flag insertion into the Ckb locus (CkbFLAG mice) and cold exposure experiment. UTR (untranslated region); CDS (coding sequence). f, Western blot from BAT of male CkbFLAG or wild-type mice (6-8 weeks of age), reared at 22°C, housed at 30°C for 5 days, then single-housed at 30°C or 6°C for 48 hours. g, Western blot of protease-protected BAT mitochondria from CkbFLAG mice (FL) or non-protease-treated wild-type (WT) mice housed at 22°C. h, TargetP probability plotted along the CKB amino acid sequence. i, Western blot of protease-treated mitochondria. j, Creatine kinase activity from protease-treated mitochondrial extracts. Ckbfl/fl (n =4), Ckb−/− brown adipocytes infected with FLAG-tagged GFP (n = 3), CKBWT (n = 4), CKBΔiMTS-L (n = 4), or CKBC283S (n = 3). k, Futile creatine cycle model. OXPHOS (oxidative phosphorylation); Pi (inorganic phosphate); PCr-ase (protein catalyzing PCr hydrolysis). l, Representative oxygen consumption trace. ADP (70 nmoles) addition is indicated by the arrow. Change in % O2 is relative to ADP (set to 100%). m, Super-stoichiometric action of creatine on ADP-dependent respiration (CKB-positive: n = 5; CKB-negative: n = 6). For gel source data, see Supplementary Figure 1. Data are presented as mean ± s.e.m. of biologically independent samples. b, multiple two-tailed Student’s t-tests; c, j, one-way ANOVA (Fisher’s LSD); m, two-tailed Student’s t-test. Cartoons were created with biorender.com.
Fig. 4:
Fig. 4:. Fat-selective deletion of Ckb decreases energy expenditure and predisposes mice to obesity.
a, CL-dependent (a, top) and saline-dependent (a, bottom) energy expenditure (EE) of male mice at 30°C (n = 8 per group). b, CL-dependent (b, top) and saline-dependent (b, bottom) EE of Ckbfl/fl (n = 7) and CkbAdipoQ-Cre (n = 9) male mice at 30°C. c, Body weight during high-fat feeding of female mice at 22°C (CkbAdipoQ-Cre(ad-lib): n = 12; Ckbfl/fl (ad-lib): n = 12; Ckbfl/fl (pair-fed): n = 10). d, Representative histological images of BAT and SAT from female mice following 16 weeks high fat feeding. Scale bar (black line), 50 μm for BAT and 125 μm for SAT. e, glucose tolerance test (e, left) and area under the curve (AUC) of glucose tolerance test (e, right) following 12 weeks of high-fat feeding of female mice at 22°C (CkbAdipoQ-Cre(ad-lib): n = 11; Ckbfl/fl (ad-lib): n = 9; Ckbfl/fl (pair-fed): n = 10). f, insulin tolerance test following 16 weeks of high-fat feeding of female mice at 22°C (CkbAdipoQ-Cre(ad-lib): n = 12; Ckbfl/fl (ad-lib): n = 11; Ckbfl/fl (pair-fed): n = 10). P values indicate comparison to CkbAdipoQ-Cre(ad-lib) mice. Data are presented as mean ± s.e.m. of biologically independent samples. a-c, e(left), f, two-way ANOVA (Fisher’s LSD); e(right), one-way ANOVA (Fisher’s LSD).
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References

    1. Ward ZJ et al. Projected U.S. State-Level Prevalence of Adult Obesity and Severe Obesity. N Engl J Med 381, 2440–2450, doi:10.1056/NEJMsa1909301 (2019). - DOI - PubMed
    1. Bartelt A et al. Brown adipose tissue activity controls triglyceride clearance. Nat Med 17, 200–205, doi:10.1038/nm.2297 (2011). - DOI - PubMed
    1. Carpentier AC et al. Brown Adipose Tissue Energy Metabolism in Humans. Front Endocrinol (Lausanne) 9, 447, doi:10.3389/fendo.2018.00447 (2018). - DOI - PMC - PubMed
    1. Kazak L et al. A creatine-driven substrate cycle enhances energy expenditure and thermogenesis in beige fat. Cell 163, 643–655, doi:10.1016/j.cell.2015.09.035 (2015). - DOI - PMC - PubMed
    1. Challa TD et al. A Genetic Model to Study the Contribution of Brown and Brite Adipocytes to Metabolism. Cell Rep 30, 3424–3433 e3424, doi:10.1016/j.celrep.2020.02.055 (2020). - DOI - PubMed

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