A creatine-driven substrate cycle enhances energy expenditure and thermogenesis in beige fat

Abstract

Thermogenic brown and beige adipose tissues dissipate chemical energy as heat, and their thermogenic activities can combat obesity and diabetes. Herein the functional adaptations to cold of brown and beige adipose depots are examined using quantitative mitochondrial proteomics. We identify arginine/creatine metabolism as a beige adipose signature and demonstrate that creatine enhances respiration in beige-fat mitochondria when ADP is limiting. In murine beige fat, cold exposure stimulates mitochondrial creatine kinase activity and induces coordinated expression of genes associated with creatine metabolism. Pharmacological reduction of creatine levels decreases whole-body energy expenditure after administration of a β3-agonist and reduces beige and brown adipose metabolic rate. Genes of creatine metabolism are compensatorily induced when UCP1-dependent thermogenesis is ablated, and creatine reduction in Ucp1-deficient mice reduces core body temperature. These findings link a futile cycle of creatine metabolism to adipose tissue energy expenditure and thermal homeostasis. PAPERCLIP.

Figure 1. Characterization of Mitochondria from Brown and Beige Adipose Tissues

(A) Schematic of mitochondrial purification and quantitative proteomics workflow. (B) Heatmap of beige fat and brown fat mitochondrial proteomics data (from Table S1). Beige-enriched proteins are shown in the subset heatmap. (C) Principal component analysis of the mitochondrial proteomics dataset. (D) Kegg pathway analysis of beige fat-selective mitochondrial proteins from Figure 1B. (E) Heatmap of beige fat and brown fat mitochondrial proteomics data (from Table S2) after selecting proteins on the basis of an expression ratio greater than 1 in pure/crude mitochondria. (F) Kegg pathway analysis of significantly enriched beige fat mitochondrial proteins after cross-referencing Table S1 and S2. (G) Schematic of creatine synthesis and byproduct removal proteins identified by mass spectrometry. Red circles, proteins identified by mass spectrometry; grey circles, proteins not identified. Gly, glycine; Arg, arginine; Met, methionine; PCr, phosphocreatine; P5C, 1-Pyrroline-5-carboxylic acid; Mi-CK, mitochondrial creatine kinase. (H) Western blot after treatment of beige and brown fat mitochondria with trypsin (0, 10, 25, 50, and 100 µg ml⁻¹). (I) Creatine Kinase activity of mitochondria from 129SVE mice housed at 30°C or 4°C for 7 days. (J) Quantitative RT-PCR (qRT-PCR) from C57BL/6 mice housed at 30°C or 4°C for 6 hours (4°C-6h) or 7 days (4°C-7d), n = 3 to 4 mice per group. (K) PCr to creatine (Cr) ratio in iWAT and BAT from 129SVE mice housed at 30°C or 4°C for 7 days. Data are presented as mean ± SEM. *p < 0.05, ***p < 0.01.

Figure 2. Creatine Stimulates Respiration in Beige Fat Mitochondria When ADP is Limiting

(A) Model of creatine-based substrate cycling. IMS, intermembrane space; AAC, ADP/ATP Carrier. (B) Western blot of mitochondrial proteins. N = 2 mitochondrial preparations, 15 mice per cohort. (C) Oxygen consumption by beige fat mitochondria treated with and without creatine (0.01 mM) in the presence of 0.2 mM ADP. Vertical dashed line, State 3 to State 4 transition. N = 9 mitochondrial preparations, 15 mice per cohort. (D) State 3 and ADP-limiting Oxygen consumption rate (OCR) of beige fat mitochondria treated with and without creatine in the presence of 0.2 mM ADP. N = 9 mitochondrial preparations, 15 mice per cohort. (E) State 3 and ADP-limiting OCR of brown fat mitochondria treated as in D. N = 3 mitochondrial preparations, 15 mice per cohort. (F) State 3 and ADP-limiting OCR of heart mitochondria treated as in D. N = 3 mitochondrial preparations, 8 mice per cohort. (G) State 3 and ADP-limiting OCR of kidney mitochondria treated as in D. N = 3 mitochondrial preparations, 15 mice per cohort. (H) State 3 and ADP-limiting OCR of liver mitochondria treated as in D. N = 3 mitochondrial preparations, 2 mice per cohort. (I) Western blot of mitochondrial proteins from beige fat, brown fat, and heart. N = 2 mitochondrial preparations, 15 mice per cohort. (J) Mitochondrial creatine concentration in beige fat, brown fat, and heart. Data are presented as mean ± SEM. *p < 0.05.

Figure 3. Creatine Metabolism in Adipose Tissue Contributes to Energy Expenditure and Thermal Homeostasis in vivo

(A) Creatine concentration in iWAT (beige), BAT, and Gastrocnemius muscle (Gstrc) from cold-exposed C57BL/6 mice treated with four daily injections of vehicle or β-GPA (0.4 g kg⁻¹), n = 5 mice per group. (B) Western blot of iWAT (beige) and BAT from animals treated as in A, n = 3 mice per group. (C) Movement, n= 8 mice per group. CL (0.2 mg kg⁻¹) was co-injected intraperitonealy with vehicle (saline) or β-GPA (0.4 g kg⁻¹). (D) Food intake, n = 8 mice per group, treated as in C. (E) Oxygen consumption, n = 8 mice per group, treated as in C. (F) OCR of minced tissues following four daily injections of vehicle, β-GPA (0.4 g kg⁻¹), CL (0.2 mg kg⁻¹), or β-GPA + CL, n = 5 mice per group. (G) OCR of minced tissues from Prdm16^lox/lox and Adipo-PRDM16 KO mice after 7 days cold exposure. β-GPA was administered on the last four days of cold exposure, n = 5 to 7 mice per group. (H) qRT-PCR of Ckmt1 and Ckmt2 in two human brown adipocyte lines (#11-1 and 11-3) and one human white adipocyte line (#11). Raw ct values are embedded in the bars. (I) OCR of human brown adipocytes treated with vehicle, β-GPA (2 mM), and creatine (5 mM). (J) OCR of human brown adipocytes transfected with si-Ctrl or si-Ckmt1. Creatine used at 5 mM. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.0001.

Figure 4. Creatine Metabolism Regulates UCP1-Independent Thermal Homeostasis in vivo

(A) qRT-PCR from iWAT of 4°C-acclimated Ucp1^+/+ and Ucp1^−/− mice, n = 5 mice per group. (B) qRT-PCR of creatine metabolism genes from same samples as A. (C) qRT-PCR from iWAT of 4°C-acclimated mice treate d with four daily injections of vehicle or β-GPA (0.4 g kg⁻¹), n = 5 mice per group. (D) Western blot from iWAT and BAT from mice treated as in C. Vinculin (VCL), loading control, n = 3 mice per group. (E) qRT-PCR of clonal human brown adipocytes, (line #11-1). Small interfering RNAs (siRNAs) targeted against control (si-Ctrl) or Ckmt1 (si-Ckmt1). Forskolin was used at 10 µM for 4 hours. (The data showing si-Ctrl and si- Ckmt1, without forskolin treatment, is the same data shown in Figure S3D), n = 3 per group. (F) Body temperature of Ucp1^+/+ and Ucp1^−/− mice treated with vehicle or β-GPA (0.4 g kg⁻¹), n = 7 to 8 mice per group. (G) Representative electromyogram (EMG) traces, measured at 4°C, of 4°Cacclimated Ucp1^−/− mice treated as in F. (H) Frequency of shivering bursts quantified from data in G. (I) Representative EMG traces, at 30°C and followin g 15 – 45 minutes at 4°C, of 30°C-acclimated wild type C57BL/6 mice treated a s in F. (J) Frequency of shivering bursts quantified from data in I. (K) OCR of minced tissues from 4°C-acclimated Ucp1^−/− mice treated as in F. N = 16 to 17 mice per group (iWAT and BAT), n = 5 to 6 mice per group (PgWAT and Gstrc). Data are presented as mean ± SEM. *p < 0.05, ***p < 0.0001.

Figure 5. A Screen of Mitochondrial Phosphatases with Ucp1-deficient Mice Identifies PHOSPHO1 as a Regulator of Adipocyte Respiration

(A) qRT-PCR of candidate phosphatases in iWAT of 4°C-acclimated Ucp1^+/+ and Ucp1^−/− mice, n = 5 mice per group. (B) Western blot of PHOSPHO1 and PHOSPHO2 from 4°C- acclimated Ucp1^+/+ and Ucp1^−/− mice, treated with vehicle or β-GPA (0.4 g kg⁻¹). Vinculin (VCL), loading control. (C) qRT-PCR of candidate phosphatases in iWAT of 4°C-ac climated wild type mice treated as in B, n = 5 mice per group. (D) qRT-PCR of clonal human brown adipocytes, (line #11-1) treated with si-Ctrl or si-Phospho1, n = 3 per group. (E) qRT-PCR of primary mouse inguinal adipocytes after Phospho1 knockdown (shPhospho1-#1 and shPhospho1-#2), n = 4 to 5 per group. (F) OCR of primary mouse inguinal adipocytes treated as in E, n = 5 to 7 per group. (G) Coomassie stain of SDS-PAGE demonstrating PHOSPHO1 protein expression and purification. M, molecular weight marker; NI, non-induced; I, induced; FT, flow-through; W, wash; E1 – E3, elutions 1 – 3. Arrows indicate recombinant PHOSPHO1. (H) Specific activity of PHOSPHO1 toward PCho (0.5 mM) and PCr (0.5 to 10 mM) exposed to various buffer pH and divalent metals. Data are presented as mean ± SEM. *p < 0.05, ***p < 0.0001.

Figure 6. Models of Creatine-Driven Futile Substrate Cycling

(A) Model of creatine-driven futile substrate cycling based on direct hydrolysis of PCr. (B) Model of creatine-driven futile substrate cycling based on multiple phosphotransfer events catalyzed by multiple enzymes (Enz₁ – Enz_n).