Journal: Cell Metabolism

Article Title: Cardiolipin Synthesis in Brown and Beige Fat Mitochondria Is Essential for Systemic Energy Homeostasis

doi: 10.1016/j.cmet.2018.05.003

Figure Lengend Snippet: Cardiolipins Are Robustly Increased in Activated Brown and Beige Fat (A) Ten most cold-induced lipids from lipidomic analysis of iBAT from mice housed at thermoneutrality or exposed to 5°C cold for 3 hr, 3 days, or 3 weeks (fold change relative to thermoneutral, 29°C = 1; n = 4 per group, one-way ANOVA). (B) Top ten cold-induced lipids from lipidomic analysis of scWAT from mice housed at thermoneutrality or exposed to 5°C cold for 3 weeks (fold change of absolute quantities normalized to protein content, 29°C = 1; n = 4 per group, t test). CL, cardiolipin; PG, phosphatidylglycerol; CAR, acyl carnitine. (C) Schematic of cardiolipin synthesis and remodeling; CDP-DAG, cytidine diphosphate diacylglycerol; CMP, cytidine monophosphate. (D) Heatmaps of log 2 (fold change cold treated/thermoneutrality) for CL species in iBAT and scWAT (n = 4 per group, one-way ANOVAs for iBAT and t tests for scWAT; gray, not detected). (E) Relative iBAT protein levels of cardiolipin de novo synthesis and remodeling pathway enzymes from proteomic analysis presented in Figure 1 A (n = 4 for 29°C and 5°C 3 weeks, n = 3 for other groups; one-way ANOVA). (F) Tissue distribution of Crls1 from mice housed at thermoneutrality or cold exposed for 3 weeks (n = 6 per group; t test). (G and H) (G) CRLS1 levels in human subcutaneous fat (n = 8) and brown fat with high UCP1 mRNA expression (n = 11, UCP1 CT values between 19 and 27; t test) and (H) correlation with UCP1 in scWAT (n = 8; white), low- UCP1 BAT (n = 8, UCP1 CT values between 27 and 37; yellow), and high- UCP1 BAT (n = 11; orange; Pearson R 2 and p value shown). Data are presented as means ±SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also Figure S2 .

Article Snippet: Custom rabbit polyclonal anti-CRLS1 antibodies were ordered from Genscript (PolyExpress Gold Package; SC1649).

Techniques: Expressing

Journal: Cell Metabolism

Article Title: Cardiolipin Synthesis in Brown and Beige Fat Mitochondria Is Essential for Systemic Energy Homeostasis

doi: 10.1016/j.cmet.2018.05.003

Figure Lengend Snippet: CRLS1 Enhances Inducible Uncoupling in Thermogenic Fat Cells (A–D) Gene expression and NE-induced respiration from primary brown (A) and subcutaneous (B) adipocytes electroporated with either empty pcDNA vector or vector expressing Crls1 (one-way ANOVA). Oxygen consumption profiles from control and Crls1 siRNA-treated (C) primary brown and (D) subcutaneous adipocytes following addition of oligomycin and NE stimulation (repeated measures two-way ANOVA). Quantified levels of basal, ATP synthesis-coupled, NE-induced uncoupled, and maximal respiration are provided to the right (t tests). (E and F) (E) Crls1 mRNA levels and cardiolipin (CL) labeling for 6 hr and (F) oxygen consumption profiles of primary brown adipocytes from control Crls1 f/f and Rosa26ERT2- Cre / Crls1 f/f mice treated with tamoxifen (TAM). Data are presented as means ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also Figure S3 .

Article Snippet: Custom rabbit polyclonal anti-CRLS1 antibodies were ordered from Genscript (PolyExpress Gold Package; SC1649).

Techniques: Gene Expression, Plasmid Preparation, Expressing, Control, Labeling

Journal: Cell Metabolism

Article Title: Cardiolipin Synthesis in Brown and Beige Fat Mitochondria Is Essential for Systemic Energy Homeostasis

doi: 10.1016/j.cmet.2018.05.003

Figure Lengend Snippet: CRLS1 Is Critically Required for the Adipose Thermogenic Program (A) Crls1 mRNA levels in tissues from control and AdCKO mice (n = 6–7; t tests). (B) Liquid chromatography-mass spectrometry (LC-MS) based quantification of cardiolipin species from control and AdCKO iBAT (n = 6 per group, t tests) and photos of control and AdCKO iBAT (B; inset). (C) Representative transmission electron microscopy (TEM) images of control and AdCKO mitochondrial structure. (D and E) Three-dimensional reconstructions of a (D) control and (E) AdCKO adipocyte generated from dual-beam focused ion beam scanning electron microscopy stacks. (F) Rectal temperature of control and AdCKO mice subjected to a 22°C–4°C cold challenge for 1.5 hr (n = 8 per group; two-way ANOVA). (G–K) (G) Oxygen consumption, (H) schematic of BAT thermogenesis assessment, and (I) interscapular temperature from control and AdCKO mice following anesthetization and 1 mg/kg NE subcutaneous administration (n = 6 per group, two-way ANOVA). Ucp1 mRNA in (J) iBAT and (K) scWAT from control and AdCKO mice before and after 3 weeks of cold adaptation. (L and M) (L) Quantified mean glucose uptake and (M) representative images of 18 F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) from control and AdCKO mice following intraperitoneal administration of CL-316,243. Data are presented as means ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also and and , , , and .

Article Snippet: Custom rabbit polyclonal anti-CRLS1 antibodies were ordered from Genscript (PolyExpress Gold Package; SC1649).

Techniques: Control, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transmission Assay, Electron Microscopy, Generated, Positron Emission Tomography, Computed Tomography, Positron Emission Tomography-Computed Tomography

Journal: Cell Metabolism

Article Title: Cardiolipin Synthesis in Brown and Beige Fat Mitochondria Is Essential for Systemic Energy Homeostasis

doi: 10.1016/j.cmet.2018.05.003

Figure Lengend Snippet: Mitochondrial CL Deficiency Suppresses Nuclear-Encoded Genes through the ER Stress Response Factor CHOP-10 (A) Reactome pathways downregulated in AdCKO iBAT compared with control (RNA sequencing [RNA-seq] data; n = 5 per group; housed at 22°C; dashed line indicates adjusted p value <0.05). (B) Volcano plot of RNA-seq data from AdCKO and control iBAT (n = 5 per group). (C) mRNA levels of known CHOP-10 targets from RNA-seq data (n = 5 per group). (D) mRNA levels from brown adipocytes treated with control, Crls1 , Chop10 , or both Crls1 and Chop10 siRNA. Data are presented as means ± SEM. ∗ p < 0.05; ∗∗∗ p < 0.001.

Article Snippet: Custom rabbit polyclonal anti-CRLS1 antibodies were ordered from Genscript (PolyExpress Gold Package; SC1649).

Techniques: Control, RNA Sequencing

Journal: Cell Metabolism

Article Title: Cardiolipin Synthesis in Brown and Beige Fat Mitochondria Is Essential for Systemic Energy Homeostasis

doi: 10.1016/j.cmet.2018.05.003

Figure Lengend Snippet: Adipose CRLS1 Is Linked to Whole-Body Insulin Sensitivity in Humans and Augments Human Fat Cell Energy Expenditure (A) Genetic link between a rare CRLS1 variant and metabolic disease from Danish population-based cohorts. (B–G) CRLS1 (B) and CS (C) mRNA levels in the scWAT of normal glucose tolerant (NGT), impaired glucose tolerant (IGT), and type 2 diabetic mellitus (T2DM) donors (n = 70, 49, and 52 per group, respectively, one-way ANOVA). Pearson correlations between scWAT CRLS1 (D) and CS (E) mRNA levels and homeostatic model assessment two insulin resistance (HOMA2-IR, calculated by insulin and c-peptide), HOMA2 insulin sensitivity (%S, calculated by insulin and c-peptide) and adiposity parameters (F and G). Pearson R 2 values and significance are shown. W/H, weight/height. (H) Schematic for CRISPRa synergistic activation mediator (SAM) targeting of CRLS1 in human white adipocytes. (I) Gene expression and forskolin (Fsk)-induced respiration (t test) from human white adipocytes transduced with empty vector or vector delivering a single-guide RNA directed to the −101 position upstream of the CRLS1 transcriptional start site. Data are presented as means ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also Figure S6 and .

Article Snippet: Custom rabbit polyclonal anti-CRLS1 antibodies were ordered from Genscript (PolyExpress Gold Package; SC1649).

Techniques: Variant Assay, Activation Assay, Gene Expression, Transduction, Plasmid Preparation

Journal: Cell Metabolism

Article Title: Cardiolipin Synthesis in Brown and Beige Fat Mitochondria Is Essential for Systemic Energy Homeostasis

doi: 10.1016/j.cmet.2018.05.003

Figure Lengend Snippet:

Article Snippet: Custom rabbit polyclonal anti-CRLS1 antibodies were ordered from Genscript (PolyExpress Gold Package; SC1649).

Techniques: Recombinant, Protease Inhibitor, In Vivo, Western Blot, Immunodetection, In Vitro, Transfection, Bicinchoninic Acid Protein Assay, Reverse Transcription, Control, Mouse Assay, Quantitative RT-PCR, Software, Variant Assay