anti hsl  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Copper Import via CTR1 Supports the β3-Adrenergic Thermogenic Program

doi: 10.64898/2026.03.24.713962

Figure Lengend Snippet: (a) Volcano plot of differentially expressed proteins in BAT from Floxed and ACKO mice after 6 h CE (n = 3 per group). Downregulated proteins were subjected to KEGG pathway enrichment analysis (bottom). (b) Immunoblot analysis of OXPHOS complex subunits (ATP5A, UQCRC2, MTCO1, SDHB, NDUFB8), UCP1, and ACTIN in BAT from Floxed and ACKO mice housed at RT or exposed to cold (CE). (c) Oxygen consumption rate (OCR) traces of immortalized pre-brown adipocytes derived from Floxed and ACKO mice treated with vehicle or CL (10 μM). (d) Quantification of respiratory parameters derived from (c), including basal respiration, ATP-linked respiration, proton leak, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration. (e) Relative mRNA expression of Ctr1 and thermogenic genes ( Ucp1 , Prdm16 , Dio2 , Cidea ) in BAT from Floxed and ACKO mice under RT or CE. (f) Representative H&E staining of BAT from Floxed and ACKO mice under RT or CE. Scale bar, 50 μm. (g) TG content in BAT from Floxed and ACKO mice under RT or CE. (h, i) Immunoblot analysis of phosphorylated HSL (Ser660) and total HSL in BAT (h) and iWAT (i) from Floxed and ACKO mice treated with saline or CL (1 mg kg⁻¹, 15 min). (j) Immunoblot analysis of CTR1 and UCP1 in iWAT from Floxed and ACKO mice treated with saline or CL. (k) Relative mRNA expression of Ppargc1a , Ucp1, and Ctr1 in iWAT from Floxed and ACKO mice treated with saline or CL. (l) Cu content in iWAT from Floxed and ACKO mice treated with saline or CL. (m) Representative H&E staining of iWAT from Floxed and ACKO mice treated with saline or CL (1 mg kg⁻¹ day⁻¹, once daily for 10 consecutive days). Scale bar, 50 μm. (n) Relative mtDNA content in iWAT following 10 days of CL treatment. Data are presented as mean ± SEM. Statistical significance for panels (d, e, k, l, n) was determined by one-way ANOVA with Tukey’s post hoc test for each parameter analyzed independently. Groups not sharing a common letter are significantly different (P < 0.05).

Article Snippet: Primary antibodies included CTR1 , UCP1 (Abcam, ab23841), ATP7A (from Dr. S. Kaler’s laboratory ), CCS (Santa Cruz Biotechnology, sc-55561), OXPHOS cocktail (Abcam, ab110413), HSL (Abcam, ab45422), phospho-HSL (Ser660; Cell Signaling, #45804), CoxIV (Thermo Fisher Scientific, A21348), β-actin (Thermo Fisher Scientific, MA515739), and GAPDH (Thermo Fisher Scientific, MA515738).

Techniques: Western Blot, Derivative Assay, Expressing, Staining, Saline

Journal: bioRxiv

Article Title: Copper Import via CTR1 Supports the β3-Adrenergic Thermogenic Program

doi: 10.64898/2026.03.24.713962

Figure Lengend Snippet: (a) Rectal body temperature of Ctr1 -floxed and ACKO mice treated with vehicle (Veh) or elesclomol (ES) during acute cold exposure (4°C). ES treatment improved cold tolerance (two-way ANOVA, treatment effect *P < 0.05; n = 3–4 per group). (b) Representative H&E staining of BAT from Ctr1-floxed and ACKO mice treated with Veh or ES under CE. Scale bar, 50 μm. (c) Immunoblot analysis of UCP1, OXPHOS complex subunits (ATP5A, UQCRC2, MTCO1, SDHB, NDUFB8), CCS, and GAPDH in BAT from Ctr1 -floxed and ACKO mice treated with Veh or ES. (d, e) Mitochondrial respiration analysis in immortalized pre-brown adipocytes derived from Ctr1 -floxed and ACKO mice treated with ES (10 nM) for 24 h. (d) Oxygen consumption rate (OCR) traces. (e) Quantification of basal respiration, ATP-linked respiration, proton leak, spare respiratory capacity, maximal respiration, and non-mitochondrial respiration. Data are presented as mean ± SEM. Statistical significance was determined by two-way ANOVA with Tukey’s post hoc test unless otherwise indicated. Groups not sharing a common letter are significantly different (P < 0.05, one-way ANOVA with Tukey’s post hoc test). (f) Working model illustrating the crosstalk between CTR1-mediated Cu import and adaptive thermogenesis , . Upon cold exposure, norepinephrine (NE) activates β3-adrenergic receptor (β3-AR) signaling, increasing cAMP levels and activating protein kinase A (PKA). PKA promotes thermogenic gene expression (via PGC-1α and CREB) and phosphorylates hormone-sensitive lipase (HSL) to stimulate lipolysis. Released free fatty acids (FFAs) activate UCP1 and provide substrates for mitochondrial oxidative phosphorylation (OXPHOS). CTR1-dependent Cu import supports mitochondrial OXPHOS capacity and thermogenic output, whereas Cu delivery by elesclomol (ES) partially restores oxidative function in Ctr1 -deficient adipocytes. The model highlights outstanding questions, including whether β3-AR signaling regulates CTR1 activity and whether mechanisms exist that prioritize Cu delivery to mitochondria during thermogenic activation.

Article Snippet: Primary antibodies included CTR1 , UCP1 (Abcam, ab23841), ATP7A (from Dr. S. Kaler’s laboratory ), CCS (Santa Cruz Biotechnology, sc-55561), OXPHOS cocktail (Abcam, ab110413), HSL (Abcam, ab45422), phospho-HSL (Ser660; Cell Signaling, #45804), CoxIV (Thermo Fisher Scientific, A21348), β-actin (Thermo Fisher Scientific, MA515739), and GAPDH (Thermo Fisher Scientific, MA515738).

Techniques: Staining, Western Blot, Derivative Assay, Gene Expression, Phospho-proteomics, Activity Assay, Activation Assay