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ctr1 flox flox mice  (Jackson Laboratory)

 
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    Jackson Laboratory ctr1 flox flox mice
    (a) Immunoblot analysis of CCS and UCP1 protein levels in brown adipose tissue (BAT) from Cu-adequate (Cu-A) and Cu-deficient (Cu-D) mice. GAPDH served as a loading control. (b, c) Rectal body temperature of Cu-A (n = 14) and Cu-D (n = 18) mice maintained at (b) room temperature (RT) or exposed to (c) acute cold (CE, 4°C) for 12 h. (d) Kaplan–Meier survival analysis during 12 h of cold exposure (CE) for Cu-A (n = 14) and Cu-D (n = 18) mice (log-rank test, P = 0.647). (e) Immunoblot analysis of ATP7A and <t>CTR1</t> in BAT from WT mice housed at RT or exposed to cold (CE, 4°C) for 12 h. Arrowheads indicate glycosylated full-length (g) and truncated (t) CTR1 species. (f) Relative mRNA expression of Ucp1 , Ppargc1a , and Slc31a1 ( Ctr1 ) in BAT under RT and CE conditions (n = 4). (g) ICP–MS quantification of Cu levels in tissues from WT mice housed at RT or CE (n = 11 per condition). (h) Representative Laser Ablation (LA)–ICP–MS maps of Cu and Zn distribution in BAT from male WT mice housed at RT or CE (10 h). Scale bar, 500 μm. (i) Immunoblot analysis of ATP7A, CTR1, and UCP1 in inguinal white adipose tissue (iWAT) from WT mice treated with saline or CL316,243 (CL; 1 mg kg⁻¹ day⁻¹, i.p., 10 days). (j) Relative mRNA expression of Ucp1 , Ppargc1a , and Slc31a1 ( Ctr1 ) in iWAT following saline or CL treatment (n = 5). (k) ICP–MS analysis of Cu levels in tissues from saline-treated (n = 9) or CL-treated (n = 11) mice. Unless otherwise indicated, mice were analyzed as mixed sex with balanced male and female representation across groups. Data are presented as mean ± SEM. Statistical significance was determined by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
    Ctr1 Flox Flox Mice, supplied by Jackson Laboratory, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
    ctr1 flox flox mice - by Bioz Stars, 2026-07
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    1) Product Images from "Copper Import via CTR1 Supports the β3-Adrenergic Thermogenic Program"

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

    Journal: bioRxiv

    doi: 10.64898/2026.03.24.713962

    (a) Immunoblot analysis of CCS and UCP1 protein levels in brown adipose tissue (BAT) from Cu-adequate (Cu-A) and Cu-deficient (Cu-D) mice. GAPDH served as a loading control. (b, c) Rectal body temperature of Cu-A (n = 14) and Cu-D (n = 18) mice maintained at (b) room temperature (RT) or exposed to (c) acute cold (CE, 4°C) for 12 h. (d) Kaplan–Meier survival analysis during 12 h of cold exposure (CE) for Cu-A (n = 14) and Cu-D (n = 18) mice (log-rank test, P = 0.647). (e) Immunoblot analysis of ATP7A and CTR1 in BAT from WT mice housed at RT or exposed to cold (CE, 4°C) for 12 h. Arrowheads indicate glycosylated full-length (g) and truncated (t) CTR1 species. (f) Relative mRNA expression of Ucp1 , Ppargc1a , and Slc31a1 ( Ctr1 ) in BAT under RT and CE conditions (n = 4). (g) ICP–MS quantification of Cu levels in tissues from WT mice housed at RT or CE (n = 11 per condition). (h) Representative Laser Ablation (LA)–ICP–MS maps of Cu and Zn distribution in BAT from male WT mice housed at RT or CE (10 h). Scale bar, 500 μm. (i) Immunoblot analysis of ATP7A, CTR1, and UCP1 in inguinal white adipose tissue (iWAT) from WT mice treated with saline or CL316,243 (CL; 1 mg kg⁻¹ day⁻¹, i.p., 10 days). (j) Relative mRNA expression of Ucp1 , Ppargc1a , and Slc31a1 ( Ctr1 ) in iWAT following saline or CL treatment (n = 5). (k) ICP–MS analysis of Cu levels in tissues from saline-treated (n = 9) or CL-treated (n = 11) mice. Unless otherwise indicated, mice were analyzed as mixed sex with balanced male and female representation across groups. Data are presented as mean ± SEM. Statistical significance was determined by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
    Figure Legend Snippet: (a) Immunoblot analysis of CCS and UCP1 protein levels in brown adipose tissue (BAT) from Cu-adequate (Cu-A) and Cu-deficient (Cu-D) mice. GAPDH served as a loading control. (b, c) Rectal body temperature of Cu-A (n = 14) and Cu-D (n = 18) mice maintained at (b) room temperature (RT) or exposed to (c) acute cold (CE, 4°C) for 12 h. (d) Kaplan–Meier survival analysis during 12 h of cold exposure (CE) for Cu-A (n = 14) and Cu-D (n = 18) mice (log-rank test, P = 0.647). (e) Immunoblot analysis of ATP7A and CTR1 in BAT from WT mice housed at RT or exposed to cold (CE, 4°C) for 12 h. Arrowheads indicate glycosylated full-length (g) and truncated (t) CTR1 species. (f) Relative mRNA expression of Ucp1 , Ppargc1a , and Slc31a1 ( Ctr1 ) in BAT under RT and CE conditions (n = 4). (g) ICP–MS quantification of Cu levels in tissues from WT mice housed at RT or CE (n = 11 per condition). (h) Representative Laser Ablation (LA)–ICP–MS maps of Cu and Zn distribution in BAT from male WT mice housed at RT or CE (10 h). Scale bar, 500 μm. (i) Immunoblot analysis of ATP7A, CTR1, and UCP1 in inguinal white adipose tissue (iWAT) from WT mice treated with saline or CL316,243 (CL; 1 mg kg⁻¹ day⁻¹, i.p., 10 days). (j) Relative mRNA expression of Ucp1 , Ppargc1a , and Slc31a1 ( Ctr1 ) in iWAT following saline or CL treatment (n = 5). (k) ICP–MS analysis of Cu levels in tissues from saline-treated (n = 9) or CL-treated (n = 11) mice. Unless otherwise indicated, mice were analyzed as mixed sex with balanced male and female representation across groups. Data are presented as mean ± SEM. Statistical significance was determined by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

    Techniques Used: Western Blot, Control, Expressing, Saline, Two Tailed Test

    (a) Immunoblot analysis of ATP7A, CTR1, and CCS in BAT, iWAT, eWAT, and liver from Ctr1 -floxed (Floxed) and adipose-specific Ctr1 knockout (ACKO; Ctr1 fl/fl ; Adipoq -Cre) mice. GAPDH served as a loading control. (b) ICP–MS quantification of Cu levels in tissues from Floxed (n = 5) and ACKO (n = 6) mice. (c) Rectal body temperature in Floxed (n = 6) and ACKO (n = 8) mice during acute cold exposure (CE, 4°C). Food, but not water, was removed at the onset of CE from RT (∼22°C). (d) Kaplan–Meier survival analysis during CE showing reduced survival in ACKO (n = 9) compared with Floxed (n = 8) mice (log-rank test, ****P < 0.0001). Mice were euthanized upon reaching the endpoint criterion (rectal temperature <28°C). (e–g) Indirect calorimetry measurements during gradual transition (∼80 min) from 22°C to 4.5°C. Food, but not water, was removed at the onset of cooling. Core body temperature (e), cold-induced energy expenditure (f), and respiratory exchange ratio (RER) (g) in Floxed (n = 6) and ACKO (n = 6) mice. (h–j) Core body temperature (h), CL-induced energy expenditure (i), and RER (j) before and after intraperitoneal injection of CL (1 mg kg⁻¹) in Floxed (n = 6) and ACKO (n = 6) mice. Data are presented as mean ± SEM. Statistical significance was determined by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus Floxed controls. Unless otherwise indicated, experiments were performed in ∼10-week-old male mice.
    Figure Legend Snippet: (a) Immunoblot analysis of ATP7A, CTR1, and CCS in BAT, iWAT, eWAT, and liver from Ctr1 -floxed (Floxed) and adipose-specific Ctr1 knockout (ACKO; Ctr1 fl/fl ; Adipoq -Cre) mice. GAPDH served as a loading control. (b) ICP–MS quantification of Cu levels in tissues from Floxed (n = 5) and ACKO (n = 6) mice. (c) Rectal body temperature in Floxed (n = 6) and ACKO (n = 8) mice during acute cold exposure (CE, 4°C). Food, but not water, was removed at the onset of CE from RT (∼22°C). (d) Kaplan–Meier survival analysis during CE showing reduced survival in ACKO (n = 9) compared with Floxed (n = 8) mice (log-rank test, ****P < 0.0001). Mice were euthanized upon reaching the endpoint criterion (rectal temperature <28°C). (e–g) Indirect calorimetry measurements during gradual transition (∼80 min) from 22°C to 4.5°C. Food, but not water, was removed at the onset of cooling. Core body temperature (e), cold-induced energy expenditure (f), and respiratory exchange ratio (RER) (g) in Floxed (n = 6) and ACKO (n = 6) mice. (h–j) Core body temperature (h), CL-induced energy expenditure (i), and RER (j) before and after intraperitoneal injection of CL (1 mg kg⁻¹) in Floxed (n = 6) and ACKO (n = 6) mice. Data are presented as mean ± SEM. Statistical significance was determined by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus Floxed controls. Unless otherwise indicated, experiments were performed in ∼10-week-old male mice.

    Techniques Used: Western Blot, Knock-Out, Control, Injection, Two Tailed Test

    (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).
    Figure Legend 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).

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

    (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.
    Figure Legend 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.

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



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    ctr1 flox flox mice  (Jackson Laboratory)
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    Jackson Laboratory ctr1 flox flox mice
    (a) Immunoblot analysis of CCS and UCP1 protein levels in brown adipose tissue (BAT) from Cu-adequate (Cu-A) and Cu-deficient (Cu-D) mice. GAPDH served as a loading control. (b, c) Rectal body temperature of Cu-A (n = 14) and Cu-D (n = 18) mice maintained at (b) room temperature (RT) or exposed to (c) acute cold (CE, 4°C) for 12 h. (d) Kaplan–Meier survival analysis during 12 h of cold exposure (CE) for Cu-A (n = 14) and Cu-D (n = 18) mice (log-rank test, P = 0.647). (e) Immunoblot analysis of ATP7A and <t>CTR1</t> in BAT from WT mice housed at RT or exposed to cold (CE, 4°C) for 12 h. Arrowheads indicate glycosylated full-length (g) and truncated (t) CTR1 species. (f) Relative mRNA expression of Ucp1 , Ppargc1a , and Slc31a1 ( Ctr1 ) in BAT under RT and CE conditions (n = 4). (g) ICP–MS quantification of Cu levels in tissues from WT mice housed at RT or CE (n = 11 per condition). (h) Representative Laser Ablation (LA)–ICP–MS maps of Cu and Zn distribution in BAT from male WT mice housed at RT or CE (10 h). Scale bar, 500 μm. (i) Immunoblot analysis of ATP7A, CTR1, and UCP1 in inguinal white adipose tissue (iWAT) from WT mice treated with saline or CL316,243 (CL; 1 mg kg⁻¹ day⁻¹, i.p., 10 days). (j) Relative mRNA expression of Ucp1 , Ppargc1a , and Slc31a1 ( Ctr1 ) in iWAT following saline or CL treatment (n = 5). (k) ICP–MS analysis of Cu levels in tissues from saline-treated (n = 9) or CL-treated (n = 11) mice. Unless otherwise indicated, mice were analyzed as mixed sex with balanced male and female representation across groups. Data are presented as mean ± SEM. Statistical significance was determined by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
    Ctr1 Flox Flox Mice, supplied by Jackson Laboratory, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    (a) Immunoblot analysis of CCS and UCP1 protein levels in brown adipose tissue (BAT) from Cu-adequate (Cu-A) and Cu-deficient (Cu-D) mice. GAPDH served as a loading control. (b, c) Rectal body temperature of Cu-A (n = 14) and Cu-D (n = 18) mice maintained at (b) room temperature (RT) or exposed to (c) acute cold (CE, 4°C) for 12 h. (d) Kaplan–Meier survival analysis during 12 h of cold exposure (CE) for Cu-A (n = 14) and Cu-D (n = 18) mice (log-rank test, P = 0.647). (e) Immunoblot analysis of ATP7A and CTR1 in BAT from WT mice housed at RT or exposed to cold (CE, 4°C) for 12 h. Arrowheads indicate glycosylated full-length (g) and truncated (t) CTR1 species. (f) Relative mRNA expression of Ucp1 , Ppargc1a , and Slc31a1 ( Ctr1 ) in BAT under RT and CE conditions (n = 4). (g) ICP–MS quantification of Cu levels in tissues from WT mice housed at RT or CE (n = 11 per condition). (h) Representative Laser Ablation (LA)–ICP–MS maps of Cu and Zn distribution in BAT from male WT mice housed at RT or CE (10 h). Scale bar, 500 μm. (i) Immunoblot analysis of ATP7A, CTR1, and UCP1 in inguinal white adipose tissue (iWAT) from WT mice treated with saline or CL316,243 (CL; 1 mg kg⁻¹ day⁻¹, i.p., 10 days). (j) Relative mRNA expression of Ucp1 , Ppargc1a , and Slc31a1 ( Ctr1 ) in iWAT following saline or CL treatment (n = 5). (k) ICP–MS analysis of Cu levels in tissues from saline-treated (n = 9) or CL-treated (n = 11) mice. Unless otherwise indicated, mice were analyzed as mixed sex with balanced male and female representation across groups. Data are presented as mean ± SEM. Statistical significance was determined by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

    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) Immunoblot analysis of CCS and UCP1 protein levels in brown adipose tissue (BAT) from Cu-adequate (Cu-A) and Cu-deficient (Cu-D) mice. GAPDH served as a loading control. (b, c) Rectal body temperature of Cu-A (n = 14) and Cu-D (n = 18) mice maintained at (b) room temperature (RT) or exposed to (c) acute cold (CE, 4°C) for 12 h. (d) Kaplan–Meier survival analysis during 12 h of cold exposure (CE) for Cu-A (n = 14) and Cu-D (n = 18) mice (log-rank test, P = 0.647). (e) Immunoblot analysis of ATP7A and CTR1 in BAT from WT mice housed at RT or exposed to cold (CE, 4°C) for 12 h. Arrowheads indicate glycosylated full-length (g) and truncated (t) CTR1 species. (f) Relative mRNA expression of Ucp1 , Ppargc1a , and Slc31a1 ( Ctr1 ) in BAT under RT and CE conditions (n = 4). (g) ICP–MS quantification of Cu levels in tissues from WT mice housed at RT or CE (n = 11 per condition). (h) Representative Laser Ablation (LA)–ICP–MS maps of Cu and Zn distribution in BAT from male WT mice housed at RT or CE (10 h). Scale bar, 500 μm. (i) Immunoblot analysis of ATP7A, CTR1, and UCP1 in inguinal white adipose tissue (iWAT) from WT mice treated with saline or CL316,243 (CL; 1 mg kg⁻¹ day⁻¹, i.p., 10 days). (j) Relative mRNA expression of Ucp1 , Ppargc1a , and Slc31a1 ( Ctr1 ) in iWAT following saline or CL treatment (n = 5). (k) ICP–MS analysis of Cu levels in tissues from saline-treated (n = 9) or CL-treated (n = 11) mice. Unless otherwise indicated, mice were analyzed as mixed sex with balanced male and female representation across groups. Data are presented as mean ± SEM. Statistical significance was determined by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

    Article Snippet: Adipocyte-specific and brown adipocyte–specific Ctr1 knockout mice were generated by crossing Ctr1 flox/flox mice with Adipoq -Cre and Ucp1 -Cre transgenic mice (The Jackson Laboratory, strains 028020 and 024670) to produce adipocyte-specific knockout (ACKO; Ctr1 flox/flox ; Adipoq -Cre) and BAT-specific knockout (BCKO; Ctr1 flox/flox ; Ucp1 -Cre) mice, respectively.

    Techniques: Western Blot, Control, Expressing, Saline, Two Tailed Test

    (a) Immunoblot analysis of ATP7A, CTR1, and CCS in BAT, iWAT, eWAT, and liver from Ctr1 -floxed (Floxed) and adipose-specific Ctr1 knockout (ACKO; Ctr1 fl/fl ; Adipoq -Cre) mice. GAPDH served as a loading control. (b) ICP–MS quantification of Cu levels in tissues from Floxed (n = 5) and ACKO (n = 6) mice. (c) Rectal body temperature in Floxed (n = 6) and ACKO (n = 8) mice during acute cold exposure (CE, 4°C). Food, but not water, was removed at the onset of CE from RT (∼22°C). (d) Kaplan–Meier survival analysis during CE showing reduced survival in ACKO (n = 9) compared with Floxed (n = 8) mice (log-rank test, ****P < 0.0001). Mice were euthanized upon reaching the endpoint criterion (rectal temperature <28°C). (e–g) Indirect calorimetry measurements during gradual transition (∼80 min) from 22°C to 4.5°C. Food, but not water, was removed at the onset of cooling. Core body temperature (e), cold-induced energy expenditure (f), and respiratory exchange ratio (RER) (g) in Floxed (n = 6) and ACKO (n = 6) mice. (h–j) Core body temperature (h), CL-induced energy expenditure (i), and RER (j) before and after intraperitoneal injection of CL (1 mg kg⁻¹) in Floxed (n = 6) and ACKO (n = 6) mice. Data are presented as mean ± SEM. Statistical significance was determined by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus Floxed controls. Unless otherwise indicated, experiments were performed in ∼10-week-old male mice.

    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) Immunoblot analysis of ATP7A, CTR1, and CCS in BAT, iWAT, eWAT, and liver from Ctr1 -floxed (Floxed) and adipose-specific Ctr1 knockout (ACKO; Ctr1 fl/fl ; Adipoq -Cre) mice. GAPDH served as a loading control. (b) ICP–MS quantification of Cu levels in tissues from Floxed (n = 5) and ACKO (n = 6) mice. (c) Rectal body temperature in Floxed (n = 6) and ACKO (n = 8) mice during acute cold exposure (CE, 4°C). Food, but not water, was removed at the onset of CE from RT (∼22°C). (d) Kaplan–Meier survival analysis during CE showing reduced survival in ACKO (n = 9) compared with Floxed (n = 8) mice (log-rank test, ****P < 0.0001). Mice were euthanized upon reaching the endpoint criterion (rectal temperature <28°C). (e–g) Indirect calorimetry measurements during gradual transition (∼80 min) from 22°C to 4.5°C. Food, but not water, was removed at the onset of cooling. Core body temperature (e), cold-induced energy expenditure (f), and respiratory exchange ratio (RER) (g) in Floxed (n = 6) and ACKO (n = 6) mice. (h–j) Core body temperature (h), CL-induced energy expenditure (i), and RER (j) before and after intraperitoneal injection of CL (1 mg kg⁻¹) in Floxed (n = 6) and ACKO (n = 6) mice. Data are presented as mean ± SEM. Statistical significance was determined by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus Floxed controls. Unless otherwise indicated, experiments were performed in ∼10-week-old male mice.

    Article Snippet: Adipocyte-specific and brown adipocyte–specific Ctr1 knockout mice were generated by crossing Ctr1 flox/flox mice with Adipoq -Cre and Ucp1 -Cre transgenic mice (The Jackson Laboratory, strains 028020 and 024670) to produce adipocyte-specific knockout (ACKO; Ctr1 flox/flox ; Adipoq -Cre) and BAT-specific knockout (BCKO; Ctr1 flox/flox ; Ucp1 -Cre) mice, respectively.

    Techniques: Western Blot, Knock-Out, Control, Injection, Two Tailed Test

    (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).

    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: Adipocyte-specific and brown adipocyte–specific Ctr1 knockout mice were generated by crossing Ctr1 flox/flox mice with Adipoq -Cre and Ucp1 -Cre transgenic mice (The Jackson Laboratory, strains 028020 and 024670) to produce adipocyte-specific knockout (ACKO; Ctr1 flox/flox ; Adipoq -Cre) and BAT-specific knockout (BCKO; Ctr1 flox/flox ; Ucp1 -Cre) mice, respectively.

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

    (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.

    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: Adipocyte-specific and brown adipocyte–specific Ctr1 knockout mice were generated by crossing Ctr1 flox/flox mice with Adipoq -Cre and Ucp1 -Cre transgenic mice (The Jackson Laboratory, strains 028020 and 024670) to produce adipocyte-specific knockout (ACKO; Ctr1 flox/flox ; Adipoq -Cre) and BAT-specific knockout (BCKO; Ctr1 flox/flox ; Ucp1 -Cre) mice, respectively.

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