Fig 1: Adipocyte‐specific caspase‐6 knockout protects against diet‐induced obesity and insulin resistance. Flox and adipocyte‐specific caspase‐6 knockout (ACKO) mice fed 60% HFD for 12 weeks. (A) Schematic diagram of generating Casp6 flox (Flox) mice and adipocyte‐specific Casp6 knockout (ACKO) mice. (B) Immunoblot of caspase‐6 protein in adipose tissues and livers. (C) Body weight (n = 9‐11). (D‐F) Tissue weights (n = 9‐11): eWAT (D), iWAT (E), BAT (F). Two tailed unpaired Student's t‐test. (G–I) Indirect calorimetry (n = 6): oxygen consumption rate (G), carbon dioxide production (H), and energy expenditure (I). ANCOVA analysis with body weight as a covariate. (J, K) GTT (J, n = 9‐10) and ITT (K, n = 8‐9) with AUC quantification. GTT/ITT: two‐way ANOVA followed by Šídák's‐corrected post hoc test. (L) Immunoblots for the expression levels of PPARγ, SP1 and ATGL in eWAT and iWAT of Flox and ACKO mice fed HFD for 12 weeks. (M) Graphical summary. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 2: Caspase‐6 deletion upregulates ATGL and alters systemic lipid metabolism. WT and C6KO mice fed 60% HFD for 12 weeks. (A) Relative expression values (Z‐scaled log2 [TPM+1]) of ATGL (Pnpla2) in eWAT, iWAT, and BAT from RNA‐seq (n = 3). (B, C) Western blot and quantification of ATGL and caspase‐6 in eWAT (B) and iWAT (C). (n = 4) Two tailed unpaired Student's t‐test. (D‐G) Glycerol and NEFA release from white and brown adipocytes with/without CL‐316243 treatment (n = 6). Two tailed unpaired Student's t‐test. (H, I) Lipidomic analysis of triglyceride (H) and FAHFA (I) species in eWAT (n = 6). For each species: two tailed unpaired Student's t‐test. (J‐L) Indirect calorimetry on mice i.p. injected 800 µmol/kg Atglistatin (n = 3). Oxygen consumption rate (J), Carbon dioxide production (K), and energy expenditure (L). ANCOVA analysis with body weight as a covariate. (M, N) Pearson correlation between expression of CASP6 and ATGL (PNPLA2) in human adipose tissue GSE59034 (M, n = 32) and GSE245948 (N, n = 76). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 3: Caspase‐6 regulates mitochondrial function in diet‐induced obesity. WT and C6KO mice fed 60% HFD for 12 weeks. (A, B) Ucp1 mRNA levels in eWAT (A) and iWAT (B) (n = 4). Two tailed unpaired Student's t‐test. (C) Immunoblot of OXPHOS complexes I‐V in white and brown adipocytes. (D, E) Quantification of mitochondrial complexes (n = 3). Two tailed unpaired Student's t‐test. (F) Seahorse assay on primary white adipocytes (n = 4). Two‐way ANOVA followed by Šídák's‐corrected post hoc test. (G‐J) Basal (G), maximal (H), spare respiratory capacity (I), and non‐respiratory oxygen consumption (J) from Seahorse assay (n = 4). Two tailed unpaired Student's t‐test. (K, L) Rectal temperature at ambient temperature (K) and after 4°C cold exposure (L) (n = 8‐10). Two tailed unpaired Student's t‐test for K; two‐way ANOVA followed by Šídák's‐corrected post hoc test for L. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 4: Caspase‐6 deficiency protects against HFD‐induced insulin resistance and inflammation. WT and C6KO mice fed 60% HFD for 12 weeks (A‐M). (A) Glucose tolerance test (GTT) and AUC quantification (n = 9‐10). (B) Insulin tolerance test (ITT) and AUC quantification (n = 9‐10). GTT/ITT: two‐way ANOVA followed by Šídák's‐corrected post hoc test. (C‐F) Expression of Tnf (C), Il6 (D), Il1b (E), and Ifng (F) in eWAT. (G‐J) Expression of Tnf (G), Il6 (H), Il1b (I), and Ifng (J) in iWAT. (n = 4) Two tailed unpaired Student's t‐test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (K‐M) Relative expression values (Z‐scaled log2 [TPM + 1]) for inflammatory response genes in iWAT (K), eWAT (L), and BAT (M) from RNA‐seq (n = 3).
Fig 5: Caspase‐6 cleaves PPARγ and SP1 to control ATGL expression in adipocytes. (A, B) Immunoblot of PPARγ and ATGL proteins in eWAT (A) and iWAT (B) of WT and C6KO mice fed 60% HFD for 12 weeks (n = 4). Two tailed unpaired Student's t‐test. (C) In vitro cleavage of recombinant PPARγ by active caspase‐6. Immunoblot of PPARγ. (D) Immunoblot of lysates from 3T3‐L1 adipocytes treated with cycloheximide (5 µg/ml) and TNFα (25 ng/ml) for indicated time with or without pretreatment of Emricasan (50 µg/ml) for 1 hr. (E, F) Immunoblot SP1 protein in eWAT (E) and iWAT (F) of WT and C6KO mice fed HFD for 12 weeks (n = 3). Two tailed unpaired Student's t‐test. (G) In vitro cleavage of recombinant SP1 by active caspase‐6. Coomassie blue staining of gel. (H) Immunoblot of lysates from 3T3‐L1 adipocytes treated with cycloheximide (CHX, 5 µg/ml) and TNFα (25 ng/ml) for indicated time with or without pretreatment of Emricasan (50 µg/ml) for 1 hr. (I) Immunoblot of lysates from ex vivo cultured eWAT from WT or C6KO, treated with CHX (15 µg/ml) and TNFα (75 ng/ml) for indicated time. (J) Pnpla2 expression in 3T3‐L1 adipocytes treated with Rosiglitazone (5µM) or T0070907 (100 nM) in the presence or absence of Mithramycin (10 µM) for 6 h (n = 3). Two tailed unpaired Student's t‐test. (K) In vitro cleavage of WT and mutant (D69E) PPARγ2 expressed in HEK293T by recombinant active caspase‐6. (L) In vitro cleavage of WT and mutant (D185E) SP1 expressed in HEK293T by recombinant active caspase‐6. (M) PPARγ activity reporter assay in HEK293T cell expressing PPRE‐H2B‐eGFP along with WT PPARγ or PPARγ D69E mutant in the absence or presence of CHX‐TNFα treatment (n = 8). Two tailed unpaired Student's t‐test. (N‐V) Pearson correlation between CASP6 and PPARγ target genes in human adipose tissues (GSE245948): Correlation matrix (N), AQP7 (O), CPT1B (P), FADS2 (Q), ME3 (R), PLIN4 (S), PCK2 (T), SLC27A1 (U), and SLC27A4 (V). (n = 76). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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