BK channel deficiency promotes lipolysis via an AKT-independent activation of the cAMP/PKA/HSL pathway

Figures

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  Fig. 1. Systemic knockout of BK channels significantly decreases WAT mass. A: Body weight changes in BK KO rats and WT rats measured weekly from 4 to 12 weeks of age (n = 5). B: Representative photographs of BK KO and WT rats at 12 weeks of age. Scale bars = 5 cm. C and D: Food intake (C) and water intake (D) assessed over a 24 h period using indirect calorimetry (n = 4). E and F: Body composition analysis by EchoMRI, showing total fat mass (E) and lean mass (F) in BK KO and WT rats (n = 4). G and H: Fat weight (G) and lean weight (H) normalized to total body weight in BK KO and WT rats (n = 4). I: Ratio of fat mass to lean mass in BK KO and WT rats (n = 4). J: Representative images of the gross morphology of iWAT, eWAT, and BAT dissected from BK KO and WT rats. Scale bars = 2 cm. K and L: Absolute weights of iWAT, eWAT, and BAT depots (K) and their weights expressed as a percentage of total body weight (L) in BK KO and WT rats (n = 4). Data are presented as mean ± SEM. Statistical analysis was performed using two-way ANOVA for (A), and an unpaired two-tailed Student's t test for (C-I, K and L). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. ns, not significant.

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  Fig. 2. BK channel deficiency enriches pathways related to lipolysis. A: Standardization of ADSCs. The x-axis label represents the sample symbol and the y-axis label represents the gene expression values. The bar above represents the data before normalization, and the bar below represents the data after normalization. B: Volcano plot of DEGs in WT and KO ADSCs (n = 5). Red dots represent upregulated genes, and blue dots represent downregulated genes. The differences were set as |log2FC| > 1 and P value < 0.05. C: Clustering heatmap of DEGs in each group of rats. Legend on the top right indicates the log2FC of the genes. D: Bar chart showed the top 10 enriched by KEGG enrichment analysis. The x-axis label represents the -log10 (P Value) and the y-axis label represents pathway. E: Bar chart displayed the top 12 terms enriched by GO enrichment of biological process (BP). The x-axis label represented the -log10 (P Value), and the y-axis label represented GO terms.

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  Fig. 3. Increase of fasting-induced lipolysis in adipocyte of BK KO rats. A: Body weight changes during 24 h (fed, fast) or 30 h (refed; 24 h fasting followed by 6h refeeding) in 12-week-old male WT and BK KO rats fed with chow diet (n = 6). B: The ratio of body weight changes to body weight after 24 h fasting was statistical (n = 6). C–E: Weight of iWAT (C), eWAT (D), and BAT (E) from BK KO and WT rats were measured (n = 6). F–H: Weights of iWAT (F), eWAT (G), and BAT (H) presented as a percentage of total body weight (n = 6). I: H&E staining was performed on eWAT from WT and BK KO rats in fed and fasted states. Scale bars = 200 μm. J: Areas of adipocytes (WT - Fed, n = 233 from six rats [30 - 45 adipocytes/rat]; WT - Fast, n = 194 from six rats [30 - 40 adipocytes/rat]; KO - Fed, n = 158 from six rats [25 - 30 adipocytes/rat]; KO - Fast, n = 256 from six rats [30 - 45 adipocytes/rat]). K: Diameter of adipocytes (WT - Fed, n = 225 from six rats [30 - 45 adipocytes/rat]; WT - Fast, n = 156 from six rats [25 - 30 adipocytes/rat]; KO - Fed, n = 158 from six rats [25 - 30 adipocytes/rat]; KO - Fast, n = 234 from six rats [30 - 45 adipocytes/rat]). L and M: Representative western blots (L)…

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  Fig. 4. HFD feeding of BKadipo−/− mice can promote lipolysis. A: Body weight changes in male BKadipo+/+ (n = 7) and BKadipo−/− mice (n = 6) over 16 weeks of HFD feeding. B: Representative photographs of BKadipo+/+ and BKadipo−/− mice after 16 weeks of HFD feeding. Scale bars = 2 cm. C and D: Total body weight gain (C) and body weight gain normalized to initial body weight (D) in BKadipo+/+ and BKadipo−/− mice over the 16-weeks HFD feeding period (n = 7). E: H&E staining was performed of eWAT from BKadipo+/+ and BKadipo−/− mice over 16 weeks of HFD feeding states. Scale bars = 200 μm. F: Areas of adipocytes (BKadipo+/+, n = 356 from six rats [55 - 65 adipocytes/rat]; BKadipo−/−, n = 357 from six rats [55 - 65 adipocytes/rat]). G: Diameter of adipocytes (BKadipo+/+, n = 380 from six rats [55 - 70 adipocytes/rat]; BKadipo−/−, n = 342 from six rats [55 - 65 adipocytes/rat]). H and I: Representative western blots (H) and quantitative analysis (I) of lipolysis-related protein levels in eWAT from BKadipo+/+ and BKadipo−/− mice after 16 weeks of HFD feeding (n = 5). J and K: Plasma levels of glycerol (J) and FFA (K) were analyzed (n = 4). Data are presented as means ± SEM. Statistical analysis…

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  Fig. 5. BK channel deficiency or inhibition promotes CL-316243 stimulation-induced lipolysis in adipocytes. A: Schematic illustration of the in vitro differentiation protocol from ADSCs to mature adipocytes. B and C: The levels of glycerol in the culture medium stimulated by different drugs as measured using the glycerol assay kit. Adipocytes were directly stimulated with 1 μM CL-316243 for 1 h (B) or preincubated with 3 μM PAX or 20 μM NS1619 for 1 h, followed by stimulation with 1 μM CL-316243 for 1 h (C) (n = 5–15). D and E: Representative western blots (D) and quantitative analysis (E) of the phosphorylation levels of key lipolytic regulatory proteins in adipocytes following drug treatments (n = 5). F: Relative mRNA expression levels of lipolysis-related genes in adipocytes, determined by qRT-PCR (n = 3). G and H: Representative western blots (G) and quantitative analysis (H) of HSL and phospho-HSL (Ser660) levels. Adipocytes were preincubated with 3 μM PAX or 20 μM NS1619 for 1 h, followed by stimulation with 1 μM CL-316243 for 1 h (n = 7). I: Relative mRNA expression levels of lipolysis-related genes, including HSL and ATGL, in adipocytes subjected to the same treatment conditions…

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  Fig. 6. BK channel deficiency enhances lipolysis via the cAMP/PKA/HSL signaling pathway. A and B: Representative western blots (A) and quantitative analysis (B) of PKA substrate phosphorylation levels in eWAT from the indicated mouse models (n = 5). C: Glycerol release from adipocytes measured under basal conditions or following stimulation with 1 μM CL-316243 for 1 h. Cells were preincubated with or without the PKA inhibitor H89 (10 μM) for 1 h prior to stimulation (n = 6). D and E: Representative western blots (D) and quantitative analysis (E) of the phosphorylation levels of key lipolytic regulatory proteins in adipocytes following the indicated treatments (n = 5). F: Glycerol release from mature adipocytes treated with 10 μM forskolin or 1 mM 8-bromo-cAMP for 1 h, measured using a glycerol assay kit (n = 4). G and H: Representative western blots (G) and quantitative analysis (H) of lipolysis-related protein phosphorylation levels in mature adipocytes treated as described in (F) (n = 4). I and J: The average (I) and Δfold change in fluo-4 fluorescence intensity (J) in mature adipocytes treated with 1 μM CL-316243 (n = 4). Data are presented as mean ± SEM. Statistical analysis was…

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  Fig. 7. BK channel deficiency facilitates AKT-independent activation of cAMP-PKA pathway to promote lipolysis. A and B: Representative western blots (A) and quantitative analysis (B) of AKT phosphorylation at Ser473 in eWAT from the indicated mouse models (n = 8). C and D: Representative western blots (C) and quantitative analysis (D) of AKT phosphorylation at Ser473 in differentiated adipocytes subjected to the indicated treatments (n = 4). Adipocytes were directly stimulated with 1 μM CL-316243 for 1 h or preincubated with 3 μM PAX or 20 μM NS1619 for 1 h, followed by stimulation with 1 μM CL-316243 for 1 h. Data are presented as mean ± SEM. Statistical analysis was performed using two-way ANOVA for (B), and one-way ANOVA followed for (D). ns, not significant.

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  Supplementary Figure S1. Deletion of BK channel promotes lipolysis in eWAT. A: Volcano plot of DEGs in WT and KO eWAT (n = 4). Red dots represent upregulated genes, and blue dots represent downregulated genes. The differences were set as |log2FC| > 1 and P value < 0.05. B: Clustering heatmap of DEGs in each group of rats. Legend on the top right indicates the log2FC of the genes. C: GSEA revealed a significant enrichment of the two pathways in KO rats. Picture above: metabolic pathways. Picture below: cAMP signaling pathway. The colors (red to blue) represent the decreasing trend of log2FC of genes. NES, normalized enrichment score; WT, wild-type.

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  Supplementary Figure S2. Generation of adipocyte-specific BK knockout mice (BKadipo−/−). A: Schematic diagram of BKadipo−/− mouse model construction. B: Genotyping identification of BKadipo+/+ and BKadipo−/− mice by PCR analysis of genomic DNA. C: Relative expression level of BK in eWAT of BKadipo+/+ and BKadipo−/− mice detected by qRT-PCR. Data are presented as means ± SEM. Statistical analysis was performed using an unpaired two-tailed Student's t-test for (C). ∗∗P < 0.01. PCR, polymerase chain reaction; SEM, standard error of the mean.

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  Supplementary Figure S3. BKadipo−/− mice can resist HFD-induced obesity. A and B: Body composition analysis by EchoMRI, showing total fat mass (A) and lean mass (B) in BKadipo+/+ and BKadipo−/− mice after 16 weeks of HFD feeding (n = 7). C and D: Fat mass (C) and lean mass (D) normalized to total body weight in BKadipo+/+ and BKadipo−/− mice (n = 7). E: Ratio of fat mass to lean mass in BKadipo+/+ and BKadipo−/− mice (n = 7). F: Representative images of iWAT, eWAT, and BAT dissected from BKadipo+/+ and BKadipo−/− mice after 16 weeks of HFD feeding. Scale bars = 2 cm. G and H: Food intake (G) and water intake (H) assessed over a 24-h period using indirect calorimetry (n = 4). I-L: Serum concentrations of total cholesterol (TC; I), triglycerides (TG; J), high-density lipoprotein (HDL; K), and low-density lipoprotein (LDL; L) in BKadipo+/+ and BKadipo−/− mice (n = 4). M and N: Intraperitoneal glucose tolerance test (GTT) (M) and quantification of the area under the curve (AUC; N) in BKadipo+/+ and BKadipo−/− mice (n = 4). O and P: Intraperitoneal insulin tolerance test (ITT) (O) and quantification of the AUC (P) in BKadipo+/+ (n = 5) and BKadipo−/− mice (n = 4). Q-S: Indirect calorimetry was performed using…

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  Supplementary Figure S4. BKadipo−/− mice showed no difference in body weight under CD feeding condition. A: Body weight changes in male BKadipo+/+ (n = 9) and BKadipo−/− mice (n = 5) over 16 weeks of CD feeding. B: Representative gross morphology images of BKadipo+/+ and BKadipo−/− mice after 16 weeks of CD feeding. Scale bars = 2 cm. C and D: Total body weight gain (C) and body weight gain normalized to initial body weight (D) in BKadipo+/+ (n = 9) and BKadipo−/− mice (n = 5) over the 16-week CD feeding period. E and F: Body composition analysis by EchoMRI, showing total fat mass (E) and lean mass (F) in BKadipo+/+ (n = 9) and BKadipo−/− mice (n = 6). G and H: Fat mass (G) and lean mass (H) normalized to total body weight in BKadipo+/+ (n = 9) and BKadipo−/− mice (n = 6). I: Ratio of fat mass to lean mass in BKadipo+/+ (n = 9) and BKadipo−/− mice (n = 6). J: Representative images of iWAT, eWAT, and BAT dissected from BKadipo+/+ and BKadipo−/− mice after 16 weeks of CD feeding. Scale bars = 2 cm. K and L: Food intake (K) and water intake (L) assessed over a 24 h period using indirect calorimetry (BKadipo+/+: n = 3; BKadipo−/−: n = 4). M-P: Serum concentrations of TC (M), TG (N), HDL (O), and LDL (P) in…

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  Supplementary Figure S5. BKadipo−/− mice had no effect on lipolysis under CD feeding condition. A: H&E staining was performed on eWAT from BKadipo+/+ and BKadipo−/− mice over 16 weeks of CD feeding states. Scale bars = 200 μm. B: Areas of adipocytes (BKadipo+/+, n = 281 from six mice [40 - 55 adipocytes/mouse]; BKadipo−/−, n = 280 from six mice [40 - 55 adipocytes/mouse]). C: Diameter of adipocytes (BKadipo+/+, n = 204 from six mice [25 - 40 adipocytes/mouse]; BKadipo−/−, n = 197 from six mice [25 - 40 adipocytes/mouse]). D and E: Representative western blots (D) and quantitative analysis (E) of lipolysis-related protein levels in eWAT from BKadipo+/+ and BKadipo−/− mice (n = 5). F and G: Plasma concentrations of glycerol (F) and FFA (G) in BKadipo+/+ (n = 7) and BKadipo−/− mice (n = 4). H-J: Metabolic cage analysis showing VO2 (H), VCO2 (I), and EE (J) in BKadipo+/+ and BKadipo−/− mice (n = 4). Data are presented as mean ± SEM. Statistical analysis was performed using the Mann-Whitney U test for (B and C), an unpaired two-tailed Student's t-test for (E-G) and ANCOVA analysis for (H-J). ns, not significant; SEM, standard error of the mean.