Activation of Transcription Factor EB Is Associated With Adipose Tissue Lipolysis in Dairy Cows With Subclinical Ketosis

Figures

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  Figure 1. Transcriptional activity of transcription factor EB (TFEB) in white adipose tissue (WAT). (A) Western blot analysis of TFEB in the nucleus and cytoplasm of WAT of control cows (n = 15) and dairy cows with subclinical ketosis (SCK; n = 15). Representative blots are shown. (B,C) Quantification of protein abundance of nuclear and cytoplasmic TFEB, respectively. (D) Western blot analysis of phosphorylated TFEB (p-TFEB) and total TFEB in WAT of control cows (n = 15) and dairy cows with SCK (n = 15). Representative blots are shown. (E,F) Quantification of ratio of p-TFEB/TFEB and protein abundance of TFEB, respectively. (G,H) Relative mRNA abundance of TFEB and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) in WAT of control cows (n = 15) and dairy cows with SCK (n = 15). Data were analyzed using unpaired t-tests and expressed as mean ± SEM.

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  Figure 2. Isoproterenol (ISO) and Torin1 treatment activate transcription factor EB (TFEB) in calf adipocytes. Calf adipocytes were stimulated with 10 μM ISO for 0, 1, 2, or 3 h, or treated with 250 nM Torin1 for 3 h. (A) Western blot analysis of TFEB in the nucleus and cytoplasm of calf adipocytes. Representative blots are shown. (B,C) Quantification of protein abundance of nuclear and cytoplasmic TFEB, respectively. (D) Western blot analysis of phosphorylated TFEB (p-TFEB) and total TFEB in calf adipocytes. Representative blots are shown. (E,F) Quantification of ratio of p-TFEB/TFEB and protein abundance of TFEB, respectively. (G,H) Relative mRNA abundance of TFEB and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) in calf adipocytes. Data were analyzed using one-way ANOVA with Tukey's tests for data meeting homogeneity of variance or with Tamhane's T2 analysis for data of heteroscedasticity and expressed as mean ± SEM. The same letter (a-e) indicates no significant difference (P > 0.05), whereas different letters indicate a significant difference (P < 0.05).

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  Figure 3. Isoproterenol (ISO) and Torin1 treatment induce lipolysis in calf adipocytes. Calf adipocytes were stimulated with 10 μM ISO for 0, 1, 2, or 3 h, or treated with 250 nM Torin1 for 3 h. (A) Western blot analysis of phosphorylated hormone sensitive lipase (p-HSL), HSL and adipose triacylglycerol lipase (ATGL) in calf adipocytes. Representative blots are shown. (B,C) Quantification of ratio of p-HSL/HSL and protein abundance of ATGL, respectively. (D) Relative mRNA abundance of ATGL in calf adipocytes. (E) The content of glycerol (GC) in the supernatant of calf adipocytes. (F) The triglyceride (TG) content in calf adipocytes. (G) Oil Red O staining. Scale bar = 20 μm. Data were analyzed using one-way ANOVA with Tukey's tests for data meeting homogeneity of variance or with Tamhane's T2 analysis for data of heteroscedasticity and expressed as mean ± SEM. The same letter (a-e) indicates no significant difference (P > 0.05), whereas different letters indicate a significant difference (P < 0.05).

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  Figure 4. Knockdown of transcription factor EB (TFEB) reduces isoproterenol (ISO)-induced lipolysis in calf adipocytes. The cells were divided into 4 groups as followed: an siControl group [transfected with negative control of small interfering RNA (siRNA) for 48 h], and an siTFEB group (transfected with siRNA for TFEB for 48 h), an siControl + ISO group (transfected with negative control of siRNA for 48 h and then treated with 10 μM ISO for 3 h), and an siTFEB + ISO group (transfected with siRNA for TFEB for 48 h and then treated with 10 μM ISO for 3 h). (A) Western blot analysis of TFEB, phosphorylated hormone sensitive lipase (p-HSL), HSL and adipose triacylglycerol lipase (ATGL) in bovine adipocytes. Representative blots are shown. (B–D) Quantification of protein abundance of TFEB, ratio of p-HSL/HSL and protein abundance of ATGL, respectively. (E) Relative mRNA abundance of ATGL in calf adipocytes. (F) The content of glycerol (GC) in the supernatant of calf adipocytes. (G) The triglyceride (TG) content in calf adipocytes. (H) Oil Red O staining. Scale bar = 20 μm. Data were analyzed using one-way or two-way ANOVA with Tukey's tests for data meeting homogeneity of variance or with…