Adipose triglyceride lipase protein abundance and translocation to the lipid droplet increase during leptin-induced lipolysis in bovine adipocytes  D.A. Koltes, M.E. Spurlock, D.M. Spurlock  Domestic Animal Endocrinology  Volume 61, Pages 62-76 (October 2017) DOI: 10.1016/j.domaniend.2017.06.001 Copyright © 2017 The Authors Terms and Conditions

Fig. 1 Demonstration of image assessment. The figure illustrates how translocation was measured for a lipid droplet using the RGB Profiles Plot plug in ImageJ [36]. The white lines represent where lines would have been drawn in ImageJ. The upper inset box is a representation of the data produced by RGB Profiles Plot plug in ImageJ. The intensity values along each pixel in the line were exported, averaged at either 0 and 0.89 μm for the average fluorescence at the lipid droplet, or 2.70 and 3.30 μm for the average fluorescence in the cytosol, and utilized for statistical analysis. Green represents the lipid droplet, blue and red represent ATGL and ABHD5, respectively. ABHD5, α/β hydroxylase domain containing 5; ATGL, adipose triglyceride lipase. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Domestic Animal Endocrinology 2017 61, 62-76DOI: (10.1016/j.domaniend.2017.06.001) Copyright © 2017 The Authors Terms and Conditions

Fig. 2 Effects of leptin on lipolysis in bovine adipocytes. N = 8 cows per treatment. (A) Glycerol concentration normalized to protein concentrations. (B) NEFA concentration normalized to protein concentrations. (C and D) Protein abundance normalized to β-actin expressed relative to control abundance. Representative Western blots are shown under the graph for the corresponding antibody. Lane 1: 0 ng/mL of leptin (control), lane 2: 10 ng/mL of leptin, lane 3: 100 ng/mL of leptin, lane 4: 200 ng/mL of leptin. STAT3, signal transducer and activator of transcription 3; HSL, hormone sensitive lipase; PSTAT3 Try705, phosphorylated STAT 3 at tyrosine 705; ATGL, adipose triglyceride lipase; PHSL ser563, phosphorylated HSL at serine 563; PHSL ser565, phosphorylated HSL at serine 565. ∗Represents P < 0.05 compared with control where the overall effect of leptin was P < 0.05. §Represents P < 0.05 compared with control where the overall effect of leptin was P < 0.11. Error bars represent SEM. Domestic Animal Endocrinology 2017 61, 62-76DOI: (10.1016/j.domaniend.2017.06.001) Copyright © 2017 The Authors Terms and Conditions

Fig. 3 Comparison of control and isoproterenol treated bovine adipocytes from cows in the leptin experiment. N = 8 cows per treatment. (A) Glycerol concentration normalized to protein concentrations. (B) NEFA concentration normalized to protein concentrations. (C) Protein abundance normalized to β-actin and expressed relative to control. Representative Western blots are shown under the graph for the corresponding antibody. Lane 1: Control samples, Lane 2: Adipocytes treated with 100 nM isoproterenol. STAT3, signaling transducer and activator of transcription 3; HSL, hormone sensitive lipase; PSTAT3 Try705, phosphorylated STAT 3 at tyrosine 705; ATGL, adipose triglyceride lipase; PHSL ser563, phosphorylated HSL at serine 563; PHSL ser565, phosphorylated HSL at serine 565. ∗Represents P < 0.05 compared with control. §Represents P < 0.1. Error bars represent SEM. Domestic Animal Endocrinology 2017 61, 62-76DOI: (10.1016/j.domaniend.2017.06.001) Copyright © 2017 The Authors Terms and Conditions

Fig. 4 Translocation of hormone sensitive lipase (HSL) to the lipid droplet after 2 h of exposure. N = 12–15 lipid droplets from 2 cultures per treatment. (A) Representative images of SVC-derived bovine adipocytes treated with media (control), 100 nM of isoproterenol, or 100 ng/mL of leptin. Treatment is listed on the left hand side of the image. In the merged image, HSL is blue and bodipy is green. The white line in the lower right hand image is 10 μm relative to the view. (B) Average translocation of HSL to the lipid droplet ±SEM. (C) Average lipid droplet diameter size ±SEM. Differences in subscripts were given when P < 0.05 and indicate pairwise differences of P < 0.05. SEM, standard error of the mean. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Domestic Animal Endocrinology 2017 61, 62-76DOI: (10.1016/j.domaniend.2017.06.001) Copyright © 2017 The Authors Terms and Conditions

Fig. 5 Translocation of adipose triglyceride lipase (ATGL) to the lipid droplet after 2 h of exposure. N = 12–15 lipid droplets from 2 cultures per treatment. (A) Representative images of SVC-derived bovine adipocytes treated with media (control), 100 nM of isoproterenol, or 100 ng/mL of leptin. Treatment is listed on the left hand side of the image. In the merged image, ATGL is blue and bodipy is green. The white line in the lower right hand image is 10 μm relative to the view. (B) Average translocation of ATGL to the lipid droplet ±SEM. (C) Average lipid droplet diameter size ±SEM. Differences in subscripts were given when P < 0.05 and indicate pairwise differences of P < 0.05. ATGL, adipose triglyceride lipase; SEM, standard error of the mean. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Domestic Animal Endocrinology 2017 61, 62-76DOI: (10.1016/j.domaniend.2017.06.001) Copyright © 2017 The Authors Terms and Conditions

Fig. 6 Colocalization of phosphorylated signal transducer and activator of transcription (STAT) 3 with nuclei. N = 5 images from 2 cultures per treatment. (A) Representative images of SVC-derived bovine adipocytes treated with 100 ng/mL of leptin over a 4-h time course. Time of exposure is listed on the left hand side. In the merged image, blue represents nuclei and green represents phosphorylated STAT3. (B) Average Pearson's correlation coefficients for the colocalization nuclear DNA and phosphorylated STAT3. Differences in lowercase subscripts were given when P < 0.05 and indicate pairwise differences of P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Domestic Animal Endocrinology 2017 61, 62-76DOI: (10.1016/j.domaniend.2017.06.001) Copyright © 2017 The Authors Terms and Conditions

Fig. 7 Time course of adipose triglyceride lipase (ATGL) and G0/G1 switch protein 2 (G0S2) translocation. N = 12–15 lipid droplets from 2 cultures per treatment. (A) Representative images of SVC-derived adipocytes treated with 100 ng/mL of leptin over the course of 2 h. Time of exposure is listed on the left hand side. In the merged image, blue represents ATGL, green represents lipid droplets, and red represents G0S2. (B) Average lipid droplet diameter ±SEM. (C) Average translocation of ATGL and G0S2 to the lipid droplet ±SEM. The white line in the lower right hand image is 10 μm relative to the view. Differences in subscripts were given when P < 0.05 and indicate pairwise differences of P < 0.05. SEM, standard error of the mean. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Domestic Animal Endocrinology 2017 61, 62-76DOI: (10.1016/j.domaniend.2017.06.001) Copyright © 2017 The Authors Terms and Conditions

Fig. 8 Time course of adipose triglyceride lipase (ATGL) and α/β hydroxylase domain containing 5 (ABHD5) translocation. N = 12–15 lipid droplets from 2 cultures per treatment. (A) Representative images of SVC-derived adipocytes treated with 100 ng/mL of leptin over the course of 2 h. Time of exposure is listed on the left hand side. In the merged image, blue represents ATGL, green represents bodipy, and red represents ABHD5. (B) Average translocation of ATGL to the lipid droplet for both images that contained ABHD5 and G0S2 ±SEM. The white line in the lower right hand image is 10 μm relative to the view. Differences in subscripts were given when P < 0.05 and indicate pairwise differences of P < 0.05. SEM, standard error of the mean. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Domestic Animal Endocrinology 2017 61, 62-76DOI: (10.1016/j.domaniend.2017.06.001) Copyright © 2017 The Authors Terms and Conditions