(A) Schematic of ubb:plin2-tdTomato construct injected into zebrafish and an adipocyte lipid droplet labeled with PLIN2-tdTOMATO fusion protein. Widefield microscope images of adult (B) wild-type sibling and (C) Tg(-3.5ubb:plin2-tdTomato) zebrafish. Box shows zoomed images of the fish tail with panels for brightfield, BODIPY, PLIN2-tdTOMATO, and merge. (D) Confocal images of fish tail adipocytes of adult wild-type sibling and Tg(-3.5ubb:plin2-tdTomato) zebrafish. Panels show brightfield, BODIPY, PLIN2-tdTOMATO, and merge. (E) Adult casper and Tg(-3.5ubb:plin2-tdTomato) zebrafish tails were fixed and immunohistochemistry was conducted for tdTOMATO expression of tail adipocytes.

(A) Schematic of visceral adipose tissue development in the 21 days post-fertilization (dpf) zebrafish. Abdominal visceral adipocytes (orange) develop around the swim bladder (gray) and pancreatic visceral adipocytes (red) develop ventrally around the pancreas. (B) Brightfield image of Tg(-3.5ubb:plin2-tdTomato) at 21 dpf. Red box indicates position of higher magnification images to visualize abdominal visceral adipocytes. (C) Widefield microscope images of 21 dpf wild-type casper and Tg(-3.5ubb:plin2-tdTomato) visceral adipocytes around the posterior swim bladder (SB) costained with BODIPY. Panels show brightfield, BODIPY, tdTOMATO, and merge. BODIPY stained adipose tissue was imaged and analyzed for (D) area, (E) standard length, and (F) area/standard length. Points indicate individual fish for N = 3 independent experiments; wild-type sibling, Data values for (D-F). n=35; Tg(-3.5ubb:plin2-tdTomato), n = 28. Bars indicate mean and SEM. Significance calculated via Mann-Whitney test. (G) 21 dpf wild-typecasperandTg(-3.5ubb:plin2-tdTomato)zebrafish were fixed and immunohistochemistry conducted for tdTOMATO expression of abdominal (AVA) and pancreatic visceral adipocytes (PVA).

(A) Heatmap of adipose depot presence during zebrafish development in Tg(-3.5ubb:plin2-tdTomato) and wild-type siblings. Shown is the percent of fish scored for presence of the depot using either BODIPY GFP signal or tdTOMATO expression at each time point. (B) Representative whole-mount images demonstrating the timing of adipose tissue development in Tg(-3.5ubb:plin2-tdTomato) and wild-type siblings using visceral adipose tissue as an example. Data points are an average of N = 3 independent experiments for each time point, excluding 14 days post-fertilization (dpf), where N = 4, and the adults, where N = 5 (5 dpf WT n = 32, 5 dpf Tg(-3.5ubb:plin2-tdTomato) n = 38, 14 dpf WT n = 32, 14 dpf Tg(-3.5ubb:plin2-tdTomato) n = 48, 21 dpf WT n = 30, 21 dpf Tg(-3.5ubb:plin2-tdTomato) n = 40, 42 dpf WT n = 48, 42 dpf Tg(-3.5ubb:plin2-tdTomato) n = 43, adult WT n = 38, adult Tg(-3.5ubb:plin2-tdTomato) n = 29).

(A) Schematic of experimental set-up for fasting experiment. 21 days post-fertilization (dpf) wild-type casper and Tg(-3.5ubb:plin2-tdTomato) zebrafish were fed or fasted for 7 days and imaged to measure standard length and adipose area. (B) Representative images of zebrafish fed or fasted after 7 days. Panels show merged images of brightfield and BODIPY-stained visceral adipose tissue. BODIPY-stained adipocytes were imaged to measure (C) area, (D) standard length, and (E) area/standard length. Data points indicate individual fish for N = 3 independent experiments; bars indicate mean and 95% CI. Fed wild-type n = 24; fed Tg(-3.5ubb:plin2-tdTomato) n = 24; fasted wild-type n = 29; fasted Tg(-3.5ubb:plin2-tdTomato) n = 20. Significance calculated via Kruskal-Wallis with Dunn?s multiple comparisons test; ****p<0.0001. (F) Representative image of computational segmentation of Tg(-3.5ubb:plin2-tdTomato) adipocytes. PLIN2-tdTOMATO was background subtracted with GFP fluorescence. Bottom panels show brightfield, segmented adipocytes, and segmentation overlaid on brightfield. (G) Schematic of experimental set-up for repeated imaging of 21 dpf Tg(-3.5ubb:plin2-tdTomato) zebrafish, which were fed or fasted for 7 days. Adipose tissue was imaged and analyzed for (H) area, (I) standard length, and (J) area/standard length. Points indicate mean and error bars indicate 95% CI for N = 3 independent experiments; by day 7, fed n = 46 and fasted n = 57. Significance calculated via Mann-Whitney test; **p<0.01, ****p<0.0001.

(A) Schematic of experimental set-up for Forskolin drug treatment. 21 days post-fertilization (dpf) Tg(-3.5ubb:plin2-tdTomato) zebrafish were individually placed in six-well plates with either dimethyl sulfoxide (DMSO) or 5 µM Forskolin for 24 hr. Adipose tissue was imaged and analyzed for (B) area, (C) standard length, and (D) area/standard length. Points indicate individual fish for N = 5 independent experiments; DMSO, n = 53; Forskolin, n = 55. Bars indicate mean and SEM. Significance calculated via Mann-Whitney test; *p<0.05.

(A) Percent breakdown of nutritional content for control feed and high-fat diet (HFD). (B) Schematic of experimental set-up for HFD experiment. 21 days post-fertiization (dpf) Tg(-3.5ubb:plin2-tdTomato) zebrafish were fed control feed or HFD for 14 days and imaged to measure standard length and adipose area. Image analysis pipeline resulted in measurements of adipose tissue (C) area, (D) standard length, and (E) area/standard length. Points indicate mean and error bars indicate 95% CI for N = 3 independent experiments; by day 14, control feed n = 57; HFD n = 61. Significance calculated via Mann-Whitney test; *p<0.05, **p<0.01, ****p<0.0001.

(A) Schematic of pharmacologic lipolysis screen in 3T3-L1 adipocytes using a glycerol release assay. Normalized log2 transformed values for top 10 drugs that inhibit lipolysis are shown. Magenta indicates compounds that modulate nitric oxide. (B) Normalized log2 transformed values for lipolysis inhibition in 3T3-L1 adipocytes using either dimethyl sulfoxide (DMSO) or 100 µM Atglistatin. N = 5 independent experiments. Bars indicate mean and SEM. Significance calculated via Welch?s t-test; ****p<0.0001. (C) Schematic of experimental set-up for drug treatment. 21 days post-fertilization (dpf) Tg(-3.5ubb:plin2-tdTomato) zebrafish were individually placed in six-well plates with either DMSO, 40 µM Atglistatin, 1 µM Auranofin, or 1 µM JS-K for 24 hr. Adipose tissue was imaged and analyzed for (C) area, (D) standard length, and (E) area/standard length. Data points indicate individual fish for N = 4 independent experiments; DMSO n = 47; Atglistatin n = 44; Auranofin n = 42; JS-K n = 44. Bars indicate mean and SEM. Significance calculated via Kruskal-Wallis with Dunn?s multiple comparisons test; *p<0.05, ****p<0.0001.

Viability of 21 days post-fertilization (dpf) Tg(-3.5ubb:plin2-tdTomato) zebrafish was measured after 24 hr of treatment with dimethyl sulfoxide (DMSO) or increasing concentrations of (A) Atglistatin, (B) Auranofin, or (C) JS-K. Percent larval viability measured from N = 3 independent experiments. After 24 hr of drug treatment, 21 dpf zebrafish were imaged to measure (D) area, (E) standard length, and (F) area/standard length. Data points indicate individual fish for N = 5 independent experiments; DMSO n = 29; 1 µM n = 32; 10 µM n = 28; 40 µM n = 31. Bars indicate mean and SEM. Significance calculated via Kruskal-Wallis with Dunn’s multiple comparisons test; *p<0.05.

(A) Schematic of zebrafish melanoma cell line with lipid droplet reporter (ZMEL-LD) with lipid droplet labeled by PLIN2-tdTOMATO. (B) Confocal images of ZMEL-LD cells after 24 hr of oleic acid treatment. Panels show fluorescence signals in PLIN2-tdTOMATO, MDH (lipid droplet dye) staining, and merge of images with cytoplasmic GFP. Red box indicates position of higher magnification image of lipid droplets. (C) ZMEL-LD cells were treated with either bovine serum albumin (BSA) or oleic acid with dimethyl sulfoxide (DMSO) for 72 hr and then analyzed by Fluorescence Activated Cell Sorting (FACS) for PLIN2-tdTOMATO expression. (D) Representative histogram of PLIN2-tdTOMATO expression of ZMEL-GFP and GFP+ZMEL-LD cells with indicated drugs. Dashed line shows threshold for PLIN2-tdTOMATO expression. (E) Quantification of percent of GFP+ZMEL-LD cells with lipid droplets. Lipid droplet low and high controls were ZMEL-LD cells treated with BSA or oleic acid for 72 hr. For drug treatments, ZMEL-LD cells were pulsed with oleic acid for 24 hr and then given DMSO, 40 µM Atglistatin, 0.5 µM Auranofin, or 0.5 µM JS-K for 48 hr. N = 3 independent experiments. Bars indicate mean and SEM. Significance calculated via one-way ANOVA with Dunnett’s multiple comparisons test; *p<0.05, ***p<0.001, ****p<0.0001.

(A) Confocal images of ZMEL-GFP and ZMEL-LD after 24 hr of 150 µM oleic acid treatment. Cells were fixed and stained, with panels showing fluorescence signals for PLIN2-tdTOMATO, Lipidtox (lipid droplet dye), and merge of images with cytoplasmic GFP. ZMEL-GFP and ZMEL-LD cells were treated with bovine serum albumin (BSA) or increasing concentrations of oleic acid for 24 hr and then analyzed by FACS. (B) Comparison of Lipidtox median fluorescence intensity (MFI) between ZMEL-GFP and ZMEL-LD. Significance calculated via Welch?s t-test with Bonferroni correction. (C) Representative histogram of PLIN2-tdTOMATO expression of ZMEL-GFP and ZMEL-LD cells. Dashed line shows the threshold for PLIN2-tdTOMATO expression. (D) Quantification of percent of GFP+ ZMEL-LD cells with lipid droplets. Significance calculated via one-way ANOVA with Dunnett?s multiple comparison test. (E) Representative histogram of Lipidtox expression of ZMEL-LD cells. (F) Quantification of Lipidtox MFI relative to BSA control of GFP+ZMEL-LD cells. Significance calculated via one-way ANOVA with Dunnett?s multiple comparison test. N = 3 independent experiments. Bars indicate mean and SEM. *p<0.05, **p<0.01, ****p<0.0001.