Anti-Obesity Natural Products Tested in Juvenile Zebrafish Obesogenic Tests and Mouse 3T3-L1 Adipogenesis Assays

Abstract

(1) Background: The obesity epidemic has been drastically progressing in both children and adults worldwide. Pharmacotherapy is considered necessary for its treatment. However, many anti-obesity drugs have been withdrawn from the market due to their adverse effects. Instead, natural products (NPs) have been studied as a source for drug discovery for obesity, with the goal of limiting the adverse effects. Zebrafish are ideal model animals for in vivo testing of anti-obesity NPs, and disease models of several types of obesity have been developed. However, the evidence for zebrafish as an anti-obesity drug screening model are still limited. (2) Methods: We performed anti-adipogenic testing using the juvenile zebrafish obesogenic test (ZOT) and mouse 3T3-L1 preadipocytes using the focused NP library containing 38 NPs and compared their results. (3) Results: Seven and eleven NPs reduced lipid accumulation in zebrafish visceral fat tissues and mouse adipocytes, respectively. Of these, five NPs suppressed lipid accumulation in both zebrafish and 3T3-L1 adipocytes. We confirmed that these five NPs (globin-digested peptides, green tea extract, red pepper extract, nobiletin, and Moringa leaf powder) exerted anti-obesity effects in diet-induced obese adult zebrafish. (4) Conclusions: ZOT using juvenile fish can be a high-throughput alternative to ZOT using adult zebrafish and can be applied for in vivo screening to discover novel therapeutics for visceral obesity and potentially also other disorders.

Keywords: diabetes; drug discovery; metabolic syndrome; screening.

Figure 1

Zebrafish obesogenic test (ZOT) identifies seven anti-obese natural products (NPs). (a) Experimental design for ZOT. (b) Short term high-fat diet (HFD) increased the amount of visceral adipose tissue (VAT). Right panel indicates typical images of the control and HFD groups. Red indicates Nile Red (NR)-stained VAT. sb; swimming bladder, vat; visceral adipose tissue. Left panel indicates quantification of NR stained area. Values presented are means. Error bars indicate SD. n = 5, ** p < 0.01, Student’s t-test. (c) ZOT result. Dotted lines indicate 1 and 0.6 ratio to HFD alone group (HFD). NPs with asterisks were administered at lower volumes (10 µg/mL) because of their toxicity. The others were administered at 100 µg/mL in the breeding water. Values presented are means. Error bars indicate SD. n = 5–10, * p < 0.05, ** p < 0.01 vs. control, one-way ANOVA with Bonferroni–Dunn multiple comparison. nd indicates not determined because of high toxicity (NP18). (d) Typical image of effective NPs in (c).

Figure 2

Mouse 3T3-L1 preadipocyte differentiation assay identified ten lipid-lowering NPs. (a) Experimental design for 3T3-L1 assay. (b) Result of 3T3-L1 screening. NPs without data (not determined: ND) were due to inability to extract compounds with ethanol extraction. Values presented are means. Error bars indicate SD. n = 8, * p < 0.05, ** p < 0.01 vs. control l, one-way ANOVA with Bonferroni–Dunn multiple comparison. (c) Representative images of differentiated 3T3-L1 cells with the same NPs as in Figure 1d. Red indicates lipid accumulation. (d) Venn diagram analysis of the effective NPs in both the ZOT (Figure 1) and 3T3-L1 cell-based assay. * While NP01 (globin digested peptides [GDP]) could not be evaluated in the 3T3-L1 assay, NP02 (GDP with magnesium) reduced lipid accumulation in 3T3-L1 cells. Thus, we categorized NP01 as effective in both assays.

Figure 3

NP administration in adult diet-induced obese (DIO) zebrafish. (a) Experimental design for DIO-zebrafish experiment. (b) Body weight increases in DIO-zebrafish with or without NPs. (c–e) Plasma triglycerides (c), total cholesterol (d), and fasting blood glucose (e) at the end of the experiment. Values presented are means. Error bars indicate SD. n = 5–10, * p < 0.05, ** p < 0.01 vs. control, one-way analysis of variance (ANOVA) with Bonferroni–Dunn multiple comparison.