Genistein Induces Adipogenic and Autophagic Effects in Rainbow Trout (Oncorhynchus mykiss) Adipose Tissue: In Vitro and In Vivo Models

Abstract

Soybeans are one of the most used alternative dietary ingredients in aquafeeds. However, they contain phytoestrogens like genistein (GE), which can have an impact on fish metabolism and health. This study aimed to investigate the in vitro and in vivo effects of GE on lipid metabolism, apoptosis, and autophagy in rainbow trout (Oncorhynchus mykiss). Primary cultured preadipocytes were incubated with GE at different concentrations, 10 or 100 μM, and 1 μM 17β-estradiol (E2). Furthermore, juveniles received an intraperitoneal injection of GE at 5 or 50 µg/g body weight, or E2 at 5 µg/g. In vitro, GE 100 μM increased lipid accumulation and reduced cell viability, apparently involving an autophagic process, indicated by the higher LC3-II protein levels, and higher lc3b and cathepsin d transcript levels achieved after GE 10 μM. In vivo, GE 50 µg/g upregulated the gene expression of fatty acid synthase (fas) and glyceraldehyde-3-phosphate dehydrogenase in adipose tissue, suggesting enhanced lipogenesis, whereas it increased hormone-sensitive lipase in liver, indicating a lipolytic response. Besides, autophagy-related genes increased in the tissues analyzed mainly after GE 50 µg/g treatment. Overall, these findings suggest that an elevated GE administration could lead to impaired adipocyte viability and lipid metabolism dysregulation in rainbow trout.

Keywords: 17β-estradiol; apoptosis; autophagy; genistein; lipid metabolism; phytoestrogens.

Figure 1

In vitro effects on pre-confluent rainbow trout preadipocytes incubated at day 5 of culture for 24 h with GE at different concentrations (10 and 100 μM), E2 (1 μM), or vehicle (0.1% DMSO) as CT. (A) Representative immunofluorescence images stained with Hoechst for nuclei and (B) quantification of the nuclear area factor (NAF). (C) Quantification of cell viability using a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Scale bar 100 μm. Data are shown as mean + SEM (n = 4–6). Significant differences among treatments were determined by one-way ANOVA and are indicated by different letters (p < 0.05). When two groups share at least one letter, they are not statistically different. CT: Control; GE: Genistein; E2: 17β-estradiol.

Figure 2

In vitro effects on pre-confluent rainbow trout preadipocytes incubated at day 5 of culture for 24 h with GE at different concentrations (10 and 100 μM), E2 (1 μM), or vehicle (0.1% DMSO) as CT. Representative Western blots and quantification of LC3-II protein levels normalized to β-tubulin. Data are shown as mean + SEM (n = 4–6). Significant differences among treatments were determined by one-way ANOVA and are indicated by different letters (p < 0.05). CT: Control; GE: Genistein; E2: 17β-estradiol.

Figure 3

In vitro effects on confluent rainbow trout preadipocytes incubated at day 7 of culture for 72 h with GE (10 μM), E2 (1 μM), or vehicle (0.1% DMSO) as CT over the expression of apoptosis- and autophagy-related genes. Relative mRNA expression normalized to ef1α and β-actin of casp3, casp8, p53, lc3b, atg4b, atg12l, ctsd, and ctsl. Data are shown as mean + SEM (n = 6). Significant differences among treatments for each gene were determined by one-way ANOVA and are indicated by different letters (p < 0.05). When two groups share at least one letter, they are not statistically different. CT: Control; GE: Genistein; E2: 17β-estradiol.

Figure 4

In vitro effects on confluent rainbow trout preadipocytes incubated at day 7 of culture for 72 h with GE at different concentrations (10 and 100 μM), E2 (1 μM), or vehicle (0.1% DMSO) as CT. (A) Representative phase-contrast images of cells after staining with ORO and (B) quantification of lipid content. (C) Glycerol and (D) NEFA culture media levels. Scale bar 100 μm. Data are shown as mean + SEM (n = 4–6). Significant differences among treatments were determined by one-way ANOVA and are indicated by different letters (p < 0.05). When two groups share at least one letter, they are not statistically different. CT: Control; GE: Genistein; E2: 17β-estradiol; ORO: Oil red O; NEFA: Non-esterified fatty acids.

Figure 5

In vivo effects on rainbow trout intraperitoneally injected with vehicle (DMSO diluted 1:3 in sesame oil) as CT, GE 5 μg/g body weight, GE 50 μg/g, or E2 5 μg/g over the expression of lipid metabolism, and apoptosis- and autophagy-related genes in adipose tissue. Relative mRNA expression normalized to ef1α and β-actin of (A) fas, lpl, hsl, pparα, pparβ, gapdh, and lxr, and (B) casp3, casp8, p53, lc3b, atg4b, atg12l, ctsd, and ctsl. Data are shown as mean + SEM (n = 6–9). Significant differences among treatments for each gene were determined by one-way ANOVA and are indicated by different letters (p < 0.05). When two groups share at least one letter, they are not statistically different. CT: Control; GE: Genistein; E2: 17β-estradiol.

Figure 6

In vivo effects on rainbow trout intraperitoneally injected with vehicle (DMSO diluted 1:3 in sesame oil) as CT, GE 5 μg/g, GE 50 μg/g, or E2 5 μg/g over the expression of lipid metabolism, and apoptosis- and autophagy-related genes in liver. Relative mRNA expression normalized to ubq and β-actin of (A) fas, lpl, hsl, pparα, pparβ, gapdh, and lxr, and (B) casp3, casp8, p53, lc3b, atg4b, atg12l, ctsd, and ctsl. Data are shown as mean + SEM (n = 6–9). Significant differences among treatments for each gene were determined by one-way ANOVA and are indicated by different letters (p < 0.05). When two groups share at least one letter, they are not statistically different. CT: Control; GE: Genistein; E2: 17β-estradiol.

Figure 7

In vivo effects on rainbow trout intraperitoneally injected with vehicle (DMSO diluted 1:3 in sesame oil) as CT, GE 5 μg/g, GE 50 μg/g, or E2 5 μg/g over the expression of lipid metabolism, and apoptosis- and autophagy-related genes in white muscle. Relative mRNA expression normalized to ubq and ef1α of (A) fas, lpl, hsl, pparα, pparβ, gapdh, and lxr, and (B) casp3, casp8, p53, lc3b, atg4b, atg12l, ctsd, and ctsl. Data are shown as mean + SEM (n = 6–9). Significant differences among treatments for each gene were determined by one-way ANOVA and are indicated by different letters (p < 0.05). When two groups share at least one letter, they are not statistically different. CT: Control; GE: Genistein; E2: 17β-estradiol.

Figure 8

Comparative heat maps showing the changes in (A) lipid metabolism and (B) apoptosis- and autophagy-related genes expression among adipose tissue, liver, and white muscle in rainbow trout intraperitoneally injected with GE 5 μg/g, GE 50 μg/g, or E2 5 μg/g. Gene expression was first calculated relative to the geometric mean of the corresponding reference genes for each tissue, and then it was standardized following a standard score normalization (log2) against the corresponding control (CT) samples. Shades of red and blue, respectively, indicate the highest and lowest expression levels, as specified in the scale bar of the figure. GE: Genistein; E2: 17β-estradiol.