Resveratrol Modulates the Redox Response and Bile Acid Metabolism to Maintain the Cholesterol Homeostasis in Fish Megalobrama amblycephala Offered a High-Carbohydrate Diet

Abstract

This study aimed to characterize the effects of resveratrol on the redox balance, cholesterol homeostasis and bile acid metabolism of Megalobrama amblycephala offered a high-carbohydrate diet. Fish (35.0 ± 0.15 g) were fed four diets including one control diet (32% nitrogen-free extract), one high-carbohydrate diet (45% nitrogen-free extract, HC), and the HC diet supplemented with different levels (0.04%, HCR1; 0.08%, HCR2) of resveratrol for 12 weeks. The HC diet-induced redox imbalance is characterized by increased MDA content and decreased T-SOD and CAT activities in the liver. Resveratrol attenuated this by up-regulating the transcription of Cu/Zn-sod, and increasing the activities of T-SOD, CAT, and GPX. The HC diet enhanced the cholesterol synthesis, but decreased the bile acid synthesis via up-regulating both hmgcr and acat2, and down-regulating cyp7a1, thus resulting in excessive cholesterol accumulation. Resveratrol supplement decreased cholesterol synthesis, and increased cholesterol uptake in the liver by down-regulating both hmgcr and acat2, and up-regulating ldlr. It also increased bile acid synthesis and biliary excretion by up-regulating cyp7a1, and down-regulating mrp2, oatp1, and oatp4 in the hindgut, thereby decreasing cholesterol accumulation. In conclusion, resveratrol improves the cholesterol homeostasis of Megalobrama amblycephala fed a high-carbohydrate diet by modulating the redox response and bile acid metabolism.

Keywords: bile acid metabolism; carbohydrate utilization; cholesterol homeostasis; fish culture; redox balance; resveratrol.

Figure 1

Liver T-SOD (A), CAT (B), and GPX (C) activities as well as GSH (D) and MDA (E) contents. Each data point represents the mean of four replicates (4 individuals per replicate). * Means p < 0.05, compared between the control and the HC group. ^ab Compared among the HC, HCR1, and HCR2 group. Bars assigned with different superscripts are significantly different (p < 0.05). Control, diet with 32% nitrogen-free extract; HC, diet with 45% nitrogen-free extract; HCR1, diet with 45% nitrogen-free extract and 0.04% resveratrol; HCR2, diet with 45% nitrogen-free extract and 0.08% resveratrol. MDA, malondialdehyde; CAT, catalase; GSH, L-glutathione reduced; GPX, glutathione peroxidase; T-SOD, total superoxide dismutase.

Figure 2

Plasma concentrations of glucose (A), TC (B), TG (C), TBA (D), LDL-C (E), and HDL-C (F). Each data point represents the mean of four replicates (4 individuals per replicate). * Means p < 0.05, compared between the control and the HC group. ^ab Compared among the HC, HCR1, and HCR2 groups. Bars assigned with different superscripts are significantly different (p < 0.05). Control, diet with 32% nitrogen-free extract; HC, diet with 45% nitrogen-free extract; HCR1, diet with 45% nitrogen-free extract and 0.04% resveratrol; HCR2, diet with 45% nitrogen-free extract and 0.08% resveratrol. TC, total cholesterol; TG, total triglycerides; TBA, total bile acid; HDL-C, high-density lipoproteins cholesterol; LDL-C, low-density lipoproteins cholesterol.

Figure 3

Liver TC (A), TG (B), and TBA (C) contents as well as hindgut TC (D), TG (E), and TBA (F) contents. Each data point represents the mean of four replicates (4 individuals per replicate). * Means p < 0.05, compared between the control and the HC group. ^ab Compared among the HC, HCR1, and HCR2 group. Bars assigned with different superscripts are significantly different (p < 0.05). Control, diet with 32% nitrogen-free extract; HC, diet with 45% nitrogen-free extract; HCR1, diet with 45% nitrogen-free extract and 0.04% resveratrol; HCR2, diet with 45% nitrogen-free extract and 0.08% resveratrol. TC, total cholesterol; TG, total triglycerides; TBA, total bile acid.

Figure 4

Hepatic transcriptions of the genes involved in the antioxidant defense. The mRNA levels of catalase (cat) (A), copper/zinc superoxide dismutase (Cu/Zn-sod) (B), manganese superoxide dismutase (Mn-sod) (C), glutathione peroxidase (gpx) (D), sirtuin 1 (sirt1) (E), kelch-like ECH associating protein 1 (keap1) (F) were evaluated using real-time RT-PCR. Data are referred to the values (relative units, RU) obtained in fish fed the control diet. Each data point represents the mean of four replicates (4 individuals per replicate). ^ab Compared among the HC, HCR1, and HCR2 groups. Bars assigned with different superscripts are significantly different (p < 0.05). The first and third quartiles are represented by the top and bottom of the box, while the minimum and maximum values are represented by the whiskers. Control, diet with 32% nitrogen-free extract; HC, diet with 45% nitrogen-free extract; HCR1, diet with 45% nitrogen-free extract and 0.04% resveratrol; HCR2, diet with 45% nitrogen-free extract and 0.08% resveratrol.

Figure 5

Hepatic transcriptions of the genes involved in cholesterol metabolism. The mRNA levels of sterol regulatory element-binding protein 2 (srebp2) (A), farnesoid X receptor α (fxrα) (B), liver X receptor α (lxrα) (C), 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (hmgcr) (D), low-density lipoproteins receptor (ldlr) (E), acyl coenzyme A: cholesterol acyltransferase 2 (acat2) (F) were evaluated using real-time RT-PCR. Data are referred to the values (relative units, RU) obtained in fish fed the control diet. Each data point represents the mean of four replicates (4 individuals per replicate). * Means p < 0.05, compared between the control and the HC group. ^abc Compared among the HC, HCR1, and HCR2 groups. Bars assigned with different superscripts are significantly different (p < 0.05). The first and third quartiles are represented by the top and bottom of the box, while the minimum and maximum values are represented by the whiskers. Control, diet with 32% nitrogen-free extract; HC, diet with 45% nitrogen-free extract; HCR1, diet with 45% nitrogen-free extract and 0.04% resveratrol; HCR2, diet with 45% nitrogen-free extract and 0.08% resveratrol.

Figure 6

Hepatic transcriptions of the genes involved in bile acid metabolism. The mRNA levels of cholesterol 7α-hydroxylase (cyp7a1) (A), sterol 12α-hydroxylase (cyp8b1) (B), multidrug resistance associated protein 2 (mrp2) (C), organic anion-transporting polypeptide 1 (oatp1) (D), organic anion-transporting polypeptide 4 (oatp4) (E), takeda G-protein-coupled BA receptor (tgr5) (F), Na⁺-taurocholate cotransporter (ntcp) (G), bile salt export pump (bsep) (H) were evaluated using real-time RT-PCR. Data are referred to the values (relative units, RU) obtained in fish fed the control diet. Each data point represents the mean of four replicates (4 individuals per replicate). * Means p < 0.05, compared between the control and the HC group. ^ab Compared among the HC, HCR1, and HCR2 groups. Bars assigned with different superscripts are significantly different (p < 0.05). The first and third quartiles are represented by the top and bottom of the box, while the minimum and maximum values are represented by the whiskers. Control, diet with 32% nitrogen-free extract; HC, diet with 45% nitrogen-free extract; HCR1, diet with 45% nitrogen-free extract and 0.04% resveratrol; HCR2, diet with 45% nitrogen-free extract and 0.08% resveratrol.

Figure 7

Relative transcriptions of the genes involved in cholesterol and bile acid metabolism in the hindgut. The mRNA levels of farnesoid X receptor α (fxrα) (A), liver X receptor α (lxrα) (B), multidrug resistance associated protein 2 (mrp2) (C), organic anion-transporting polypeptide 1 (oatp1) (D), organic anion-transporting polypeptide 4 (oatp4) (E), takeda G-protein-coupled BA receptor (tgr5) (F) were evaluated using real-time RT-PCR. Data are referred to the values (relative units, RU) obtained in fish fed the control diet. Each data point represents the mean of four replicates (4 individuals per replicate). * Means p < 0.05, compared between the control and the HC group. ^ab Compared among the HC, HCR1, and HCR2 groups. Bars assigned with different superscripts are significantly different (p < 0.05). The first and third quartiles are represented by the top and bottom of the box, while the minimum and maximum values are represented by the whiskers. Control, diet with 32% nitrogen-free extract; HC, diet with 45% nitrogen-free extract; HCR1, diet with 45% nitrogen-free extract and 0.04% resveratrol; HCR2, diet with 45% nitrogen-free extract and 0.08% resveratrol.

Figure 8

Protein levels of Srebp2 (A), Hmgcr (B), Ldlr (C), Fxrα (D), Lxrα (E), and Cyp7a1 (F) as well as the CYP7A1 (G) activity in the liver. Each data point represents the mean of four replicates (4 individuals per replicate). * Means p < 0.05, compared between the control and the HC group. ^ab Compared among the HC, HCR1, and HCR2 groups. Bars assigned with different superscripts are significantly different (p < 0.05). Control, diet with 32% nitrogen-free extract; HC, diet with 45% nitrogen-free extract; HCR1, diet with 45% nitrogen-free extract and 0.04% resveratrol; HCR2, diet with 45% nitrogen-free extract and 0.08% resveratrol. Srebp2, sterol regulatory element-binding protein 2; Hmgcr, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase; Ldlr, low-density lipoproteins receptor; Fxrα, farnesoid X receptor α; Lxrα, liver X receptor; Cyp7a1, cholesterol 7α-hydroxylase.