Resveratrol Reduces Glucolipid Metabolic Dysfunction and Learning and Memory Impairment in a NAFLD Rat Model: Involvement in Regulating the Imbalance of Nesfatin-1 Abundance and Copine 6 Expression

Abstract

Resveratrol (RES) is a polyphenolic compound, and our previous results have demonstrated its neuroprotective effect in a series of animal models. The aim of this study was to investigate its potential effect on a nonalcoholic fatty liver disease (NAFLD) rat model. The parameters of liver function and glucose and lipid metabolism were measured. Behavior performance was observed via the open field test (OFT), the sucrose preference test (SPT), the elevated plus maze (EPM), the forced swimming test (FST), and the Morris water maze (MWM). The protein expression levels of Copine 6, p-catenin, catenin, p-glycogen synthase kinase-3beta (GSK3β), GSK3β, and cyclin D1 in the hippocampus and prefrontal cortex (PFC) were detected using Western blotting. The results showed that RES could reverse nesfatin-1-related impairment of liver function and glucolipid metabolism, as indicated by the decreased plasma concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), glucose, insulin, and nesfatin-1; increase the plasma level of high-density lipoprotein cholesterol (HDL-C); and reduce hepatocyte steatosis in NAFLD rats. Although there was no significant difference among groups with regard to performance in the OFT, EPM, and FST tasks, RES-treated NAFLD rats showed an increased sucrose preference index in the SPT and improved learning and memory ability in the MWM task. Furthermore, the imbalanced protein expression levels of Copine 6, p-catenin, and p-GSK3β in the hippocampus and PFC of NAFLD rats were also restored to normal by treatment with RES. These results suggested that four consecutive weeks of RES treatment not only ameliorated glucolipid metabolic impairment and liver dysfunction in the NAFLD rat model but also mitigated the attendant behavioral and cognitive impairments. In addition to the mediating role of nesfatin-1, the mechanism underlying the therapeutic effect of RES on NAFLD might be associated with its ability to regulate the imbalanced expression level of Copine 6 and the Wnt signaling pathway in the hippocampus and PFC.

Keywords: Copine 6; Morris water maze; glycogen synthase kinase-3beta (GSK3β); nesfatin-1; nonalcoholic fatty liver disease (NAFLD); resveratrol.

Figure 1

The outline of the experimental design.

Figure 2

Effect of RES on the bodyweight, liver weight, and liver index of NAFLD rats. The data are presented as the mean ± SEM, with five rats in the CON + RES group and eight rats in each of the other groups. The bodyweight is shown in panel (A), and results of the repeated-measures ANOVA showed a significant effect of time but not treatment on the bodyweight, with a significant interaction between treatment and week. The NAFLD model rats showed greater liver weight (B) and a higher liver index (C) than the control or CON + RES rats. Treatment with RES reversed the increase in liver weight and liver index but had no significant effect on bodyweight.

Figure 3

Effect of RES on the macroscopic and histological liver changes in NAFLD rats. The livers of the NAFLD rats appeared yellow or pale, greasy, and brittle. Inflammatory cells and numerous lipid droplets were detected in the livers of NAFLD rats via HE staining (A). The gross and cellular scores of the liver histological changes in the NAFLD model rats were significantly increased (B). Compared with those in the NAFLD group, the color and texture of the livers of the RES-treated rats were closer to normal, and the gross and cellular scores were significantly decreased.

Figure 4

Effect of RES on the plasma concentrations of insulin, leptin, and nesfatin-1 in NAFLD rats. The data are presented as the mean ± SEM, with five rats in the CON + RES group and eight rats in each of the other groups. Compared with the control and CON + RES rats, the NAFLD group showed increased plasma insulin (A) and nesfatin-1 (C) levels, while the plasma leptin (B) level was decreased. Treatment with RES decreased the plasma concentrations of insulin and nesfatin-1 but had no effect on the plasma leptin level.

Figure 5

Effect of RES on the behavioral performance of NAFLD rats in the OFT, EPM, FST, and SPT. The data are presented as the mean ± SEM, with five rats in the CON + RES group and eight rats in each of the other groups. In the OFT, there was no significant difference among groups with respect to the total distance traveled (A), the distance traveled in the center (B), or the frequency of rearing or grooming (C). There was no change in performance in the EPM (D) or FST (E) among the groups. Compared with those of the control and CON + RES groups, the SPI of the rats in NAFLD model group was reduced (F), and this reduction could be reversed by treatment with RES, STG, or FLX (F).

Figure 6

Effect of RES on the behavioral performance of NAFLD rats in the MWM. The data are presented as the mean ± SEM, with five rats in the CON + RES group and eight rats in each of the other groups. In the acquisition phase, the escape latency of the NAFLD rats was longer than that of the control or CON + RES rats on all 4 days (A). In the probe trial, NAFLD rats spent less time than control rats in the target quadrant (B). These abnormalities could be reversed by treatment with RES.

Figure 7

Effect of RES on the protein expression level of Copine 6, p-β-catenin, β-catenin, p-GSK3β, GSK3β, and cyclin D1 in the hippocampus and PFC of NAFLD rats. (A) shows a typical graph, and (B–E) show a statistical analysis of the Western blotting results. The data in panels (B,C) are presented as the mean ± SEM, with n = 3 for each group. The protein expression level of Copine 6 and p-catenin/catenin was decreased, whereas the protein expression level of p-GSK3β/GSK3β was increased in the hippocampus and PFC of NAFLD rats. The expression level of cyclin D1 was increased in the hippocampus but decreased in the PFC of NAFLD rats. Treatment with RES restored the imbalanced expression level of Copine 6 and p-GSK3β/GSK3β to normal in both the hippocampus and PFC and normalized the expression level of p-catenin/catenin in the hippocampus, without any notable effect on that of cyclin D1. 1 control; 2 CON + RES; 3 NAFLD; 4 NAFLD + DNP; 5 NAFLD + STG; 6 NAFLD + RSG; 7 NAFLD + FLX; 8 NAFLD + RES.

Figure 8

Summary of the therapeutic effect of RES on the peripheral metabolic syndrome and behavioral and cognitive impairments associated with NAFLD in a rat model. The red description and arrows indicate the dysfunction induced by 8 weeks on a high-fat diet, and the blue ones show the effects of four consecutive weeks of treatment with RES. The up arrows indicate increase, the down arrows indicate decrease, and the symbol “—” indicates that there is no significant change.