The Combined Intervention with Germinated Vigna radiata and Aerobic Interval Training Protocol Is an Effective Strategy for the Treatment of Non-Alcoholic Fatty Liver Disease (NAFLD) and Other Alterations Related to the Metabolic Syndrome in Zucker Rats

Abstract

Metabolic syndrome (MetS) is a group of related metabolic alterations that increase the risk of developing non-alcoholic fatty liver disease (NAFLD). Several lifestyle interventions based on dietary treatment with functional ingredients and physical activity are being studied as alternative or reinforcement treatments to the pharmacological ones actually in use. In the present experiment, the combined treatment with mung bean (Vigna radiata), a widely used legume with promising nutritional and health benefits that was included in the experimental diet as raw or 4 day-germinated seed flour, and aerobic interval training protocol (65-85% VO₂ max) has been tested in lean and obese Zucker rats following a 2 × 2 × 2 (2 phenotypes, 2 dietary interventions, 2 lifestyles) factorial ANOVA (Analysis of Variance) statistical analysis. Germination of V. radiata over a period of four days originated a significant protein hydrolysis leading to the appearance of low molecular weight peptides. The combination of 4 day-germinated V. radiata and aerobic interval training was more efficient compared to raw V. radiata at improving the aerobic capacity and physical performance, hepatic histology and functionality, and plasma lipid parameters as well as reverting the insulin resistance characteristic of the obese Zucker rat model. In conclusion, the joint intervention with legume sprouts and aerobic interval training protocol is an efficient treatment to improve the alterations of glucose and lipid metabolism as well as hepatic histology and functionality related to the development of NAFLD and the MetS.

Keywords: Vigna radiata; aerobic interval training; diet; exercise; germination; legume protein hydrolyzate; liver histology; metabolic syndrome; non-alcoholic fatty liver disease; obesity.

Figure 1

Detection processes of V. radiata protein digestion by germination process.Band pattern in SDS-PAGE. M, molecular weight markers; 1, raw V. radiata protein extract; 2, 4 day-germinated protein extract. The amount of Kjeldahl-N loaded per lane was 2.0 µg. The figure is representative of 3 independent analyses.

Figure 2

(A) Chromatogram of raw V. radiata protein and Mass Spectrum of major peak (13.270 min) of chromatogram; (B) Chromatogram of 4 day-germinated V. radiata protein after hydrolysis and Mass Spectrum of hydrolysis-derived peaks (0.985, 1.202, and 1.885 min, respectively) of chromatogram.

Figure 3

Effect of dietary treatment with raw or 4 day-germinated V. radiata and aerobic interval training protocol on food intake (A) and body weight gain (B) of lean and obese Zucker rats. (A) Weekly food intake (g DM/day); (B) Weekly body weight gain (g/day). DM, dry matter, RVR, raw V. radiata, GVR, 4 day-germinated V. radiata. Groups: LS, Lean (fa/+) sedentary rats, LE, Lean (fa/+) rats performing aerobic interval exercise, OS, Obese (fa/fa) sedentary rats, OE, Obese (fa/fa) rats performing aerobic interval exercise. W, week. Values are means ± SEM depicted by vertical bars (n = 10).

Figure 4

Effect of dietary treatment with raw or 4 day-germinated V. radiata and aerobic interval training protocol on blood glucose levels of lean and obese Zucker rats prior to or at different time points after oral glucose overload. (A) raw V. radiata (RVR), (B) 4 day-germinated V. radiata (GVR). Groups: LS, Lean (fa/+) sedentary rats, LE, Lean (fa/+) rats performing aerobic interval exercise, OS, Obese (fa/fa) sedentary rats, OE, Obese (fa/fa) rats performing aerobic interval exercise. Values are means ± SEM depicted by vertical bars (n = 10). The following notation is used to express significant differences (p < 0.05) between groups pointed out by Dunnet’s t-test: a, OS vs. LS, b, OE vs. LS, c, LE vs. LS.

Figure 5

Effect of dietary treatment with raw or 4 day-germinated V. radiata and AIT protocol on liver histology (hematoxylin-eosin stain) of lean and obese Zucker rats. (A) RVR-LS, (B) RVR-LE, (C) RVR-OS, (D) RVR-OE, (E) GVR-LS, (F) GVR-LE, (G) GVR-OS, (H) GVR-OE. RVR, raw V. radiata, GVR, 4 day-germinated V. radiata. Groups: LS, Lean (fa/+) sedentary rats, LE, Lean (fa/+) rats performing aerobic interval exercise, OS, Obese (fa/fa) sedentary rats, OE, Obese (fa/fa) rats performing aerobic interval exercise. Photographs are representative of livers of 10 different rats for each experimental group. nc, necrosis, mv, microvesicular steatosis, fd, fatty droplet accumulation.

Figure 6

Effect of dietary treatment with raw or 4 day-germinated V. radiata and aerobic interval training protocol on AMPKα/pAMPKα protein expression in the liver of lean and obese Zucker rats measured by Western blot analysis. AMPK, 5′-AMP-activated protein kinase; phosphorylated-AMPK, pAMPK. RVR, raw V. radiata, GVR, 4 day-germinated V. radiata. Groups: LS, Lean (fa/+) sedentary rats, LE, Lean (fa/+) rats performing aerobic interval exercise, OS, Obese (fa/fa) sedentary rats, OE, Obese (fa/fa) rats performing aerobic interval exercise. Immunoblots are representative of liver homogenates of eight different rats for each experimental group; all samples were derived at the same time and processed in parallel. The amount of sample loaded per lane was 100 µg of protein. Levels of pAMPK were normalized to the total AMPK. Densitometric analysis values represented in the graphs are means ± SEM depicted by vertical bars (n = 8). Means without a common letter differ, p < 0.05.