Mitigation of MAFLD in High Fat-High Sucrose-Fructose Fed Mice by a Combination of Genistein Consumption and Exercise Training

Abstract

Purpose: Metabolic dysfunction-associated fatty liver disease (MAFLD) is fueled by escalations in both sedentary behavior and caloric intake and is noted in obese type 2 diabetic (T2DM) patients. This study aimed to examine the effects of exercise and the phytoestrogen genistein in mice fed a high fat (60% fat) high sugar (55% fructose with 45% sucrose), HFHS diet.

Methods: Male C57BL/6J mice were assigned to five groups: HFHS, HFHS with genistein (600 mg/kg diet, HFHS+Gen), HFHS with moderate exercise (HFHS+Ex), and HFHS with combined genistein and moderate exercise (HFHS-Gen+Ex). Control lean mice were fed standard chow and water. Exercise consisted of 30-minute sessions of treadmill running five days/week for the 12-week study duration. Body weight was assessed weekly. Liver, kidney, fecal pellets and serum were extracted at the end of the study and maintained at -80°C.

Results: After 12 weeks of treatment, mice in the HFHS group had the highest hepatic lipid content. Plasma levels of glucose, insulin, leptin, cholesterol, amylin, and total fat content were significantly elevated in HFHS mice compared to control mice. HFHS feeding increased protein expression of carnitine palmitoyltransferase 1b (CPT-1b isoform) in gastrocnemius, CPT1a, glucose transporter protein 2 (GLUT2), glucocorticoid receptor (GR), and fructose 1,6-bisphosphate 1 (FBP1) expression in liver. Exercise alone had minor effects on these metabolic abnormalities. Genistein alone resulted in improvements in body weight, fat content, amylin, insulin sensitivity, and liver histopathology, GR, FBP1, and acetyl-CoA carboxylase 1 (ACC1). Combination treatment resulted in additional metabolic improvements, including reductions in hepatic lipid content and lipid area, alanine transferase activity, CPT1b, and CPT1a.

Conclusion: Our results indicate that a HFHS diet is obesogenic, inducing metabolic perturbations consistent with T2DM and MAFLD. Genistein alone and genistein combined with moderate intensity exercise were effective in reducing MAFLD and the aberrations induced by chronic HFHS feeding.

Keywords: Soy isoflavone; diabetes; exercise; genistein; hepatic steatosis; western diet.

Figure 1

Effects of 12 weeks of genistein treatment, exercise training, and combined treatment on glucose tolerance. (A) Glucose tolerance tests, GTTs, were performed in overnight fasted mice at week 11 of the study. Following an intraperitoneal bolus of glucose (2 mg/g body weight), glucose readings were obtained from the tail vein at time 15, 30, 60, and 120 minutes. Excursions in blood glucose for 120 minutes following bolus of glucose. (B) Average area under the curve (AUC) calculated from the GTT data shown in 1A. Values are reported as mean ± SEM for 4–5 mice in each group. *Significant difference from lean control mice, ^#Significant treatment effect, P < 0.05.

Figure 2

Effects of 12 weeks of genistein treatment, exercise training, and combined treatment on hepatic steatosis. (A) Representative histology liver sections stained with Oil Red O staining. Images are at 20x magnification and scale bar is 50 µm. (B) Average fat droplet size in liver. (C) Average fat area in liver per given area evaluated. (D) Serum alanine aminotransferase, ALT, a marker for hepatic injury. Values are reported as mean ± SEM for 7–10 mice in each group. *Significant difference from lean control mice, ^#Significant treatment effect, P < 0.05.

Figure 3

Effects of 12 weeks of genistein treatment, exercise training, and combined treatment on expression of key hepatic and gastrocnemius proteins. (A) Expression of carnitine palmitoyl transferase, CPT1b, in gastrocnemius. (B) Expression of GLUT4 in gastrocnemius. (C) Expression of glucose transporter protein, GLUT2, in liver. (D) Expression of glucocorticoid receptor, GR, in liver. (E) Expression of fructose-1,6-bisphosphate, FBP1, in liver. Protein expression was determined by Western blot analysis. Protein expression was normalized using either GAPDH or actin as the housekeeping gene. (F) Fecal corticosterone level. Fecal matter was collected from mice at the end of the study for the measurement of corticosterone levels (n = 7–8 samples/group). Values are reported as mean ± SEM for 2–3 independent experiments for each protein of interest performed on 4–8 samples per group. *Significant difference from lean, ^#Significant treatment effect, P < 0.05.

Figure 4

Effects of 12 weeks of genistein treatment, exercise training, and combined treatment on the expression of key hepatic proteins relating to fatty acid metabolism. (A) Expression of acetyl-CoA carboxylase, ACC1, in liver. (B) Expression of carnitine palmitoyl transferase, CPT1a, in liver. (C) Expression of fatty acid synthase, FAS, in liver. Protein expression was determined by Western blot analysis. Protein expression was normalized to actin. Values are reported as mean ± SEM for 2–3 independent experiments for each protein of interest performed on 4–8 samples per group. *Significant difference from lean, ^#Significant treatment effect, P < 0.05.