Gallic acid ameliorated impaired glucose and lipid homeostasis in high fat diet-induced NAFLD mice

Abstract

Gallic acid (GA), a naturally abundant plant phenolic compound in vegetables and fruits, has been shown to have potent anti-oxidative and anti-obesity activity. However, the effects of GA on nonalcoholic fatty liver disease (NAFLD) are poorly understood. In this study, we investigated the beneficial effects of GA administration on nutritional hepatosteatosis model by a more "holistic view" approach, namely 1H NMR-based metabolomics, in order to prove efficacy and to obtain information that might lead to a better understanding of the mode of action of GA. Male C57BL/6 mice were placed for 16 weeks on either a normal chow diet, a high fat diet (HFD, 60%), or a high fat diet supplemented with GA (50 and 100 mg/kg/day, orally). Liver histopathology and serum biochemical examinations indicated that the daily administration of GA protects against hepatic steatosis, obesity, hypercholesterolemia, and insulin resistance among the HFD-induced NAFLD mice. In addition, partial least squares discriminant analysis scores plots demonstrated that the cluster of HFD fed mice is clearly separated from the normal group mice plots, indicating that the metabolic characteristics of these two groups are distinctively different. Specifically, the GA-treated mice are located closer to the normal group of mice, indicating that the HFD-induced disturbances to the metabolic profile were partially reversed by GA treatment. Our results show that the hepatoprotective effect of GA occurs in part through a reversing of the HFD caused disturbances to a range of metabolic pathways, including lipid metabolism, glucose metabolism (glycolysis and gluconeogenesis), amino acids metabolism, choline metabolism and gut-microbiota-associated metabolism. Taken together, this study suggested that a 1H NMR-based metabolomics approach is a useful platform for natural product functional evaluation. The selected metabolites are potentially useful as preventive action biomarkers and could also be used to help our further understanding of the effect of GA in hepatosteatosis mice.

Figure 1. Gallic acid (GA) protects against hepatic steatosis and insulin resistance in high-fat diet-fed mice.

(A) Effect of a high fat diet and gallic acid treatment on liver weight. (B–C) Administration of gallic acid for 16 weeks effectively improves glucose and insulin concentrations in mice fed the high fat diet. Serum insulin levels and blood glucose were assessed in mice fed a normal chow diet (normal group, n = 10), a high fat diet (HFD group, n = 11), and a high fat diet supplemented with GA (treatment group, high fat diet+GA, 50 (the number of mice used for the serum insulin analysis, n = 8; other experiments, n = 10) and 100 (the number of mice used for the serum insulin analysis, n = 8; other experiments, n = 10) mg/kg/day, orally). The data are presented as the mean ± SEM. #p<0.05, versus normal diet mice; *p<0.05, versus high fat diet-fed mice. (D and E) The gross morphology of the mouse livers and H&E staining of liver sections in different groups.

Figure 2. Gallic acid (GA) ameliorates changes in the serum biochemical parameters of mice with hepatic steatosis induced by high fat-diet feeding.

(A) Serum triglyceride. (B) Serum cholesterol. (C) Serum high-density lipoprotein (HDL). (D) Aspartate aminotransferase (AST). (E) Alanine aminotransferase (ALT). The serum biochemical parameters were assessed in mice fed a normal chow diet (normal group, n = 10), a high fat diet (HFD group, n = 11), and a high fat diet supplemented with GA (treatment group, high fat diet+GA, 50 (the number of mice used for the AST and ALT analysis, n = 9; other experiments, n = 10) and 100 (the number of mice used for the AST and ALT analysis, n = 9; other experiments, n = 10) mg/kg/day). The data are presented as the mean ± SEM. #p<0.05, versus normal diet mice; *p<0.05, versus high fat diet-fed mice.

Figure 3. GA Gallic acid (GA) reduced liver lipid accumulation in high-fat diet-fed mice.

(A) Liver cholesterol; (B) Liver triglyceride; (C) Liver fatty acids; (D) PUFA/MUFA ratio. The relative integrals of the liver cholesterol, liver triglyceride and liver fatty acids were calculated from the spectral regions at δ 0.670−0.695 for liver cholesterol (C18-H₃), at δ 4.120−4.170 for liver triglyceride (Glycerol (C1-H^u) and (C3-H^u)) and at δ 0.81−0.93 for methyl groups of all fatty acids (−CH₃). The PUFA-to-MUFA ratio was calculated from the spectral regions at δ 5.29−5.44 for UFA (−CH = CH−), at δ 2.73−2.88 for PUFA (−C = C−CH₂−C = C−) and at δ 0.81−0.93 for methyl groups of all fatty acids (−CH₃) . The data are presented as the mean ± SEM. #p<0.05, versus normal diet mice; *p<0.05, versus high fat diet-fed mice. The relative integrals were normalized against the weight of the wet tissue used for liver the extract.

Figure 4. Typical 600 MHz ¹H CPMG (A), NOESY (B), projected J-resolved (C),and BPP-LED (D) spectra of serum samples and ¹H NOESY (E) and projected J-resolved (F) spectra of urine samples collected from the mice fed a normal chow diet at the 16 weeks.

The keys metabolites in the serum and urine were assigned. The chemical shifts and peak multiplicity are described in Table S1 and Table S2.

Figure 5. O-PLS-DA results for normal chow diet and high fat diet-fed mice derived from ¹H NMR CPMG spectra of serum (A, B, C), BPP-LED spectra of serum (D, E, F), and NOESY spectra of urine (G, H, I).

O-PLS-DA scores plots (A, D, G), coefficient-coded loadings plots (B, E, H), and the S-plot combined with the VIP plot and color coefficient scale bar (C, F, I) for the models discriminating the normal group (black filled dots) and HFD groups (red filled dots) based on data for plasma and urine. Metabolite keys to the number are shown in Table 1 and Table 2 . Nor, normal group; HFD, high fat diet group.

Figure 6. PLS-DA scores plots for (A) standard 1D CPMG spectra of serum, (B) NOESY spectra of serum, (C) BPP-LED spectra of serum, and (D) NOESY spectra of urine from normal group, high fat diet group, and GA treatment group.

Nor, normal group; HFD, high fat diet group; GAH and GAL, high and low dose of GA treatment group.

Figure 7. Disturbed metabolic pathways in high-fat diet-induced hepatosteatosis mice.

The metabolic pathways where it was that gallic acid treatment was able to intervene are indicated by blue arrows. ↑, Up-regulated; ↓, down-regulated; red color, serum; blue color, urine. BCAA, Branched-chain amino acids; TCA cycle: tricarboxylic acid-cycle; TMA: Trimethylamine; DMA: Dimethylamine.