Effect of dietary monosodium glutamate on trans fat-induced nonalcoholic fatty liver disease

Abstract

The effects of dietary monosodium glutamate (MSG) on trans-fatty acid (TFA)-induced nonalcoholic fatty liver disease (NAFLD) are addressed in an animal model. We used Affymetrix microarray analysis to investigate hepatic gene expression and the contribution of visceral white adipose tissue (WAT) to diet-induced NAFLD. Trans-fat feeding increased serum leptin, FFA, HDL-cholesterol (HDL-C), and total cholesterol (T-CHOL) levels, while robustly elevating the expression of genes involved in hepatic lipogenesis, including the transcription factor sterol-regulatory element binding protein 1c. Histological examination revealed hepatic macrosteatosis in TFA-fed animals. Conversely, dietary MSG at doses similar to human average daily intake caused hepatic microsteatosis and the expression of beta-oxidative genes. Serum triglyceride, FFA, and insulin levels were elevated in MSG-treated animals. The abdominal cavities of TFA- or MSG-treated animals had increased WAT deposition compared with controls. Microarray analysis of WAT gene expression revealed increased lipid biosynthetic gene expression, together with a 50% decrease in the key transcription factor Ppargc1a. A combination of TFA+MSG resulted in the highest levels of serum HDL-C, T-CHOL, and leptin. Microarray analysis of TFA+MSG-treated livers showed elevated expression of markers of hepatic inflammation, lipid storage, cell damage, and cell cycle impairment. TFA+MSG mice also had a high degree of WAT deposition and lipogenic gene expression. Levels of Ppargc1a were further reduced to 25% by TFA+MSG treatment. MSG exacerbates TFA-induced NAFLD.

Fig. 1.

Diet-induced changes in liver histology and TG content. A: Increased lipid deposition, as indicated by Oil-Red-O lipid staining in liver sections from mice on the MSG, TFA, and TFA+MSG diet groups at 32 weeks, compared with control. Red staining indicates lipid deposition. Scale is ×790. Micrographs are representative of five different experiments per diet group. B: Microvesicular steatosis in HE-stained liver sections. Scale is ×790. Micrographs are representative of five different experiments per diet group. C: Higher-magnification micrographs. D: Liver weight differences among the four different diet groups. Results are mean ± SEM (P ≤ 0.05, n = 6). E: Quantitation of hepatic TG content from mice in the control (standard chow), MSG, TFA, and TFA+MSG diet groups. Results are mean ± SEM (P ≤ 0.05, n = 6).

Fig. 2.

Liver expression heat map and unsupervised hierarchical clustering analysis. Red pseudocolor and blue represents upregulated and downregulated genes, respectively. Each row represents a differentially expressed gene within livers from mice in the four diet groups (P ≤ 0.05). Each column represents the average from four samples in each diet group. For the hierarchical tree clustering of differentially expressed genes from liver, both genes and samples are clustered using Pearson dissimilarity.

Fig. 3.

Validation of Affymetrix microarray analysis of gene expression using qRT-PCR. A: qRT-PCR ratios of cyp7a1 (NM_ 007824), GADD45b (NM_008655), SREBP1c (NM_011480), CIDEC (NM_178373), and MTTP (NM_008642) relative to actin in 16-week-old mouse liver mRNA from the four different diet groups (control, MSG, TFA, and TFA+MSG; n = 4 per diet group). B: PCR products run on 2% agarose gel and stained with ethidium bromide. C: Concordance of qRT-PCR (light bars) versus microarray data (dark bars) for Cyp7a1, GADD45b, SREBP1c, CIDEC, and MTTP (signal intensity expressed as a percentage of control SD, n = 4 per diet group). Pearson correlation coefficients (r) are indicated for each bar chart. Results are representative of a minimum of two separate qRT-PCR experiments performed in triplicate.

Fig. 4.

Effect of diet on hepatic SREBP1c activity: SREBP1c DNA binding activity was determined using ELISA-based SREBP1c activation kit and quantified in triplicate by colorimetry. The levels of hepatic SREBP1c activity increased by 2.9-fold in the livers of TFA diet mice compared with control (dark gray bars, mean ± SEM, P<0.01, n = 4 per diet group). Inclusion of a wild-type consensus oligonucleotide (light gray bars) attenuated the binding of nuclear SREBP1c, indicative of the specificity of the assay.

Fig. 5.

Diet-induced changes in visceral fat histology. A: Increased abdominal adipose distribution in 32-week-old mice from MSG, TFA, and TFA+MSG diet groups compared with control. B: HE-stained micrograph depicting increased adipocyte volume in visceral adipose tissues from MSG, TFA, and TFA+MSG treated mice compared with control. Results are representative of four separate experiments. C: Quantitation of changes in adipocyte area. The data are expressed as the means ± SEM of at least 30 cells per diet group (P ≤ 0.001).

Fig. 6.

WAT expression heat map and unsupervised hierarchical clustering analysis. Red pseudocolor and blue represents upregulated and downregulated genes, respectively. Each row represents a differentially expressed gene within livers from mice in the four diet groups (P ≤ 0.05). Each column represents the average from four samples in each diet group. For the hierarchical tree clustering of differentially expressed genes from liver, both genes and samples are clustered using Pearson dissimilarity.