Branched chain amino acid metabolism profiles in progressive human nonalcoholic fatty liver disease

Abstract

Nonalcoholic fatty liver disease (NAFLD) is a globally widespread disease of increasing clinical significance. The pathological progression of the disease from simple steatosis to nonalcoholic steatohepatitis (NASH) has been well defined, however, the contribution of altered branched chain amino acid metabolomic profiles to the progression of NAFLD is not known. The three BCAAs: leucine, isoleucine and valine are known to mediate activation of several important hepatic metabolic signaling pathways ranging from insulin signaling to glucose regulation. The purpose of this study is to profile changes in hepatic BCAA metabolite levels with transcriptomic changes in the progression of human NAFLD to discover novel mechanisms of disease progression. Metabolomic and transcriptomic data sets representing the spectrum of human NAFLD (normal, steatosis, NASH fatty, and NASH not fatty livers) were utilized for this study. During the transition from steatosis to NASH, increases in the levels of leucine (127% of normal), isoleucine (139%), and valine (147%) were observed. Carnitine metabolites also exhibited significantly elevated profiles in NASH fatty and NASH not fatty samples and included propionyl, hexanoyl, lauryl, acetyl and butyryl carnitine. Amino acid and BCAA metabolism gene sets were significantly enriched among downregulated genes during NASH. These cumulative alterations in BCAA metabolite and amino acid metabolism gene profiles represent adaptive physiological responses to disease-induced hepatic stress in NASH patients.

Fig. 1

Principal components analysis of amino acid genes and metabolites in human NAFLD. In a the first principal component distinguishes NASH from normal and steatosis amino acid gene expression. This plot demonstrates that gene expression differences among amino acid genes are able to differentiate between samples with a NASH diagnosis versus those designated as normal or steatosis. b The metabolomics PCA profile of amino acid and acylcarnitine metabolites distinguishes between sample diagnosis groups although diagnosis is shown to be correlated more with the second and not the first principal component in this plot

Fig. 2

Hierarchical clustering analysis of BCAA and MTOR signaling genes. In a the heatmap shown for BCAA metabolism, transport, and signaling genes demonstrates the clustering of samples diagnosed as NASH fatty or NASH not fatty according to gene expression changes that distinguishes NASH liver samples as a whole from normal and steatosis samples. In b the MTOR signaling gene category also shows that samples diagnosed as NASH cluster together within the heatmap while steatosis and normal samples do not. Upregulation of genes is represented by red squares within the heat-map while downregulation is represented by blue squares and genes that are unchanged are represented by yellow. Diagnosis groups are represented along the tops of the heatmap as black (normal), blue (steatosis), yellow (NASH fatty), and orange (NASH not fatty) (color figure online)

Fig. 3

Metabolomic composition of hepatic amino acids in progressive stages of human NAFLD. The metabolomics profiling results for the BCAAs leucine, isoleucine and valine with the amino acids tyrosine, alanine, and phenylalanine are shown as log-transformed area under the curve (AUC) mass spectrometry units normalized to the median value of the normal samples ± the standard deviation. Significance from normal is shown by asterisk and significance from steatosis is represented by pound. Significance set at p ≤ 0.05

Fig. 4

Metabolomic composition of acylcarnitine levels in progressive stages of human NAFLD. Short, medium, and long chain hepatic acylcarnitine levels are shown as log-transformed AUC mass spectrometry units normalized to the median value of the normal samples ± the standard deviation. Significance from normal is shown by asterisk and significance from steatosis is represented by pound. Significance was set at p ≤ 0.05

Fig. 5

BCAA metabolizing enzyme, transporter, and mTOR signaling genes in progressive human NAFLD. Gene expression values of BCAT1, BCKDK, SLC6A15, SLC16A10, SLC43A1, MTOR, AKT1, RPS6KA5, and RPS6KB2 are shown for normal, steatosis, NASH fatty and NASH not fatty samples. Log2-transformed values ± the standard deviation are normalized to the median of the normal samples. Significance from normal is shown by asterisk and significance from steatosis is represented by pound. Significance was set at p ≤ 0.05

Fig. 6

Hepatic BCAA enzyme protein expression in progressive human NAFLD. Relative protein expression levels ± the standard deviation of BCKDK and BCAT1 are shown for normal (n = 7), steatosis (n = 7), NASH fatty (n = 11), and NASH not fatty (n = 13) samples. Representative blots of normal (n = 3), steatosis (n = 3), NASH fatty (n = 4), and NASH not fatty (n = 4) samples are shown. Total ERK was utilized as control protein. Significance from normal is shown by asterisk and significance from steatosis is represented by pound. Significance was set at p ≤ 0.05