The metabolic responses to hepatitis B virus infection shed new light on pathogenesis and targets for treatment

Abstract

Chronic infection caused by the hepatitis B virus (HBV), is strongly associated with hepatitis, fatty liver and hepatocellular carcinoma. To investigate the underlying mechanisms, we characterize the metabolic features of host cells infected with the virus using systems biological approach. The results show that HBV replication induces systematic metabolic alterations in host cells. HBV infection up-regulates the biosynthesis of hexosamine and phosphatidylcholine by activating glutamine-fructose-6-phosphate amidotransferase 1 (GFAT1) and choline kinase alpha (CHKA) respectively, which were reported for the first time for HBV infection. Importantly suppressing hexosamine biosynthesis and phosphatidylcholine biosynthesis can inhibit HBV replication and expression. In addition, HBV induces oxidative stress and stimulates central carbon metabolism and nucleotide synthesis. Our results also indicate that HBV associated hepatocellular carcinoma could be attributed to GFAT1 activated hexosamine biosynthesis and CHKA activated phosphatidylcholine biosynthesis. This study provides further insights into the pathogenesis of HBV-induced diseases, and sheds new light on drug target for treating HBV infection.

Figure 1. The alterations of metabolic profiles between HepG2.2.15 and HepG2 cells.

(A) Validated OPLS-DA scores and coefficient plots showing the discriminations of metabolic profiles of extracts of HepG2.2.15 (T) from HepG2 (W) cells. Here the resonance peaks pointing upward indicate an increase of metabolites in HepG2.2.15 cells and downwards a decrease. The color of the peaks represents correlation coefficient of a metabolite with the cutoff value of |r| is 0.602 (n = 10, P < 0.05). Detailed data are shown in Table S2. Keys: 7, adenosine; 8, adenosine 5′-diphosphate; 9, adenosine 5′-monophosphate; 11, Aspartate; 12, choline; 16, fumarate; 19, GSH; 22, guanosine; 24, inosine; 25, inosine 5′-monophosphate; 27, lactate; 33, N-acetyl-glucosamine; 37, phosphocholine; 47, UDP-glucuronate; 48, UDP-N-acetyl galactosamine; 49, UDP-N-acetyl glucosamine; 51, uridine; 52, uridine 5′-diphosphate. (B) Metabolites consumed by HepG2.2.15 and HepG2 cells, which are derived by subtracting levels of metabolites in the spent medium from fresh medium. Hence, higher levels of metabolites indicate more of the respective metabolites being consumed. (C) Metabolites secreted to cell medium. 4HPPA, 4-hydroxyphenylpyruvate. Data are shown as mean ± standard deviation (s.d.), n = 10, t-test, ***P < 0.001. The details of the metabolite assignment are shown in Figure S1 and Table S1.

Figure 2. Promotion of hexosamine biosynthesis is necessary for HBV replication.

(A) mRNA levels of GFAT1 in HepG2.2.15 and HepG2 cells. (B) Relative levels of UDP-N-acetyl glucosamine (UDP-GlcNAc) and UDP-N-acetyl galactosamine (UDP-GalNAc) in HepG2.2.15 and HepG2 cells. (C-E) The effects of inhibiting GFAT1 by culturing HepG2.2.15 cells with 2 mM DON. (C) Relative levels of HBsAg expression; (D) Relative levels of HBeAg expression; (E) Relative core-associated HBV DNA levels. (F and G) HepG2.2.15 cells were transfected by siRNA targeting GFAT1 (siGFAT1) or no sense control (siNC). (F) The transcriptional levels of GFAT1, data was normalized to actin; (G) Relative core-associated HBV DNA levels. For all graphs, data are shown as mean ± s.d., n = 3, t-test, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. The relationship between HBV replication and phosphatidylcholine synthesis.

(A) The mRNA levels of the enzymes involved in the phosphatidylcholine synthesis. (B) The levels of choline and phosphocholine in HepG2.2.15 and HepG2 cells. (C and D) HepG2.2.15 cells were transfected by siRNA targeting CHKA (siCHKA) or no sense control (siNC). (C) Relative mRNA levels of CHKA. (D) Relative core-associated HBV DNA levels. For all graphs, data are shown as mean ± s.d., n = 3, t-test, *P < 0.05, **P < 0.01.

Figure 4. The alterations of fatty acid composition between HepG2.2.15 and HepG2 cells.

TFA, total fatty acids. Data are shown as mean ± s.d., n = 10, t-test, ***P < 0.001. More information can be found in Table S3.

Figure 5. HBV replication induces oxidative stress as manifested by consumption of GSH.

(A) The levels of GSH in HepG2.2.15 and HepG2 cells, data are shown as mean ± s.d., n = 10, t-test, ***P < 0.001. (B) The mRNA levels of enzymes involved in GSH biosynthesis in HepG2.2.15 and HepG2 cells, Data are shown as mean ± s.d., n = 3, t-test, *P < 0.05, **P < 0.01. (C) The relative levels of glycine and glutamate in the cell detected by ¹H NMR, data are shown as mean ± s.d., n = 10, t-test, P > 0.05.

Figure 6. Summary of the changes of metabolic pathways caused by HBV replication.

Metabolites, enzymes and mRNAs of enzymes are shown in red when their levels are higher in HepG2.2.15 or in blue when lower. The enzymes are display in roman type and mRNAs of the genes are shown in italic type. The full names of the enzymes are shown in Table 1. The dashed line means that the reactions involved in the transformation between metabolites need more than one step. Notes: G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F1,6-2P, fructose-1,6-diphosphate; R5P, Ribulose-5P; Glu6P, Gluconate-6 phosphate; GC1,3-2P, Glyceric acid 1,3-biphosphate; GC3P, 3-Phosphoglyceric acid; GlcA6P, glucosamine-6-phosphate; PEP, phosphoenolpyruvate; PGO, glycerone phosphate; 3PGA, glyceraldehyde-3-phosphate; PRPP, phosphoribosyl pyrophosphate; OAA, oxaloacetate; SCoA, succinyl CoA. ^aThe consumed metabolites; ^bResults from references.