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Discussion
The present study is the first targeted and biology driven metabolomics profiling of chronic HBV infection, characterizing the natural progression through its distinct clinical phases. With the liver being the central organ in nutritional regulation and metabolism, it is not surprising that chronic HBV infections have been shown to induce multiple metabolic alterations in lipid metabolism of the host [15, 16, 17, 18, 19, 20, 21]. However, these studies have not addressed and positioned metabolic changes in relation to the progression of chronic HBV disease.
The IT phase is clinically characterized by high levels of HBV replication and minimal hepatic injury. We now show that this phase exhibits major lipid alterations, with increased free fatty acids, acyl-carnitines, and plasmalogens concurrent with decreased triglyceride, phospholipid (ester-linked), and sphingomyelin levels (Fig. 4). The glycolysis intermediate dihydroxyacetone phosphate (DHAP) is the precursor metabolite via glycerol-3-phosphate (G3P) for the de novo glycerophospholipids and triglyceride synthesis pathways. Alternatively, DHAP can also be transported to the peroxisomes where it is the precursor for the synthesis of vinyl ether-linked plasmalogen phospholipids. The DHAP to G3P reaction is catalyzed by glycerol-3-phosphate dehydrogenase (GPDH) and is known as the G3P–NADH shuttle, simultaneously converting NADH to NAD+ necessary during the glycolysis cycle. Our results suggest that HBV hijacks this G3P–NADH shuttle, resulting in reduced levels of glycerophospholipids, lysoglycerophospholipids, triglycerides, and sphingomyelins. The secretion of glycerophospholipids from the liver is the main contributor to their serum levels, substantiating the hepatic metabolic fingerprint and the link to HBV activity [36]. Plasmalogen phosphatidylcholine has been identified as the preferred lipid species in the viral envelope and surface antigen particles of HBV, accounting for approximately 60 % of total lipid content [37]. Additionally, Li et al. [16] reported the upregulation of mRNA transcripts in the phosphatidylcholine biosynthesis pathway during HBV infection in HepG2 cell lines and its necessity for HBV replication. Collectively, the observed increase in choline plasmalogen levels reveals the metabolic engineering capacity of HBV and supports our hypothesis of HBV hijacking of the G3P–NADH shuttle. Importantly, this altered lipid profile of down-regulated phospholipids and increased plasmalogens persists during the progression of chronic HBV infection (Fig 3a, b), even during the IC phase where virtually no HBV replication is taking place, implying a HBV-induced metabolic imprinting effect. Although other reasons could be used to explain metabolic observations (such as an altered metabolic flux or consumption rate of metabolites and even diet), the use of pattern analyses and the progressive nature of our clinical phase-defined chronic HBV samples provide concrete evidence to illustrate an altered metabolic state.
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Fig. 4
Metabolic alterations identified in the IT phase of chronic HBV. Increased levels of DHAP-derived plasmalogen phospholipid species, free fatty acids, and acyl carnitines were found. A significant decrease in glycerophospholipids, triglycerides, and sphingomyelins was found, suggesting the HBV hijacking of the cytosolic glycerol-3-phosphate dehydrogenase (GPDH) enzyme, favoring the synthesis of plasmalogen lipid species. Detected metabolic species are highlighted in red with the arrow indicating its trend. DHAP dihydroxyacetone phosphate, G3P glycerol-3-phosphate, LPA lysophosphatidic acid, PA phosphatidic acid, DG diacylglycerol, PE phosphatidylethanolamine, PC phosphatidylcholine, LPE lysophosphatidylethanolamine, LPC lysophosphatidylcholine, TG triglyceride, FFA free fatty acids, pPE plasmalogen phosphatidylethanolamine, pPC plasmalogen phosphatidylcholine, pLPE plasmalogen lysophosphatidylethanolamine, pLPC plasmalogen lysophosphatidylcholine, GAPDH glyceraldehyde-3-phosphate dehydrogenase, TPI triosephosphate isomerase, cGPDH cytosolic glycerol-3-phosphate dehydrogenase, mGPDH mitochondrial glycerol-3-phosphate dehydrogenase
During the natural progression of chronic HBV, we measured increased choline, methionine, and very long acyl chain triglyceride levels together with reduced phosphatidylcholine and lysophosphatidylcholine levels, indicating a perturbed choline metabolism. Dietary choline and methionine depravation is strongly linked to the development of steatosis, non-alcoholic fatty liver disease, cirrhosis, and HCC [38, 39, 40, 41]. During conditions of choline restriction, the reduced levels of phosphatidylcholine, a critical component of the very low density lipoprotein particle, impair hepatic lipoprotein synthesis and result in the accumulation of free triglycerides within hepatocytes [42, 43]. Decreased phosphatidylcholine species in the presence of high choline levels during the IA, IC, and ENEG phases support the permanent G3P–NADH shuttle hijacking hypothesis, impairing lipoprotein synthesis during chronic HBV, while also explaining the accumulation of triglycerides. Even with decreased levels of long chain free fatty acids in the IC phase, no attenuation of long chain triglyceride levels was observed in this phase. The stable elevated levels of betaine, sarcosine, and methionine indicate enhanced choline catabolism, while increased levels of methionine are also reflective of hepatic injury [32, 33]. Previous studies demonstrated that host factors were responsible for the development of steatosis rather than viral factors [44, 45]. Our metabolomics data suggest that initiation of steatosis may be a consequence of HBV hijacking of the host’s glycerophopholipid metabolism, as liver fat content closely correlates with serum triglyceride levels [46, 47].
Another metabolic pathway with increased activity reflective of the natural progression of chronic HBV infection is composed of urea cycle intermediates: enhanced levels of citrulline and ornithine were detected in the IC and ENEG phases, respectively. The urea cycle is predominantly active in hepatocytes and responsible for detoxifying ammonia. Moreover, it has an intimate relationship with the aspartate–malate NADH shuttle functioning across the mitochondrial membrane. One could speculate that the HBV hijacking of the G3P–NADH shuttle, as explained above, will affect the redox status (NADH/NAD+) of the cell and cause an up-regulation/stress of the malate–aspartate NADH/NAD+ shuttle to rectify this imbalance (Fig. 5). The mitochondrial transporter citrin (AGC), encoded by the gene SLC25A13, is responsible for the transport of aspartate and glutamate during aspartate–malate NADH shuttling. Cytosolic aspartate binds to citrulline to form the urea cycle intermediate argininosuccinate, which can be converted to arginine, which in turn is converted to ornithine with the release of urea. We detected increased levels of citrulline, ornithine and glutamate, all implicating impaired aspartate transport, and may reflect reduced integrity and functioning of the mitochondrial citrin transporter. A dysregulated urea cycle precedes the histological manifestations of irreversible liver damage [48], and thus might be a prediction marker for chronic HBV progression and severity.
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Fig. 5
Cellular NADH shuttles and the urea cycle. The interplay between a the reduced G3P–NADH shuttle and b a stressed aspartate–malate NADH shuttle and its influence on c the urea cycle. The aspartate transporter AGC (aka citrin) facilitates the transport of aspartate across the mitochondrial membrane to the cytosol where it binds to citrulline in the urea cycle to help detoxify ammonium. Ineffective aspartate transport will lead to the accumulation of glutamate, citrulline, and ornithine identified in the IC and ENEG clinical phases of HBV infection
Collectively, the data presented here comprise the first metabolic study on the natural progression of chronic HBV infection using patient samples. We found impaired choline glycerophospholipid metabolism across the four chronic HBV clinical phases, together with increasing triglyceride and urea cycle intermediate levels as a liver metabolic fingerprint of the progression of chronic HBV infection. This metabolic fingerprint relates to HBV’s hijacking of the G3P–NADH shuttle, a key player in the plasmalogen, choline, and glycerophospholipid metabolic pathways, and its potential as a therapeutic target deserves further investigation. Elegant work done by Zeissig et al. [34] demonstrated the role of lysophospholipids as endogenous antigenic lipid species able to illicit protective immunological responses, which enhanced HBV clearance during acute infection. This may imply that, in addition to redirecting host lipid metabolism to produce the plasmalogens, hijacking of glycerophospholipid metabolism during acute HBV infection could act as a switch to determine HBV clearance or persistence. Furthermore, the diabetes drug metformin, which inhibits mitochondrial GPDH [49], part of the G3P–NADH shuttle, was found to inhibit HBV protein production and replication [50]. These findings substantiate the therapeutic value of the G3P–NADH shuttle in chronic HBV infection.
Conclusions
The present study provides many insights and leads to design follow-up studies and, at the same time, highlights the need for a systems biology approach to better understand chronic HBV infection. We identified liver-related metabolic and injury perturbations, which reflect the natural progression of the disease. The altered glycerophospholipid metabolism in the IT phase attributed to the HBV hijacking of the G3P–NADH shuttle has an intimate relationship with the persistent lipid dysregulation observed in the IA, IC, and ENEG clinical phases. Increased levels of the very long chain triglycerides in the IA phase and urea cycle intermediates in the IC phase highlight the risk for developing secondary liver complications during chronic HBV infection. These metabolites might prove useful as markers of disease progression and severity.
Abbreviations
ALT, alanine aminotransferase; DHAP, dihydroxyacetone phosphate; ENEG, HBeAg-negative; G3P, glycerol-3-phosphate; GPDH, glycerol-3-phosphate dehydrogenase; HBeAg, HBV envelope antigen; HBsAg, HBV surface antigen; HBV, hepatitis B virus; HC, healthy control; HCC, hepatocellular carcinoma; IA, immune active; IC, inactive carrier; IDO, indoleamine 2,3-dioxygenase; IT, immune tolerant; LC–MS, liquid chromatography–mass spectrometry; QC, quality control.
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发表于 2016-7-5 15:52