Liver receptor homolog 1 and transmethylation fluxes in nonalcoholic steatohepatitis.

Abstract

Potential conflict of interest: Dr. Mato consults and owns stock in Owl. He consults for Abbott. See Article on Page 95 In the 1930s, the American biochemist Vincent du Vigneaud, who won a Nobel Prize in Chemistry in 1955 for his work on the metabolism of sulfur compounds, discovered, through a series of elegant rat feeding experiments, that only the sulfur atom of methionine, not its carbon chain, was utilized for the biosynthesis of cysteine, a process that became known as “transsulfuration” (Fig. 1). Du Vigneaud also observed that rats fed a methionine‐cysteine–free diet that was supplemented with homocysteine failed to grow and developed fatty liver. Du Vigneaud, who knew of the work of Charles Best (the codiscoverer of insulin) showing that rats fed a choline‐deficient diet developed fatty liver, hypothesized that choline, a molecule rich in methyl groups, was the factor missing in his diet. After demonstrating that choline could act as the methyl donor for the synthesis of methionine from homocysteine in his experiments, Du Vigneaud also observed that rats grew well and failed to develop fatty liver when fed a choline‐free diet that was supplemented with methionine, which led him to speculate that methionine could serve as a methyl source for choline synthesis (Fig. 1). Du Vigneaud coined the term “transmethylation” to refer to the transfer of methyl groups from methionine to choline and from choline to methionine. He also found that the biosynthesis of creatine from guanidinoacetic acid involved the transfer of a methyl group derived from methionine. Moreover, he hypothesized that to transfer its methyl group to synthesize creatine, the bond between the methyl group and the sulfur atom in methionine needed to be weakened and that “the formation of a sulfonium ion would be expected to effect such a labilization” (Fig. 1). This hypothetical sulfonium ion received the name of “active methyl donor” and it was Giulio Cantoni who, in 1953, demonstrated that (1) methionine combines with adenosine triphosphate (ATP) to generate S‐adenosylmethionine (SAMe), a sulfonium ion, through a reaction catalyzed by the enzyme, methionine adenosyltransferase (MAT), and (2) SAMe reacted with guanidinoacetic acid to form creatine through a reaction catalyzed by the enzyme, guanidinoacetic acid N‐methyltransferase (GAMT). In 1959, Bremer and Greenberg observed that (1) phosphatidylcholine (PC) could be synthesized from phosphatidylethanolamine (PE) by three consecutive methylation reactions, with SAMe as the methyl donor, and (2) PC could be cleaved with formation of choline and diacylglycerol (Fig. 1). The discovery of this reaction, known as PE N‐methyltransferase (PEMT), provided the final proof of the transfer of methyl groups from methionine to choline as well as the rationale to feed rats and mice a diet deficient in methyl groups (methionine and choline) to induce nonalcoholic steatohepatitis (NASH).Figure 1: Methionine (Met), an essential amino acid, combines with ATP (not shown) to generate SAMe, (a sulfonium ion) through a reaction catalyzed by the enzyme, MAT. A variety of specific methyltransferases (MT) transfer SAMe methyl group (CH3 in blue) to a large variety of substrates (DNA, histones, guanidinoacetic acid, lipids, and so on). PEMT catalyzes the synthesis of PC from PE by three consecutive N‐methylation reactions, with SAMe as the methyl donor. Of all liver MTs, GNMT, which converts glycine in sarcosine, is the most abundant and acts as a chemical rheostat that maintains hepatic SAMe content constant. The side product of all transmethylation reactions is S‐adenosylhomocysteine (SAH), which is hydrolyzed through a reversible reaction catalyzed by the enzyme, SAH hydrolase (SAHH), to generate homocysteine (Hcy; which conserves the sulfur atom of methionine) and adenosine (not shown). Hcy is a branch‐point metabolite that may react with serine (not shown) to enter the trans‐sulfuration pathway to yield cysteine (Cys) and its downstream metabolites, taurine (Tau) and glutathione (GSH). Only the sulfur atom of methionine (in red), not its carbon chain, is utilized for the synthesis of Cys, Tau, and GSH. The sulfur atom of methionine is finally secreted in urine as sulfate. PC can be cleaved with formation of diacylglycerol (DG) and choline, which, after being converted to betaine (not shown), transfers a methyl group to Hcy to form Met through a reaction catalyzed by the enzyme, betaine homocysteine S‐methyltransferase (BHMT). Alternatively, Hcy may receive a methyl group from methyl‐tetrahydrofolate (CH3‐THF) to generate Met through a reaction catalyzed by Met synthase (MS). The pathway linking serine (Ser) to CH3‐THF is known as methyl‐neogenesis. The nuclear receptor, LRH‐1, may be activated by DLPC7 and labile methyl groups (LMG),6 leading to down‐regulation of SREBP‐1c and inhibition of lipogenesis, as well as activation of GNMT and reduction of SAMe content. TG, triacylglycerol.Studies carried out by Laurence Kinsell in the mid‐to‐late 1940s established the essential role of the liver in methionine and sulfur metabolism as well as the impaired clearance of this amino acid in patients with liver injury. In 1988, Mato's laboratory observed that liver MAT and PEMT activities were markedly reduced in patients with liver cirrhosis.1 On the basis of these findings, showing in essence that labile methyl balance and transmethylation fluxes were deficient in patients with liver injury, Lu and Mato speculated that SAMe deficiency could be sufficient to induce NASH. This hypothesis was proven to be correct by showing that deletion of Mat1a, the main gene involved in hepatic SAMe synthesis, reduced SAMe content leading to spontaneous development of NASH and hepatocellular carcinoma (HCC).2 Of all liver methyltransferases, glycine N‐methyltransferase (GNMT) is particularly important given that it acts as a chemical rheostat that maintains cellular SAMe content constant (Fig. 1). Accordingly, Gnmt deletion in mice was shown to induce the accumulation of hepatic SAMe.4Gnmt−/− mice also developed NASH and HCC, indicating that total transmethylation fluxes need to be tightly regulated.4 However, little is known on the transcriptional regulation of hepatic transmethylation fluxes. The study of Wagner et al. in this issue of Hepatology6 describing the function of the nuclear receptor liver receptor homolog 1 (LRH‐1)/NR5A2 as a direct regulator of Gnmt unveils a novel episode in the regulation of labile methyl balance. Wagner et al. observed that Lrh‐1−/− mice fed a methionine‐ and choline‐deficient diet (MCD), although having developed hepatic steatosis, were more resistant to develop liver injury (examined by measuring liver enzymes, inflammatory markers, and fibrosis) than wild‐type (WT) animals. The investigators found that MCD‐fed Lrh‐1−/− mice livers showed higher SAMe content and SAMe/S‐adenosylhomocysteine ratio (an estimation of the cell's transmethylation capacity) than WT controls. GNMT messenger RNA (mRNA) and protein content was markedly decreased in Lrh‐1−/− mice, which led the investigators to propose that a reduced flux of SAMe through the GNMT pathway may contribute to reduced injury in MCD‐fed Lrh‐1−/− mice by saving SAMe methyl groups for choline synthesis, through the PEMT pathway, as well as for other essential transmethylation reactions (Fig. 1). Hepatic PC/PE ratio, which is critical to maintain membrane integrity and function, was markedly decreased in MCD‐fed WT mice and this response was blunted in MCD‐fed Lrh‐1−/− mice. Mdr2/Abcb4, a flippase that shuttles phospholipids from hepatocytes into bile, was markedly reduced in Lrh‐1−/− mice, which led the investigators to speculate that a reduced transport of PC molecules through Mdr2 may also contribute to reduced injury in MCD‐fed Lrh‐1−/− mice by saving PC molecules. The investigators also determined whether the decrease in Gnmt and Mdr2 mRNA content was a direct result of LRH‐1 loss. Small interfering RNA–mediated knockdown of LRH‐1 in AML2 cells, a murine‐derived hepatocyte cell line, resulted in reduction of Gnmt and Mdr2 mRNA levels, and the administration of didodecanoyl‐PC (PC12:0/12:0 or DLPC), an LRH‐1 ligand, markedly increased Gnmt and Mdr2 mRNA content. Through a series of well‐designed experiments, Wagner et al. concluded that LRH‐1 is a direct transcriptional regulator of Gnmt, whereas regulation of Mdr2 transcription may be mediated by other factors, such as bile acids. Finally, the investigators determined the effect of depletion of labile methyl groups on LRH‐1 activity. They observed that, in C3HepG2 cells, incubation with MCD media induced a time‐ and dose‐dependent decrease in LRH‐1 luciferase activity. The mRNA content of the canonic LRH‐1 target genes, CYP8B1 and CYP7A1, and of the methyltransferases, GNMT and GAMT, was significantly reduced in C3HepG2 cells incubated with MCD media. In essence, Wagner et al. have made the novel observation that LRH‐1 is a transcriptional regulator of key enzymes that control hepatic total transmethylation fluxes and labile methyl balance, and that methyl‐pool depletion induces an LRH‐1 response that promotes maintenance of SAMe content and PC/PE ratio (Fig. 1). Although these results clearly demonstrate that LRH‐1 ablation protects MCD‐induced liver injury and stress the therapeutic potential in NASH of controlling transmethylation fluxes and labile methyl balance, it is important to emphasize that LRH‐1 inactivation in this model did not prevent steatosis, which agrees with earlier work showing that LRH‐1 agonists, such as DLPC, have lipotropic and antidiabetic effects in mouse models of insulin resistance, an effect that seems to be mediated through down‐regulation of sterol regulatory element‐binding protein 1c (SREBP‐1c) expression and its downstream lipogenic targets.7 Together, these findings indicate that LRH‐1 can control hepatic fatty acid homeostasis at two different levels, tuning SREBP‐1c activity and adjusting SAMe content (Fig. 1). The role of LRH‐1 signaling in human NASH remains to be examined, and whether targeting this pathway could be a therapeutic strategy is unclear. It is tempting to suggest that whereas some NASH patients may profit from activation of the LRH‐1 pathway, others may benefit from its down‐regulation.