DIO in mice leads to NAFLD that is characterized by hepatic accumulation of fat which could result from increased fatty acid uptake, increased hepatic de novo
fatty acid synthesis, impaired oxidation of fatty acids or a reduced VLDL-mediated TG export 
. The latter is dependent on provision of sufficient PC for assembly of VLDL particles. It has been estimated that up to 30% of the hepatic PC biosynthesis originates from the PEMT pathway by a PEMT-mediated methylation of PE using SAM as a methyl-donor 
. Previously, we have shown that feeding C57BL/6N mice a beef tallow-based HF diet resulted in hyperglycemia, hyperinsulinemia, reduced glucose tolerance and hepatic TG accumulation 
. Furthermore, we have identified changes in the hepatic PL content and PC signature suggesting a DIO-based modulation of PC 
. Recently, Rubio-Aliaga et al.
also described alterations of hepatic C1-metabolism in mice upon HF diet feeding 
demonstrating a general link between DIO- induced NAFLD and the C1-metabolism.
We here extend these observations and provide evidence that hepatic methionine homeostasis is conserved in C57BL/6N mice on a HF diet based on a PPARα-mediated downregulation of the hepatic transsulfuration pathway and an enhanced Hcy remethylation capacity associated with an elevated taurine production despite repression of hepatic transsulfuration (). These findings are compatible with data of Finkelstein et al
. using rats fed a low methionine diet (0.25% w/w) with provision of extra dietary cystine (0.8% or 1.3% w/w) 
. In these rats, a hepatic methionine-sparing effect with reduced CBS activity accompanied with increased BHMT activity was observed. The lower CBS activity could only be detected under a low dietary methionine supply 
. Our findings in mice fed a HF diet with sufficient methionine (0.86%, w/w) and cystine (0.46%, w/w) suggest that, despite an adequate methionine supply, the transsulfuration pathway is downregulated whereas BHMT-dependent Hcy-remethylation is increased. Furthermore, we detected lower mRNA levels of CBS and an increase of BHMT mRNA with corresponding changes on protein level. Tang et al.
also identified a comparable mechanism in methionine-deprived C57BL/6 mice with a proposed post-transcriptional downregulation of hepatic CBS and increased BHMT enzyme activity associated with a transient increase in plasma Hcy level 
. Interestingly, we detected a reduced Hcy transsulfuration and an increased Hcy remethylation capacity. This finding is supported by reduced hepatic betaine concentration, increased BHMT protein expression and increased [DMG]/[betaine] ratio in HF mice, suggesting increased BHMT activity. In rats, Finkelstein et al
. showed that hepatic betaine content is influenced by Hcy remethylation and is dependent on dietary protein level 
, whereas the protein intake of control and HF mice in our study was not significantly different (Table S2
). Our finding of reduced hepatic betaine concentrations in obese mice is in line with data of Kim et al
. reporting significantly lower betaine levels in livers from DIO mice 
. In general, the increased hepatic BHMT protein expression, [DMG]/[betaine] ratio and reduced betaine content in our HF mice are indicative for an enhanced BHMT mediated Hcy-remethylation, however we did not find any evidence for changes in the hepatic methionine cycle in obese mice. This implies that maintaining methionine levels by reduced Hcy transsulfuration and enhanced Hcy remethylation is of prime importance for hepatocytes in NAFLD despite elevated taurine levels derived from either hepatic or extrahepatic cysteine. Since methionine is an essential amino acid, a reduction in methionine levels would critically affect protein synthesis capacity for the maintenance of the hepatic proteome as well as its protein secretion function.
Schematic presentation of analyzed changes in hepatic C1-metabolism after HF feeding in C57BL/6N mice.
The development of liver steatosis upon HF feeding is known to be associated with hepatic insulin resistance 
and enhanced rates of gluconeogenesis. HF mice analyzed in the present study exhibited similar characteristic phenotypic alterations as described previously 
. In Zucker diabetic fatty rats representing a type 2 diabetes model and in a type 1 diabetes animal model with streptozotocin an impaired insulin secretion and signaling and an increased hepatic gene expression of CBS and BHMT representing the branch-point enzymes of C1-metabolism have been reported 
. This is to some extent in line with our data suggesting increased Hcy remethylation and improved VLDL secretion due to increased BHMT expression in obese animals 
. Furthermore, the implication of BHMT in lipid and PL metabolism of the liver and adipose tissue was recently shown by Teng et al.
. However, the identified downregulation of CBS in our study contradicts a disturbed insulin signaling as described by others 
indicating that other mechanisms may have caused the observed decrease of CBS expression. In this context, reduced hepatic Cbs mRNA level and a reduced enzyme activity were also reported for rats fed a HF diet 
or for rats on HF diets treated with the PPARα agonist WY14,643 
. Our data also provide evidence for alterations in PPARα signaling in the liver of obese mice as shown by elevated mRNA expression of Pparα in conjunction with enhanced expression of prototypical PPARα target genes such as Cpt1a, Ucp2 or Acox1 (). Although PPARα is primarily considered to affect genes controlling lipid metabolism, it is also a regulator of hepatic amino acid metabolism 
. For example, Kersten et al.
have shown that PPARα can mediate suppressive effect on urea cycle enzymes 
, and inhibitory effects of fatty acids, which are ligands of PPARα, on ureagenesis 
and ammonia detoxification 
have been described. In this context, interestingly, we detected higher concentrations for glutamine and lower citrulline and ornithine concentrations in the liver of HF mice than in controls pointing to changes in ureagenesis and ammonia detoxification upon prolonged HF feeding. In addition, glutamine is known to influence fatty acid oxidation, lipolysis and glutathione biosynthesis 
. Regarding to the mechanistic role of PPARα in our study, we could demonstrate in hepatoma cells using the PPARα agonist WY14,643 that Cbs is a target gene of the PPARα signaling pathway () leading to a downregulation of the transsulfuration pathway comparably as shown by low CBS levels in HF mice. Interestingly, in vitro
studies in HepG2 cells show decreased PPARα expression dependent on Hcy concentration, establishing a possible feedback signaling of the methionine cycle on PPARα signaling via Hcy 
. Findings from CBS-deficient mice 
also corroborate the prominent role of the transsulfuration pathway and especially of CBS in the development of a fatty liver.
Taken together, mice fed a HF diet display an altered equilibrium between the transmethylation (methionine cycle) and processes that relate to the transsulfuration pathway such as glutathione and taurine synthesis which seem at least partially controlled by PPARα. The decrease in hepatic L-α-amino-n-butyrate levels found in our DIO mice suggest that α-ketobutyrate production from cystathionine in the transsulfuration pathway is also reduced which could affect also hepatic ophthalmic acid levels. This alternative non-thiol is produced by glutathione-synthetase when using L-α-amino-n-butyrate rather than cysteine as a substrate 
. Reduced levels of L-α-amino-n-butyrate would promote increased glutathione levels for proper redox-balance in liver. The increased taurine levels observed in DIO mice may also be indicative for changes in stress-response. The observed downregulation of Got1 gene expression known to be regulated by PPARα activity 
together with increased gene expression of Csad and elevated hepatic taurine concentrations in obese mice points to a reduced sulfate production from cysteine compensating reduced Hcy transsulfuration in favor of increased taurine synthesis. These changes may possibly improve osmoregulatory, cytoprotective and antioxidant capacities in the steatotic liver 
. In the nucleus, the principal methyl-donor, SAM, provides methyl-groups for DNA methylation and histone modification which are important for epigenetic gene expression regulation, chromatin condensation and genome integrity 
. Although we observed a downregulation of hepatic de novo
Dnmts, this did not seem to influence the global DNA methylation state in our DIO mice. Although, in male Sprague-Dawley rats, diabetes-mediated perturbations in C1-metabolism resulted in hepatic DNA-hypomethylation we could not confirm this in our DIO mice 
. The analysis of CpG island DNA methylation of the Cbs gene by MS-qPCR and analyzing 115 bp of a Cbs promoter CpG island (−270 until −155) containing a putative insulin response element (PEPCK-like [TGTTTGT] motif
by bisulfite conversion/pyrosequencing of liver genomic DNA from DIO mice compared with controls revealed no obvious changes in DNA methylation in the Cbs promoter or intragenic region. However, our DNA methylation analysis along the Cbs gene by MS-qPCR may have not detected DNA methylation due to the limited detection sensitivity of the MS-qPCR method at CpG sites in specific DNA sequence areas or in case of bisulfite conversion/pyrosequencing anaylsis with lower methylation frequency compared to bisulfite-sequencing shown by Uekawa et al.
In conclusion, our data demonstrate that HF diet feeding in mice can induce a suppression of gene and protein expression of enzymes operating in the hepatic transsulfuration pathway in favor of an increased remethylation of Hcy to methionine. In addition, we show that the PPARα pathway is involved in the downregulation of CBS. Based on our findings, we postulate the primacy of methionine homeostasis mediated by remethylation of Hcy to methionine in liver physiology and in pathological situations such as NAFLD to ensure the maintenance of basic liver functions such as synthesis of hepatic and plasma proteins despite previously reported altered PL homeostasis and PC signature. High dietary fat intake associated with the development of hepatic steatosis is consequently linked to major changes in hepatic C1-metabolism that secondarily may also translate into changes of hepatic redox-status and cellular osmolyte levels.