Alcoholic liver disease (ALD) is a major healthcare problem with worldwide prevalence. Our research group and others have modelled ALD by feeding animals a proportion of calories as ethanol resulting in the reproduction of many of the clinical manifestations seen in ALD; including the ethanol-induced disruption of liver enzyme activities and metabolite production associated with the methionine metabolic pathway ^[1?9], depletion of the major endogenous antioxidant glutathione, production of reactive oxygen species and aldehydic byproduct damage ^[10?15], and apoptosis ^{[4,10,11,14,16]}.

Previous research has established that prolonged ethanol feeding affects both pathways that catalyse the remethylation of homocysteine to form methionine in the liver methionine metabolic pathway ^[1,5]; resulting in a lowering of hepatocyte S-adenosylmethionine (SAM) levels ^[1,7]. Additionally, ethanol-induced impaired homocysteine remethylation drives the thermodynamically favoured conversion of homocysteine (with adenosine) to S-adenosylhomocysteine (SAH); resulting in an elevation of hepatocyte SAH levels ^[6,7]. The consequential alteration in the hepatocellular SAM to SAH ratio can compromise activities of many methyltransferases, including protein isoaspartyl methyltransferase (PIMT), to result in an accumulation of damaged proteins bearing isoaspartate residues ^[9,17?20].

We and other research groups have shown that betaine diet supplementation has therapeutic potential to abrogate some of the ill effects of ethanol consumption ^{[3,6,8,9,11,12,15]}; including an ability to restore a normal SAM:SAH ratio ^[3,6,8,9] to protect the liver from ethanol-induced fatty infiltration ^[3,8], and prevent accumulation of isoaspartate damage in cytosolic proteins ^[9]. In order to further examine the influences of ethanol and betaine on other components of the methionine metabolic pathway and their molecular interactions, we adopted a proteomic approach to identify gross cytosolic liver protein changes in livers of rats fed control or ethanol diets in the presence and absence of betaine supplementation.

Animal treatment. Animals were fed Lieber DeCarli control and ethanol liquid diets with or without 1% betaine supplementation for 4?weeks as previously described ^[8]. At the time of sacrifice the liver was removed and immediately used for preparing subcellular fractions ^[8]. The care, use, and procedures performed on these rats were approved by the Institutional Animal Care and Use Committee at the Omaha Veterans Affairs Medical Center, USA.

Protein concentrations. Protein concentrations of liver homogenates were measured using the DC protein assay (Bio-Rad) using bovine serum albumin as a protein standard.

One dimensional polyacrylamide gel electrophoresis (1D PAGE). Liver homogenate proteins (20??g/gel lane) were separated on 4?12% Bis?Tris NuPAGE Novex pre-cast gels run with MES running buffer as described previously ^[20]. Resolved proteins were either stained with Coomassie and photographed using a Fugi E900 digital camera followed by quantification using a GS-710 imaging densitometer (Bio-Rad) utilising Quantity One densitometric quantitation software (Bio-Rad), or transferred at 80?V for 2?h to a polyvinylidene difluoride (PVDF) membrane for Western blotting.

Carbonic anhydrase-III (CA-III) Western blotting. PVDF membranes were washed in a buffer of 20?mM K-MES, pH 6.2 containing 150?mM NaCl and 0.05% (v/v) Tween 20, blocked for 1?h at room temperature with 5% (w/v) milk fat in wash buffer, and then incubated overnight at 4??C with a goat polyclonal CA-III primary antibody (Santa-Cruz, sc-50715) at a 1:250 dilution in wash buffer. Following blot washing and incubation with a secondary antibody (polyclonal rabbit anti-goat immunoglobulins-horseradish peroxide-conjugated (Dako, P0449) at 1:2000 dilution in wash buffer, CA-III localisation was visualised using SuperSignal West Pico Chemiluminescent substrate (Pierce) with the light generated captured on CL-Xposure X-ray film (Pierce). The levels of CA-III were quantified using a GS-70 densitometer and Quantity One software.

One dimensional isoelectric focussing (1D IEF). Cytosolic liver proteins (20??g/gel lane) and IEF standards (Serva IEF markers, Invitrogen) were loaded onto vertical Novex, pH 3?10, IEF gels, and proteins resolved at constant voltage, initially for 1?h at 100?V, and then 1.5?h at 200?V. Proteins were fixed by washing gels in 12% (w/v) trichloroacetic acid, stained with Coomassie, and then photographed.

Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. For each analysis, six hundred ?g of cytosolic liver protein was resolved by 2D-PAGE and separated proteins Coomassie stained and then photographed. Protein bands or spots from either, 1D PAGE, 1D IEF, or 2D-PAGE gels were excised and identified by matrix assisted laser-desorption ionisation-time of flight (MALDI-TOF) mass spectrometry or liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS). Procedures were similar to those described in a previous publication ^[20] and have been included as Supplementary Material.

Models of ALD provide both a molecular insight into ethanol-induced cellular and protein dysfunction, and also afford a viable means to evaluate the usefulness of therapeutic agents aimed at prevention of disease progression. Here we have adopted proteomic approaches to evaluate the most visually prominent protein level changes in rats fed ethanol and betaine supplemented diets. Other minor protein level changes that may arise from these diet regimens were not examined and considered beyond the scope of this study.

From protein profiling of liver cytosolic proteins we were able to characterise upregulation of BHMT-1, MAT-1, and GNMT, with highest increases with the ethanol-fed betaine supplemented rats. This elevation in BHMT-1 protein levels we would expect to drive the remethylation of homocysteine to generate methionine, and this in turn to promote upregulation of MAT-1 and a subsequent increase in SAM levels; levels which are monitored and utilised by GNMT in the methylation of glycine to sarcosine ^[21] ? refer to Fig. 2.

Thus our proteomic study results suggest a hypothesis that betaine supplementation to ethanol-fed rats exerts a concerted influence upon the protein levels of the interconnected methionine metabolic pathway enzymes to drive a normalisation of the SAM to SAH ratio. Noteworthy is that the health benefits of a concerted upregulation of BHMT-1, MAT-1, and GNMT by betaine treatment could counter their collective downregulation in human hepatocellular carcinoma ^[22].

Our proteomic profiling also fortuitously revealed a dramatic ethanol-induced downregulation of CA-III protein, the levels of which were not restored by betaine treatment. CA-III is a member of the carbonic anhydrase family of zinc metalloenzymes (E.C. 4.2.1.1). Their general attributed function is the reversible catalysis of carbon dioxide hydration to generate bicarbonate and hydrogen ions for maintenance of pH homeostasis, and to aid in the transport of carbon dioxide out of tissues.

However, CA-III is somewhat distinct from the other two cytosolic CA family members (CA-I and CA-II) due to its relatively low carbon dioxide hydratase specific activity, and resistance to inhibition by the sulphonamide, acetazolamide ^[23]; indicating that CA-III may have other or additional functions. Indeed, CA-III has two reactive sulfhydryl groups (Cys181 and Cys186 that are absent in CA-I or CA-II) that readily form disulphide linkages with glutathione, an active process, termed S-(gluta)thiolation, under conditions of redox stress ^[24?29]. Furthermore, CA-III (but not CA-II) has been shown to protect cells from hydrogen peroxide-induced apoptosis ^[26]. Taken together, these observations suggest that while CA-III is particularly susceptible to oxidative damage, it may function in hepatocytes and skeletal muscle as an oxyradical scavenger to protect cells from oxidative damage ^[24?28].

Since ethanol consumption results in cellular redox stress via depletion of glutathione and generation of reactive oxygen species and aldehydes ^[10?15], we propose that this ethanol-induced downregulation of CA-III levels could further exacerbate any subsequent or sustained ethanol-induced redox stress.

In summary, the health benefits of betaine in part arise through concerted upregulation of methionine metabolic pathway enzymes, but do not extend to maintenance of CA-III levels and its function to resist redox stress.

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2009.02.082.

Supplementary data
Click here for additional data file (mmc1.doc)

We gratefully acknowledge the financial support of the following: Department of Veterans Affairs National Merit Review grant to K.K.K. and a NIH R21 AA017296-A1 grant to K.K.K. and W.G.C.; Medical Research Council programme grant to D.E.R.; Wellcome Trust summer scholarship to A.U.N. We also thank Mr. M. Liaque Latif for assistance with densitometry, and Dr. Michael Garle for a manuscript review.

The authors who have taken part in this study confirm that they have no commercial associations that pose a conflict of interest in connection with this submitted article.