Variations in hepatic lipid species of age-matched male mice fed a methionine-choline-deficient diet and housed in different animal facilities.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a common disease and feeding mice a methionine-choline-deficient (MCD) diet is a frequently used model to study its pathophysiology. Genetic and environmental factors influence NASH development and liver lipid content, which was studied herein using C57BL/6 J mice bred in two different animal facilities. METHODS: Age-matched male C57BL/6 J mice bred in two different animal facilities (later on referred to as WT1 and WT2) at the University Hospital of Regensburg were fed identical MCD or control chows for 2 weeks. Hepatic gene and protein expression and lipid composition were determined. RESULTS: NASH was associated with increased hepatic triglycerides, which were actually higher in WT1 than WT2 liver in both dietary groups. Cholesterol contributes to hepatic injury but was only elevated in WT2 NASH liver. Ceramides account for insulin resistance and cell death, and ceramide species d18:1/16:0 and d18:1/18:0 were higher in the NASH liver of both groups. Saturated sphingomyelins only declined in WT1 NASH liver. Lysophosphatidylcholine concentrations were quite normal in NASH and only one of the 12 altered phosphatidylcholine species declined in NASH liver of both groups. Very few phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol species were comparably regulated in NASH liver of both animal groups. Seven of these lipid species declined and two increased in NASH. Notably, hepatic mRNA expression of proinflammatory (F4/80, CD68, IL-6, TNF and chemerin) and profibrotic genes (TGF beta and alpha SMA) was comparable in WT1 and WT2 mice. CONCLUSIONS: Mice housed and bred in different animal facilities had comparable disease severity of NASH whereas liver lipids varied among the groups. Thus, there was no specific lipid signature for NASH in the MCD model.

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury.

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

p16 deficiency promotes nonalcoholic steatohepatitis via regulation of hepatic oxidative stress.

Nonalcoholic steatohepatitis (NASH) is characterized by excess accumulation of lipids in liver, accompanied with hepatocyte injury, cell death and inflammation. Although p16 is known as tumor suppressor in multiple cancer types, it remains unclear whether p16 plays a critical role in NASH. To determine whether p16 could play a role in the pathogenesis of NASH, wild-type mice and p16-/- mice were fed on a methionine and choline-deficient (MCD) diet for 3 weeks, and liver steatosis, fibrosis, and inflammation were evaluated. Our data show that p16-/- mice fed with MCD diet displayed more significant hepatic steatosis, hepatocyte damage, increased oxidative stress and inflammatory cell infiltration compared to MCD-fed WT mice. It was also clear that the increased ROS and the accumulation of lipid in BEL-7402 cells occurred when p16 expression was depleted with siRNA. These findings indicate that p16 may play a critical role in the development of NASH by reining in ROS production and by inhabiting inflammatory response.

Elevation of liver endoplasmic reticulum stress in a modified choline-deficient l-amino acid-defined diet-fed non-alcoholic steatohepatitis mouse model.

Endoplasmic reticulum (ER) stress caused by accumulation of misfolded proteins is observed in several kinds of diseases. Since ER stress is reported to be involved in the progression of non-alcoholic steatohepatitis (NASH), highly sensitive and simple measurement methods are required for research into developing novel therapy for NASH. To investigate the involvement of ER stress in NASH pathogenesis in a mouse model, an assay for liver ER stress was developed using ER stress activated indicator-luciferase (ERAI-Luc) mice. To establish the assay method for detection of ER stress in the liver, tunicamycin (TM) (0.3 mg/kg i. p.) was administered to ERAI-Luc mice, and the luciferase activity was measured in ex vivo and in vivo. To evaluate ER stress in the NASH model, ERAI-Luc mice were fed a modified choline-deficient l-amino acid-defined (mCDAA) diet for 14 weeks. After measurement of ER stress by luminescence imaging, levels of liver lipids and pro-fibrotic and pro-inflammatory gene expression were measured as NASH-related indexes. In non-invasive whole-body imaging, TM elevated luciferase activity in the liver, induced by activation of ER stress. The highest luminescence in the liver was confirmed by ex vivo imaging of isolated tissues. In parallel with progression of NASH, elevated luminescence induced by ER stress in liver was observed in mCDAA diet-fed ERAI-Luc mice. Luciferase activity was significantly and positively correlated to levels of triglyceride and free cholesterol in the liver, as well as to the mRNA expression of type 1 collagen α1 chain and tumor necrosis factor α. These data indicated that the use of ERAI-Luc mice was effective in the detection of ER stress in the liver. Moreover, the NASH model using ERAI-Luc mice can be a useful tool to clarify the role of ER stress in pathogenesis of NASH and to evaluate effects of drugs targeted against ER stress.

Restriction of dietary methyl donors limits methionine availability and affects the partitioning of dietary methionine for creatine and phosphatidylcholine synthesis in the neonatal piglet.

Methionine is required for protein synthesis and provides a methyl group for >50 critical transmethylation reactions including creatine and phosphatidylcholine synthesis as well as DNA and protein methylation. However, the availability of methionine depends on dietary sources as well as remethylation of demethylated methionine (i.e., homocysteine) by the dietary methyl donors folate and choline (via betaine). By restricting dietary methyl supply, we aimed to determine the extent that dietary methyl donors contribute to methionine availability for protein synthesis and transmethylation reactions in neonatal piglets. Piglets 4-8 days of age were fed a diet deficient (MD-) (n=8) or sufficient (MS+) (n=7) in folate, choline and betaine. After 5 days, dietary methionine was reduced to 80% of requirement in both groups to elicit a response. On day 8, animals were fed [(3)H-methyl]methionine for 6h to measure methionine partitioning into hepatic protein, phosphatidylcholine, creatine and DNA. MD- feeding reduced plasma choline, betaine and folate (P<.05) and increased homocysteine ~3-fold (P<.05). With MD- feeding, hepatic phosphatidylcholine synthesis was 60% higher (P<.05) at the expense of creatine synthesis, which was 30% lower during MD- feeding (P<.05); protein synthesis as well as DNA and protein methylation were unchanged. In the liver, ~30% of dietary label was traced to phosphatidylcholine and creatine together, with ~50% traced to methylation of proteins and ~20% incorporated in synthesized protein. Dietary methyl donors are integral to neonatal methionine requirements and can affect methionine availability for transmethylation pathways.

MicroRNA-21 is associated with fibrosis in a rat model of nonalcoholic steatohepatitis and serves as a plasma biomarker for fibrotic liver disease.

Evidence indicates that hepatic fibrosis is the initial lesion of cirrhosis or hepatocellular carcinoma in diseases such as nonalcoholic steatohepatitis (NASH). To induce NASH, we fed rats a choline-deficient and iron-supplemented L-amino acid-defined (CDAA) diet. Histopathological examination revealed that fibrosis appeared from week 4 and progressed to bridging fibrosis from week 12. Using qRT-PCR assays, we detected increased expression of miR-21, Mmp-9, and Timp-1 in liver that peaked during week 4, when fibrosis was first detected. The expression pattern of miR-21 in plasma paralleled that in liver. Fibrosis tended to be resolved within 12 weeks of a recovery period after 12 weeks of feeding, and the expression of miR-21, Timp-1, and Mmp-9 decreased in liver. Comprehensive analyses of miRNA and mRNA expression in the liver using samples acquired at week 4 detected 16 miRNAs and 11 mRNAs that are mutually-interacting fibrosis-related factors. We therefore conclude that miR-21 was closely associated with fibrosis in a rat model of NASH and has potential as a plasma biomarker for hepatic fibrosis.

Choline Supplementation With a Structured Lipid in Children With Cystic Fibrosis: A Randomized Placebo-Controlled Trial.

BACKGROUND: Choline depletion is seen in cystic fibrosis (CF) and pancreatic insufficiency in spite of enzyme treatment and may result in liver, fatty acid, and muscle abnormalities. This study evaluated the efficacy and safety of an easily absorbed choline-rich structured lipid (LYM-X-SORB™ [LXS]) to improve choline status. METHODS: Children with CF and pancreatic insufficiency were randomized to LXS or placebo in a 12-month double blind trial. Dietary choline intake, plasma cholines, plasma and fecal phospholipids, coefficient of fat absorption, pulmonary function, growth status, body composition, and safety measures were assessed. Magnetic resonance spectroscopy for calf muscle choline and liver fat were assessed in a subgroup and compared with a healthy comparison group matched for age, sex, and body size. RESULTS: A total of 110 subjects were enrolled (age 10.4 ±â€Š3.0 years). Baseline dietary choline, 88% recommended, increased 3-fold in the LXS group. Plasma choline, betaine, and dimethylglycine increased in the LXS but not placebo (P = 0.007). Plasma lysophosphatidylcholine and phosphatidylcholine increased, and fecal phosphatidylcholine/phosphatidylethanolamine ratio decreased (P ≤ 0.05) in LXS only, accompanied by a 6% coefficient of fat absorption increase (P = 0.001). Children with CF had higher liver fat than healthy children and depleted calf muscle choline at baseline. Muscle choline concentration increased in LXS and was associated with improvement in plasma choline status. No relevant changes in safety measures were evident. CONCLUSIONS: LXS had improved choline intake, plasma choline status, and muscle choline stores compared with placebo group. The choline-rich supplement was safe, accepted by participants, and improved choline status in children with CF.

Plasma free choline, betaine and cognitive performance: the Hordaland Health Study.

Choline and betaine are nutrients involved in one-carbon metabolism. Choline is essential for neurodevelopment and brain function. We studied the associations between cognitive function and plasma concentrations of free choline and betaine. In a cross-sectional study, 2195 subjects (55 % women), aged 70-74 years, underwent extensive cognitive testing including the Kendrick Object Learning Test (KOLT), Trail Making Test (part A, TMT-A), modified versions of the Digit Symbol Test (m-DST), Block Design (m-BD), Mini-Mental State Examination (m-MMSE) and Controlled Oral Word Association Test (COWAT). Compared with low concentrations, high choline (>8·4 µmol/l) was associated with better test scores in the TMT-A (56·0 v. 61·5, P=0·004), m-DST (10·5 v. 9·8, P=0·005) and m-MMSE (11·5 v. 11·4, P=0·01). A generalised additive regression model showed a positive dose-response relationship between the m-MMSE and choline (P=0·012 from a corresponding linear regression model). Betaine was associated with the KOLT, TMT-A and COWAT, but after adjustments for potential confounders, the associations lost significance. Risk ratios (RR) for poor test performance roughly tripled when low choline was combined with either low plasma vitamin B12 (≤257 pmol/l) concentrations (RR(KOLT)=2·6, 95 % CI 1·1, 6·1; RR(m-MMSE)=2·7, 95 % CI 1·1, 6·6; RR(COWAT)=3·1, 95 % CI 1·4, 7·2) or high methylmalonic acid (MMA) (≥3·95 µmol/l) concentrations (RR(m-BD)=2·8, 95 % CI 1·3, 6·1). Low betaine (≤31·1 µmol/l) combined with high MMA was associated with elevated RR on KOLT (RR(KOLT)=2·5, 95 % CI 1·0, 6·2). Low plasma free choline concentrations are associated with poor cognitive performance. There were significant interactions between low choline or betaine and low vitamin B12 or high MMA on cognitive performance.

Choline and betaine in health and disease.

Choline is an essential nutrient, but is also formed by de novo synthesis. Choline and its derivatives serve as components of structural lipoproteins, blood and membrane lipids, and as a precursor of the neurotransmitter acetylcholine. Pre-and postnatal choline availability is important for neurodevelopment in rodents. Choline is oxidized to betaine that serves as an osmoregulator and is a substrate in the betaine-homocysteine methyltransferase reaction, which links choline and betaine to the folate-dependent one-carbon metabolism. Choline and betaine are important sources of one-carbon units, in particular, during folate deficiency. Choline or betaine supplementation in humans reduces concentration of total homocysteine (tHcy), and plasma betaine is a strong predictor of plasma tHcy in individuals with low plasma concentration of folate and other B vitamins (B2, B6, and B12) in combination TT genotype of the methylenetetrahydrofolate reductase 677 C->T polymorphism. The link to one-carbon metabolism and the recent availability of food composition data have motivated studies on choline and betaine as risk factors of chronic diseases previously studied in relation to folate and homocysteine status. High intake and plasma level of choline in the mother seems to afford reduced risk of neural tube defects. Intake of choline and betaine shows no consistent relation to cancer or cardiovascular risk or risk factors, whereas an unfavorable cardiovascular risk factor profile was associated with high choline and low betaine concentrations in plasma. Thus, choline and betaine showed opposite relations with key components of metabolic syndrome, suggesting a disruption of mitochondrial choline oxidation to betaine as part of the mitochondrial dysfunction in metabolic syndrome.

Choline-deprivation alters crucial brain enzyme activities in a rat model of diabetic encephalopathy.

Diabetic encephalopathy describes the moderate cognitive deficits, neurophysiological and structural central nervous system changes associated with untreated diabetes. It involves neurotoxic effects such as the generation of oxidative stress, the enhanced formation of advanced glycation end-products, as well as the disturbance of calcium homeostasis. Due to the direct connection of choline (Ch) with acetylcholine availability and signal transduction, a background of Ch-deficiency might be unfavorable for the pathology and subsequently for the treatment of several metabolic brain diseases, including that of diabetic encephalopathy. The aim of this study was to shed more light on the effects of adult-onset streptozotocin (STZ)-induced diabetes and/or Ch-deprivation on the activities of acetylcholinesterase (AChE) and two important adenosine triphosphatases, namely Na(+),K(+)-ATPase and Mg(2+)-ATPase. Male adult Wistar rats were divided into four main groups, as follows: control (C), diabetic (D), Ch-deprived (CD), and Ch-deprived diabetic (D+CD). Deprivation of Ch was provoked through the administration of Ch-deficient diet. Both the induction of diabetes and the beginning of dietary-mediated provoking of Ch-deprivation occurred at the same day, and rats were killed by decapitation after 30 days (1 month; groups C1, D1, CD1 and D1+CD1) and 60 days (2 months; groups C2, D2, CD2 and D2+CD2, respectively). The adult rat brain AChE activity was found to be significantly increased by both diabetes (+10%, p < 0.001 and +11%, p < 0.01) and Ch-deprivation (+19%, p < 0.001 and +14%, p < 0.001) when compared to the control group by the end of the first (C1) and the second month (C2), respectively. However, the Ch-deprived diabetic rats' brain AChE activity was significantly altered only after a 60-day period of exposure, resulting in a +27% increase (D2+CD2 vs. C2, p < 0.001). Although the only significant change recorded in the brain Na(+),K(+)-ATPase activity after the end of the first month is attributed to Ch-deprivation (+21%, p < 0.05, CD1 vs. C1), all groups of the second month exhibited a statistically significant decrease in brain Na(+),K(+)-ATPase activity (-24%, p < 0.01, D2 vs. C2; -21%, p < 0.01, CD2 vs. C2; -22%, p < 0.01, D2+CD2 vs. C2). As concerns Mg(2+)-ATPase, the enzyme's activity demonstrates no significant changes, with the sole exception of the D2+CD2 group (+21%, p < 0.05, D2+CD2 vs. C2). In addition, statistically significant time-dependent changes concerning the brain Mg(2+)-ATPase activity were recorded within the diabetic (p < 0.05, D2 vs. D1) and the Ch-deprived (p < 0.05, CD2 vs. CD1) rat groups. Our data indicate that Ch-deprivation seems to be an undesirable background for the above-mentioned enzymatic activities under untreated diabetes, in a time-evolving way. Further studies on the issue should focus on a region-specific reevaluation of these crucial enzymes' activities as well as on the possible oxidative mechanisms involved.

Reduced brain choline in homocystinuria due to remethylation defects.

OBJECTIVE: To investigate whether secondary impairment of the transmethylation pathway is a mechanism underlying the neurologic involvement in homocystinuria due to remethylation defects. METHODS: Twelve patients with neurologic disease due to remethylation defects were examined by brain magnetic resonance spectroscopic imaging ((1)H MRSI). Brain N-acetylaspartate, choline-containing compounds (Cho), and creatine (Cr) were quantified and compared to with controls. Metabolites of remethylation cycle and creatine biosynthesis pathway were measured in plasma and urine. RESULTS: MRSI revealed isolated Cho deficiency in all regions examined (mean concentration units +/- SD, patients vs controls): frontal white matter (0.051 +/- 0.010 vs 0.064 +/- 0.010; p = 0.001), lenticular nucleus (0.056 +/- 0.011 vs 0.069 +/- 0.009; p < 0.001), and thalamus (0.063 +/- 0.010 vs 0.071 +/- 0.007; p = 0.006). In contrast to controls, the Cho/Cr ratio decreased with age in patients in the three brain regions examined. Low creatine urinary excretion (p < 0.005), normal urine and plasma guanidinoacetate, and a paradoxical increase in plasma S-adenosylmethionine (p < 0.005) concentrations were observed. CONCLUSION: Patients with homocystinuria due to remethylation defects have an isolated brain choline deficiency, probably secondary to depletion of labile methyl groups produced by the transmethylation pathway. Although biochemical studies suggest mild peripheral creatine deficiency, brain creatine is in the reference range, indicating a possible compartmentation phenomenon. Paradoxical increase of S-adenosylmethionine suggests that secondary inhibition of methylases contributes to the transmethylation defect in these conditions.

Iron preloading aggravates nutritional steatohepatitis in rats by increasing apoptotic cell death.

BACKGROUND/AIMS: High serum ferritin and liver iron concentrations were found in some patients with NASH, suggesting a role for iron as a co-factor that aggravates liver injury. The aim of this study is to investigate the effects of parenteral iron in a rat model of NASH induced by a methionine choline deficient diet (MCDD). METHODS: Wistar rats were divided into 1 - Control, 2 - Iron (Fe), 3 - MCDD, 4 - MCDD&Fe groups. Iron dextran 100mg/kg was administered intra-muscularly in groups 2 and 4. All rats were fed MCDD, Groups 1 and 2 were supplied with choline and methionine. Blood and tissue samples were obtained after 4weeks. RESULTS: The iron injection alone did not affect the liver whereas MCDD led to steatohepatitis. Iron worsened steatosis without any obvious effect on accompanying inflammation. It aggravated tissue injury by increasing apoptosis. Liver fibrosis was observed only in 3 out of 10 rats in the MCDD&Fe group. CONCLUSIONS: Observation of liver fibrosis only in the MCDD&Fe group suggests that iron induced increase in apoptosis contributes to the development of fibrosis at an earlier time than expected.

Temporal correlation of pathology and DNA damage with gene expression in a choline-deficient model of rat liver injury.

Hepatocellular carcinoma (HCC) is the terminal event in chronic liver diseases with repeated cycles of cellular injury and regeneration. Although much is known about the cellular pathogenesis and etiological agents leading to HCC, the molecular events are not well understood. The choline-deficient (CD) model of rodent HCC involves the consecutive emergence of a fatty liver, apoptosis, compensatory proliferation, fibrosis, and cirrhosis that is markedly similar to the sequence of events typified by human HCC. Moreover, oxidative stress is thought to play a pivotal role in the progression of the disease. Here, we hypothesize that gene expression profiling can temporally mirror the histopathology and oxidative DNA damage observed with this model. We show that clusters of highly co-regulated genes representing distinct cellular pathways for lipid biosynthesis and metabolism, apoptosis, cell proliferation, and tissue remodeling temporally correlate with the well-defined sequential emergence of pathological alterations in the progression of liver disease. Additionally, an oxidative stress signature was observed that was corroborated in a time-dependent manner with increases in oxidized purines and abasic sites in DNA. Collectively, expression patterns were strongly driven by pathology, demonstrating that patterns of gene expression in advanced stages of liver disease are primarily driven by histopathological changes and to a much lesser degree by the original etiological agent. In conclusion, gene expression profiling coupled with the CD model of HCC provides a unique opportunity to unveil the molecular events associated with various stages of liver injury and carcinogenesis and to distinguish between causal and consecutive changes.