The necroptosis-inducing kinase RIPK3 dampens adipose tissue inflammation and glucose intolerance.

Receptor-interacting protein kinase 3 (RIPK3) mediates necroptosis, a form of programmed cell death that promotes inflammation in various pathological conditions, suggesting that it might be a privileged pharmacological target. However, its function in glucose homeostasis and obesity has been unknown. Here we show that RIPK3 is over expressed in the white adipose tissue (WAT) of obese mice fed with a choline-deficient high-fat diet. Genetic inactivation of Ripk3 promotes increased Caspase-8-dependent adipocyte apoptosis and WAT inflammation, associated with impaired insulin signalling in WAT as the basis for glucose intolerance. Similarly to mice, in visceral WAT of obese humans, RIPK3 is overexpressed and correlates with the body mass index and metabolic serum markers. Together, these findings provide evidence that RIPK3 in WAT maintains tissue homeostasis and suppresses inflammation and adipocyte apoptosis, suggesting that systemic targeting of necroptosis might be associated with the risk of promoting insulin resistance in obese patients.

Effects of Nonalcoholic Fatty Liver Disease on Hepatic CYP2B1 and in Vivo Bupropion Disposition in Rats Fed a High-Fat or Methionine/Choline-Deficient Diet.

Nonalcoholic fatty liver disease (NAFLD) refers to hepatic pathologies, including simple fatty liver (SFL), nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis, that may progress to hepatocellular carcinoma. These liver disease states may affect the activity and expression levels of drug-metabolizing enzymes, potentially resulting in an alteration in the pharmacokinetics, therapeutic efficacy, and safety of drugs. This study investigated the hepatic cytochrome P450 (CYP) 2B1-modulating effect of a specific NAFLD state in dietary rat models. Sprague-Dawley rats were given a methionine/choline-deficient (MCD) or high-fat (HF) diet to induce NASH and SFL, respectively. The induction of these disease states was confirmed by plasma chemistry and liver histological analysis. Both the protein and mRNA levels of hepatic CYP2B1 were considerably reduced in MCD diet-fed rats; however, they were similar between the HF diet-fed and control rats. Consistently, the enzyme-kinetic and pharmacokinetic parameters for CYP2B1-mediated bupropion metabolism were considerably reduced in MCD diet-fed rats; however, they were also similar between the HF diet-fed and control rats. These results may promote a better understanding of the influence of NAFLD on CYP2B1-mediated metabolism, which could have important implications for the safety and pharmacokinetics of drug substrates for the CYP2B subfamily in patients with NAFLD.

Purified anthocyanins from bilberry and black currant attenuate hepatic mitochondrial dysfunction and steatohepatitis in mice with methionine and choline deficiency.

The berries of bilberry and black currant are a rich source of anthocyanins, which are thought to have favorable effects on nonalcoholic steatohepatitis (NASH). This study was designed to examine whether purified anthocyanins from bilberry and black currant are able to limit the disorders related to NASH induced by a methionine-choline-deficient (MCD) diet in mice. The results showed that treatment with anthocyanins not only alleviated inflammation, oxidative stress, steatosis, and even fibrosis but also improved depletion of mitochondrial content and damage of mitochondrial biogenesis and electron transfer chain developed concomitantly in the liver of mice fed the MCD diet. Furthermore, anthocyanins treatment promoted activation of AMP-activated protein kinase (AMPK) and expression of peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α). These data provide evidence that anthocyanins possess significant protective effects against NASH and mitochondrial defects in response to a MCD diet, with a mechanism maybe through affecting the AMPK/PGC-1α signaling pathways.

Lipid metabolism, oxidative stress and cell death are regulated by PKC delta in a dietary model of nonalcoholic steatohepatitis.

Steatosis, oxidative stress, and apoptosis underlie the development of nonalcoholic steatohepatitis (NASH). Protein kinase C delta (PKCδ) has been implicated in fatty liver disease and is activated in the methionine and choline-deficient (MCD) diet model of NASH, yet its pathophysiological importance towards steatohepatitis progression is uncertain. We therefore addressed the role of PKCδ in the development of steatosis, inflammation, oxidative stress, apoptosis, and fibrosis in an animal model of NASH. We fed PKCδ(-/-) mice and wildtype littermates a control or MCD diet. PKCδ(-/-) primary hepatocytes were used to evaluate the direct effects of fatty acids on hepatocyte lipid metabolism gene expression. A reduction in hepatic steatosis and triglyceride levels were observed between wildtype and PKCδ(-/-) mice fed the MCD diet. The hepatic expression of key regulators of ß-oxidation and plasma triglyceride metabolism was significantly reduced in PKCδ(-/-) mice and changes in serum triglyceride were blocked in PKCδ(-/-) mice. MCD diet-induced hepatic oxidative stress and hepatocyte apoptosis were reduced in PKCδ(-/-) mice. MCD diet-induced NADPH oxidase activity and p47(phox) membrane translocation were blunted and blocked, respectively, in PKCδ(-/-) mice. Expression of pro-apoptotic genes and caspase 3 and 9 cleavage in the liver of MCD diet fed PKCδ(-/-) mice were blunted and blocked, respectively. Surprisingly, no differences in MCD diet-induced fibrosis or pro-fibrotic gene expression were observed in 8 week MCD diet fed PKCδ(-/-) mice. Our results suggest that PKCδ plays a role in key pathological features of fatty liver disease but not ultimately in fibrosis in the MCD diet model of NASH.

Carnitine modulates crucial myocardial adenosine triphosphatases and acetylcholinesterase enzyme activities in choline-deprived rats.

Choline is an essential nutrient, and choline deficiency has been associated with cardiovascular morbidity. Choline is also the precursor of acetylcholine (cholinergic component of the heart's autonomic nervous system), whose levels are regulated by acetylcholinesterase (AChE). Cardiac contraction-relaxation cycles depend on ion gradients established by pumps like the adenosine triphosphatases (ATPases) Na(+)/K(+)-ATPase and Mg(2+)-ATPase. This study aimed to investigate the impact of dietary choline deprivation on the activity of rat myocardial AChE (cholinergic marker), Na(+)/K(+)-ATPase, and Mg(2+)-ATPase, and the possible effects of carnitine supplementation (carnitine, structurally relevant to choline, is used as an adjunct in treating cardiac diseases). Adult male albino Wistar rats were distributed among 4 groups, and were fed a standard or choline-deficient diet for one month with or without carnitine in their drinking water (0.15% w/v). The enzyme activities were determined spectrophotometrically in the myocardium homogenate. Choline deficiency seems to affect the activity of the aforementioned parameters, but only the combination of choline deprivation and carnitine supplementation increased myocardial Na(+)/K(+)-ATPase activity along with a concomitant decrease in the activities of Mg(2+)-ATPase and AChE. The results suggest that carnitine, in the setting of choline deficiency, modulates cholinergic myocardial neurotransmission and the ATPase activity in favour of cardiac work efficiency.

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.

c-Jun N-terminal kinase-1 from hematopoietic cells mediates progression from hepatic steatosis to steatohepatitis and fibrosis in mice.

BACKGROUND & AIMS: c-Jun N-terminal kinase (JNK) plays a pivotal role in the development of the metabolic syndrome including nonalcoholic fatty liver disease. However, the mechanism underlying the contribution of JNK to the progression from simple steatosis to steatohepatitis and liver fibrosis is unresolved. METHODS: Hepatic steatosis, inflammation, and fibrosis were examined in wild-type, jnk1(-/-), or jnk2(-/-) mice fed a choline-deficient L-amino acid-defined (CDAA) diet for 20 weeks. The functional contribution of JNK isoforms in Kupffer cells was assessed in vitro and in vivo using chimeric mice in which the hematopoietic compartment including Kupffer cells was replaced by wild-type, jnk1(-/-), or jnk2(-/-) cells. RESULTS: CDAA diet induced significantly less hepatic inflammation and less liver fibrosis despite a similar level of hepatic steatosis in jnk1(-/-) mice as compared with wild-type or jnk2(-/-) mice. CDAA diet-induced hepatic inflammation was chronic and mediated by Kupffer cells. Pharmacologic inhibition of JNK or gene deletion of jnk1 but not jnk2 repressed the expression of inflammatory and fibrogenic mediators in primary Kupffer cells. In vivo, CDAA diet induced less hepatic inflammation and liver fibrosis despite an equivalent level of hepatic steatosis in chimeric mice with jnk1(-/-) hematopoietic cells as compared with chimeric mice with wild-type or jnk2(-/-) hematopoietic cells. CONCLUSIONS: jnk1(-/-) mice are resistant to diet-induced steatohepatitis and liver fibrosis. JNK1 in hematopoietic cells, especially in Kupffer cells, contributes to the development of liver fibrosis by inducing chronic inflammation.

Mechanisms of hepatic steatosis in mice fed a lipogenic methionine choline-deficient diet.

The methionine choline-deficient (MCD) diet results in liver injury similar to human nonalcoholic steatohepatitis (NASH). The aims of this study were to define mechanisms of MCD-induced steatosis in insulin-resistant db/db and insulin-sensitive db/m mice. MCD-fed db/db mice developed more hepatic steatosis and retained more insulin resistance than MCD-fed db/m mice. Both subcutaneous and gonadal fat were reduced by MCD feeding: gonadal fat decreased by 23% in db/db mice and by 90% in db/m mice. Weight loss was attenuated in the db/db mice, being only 13% compared with 35% in MCD-fed db/db and db/m mice, respectively. Both strains had upregulation of hepatic fatty acid transport proteins as well as increased hepatic uptake of [14C]oleic acid: 3-fold in db/m mice (P < 0.001) and 2-fold in db/db mice (P < 0.01) after 4 weeks of MCD feeding. In both murine strains, the MCD diet reduced triglyceride secretion and downregulated genes involved in triglyceride synthesis. Therefore, increased fatty acid uptake and decreased VLDL secretion represent two important mechanisms by which the MCD diet promotes intrahepatic lipid accumulation in this model. Feeding the MCD diet to diabetic rodents broadens the applicability of this model for the study of human NASH.

Rho/Rho kinase is a key enzyme system involved in the angiotensin II signaling pathway of liver fibrosis and steatosis.

BACKGROUND AND AIM: The molecular mechanisms underlying the involvement of the renin-angiotensin system in hepatic fibrosis are unclear. Recently, it was reported that a Rho kinase inhibitor prevented fibrosis of various tissues and that the Rho/Rho kinase pathway was involved in the renin-angiotensin system of vascular smooth muscle cells. In this study, the involvement of the Rho/Rho kinase pathway on angiotensin II signaling in liver fibrogenesis and generation of steatosis was investigated. METHODS: Rats were fed a choline-deficient/L-amino acid-defined (CDAA) diet continuously and treated with a Rho kinase inhibitor, Y-27632, and an angiotensin II receptor blocker, TCV-116. Liver histology and hepatic stellate cell activation were analyzed. Free radical production was detected by 4-hydroxynonenal and 8-hydroxy-2'-deoxyguanosine immunostaining and the expression of tumor necrosis factor-alpha was examined. Isolated hepatic stellate cells were pretreated with a Rho kinase inhibitor, Y-27632, or an angiotensin II receptor blocker, CV-11974, and stimulated with angiotensin II, and mRNA expression of transforming growth factor-beta and alpha-smooth muscle actin was analyzed. RESULTS: Both the angiotensin II receptor blocker and the Rho kinase inhibitor improved fibrosis and steatosis of the liver in CDAA-fed rats. The increase in the number of hepatocytes positive for 4-hydroxynonenal and 8-hydroxy-2'-deoxyguanosine in CDAA-fed rats was significantly prevented by the angiotensin II receptor blocker and the Rho kinase inhibitor. The levels of tumor necrosis factor-alpha mRNA in the liver of CDAA-fed rats were significantly increased and this increase was significantly inhibited by treatment with the angiotensin II receptor blocker and the Rho kinase inhibitor. mRNA expression of transforming growth factor-beta and alpha-smooth muscle actin stimulated by angiotensin II was also significantly suppressed by these two drugs. CONCLUSION: These results suggest that the Rho/Rho kinase pathway is at least partly involved in the renin-angiotensin system and plays an important role in hepatic fibrosis and steatosis.

Lymphocyte gene expression in subjects fed a low-choline diet differs between those who develop organ dysfunction and those who do not.

BACKGROUND: Some humans fed a low-choline diet develop hepatosteatosis, liver and muscle damage, and lymphocyte apoptosis. The risk of developing such organ dysfunction is increased by the presence of single-nucleotide polymorphisms (SNPs) in genes involved in folate and choline metabolism. OBJECTIVE: We investigated whether these changes that occur in the expression of many genes when humans are fed a low-choline diet differ between subjects who develop organ dysfunction and those who do not. We also investigated whether expression changes were dependent on the presence of the SNPs of interest. DESIGN: Thirty-three subjects aged 20-67 y were fed for 10 d a baseline diet containing the recommended adequate intake of choline. They then were fed a low-choline diet for up to 42 d or until they developed organ dysfunction. Blood was collected at the end of each phase, and peripheral lymphocytes were isolated and used for genotyping and for gene expression profiling with the use of microarray hybridization. RESULTS: Feeding a low-choline diet changed the expression of 259 genes, and the profiles of subjects who developed and those who did not develop signs of organ dysfunction differed. Group clustering and gene ontology analyses found that the diet-induced changes in gene expression profiles were significantly influenced by the SNPs of interest and that the gene expression phenotype of the variant gene carriers differed significantly even with the baseline diet. CONCLUSION: These findings support our hypothesis that a person's susceptibility to organ dysfunction when fed a low-choline diet is modulated by specific SNPs in genes involved in folate and choline metabolism.

Phosphatidylethanolamine N-methyltransferase (PEMT) gene expression is induced by estrogen in human and mouse primary hepatocytes.

Choline is an essential nutrient for humans, though some of the requirement can be met by endogenous synthesis catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). Premenopausal women are relatively resistant to choline deficiency compared with postmenopausal women and men. Studies in animals suggest that estrogen treatment can increase PEMT activity. In this study we investigated whether the PEMT gene is regulated by estrogen. PEMT transcription was increased in a dose-dependent manner when primary mouse and human hepatocytes were treated with 17-beta-estradiol for 24 h. This increased message was associated with an increase in protein expression and enzyme activity. In addition, we report a region that contains a perfect estrogen response element (ERE) approximately 7.5 kb from the transcription start site corresponding to transcript variants NM_007169 and NM-008819 of the human and murine PEMT genes, respectively, three imperfect EREs in evolutionarily conserved regions and multiple imperfect EREs in nonconserved regions in the putative promoter regions. We predict that both the mouse and human PEMT genes have three unique transcription start sites, which are indicative of either multiple promoters and/or alternative splicing. This study is the first to explore the underlying mechanism of why dietary requirements for choline vary with estrogen status in humans.

Expression of inducible nitric oxide (NO) synthase but not prevention by its gene ablation of hepatocarcinogenesis with fibrosis caused by a choline-deficient, L-amino acid-defined diet in rats and mice.

Expression of inducible nitric oxide synthase (iNOS) and effects of iNOS gene ablation on the hepatocarcinogenesis associated with fibrosis caused by a choline-deficient, L-amino acid-defined (CDAA) diet, were examined in male F344 rats and C57BL/6J wild-type and iNOS-/- mice. Western blot, RT-PCR and immunohistochemical analyses revealed increased expression of iNOS protein and mRNA in the livers of rats and wild-type mice fed a CDAA diet for 12-80 weeks, associated with elevated serum NO(x) and liver nitrotyrosine levels. iNOS-/- mice demonstrated greater liver injury and fibrosis in the early stage than their wild-type counterparts, but this did not significantly affect the incidence and multiplicity of altered foci, adenomas and hepatocellular carcinomas in spite of immunohistochemical iNOS expression in these lesions. Results suggested no major determinant roles of the expressed iNOS in the development of liver tumors caused by the CDAA diet.

Elevated serum creatine phosphokinase in choline-deficient humans: mechanistic studies in C2C12 mouse myoblasts.

BACKGROUND: Choline is a required nutrient, and humans deprived of choline develop liver damage. OBJECTIVE: This study examined the effect of choline deficiency on muscle cells and the release of creatine phosphokinase (CPK) as a sequela of that deficiency. DESIGN: Four men were fed diets containing adequate and deficient amounts of choline, and serum was collected at intervals for measurement of CPK. C2C12 mouse myoblasts were cultured in a defined medium containing 0 or 70 micromol choline/L for up to 96 h, and CPK was measured in the media; choline and metabolites were measured in cells. Apoptosis was assessed by using terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling and activated caspase-3 immunohistochemistry. Cell fragility in response to hypo-osmotic stress was also assessed. RESULTS: Three of 4 humans fed a choline-deficient diet had significantly elevated serum CPK activity derived from skeletal muscle (up to 66-fold; P < 0.01) that resolved when choline was restored to their diets. Cells grown in choline-deficient medium for 72 h leaked 3.5-fold more CPK than did cells grown in medium with 70 micromol choline/L (control medium; P < 0.01). Apoptosis was induced in cells grown in choline-deficient medium. Phosphatidylcholine concentrations were diminished in choline-deficient cells (to 43% of concentrations in control cells at 72 h; P < 0.01), as were concentrations of intracellular choline, phosphocholine, and glycerophosphocholine. Cells grown in choline-deficient medium had greater membrane osmotic fragility than did cells grown in control medium. CONCLUSIONS: Choline deficiency results in diminished concentrations of membrane phosphatidylcholine in myocytes, which makes them more fragile and results in increased leakage of CPK from cells. Serum CPK may be a useful clinical marker for choline deficiency in humans.

Seizure-induced memory impairment is reduced by choline supplementation before or after status epilepticus.

Prenatal choline supplementation can protect rats against cognitive deficits induced by status epilepticus induced by the cholinergic agent pilocarpine [J. Neurosci. 20 (2000) 1]. In the present day, we have extended this novel finding by investigating the effects of pre- and postnatal choline supplementation in memory deficits associated with status epilepticus induced with kainic acid (KA). In the first experiment pregnant rats received a normal, choline-supplemented, or choline deficient diet starting on the 11th day of gestation and continuing until postnatal (P) 7. At P42, rats were given a convulsant dosage of KA. Two weeks following the KA-induced status epilepticus rats underwent testing of visual-spatial memory using the Morris water maze test. Rats receiving supplemental choline performed better in the water maze than the deficient and control groups. Moreover, the activity of hippocampal choline acetyltransferase was 18% lower in the choline deficient animals as compared with the other two groups. In the second experiment we administered KA to P35 rats that had been given a normal diet. Following the status epilepticus the rats were given a choline-supplemented or control diet for 4 weeks and then tested in the water maze. Rats receiving choline supplementation performed far better than rats receiving a regular diet. This study demonstrates that choline supplementation prior to or following KA-induced status epilepticus can protect rats from memory deficits induced by status epilepticus.

Choline deficiency-induced liver damage is reversible in Pemt(-/-) mice.

Hepatic tissue has two pathways for phosphatidylcholine (PC) synthesis, i.e., the cytidinediphosphocholine (CDP-choline) pathway and the methylation pathway, which utilizes phosphatidylethanolamine-N-methyltransferase (PEMT). Fatal liver damage occurs in Pemt(-/-)mice fed a choline-deficient (CD) diet. We investigated whether liver damage can be reversed by the addition of dietary choline. Mice (8 wk old) were fed the CD purified diet for 4 d, a choline-supplemented (CS) diet (CD diet + 0.4% choline chloride) for 4 d, or the CD diet for 3 d and a CS diet for 1 d (CD/CS). Pemt(-/-)mice fed the CD diet for 3 d exhibited liver damage as assayed by plasma aminotransferase levels. The livers appeared normal after subsequent feeding of the CS diet for 1 d (CD/CS). The activities of plasma aminotransferases of CD/CS fed mice were comparable to Pemt(-/-)mice fed the CS diet. Hepatic PC and triacylglycerol levels as well as plasma PC levels in the CD/CS-fed Pemt(-/-)mice were lower than those of mice fed the CD diet and began to approach normal levels. Although the CD diet induces liver damage in Pemt(-/-)mice, this damage can be rapidly reversed by the addition of dietary choline.

Application of the PKCYP-test in cases of altered CYP1A2 for multiple CYP systems in rat models of disease.

Previously, we established a method to assess drug metabolism capacity based on a pharmacokinetic estimation of the quantity of cytochrome P450 (CYP) in vivo (PKCYP-test) by introducing an apparent liver-to-blood free concentration gradient in vivo (qg). The qg values were determined as the ratio of in vivo-in vitro clearance. In this study, we examined the application of the PKCYP-test to the clearance of acetanilide and caffeine mediated by CYP1A2 using rat models in which the levels of CYP enzymes were reduced. Rats fed a choline-deficient diet (CD-fed) and aged rats were used as models for a low level of CYP in the liver. In both rat models, the contribution (fCYP) of CYP1A2 to the in vivo intrinsic clearance values (CLint) of acetanilide and caffeine metabolism was less than unity, suggesting that other metabolic pathways are involved in the CLint. The in vivo clearance for CYP1A2 was estimated by multiplying fCYP by CLint, then the value of qg was determined as the ratio of in vivo-in vitro clearance. We predicted the level of CYP1A2 in CD-fed and aged rats, based on the clearance of acetanilide mediated by CYP1A2, using the qg value of control rats. The clearance of caffeine mediated by CYP1A2 in CD-fed and aged rats, as estimated from the predicted level of CYP1A2, correlated with the observed values. In conclusion, we have demonstrated that the PKCYP-test can be applied to CYP1A2 for drugs metabolized by multiple CYP isozymes, and/or to models involving reduced CYP.

Starvation impairs antioxidant defense in fatty livers of rats fed a choline-deficient diet.

Although fatty liver (FL) is considered an innocuous condition, the frequent incidence of graft failure when FL are transplanted has renewed interest in the intracellular disorders causative of or consequent to fatty degeneration. Oxidative stress and nutritional status modulate the tolerance to reperfusion injury in control livers (CL), but very little is known in the case of FL. This study was designed to compare the oxidative balance in CL and FL from fed and food-deprived rats. Serum and liver samples were collected from fed and starved (18 h) rats with CL or FL induced by a choline-deficient diet. Hepatic injury was assessed by transaminase activities and histology. The hepatic concentrations of glutathione (GSH), vitamin C, alpha-tocopherol, thiobarbituric acid-reactive substances (TBARS) and protein carbonyls (PC) were measured. Fed rats with FL had significantly greater TBARS and lower alpha-tocopherol and vitamin C levels than those with CL, whereas GSH and PC concentrations were not affected. Starvation impaired the oxidative balance in both groups. However, compared with the other groups, FL from food-deprived rats generally had the lowest hepatic concentrations of alpha-tocopherol, vitamin C and GSH. Unlike in CL, protein oxidation occurred in FL. These data indicate that fatty liver induced by consumption of a choline-deficient diet is associated with a lower level of antioxidants, which results in lipid peroxidation. Starvation further affects these alterations and extends the damage to proteins. In conclusion, steatosis and starvation may act synergistically on the depletion of antioxidants, predisposing fatty livers to a reduced tolerance to oxidative injury.

Retrospective evaluation of serum ornithine carbamyltransferase activity as an index of hepatotoxicity in toxicological studies with rats.

The sensitivity of elevated serum ornithine carbamyltransferase (OCT) as an index of hepatotoxicity in rats was assessed in different studies conducted over a number of years and originally designed to examine the toxicity or carcinogenicity of a variety of test chemicals and diets. Changes in serum OCT activities were compared with the more widely used clinical endpoints, alanine aminotranserase (ALT) and aspartate aminotransferase (AST). In the first study, rats received a single oral dose of the hepatotoxic and hepatocarcinogenic fungal toxin aflatoxin B(1) (AFB(1)). The increase in enzyme levels between control and AFB(1)-treated rats was greater for serum OCT than for ALT or AST. This response was similar to the changes in serum enzyme levels in studies where rats ingested a hepatotoxic and hepatocarcinogenic choline deficient (CD) diet. When rats were exposed to the hepatotoxic and nephrotoxic fungal toxin fumonisin B(1) (FB(1)) by intraperitoneal injection for 6 days, serum AST and ALT were significantly elevated above control levels while OCT was unaffected. The peroxisome proliferator ciprofibrate caused elevated ALT and AST but not OCT at week 52 of dietary exposure, after the development of liver nodules and tumours. Of the two liver-specific enzymes examined in all of the studies, ALT was more consistently predictive of hepatotoxicity than OCT.

Elevated expression and altered pattern of activity of DNA methyltransferase in liver tumors of rats fed methyl-deficient diets.

DNA methyltransferase (MTase) activity in nuclear extracts from neoplastic and preneoplastic livers of rats fed a methyl-deficient diet (MDD) is elevated compared with that seen in the livers of control rats. Nuclear proteins were prepared in the presence of protease inhibitors including trans-epoxy succinyl-L-leucylamido-(4-guanido)butane and were fractionated by isoelectric focusing. In normal, control liver, two distinct MTase fractions were observed. In MDD-induced malignant liver, a third fraction, in addition to the previous two, was also seen. Both the DNA substrate and the cytosine site specificities of the third MTase fraction differ from those of the other two fractions. The distinct MTase activity in liver tumor has significantly more de novo MTase activity than do the MTase fractions of normal, control liver. Thus, normal and neoplastic rat livers differ in DNA MTase fractionation patterns and site specificities. The altered DNA MTase activity observed in rat liver tumors caused by MDDs may be one of the critical factors contributing to cancer formation through abnormal DNA methylation.

Sexually dimorphic activation of liver and brain phosphatidylethanolamine N-methyltransferase by dietary choline deficiency.

Phosphatidylethanolamine N-methyltransferase (PEMT) activity was measured by a radioenzymatic assay in homogenates of brain and liver obtained from Sprague Dawley rats fed a choline-free or control (0.3 g/kg of choline chloride) diet for seven days. Choline deficiency increased PEMT activity in the liver of male rats by 34% but had no effect on hepatic PEMT in females. In contrast, brain PEMT activity was increased in brain of choline deficient females (by 49%) but was unaltered in males. Activation of the PE methylation pathway in female brain may constitute a compensatory mechanism to sustain PC synthesis during choline deficiency.