Metabolic profiling reveals the protective effect of diammonium glycyrrhizinate on acute hepatic injury induced by carbon tetrachloride

Diammonium glycyrrhizinate (DG), a constitutent of Glycyrrhiza uralensis, has a protective effect on hepatic injury, hepatisis and cirrhosis. To date, the mechanism has been poorly understood, especially at the metabolic level. A metabolomic profiling study was performed to characterize the carbon tetrachloride (CCl₄) induced global metabolic alteration and the protective effects of DG in Sprague-Dawley rats. Urinary and hepatic tissue metabolic profiling revealed that CCl₄ perturbed the amino acid metabolism (alanine, glycine, leucine), tricarboxylic acid cycle (citrate), lipid metabolism (unsaturated fatty acids) and gut microbiota related metabolites. Our results also indicated that DG was able to attenuate CCl₄ perturbed metabolic pathways and ameliorated biochemical markers of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and Total cholesterol (TCHO). This global metabolomic approach also revealed full metabolic recovery takes longer than apparent and conventional histological and biochemical markers.     

Urinary metabolomic phenotyping of nickel induced acute toxicity in rat: an NMR spectroscopy approach

The metabolomic approach has been widely used in toxicology to investigate mechanisms of toxicity. To understand the mammalian system??s response to nickel exposure, we analysed the NiCl₂ induced metabolomic changes in urine of rats using ¹H nuclear magnetic resonance (¹H NMR) spectroscopy together with clinically relevant biochemical parameters. Male Sprague?CDawley rats were administered intraperitoneally with NiCl₂ at doses of 4, 10 and 20?mg/kg body weight. Urine samples were collected at 8, 16, 24, 72, 96 and 120?h post treatment. The metabolomic profile of rat urine showed prominent changes in citrate, dimethylamine, creatinine, choline, trimethylamine oxide (TMAO), phenyl alanine and hippurate at all doses. Principal component analysis of urine ¹H NMR spectra demonstrated the dose and time dependent development of toxicity. The metabolomic time trajectory, based on pattern recognition analysis of ¹H NMR spectra of urine, illustrated clear separation of pre and post treatments (temporal). Only animals treated with a low dose of NiCl₂ returned to normal physiology. The ¹H NMR spectral data correlated well with the clinically relevant nephrotoxic biomarkers. The urinary metabolomic phenotyping for NiCl₂ induced nephrotoxicity was defined according to the predictive ability of the known metabolite biomarkers, creatinine, citrate and TMAO. The current approach demonstrates that metabolomics, one of the most important platform in system biology, may be a promising tool for identifying and characterizing biochemical responses to toxicity.     

Concomitant Increases in the Levels of Choline and Free Fatty Acids in Rat Brain: Evidence Supporting the Seizure-Induced Hydrolysis of Phosphatidylcholine

The main objective of this study was to determine whether the excitotoxic cholinesterase inhibitor soman increases the catabolism of phospholipids in rat brain. Injections of soman (70 micrograms/kg, s.c.), at a dose that produced toxic effects, increased the levels of both free fatty acids (175-250% of control) and free choline (250% of control) in rat cerebrum 1 h after administration. All fatty acids contained in brain phosphatidylcholine were elevated significantly including palmitic (16:0), stearic (18:0), oleic (18:1), arachidonic (20:4), and docosahexaenoic (22:6) acids. The changes observed were consistent with those reported to occur following ischemia and the administration of other convulsants. Pretreatment of rats with the anticonvulsant diazepam (4 mg/kg, i.p.) prevented both the signs of soman toxicity and the soman-induced increase of choline and free fatty acids. Diazepam alone did not affect the levels of choline or free fatty acids, cholinesterase activity, or soman-induced cholinesterase inhibition, suggesting that soman toxicity involves a convulsant-mediated increase in phosphatidylcholine catabolism. In addition, administration of the convulsant bicuculline, at a dose that produces seizures and increases the levels of free fatty acids in brain, significantly increased the levels of choline. Results suggest that excitotoxic events enhance the hydrolysis of phosphatidylcholine in brain as evidenced by a concomitant increase in the levels of choline and free fatty acids.     

Lipid peroxidation in plasma of rats treated with ferric-nitrilotriacetate, in relation to kidney and liver modifications

Intraperitoneal injection of the iron chelate ferric-nitrilotriacetate (Fe-NTA) induces in rodents renal and hepatic suffering, associated with oxidative damage. We investigated the oxidation pattern in plasma of treated rats in relation to liver and kidney, monitoring the variation of the lipid components more susceptible to oxidation, unsaturated fatty acids (UFA) and alpha-tocopherol, as biomarkers of the oxidative damage. A sublethal dose of Fe-NTA induced a strong and extremely significant decrease of UFA levels at 1 h after injection in the plasma compartment and at 3 h in the kidney, with reductions up to 40-50% of the control values, together with an increase of conjugated dienes fatty acids hydroperoxides and a consumption of alpha-tocopherol. The same modifications were observed in the liver, but to a lesser extent. Histological observation proved that biochemical changes in the lipid fraction were a direct consequence of an ongoing membrane lipid peroxidation process. Our data show that oxidative damage to the lipid fraction is initially evident in the plasma compartment, where Fe-NTA toxicity is assumed to be caused by the elevation of serum free iron concentration, and proceeds with different speed and severity in the kidney and liver.     

Hepatic metabolic adaptation and adipose tissue expansion are altered in mice with steatohepatitis induced by high-fat high sucrose diet

Background: Obesity is a chronic progressive disease with several metabolic alterations. Nonalcoholic fatty liver disease (NAFLD) is an important comorbidity of obesity that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis or hepatocarcinoma. This study aimed at clarifying the molecular mechanisms underlying the metabolic alterations in hepatic and adipose tissue during high-fat high-sucrose diet-induced NAFLD development in mice. Methods: Twenty-four male mice (C57BL/6J) were randomly allocated into 3 groups (n = 8 mice per group) to receive a chow diet, a high-fat diet (HFD), or a high-fat high-sucrose diet (HF-HSD) for 20 weeks. At sacrifice, liver and adipose tissue were obtained for histopathological, metabolomic, and protein expression analyses. Results: HF-HSD (but not HFD) was associated with NASH and increased oxidative stress. These animals presented an inhibition of hepatic autophagy and alterations in AMP-activated protein kinase/mammalian target of rapamycin activity. We also observed that the ability of metabolic adaptation was adversely affected by the increase of damaged mitochondria. NASH development was associated with changes in adipose tissue dynamics and increased amounts of saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids in visceral adipose tissue. Conclusion: HF-HSD led to a metabolic blockage and impaired hepatic mitochondria turnover. In addition, the continuous accumulation of fatty acids produced adipose tissue dysfunction and hepatic fat accumulation that favored the progression to NASH.     

Metabolomic analysis of liver and skeletal muscle tissues in C57BL/6J and DBA/2J mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated to have the adverse effects on human health. In this study, we applied a metabolomic approach in conjunction with unsupervised and supervised machine learning methods to investigate the toxic effects of TCDD. By using liquid chromatography/quadrupole time-of-flight mass spectrometry, non-targeted metabolomic analysis revealed the metabolic signatures of the toxicity in aryl hydrocarbon receptor (AhR)-high affinity C57BL/6J (C6) mice as well as low affinity strain-DBA/2J (D2) mice. Lysophospholipids and long chain fatty acids were strikingly elevated in the C6 mice exposed to TCDD in both liver and skeletal muscle tissues. Meanwhile, the level of palmitoylcarnitine, which is one of the important indicators in fatty acid β-oxidation, increased significantly. Moreover, several nucleosides and amino acids decreased markedly. On the other hand, much less differentiating metabolites were highlighted in another strain-D2 mouse model. Taking liver and skeletal muscle tissues together, the levels of inosine, valine and glutamine decreased significantly. One lysophospholipid and two fatty acids were found to be enhanced. The principal components analysis and support vector machine clustering results also exhibited discriminations in the liver and skeletal muscle tissues of the mice. The obtained results indicated that TCDD could disrupt several metabolic pathways, including fatty acid biosynthesis and amino acid metabolism in both C6 and D2 mice. The increased rate of fatty acid beta-oxidation, however, was only observed in the liver and skeletal muscle tissues of C6 mice. The perturbation of the tricarboxylic acid (TCA) cycle was testified in two strains but the change was much slighter in D2 mice. It was of particular interest to note that the succinate level was enhanced in the liver tissues of both strains, and particularly, the change was up to 11.49-fold in the liver of C6 mice treated with TCDD. Collectively, the discrimination of D2 mice was not as distinct as that of C6 mice when exposed to the same dosage. Furthermore, D2 was confirmed to be less-sensitive rather than resistant to a high dose of TCDD.     

Dietary leucine--an environmental modifier of insulin resistance acting on multiple levels of metabolism

Environmental factors, such as the macronutrient composition of the diet, can have a profound impact on risk of diabetes and metabolic syndrome. In the present study we demonstrate how a single, simple dietary factor--leucine--can modify insulin resistance by acting on multiple tissues and at multiple levels of metabolism. Mice were placed on a normal or high fat diet (HFD). Dietary leucine was doubled by addition to the drinking water. mRNA, protein and complete metabolomic profiles were assessed in the major insulin sensitive tissues and serum, and correlated with changes in glucose homeostasis and insulin signaling. After 8 weeks on HFD, mice developed obesity, fatty liver, inflammatory changes in adipose tissue and insulin resistance at the level of IRS-1 phosphorylation, as well as alterations in metabolomic profile of amino acid metabolites, TCA cycle intermediates, glucose and cholesterol metabolites, and fatty acids in liver, muscle, fat and serum. Doubling dietary leucine reversed many of the metabolite abnormalities and caused a marked improvement in glucose tolerance and insulin signaling without altering food intake or weight gain. Increased dietary leucine was also associated with a decrease in hepatic steatosis and a decrease in inflammation in adipose tissue. These changes occurred despite an increase in insulin-stimulated phosphorylation of p70S6 kinase indicating enhanced activation of mTOR, a phenomenon normally associated with insulin resistance. These data indicate that modest changes in a single environmental/nutrient factor can modify multiple metabolic and signaling pathways and modify HFD induced metabolic syndrome by acting at a systemic level on multiple tissues. These data also suggest that increasing dietary leucine may provide an adjunct in the management of obesity-related insulin resistance.     

Evaluation of kidney and liver subacute toxicity induced by Bezalip-Pravastatin-Lopid antihyperlipidaemic compounds in rats

Renal and hepatic subacute toxicity induced by the antihyperlipidaemic drugs: Bezalip-Pravastatin and Lopid was investigated in rats using serum biochemical parameters. Toxicological evaluation was performed in serum samples following the administration of the therapeutic dose regimens of the compounds that were previously shown to be effective in inhibition of 3-hydroxy-methylglutaryl coenzyme A (HMG CoA) reductase, the enzyme controlling the rate-limiting step in the synthesis of cholesterol, and acyl-CoA cholesterol acyl transferase (ACAT) which converts intracellular free cholesterol to cholesterol ester. Renal and hepatic subacute toxicity was evaluated by measuring enzyme activity or concentrations of: alanine aminotransferace, alkaline phosphatase, aspartate aminotransferase, gamma-glutamyltransferase, glucose, potassium, sodium, blood urea nitrogen, uric acid and creatinine. The use of the above serum biochemical parameters indicated that the overall toxicity impact of antihyperlipidaemic drugs was Bezalip = Pravastatin < Lopid. We have found that the Pravastatin--in contrast to the above antihyperlipidaemic drugs--only transiently affects the biochemical parameters associated with toxicity, but, it affects some of the biochemical parameters associated with hepatic and renal toxicity, up to a significantly lower extent than the antihyperlipidaemic drugs.     

Metabolomic analysis of resveratrol-induced effects in the human breast cancer cell lines MCF-7 and MDA-MB-231

Resveratrol is a naturally occurring anticancer compound present in grapes and wine with antiproliferative properties against breast cancer cells and xenografts. Our objective was to investigate the metabolic alterations that characterize the effects of resveratrol in the human breast cancer cell lines MCF-7 and MDA-MB-231 using high-throughput liquid chromatography-based mass spectrometry. In both cell lines, growth inhibition was dose dependent and accompanied by substantial metabolic changes. For all 21 amino acids analyzed levels increased more than 100-fold at a resveratrol dose of 100?μM with far lower concentrations in MDA-MB-231 compared to MCF-7 cells. Among the biogenic amines and modified amino acids (n?=?16) resveratrol increased the synthesis of serotonin, kynurenine, and spermindine in both cell lines up to 61-fold indicating that resveratrol strongly interacts with cellular biogenic amine metabolism. Among the eicosanoids and oxidized polyunsaturated fatty acids (n?=?17) a pronounced increase in arachidonic acid and its metabolite 12S-HETE was observed in MDA-MB-231 and to a lesser extent in MCF-7 cells, indicating release from cell membrane phospholipids upon activation of phospholipase A? and subsequent metabolism by 12-lipoxygenase. In conclusion, metabolomic analysis elucidated several small molecules as markers for the response of breast cancer cells to resveratrol.     

Metabolic factors underlying high serum triglycerides in the normal hamster

Comparative lipid metabolism of rats and hamsters was investigated to determine the metabolic basis for the relatively high concentrations of serum triglycerides in the hamster. It was found that serum free fatty acids (FFA) in the hamster are higher than in the rat in the fed condition. In addition, a higher percentage of the fatty acids esterified in the liver of the hamster is utilized for triglyceride synthesis. These factors combine to elevate hepatic triglyceride synthesis in the hamster. However, triglyceride does not accumulate in the liver in these animals in the fed state. In fact, liver triglycerides are lower in the fed hamster than in the fed rat, and the hamster stores much less triglyceride in liver lipid droplets than does the rat in this nutritional state. Most of the liver triglyceride in fed hamsters is present in dense particles corresponding to vesicular lipoprotein triglyceride in the secretory pool. In isolated organ perfusion experiments hamsters livers exhibited greater net triglyceride secretion than did rat livers. Serum triglycerides in the hamster remain elevated in the fasting state. In this condition the high proportion of free fatty acids utilized for liver triglyceride synthesis, relative to that incorporated into hepatic phospholipids, persists in the hamster and marked liver triglyceride accumulation occurs. Lipid droplets are extremely abundant in these livers. The present study implicates increased conversion of free fatty acids to triglyceride in the liver and increased hepatic production of very low density lipoproteins (VLDL) in the hamster in the genesis of the hyperglyceridemia characteristic of this species.     

Lipid metabolome-wide effects of the PPARgamma agonist rosiglitazone

Successful therapy for chronic diseases must normalize a targeted aspect of metabolism without disrupting the regulation of other metabolic pathways essential for maintaining health. Use of a limited number of single molecule surrogates for disease, or biomarkers, to monitor the efficacy of a therapy may fail to predict undesirable side effects. In this study, a comprehensive metabolomic assessment of lipid metabolites was employed to determine the specific effects of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist rosiglitazone on structural lipid metabolism in a new mouse model of Type 2 diabetes. Dietary supplementation with rosiglitazone (200 mg/kg diet) suppressed Type 2 diabetes in obese (NZO x NON)F1 male mice, but chronic treatment markedly exacerbated hepatic steatosis. The metabolomic data revealed that rosiglitazone i) induced hypolipidemia (by dysregulating liver-plasma lipid exchange), ii) induced de novo fatty acid synthesis, iii) decreased the biosynthesis of lipids within the peroxisome, iv) substantially altered free fatty acid and cardiolipin metabolism in heart, and v) elicited an unusual accumulation of polyunsaturated fatty acids within adipose tissue. These observations suggest that the phenotypes induced by rosiglitazone are mediated by multiple tissue-specific metabolic variables. Because many of the effects of rosiglitazone on tissue metabolism were reflected in the plasma lipid metabolome, metabolomics has excellent potential for developing clinical assessments of metabolic response to drug therapy.     

Metabolomics identifies changes in fatty acid and amino acid profiles in serum of overweight older adults following a weight loss intervention

The application of metabolomics in nutritional research may be a useful tool to analyse and predict the response to a dietary intervention. The aim of this study was to examine metabolic changes in serum samples following exposure to an energy-restricted diet (?15 % of daily energy requirements) over a period of 8 weeks in overweight and obese older adults (n?=?22) using a gas chromatography/mass spectrometry (GC/MS) metabolomic approach. After 8 weeks, there were significant reductions in weight (7 %) and metabolic improvement (glucose and lipid profiles). Metabolomic analysis found that total saturated fatty acids (SFAs), including palmitic acid (C16:0) and stearic acid (C18:0) and monounsaturated fatty acids (MUFAs), were significantly decreased after the 8-week intervention. Furthermore, palmitoleic acid (C16:1) was found to be a negative predictor of change in body fat loss. Both the total ω-6 and ω-3 polyunsaturated fatty acids (PUFAs) significantly decreased, although the overall total amounts of PUFAs did not. The branched chain amino acid (BCAA) isoleucine significantly decreased in the serum samples after the intervention. In conclusion, this study demonstrated that the weight loss intervention based on a hypocaloric diet identified changes in the metabolic profiles of serum in overweight and obese older adults, with a reduction in anthropometric and biochemical parameters also found.     

A genomics and multi-platform metabolomics approach to identify new traits of rice quality in traditional and improved varieties

Using a novel approach combining four complementary metabolomic and mineral platforms with genome-wide genotyping at 1536 single nucleotide polymorphism (SNP) loci, we have investigated the extent of biochemical and genetic diversity in three commercially-relevant waxy rice cultivars important to food production in the Lao People??s Democratic Republic (PDR). Following cultivation with different nitrogen fertiliser regimes, multiple metabolomic data sets, including minerals, were produced and analysed using multivariate statistical methods to reveal the degree of similarity between the genotypes and to identify discriminatory compounds supported by multiple technology platforms. Results revealed little effect of nitrogen supply on metabolites related to quality, despite known yield differences. All platforms revealed unique metabolic signatures for each variety and many discriminatory compounds could be identified as being relevant to consumers in terms of nutritional value and taste or flavour. For each platform, metabolomic diversity was highly associated with genetic distance between the varieties. This study demonstrates that multiple metabolomic platforms have potential as phenotyping tools to assist breeders in their quest to combine key yield and quality characteristics. This better enables rice improvement programs to meet different consumer and farmer needs, and to address food security in rice-consuming countries.     

Metabolite profiles correlate closely with neurobehavioral function in experimental spinal cord injury in rats

Traumatic spinal cord injury (SCI) results in direct physical damage and the generation of local factors contributing to secondary pathogenesis. Untargeted metabolomic profiling was used to uncover metabolic changes and to identify relationships between metabolites and neurobehavioral functions in the spinal cord after injury in rats. In the early metabolic phase, neuronal signaling, stress, and inflammation-associated metabolites were strongly altered. A dynamic inflammatory response consisting of elevated levels of prostaglandin E2 and palmitoyl ethanolamide as well as pro- and anti-inflammatory polyunsaturated fatty acids was observed. N-acetyl-aspartyl-glutamate (NAAG) and N-acetyl-aspartate (NAA) were significantly decreased possibly reflecting neuronal cell death. A second metabolic phase was also seen, consistent with membrane remodeling and antioxidant defense response. These metabolomic changes were consistent with the pathology and progression of SCI. Several metabolites, including NAA, NAAG, and the ω-3 fatty acids docosapentaenoate and docosahexaenoate correlated greatly with the established Basso, Beattie and Bresnahan locomotive score (BBB score). Our findings suggest the possibility of a biochemical basis for BBB score and illustrate that metabolites may correlate with neurobehavior. In particular the NAA level in the spinal cord might provide a meaningful biomarker that could help to determine the degree of injury severity and prognosticate neurologic recovery.     

Abcb11 deficiency induces cholestasis coupled to impaired β-fatty acid oxidation in mice

The bile salt export pump (BSEP) is an ATP-binding cassette transporter that serves as the primary system for removing bile salts from the liver. In humans, deficiency of BSEP, which is encoded by the ABCB11 gene, causes severe progressive cholestatic liver disease from early infancy. In previous studies of Abcb11 deficiency in mice generated on a mixed genetic background, the animals did not recapitulate the human disease. We reasoned that ABCB11 deficiency may cause unique changes in hepatic metabolism that are predictive of liver injury. To test this possibility, we first determined that Abcb11 knock-out (KO) C57BL/6J mice recapitulate human deficiency. Before the onset of cholestasis, Abcb11 KO mice have altered hepatic lipid metabolism coupled with reduced expression of genes important in mitochondrial fatty acid oxidation. This was associated with increased serum free-fatty acids, reduced total white adipose, and marked impairment of long-chain fatty acid β-oxidation. Importantly, metabolomic analysis confirmed that Abcb11 KO mice have impaired mitochondrial fatty acid β-oxidation with the elevated fatty acid metabolites phenylpropionylglycine and phenylacetylglycine. These metabolic changes precede cholestasis but may be of relevance to cholestatic disease progression because altered fatty acid metabolism can enhance reactive oxygen species that might exacerbate cholestatic liver damage.     

Metabolomics of prolonged fasting in humans reveals new catabolic markers

Fasting is one of the simplest metabolic challenges that can be performed in humans. We here report for the first time a comprehensive analysis of the human ??fasting metabolome?? obtained from analysis of plasma and urine samples in a small cohort of healthy volunteers, using nuclear magnetic resonance (NMR), gas chromatography- and liquid chromatography-mass spectrometry (GC-MS and LC-MS). Intra- and inter-individual variation of metabolites was on measurement of four overnight fasting samples collected from each volunteer over a four week period. One additional sample per volunteer was collected following a prolonged fasting period of 36?h. Amongst a total of 377 quantified entities in plasma around 44% were shown to change significantly in concentration when volunteers extended fasting from 12 to 36?h. In addition to known markers (plasma free fatty acids, glycerol, ketone bodies) that reflect changes in the body??s fuel management under fasting conditions a wide range of ??new?? entities such as ??-aminobutyrate as well as other amino and keto acids were identified as fasting markers. Based on multiple correlations amongst the metabolites and selected hormones in plasma such as leptin or insulin-like-growth-factor-1 (IGF-1), a robust metabolic network with coherent regulation of a wide range of metabolites could be identified. The metabolomics approach described here demonstrates the plasticity of human metabolism and identifies new and robust markers of the fasting state.     

Toxicogenomic analysis of gene expression changes in rat liver after a 28-day oral benzene exposure

Benzene is an industrial chemical, component of automobile exhaust and cigarette smoke. After hepatic bioactivation benzene induces bone marrow, blood and hepatic toxicity. Using a toxicogenomics approach this study analysed the effects of benzene at three dose levels on gene expression in the liver after 28 daily doses. NMR based metabolomics was used to assess benzene exposure by identification of characteristic benzene metabolite profiles in urine. The 28-day oral exposure to 200 and 800 mg/kg/day but not 10 mg/kg/day benzene-induced hematotoxicity in male Fisher rats. Additionally these upper dose levels slightly reduced body weight and increased relative liver weights. Changes in hepatic gene expression were identified with oligonucleotide microarrays at all dose levels including the 10 mg/kg/day dose level where no toxicity was detected by other methods. The benzene-induced gene expression changes were related to pathways of biotransformation, glutathione synthesis, fatty acid and cholesterol metabolism and others. Some of the effects on gene expression observed here have previously been observed after induction of acute hepatic necrosis with bromobenzene and acetaminophen. In conclusion, changes in hepatic gene expression were found after treatment with benzene both at the toxic and non-toxic doses. The results from this study show that toxicogenomics identified hepatic effects of benzene exposure possibly related to toxicity. The findings aid to interpret the relevance of hepatic gene expression changes in response to exposure to xenobiotics. In addition, the results have the potential to inform on the mechanisms of response to benzene exposure.     

Role of white adipose lipolysis in the development of NASH induced by methionine- and choline-deficient diet

Methionine- and choline-deficient diet (MCD) is a model for nonalcoholic steatohepatitis (NASH) in rodents. However, the mechanism of NASH development by dietary methionine/choline deficiency remains undetermined. To elucidate the early metabolic changes associated with MCD-NASH, serum metabolomic analysis was performed using mice treated with MCD and control diet for 3 days and 1  week, revealing significant increases in oleic and linoleic acids after MCD treatment. These increases were correlated with reduced body weight and white adipose tissue (WAT) mass, increased phosphorylation of hormone-sensitive lipase, and up-regulation of genes encoding carboxylesterase 3 and β2-adrenergic receptor in WAT, indicating accelerated lipolysis in adipocytes. The changes in serum fatty acids and WAT by MCD treatment were reversed by methionine supplementation, and similar alterations were detected in mice fed a methionine-deficient diet (MD), thus demonstrating that dietary methionine deficiency enhances lipolysis in WAT. MD treatment decreased glucose and increased fibroblast growth factor 21 in serum, thus exhibiting a similar metabolic phenotype as the fasting response. Comparison between MCD and choline-deficient diet (CD) treatments suggested that the addition of MD-induced metabolic alterations, such as WAT lipolysis, to CD-induced hepatic steatosis promotes liver injury. Collectively, these results demonstrate an important role for dietary methionine deficiency and WAT lipolysis in the development of MCD-NASH.     

Utilization of exogenous free fatty acids for the production of very low density lipoprotein triglyceride by livers of carbohydrate-fed rats

High carbohydrate diets enhance the hepatic output of very low density lipoprotein triglycerides. The fatty acids of these triglycerides could come from exogenous sources (i.e., diet or adipose tissue) or from de novo fatty acid synthesis in the liver. The role of exogenous free fatty acids was evaluated in rats fed Purina Chow or diets containing 10% fructose for up to 14 wk. In carbohydrate-fed rats, serum triglycerides were twice normal, and VLDL accounted for about 60% of the increases. Pre-beta-lipoprotein was increased and alpha- and beta-lipoprotein were decreased. Phospholipid and cholesterol levels were unchanged. Livers were perfused with glucose and free fatty acids. Perfusate free fatty acids rose from 180 to 1800 micro eq/liter as the infused acids increased from 0 to 992 micro eq/3 hr; simultaneously, net free fatty acid uptake rose from < 1 to 18 micro eq/g/hr and triglyceride output by the liver doubled. However, rates of secretion of triglyceride became constant, and triglyceride accumulated in liver at uptakes of free fatty acids > 13 micro eq/g/hr. More lauric and myristic acid appeared in the perfusate than was infused, suggesting the hepatic discharge of free fatty acids. Livers of fructose-fed rats secreted twice as much oleate-(14)C-labeled triglyceride as controls at all levels of free fatty acid uptake. The ratios of the specific activities of perfusate triglyceride to free oleate-(14)C were unaffected by diet and were about 0.6 and 1.0 at low and high triglyceride secretion rates, respectively. Thus, carbohydrate feeding did not result in altered uptakes of free fatty acids or preferential secretion of triglycerides containing endogenously synthesized fatty acid. Instead, the increased secretion of triglyceride was accomplished by enhanced formation of VLDL triglyceride from exogenous free fatty acids.