Regeneration of ethanol-injured rat gastric mucosa must undergo changes in major metabolic pathways to achieve DNA replication and cell proliferation. These events are highly dependent on glucose utilization and inhibited by vitamin E (VE) (α-tocopherol) administration. Therefore, the present study aimed at assessing lipid metabolism in the gastric mucosa and ethanol-induced gastric damage and the effect of α-tocopherol administration. For this, rates of fatty acid β-oxidation and lipogenesis were tested in gastric mucosa samples. Through histological analysis, we found loss of the mucosa’s superficial epithelium, which became gradually normalized during the recovery period. Proliferation of gastric mucosa occurred with augmented formation of β-oxidation by-products, diminished synthesis of triacylglycerols (TGs), as well as of phospholipids, and a reduced cytoplasmic NAD/NADH ratio, whereas the mitochondrial redox NAD/NADH ratio was much less affected. In addition, α-tocopherol increased palmitic acid utilization in the gastric mucosa, which was accompanied by the induction of ‘mirror image’ effects on the cell redox state, reflected in an inhibited cell gastric mucosa proliferation by the vitamin administration. In conclusion, the present study shows, for the first time, the role of lipid metabolism in the adaptive cell gastric mucosa changes that drive proliferation after a chronic insult. Moreover, α-tocopherol increased gastric mucosa utilization of palmitic acid associated with energy production. These events could be associated with its antioxidant properties in co-ordination with regulation of genes and cell pathways, including changes in the cell NAD/NADH redox state.

CVD (cardiovascular disease) is associated with abnormal liver enzymes, and NAFLD (non-alcoholic fatty liver disease) is independently associated with cardiovascular risk. To gain insights into the molecular events underlying the association between liver enzymes and CVD, we developed an HFD (high-fat diet)-induced NAFLD in the SHR (spontaneously hypertensive rat) and its control WKY (Wistar–Kyoto) rat strain. We hypothesized that hepatic induction of Hif1a (hypoxia-inducible factor 1α) might be the link between CVD and liver injury. Male SHRs ( n =13) and WKY rats ( n =14) at 16 weeks of age were divided into two experimental groups: standard chow diet and HFD (10 weeks). HFD-fed rats, irrespective of the strain, developed NAFLD; however, only HFD-SHRs had focus of lobular inflammation and high levels of hepatic TNFα (tumour necrosis factor α). SHRs had significantly higher liver weight and ALT (alanine aminotransferase) levels, irrespective of NAFLD. Liver abundance of Hif1a mRNA and Hif1α protein were overexpressed in SHRs ( P <0.04) and were significantly correlated with ALT levels ( R =0.50, P <0.006). This effect was not reverted by a direct acting splanchnic vasodilator (hydralazine). Angiogenesis may be induced by the HFD, but the disease model showed significantly higher hepatic Vegf (vascular endothelial growth factor) levels ( P <0.025) even in absence of dietary insult. Hif1a mRNA overexpression was not observed in other tissues. Liver mRNA of Nr1d1 (nuclear receptor subfamily 1, group D, member 1; P <0.04), Ppara [Ppar (peroxisome-proliferatoractivated receptor) α; P <0.05], Pparg (Pparγ; P <0.001) and Sirt1 (Sirtuin 1; P <0.001) were significantly upregulated in SHRs, irrespective of NAFLD. Sirt1 and Hif1a mRNAs were significantly correlated ( R =0.71, P <0.00002). In conclusion, CVD is associated with Hif1a -related liver damage, hepatomegaly and reprogramming of liver metabolism, probably to compensate metabolic demands.

NAFLD (non-alcoholic fatty liver disease) represents a spectrum of fatty liver diseases associated with an increased risk of Type 2 diabetes and cardiovascular disease. The spectrum of fatty liver diseases comprises simple steatosis, steatosis with inflammation [i.e. NASH (non-alcoholic steatohepatitis)], fatty liver disease with inflammation and fibrosis (severe NASH) and cirrhosis. The molecular mechanisms contributing to NASH are the subject of considerable investigation, as a better understanding of the pathogenesis of NASH will lead to novel therapies for a condition that hitherto remains difficult to treat. In the present issue of Clinical Science , Piguet and co-workers have investigated the effects of hypoxia in the PTEN (phosphatase and tensin homologue deleted on chromosome 10)-deficient mouse, a mouse model that develops NAFLD. The authors show that a short period (7 days) of exposure to hypoxia aggravates the NAFLD phenotype, causing changes in the liver that are in keeping with NASH with increased lipogenesis and inflammation.

The metabolic disorders that predispose patients to NASH (non-alcoholic steatohepatitis) include insulin resistance and obesity. Repeated hypoxic events, such as occur in obstructive sleep apnoea syndrome, have been designated as a risk factor in the progression of liver disease in such patients, but the mechanism is unclear, in particular the role of hypoxia. Therefore we studied the influence of hypoxia on the development and progression of steatohepatitis in an experimental mouse model. Mice with a hepatocellular-specific deficiency in the Pten (phosphatase and tensin homologue deleted on chromosome 10) gene, a tumour suppressor, were exposed to a 10% O 2 (hypoxic) or 21% O 2 (control) atmosphere for 7 days. Haematocrit, AST (aspartate aminotransferase), glucose, triacylglycerols (triglycerides) and insulin tolerance were measured in blood. Histological lesions were quantified. Expression of genes involved in lipogenesis and mitochondrial β-oxidation, as well as FOXO1 (forkhead box O1), hepcidin and CYP2E1 (cytochrome P450 2E1), were analysed by quantitative PCR. In the animals exposed to hypoxia, the haematocrit increased (60±3% compared with 50±2% in controls; P <0.01) and the ratio of liver weight/body weight increased (5.4±0.2% compared with 4.7±0.3% in the controls; P <0.01). Furthermore, in animals exposed to hypoxia, steatosis was more pronounced ( P <0.01), and the NAS [NAFLD (non-alcoholic fatty liver disease) activity score] (8.3±2.4 compared with 2.3±10.7 in controls; P <0.01), serum AST, triacylglycerols and glucose were higher. Insulin sensitivity decreased in mice exposed to hypoxia relative to controls. The expression of the lipogenic genes SREBP-1c (sterol-regulatory-element-binding protein-1c), PPAR-γ (peroxisome-proliferator-activated receptor-γ), ACC1 (acetyl-CoA carboxylase 1) and ACC2 (acetyl-CoA carboxylase 2) increased significantly in mice exposed to hypoxia, whereas mitochondria β-oxidation genes [PPAR-α (peroxisome-proliferator-activated receptor-α) and CPT-1 (carnitine palmitoyltransferase-1)] decreased significantly. In conclusion, the findings of the present study demonstrate that hypoxia alone aggravates and accelerates the progression of NASH by up-regulating the expression of lipogenic genes, by down-regulating genes involved in lipid metabolism and by decreasing insulin sensitivity.

The enzyme DGAT (acyl-CoA:diacylglycerol acyltransferase) catalyses the final step of triacylglycerol (triglyceride) synthesis. Mice overexpressing hepatic DGAT2 fed a high-fat diet develop fatty liver, but not insulin resistance, suggesting that DGAT2 induces a dissociation between fatty liver and insulin resistance. In the present study, we investigated whether such a phenotype also exists in humans. For this purpose, we determined the relationships between genetic variability in the DGAT2 gene with changes in liver fat and insulin sensitivity in 187 extensively phenotyped subjects during a lifestyle intervention programme with diet modification and an increase in physical activity. Changes in body fat composition [MR (magnetic resonance) tomography], liver fat and intramyocellular fat ( 1 H-MR spectroscopy) and insulin sensitivity [OGTT (oral glucose tolerance test) and euglycaemic–hyperinsulinaemic clamp] were determined after 9 months of intervention. A change in insulin sensitivity correlated inversely with changes in total body fat, visceral fat, intramyocellular fat and liver fat (OGTT, all P <0.05; clamp, all P ≤0.03). Changes in total body fat, visceral fat and intramyocellular fat were not different between the genotypes of the SNPs (single nucleotide polymorphisms) rs10899116 C>T and rs1944438 C>T (all P ≥0.39) of the DGAT2 gene. However, individuals carrying two or one copies of the minor T allele of SNP rs1944438 had a smaller decrease in liver fat (−17±10 and −24±5%; values are means±S.E.M.) compared with subjects homozygous for the C allele (−39±7%; P =0.008). In contrast, changes in insulin sensitivity were not different among the genotypes (OGTT, P =0.76; clamp, P =0.53). In conclusion, our findings suggest that DGAT2 mediates the dissociation between fatty liver and insulin resistance in humans. This finding may be important in the prevention and treatment of insulin resistance and Type 2 diabetes in subjects with fatty liver.

The physiological response to starvation involves increased muscle proteolysis and adipose tissue lipolysis that supply amino acids and non-esterified fatty acids (‘free fatty acids’) for gluconeogenesis, oxidation and ketogenesis. In the present issue of Clinical Science , Moller and co-workers show that, in humans, IHL (intrahepatic lipid) content, measured using 1 H-magnetic resonance spectroscopy, increases following 36 h of fasting, with a direct association with plasma levels of 3-hydroxybutyrate. The observation raises interesting questions as to how IHL levels increase in a situation of increased mitochondrial fatty acid oxidation and ketogenesis. Possible mechanisms for increased IHLs include reduced apoB-100 (apolipoprotein B-100) production and hepatic lipid export, and/or impaired mitochondrial function resulting from increased oxidative stress, with diversion of fatty acids for esterification. The accumulation of IHL during prolonged fasting may, therefore, reflect a maladaptive response to increased non-esterified fatty acid delivery to the liver that unmasks a subtle defect in mitochondrial function. This could have implications for the pathogenesis of the common human disorder of non-alcoholic fatty liver disease. The accumulation of IHLs observed with prolonged fasting may also explain exacerbations of steatohepatitis seen sometimes with rapid weight loss, anorexia nervosa and parenteral nutrition. The findings also suggest caution against promoting excessive ketogenesis with weight-loss regimens.

The impact of fasting on IHL (intrahepatic lipid) content in human subjects has not been investigated previously, but results indicate that it may change rapidly in response to metabolic cues. The aim of the present study was to measure IHL content after fasting and to correlate this with circulating lipid intermediates. A total of eight healthy non-obese young males were studied before and after 12 or 36 h of fasting. IHL content was assessed by 1 H-magnetic resonance spectroscopy, and blood samples were drawn after the fasting period. IHL content increased significantly after the 36 h fasting period [median increase 156% (range, 4–252%); P <0.05]. Furthermore, a significant positive correlation between this increase and 3-hydroxybutyrate concentration was detected ( P =0.03). No significant change in IHL content was demonstrated after the 12 h fasting period. The baseline median inter-individual variation in IHLs was 0.51% (range, 0.25–0.72%). The coefficient of variation of IHL measurements was 11.6%; 25–30% of the variation was of analytical origin and the remaining 70–75% was attributed to repositioning. In conclusion, IHL content increases in healthy male subjects during fasting, which demonstrates that nutritional status should be accounted for when assessing IHLs in clinical studies. Moreover, the increase in IHLs was positively correlated with the concentration of 3-hydroxybutyrate.

Effectively assessing subtle hepatic metabolic functions by novel non-invasive tests might be of clinical utility in scoring NAFLD (non-alcoholic fatty liver disease) and in identifying altered metabolic pathways. The present study was conducted on 39 (20 lean and 19 obese) hypertransaminasemic patients with histologically proven NAFLD {ranging from simple steatosis to severe steatohepatitis [NASH (non-alcoholic steatohepatitis)] and fibrosis} and 28 (20 lean and eight overweight) healthy controls, who underwent stable isotope breath testing ([ 13 C]methacetin and [ 13 C]ketoisocaproate) for microsomal and mitochondrial liver function in relation to histology, serum hyaluronate, as a marker of liver fibrosis, and body size. Compared with healthy subjects and patients with simple steatosis, NASH patients had enhanced methacetin demethylation ( P =0.001), but decreased ( P =0.001) and delayed ( P =0.006) ketoisocaproate decarboxylation, which was inversely related ( P =0.001) to the degree of histological fibrosis ( r =−0.701), serum hyaluronate ( r =−0.644) and body size ( r =−0.485). Ketoisocaproate decarboxylation was impaired further in obese patients with NASH, but not in patients with simple steatosis and in overweight controls. NASH and insulin resistance were independently associated with an abnormal ketoisocaproate breath test ( P =0.001). The cut-off value of 9.6% cumulative expired 13 CO 2 for ketoisocaproate at 60 min was associated with the highest prediction (positive predictive value, 0.90; negative predictive value, 0.73) for NASH, yielding an overall sensitivity of 68% and specificity of 94%. In conclusion, both microsomal and mitochondrial functions are disturbed in NASH. Therefore stable isotope breath tests may usefully contribute to a better and non-invasive characterization of patients with NAFLD.

1. The CoA and carnitine ester intermediates of mitochondrial β-oxidation have not previously been quantified in liver disease, although there is some evidence that β-oxidation is inhibited in alcoholic fatty liver. Mitochondria were isolated from needle liver biopsies from normal subjects, from patients with alcoholic fatty liver and patients with fatty liver of other aetiologies, incubated with 60 μmol/l [U- 14 C]hexadecanoate and the resultant CoA and carnitine esters were measured. 2. Although there was no significant difference in β-oxidation flux between the patient groups, there was a significant rise in the proportion of 3-hydroxyacyl-CoA and 2-enoyl-CoA esters in patients with alcoholic fatty liver compared with normal subjects, and in patients with non-alcoholic fatty liver, suggesting an inhibition at the level of 3-hydroxyacyl-CoA dehydrogenase activity. 3. In alcoholic patients this difference could not be accounted for on the basis of the measured activity of short and long-chain 3-hydroxyacyl-CoA dehydrogenases, and it is suggested that either an inhibition of complex I activity or diminished amounts of ubiquinone are likely to be responsible for the observed accumulation of CoA and carnitine esters, which may contribute to the accumulation of triacylglycerols in alcoholic steatosis. In fatty liver of other aetiologies, short- and long-chain 3-hydroxyacyl-CoA dehydrogenase activities were decreased.

1. Chronic alcohol feeding with a low-fat diet (4.4% total calories) produced a two- to three-fold increase in hepatic triacylglycerol and esterified cholesterol compared with pair-fed low-fat diet controls. Plasma lipids were similar in both groups. 2. Hepatic fatty acid synthesis rates measured in vivo with 3 H 2 O were significantly lower in the alcohol-fed animals than in controls. Activities of hepatic fatty acid synthase (EC 2.3.1.85) and acetyl-CoA carboxylase (EC 6.4.1.2) were reduced in the alcohol-fed rats. 3. These results indicate that enhanced hepatic fatty acid synthesis does not occur in rats fed alcohol and a low-fat diet for 4 weeks, and is thus not implicated in the pathogenesis of alcohol-induced fatty liver.

1. Plasma and urine free and total carnitine and acylcarnitine levels were assayed in 12 control subjects and 20 chronic alcoholics with fatty liver. Although the alcoholics had a wider range of values than the controls, there was no significant difference between the two groups. 2. Hepatic free and total carnitine and long- and short-chain acylcarnitines were assayed by a radioenzymatic method in samples from seven control subjects and seven alcoholics. No significant differences in any of the indices were noted between the patient and control groups and it was concluded that carnitine deficiency did not contribute to alcoholic fatty liver in patients without cirrhosis. 3. Skeletal muscle free and total carnitine and long-and short-chain acylcarnitines were assayed in eight alcoholics and seven control subjects. The alcoholics had significantly higher total and free carnitine levels. It is suggested that this reflects a selective enrichment of the biopsy sample with type I carnitine-rich fibres due to the type II fibre atrophy found in approximately half the patients.

1. Chronic (5 weeks) alcohol-fed and isocaloric glucose pair-fed control rats had similar body weights, liver weights and liver protein contents. 2. Hepatic esterified cholesterol and triacylglycerol levels were two- to three-fold higher in alcohol-fed rats than in controls. 3. Hepatic cholesterol synthesis rates measured in vivo with 3 H 2 O were significantly reduced in alcohol-fed rats. 4. Hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (NADPH) (EC 1.1.1.34) activity was increased and the apparent K m for 3-hydroxymethyl-3-glutaryl-CoA was decreased in alcohol-fed rats. 5. Hepatic acyl-CoA:cholesterol acyltransferase (cholesterol acyltransferase; EC 2.3.1.26) activity was significantly increased in alcohol-fed rats. 6. These results indicate that there is no direct relationship between 3-hydroxy-3-methylglutaryl-CoA reductase activity and sterol synthesis in liver of alcohol-fed rats.

1. Liver slices from chronically alcohol-fed rats incubated with 3 H 2 O showed less than half the fatty acid synthesis rates of pair-fed controls. Addition of 50 mmol/l ethanol or of 10 mmol/l lactate and 1 mmol/l pyruvate to the incubation medium did not alter the fatty acid synthesis rates in either groups. Hepatic fatty acid synthesis rates measured in vivo with 3 H 2 O were also significantly reduced in alcohol-fed rats. 2. Time-course experiments showed that after 1 week on the ethanol diet hepatic fatty acid synthesis rates in vitro were similar to control rats, although the liver triacylglycerol content was significantly increased. From the second week of feeding, fatty acid synthesis rates were significantly lower in alcohol-fed rats and the liver triacylglycerol content progressively increased compared with controls. 3. Fatty acid synthase activity in liver cytosolic fractions were similar to controls in the alcohol-fed group after 1 week of feeding but were significantly lower in alcohol-fed rats from the second week onwards. 4. These results indicate that hepatic triacylglycerol accumulation after alcohol feeding is not due to increased fatty acid synthesis. The reduced fatty acid synthesis observed is a consequence of triacylglycerol accumulation.

1. A micro-technique was developed to measure fatty acid oxidation in vitro and to investigate its possible derangement in alcoholic fatty liver disease. 2. Percutaneous liver biopsy specimens were obtained from nine control subjects and 28 alcoholic patients with mild to severe fatty liver. Fresh tissue (10–15 mg) was incubated at 37°C for 90 min in a sealed reaction flask containing 1.92 mmol/l [l- 14 C]palmitic acid (1–2 μCi) and 1% essentially fatty acid free albumin in Krebs-Henseleit buffer, pH 7.4. Radiolabelled CO 2 and perchloric acid-soluble ketone bodies were isolated and counted. 3. CO 2 production was markedly reduced in alcoholic patients with mild and severe fatty liver compared with controls. This depression was reversed by the addition of malate to the reaction flask but not by carnitine or coenzyme A. 4. Ketone body production was similar in controls and patients with mild and severe fatty liver. 5. After the incubation in vitro , the tissue was extracted with chloroform/methanol and the triglyceride fraction isolated by thin layer chromatography and counted for radioactivity. The rate of palmitic acid incorporation into triglyceride was higher in alcoholic patients, particularly those with severe fatty infiltration, compared with controls. 6. It is suggested that alcoholic fatty liver is accompanied by a progressive reduction in palmitic acid oxidation with the major defect occurring in the tricarboxylic acid cycle. In contrast, the rate of palmitic acid esterification into ‘triglyceride is enhanced.

1. Human hepatic lipase activities were studied in needle biopsy specimens by fluorogenic and radioisotopic assay methods. Using analytical subcellular fractionation by sucrose density gradient centrifugation a lysosomal acid lipase, with pH optimum of 4.0, and an endoplasmic reticulum alkaline lipase, with pH optimum of 8.5, were demonstrated with the fluorogenic assay. 2. The apparent K m of the acid lipase was 17 μmol/l by the fluorogenic method and 23 mmol/l by the radioisotopic method. The values for alkaline lipase were 94 μmol/l and 1.4 mmol/l respectively. 3. Assay of these activities in biopsies from 16 control subjects and 42 chronic alcoholics showed increasing activity with increasing hepatic fatty infiltration when the fluorogenic assay was used: no differences were demonstrated with radioisotopic assay. These results suggest that depressed lipolysis due to a relative deficiency of triglyceride lipase is not a causal factor in triglyceride accumulation in chronic alcoholic fatty liver.

1. Needle biopsy specimens of liver were obtained from six control subjects with histologically normal liver and 11 chronic alcoholics with fatty liver. 2. Micro- and macro-lipid droplet fractions were isolated by differential flotation. These fractions, together with the sedimenting membranes, were assayed for cholesterol, cholesteryl ester, phospholipid, free fatty acids and triglyceride. 3. Electron microscopy demonstrated marked differences in the range of lipid droplet sizes in the two fractions and biochemical analysis suggested that the microdroplet lipid corresponded to pre-very low density lipoprotein (VLDL) particles. 4. Studies on biopsies from patients with alcoholic fatty liver showed a 2–3-fold increase in triglyceride in both lipid droplet fractions but most of the accumulating triglyceride was sedimentable and membrane-bound. 5. Needle biopsy specimens from two patients with alcoholic fatty liver were fractionated with a vertical pocket re-orientating rotor. The principal organelles were separated and the subcellular distribution of triglyceride, phospholipid and free cholesterol determined. Triglyceride showed a bimodal distribution to a particulate fraction tentatively located to Golgi particles and to droplet-lipid remaining in the sample layer.

1. Percutaneous needle biopsy specimens of liver were obtained from alcoholic, diabetic and control patients. Micro-methods of lipid separation and quantification were employed to determine the detailed nature of hepatic lipid. 2. Triglyceride is the major accumulating liver lipid in both alcoholic and diabetic patients. Cholesteryl ester levels were raised in both alcoholic and diabetic patients but only diabetic patients had significantly increased free cholesterol and phospholipid levels. Determination of phospholipid/free cholesterol ratios revealed a significant decrease in alcoholic cirrhosis compared with controls. 3. Fatty acid ester analysis of hepatic phospholipid and triglyceride revealed significant differences between alcoholic patients and controls but not between diabetic patients and controls. An increased ratio of non-essential/essential fatty acids was found in the patients with alcoholic liver disease whereas those of diabetic patients were similar to the controls.

1. The mechanism of development of acute alcoholic fatty liver in the Rhesus monkey was investigated by studying the effect of Triton WR-1339 on plasma triglycerides. 2. After ethanol infusion, the rise in plasma triglyceride produced by Triton was greatly reduced and there was an accumulation of triglyceride in the liver. 3. Thus findings suggest that acute alcoholic fatty liver in Rhesus monkeys is probably due to a defect in the release of triglyceride from the liver.

1. The effect of intravenous infusion of ethanol on the concentration of plasma free fatty acids (FFA) and triglycerides (TG) and on hepatic triglyceride content was studied in Rhesus monkeys. 2. A minor fall in the plasma FFA was observed after the infusion of ethanol, followed by a rise in FFA which was prevented by a beta-receptor blocking agent. 3. Ethanol administration increased the liver triglyceride level. 4. Prior treatment with a beta-receptor blocking agent which apparently blocked the adrenergic process did not prevent accumulation of triglycerides in the ethanol-treated animals.