Fatty liver disease

Lifestyle factors other than alcohol intake can lead to insidious outcomes from this surprisingly common condition. Assoc Prof David Cameron-Smith reviews current and potential management strategies.

Liver cell transplantation leads to repopulation and functional correction in a mouse model of Wilson's disease

Background and Aim: The toxic milk (tx) mouse is a non-fatal animal model for the metabolic liver disorder, Wilson's disease. The tx mouse has a mutated gene for a copper-transporting protein, causing early copper accumulation in the liver and late accumulation in other tissues. The present study investigated the efficacy of liver cell transplantation (LCT) to correct the tx mouse phenotype.

Methods: Congenic hepatocytes were isolated and intrasplenically transplanted into 3–4-month-old tx mice, which were then placed on various copper-loaded diets to examine its influence on repopulation by transplanted cells. The control animals were age-matched untransplanted tx mice. Liver repopulation was determined by comparisons of restriction fragment length polymorphism ratios (DNA and mRNA), and copper levels were measured by atomic absorption spectroscopy.

Results: Repopulation in recipient tx mice was detected in 11 of 25 animals (44%) at 4 months after LCT. Dietary copper loading (whether given before or after LCT, or both) provided no growth advantage for donor cells, with similar repopulation incidences in all copper treatment groups. Overall, liver copper levels were significantly lower in repopulated animals (538 ± 68 µg/g, n = 11) compared to non-repopulated animals (866 ± 62 µg/g, n = 14) and untreated controls (910 ± 103 µg/g, n = 6; P < 0.05). This effect was also seen in the kidney and spleen. Brain copper levels remained unchanged.

Conclusion: Transplanted liver cells can proliferate and correct a non-fatal metabolic liver disease, with some restoration of hepatic copper homeostasis after 4 months leading to reduced copper levels in the liver and extrahepatic tissues, but not in the brain.

Effect of Glucocorticoid pretreatment on oxidative liver injury and survival in jaundiced rats with Endotoxing Cholangitis

Introduction: Biliary tract infection is associated with high mortality. This study investigated the effect of glucocorticoid pretreatment on lipopolysaccharide (LPS)-induced cholangitis. Methods: Rats undergoing either sham operation or ligation of the extrahepatic bile duct (BDL) for 2 weeks were randomly assigned to receive intravenous injections of dexamethasone (DX) or normal saline (NS) prior to infusing LPS into the biliary tract. The plasma levels of tumor necrosis factor-α (TNFα), chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) as well as liver mRNA expression of MCP-1 and MIP-2 were determined. Infiltration of monocytes, Kupffer cells, and neutrophils in rat liver were studied with immunohistochemistry. Oxidative liver injury was measured by the malondialdehyde (MDA) content. Results: Dexamethasone pretreatment resulted in significantly decreased plasma levels of TNFα at 1 hour, MCP-1 and MIP-2 at 2 and 3 hours, and decreased liver MCP-1 mRNA expression at 3 hours following LPS infusion in BDL-DX rats than in BDL-NS rats. The number of inflammatory cells in the liver was significantly different between sham- and BDL-treated rats but was not affected by DX pretreatment. Pretreatment with DX resulted in significantly decreased liver MDA contents in the BDL-DX group than that in the BDL-NS group. Jaundiced rats pretreated with 5 mg DX prior to infusion of 1 g of LPS were 6.8 times more likely to survive than those that were not pretreated. Conclusions: Pretreatment of jaundiced, LPS-treated rats with a  supraphysiological dose of dexamethasone may rescue their lives by suppression of chemokine expression and alleviation of oxidative liver injury.

Biliary intervention augments chemotactic reaction and aggravates cholestatic liver injury in rats

Background
Intervention of the biliary system is frequently done in patients with obstructive jaundice and is associated with significant morbidity and mortality. The pathogenesis is unknown.
Materials and methods
A rat model of bile duct ligation (BDL) for 2 weeks was established in which biliary intervention was feasible by injection of normal saline through an indwelling catheter in the bile ducts. Plasma levels of C-C chemokine MCP-1 and C-X-C chemokine MIP-2 were measured by using ELISA. Blood monocytes, Kupffer cells, and neutrophils in the liver were characterized with antibodies to ED1, ED2, and myeloperoxidase (MPO). Lipid peroxidation was measured by malondialdehyde contents and apoptosis by TUNEL stain of the liver.
Results
Biliary intervention resulted in an increase of plasma MCP-1 and MIP-2 proteins by 1 h, which declined to normal level by 3 h in both sham and BDL rats. The levels in BDL rats were significantly higher than in sham at most points. There was a transient increase of ED1- and ED2-positive cells and MPO-staining cells in sham rat liver by 1 h after intervention. ED2-positive cells increased significantly by 1 h, while ED1- and MPO-positive cells decreased, yet insignificantly after intervention in BDL rats. The cell counts in BDL were constantly higher than in sham. Malondialdehyde increased precipitously in BDL by 3 h and was significantly higher than in sham throughout the study period. Parenchymal liver injury, manifested by elevated ALT, as well as apoptosis and necrosis of liver cells, was significantly increased in BDL rats, but not in sham rats.
Conclusion
Biliary intervention augments chemokine expression, precipitates lipid peroxidation, and aggravates liver injury in cholestatic rats.

Changes in bacterial concentration in the liver correlate with that in the hepaticojejunostomy after bile duct reconstruction: implication in the pathogenesis of postoperative cholangitis

Postoperative cholangitis is a frequent and unpredictable complication of unknown etiology following bile duct reconstruction (BDR), particularly for biliary atresia. This study was undertaken to correlate the growth of bacteria in the hepaticojejunostomy with that in the liver after BDR. Quantitative bacterial culture was done on the specimens taken from the liver and from the hepaticojejunostomy at 1 week (group 1, n = 7), 1 month (group 2, n = 7), and 2 months (group 3, n = 7) following BDR with Roux-en-Y hepaticojejunostomy in piglets after 2 weeks of common bile duct ligation. The histological examination of the liver and the hepaticojejunostomy, as well as serial monitoring of hemogram and liver function tests, were performed to correlate the findings with the bacterial concentration of the liver and the hepaticojejunostomy following BDR. The bacterial concentration of the hepaticojejunostomy, expressed as log10 colony-forming units per gram (log10 CFU/g) of the hepaticojejunostomy, showed a progressive decrease from 8.38 ± 1.36 in group 1, 7.07 ± 2.54 in group 2, to 3.56 ± 1.31 in group 3 (p = 0.001). The log10 CFU/g of the liver also showed a progressive decrease from 5.02 ± 1.59 in group 1, 3.16 ± 1.56 in group 2, to 2.19 ± 1.09 in group 3 (p = 0.006). There was a significant positive correlation of the log10 CFU/g of the liver (n = 21) with that of the hepaticojejunostomy (n = 21) following BDR (r = 0.600, p = 0.004). Most of the infectious pathogens isolated from the liver were also isolated from the hepaticojejunostomy. The changes in hemoglobin, bilirubin, albumin, and ammonia significantly correlated with the changes of the bacterial concentration of the liver. The results of the study suggests that hepatic bacterial proliferation after BDR is significantly affected by microbial overgrowth in the bilioenteric anastomosis and is associated with deteriorated liver function and hemogram.

Differential effects of dietary fatty acids on genes associated with liver fat metabolism

Background – It has been recognized that specific fatty acids have the ability to directly influence the abundance of gene transcripts in organs such as the liver. However little comparison has been made between the effects of common dietary of fatty acids and there influence on gene expression.
Objectives – To determine the effect of diets rich saturated, monounsaturated and polyunsaturated on gene transcripts associated with liver fat metabolism. Specifically how these three classes of fatty acids influence mRNA levels of key transcriptional regulators (PGC1a, PPARa, PPARd, SREBP1C & ChREBP), fat oxidative (ACO, LCPT1, HMG-CoA lyase & UCP-2) and fat synthetic (ACC, MCD, GPAT & malic enzyme) genes were investigated.
Design - Rats (n=32) were evenly divided into four groups; a saturated fat diet, a monounsaturated fat diet, a polyunsaturated fat diet (each diet contained 23% fat) and standard rat chow (7% fat) diet and fed for 12 weeks. Real-time PCR analysis was performed on liver tissue.
Outcomes – PGC1a and SREBP1C increased 1.9 fold or greater in all groups. Conversely, PPARa, PPARd and ChREBP demonstrated variable changes with diet composition. Monounsaturated and polyunsaturated fat increased HMG-CoA lyase 2.8 fold, a response that was absent in the saturated fat fed animals. UCP-2 was decrease 3.0 fold by all dietary treatments. Malic enzyme was increased 2.8 and 2.4 fold with saturated and polyunsaturated diets respectively, yet was unaltered by the monounsaturated fat diet.
Conclusion – Modifications in common dietary fat composition initiated divergent gene responses in liver. These alterations were complex, with no uniform alteration in transcription factors with closely related functions (PPARfamily) and genes encoding proteins within the same metabolic pathway (fat oxidation or fat synthesis). Further studies are necessary to identify the predominant mechanisms regulating these differences in gene expression.

Squalene supplementation alters genes associated with liver cholesterol metabolism

Background – Squalene is a component of shark liver oil and has been speculated to have cholesterol reducing properties. High levels of total and LDL cholesterol have been shown to contribute to the development of chronic heart disease. The liver is central to the regulation of cholesterol metabolism and dietary intervention has long been recognized as a primary means to reduce the risks of chronic heart disease and related ailments.
Objectives – To determine the effect of dietary squalene supplementation on gene transcripts associated with liver cholesterol metabolism. Specifically the effect of squalene supplementation on mRNA levels for proteins that
regulate cholesterol biosynthesis (HMDH & ERG1), cholesterol elimination (SRB1), bile synthesis (CP7A1 & CP27A) and cholesterol excretion by the liver into bile (ABCG5 & ABCG8) was investigated.
Design – Rats (n=32) were divided into four groups and supplemented for 12 weeks. Groups one and two were fed a cholesterol rich diet for six weeks followed by six weeks of a cholesterol rich diet plus 1.75mg/day of squalene or 3.5 mg/day. Group three was fed a cholesterol rich diet for 12 weeks and group four was fed standard rat chow for 12 weeks. Blood lipid levels were monitored during the study and liver gene expression was determined at the
conclusion of the feeding trial via RT-PCR.
Outcomes – 3.5 mg/day of squalene lowered total and LDL cholesterol in rats consuming a cholesterol rich diet. This dose of squalene also resulted in constant levels of HMDH and ERG1 whereas the cholesterol rich diet halved mRNA levels of these enzymes. Furthermore 3.5 mg/day of squalene caused a greater than 3.0 fold increase in mRNA levels of the proteins SRB1, CP7A1, CP27A and ABCG5.
Conclusion – Dietary squalene supplementation at a dose of 3.5 mg/day lowers total and LDL cholesterol in rats consuming a cholesterol rich diet. These reductions in cholesterol levels may be due to increased cholesterol
elimination, bile synthesis and cholesterol excretion by the liver into bile mediated by changes in gene expression of key enzymes involved in these metabolic pathways

Characterization of the drug binding specificity of rat liver fatty acid binding protein

Liver-fatty acid binding protein (L-FABP) is found in high levels in enterocytes and is involved in the cytosolic solubilization of fatty acids during fat absorption. In the current studies, the interaction of L-FABP with a range of lipophilic drugs has been evaluated to explore the potential for L-FABP to provide an analogous function during the absorption of lipophilic drugs. Binding affinity for L-FABP was assessed by displacement of a fluorescent marker, 1-anilinonaphthalene-8-sulfonic acid (ANS), and the binding site location was determined via nuclear magnetic resonance chemical shift perturbation studies. It was found that the majority of drugs bound to L-FABP at two sites, with the internal site generally having a higher affinity for the compounds tested. Furthermore, in contrast to the interaction of L-FABP with fatty acids, it was demonstrated that a terminal carboxylate is not required for specific binding of lipophilic drugs at the internal site of L-FABP.

Probing the fibrate binding specificity of rat liver fatty acid binding protein

Liver-fatty acid binding protein (L-FABP) is found in high levels in enterocytes and is involved in cytosolic solubilization of fatty acids. In addition, L-FABP has been shown to bind endogenous and exogenous lipophilic compounds, suggesting that it may also play a role in modulating their absorption and disposition within enterocytes. Previously, we have described binding of L-FABP to a range of drugs, including a series of fibrates. In the present study, we have generated structural models of L-FABP-fibrate complexes and undertaken thermodynamic analysis of the binding of fibrates containing either a carboxylic acid or ester functionality. Analysis of the current data reveals that both the location and the energetics of binding are different for fibrates that contain a carboxylate compared to those that do not. As such, the data presented in this study suggest potential mechanisms that underpin molecular recognition and dictate specificity in the interaction between fibrates and L-FABP.

An improved method for the purification of rat liver-type fatty acid binding protein from Escherichia coli

We have developed expedient and reliable methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins. We have examined enzyme purification strategies suited to large-scale processing and present a chromatographic sequence that serves as a pilot model for industrial scale preparation of cyclosporin synthetase from cyclosporin producing fungi. A chromatographic sequence consisting of ammonium sulfate precipitation → gel filtration → hydrophobic interaction chromatography → anion exchange chromatography, yielded an electrophoretically homogeneous cyclosporin synthetase preparation (Coomassie G-250 brilliant blue staining). Furthermore, a native polyacrylamide gel electrophoresis system was developed for the isolation of active cyclosporin synthetase enzyme from crude extracts of cyclosporin producing fungi. The environmental factors affecting enzyme stability and the continuity of the in vitro cyclosporin biosynthetic reaction-temperature, pH, and substrate depletion were assessed and manageable conditions have been defined for sustainable cyclosporin biosynthesis with enzyme isolates. Cyclosporin synthetase exhibited an optimal temperature range of 24–29 °C and a pH optimum of 7.6. The native enzyme displayed a pI of 5.7, as determined by isoelectric focusing. The industrial implementation of an in vitro biosynthetic approach could potentially prove useful for the production of important therapeutic cyclosporins which occur as only minor fermentation by-products.

Novel genes in the liver of diabetic Psammomys obesus

Type 2 diabetes mellitus is a metabolic disease characterised by defects in insulin secretion and insulin action and disturbances in carbohydrate, fat and protein metabolism. Hepatic insulin resistance contributes to hyperglycemia and also leads to disturbances in fat metabolism in type 2 diabetes. Psammomys obesus is a unique poly genie animal model of type 2 diabetes and obesity, ideally suited for studies examining physiological and genetic aspects of these diseases. To identify metabolic abnormalities potentially contributing to the obesity and diabetes phenotype in P. obesus, indirect calorimetry was used to characterise whole body energy expenditure and substrate utilisation. Lean-NGT, obese-IGT and obese-diabetic animals were examined in fed and fasted states and following 14 days of dietary energy restriction. Energy expenditure and fat oxidation were elevated in the obese-IGT and obese-diabetic groups in proportion to body weight. Glucose oxidation was not different between groups. Obese-diabetic P. obesus displayed elevated nocturnal blood glucose levels and fat oxidation. Following 14 days of dietary energy restriction, body weight was reduced and plasma insulin and blood glucose levels were normalised in all groups. Glucose oxidation was reduced and fat oxidation was increased. After 24 hours of fasting, plasma insulin and blood glucose levels were normalised in all groups. Energy expenditure and glucose oxidation were greatly reduced and fat oxidation was increased. Following either dietary energy restriction or fasting, energy expenditure, glucose oxidation and fat oxidation were not different between groups of P. obesus. Energy expenditure and whole body substrate utilisation in P. obesus was similar to that seen in humans. P. obesus responded normally to short term fasting and dietary energy restriction. Elevated nocturnal fat oxidation rates and plasma glucose levels in obese-diabetic P. obesus may be an important factor in the pathogenesis of obesity and type 2 diabetes in these animals. These studies have further validated P. obesus as an ideal animal model of type 2 diabetes and obesity. It was hypothesised that many genes in the liver of P. obesus involved in glucose and fat metabolism would be differentially expressed between lean-NGT and obese-diabetic animals. These genes may represent significant factors in the pathophysiology of type 2 diabetes. Two gene discovery experiments were conducted using suppression subtractive hybridisation (SSH) to enrich a cDNA library for differentially expressed genes. Experiment 1 used cDNA dot blots to screen 576 clones with cDNA derived from lean-NGT and obese-diabetic animals. 6 clones were identified as overexpressed in lean-NGT animals and 6 were overexpressed in obese-diabetic animals. These 12 clones were sequenced and SYBR-Green PCR was used to confirm differential gene expression. 4 genes were overexpressed (≥1.5 fold) in lean-NGT animals and 4 genes were overexpressed (≥1.5 fold) in obese-diabetic animals. To explore the physiological role of these genes, hepatic gene expression was examined in several physiological conditions. One gene, encoding thyroxine binding globulin (TBG), was confirmed as overexpressed in lean-NGT P. obesus with ad libitum access to food, relative to both obese-IGT and obese-diabetic animals. TBG expression decreased with fasting in all animals. Fasting TBG expression remained greater in lean-NGT animals than obese-IGT and obese-diabetic animals. TBG expression was not significantly affected by dietary energy restriction. TBG is involved in thyroid metabolism and is potentially involved in the regulation of energy expenditure. Fasting increased hepatic site 1 protease (SIP) expression in lean-NGT animals but was not significantly affected in obese-IGT and obese-diabetic animals. SIP expression was not significantly affected by dietary energy restriction. SIP is involved in the proteolytic processing of steroid response element binding proteins (SREBP). SREBPs are insulin responsive and are known to be involved in lipid metabolism. Gene expression studies found TBG and SIP were associated with obesity and diabetes. Future research will determine whether TBG and SIP are important in the pathogenesis of these diseases. Experiment 2 used SSH and cDNA microarray to screen 8064 clones. 223 clones were identified as overexpressed in lean-NGT P. obesus and 274 clones were overexpressed in obese-diabetic P. obesus (p ≤0.05). The 9 most significantly differentially expressed clones identified from the microarray screen were sequenced (p ≤0.01). 7 novel genes were identified as well as; sulfotransferase related protein and albumin. These 2 genes have not previously been associated with either type 2 diabetes or obesity. It is unclear why hepatic expression of these genes may differ between lean-NGT and obese-diabetic groups of P. obesus. Subsequent studies will explore the potential role of these novel and known genes in the pathophysiology of type 2 diabetes.