Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor (TGF)-β superfamily. The rejuvenative effect of GDF11 has been called into question recently, and its role in liver regeneration is unclear. Here, we investigated the pathophysiologic role of GDF11, as well as its plausible signaling mechanisms in a mouse model of partial hepatectomy (PH). We demonstrated that both serum and hepatic GDF11 protein expression increased following PH. Treatment with adeno-associated viruses-GDF11 and recombinant GDF11 protein severely impaired liver regeneration, whereas inhibition of GDF11 activity with neutralizing antibodies significantly improved liver regeneration after PH. In vitro , GDF11 treatment significantly delayed cell proliferation and induced cell-cycle arrest in α mouse liver 12 (AML12) cells. Moreover, GDF11 activated TGF-β-SMAD2/3 signaling pathway. Inhibition of GDF11-induced SMAD2/3 activity significantly blocked GDF11-mediated reduction in cell proliferation both in vivo and in vitro . In the clinical setting, GDF11 levels were significantly elevated in patients after hepatectomy. Collectively, these results indicate that rather than a ‘rejuvenating’ agent, GDF11 impairs liver regeneration after PH. Suppression of cell-cycle progression via TGF-β-SMAD2/3 signaling pathway may be a key mechanism by which GDF11 inhibits liver regeneration.

Ubiquitin-specific peptidase 4 (USP4) protein is a type of deubiquitination enzyme that is correlated with many important biological processes. However, the function of USP4 in hepatic ischaemia/reperfusion (I/R) injury remains unknown. The aim of the present study was to explore the role of USP4 in hepatic I/R injury. USP4 gene knockout mice and primary hepatocytes were used to construct hepatic I/R models. The effect of USP4 on hepatic I/R injury was examined via pathological and molecular analyses. Our results indicated that USP4 was significantly up-regulated in liver of mice subjected to hepatic I/R injury. USP4 knockout mice exhibited exacerbated hepatic I/R injury, as evidenced by enhanced liver inflammation via the nuclear factor κB (NF-κB) signalling pathway and increased hepatocyte apoptosis. Additionally, USP4 overexpression inhibited hepatocyte inflammation and apoptosis on hepatic I/R stimulation. Mechanistically, our study demonstrates that USP4 deficiency exerts its detrimental effects on hepatic I/R injury by inducing activation of the transforming growth factor β-activated kinase 1 (TAK1)/JNK signalling pathways. TAK1 was required for USP4 function in hepatic I/R injury as TAK1 inhibition abolished USP4 function in vitro . In conclusion, our study demonstrates that USP4 deficiency plays a detrimental role in hepatic I/R injury by promoting activation of the TAK1/JNK signalling pathways. Modulation of this axis may be a novel strategy to alleviate the pathological process of hepatic I/R injury.

Increased glucose production and reduced hepatic glycogen storage contribute to metabolic abnormalities in diabetes. Irisin, a newly identified myokine, induces the browning of white adipose tissue, but its effects on gluconeogenesis and glycogenesis are unknown. In the present study, we investigated the effects and underlying mechanisms of irisin on gluconeogenesis and glycogenesis in hepatocytes with insulin resistance, and its therapeutic role in type 2 diabetic mice. Insulin resistance was induced by glucosamine (GlcN) or palmitate in human hepatocellular carcinoma (HepG2) cells and mouse primary hepatocytes. Type 2 diabetes was induced by streptozotocin/high-fat diet (STZ/HFD) in mice. In HepG2 cells, irisin ameliorated the GlcN-induced increases in glucose production, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) expression, and glycogen synthase (GS) phosphorylation; it prevented GlcN-induced decreases in glycogen content and the phosphoinositide 3-kinase (PI3K) p110α subunit level, and the phosphorylation of Akt/protein kinase B, forkhead box transcription factor O1 (FOXO1) and glycogen synthase kinase-3 (GSK3). These effects of irisin were abolished by the inhibition of PI3K or Akt. The effects of irisin were confirmed in mouse primary hepatocytes with GlcN-induced insulin resistance and in human HepG2 cells with palmitate-induced insulin resistance. In diabetic mice, persistent subcutaneous perfusion of irisin improved the insulin sensitivity, reduced fasting blood glucose, increased GSK3 and Akt phosphorylation, glycogen content and irisin level, and suppressed GS phosphorylation and PEPCK and G6Pase expression in the liver. Irisin improves glucose homoeostasis by reducing gluconeogenesis via PI3K/Akt/FOXO1-mediated PEPCK and G6Pase down-regulation and increasing glycogenesis via PI3K/Akt/GSK3-mediated GS activation. Irisin may be regarded as a novel therapeutic strategy for insulin resistance and type 2 diabetes.

Sorafenib is the standard treatment for patients with advanced hepatocellular carcinoma (HCC). However, the median overall survival (OS) benefit is only ~3 months, and sufficient biomarkers predicting treatment response are not available. The aim of the present study was to evaluate miRNA expression patterns from HCC tissue biopsies as potential biomarkers in patients under sorafenib treatment. Nineteen patients with advanced HCC treated with sorafenib were included. RNA was extracted from formalin-fixed paraffin-embedded (FFPE) liver biopsies. miRNA expression profiling of 818 mature miRNAs was performed using GeneChip® miRNA Array 2.0 (Affymetrix). Global miRNA patterns were assessed using unsupervised hierarchical clustering analysis (UCA), and specific miRNAs with correlation with disease control rate (DCR) or good OS were evaluated by pairwise supervised analyses. UCA divided the patients into three distinct groups by their miRNA expression patterns. However, DCR or OS did not correlate with these sub-groups. We have identified several miRNAs that correlated with either DCR or OS ( P <0.05). However, with correction for multiple testing, these results did not reach statistical significance in this small cohort. Global miRNA analysis from very low input RNA deriving from liver biopsies showed distinctive clustering of molecular sub-groups in patients with intermediate and advanced HCC. Clinical response including OS under sorafenib did not correlate with global miRNA expression patterns, but we have identified candidate miRNAs for the prediction of DCR and OS to be evaluated in prospective studies and larger patient cohorts.

The biological and pharmacological effects of BK (bradykinin) are mediated by two receptors: the constitutive B 2 R (B 2 receptor) and the inducible B 1 R (B 1 receptor). BK plays a role in the hepatic microcirculation by inducing the PHR (portal hypertensive response) via B 2 R, whereas DABK (des-Arg 9 -BK), a B 1 R agonist, does not elicit the response. During IRI (ischaemia/reperfusion injury), important changes occur in the microcirculation, and cell death by necrosis and apoptosis is involved in poor graft function. The aim of the present study was to analyse the role of B 1 R and B 2 R in liver cell death induced by IRI. Livers from Wistar rats were submitted to ischaemia (4°C) for 4 or 24 h. After this period, livers were reperfused ex vivo with Krebs–Henseleit solution (37°C). BK or DABK was then injected as a bolus during reperfusion in the absence or presence of HOE-140 (a B 2 R antagonist) or DALBK (des-Arg 9 -Leu 8 -BK) (a B 1 R antagonist) respectively. Liver viability was analysed by glucose release and bile secretion. The PHR to kinins did not change. Cell death was higher in the DABK group and its antagonist significantly decreased cell death. Interestingly, the B 1 R antagonist did not alter the number of necrotic cells, but it decreased the number of apoptotic cells. On the other hand, the B 2 R antagonist decreased the number of necrotic cells, but did not alter the number of apoptotic cells. Therefore B 1 R may participate in apoptotic cell death signalling, and B 2 R may be involved in necrotic cell death.

RFA (radiofrequency ablation) is an established therapy for HCC (hepatocellular carcinoma). The multikinase inhibitor sorafenib prolongs survival in advanced HCC. We examined the effects of RFA alone and in combination with sorafenib on a bystanding tumour in a two-tumour rat model of HCC. A total of 80 rats were implanted with two liver tumours and randomized to four treatment groups: vehicle and sham operation (control), sorafenib and sham operation (Sora/Sham), vehicle and RFA (Vh/RFA), and sorafenib and RFA (Sora/RFA) ( n =10/group per time point). RFA or sham-operation was performed on the left lobe tumour on day 15. Animals were killed at day 18 and day 30. Non-RFA-targeted right lobe tumours were analysed for angiogenesis, growth factors [HGF (hepatocyte growth factor), EGF (epidermal growth factor) and VEGF (vascular endothelial growth factor)] and infiltrating immune cells (CD3 and CD68). At day 30, the non-RFA-targeted tumours were significantly smaller in all three treatment groups compared with control (Sora/Sham P ≤0.0001, Vh/RFA P =0.005 and Sora/RFA P ≤0.0001). The smallest tumours were observed in animals treated with a combination of sorafenib and RFA, whereas the size reduction seen in the RFA-only group indicated an RFA-mediated distant suppression of tumour growth. Growth factor measurement revealed transiently decreased EGF levels after RFA ( P =0.008), whereas sorafenib treatment decreased HGF levels ( P =0.001). MVD (microvessel density) was reduced by sorafenib ( P =0.002) despite increased VEGF levels ( P ≤0.0001). The immune parameters revealed augmented T-cells and IL-10 (interleukin 10) levels in all three treatment groups; sorafenib additionally increased macrophage numbers ( P ≤0.0001). RFA and sorafenib alone resulted in significant volume reduction of the non-RFA-targeted tumour; this effect was enhanced when both modalities were combined.

In the present Hypothesis article, we summarize and present data from the literature that support our hypothesis on the potential mechanisms by which UPS (ubiquitin–proteasome system) inhibitors reduce I/R (ischaemia/reperfusion) injury in the liver. I/R is the main cause of primary liver failure and, consequently, minimizing the detrimental effects of this process could increase the number of suitable transplantation grafts and also enhance the survival rate of patients after liver transplantation. A potential strategy to reduce I/R injury is the use of UPS inhibitors either as additives to preservation solutions or as drugs administered to patients. However, there is still controversy over whether the use of UPS inhibitors is beneficial or deleterious with regard to liver injury. From our experience and the few studies that have investigated the role of UPS in hepatic I/R, we believe that the use of UPS inhibitors is a potential strategy to reduce I/R injury in liver transplantation and graft preservation. We hypothesize that one of the main mechanisms of action of UPS inhibitors may be the up-regulation of AMPK (AMP-activated protein kinase) activity and the consequent down-regulation of mTOR (mammalian target of rapamycin), which may finally influence autophagy and preserve the energy state of the cell.

BA (bile acid) formation is considered an important final step in RCT (reverse cholesterol transport). HDL (high-density lipoprotein) has been reported to transport BAs. We therefore investigated the effects of monogenic disturbances in human HDL metabolism on serum concentrations and lipoprotein distributions of the major 15 BA species and their precursor C4 (7α-hydroxy-4-cholesten-3-one). In normolipidaemic plasma, approximately 84%, 11% and 5% of BAs were recovered in the LPDS (lipoprotein-depleted serum), HDL and the combined LDL (low-density lipoprotein)/VLDL (very-low-density lipoproteins) fraction respectively. Conjugated BAs were slightly over-represented in HDL. For C4, the respective percentages were 23%, 21% and 56% (41% in LDL and 15% in VLDL) respectively. Compared with unaffected family members, neither HDL-C (HDL-cholesterol)-decreasing mutations in the genes APOA1 [encoding ApoA-I (apolipoprotein A-I], ABCA1 (ATP-binding cassette transporter A1) or LCAT (lecithin:cholesterol acyltransferase) nor HDL-C-increasing mutations in the genes CETP (cholesteryl ester transfer protein) or LIPC (hepatic lipase) were associated with significantly different serum concentrations of BA and C4. Plasma concentrations of conjugated and secondary BAs differed between heterozygous carriers of SCARB1 (scavenger receptor class B1) mutations and unaffected individuals ( P <0.05), but this difference was not significant after correction for multiple testing. Moreover, no differences in the lipoprotein distribution of BAs in the LPDS and HDL fractions from SCARB1 heterozygotes were observed. In conclusion, despite significant recoveries of BAs and C4 in HDL and despite the metabolic relationships between RCT and BA formation, monogenic disorders of HDL metabolism do not lead to altered serum concentrations of BAs and C4.

TLRs (Toll-like receptors), as evolutionarily conserved germline-encoded pattern recognition receptors, have a crucial role in early host defence by recognizing so-called PAMPs (pathogen-associated molecular patterns) and may serve as an important link between innate and adaptive immunity. In the liver, TLRs play an important role in the wound healing and regeneration processes, but they are also involved in the pathogenesis and progression of various inflammatory liver diseases, including autoimmune liver disease, alcoholic liver disease, non-alcoholic steatohepatitis, fibrogenesis, and chronic HBV (hepatitis B virus) and HCV (hepatitis C virus) infection. Hepatitis viruses have developed different evading strategies to subvert the innate immune system. Thus recent studies have suggested that TLR-based therapies may represent a promising approach in the treatment in viral hepatitis. The present review focuses on the role of the local innate immune system, and TLRs in particular, in the liver.

Hepatic IR (ischaemia/reperfusion) injury is an important clinical problem complicating liver surgery and transplantation. IPC (ischaemic preconditioning) is a strategy whereby brief episodes of IR in an organ can induce an adaptive response to protect against subsequent prolonged IR injury. However, trauma to vessels supplying the target organ is unavoidable using the technique of direct IPC. One amenable strategy would be to apply the protective preconditioning stimulus to an organ distant or remote from the target organ of interest, a technique known as RIPC (remote IPC). In the present issue of Clinical Science , Abu-Amara and co-workers utilize hindlimb RIPC as a novel therapeutic strategy against liver IR injury and investigate the mechanistic contribution of NO to hepatoprotection by administering C-PTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt], an NO scavenger. Their experiments set the stage for more definitive studies to demonstrate a discernible benefit for the utility of RIPC in liver surgery and transplantation.

NO (nitric oxide) may protect the liver from IR (ischaemia/reperfusion) injury. RIPC (remote ischaemic preconditioning) also protects against liver IR injury; however, the molecular mediator(s) of RIPC are currently unknown. The aim of the present study was to assess the role of NO in hindlimb RIPC-induced protection against liver IR injury. Mice were allocated to the following groups: sham group; RIPC group (six cycles of 4×4 min IR of hindlimb); IR group [40 min lobar (70%) hepatic ischaemia and 2-h reperfusion]; RIPC+IR group (RIPC followed by IR group procedures); and C-PTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt]+RIPC+IR group [C-PTIO (a direct NO scavenger) was administered, followed by the RIPC+IR group procedure]. Hepatic MBF (microcirculatory blood flow) was measured throughout the experiment. Circulating NO x (nitrite and nitrate) levels, plasma liver transaminases, hepatic histopathological and TEM (transmission electron microscopy) studies were performed at the end of the experiment. NO x concentrations were significantly elevated ( P <0.05) in the RIPC and RIPC+IR groups. Compared with liver IR alone, RIPC+IR preserved hepatic MBF during liver reperfusion ( P <0.05). In contrast, C-PTIO+RIPC+IR reduced MBF compared with RIPC+IR ( P <0.05). RIPC+IR reduced plasma transaminases ( P <0.05), and histopathological and ultrastructural features of injury compared with IR alone. The protective effects of RIPC+IR in reducing liver IR injury were abrogated in the group that received antecedent C-PTIO (C-PTIO+RIPC+IR). In conclusion, NO is an essential mediator of the protection afforded by hindlimb RIPC against liver IR injury. The mechanisms underlying this protection involve preservation of the sinusoidal structure and maintenance of blood flow through the hepatic microcirculation.

There has been a marked increase in the number of babies born by elective CS (Caesarean section). Following CS, the lack of normal stimuli that occur at birth alters the thermogeneic response, but any effects on hepatic metabolism have not been identified. In the present study, we compared the effect of delivery on hepatic metabolism in piglets, born either by CS or VD (vaginal delivery) and fed by TPN (total parenteral nutrition), by measuring lipid metabolism and enzyme activity coupled with metabolomic and genomic approaches. Hepatic lipid in the CS piglets at 7 days post-partum was in excess of 5 mg/g of liver consistent with hepatic steatosis, whereas in the VD piglets the amount of lipid was markedly lower (3 mg/g of liver) and below the threshold for a diagnosis of steatosis. Metabolomic analysis indicated that CS resulted in higher hepatic glycerol and lower glycerol phosphate dehydrogenase activity, suggesting that CS causes a decrease in hepatic gluconeogenesis from glycerol. CS also resulted in altered cholesterol handling and gene expression, despite the same dietary intake for 7 days post-partum. Furthermore, the CS piglets had a lower expression of interferon-responsive genes, but a higher expression of markers of immature hepatocytes. In conclusion, the results suggest that VD promotes normal liver maturation and hepatic metabolism, thereby reducing the accumulation of hepatic lipid.

The global increase in the prevalence of obesity has heralded a rise in associated liver injury namely NAFLD (non-alcoholic fatty liver disease). It is estimated that 20–30% of adult populations in developed countries have NAFLD and, although high quality data is currently lacking, the condition is clearly increasing in children also. NAFLD should be suspected in those with commonly available simple clinical signs and biochemistry consistent with insulin resistance. A small number of individuals with NAFLD, often considered a relatively benign condition, will progress to more severe stages of liver disease including NASH (non-alcoholic steatohepatitis) with or without fibrosis, cirrhosis and occasionally hepatocellular carcinoma. NAFLD is also commonly associated with an increased risk of developing Type 2 diabetes and treatable features of insulin resistance such as dyslipidaemia and dysglycaemia. Histological examination of liver tissue remains the only proven method to distinguish between simple steatosis and NASH, a condition far more likely to progress to cirrhosis. Identification of an imaging technique or non-invasive marker to achieve this distinction is therefore much sought after and would allow larger clinical trials and better clinical assessment. Case series and pilot studies of lifestyle advice, insulin sensitizers and other medications have shown improvements in liver histology and serum liver enzymes but robust randomized controlled studies are needed. Furthermore, the cost/benefit ratio of any new therapies, and any potential harms, must be evaluated carefully before being clinically advocated.

Oxidative stress mediates cell injury during ischaemia/reperfusion. On the other hand, experimental findings suggest that ROS (reactive oxygen species) induce processes leading to ischaemic preconditioning. The extent and source of oxidative stress and its effect on antioxidant status in the human liver during intermittent ischaemia and reperfusion remains ill-defined. Therefore the aim of the present study was to investigate the occurrence of oxidative stress in humans undergoing liver resection. Liver biopsies, and arterial and hepatic venous blood samples were taken from ten patients undergoing hepatectomy with an intermittent Pringle manoeuvre. Plasma MDA (malondialdehyde) and hepatic GSSG levels were measured as markers of oxidative stress and plasma uric acid as a marker of xanthine oxidase activity. In addition, changes in hepatosplanchnic consumption of plasma antioxidants and hepatic levels of carotenoids and glutathione (GSH) were measured. After ischaemia, hepatosplanchnic release of MDA and increased hepatic GSSG levels were found. This was accompanied by the release of uric acid, reflecting xanthine oxidase activity. During reperfusion, ongoing oxidative stress was observed by further increases in hepatic GSSG content and hepatosplanchnic MDA release. Uric acid release was minimal during reperfusion. A gradual decrease in plasma antioxidant capacity and net hepatosplanchnic antioxidant uptake was observed upon prolonged cumulative ischaemia. Oxidative stress occurs during hepatic ischaemia in man mainly due to xanthine oxidase activity. Interestingly, the gradual decline in plasma antioxidant capacity and net hepatosplanchnic antioxidant uptake during prolonged cumulative ischaemia, preserved both hydrophilic and lipophilic hepatic antioxidant levels. Decreasing plasma levels and net hepatosplanchnic uptake of plasma antioxidants may warrant antioxidant supplementation, although it should be clarified to what extent limitation of oxidative stress compromises ROS-dependent pathways of ischaemic preconditioning.

The process of blood vessel proliferation, known as angiogenesis, is essential during embryonic development and organogenesis. In adult life, it participates in normal tissue repair, wound healing, and cyclical growth of the corpus luteum and the endometrium. Crucial as it is, angiogenesis can become pathological, and abnormal angiogenesis contributes to the pathogenesis of inflammatory and neoplasic diseases. The present review highlights the evidence for the role of angiogenesis in HCC (hepatocellular carcinoma) and discusses the increasing importance of inhibitors of angiogenesis in HCC therapy.

Ras oncoproteins are probably implicated in normal and malignant cell growth in various organs. Inhibition of Ras interferes with cell proliferation of non-hepatic cells in vitro and in vivo . A potential role for Ras in normal and malignant hepatocyte proliferation prompted us to evaluate the impact of Ras inhibition by FTS ( S -farnesylthiosalicylic acid) on hepatocyte proliferation in vitro in the human hepatic tumour cell line HepG2 and in vivo after PH (partial hepatectomy) in rats. Rats were administered with FTS intraperitoneally (1, 8 and 16 h after PH) and killed 12, 24 and 48 h after PH. Cell proliferation, phosphorlyation of members of the MAPK (mitogen-activated protein kinase) pathway and levels and activity of cell cycle effectors (cyclin D, cyclin E, Cdk2 and Cdk4) were assessed in FTS-treated rats compared with controls. FTS significantly decreased overall cell count, PCNA (proliferating-cell nuclear antigen) expression and BrdU (bromodeoxyuridine) incorporation into HepG2 cells after 7 days of culture. FTS treatment significantly reduced BrdU incorporation and PCNA expression in hepatocytes after PH. Unlike control rats, cell-membrane expression of Ras was decreased in FTS-treated animals after PH, resulting in decreased Raf membrane recruitment and phosphorylation and in reduced phosphorylation of ERK1/2 (extracellular-signal-regulated kinase 1/2). The antiproliferative effect of FTS was linked to a decrease in expression and activity of the cyclin E/Cdk2 complex, without affecting cyclin D and Cdk4. Ras inhibition by FTS significantly decreased proliferation of HepG2 cells and normal hepatocytes after a strong and highly synchronized proliferation stimulus elicited by PH. The inhibitory effect was at least partially mediated by inhibition of Ras/Raf/MAPK signalling. It appears worthwhile to evaluate the impact of Ras inhibition on the development of hepatocarcinomas in vivo in adequate animal models.

FCHL (familial combined hyperlipidaemia) is characterized by multiple phenotypes that are shaped by genes, the environment and time. A longitudinal study by Brouwers and co-workers, which appears in this issue of Clinical Science , points to the central role of the liver in defining the FCHL phenotypes and demonstrates how they vary over time in relation to energy excess. On the basis of their work and that of others, we propose that FCHL is a multiple gene/multiple pathway/multiple phenotype disease. The key feature of this model of common complex disease is that it posits testable faults in definable metabolic pathways, which supply the genetic underpinning of the disorder.

FCHL (familial combined hyperlipidaemia), an entity with many features of the metabolic syndrome, is characterized by changes in cholesterol and triacylglycerol (triglyceride) phenotype over time. The present study was conducted to investigate the relationship of ALT (alanine aminotransferase) levels, used as a surrogate marker for the amount of hepatic fat, with the switch in triacylglycerol phenotype and the increased susceptibility to develop hypertriglyceridaemia in FCHL. BMI (body mass index), waist circumference and plasma triacylglycerols, insulin and ALT levels were measured in 145 FCHL family members and 54 spouses at baseline and after a 5-year follow-up. A switch from normotriglyceridaemia to hypertriglyceridaemia or vice versa, as observed in 22 of 145 FCHL family members, was associated with changes in plasma ALT levels ( P =0.001), but not with insulin levels or waist circumference. At 5 years of follow-up, an intra-individual relationship was observed between waist circumference and plasma triacylglycerols, insulin and ALT levels. For each waist circumference, FCHL patients, but not their NL (normolipidaemic) relatives, exhibited higher triacylglycerol and insulin levels than spouses ( P <0.001). Remarkably, both FCHL patients and the NL relatives had higher ALT levels for each waist circumference compared with spouses ( P <0.001 for FCHL patients, and P =0.035 for NL relatives). In conclusion, the present study shows that the longitudinal relationship of abdominal obesity–ALT is more specific for all FCHL family members, i.e. patients and their NL relatives, than the relationship of abdominal obesity–triacylglycerols. Additionally, the association of ALT with the switch in triacylglycerol phenotype suggests a central role of the liver in the pathogenesis of FCHL.

There is an increasing body of evidence to suggest that the RAS (renin–angiotensin system) contributes to tissue injury and fibrosis in chronic liver disease. A number of studies have shown that components of a local hepatic RAS are up-regulated in fibrotic livers of humans and in experimental animal models. Angiotensin II, the main physiological effector molecule of this system, mediates liver fibrosis by stimulating fibroblast proliferation (myofibroblast and hepatic stellate cells), infiltration of inflammatory cells, and the release of inflammatory cytokines and growth factors such as TGF (transforming growth factor)-β1, IL (interleukin)-1β, MCP (monocyte chemoattractant protein)-1 and connective tissue growth factor. Furthermore, blockade of the RAS by ACE (angiotensin-converting enzyme) inhibitors and angiotensin type 1 receptor antagonists significantly attenuate liver fibrosis in experimental models of chronic liver injury. In 2000 ACE2 (angiotensin-converting enzyme 2), a human homologue of ACE, was identified. ACE2 efficiently degrades angiotensin II to angiotensin-(1–7), a peptide which has recently been shown to have both vasodilatory and tissue protective effects. This suggests that ACE2 and its products may be part of an alternate enzymatic pathway in the RAS, which counterbalances the generation and actions of angiotensin II, the ACE2–angiotensin-(1–7)–Mas axis. This review focuses on the potential roles of the RAS, angiotensin II and ACE2 in chronic liver injury and fibrogenesis.

Liver fibrosis represents a major worldwide health care burden. The last 15 years have seen a rapid growth in our understanding of the pathogenesis of this clinically relevant model of inflammation and repair. This work is likely to inform the design of effective antifibrotic therapies in the near future. In this review, we examine how the innate and adaptive immune response interacts with other key cell types in the liver, such as the myofibroblast, regulating the process of hepatic fibrosis and, where relevant, resolution of fibrosis with remodelling. Emphasis is placed on the increasing knowledge that has been generated by the use of transgenic animals and animals in which specific cell lines have been deleted. Additionally, we review the increasing evidence that, although significant numbers of wound-healing myofibroblasts are derived from the hepatic stellate cell, significant contributions may occur from other cell lineages, including those from distant sites such as bone marrow stem cells.

The relationship between BA (bile acid) secretion (measured by GC–MS) and the expression of genes (measured by reverse transcription real-time PCR) involved in liver BA transport and metabolism was investigated at 20 and 32 weeks during rat hepatocarcinogenesis. A progressive loss of mRNA for transporters (more marked for Ntcp, Bsep and Mrp2 than for Oatp1/Oatp1a1, Oatp2/Oatp1a4 and Oatp4/Oatp1b2) was found. The mRNA levels of Cyp7a1 and the nuclear receptors FXR (farnesoid X receptor), SHP (small heterodimer partner) and FTF (α-fetoprotein transcription factor) were not modified, whereas those of Cyp8b1 were enhanced and those of Cyp27 were reduced. Biliary secretion of CA (cholic acid) remained unchanged, whereas that of CDCA (chenodeoxycholic acid) and other non-C 12 -hydroxylated BAs was diminished. The re-appearance of ‘flat-BAs’ (mainly allo -BAs at 20 weeks and Δ 4 -unsaturated-BAs at 32 weeks) probably reflects the progressive decrease observed in the expression of 3-oxo-Δ 4 -steroid 5β-reductase, together with the maintenance of steroid 5α-reductase type I. A significant correlation between the 5α-reductase/5β-reductase ratio and bile output of ‘flat-BAs’ was found. In conclusion, during rat hepatocarcinogenesis, the expression of transporters/enzymes responsible for BA homoeostasis is changed due to mechanisms other than those controlled by FXR/SHP/FTF. These modifications result in the re-appearance of ‘flat-BAs’, together with an increased CA/CDCA ratio in bile.

Cell therapy means treating diseases with the body's own cells. The ability to produce differentiated cell types at will offers a compelling new approach to cell therapy and therefore for the treatment and cure of a plethora of clinical conditions, including diabetes, Parkinson's disease and cardiovascular disease. Until recently, it was thought that differentiated cells could only be produced from embryonic or adult stem cells. Although the results from stem cell studies have been encouraging, perhaps the most startling findings have been the recent observations that differentiated cell types can transdifferentiate (or convert) into a completely different phenotype. Harnessing transdifferentiated cells as a therapeutic modality will complement the use of embryonic and adult stem cells in the treatment of degenerative disorders. In this review, we will examine some examples of transdifferentiation, describe the theoretical and practical issues involved in transdifferentiation research and comment on the long-term therapeutic possibilities.

IPC (ischaemic preconditioning) may protect the steatotic liver, which is particularly susceptible to I/R (ischaemia/reperfusion) injury. Hepatic steatosis was induced in Sprague–Dawley rats with a high-cholesterol (2%) diet for 12 weeks after which rats were subjected to I/R (ischaemia/reperfusion; 45 min of lobar ischaemia followed by 2 h of reperfusion). Rats were divided into three study groups ( n =6 each) receiving: (i) sham laparotomy alone, (ii) I/R, and (iii) IPC (5 min of ischaemia, followed by 10 min of reperfusion) before I/R. Hepatic extra- and intra-cellular oxygenation and HM (hepatic microcirculation) were measured with near-infrared spectroscopy and laser Doppler flowmetry respectively. Plasma liver enzymes and hepatic tissue ATP were measured as markers of liver injury. Histology showed moderate-grade steatosis in the livers. At the end of 2 h of reperfusion, I/R significantly decreased extra- and intra-cellular oxygenation concomitant with a failure of recovery of HM (21.1±14.4% of baseline; P <0.001 compared with sham animals). IPC increased intracellular oxygenation (redox state of the copper centre of cytochrome oxidase; P <0.05 compared with rats receiving I/R alone) and flow in HM (70.9±17.1% of baseline; P <0.001 compared with rats receiving I/R alone). Hepatocellular injury was significantly reduced with IPC compared with I/R injury alone (alanine aminotransferase, 474.8±122.3 compared with 5436.3±984.7 units/l respectively; P <0.01; aspartate aminotransferase, 630.8±76.9 compared with 3166.3±379.6 units/l respectively; P <0.01]. In conclusion, IPC has a hepatoprotective effect against I/R injury in livers with moderate steatosis. These data may have important clinical implications in liver surgery and transplantation.

The effect of sepsis on liver synthesis of albumin remains controversial, with studies in man suggesting that synthesis increases, whereas in animals increased, decreased and unaltered synthesis have been reported. To reconcile these conflicting data, total and relative albumin synthesis was measured in rats 24 h after caecal ligation and puncture (CLP) by immunoprecipitation of albumin following a flooding dose of L -[4- 3 H]phenylalanine. Following CLP, animals were starved for 18 h and then received intravenous infusions of saline or parenteral nutrition (PN) with or without glutamine for 6 h. In animals receiving PN, parenteral injections of growth hormone (GH) or saline vehicle were also administered. Fractional rate of liver total protein synthesis was elevated and total albumin synthesis rate was reduced in all CLP groups when compared with non-operated animals. Total albumin synthesis was also lower in all animals receiving PN than those receiving saline alone, although these differences did not attain statistical significance, except for the group receiving PN+GH. Relative albumin synthesis was also reduced after CLP, and was significantly lower in animals receiving PN than in those receiving saline alone. These findings suggest that in sepsis hepatic protein synthesis is reprioritized away from the production of albumin towards the production of acute-phase proteins and that this change is not influenced by the provision of nutritional support, glutamine or the administration of GH.

MOCP (maternal obstructive cholestasis during pregnancy) induces a reversible impairment in bile formation in young rats born to these mothers. The aim of the present study was to gain information on the effects of MOCP on the maturation of pathways involved in protein secretion into bile in young (4-week-old) rats. The amount of hepatic α-tubulin and the structure of the microtubular network were apparently not affected by MOCP. HRP (horseradish peroxidase) was used as a model protein, and its secretion into bile after administration through the jugular vein was measured. In adult (8-week-old) rats, two peaks of HRP output into bile were observed following administration: an early peak presumably due to paracellular transfer, and a late peak presumably due to transcytosis. In young rats (4 weeks old), the early peak was similar to that of adult animals, and was not affected by MOCP. However, the late peak was markedly smaller in young control rats, and was further reduced by MOCP. Brefeldin A decreased, whereas taurocholate did not change, the early peak, whereas both affected the transcytotic transport of HRP. Brefeldin A delayed HRP secretion (similarly in control and MOCP groups), without affecting cumulative output, whereas taurocholate accelerated the transcytotic transport of HRP in the control group, but not in the MOCP group. These results suggest that MOCP affects the maturation of hepatocyte mechanisms involved in the transcytotic secretion of HRP into bile.

Although glucose and protein metabolism have been investigated extensively in experimental models of hypodynamic sepsis, relatively little information is available regarding the compensated stage of sepsis. We investigated interorgan amino acid and glucose metabolism in a porcine model of compensated hyperdynamic sepsis. Fasting catheterized pigs received endotoxin ( Escherichia coli lipopolysaccharide; 3 µ g·h -1 ·kg -1 ; intravenous) or saline (controls) and volume resuscitation over 24h to reproduce hyperdynamic sepsis. Primed-constant infusions of p -aminohippurate and 3 H-labelled isotopes were used to measure glucose, amino acid and protein metabolism across the portal-drained viscera, liver and hindquarters (to represent muscle) at 0 and 24h of endotoxaemia. Whole-body protein and glucose flux were increased during hyperdynamic compensated sepsis. In endotoxaemic pigs, visceral protein was conserved, and hindquarter protein breakdown exceeded the increase in liver protein synthesis, resulting in net whole-body protein loss. Endotoxaemia increased hindquarter and visceral glycolysis and branched-chain amino acid transamination. The rate of efflux of glutamine and alanine from the hindquarters was higher than anticipated from protein breakdown, indicating de novo synthesis of these amino acids during endotoxaemia. In addition to the hindquarters, the portal-drained viscera provided substantial gluconeogenic amino acids and lactate to the liver. Although increased liver glutamate release constitutes an important nitrogen-sparing mechanism and carbon skeletons are effectively being cycled in glucose, net body protein is lost through increased ureagenesis during the hyperdynamic stage of sepsis. Specific amino acid requirements may develop in compensated hyperdynamic sepsis that is characterized by maintained organ perfusion and increased substrate utilization at the expense of body protein.

Preparations of isolated liver cells, either freshly prepared or in culture, have been available for many years; however, because they lack the polarization of the cell in the tissue, their application to the study of processes involved in bile formation has been very limited. The hepatocyte couplet model offers a unique opportunity to study in vitro the intracellular processes involved: not only the physiology and pathophysiology of bile formation, but also the corresponding structural and molecular disturbances underlying different experimental models of cholestasis. In this review we describe the experimental data generated from this model in our laboratory over a period of years, including application of hepatocyte couplets to the study of cytoskeletal changes within the hepatocyte and mechanisms of hepatoprotection in experimentally induced, clinically relevant models of intrahepatic cholestasis.

Electrolysis is a method of tissue ablation that creates chemical species and a pH gradient in response to direct current. Initial studies of electrolysis in animal models and humans have shown that it is a safe, predictable and effective process for destroying normal and tumour-bearing liver in a linear, dose-dependent manner. Presently, the amount of current that is applied (in coulombs) has to be calculated using historical data, with inherent inaccuracy. The present study tested whether pH could be used as a real-time monitor in order to predict more accurately the extent of necrosis. A total of 70 electrolytic lesions were created in 14 pigs, with pH monitoring of the lesion edge. The normal range of pH values was 6.5-8.7. A pH of less than 6 (at the anode) or more than 9 (at the cathode) reflected total cellular necrosis. When a pH value was recorded between 6.0 and 6.5 at the anode or between 8.7 and 9.0 at the cathode, the presence of necrosis was variable. In conclusion, during electrolytic ablation, pH measurement can monitor the extent of the induced necrosis.

Glutamine has beneficial effects on enterocytes and the immune system in sepsis, but its effects on hepatic metabolism remain unknown. The aim of the present study was to determine the effects of glutamine on hepatocyte energy metabolism under conditions of neonatal endotoxaemia. Suckling Wistar rats were injected intraperitoneally with 200 μ g/kg lipopolysaccharide. Oxygen consumption was measured polarographically in hepatocytes respiring on either palmitate (0.5mM) or palmitate plus glutamine (10mM). Total hepatocyte oxygen consumption was similar in hepatocytes from control and endotoxic rats, but this was due to a decrease in intramitochondrial and an increase in extramitochondrial oxygen consumption in the cells from endotoxic animals. The addition of glutamine to hepatocytes from endotoxic rats restored intramitochondrial oxygen consumption to control levels. Although glutamine did not reverse the inhibition of the thermogenic proton leak observed in endotoxaemia, it significantly increased oxygen consumption due to mitochondrial ATP synthesis ( P = 0.03). Glutamine significantly increased the hepatocyte ATP/ADP ratio ( P = 0.02 compared with hepatocytes from endotoxic rats). Electron microscopy revealed morphological damage to the mitochondria of hepatocytes from endotoxic rats, and a return to a normal appearance with the addition of glutamine. We conclude that glutamine reverses the inhibition of mitochondrial metabolism that is observed in endotoxaemia. The effect is primarily at the level of ATP synthesis.

To discriminate between the effects of infection and of anorexia associated with infection, liver albumin synthesis was measured in well-fed rats, in rats injected with live Escherichia coli and in pair-fed rats at different stages of the inflammatory response (1, 6 and 10 days after infection) using a large dose of l -[1- 14 C]valine. Albuminaemia and albumin mRNA levels were unchanged following food restriction. However, absolute albumin synthesis was decreased in pair-fed rats compared with control animals after 1 day of food restriction, and had returned to normal values by day 10 when food intake was restored. Infection was characterized by a decrease in the plasma albumin concentration (35%, 45% and 28% as compared with pair-fed rats at 1, 6 and 10 days after infection respectively). Albumin mRNA levels and relative albumin synthesis were reduced in infected rats as compared with both control and pair-fed animals at all stages of infection. However, during the early acute response, the albumin absolute synthesis rate was similar in infected rats and pair-fed rats, indicating no specific effect of infection at this stage. Later in the course of infection, the amount of albumin synthesized by the liver was lower in infected than in pair-fed rats, and hypoalbuminaemia was probably maintained due to a lack of stimulation of synthesis despite increased food intake.

We have investigated sequential changes in skeletal muscle and hepatic protein synthesis following sepsis, and their relationship to changes in circulating and tissue glutamine concentrations. Male Wistar rats underwent caecal ligation and puncture (CLP) or sham operation, with starvation, and were killed 24, 72 or 96 h later. A group of non-operated animals were killed at the time of surgery. Protein synthesis was determined using a flooding dose of l -[4- 3 H] phenylalanine, and glutamine concentrations were measured by an enzymic fluorimetric assay. Protein synthesis in gastrocnemius muscle fell in all groups. Gastrocnemius total protein content was reduced after CLP and at 72 and 96 h after sham operation. After CLP, protein synthesis was lower at 24 h, and total protein content was lower at 72 and 96 h, than in sham-operated animals. CLP was associated with increased liver protein synthesis at all time points, whereas there was no change after sham operation. Liver protein content did not change after CLP, but was lower at 72 and 96 h after sham operation than in non-operated animals. Plasma glutamine concentrations were reduced at 24 h after sham operation, and at 72 and 96 h after CLP. Muscle glutamine concentrations were reduced in all groups, with the decrease being greater following CLP than after sham operation. In the liver, glutamine concentrations were unchanged after CLP, but increased after sham operation. In rats with sepsis, decreases in muscle protein synthesis and content are associated with markedly reduced muscle glutamine concentrations. Plasma glutamine concentrations are initially maintained, but fall later. In liver, protein synthesis is increased, while glutamine concentrations are preserved. These results support a peripheral-to-splanchnic glutamine flux in sepsis.

Stable-isotope tracers were used to assess whether levels of phosphomonoesters (PME) and phosphodiesters (PDE) in the livers of lung cancer patients, as observed by 31 P magnetic resonance (MR) spectroscopy, reflect elevated whole-body glucose turnover and gluconeogenesis from alanine. Patients with advanced non-small-cell lung cancer without liver metastases ( n = 24; weight loss 0–24%) and healthy control subjects ( n = 13) were studied after an overnight fast. 31 P MR spectra of the liver in vivo were obtained, and glucose turnover and gluconeogenesis from alanine were determined simultaneously using primed-constant infusions of [6,6- 2 H 2 ]glucose and [3- 13 C]alanine. Liver PME concentrations were 6% higher in lung cancer patients compared with controls (not significant); PME levels in patients with ⩾ 5% weight loss were significantly higher than in patients with < 5% weight loss ( P < 0.01). PDE levels did not differ between the groups. In lung cancer patients, whole-body glucose production was 19% higher (not significant) and gluconeogenesis from alanine was 42% higher ( P < 0.05) compared with healthy subjects; turnover rates in lung cancer patients with ⩾ 5% weight loss were significantly elevated compared with both patients with < 5% weight loss and healthy subjects ( P < 0.05). PME levels were significantly correlated with glucose turnover and gluconeogenesis from alanine in lung cancer patients ( r = 0.48 and r = 0.48 respectively; P < 0.05). In conclusion, elevated PME levels in lung cancer patients appear to reflect increased glucose flux and gluconeogenesis from alanine. These results are consistent with the hypothesis that elevated PME levels are due to contributions from gluconeogenic intermediates.

1. The time course of iron overload of the pancreas in hypotransferrinaemic mice maintained on a standard rodent diet was compared with biochemical and histological markers of tissue damage. 2. Pancreatic iron levels increased linearly from weaning till 9 months of age [73.3 nmol/mg of tissue (SEM 9.9; n = 5) compared with 0.9 nmol/mg of tissue (SEM 0.1; n = 4) in age-matched controls] then decreased linearly till at least 18 months of age. 3. Investigation of tissue distribution of newly absorbed radioiron suggested that significant redistribution of iron from liver to pancreas (rather than direct dietary iron sources) must be invoked to explain the rate of pancreatic iron loading in hypotransferrinaemic mice. 4. Pancreatic epithelial cells first showed altered morphology at 9 months of age. At 12 months of age, the pancreatic epithelium had developed a micronodular appearance, with large numbers of acini replaced by atrophic, degenerated acinar cells. Increased collagen fibre deposition was evident by trichrome staining and by electron microscopy. Biochemical markers of pancreatitis (serum lipase, tissue pancreatitis-associated protein mRNA) were elevated before 9 months of age, whereas the levels of pancreatic amylase mRNA declined from 9 months of age. 5. The data suggest that iron loading of hypotransferrinaemic mouse pancreas proceeds up to a threshold level at 9 months of age followed by a progressive atrophy of secretory epithelium. The hypotransferrinaemic mouse pancreas is a useful model system for investigation of parenchymal cell damage by iron.

1. Living-related liver transplantation has some advantages in the evaluation of novel clinical protocols, since many complicated factors affecting initial graft function are almost uniform in grafts obtained from healthy donors. 2. To compare histidine—tryptophan—ketoglutarate (HTK) and University of Wisconsin (UW) solution in terms of tissue oxygenation in living-related liver transplantation, oxygen saturation of haemoglobin (SO 2 ) in hepatic tissue and its heterogeneity (CV, coefficient of variation) were measured by near-infrared spectroscopy. The HTK and UW groups consisted of 15 and 49 successful transplants respectively, in which no statistical differences in background were observed. 3. In the HTK group, hepatic SO 2 after portal vein reflow was higher ( P <0.01) than that in the UW group, as was that after hepatic artery reflow ( P <0.05). In the UW group, hepatic SO 2 remained at the lower level at the end of the operation. 4. Furthermore, the increase in CV after portal vein reflow was normalized after hepatic artery reflow in the HTK group. However, the CV remained at a high level at the end of the operation in the UW group. 5. Postoperative peak aspartate aminotransferase level in the HTK group was lower than that in the UW group ( P <0.05). 6. In cadaveric liver transplantation, higher hepatic SO 2 and lower CV of hepatic SO 2 in the early phase after reperfusion compared with the UW group ( n = 18) were also observed in the HTK group ( n = 30) ( P <0.05). 7. In conclusion, recovery of tissue oxygenation and its heterogeneity after reperfusion in HTK-preserved livers were more rapid and homogeneous than in UW-preserved livers in living-related liver transplantation. Accordingly, HTK solution may be a potential alternative to UW solution.

1. Primary biliary cirrhosis is a chronic cholestatic liver disease with an autoimmune aetiology. The classical histopathological lesion, of portal tract biliary epithelial cell damage, is accompanied by a T-cell-rich mononuclear cell infiltrate and upregulation of cell surface markers suggestive of local T-cell activation and cytokine release. This suggests that T-cell mediated mechanisms play an important role in tissue damage in primary biliary cirrhosis. 2. CD4+ T-cells specific for the E2 component of human pyruvate dehydrogenase complex (PDC-E2), a highly conserved enzyme which plays a critical role in intermediate metabolism, are present in the peripheral repertoire of the majority of patients with primary biliary cirrhosis. These cells are almost entirely absent from normal and chronic liver disease control subjects. The observations that peripheral blood PDC-E2-specific cells are most commonly seen in early stage disease, when active bile duct damage is occurring, and that PDC-E2-specific cells can be found in the portal tract infiltrate at times when this damage is occurring, suggest that these autoreactive cells may have a role to play in the aetiology of primary biliary cirrhosis. 3. T-cells specific for the whole PDC and its E1 component are seen in significant numbers of normal control subjects as well as patients with primary biliary cirrhosis. Retention of potentially autoreactive cells in the normal T-cell repertoire has been reported for a number of other autoantigens. 4. T-cell epitopes appear to be widely distributed throughout PDC-E2. This is in contrast to the B-cell epitopes which are highly restricted to the inner lipoyl binding domain of the protein.

1. We studied the effects of the non-selective, non-peptide, orally active endothelin (ET) receptor antagonist bosentan (Ro 47–0203) on rat hepatic and mesenteric vascular membrane 125 I-ET-1 binding characteristics in vitro and ex vivo (after bosentan by gavage in vivo ). 2. Bosentan caused a concentration-dependent competitive inhibition of 125 I-ET-1 binding to female rat mesenteric vascular (predominantly ETA receptors) and hepatic (predominantly ETB receptors) membranes in vitro and ex viva 3. The time course of the inhibition of binding ex vivo after administration of bosentan in vivo was 1–4 h for mesenteric vascular (predominantly ETA receptors) binding and 1–16 h for hepatic (predominantly ETB receptors) binding. 4. The time course of displacement of 125 I-ET-1 binding from mesenteric vascular and hepatic membranes by bosentan in vitro was similar. 5. Since bosentan is significantly excreted by the liver, the prolonged hepatic 125 I-ET-1 binding by bosentan presumably represents hepatic accumulation of bosentan, which may have implications for bosentan antagonizing the actions of ET in the liver.

1. To investigate the link between post-prandial thermogenesis and sympathetic nervous activation we have studied the effects of a single large meal on regional sympathetic nervous activity in healthy, lean subjects. 2. In nine male subjects, noradrenaline spillover was measured from the heart, kidney and liver using isotope dilution, both while fasting and after consumption of a high-energy liquid meal of composition 53% carbohydrate, 32% fat and 15% protein (energy value 2.64–3.51 MJ). Regional oxygen consumption, whole-body oxygen consumption and, in a subset of subjects, muscle sympathetic nerve firing (microneurography) were also measured. 3. Both whole-body oxygen consumption ( P < 0.03) and total body spillover of noradrenaline ( P < 0.01) rose after the meal, with peak increases of 24% and 56% respectively. Spillover of noradrenaline from the heart was unchanged, that from the hepatosplanchnic circulation increased marginally (0.377 nmol/min to 0.480 nmol/min, P = 0.09), while renal noradrenaline spillover more than doubled (0.440 nmol/min to 0.937 nmol/min, P < 0.05). Skeletal muscle sympathetic nerve activity (peroneal nerve) increased from 7.7 bursts/min at rest to peak at 17.9 bursts/min 60 min after the meal in the three subjects in whom stable recordings were obtained. 4. The meal increased oxygen consumption in the kidneys and liver significantly, from 11.5 ± 1.6 ml/min to 14.5 ± 1.1 ml/min and from 46 ± 7 ml/min to 57 ± 6 ml/min respectively ( P < 0.05), but not in the heart. 5. Consumption of a large meal produces a substantial and relatively selective increase in sympathetic outflow to the kidneys and skeletal muscle. While resting regional oxygen consumptions and noradrenaline spillovers were related, the changes that occurred in each were unrelated, so that no direct relationship could be demonstrated between post-prandial thermogenesis and sympathetic activity.

1. Endotoxin induces a shock-like syndrome with increased nitric oxide synthesis. To clarify the cellular source of NO in endotoxic shock we used immunohistochemistry and in situ hybridization to localize inducible NO synthase in rats given lipopolysaccharide or Corynebacterium parvum and lipopolysaccharide. Immunohistochemistry was carried out with an antibody raised against a synthetic peptide of mouse macrophage NO synthase. In situ hybridization was performed with 35 S-labelled oligonucleotide probes corresponding to cDNA sequences common to mouse macrophage inducible NO synthase and rat vascular smooth inducible NO synthase. Monocytes and macrophages were identified by immunohistochemistry with the mouse monoclonal antibody ED1. 2. After lipopolysaccharide alone, the major site of NO synthase induction was monocytes and macrophages in multiple organs, principally liver and spleen. Bronchial, bile duct, intestinal and bladder epithelium and some hepatocytes also expressed inducible NO synthase. Expression peaked at 5 h and had returned to normal by 12 h except in spleen. 3. After priming with C. parvum , lipopolysaccharide led to a similar distribution of inducible NO synthase as lipopolysaccharide alone, but in addition there was more prominent hepatocyte staining, staining in macrophage granulomas in the liver and inducible NO synthase was present in some endothelial cells in the aorta. 4. These findings provide a direct demonstration of the cellular localization of inducible NO synthase after lipopolysaccharide.

1. The Z deficiency variant of α 1 -antitrypsin predisposes the homozygote to early-onset panlobular emphysema and results in the accumulation of antitrypsin within the hepatocyte, which leads to hepatocellular damage and cirrhosis. The mechanism of this accumulation has been shown to be due to the Z mutation (Glu-342→Lys) perturbing the structure of the protein, allowing a unique interaction between the reactive-centre loop of one molecule and the A sheet of a second. This loop—sheet polymerization occurs spontaneously at 37°C in purified plasma Z but not M antitrypsin. The rate of polymerization is greatly accelerated at 41 C and is blocked by the insertion of a specific peptide into the A sheet of the antitrypsin molecule. Electron microscopy and circular dichroic spectral analysis confirm that the Z antitrypsin polymers formed in vitro have structural identity with those isolated from the liver of a Z homozygote. 2. That loop—sheet polymerization is a more general phenomenon was shown by the examination of a second deficiency variant, antitrypsin S iiyama (Ser-53→Phe), which is also associated with liver inclusions. Electron microscopy confirmed that isolated antitrypsin S iiyama from the plasma of a homozygote was present as long chains of polymers identical with those of Z antitrypsin.

1. It has recently been suggested that glutamine may be a conditionally essential nutrient rather than a non-essential amino acid. Therefore, administration of methionine sulphoximine was used to create a model of decreased arterial glutamine concentrations for 4 days. Glutamine consumption in portal-drained viscera and liver was measured after an overnight fast by determining fluxes and intracellular concentrations in normal rats, methionine sulphoximine-treated rats and pair-fed controls. Moreover, fluxes and intracellular concentrations of several other amino acids and ammonia and production of urea by the liver were determined concomitantly. 2. Methionine sulphoximine treatment for 4 days resulted in a 50% decrease in arterial glutamine concentration. Although the glutamine consumption and the intracellular glutamine concentration of the intestine were reduced by 50% at day 4, no changes in intestinal amino acid and ammonia metabolism were observed. 3. In the liver, glutamine consumption was reduced and ammonia uptake was increased, but urea synthesis was not changed. The decreased intracellular glutamine, glutamate, aspartate and ammonia concentrations coincided with a substantial reduction in liver branched-chain amino acid production. 4. These results suggest that reduced intestinal glutamine uptake does not induce marked changes in intestinal amino acid metabolism. The decreased liver branched-chain amino acid production suggests a reduction in the net liver protein degradation rate during methionine sulphoximine treatment.

1. The influence of an acute-phase reaction on the ability of protein synthesis rates in liver and three different muscles (gastrocnemius, soleus and heart) to respond to a short intravenous infusion of nutrients (glucose plus amino acids) was investigated during experimental inflammation induced by injection of human recombinant interleukin-1β or turpentine in young male rats. 2. Interleukin-1β induced a consistent increase of 3°C in body temperature between 3 and 5 h after injection, whereas turpentine induced a delayed fever, peaking by 13 h. 3. Interleukin-1β and turpentine stimulated fractional rates of protein synthesis in liver. The synthesis rate was inhibited by interleukin-1β in gastrocnemius and soleus muscle, but an elevation was seen in heart muscle. In this study there was no significant response of muscle to turpentine injection. 4. Two hours of parenteral nutrition increased fractional synthesis rates in all tissues when compared with Ringer's lactate. Somewhat larger responses to feeding were observed as a result of either interleukin-1β or turpentine injection in all tissues, but these improvements were not significant. 5. We conclude that the response of protein synthesis rates in liver and skeletal muscle to parenteral nutrition is not inhibited, and may be somewhat enhanced, during acute inflammatory conditions in the growing rat.

1. Electron paramagnetic resonance spectroscopy was used to study free-radical signals in freeze-clamped frozen liver tissue from rats after a 1 year period of dietary supplementation with alcohol, iron, or alcohol and iron. In alcohol-fed, iron-fed and alcohol- and iron-fed animals, mild histological damage was seen on light microscopy and evidence of mitochondrial and nuclear injury was identified by electron microscopy. 2. Subcellular fractionation studies showed an increase in the activity of the peroxisomal marker catalase ( P <0.01) in alcohol-fed rats compared with controls, but a fall of 82% ( P <0.001) in alcohol- and iron-fed animals. The activity of the mitochondrial marker succinate dehydrogenase rose by 7% (not significant) in alcohol-fed animals and by 17% (not significant) in iron-fed animals, but fell by 94% ( P <0.001) in alcohol- and iron-fed animals, suggesting serious impairment of mitochondrial function. 3. Iron overload was substantial in the iron-fed animals and there was an excellent correlation between liver iron concentration and iron-derived signals by electron paramagnetic resonance spectroscopy ( P <0.001). A clear free-radical signal of g = 2.003–2.005 was detected in all liver samples, but there was no significant difference in the magnitude of this signal in any study group. 4. The absence of any increase in the stable free-radical signal, even in the presence of considerable hepatic damage, does not support the hypothesis that free radicals mediate alcoholic liver disease in this animal model, although the results cannot be taken as proof against this hypothesis.

1. The short-term (120 min) kinetics of Zn turnover has been studied in control subjects and patients with alcoholic liver disease after intravenous injection of 0.5 mg of 96.5% enriched 70 ZnCl 2 . 2. The 70 Zn enrichment of plasma was found closely to obey two-compartment kinetics and the derived two-component decay equation has been used to calculate the size and turnover of the initial two rapidly exchanging pools of body Zn. 3. In normal subjects isotopic Zn appears initially to equilibrate with the whole of the plasma Zn which comprises the first metabolic compartment, pool a. This has a size of 0.72 ± 0.1 μmol/kg. 70 Zn equilibration then occurs with a second compartment, pool b, consistent with a rapidly exchanging liver Zn pool of size 3.60 ± 0.93 μmol/kg. The fractional turnover rate of pool b was found to be fivefold slower than that of pool a. 4. In the alcoholic group an expansion of pool a was observed (1.63 ± 0.39 μmol/kg), but the size of the second pool was not significantly different from that of control subjects (5.55 ± 1.0 μmol/kg), although its fractional turnover was significantly increased ( K ab : control subjects, 0.018 ± 0.002 min −1 , alcoholic patients, 0.031 ± 0.006 min −1 ). 5. These data therefore demonstrate that kinetic studies using stable isotopes of Zn can provide novel information on exchangeable Zn pools in man, but provide no support for the possibility of an underlying Zn depletion in patients with alcoholic liver disease.

1. This study was designed to test the hypothesis that three-carbon intermediates can be used in the ‘indirect’ pathway of glycogen synthesis in human liver (i.e. a route additional to the use of glucose by the ‘direct’ pathway). 2. After an overnight fast, 13 patients were given an infusion of 20% (w/v) glucose before elective abdominal operation. All received a 2.5 g bolus of 2220 kBq of selectively 3 H- and 14 C-labelled glucose before removal of a 1 g biopsy of liver. 3 H and 14 C were determined in purified glycogen as well as in glucose and lactate from samples of peripheral blood. 3. The ratio and specific activities of 3 H and 14 C in glycogen were found to be significantly lower than those in administered glucose. By calculation, 7–74% of glycogen repletion occurred by indirect pathways and not all of this was from the glucose supplied. 4. This study suggests that the operation of a direct pathway in man is not exclusive and that significant repletion of hepatic glycogen occurs by an indirect route.

1. After operation, changes in nitrogen metabolism occur. Although increased flux of amino acids from peripheral to splanchnic organs after operation has been described, substrate utilization by the individual organs in the splanchnic area is less well characterized. We were specifically interested in substrate flux across the spleen as it is an organ with important immunological functions. 2. Therefore, hindquarter, gut, spleen and liver fluxes of amino acids, ammonia, glucose, lactate and blood gases were measured for 4 days after a standard operation in pigs. In a separate control group, fluxes were measured 2–3 weeks after this operation and these values were assumed to represent the normal situation. 3. One day after operation, the hindquarter effluxes of glutamine, alanine and several essential amino acids were increased ( P <0.001), but these normalized at the end of the observation period. In the same period, liver glutamine uptake increased ( P <0.01), concomitantly with increased HCO - 3 , glucose and urea production, which also normalized. Portal drained viscera ammonia production decreased, concomitant with decreased glutamine uptake ( P <0.001). After operation, the splenic release of ammonia increased sevenfold ( P <0.05) and that of lactate increased from −158 ± 544 to 3294 ± 642 nmol min −1 kg −1 body weight ( P <0.001). Glucose uptake increased from −964 ± 632 to −3933 ± 1524 nmol min −1 kg −1 body weight and glutamine efflux (391 ± 143) reversed to uptake (−752 ± 169 nmol min −1 kg −1 body weight) ( P <0.001). 4. The experiments show that after operation in the pig, amino acid flow is from peripheral tissue to liver, probably for gluconeogenesis. The increased postoperative splenic metabolism possibly represents an indication of increased substrate utilization by the immune system.

1. Effects of endothelin-1 on systemic arterial blood pressure, heart rate and portal venous pressure were compared in normal Sprague-Dawley rats and rats with portal hypertension induced by CCl 4 and partial portal vein ligation. 2. Endothelin-1 produced biphasic effects on systemic blood pressure and portal venous pressure in all three groups of rats. However, the magnitude of the changes in blood pressure was less in portal hypertensive rats. 3. The ability of endothelin-1 to increase the portal venous pressure was also significantly diminished in portal hypertensive rats. On the other hand, the initial decrease in portal pressure was augmented in rats with partial portal vein ligation, and disappeared at higher dosage in CCl 4 -treated rats. 4. In accordance with the pressure recording in vivo , the dose-response vasoconstrictive activity of endothelin-1 was significantly attenuated in the intrahepatic vasculature. 5. The plasma immunoreactive endothelin concentration was significantly higher (5.55 ± 0.81 fmol/ml) in Sprague-Dawley rats than in CCl 4 -treated rats (2.83 ± 0.56 fmol/ml) and rats with partial portal vein ligation (2.68 ± 0.53 fmol/ml). 6. It was concluded that a lower plasma level of endothelin and a reduced vascular responsiveness may contribute, at least in part, to the hyperdynamics of portal hypertension.

1. 31 P n.m.r. spectroscopy in vivo was used to study the effect of l -alanine infusion on the concentrations of gluconeogenic intermediates in normal human liver. Studies were performed in six healthy male subjects (34–44 years, fasted overnight) using a chemical shift imaging pulse sequence on a whole-body n.m.r. system operating at 1.6T. Hepatic 31 P n.m.r. spectra were obtained from 10 min before to 70 min after intravenous administration of 0.70 ( n = 2), 1.40 ( n = 3) or 2.80 ( n = 5) nmol of l -alanine/kg body weight over 4.5 min. Concentrations of phosphomonoesters, P i and phosphodiesters relative to ATP were calculated from peak areas in the n.m.r. spectra, using the β-ATP peak as a reference. 2. Dose-dependent spectral changes were observed for [phosphomonoesters]/[ATP] and [P i ]/[ATP]. At the highest dose given, maximal changes in [phosphomonoesters]/[ATP] (mean ± sem : 98 ± 12%, P <0.005) and [P i ]/[ATP] (−33 ± 3%, P <0.001) were observed approximately 45 min after the l -alanine infusion. [Phosphodiesters]/[ATP] showed a maximal increase of 24 ± 6% ( P <0.05), which was independent of the l -alanine dose. Hepatic ATP levels and pH did not change. 3. To identify the metabolites responsible for the changes observed in vivo , male Wistar rats were infused with 11.2 mmol of l -alanine/kg body weight. After 15 min, livers were freeze-clamped and were extracted according to standard procedures. In vitro , 31 P n.m.r. spectra obtained at 8.4 or 11.7 T revealed sharp increases in the concentrations of 3-phosphoglycerate and phosphoenolpyruvate after l -alanine infusion. No significant increases in other metabolites contributing to the phosphomonoester or phosphodiester resonances in vivo were observed, suggesting that the rise in [phosphomonoesters] observed in vivo was caused by increasing concentrations of 3-phosphoglycerate, and that phosphoenolpyruvate contributed to the rise in [phosphodiesters]. 5. These results show that l -alanine infusion leads to consistent changes in the 31 P n.m.r. spectra of the human liver owing to increased concentrations of gluconeogenic intermediates. The ‘n.m.r.-alanine test’ may constitute a useful tool for studies of gluconeogenesis and hepatic biochemical pathology in vivo.

1. The hepatic metabolism of glutamine, alanine, ammonia, urea, glutathione and glucose was studied in rats made septic by caecal ligation and puncture and was compared with that in rats that had undergone sham operation (laparotomy). 2. Sepsis resulted in increases in the plasma activities of γ-glutamyltransferase ( P < 0.001), alanine aminotransferase ( P < 0.001) and aspartate aminotransferase ( P < 0.001), the serum total and direct bilirubin concentrations ( P < 0.001), and the blood lactate ( P < 0.01), glutamine ( P < 0.05), alanine ( P < 0.001) and urea ( P < 0.05) concentrations, but produced decreases in the blood ketone body ( P < 0.001) and glutathione ( P < 0.05) concentrations and in the plasma cholesterol concentration ( P < 0.05). These changes were associated with marked negative nitrogen balance in septic rats. 3. Sepsis increased total hepatic blood flow (by 22.7%) together with hepatic arterial flow (by 25.8%) and portal venous flow (by 18.7%). Sepsis resulted in marked increases in the net rates of hepatic extraction of glutamine (by 164%), alanine (by 138%) and ammonia (by 259%) with concomitant increases in the net rates of hepatic release of glutamate (by 105%), glutathione (by 87.5%), glucose (by 70.1%) and urea (by 100.4%). 4. Sepsis increased the activities of liver carbamoyl-phosphate synthase (by 16.4%), ornithine transcarbamylase (by 29.8%), argininosuccinate synthase (by 28.1%) and arginase (by 33.8%). 5. Septic rats exhibited marked increases in hepatic protein (by 46.0%), RNA (by 43.4%) and DNA (by 37.7%) contents. These changes were accompanied by marked increases in the activity of thymidine kinase (by 35.9%). 6. It is concluded that the enhanced hepatic glutamine extraction in response to sepsis could be attributed to increases in hepatic ureagenesis, gluconeogenesis, proliferative and synthetic activities, and glutathione production.

1. Little information is currently available on protein turnover during chronic protein loss situations. We have thus measured the whole-body and tissue protein fractional synthesis rates ( k s ), the whole-body fractional protein degradation rate ( k d ), the capacity for protein synthesis ( C s ) and the efficiency of protein synthesis ( k RNA ) in vivo in fed and fasted (1, 5 and about 9 days) 400 g rats. 2. One day of starvation resulted in a reduced k s and an increased k d in the whole body. k s was selectively depressed in skeletal muscles, mainly owing to a reduced k RNA , and was not modified in heart, liver and skin. The contribution of skin to whole-body protein synthesis increased by 39%. 3. During the phase of protein sparing (5 days of fasting), k d in the whole body decreased below the control fed level. k s in skeletal muscles was sustained because k RNA was restored to 82–98% of the control value. 4. Rats were in a protein-wasting phase after 9 days of starvation. k d in the whole body did not increase and was actually 78% of the value observed in fed animals. By contrast, k s in the whole body and tissues decreased to 14–34% of the control values, owing to reductions in both C s and k RNA . Whatever the duration of the fast, the contribution of the skin to whole-body protein synthesis largely exceeded that of skeletal muscle. 5. The present findings suggest that the main goal in the treatment of chronic protein loss should be to sustain protein synthesis. Our data also emphasize the importance of skin in whole-body protein synthesis in fasting and possibly in other protein loss situations.

1. It has been suggested that low levels of serum caeruloplasmin in Wilson's disease result from the failure to switch from a fetal to an adult mode of caeruloplasmin gene expression. To investigate postnatal expression of the caeruloplasmin gene, steady-state levels of caeruloplasmin messenger RNA in adult and neonatal rat liver were measured. 2. Copper parameters observed in neonatal rats were similar to those seen in Wilson's disease: hepatic copper concentration was significantly elevated (neonatal 164 ± 35 μg/g, adults 50 ± 8 μg/g, P < .001) and serum copper and caeruloplasmin levels were low (neonatal 0.5 ± 0.1 μg/ml, adults 1.3 ± 0.2 μg/ml, P < .001; neonatal 0.20 ± 0.04 arbitrary units, adults 0.69 ± 0.16 arbitrary units, P < .001), respectively. 3. Caeruloplasmin messenger RNA levels were analysed by Northern and dot blotting using a 12 P-labelled caeruloplasmin complementary DNA probe. A caeruloplasmin messenger RNA of approximately 4.4 kilobases was detected in both adult and neonatal rat liver, with no significant difference observed in steady-state levels. 4. A step subsequent to caeruloplasmin gene transcription must therefore be impaired in neonatal rats.