Non-alcoholic fatty liver disease: Immunological mechanisms and current treatments.

Non-alcoholic fatty liver disease (NAFLD) causes significant global disease burden and is a leading cause of mortality. NAFLD induces a myriad of aberrant changes in hepatocytes at both the cellular and molecular level. Although the disease spectrum of NAFLD is widely recognised, the precise triggers for disease progression are still to be fully elucidated. Furthermore, the propagation to cirrhosis is poorly understood. Whilst some progress in terms of treatment options have been explored, an incomplete understanding of the hepatic cellular and molecular alterations limits their clinical utility. We have therefore reviewed some of the key pathways responsible for the pathogenesis of NAFLD such as innate and adaptative immunity, lipotoxicity and fibrogenesis, and highlighted current trials and treatment options for NAFLD patients.

Antioxidant and Antidiabetic Properties of a Thinned-Nectarine-Based Nanoformulation in a Pancreatic ß-Cell Line.

Pancreatic ß-cells play a crucial role in maintaining glucose homeostasis, although they are susceptible to oxidative damage, which can ultimately impair their functionality. Thinned nectarines (TNs) have gained increasing interest due to their high polyphenol and abscisic acid (ABA) content, both of which possess antidiabetic properties. Nevertheless, the efficacy of these bioactive compounds may be compromised by limited stability and bioavailability in vivo. This study aimed to develop nanoformulations (NFs) containing pure ABA or a TN extract (TNE) at an equivalent ABA concentration. Subsequently, the insulinotropic and antioxidant potential of the NFs and their unformulated (free) forms were compared in MIN-6 pancreatic cells exposed to varying glucose (5.5 mM and 20 mM) and iron (100 µM) concentrations. NF-TNE treatment exhibited enhanced antioxidant activity compared to free TNE, while ABA-based groups showed no significant antioxidant activity. Moreover, MIN6 cells incubated with both high glucose and iron levels demonstrated significantly higher insulin AUC levels after treatment with all samples, with NF-TNE displaying the most pronounced effect. In conclusion, these results highlight the additional beneficial potential of TNE due to the synergistic combination of bioactive compounds and demonstrate the significant advantage of using a nanoformulation approach to further increase the benefits of this and similar phytobioactive molecules.

Alcoholic liver disease: Current insights into cellular mechanisms.

Alcoholic liver disease (ALD) due to chronic alcohol consumption is a significant global disease burden and a leading cause of mortality. Alcohol abuse induces a myriad of aberrant changes in hepatocytes at both the cellular and molecular level. Although the disease spectrum of ALD is widely recognized, the precise triggers for disease progression are still to be fully elucidated. Oxidative stress, mitochondrial dysfunction, gut dysbiosis and altered immune system response plays an important role in disease pathogenesis, triggering the activation of inflammatory pathways and apoptosis. Despite many recent clinical studies treatment options for ALD are limited, especially at the alcoholic hepatitis stage. We have therefore reviewed some of the key pathways involved in the pathogenesis of ALD and highlighted current trials for treating patients.

Excessive Iron Induces Oxidative Stress Promoting Cellular Perturbations and Insulin Secretory Dysfunction in MIN6 Beta Cells.

Exposure to high levels of glucose and iron are co-related to reactive oxygen species (ROS) generation and dysregulation of insulin synthesis and secretion, although the precise mechanisms are not well clarified. The focus of this study was to examine the consequences of exposure to high iron levels on MIN6 ß-cells. MIN6 pseudoislets were exposed to 20 µM (control) or 100 µM (high) iron at predefined glucose levels (5.5 mM and 11 mM) at various time points (3, 24, 48, and 72 h). Total iron content was estimated by a colourimetric FerroZine™ assay in presence or absence of transferrin-bound iron. Cell viability was assessed by a resazurin dye-based assay, and ROS-mediated cellular oxidative stress was assessed by estimating malondialdehyde levels. ß-cell iron absorption was determined by a ferritin immunoassay. Cellular insulin release and content was measured by an insulin immunoassay. Expression of SNAP-25, a key protein in the core SNARE complex that modulates vesicle exocytosis, was measured by immunoblotting. Our results demonstrate that exposure to high iron levels resulted in a 15-fold (48 h) and 4-fold (72 h) increase in cellular iron accumulation. These observations were consistent with data from oxidative stress analysis which demonstrated 2.7-fold higher levels of lipid peroxidation. Furthermore, exposure to supraphysiological (11 mM) levels of glucose and high iron (100 µM) at 72 h exerted the most detrimental effect on the MIN6 ß-cell viability. The effect of high iron exposure on total cellular iron content was identical in the presence or absence of transferrin. High iron exposure (100 µM) resulted in a decrease of MIN6 insulin secretion (64% reduction) as well as cellular insulin content (10% reduction). Finally, a significant reduction in MIN6 ß-cell SNAP-25 protein expression was evident at 48 h upon exposure to 100 µM iron. Our data suggest that exposure to high iron and glucose concentrations results in cellular oxidative damage and may initiate insulin secretory dysfunction in pancreatic ß-cells by modulation of the exocytotic machinery.

High omega arachidonic acid/docosahexaenoic acid ratio induces mitochondrial dysfunction and altered lipid metabolism in human hepatoma cells.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a common cause of liver disease worldwide and is a growing epidemic. A high ratio of omega-6 fatty acids to omega-3 fatty acids in the diet has been implicated in the development of NAFLD. However, the inflicted cellular pathology remains unknown. A high ratio may promote lipogenic pathways and contribute to reactive oxygen species (ROS)-mediated damage, perhaps leading to mitochondrial dysfunction. Therefore, these parameters were investigated to understand their contribution to NAFLD development. AIM: To examine the effect of increasing ratios of omega-6:3 fatty acids on mitochondrial function and lipid metabolism mediators. METHODS: HepG2-derived VL-17A cells were treated with normal (1:1, 4:1) and high (15:1, 25:1) ratios of omega-6: omega-3 fatty acids [arachidonic acid (AA): docosahexaenoic acid (DHA)] at various time points. Mitochondrial activity and function were examined via MTT assay and Seahorse XF24 analyzer, respectively. Triglyceride accumulation was determined by using EnzyChrom™ and levels of ROS were measured by fluorescence intensity. Protein expression of the mediators of lipogenic, lipolytic and endocannabinoid pathways was assessed by Western blotting. RESULTS: High AA:DHA ratio decreased mitochondrial activity (P < 0.01; up to 80%) and promoted intracellular triglyceride accumulation (P < 0.05; 40%-70%). Mechanistically, it altered the mediators of lipid metabolism; increased the expression of stearoyl-CoA desaturase (P < 0.05; 22%-35%), decreased the expression of peroxisome proliferator-activated receptor-alpha (P < 0.05; 30%-40%) and increased the expression of cannabinoid receptor 1 (P < 0.05; 31%). Furthermore, the high ratio increased ROS production (P < 0.01; 74%-115%) and reduced mitochondrial respiratory functions such as basal and maximal respiration, ATP production, spare respiratory capacity and proton leak (P < 0.01; 35%-68%). CONCLUSION: High AA:DHA ratio induced triglyceride accumulation, increased oxidative stress and disrupted mitochondrial functions. Stimulation of lipogenic and steroidal transcription factors may partly mediate these effects and contribute to NAFLD development.

Hepcidin secretion was not directly proportional to intracellular iron-loading in recombinant-TfR1 HepG2 cells: short communication.

Hepcidin is the master regulator of systemic iron homeostasis and its dysregulation is observed in several chronic liver diseases. Unlike the extracellular iron-sensing mechanisms, the intracellular iron-sensing mechanisms in the hepatocytes that lead to hepcidin induction and secretion are incompletely understood. Here, we aimed to understand the direct role of intracellular iron-loading on hepcidin mRNA and peptide secretion using our previously characterised recombinant HepG2 cells that over-express the cell-surface iron-importer protein transferrin receptor-1. Gene expression of hepcidin (HAMP) was determined by real-time PCR. Intracellular iron levels and secreted hepcidin peptide levels were measured by ferrozine assay and immunoassay, respectively. These measurements were compared in the recombinant and wild-type HepG2 cells under basal conditions at 30 min, 2 h, 4 h and 24 h. Data showed that in the recombinant cells, intracellular iron content was higher than wild-type cells at 30 min (3.1-fold, p < 0.01), 2 h (4.6-fold, p < 0.01), 4 h (4.6-fold, p < 0.01) and 24 h (1.9-fold, p < 0.01). Hepcidin (HAMP) mRNA expression was higher than wild-type cells at 30 min (5.9-fold; p = 0.05) and 24 h (6.1-fold; p < 0.03), but at 4 h, the expression was lower than that in wild-type cells (p < 0.05). However, hepcidin secretion levels in the recombinant cells were similar to those in wild-type cells at all time-points, except at 4 h, when the level was lower than wild-type cells (p < 0.01). High intracellular iron in recombinant HepG2 cells did not proportionally increase hepcidin peptide secretion. This suggests a limited role of elevated intracellular iron in hepcidin secretion.

Measurement of 4-Hydroxynonenal (4-HNE) Protein Adducts by ELISA.

Enzyme linked immunosorbent assay (ELISA) is a widely used technique for the measurement of antigens and antibodies alike. We describe here procedures, indirect ELISA and sandwich ELISA for the detection of 4-hydroxynonenal protein adducts. These adducts are stable compounds formed within cells and bodily fluids under conditions of oxidative stress. They can act as sensitive biomarkers of oxidative stress and are directly linked to disease pathology.

Iron Enhances Hepatic Fibrogenesis and Activates Transforming Growth Factor-ß Signaling in Murine Hepatic Stellate Cells.

BACKGROUND: Although excess iron induces oxidative stress in the liver, it is unclear whether it directly activates the hepatic stellate cells (HSC). MATERIALS AND METHODS: We evaluated the effects of excess iron on fibrogenesis and transforming growth factor beta (TGF-ß) signaling in murine HSC. Cells were treated with holotransferrin (0.005-5g/L) for 24 hours, with or without the iron chelator deferoxamine (10µM). Gene expressions (α-SMA, Col1-α1, Serpine-1, TGF-ß, Hif1-α, Tfrc and Slc40a1) were analyzed by quantitative real time-polymerase chain reaction, whereas TfR1, ferroportin, ferritin, vimentin, collagen, TGF-ß RII and phospho-Smad2 proteins were evaluated by immunofluorescence, Western blot and enzyme-linked immunosorbent assay. RESULTS: HSC expressed the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-export protein ferroportin. Holotransferrin upregulated TfR1 expression by 1.8-fold (P < 0.03) and ferritin accumulation (iron storage) by 2-fold (P < 0.01), and activated HSC with 2-fold elevations (P < 0.03) in α-SMA messenger RNA and collagen secretion, and a 1.6-fold increase (P < 0.01) in vimentin protein. Moreover, holotransferrin activated the TGF-ß pathway with TGF-ß messenger RNA elevated 1.6-fold (P = 0.05), and protein levels of TGF-ß RII and phospho-Smad2 increased by 1.8-fold (P < 0.01) and 1.6-fold (P < 0.01), respectively. In contrast, iron chelation decreased ferritin levels by 30% (P < 0.03), inhibited collagen secretion by 60% (P < 0.01), repressed fibrogenic genes α-SMA (0.2-fold; P < 0.05) and TGF-ß (0.4-fold; P < 0.01) and reduced levels of TGF-ß RII and phospho-Smad2 proteins. CONCLUSIONS: HSC express iron-transport proteins. Holotransferrin (iron) activates HSC fibrogenesis and the TGF-ß pathway, whereas iron depletion by chelation reverses this, suggesting that this could be a useful adjunct therapy for patients with fibrosis. Further studies in primary human HSC and animal models are necessary to confirm this.

Erratum to: HFE mRNA expression is responsive to intracellular and extracellular iron loading: short communication.

The original article has been changed to reflect the correct co-author name: Sebastien Farnaud. The original article was corrected.

HFE mRNA expression is responsive to intracellular and extracellular iron loading: short communication.

In liver hepatocytes, the HFE gene regulates cellular and systemic iron homeostasis by modulating cellular iron-uptake and producing the iron-hormone hepcidin in response to systemic iron elevation. However, the mechanism of iron-sensing in hepatocytes remain enigmatic. Therefore, to study the effect of iron on HFE and hepcidin (HAMP) expressions under distinct extracellular and intracellular iron-loading, we examined the effect of holotransferrin treatment (1, 2, 5 and 8 g/L for 6 h) on intracellular iron levels, and mRNA expressions of HFE and HAMP in wild-type HepG2 and previously characterized iron-loaded recombinant-TfR1 HepG2 cells. Gene expression was analyzed by real-time PCR and intracellular iron was measured by ferrozine assay. Data showed that in the wild-type cells, where intracellular iron content remained unchanged, HFE expression remained unaltered at low holotransferrin treatments but was upregulated upon 5 g/L (p < 0.04) and 8 g/L (p = 0.05) treatments. HAMP expression showed alternating elevations and increased upon 1 g/L (p < 0.05) and 5 g/L (p < 0.05). However, in the recombinant cells that showed higher intracellular iron levels than wild-type cells, HFE and HAMP expressions were elevated only at low 1 g/L treatment (p < 0.03) and were repressed at 2 g/L treatment (p < 0.03). Under holotransferrin-untreated conditions, the iron-loaded recombinant cells showed higher expressions of HFE (p < 0.03) and HAMP (p = 0.05) than wild-type cells. HFE mRNA was independently elevated by extracellular and intracellular iron-excess. Thus, it may be involved in sensing both, extracellular and intracellular iron. Repression of HAMP expression under simultaneous intracellular and extracellular iron-loading resembles non-hereditary iron-excess pathologies.

Repeat-measures longitudinal study evaluating behavioural and gastrointestinal symptoms in children with autism before, during and after visceral osteopathic technique (VOT).

UNLABELLED: This study investigated the influence of visceral osteopathic technique (VOT) on the behaviour and gastrointestinal (GI) symptoms of children with autism using a validated questionnaire to measure outcome. METHODS: The 49 recruited autistic children suffered GI symptoms and impaired social interaction and communication, but were otherwise healthy. Thirty minute VOT sessions were applied to the abdomens of the children over a 6 week period whilst their GI and behavioural parameters were recorded. Outcomes were measured using a modified Autism Research Institute Secretin Outcomes Survey Form, the 'S.O.S Form'. Four questionnaires were completed by parents before treatment (control period), four completed during treatment (treatment period) and one completed six weeks after the last treatment (post treatment period). Subjects acted as their own controls. RESULTS: Results from repeat ANOVA demonstrated a positive, overall significant, symptomatic improvement (p < 0.05) in 'social behaviour and communication' and 'digestive signs' subscales of the questionnaire comparing before and after VOT. Significant improvement in vomiting (p = 0.00029), poor appetite (p = 0.039) and eye contact (p = 0.035) was also demonstrated after VOT application. DISCUSSION AND CONCLUSION: The experimental hypothesis has been supported indicating a positive effect of VOT on some of the measured GI symptoms and behavioural patterns in this group of children with autism. This data indicates that the application of VOT may be of benefit to children with autism and GI disturbance.

Characterization of hepcidin response to holotransferrin in novel recombinant TfR1 HepG2 cells.

Hepcidin is the key regulator of systemic iron homeostasis. The iron-sensing mechanisms and the role of intracellular iron in modulating hepatic hepcidin secretion are unclear. Therefore, we created a novel cell line, recombinant-TfR1 HepG2, expressing iron-response-element-independent TFRC mRNA to promote cellular iron-overload and examined the effect of excess holotransferrin (5g/L) on cell-surface TfR1, iron content, hepcidin secretion and mRNA expressions of TFRC, HAMP, SLC40A1, HFE and TFR2. Results showed that the recombinant cells exceeded levels of cell-surface TfR1 in wild-type cells under basal (2.8-fold; p<0.03) and holotransferrin-supplemented conditions for 24h and 48h (4.4- and 7.5-fold, respectively; p<0.01). Also, these cells showed higher intracellular iron content than wild-type cells under basal (3-fold; p<0.03) and holotransferrin-supplemented conditions (6.6-fold at 4h; p<0.01). However, hepcidin secretion was not higher than wild-type cells. Moreover, holotransferrin treatment to recombinant cells did not elevate HAMP responses compared to untreated or wild-type cells. In conclusion, increased intracellular iron content in recombinant cells did not increase hepcidin responses compared to wild-type cells, resembling hemochromatosis. Furthermore, TFR2 expression altered within 4h of treatment, while HFE expression altered later at 24h and 48h, suggesting that TFR2 may function prior to HFE in HAMP regulation.

Characterisation of hepcidin response to holotransferrin treatment in CHO TRVb-1 cells.

Iron overload coupled with low hepcidin levels are characteristics of hereditary haemochromatosis. To understand the role of transferrin receptor (TFR) and intracellular iron in hepcidin secretion, Chinese hamster ovary transferrin receptor variant (CHO TRVb-1) cells were used that express iron-response-element-depleted human TFRC mRNA (TFRC∆IRE). Results showed that CHO TRVb-1 cells expressed higher basal levels of cell-surface TFR1 than HepG2 cells (2.2-fold; p < 0.01) and following 5 g/L holotransferrin treatment maintained constitutive over-expression at 24h and 48 h, contrasting the HepG2 cells where the receptor levels significantly declined. Despite this, the intracellular iron content was neither higher than HepG2 cells nor increased over time under basal or holotransferrin-treated conditions. Interestingly, hepcidin secretion in CHO TRVb-1 cells exceeded basal levels at all time-points (p < 0.02) and matched levels in HepG2 cells following treatment. While TFRC mRNA expression showed expected elevation (2h, p < 0.03; 4h; p < 0.05), slc40a1 mRNA expression was also elevated (2 h, p < 0.05; 4 h, p < 0.03), unlike the HepG2 cells. In conclusion, the CHO TRVb-1 cells prevented cellular iron-overload by elevating slc40a1 expression, thereby highlighting its significance in the absence of iron-regulated TFRC mRNA. Furthermore, hepcidin response to holotransferrin treatment was similar to HepG2 cells and resembled the human physiological response.

New insights into the role of the branched-chain aminotransferase proteins in the human brain.

The human cytosolic branched-chain aminotransferase (hBCATc) enzyme is strategically located in glutamatergic neurons, where it is thought to provide approximately 30% of de novo nitrogen for brain glutamate synthesis. In health, glutamate plays a dominant role in facilitating learning and memory. However, in patients with Alzheimer's disease (AD), synaptic levels of glutamate become toxic, resulting in a direct increase in postsynaptic neuronal calcium, causing a cascade of events that contributes to the destruction of neuronal integrity and cell death, pathological features of AD. Our group is the first to map the hBCAT proteins to the human brain, where cell-specific compartmentation indicates key roles for these proteins in regulating glutamate homeostasis. Moreover, increased expression of hBCAT was observed in the brains of patients with AD relative to matched controls. We reflect on the importance of the redox-active CXXC motif, which confers novel roles for the hBCAT proteins, particularly with respect to substrate channeling and protein folding. This implies that, in addition to their role in glutamate metabolism, these proteins have additional functional roles that might impact redox cell signaling. This review discusses how these proteins behave as potential neuroprotectors during periods of oxidative stress. These findings are particularly important because an increase in misfolded proteins, linked to increased oxidative stress, occurs in several neurodegenerative conditions. Together, these studies give an overview of the diverse role that these proteins play in brain metabolism, in which a dysregulation of their expression may contribute to neurodegenerative conditions such as AD.

The branched-chain aminotransferase proteins: novel redox chaperones for protein disulfide isomerase--implications in Alzheimer's disease.

AIMS: The human branched-chain aminotransferase proteins (hBCATm and hBCATc) are regulated through oxidation and S-nitrosation. However, it remains unknown whether they share common redox characteristics to enzymes such as protein disulfide isomerase (PDI) in terms of regulating cellular repair and protein misfolding. RESULTS: Here, similar to PDI, the hBCAT proteins showed dithiol-disulfide isomerase activity that was mediated through an S-glutathionylated mechanism. Site-directed mutagenesis of the active thiols of the CXXC motif demonstrates that they are fundamental to optimal protein folding. Far Western analysis indicated that both hBCAT proteins can associate with PDI. Co-immunoprecipitation studies demonstrated that hBCATm directly binds to PDI in IMR-32 cells and the human brain. Electron and confocal microscopy validated the expression of PDI in mitochondria (using Mia40 as a mitochondrial control), where both PDI and Mia40 were found to be co-localized with hBCATm. Under conditions of oxidative stress, this interaction is decreased, suggesting that the proposed chaperone role for hBCATm may be perturbed. Moreover, immunohistochemistry studies show that PDI and hBCAT are expressed in the same neuronal and endothelial cells of the vasculature of the human brain, supporting a physiological role for this binding. INNOVATION: This study identifies a novel redox role for hBCAT and confirms that hBCATm differentially binds to PDI under cellular stress. CONCLUSION: These studies indicate that hBCAT may play a role in the stress response of the cell as a novel redox chaperone, which, if compromised, may result in protein misfolding, creating aggregates as a key feature in neurodegenerative conditions such as Alzheimer's disease.

Hepatic mitochondrial dysfunction induced by fatty acids and ethanol.

Understanding the key aspects of the pathogenesis of alcoholic fatty liver disease particularly alterations to mitochondrial function remains to be resolved. The role of fatty acids in this regard requires further investigation due to their involvement in fatty liver disease and obesity. This study aimed to characterize the early effects of saturated and unsaturated fatty acids alone on liver mitochondrial function and during concomitant ethanol exposure using isolated liver mitochondria and VA-13 cells (Hep G2 cells that efficiently express alcohol dehydrogenase). Liver mitochondria or VA-13 cells were treated with increasing concentrations of palmitic or arachidonic acid (1 to 160 µM) for 24 h with or without 100 mM ethanol. The results showed that in isolated liver mitochondria both palmitic and arachidonic acids significantly reduced state 3 respiration in a concentration-dependent manner (P<0.001), implicating their ionophoric activities. Increased ROS production occurred in a dose-dependent manner especially in the presence of rotenone (complex I inhibitor), which was significantly more prominent in arachidonic acid at 80 µM (+970%, P<0.001) than palmitic acid (+40%, P<0.01). In VA-13 cells, ethanol alone and both fatty acids (40 µM) were able to decrease the mitochondrial membrane potential and cellular ATP levels and increase lipid formation. ROS production was significantly increased with arachidonic acid (+110%, P<0.001) exhibiting a greater effect than palmitic acid (+39%, P<0.05). While in the presence of ethanol, the drop in the mitochondrial membrane potential, cellular ATP levels, and increased lipid formation were further enhanced by both fatty acids, but with greater effect in the case of arachidonic acid, which also correlated with significant cytotoxicity (P<0.001). This study confirms the ability of fatty acids to promote mitochondrial injury in the development of alcoholic fatty liver disease.

S-Nitrosoglutathione inactivation of the mitochondrial and cytosolic BCAT proteins: S-nitrosation and S-thiolation.

Specific proteins with reactive thiol(ate) groups are susceptible to nitric oxide (NO) modification, which can result in S-nitrosation, S-thiolation, or disulfide bond formation. In the present study the effect of NO modification on the functionality of human mitochondrial and cytosolic branched-chain aminotransferases (hBCATm and hBCATc, respectively) was investigated. Here, the NO reactive agents, S-nitrosoglutathione (GSNO), S-nitroso-N-acetyl-dl-penacillamine, and sodium nitroprusside, inactivated both isoforms in a dose-dependent manner. Furthermore, low concentrations of GSNO caused a time-dependent loss in BCAT activity (50 +/- 3% and 77 +/- 2% for hBCATc and hBCATm, respectively) correlating with the loss of four and one to two thiol groups, respectively, confirming the thiols as targets for NO modification. Analysis of GSNO-modified hBCATc by quadrupole time-of-flight mass spectrometry identified a major peak containing three NO adducts and a minor peak equivalent to two NO adducts and one glutathione (GSH) molecule, the latter confirmed by Western blot analysis. Moreover, prolonged exposure or increased levels of GSNO caused increased S-glutathionylation and partial dimerization of hBCATc, suggesting a possible shift from regulation by NO to one of adaptation during nitrosated stress. Although GSNO inactivated hBCATm, neither S-nitrosation, S-glutathionylation, nor dimerization could be detected, suggesting differential mechanisms of regulation through NO between isoforms in the mitochondria and cytosol. Reversal of GSNO-modified hBCAT using GSH alone was only partial, and complete reactivation was only possible using the glutaredoxin/GSH system (97 +/- 4% and 91 +/- 3% for hBCATc and hBCATm, respectively), implicating the importance of a full physiological redox system for activation/inactivation. To conclude, these results clearly demonstrate distinct functional/mechanistic responses to GSNO modification between BCAT isoforms and offer intriguing comparisons between the BCAT proteins and the respective cytosolic and mitochondrial hTrx and hGrx proteins.

Effects of 4-hydroxynonenal on mitochondrial 3-hydroxy-3-methylglutaryl (HMG-CoA) synthase.

Chronic ethanol consumption causes increased production of reactive oxygen species in hepatic mitochondria accompanied by elevations in products of lipid peroxidation such as 4-hydroxynonenal (4-HNE). In the current study we investigated the effects of chronic ethanol consumption on a prominent protein-4-HNE adduct in liver mitochondria. Male Sprague-Dawley rats were fed a liquid diet for 31 days in which ethanol constituted 36% of total calories. Immunoblot analyses of liver mitochondria from ethanol-fed and control animals, using an antibody to a 4-HNE-protein adduct, demonstrated elevated 4-HNE binding (+50%) to a mitochondrial protein of approximately 55 kDa due to chronic ethanol consumption. Analysis of this protein using AspN digestion and tandem mass spectrometry identified it as the mitochondrial form of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. Activity of the activated form of this enzyme was unchanged in livers from ethanol-fed animals, but the protein level was elevated by 36%, which suggests a compensatory mechanism to maintain constant levels of synthase activity in the mitochondrion in the face of continuous inactivation by 4-HNE. Treatment of isolated mitochondria with 4-HNE demonstrated that the enzyme activity decreased as a function of 4-HNE concentration and with time of exposure. This study demonstrates that ethanol consumption increases the formation of a 4-HNE adduct with mitochondrial HMG-CoA synthase, which has the potential to inactivate the enzyme in situ.

Protein adduct species in muscle and liver of rats following acute ethanol administration.

AIMS: Previous immunohistochemical studies have shown that the post-translational formation of aldehyde-protein adducts may be an important process in the aetiology of alcohol-induced muscle disease. However, other studies have shown that in a variety of tissues, alcohol induces the formation of various other adduct species, including hybrid acetaldehyde-malondialdehyde-protein adducts and adducts with free radicals themselves, e.g. hydroxyethyl radical (HER)-protein adducts. Furthermore, acetaldehyde-protein adducts may be formed in reducing or non-reducing environments resulting in distinct molecular entities, each with unique features of stability and immunogenicity. Some in vitro studies have also suggested that unreduced adducts may be converted to reduced adducts in situ. Our objective was to test the hypothesis that in muscle a variety of different adduct species are formed after acute alcohol exposure and that unreduced adducts predominate. METHODS: Rabbit polyclonal antibodies were raised against unreduced and reduced aldehydes and the HER-protein adducts. These were used to assay different adduct species in soleus (type I fibre-predominant) and plantaris (type II fibre-predominant) muscles and liver in four groups of rats administered acutely with either [A] saline (control); [B] cyanamide (an aldehyde dehydrogenase inhibitor); [C] ethanol; [D] cyanamide+ethanol. RESULTS: Amounts of unreduced acetaldehyde and malondialdehyde adducts were increased in both muscles of alcohol-dosed rats. However there was no increase in the amounts of reduced acetaldehyde adducts, as detected by both the rabbit polyclonal antibody and the RT1.1 mouse monoclonal antibody. Furthermore, there was no detectable increase in malondialdehyde-acetaldehyde and HER-protein adducts. Similar results were obtained in the liver. CONCLUSIONS: Adducts formed in skeletal muscle and liver of rats exposed acutely to ethanol are mainly unreduced acetaldehyde and malondialdehyde species.

Liver dysfunction induced by bile duct ligation and galactosamine injection alters cardiac protein synthesis.

Liver disease has been shown to affect the cardiovascular system and may influence cardiac protein metabolism. This hypothesis was tested by measuring rates of cardiac protein synthesis in 2 models of liver disease in rats. The study consisted of 5 groups--group 1: control, injected with saline and fed ad libitum; group 2: acute liver injury, by dosage with 400 mg/kg galactosamine; group 3: injected with saline and pair-fed to group 2; group 4: chronic liver disease, using bile duct ligation; and group 5: sham-operated and pair-fed to group 4. Rates of cardiac protein synthesis were measured using the flooding dose technique. After 1 week, galactosamine injection caused the following cardiac changes, i.e. group (2) versus (3): an increased RNA content, RNA/DNA ratio, and RNA/protein ratio. However, there was no change in DNA or protein content, or protein/DNA ratio. There was an increase in the fractional rate of protein synthesis, and the absolute synthesis rate. Cellular efficiency was increased, but RNA activity remained unchanged. Comparison of groups 4 and 5 showed that bile duct ligation caused no change in any parameters measured. Although comparison of the ad libitum-fed group 1 with the bile duct ligation group 4 showed reduced cardiac weight, protein, and RNA content, with decreased right ventricular absolute synthesis rates; this was also seen in the pair-fed group 5, suggesting that these effects were due solely to reduced oral intake. Thus, although galactosamine-induced acute liver injury caused marked changes in cardiac biochemistry, bile duct ligation per se did not. This study also illustrates the importance of including a pair-fed group.