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Background: The objective of this study was to investigate the effects of a novel form of biotin (magnesium biotinate) on serum glucose, lipid profile, and hepatic lipid metabolism-related genes in rats. Methods: Forty-two rats were divided into six groups and fed a standard diet-based egg white powdered diet supplemented with either d-biotin at 0.01, 1 or 100 mg/kg BW or magnesium biotinate at 0.01, 1, or 100 mg/kg BW. Results: Serum total cholesterol and triglyceride decreased with biotin by both sources (P < 0.05). Concentrations were lower with magnesium biotinate when comparing the 1 mg/kg dose groups (P < 0.05). Serum, liver, and brain biotin, and liver cyclic guanosine monophosphate (cGMP) concentrations were greater when rats were treated with magnesium biotinate versus d-biotin, particularly when comparing the 1 mg/kg and 100 mg/kg dose groups (P < 0.05). Both forms of biotin decreased the liver SREBP‐1c and FAS and increased AMPK-α1, ACC-1, ACC-2, PCC, and MCC levels (P < 0.05). The magnitudes of responses were more emphasized with magnesium biotinate. Conclusions: Magnesium biotinate, compared with a commercial d-biotin, is more effective in reducing serum lipid concentrations and in regulating gene levels of intermediary metabolism-related biomarkers.

Autoimmune hepatitis (AIH) is a chronic inflammatory condition of the liver characterized by parenchymal destruction, hypergammaglobulinemia, specific autoantibody production, and hepatic fibrosis and necrosis. Murine models of AIH have been described; however, little is known about the immunologic mechanisms of tissue destruction. In this study, a new murine model of type 2 AIH was developed using recombinant human cytochrome P450 (CYP) 2D6 emulsified with complete Freund's adjuvant (CFA). BALB/c mice were immunized with 2 μg/mL i.p. of CYP2D6 in CFA. The control group received CFA or phosphate-buffered saline alone. Alanine aminotransferase activity, autoantibody production, IgG concentrations, histologic damage, and specific T-cell response were evaluated. Persistent AIH, characterized by cellular infiltration, hepatic fibrosis, elevated alanine aminotransferase, and the production of anti-liver kidney microsomal antibody type 1 developed in CFA/CYP2D6-immunized mice. This group presented high levels of IgG and its subclasses IgG1, IgG2a, and IgG2b against liver self-proteins. Interestingly, IL-2+ and interferon γ-positive Cyp2d6-specific T cells were present in greater concentrations in mice immunized with CFA/CYP2D6 compared with controls. Immunization with CFA, in combination with a natural human autoantigen like CYP2D6, was demonstrated to break tolerance, resulting in a chronic form of autoimmune-related liver damage. This murine model of type 2 AIH is expected to be instrumental in understanding the immunologic mechanisms of the pathogenesis of this autoimmune liver disease.

The aim of this study was to optimize the protein hydrolysates from chicken liver with xylose under Maillard reaction (MR) conditions using response surface methodology. The correlation between the browning degree, grafting degree, and the antioxidant activities of the Maillard reaction products (MRPs) was investigated. The optimal reaction conditions were achieved with a reaction temperature of 138.78°C, an initial pH of 7.99, and a reaction time of 93.14 min. The grafting degree (41.98%) and browning degree (2.582) of chicken liver protein hydrolysate MRPs (CLPHM) were notably higher (P

Experiment was carried out on 111 and 13-14 weeks old mice divergently selected for low (L) and high (C) body weight over 108 generations. In leg muscle and liver cathepsin D (CatD) was determined as pepstatin sensitive activity (PSCatD), and as pepstatin-insensitive (PIA) and leupeptin-insensitive (LIA) acid autolytic activities (AAA) were measured. In leg muscle and liver all measured activities were higher in L as compared to C and control (K) mice. The percent of inhibition of LIA was lower in L then C mice in both examined tissues. In the leg muscles, in L mice protein catabolism was mostly influenced by CatD, but in the liver CatD was mostly active in K mice. CatD, PSCatD, AAA and LIA in leg muscle of L mice as compare to K were higher in female (F) by 68 %, 68.5 %, 91.3 % and 94.5 %, respectively, and in male (M) mice by 43.4 %, 54 %, 47.1 % and 64 %, respectively. The percent of inhibition by leupeptin in AAA was higher in C mice by 61.9 % in F and by 40 % in M mice. In the liver, PIA was higher by about 45 % in L and by 28.5 % in C mice as compare to K ones. There were about 36% of 30.00-39.99 μm muscle fibrils in M mice and in males of L group 40.00-49.99 μm fibrils appeared at 25% (absent in 3 weeks mice). In female, all measured fibril diameters were in higher percent in L than in C group. RNA variables were higher by about 25% (in average) in C as compare to K and L groups. Functional cell size (FCS) was lowered in L and C group as compared to K by about 10 % (in average). These results indicate on faster proteins turnover in L than in C group of mice.

The postprandial state is characterized by a storage of nutrients in the liver, muscle, and adipose tissue for later utilization. In the case of a protein-rich meal, amino acids (AA) stimulate glucagon secretion by the α-cell. The aim of the present study was to determine the impact of the rise in glucagon on AA metabolism, particularly in the liver. We used a conscious catheterized dog model to recreate a postprandial condition using a pancreatic clamp. Portal infusions of glucose, AA, and insulin were used to achieve postprandial levels, while portal glucagon infusion was either maintained at the basal level or increased by three-fold. The high glucagon infusion reduced the increase in arterial AA concentrations compared with the basal glucagon level (−23%, P < 0.05). In the presence of high glucagon, liver AA metabolism shifted toward a more catabolic state with less protein synthesis (−36%) and increased urea production (+52%). Net hepatic glucose uptake was reduced modestly (−35%), and AA were preferentially used in gluconeogenesis, leading to lower glycogen synthesis (−54%). The phosphorylation of AMPK was increased by the high glucagon infusion (+40%), and this could be responsible for increasing the expression of genes related to pathways producing energy and lowering those involved in energy consumption. In conclusion, the rise in glucagon associated with a protein-rich meal promotes a catabolic utilization of AA in the liver, thereby, opposing the storage of AA in proteins.

In order to make the by-products generated from the porcine industry more valuable, pig livers were used in this trial to obtain protein hydrolysates. Three proteases (alcalase, bromelain, and papain) were utilized for enzymatic hydrolysis with two different durations, 4 and 8 hours. Ultrafiltration process was used for the recovery of the extracts, employing three different membrane pore sizes (30, 10, and 5 kDa). The porcine livers contained considerable amounts of protein (19.0%), considering they are almost composed of water (74.1%). The antioxidant activity of the obtained hydrolysates was investigated using four antioxidant methods (2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2-2&prime, Azino-di-[3-ethylbenzthiazoline sulfonate] (ABTS) radical scavenging activity, ferric reducing antioxidant power assay (FRAP), and oxygen radical absorbance capacity assay (ORAC)). Antibacterial properties were also measured against Gram-negative and Gram-positive bacteria. Results indicated that the three studied factors (type of enzyme, membrane pore size, and time) significantly affected the parameters evaluated. Hydrolysates obtained at 8 hours with alcalase had the best antioxidant properties. The 30 kDa alcalase extracts exhibited the highest DPPH (562 &micro, g Trolox/g), FRAP (82.9 &micro, mol Fe2+/100 g), and ORAC (53.2 mg Trolox/g) activities, while for ABTS the 10 kDa alcalase showed the higher values (1068 mg ascorbic acid/100 g). Concerning the antibacterial activity, 30 kDa hydrolysates obtained with bromelain for 4 hours exhibited the highest antimicrobial capacity, providing an inhibition of 91.7%.

Cytotoxic agents has been widely used for the treatment various human malignancies. Cisplatin and etoposide are chemotheraupeutic agents which are used for treatment of metastatic cancers, solid tumors and neoplasm. The drug is known to exert its toxic effect on hepatic and renal tissue by altering its antioxidant response of these organs. In the present study both the drugs Cisplatin and etoposide were analyzed for its effects on hepatic protein status. Cisplatin has shown to cause consequential alterations in protein levels as compared to etoposide treated hepatic tissue. The protein with molecular weight 44.46, 13.8, 51.2 kd was downregulated and other ranging from 16.98-38.9 kd did not show any expression as compared to controls. In addition etoposide studies revealed overexpression in protein level ranging from molecular weight 14.45-33.11 kd which might suggest significant role in organ protection. Some of protein bands ranging from 25.12 to 51.29 kd showed similar pattern of expression compared to controls. Present studies might provide us clues of important role of these protein in combating free radical induced oxidative stress on the tissue.

This work aimed at exploring the potential of partial substitution of the back-fat with fat mimetic, a novel polysaccharide-protein complex of xanthan gum and succinylated chicken liver protein, to improve the physicochemical characteristics and fatty acid composition of emulsified sausages. The quality characteristics of the sausages demonstrated that low-fat emulsified sausages with the addition of fat mimetic or its sunflower oil pre-emulsion presented similar microstructure and texture to high-fat sausages, while pre-emulsion increased the gel strength and whiteness of products. The rheological behavior characterization indicated that fat replacers caused a significant increase in storage modulus and loss modulus, explaining well the decreased cooking loss and the improved emulsification stability, water holding capacity (WHC) and heating stability. Although the back-fat replacement led to a great decrease in fatty acids, the nutritional values of fatty acids were optimized due to the incorporation of sunflower oil, as revealed by a 10% increase of polyunsaturated fatty acids and the decreased TBARS values within the storage. This study suggests the technological feasibility to simultaneously improve the quality and nutritional features of emulsified sausages with partial back-fat replacement by the proposed polysaccharide-protein complex, as well as the way to achieve chicken liver valorization.

The high efficiency of protein deposition during the neonatal period is driven by high rates of protein synthesis, which are maximally stimulated after feeding. Infusion of amino acids, but not insulin, reproduces the feeding-induced stimulation of liver protein synthesis. To determine whether amino acid-stimulated liver protein synthesis is independent of insulin in neonates, and to examine the role of amino acids and insulin in the regulation of translation initiation in neonatal liver, we performed pancreatic glucose-amino acid clamps in overnight-fasted 7-day-old pigs. Pigs ( n = 9–12/group) were infused with insulin at 0, 10, 22, and 110 ng·kg−0.66·min−1to achieve 0, 2, 6, and 30 μU/ml insulin, respectively. At each insulin dose, amino acids were maintained at fasting or fed levels or, in conjunction with the highest insulin dose, allowed to fall to below fasting levels. Insulin had no effect on the fractional rate of protein synthesis in liver. Amino acids increased fractional protein synthesis rates in liver at each dose of insulin, including the 0 μU/ml dose. There was a dose-response effect of amino acids on liver protein synthesis. Amino acids and insulin increased protein S6 kinase and 4E-binding protein 1 (4E-BP1) phosphorylation; however, only amino acids decreased formation of the inactive 4E-BPI·eukaryotic initiation factor-4E (eIF4E) complex. The results suggest that amino acids regulate liver protein synthesis in the neonate by modulating the availability of eIF4E for 48S ribosomal complex formation and that this response does not require insulin.

Diet-induced obesity has been linked to metabolic disorders such as cardiovascular diseases and type 2 diabetes. A factor linking diet to metabolic disorders is oxidative stress, which can damage biomolecules, especially proteins. The present study was designed to investigate the effect of marine omega-3 polyunsaturated fatty acids (PUFAs) (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) and their combination with grape seed polyphenols (GSE) on carbonyl-modified proteins from plasma and liver in Wistar Kyoto rats fed an obesogenic diet, namely high-fat and high-sucrose (HFHS) diet. A proteomics approach consisting of fluorescein 5-thiosemicarbazide (FTSC) labelling of protein carbonyls, visualization of FTSC-labelled protein on 1-DE or 2-DE gels, and protein identification by MS/MS was used for the protein oxidation assessment. Results showed the efficiency of the combination of both bioactive compounds in decreasing the total protein carbonylation induced by HFHS diet in both plasma and liver. The analysis of carbonylated protein targets, also referred to as the ‘carbonylome’, revealed an individual response of liver proteins to supplements and a modulatory effect on specific metabolic pathways and processes due to, at least in part, the control exerted by the supplements on the liver protein carbonylome. This investigation highlights the additive effect of dietary fish oils and grape seed polyphenols in modulating in vivo oxidative damage of proteins induced by the consumption of HFHS diets., Xunta de Galicia is thankfully recognized by the financial support of the postdoctoral contract to Lucía Méndez. The Spanish Ministry of Science and Innovation is also gratefully acknowledged for the doctoral fellowship to Silvia Muñoz. The authors thank Lorena Barros and María Jesús González for their excellent technical assistance and AFAMSA (Vigo, Spain) for generously providing us raw material.

Background: The phosphorylation of eukaryotic initiation factor 2 (p-eIF2) during dietary amino acid insufficiency reduces protein synthesis and alters gene expression via the integrated stress response (ISR).Objective: We explored whether a Met-restricted (MR) diet activates the ISR to reduce body fat and regulate protein balance.Methods: Male and female mice aged 3-6 mo with either whole-body deletion of general control nonderepressible 2 (Gcn2) or liver-specific deletion of protein kinase R-like endoplasmic reticulum kinase (Perk) alongside wild-type or floxed control mice were fed an obesogenic diet sufficient in Met (0.86%) or an MR (0.12% Met) diet for ≤5 wk. Ala enrichment with deuterium was measured to calculate protein synthesis rates. The guanine nucleotide exchange factor activity of eIF2B was measured alongside p-eIF2 and hepatic mRNA expression levels at 2 d and 5 wk. Metabolic phenotyping was conducted at 4 wk, and body composition was measured throughout. Results were evaluated with the use of ANOVA (P < 0.05).Results: Feeding an MR diet for 2 d did not increase hepatic p-eIF2 or reduce eIF2B activity in wild-type or Gcn2-/- mice, yet many genes transcriptionally regulated by the ISR were altered in both strains in the same direction and amplitude. Feeding an MR diet for 5 wk increased p-eIF2 and reduced eIF2B activity in wild-type but not Gcn2-/- mice, yet ISR-regulated genes altered in both strains similarly. Furthermore, the MR diet reduced mixed and cytosolic but not mitochondrial protein synthesis in both the liver and skeletal muscle regardless of Gcn2 status. Despite the similarities between strains, the MR diet did not increase energy expenditure or reduce body fat in Gcn2-/- mice. Finally, feeding the MR diet to mice with Perk deleted in the liver increased hepatic p-eIF2 and altered body composition similar to floxed controls.Conclusions: Hepatic activation of the ISR resulting from an MR diet does not require p-eIF2. Gcn2 status influences body fat loss but not protein balance when Met is restricted.

This study was undertaken to elucidate the specific effects of growth hormone (GH) on liver protein metabolism in humans during surgery. Otherwise healthy patients scheduled for elective laparoscopic cholecystectomy were randomized into controls ( n = 9) or pretreatment with 12 units of GH for 1 day (GH 1, n = 9) or daily for 5 days (GH 5, n = 10). The fractional synthesis rate of liver proteins, as assessed by flooding with [2H5]phenylalanine, was higher in the GH 5 group (22.0 ± 6.9%/day, mean ± SD, P < 0.05) than in the control (16.1 ± 3.1%/day) and GH 1 (16.5 ± 5.5%/day) groups. During surgery, the fraction of polyribosomes in the liver, as assessed by ribosome analysis, decreased in the control group by ∼12% ( P < 0.01) but did not decrease in the GH-treated groups. In addition, the concentrations of the essential amino acids and aspartate in the liver decreased in response to GH treatment. In conclusion, GH pretreatment decreases hepatic free amino acid concentrations and preserves liver protein synthesis during surgery.

International audience; Objectives Severely low-protein diets (LP) induce behavioral and metabolic changes including a decrease in body weight, an increase in relative food intake (FI) and alterations in hepatic metabolism. During such protein restriction, changes in hepatic anabolic and catabolic protein pathways could transitory participate to compensate for amino acid (AA) deficiency. In the present study, liver expression of gene involved in proteosynthesis and proteolysis pathways, were related to FI, blood AA levels and body composition in rats fed LP diet. Methods Growing rats were fed for three weeks different diets containing 3-5-8-12-15 or 20% energy of milk protein. Body weight and FI were measured daily. At the end of the experiment, tissues and biological fluids were removed for gene expression measurement and blood AA UPLC analysis. Statistical analysis was done by 1- or 2-factor ANOVA, when data were repeated. Results Despite an increase in relative food intake under P3 and P5% diets, P3, P5 and P8% diets resulted in significant growth retardation compared to other groups. Lean mass was significantly decreased in rats under P3, P5 and P8% compared to P12, P15 and P20% diets, while there was no difference in fat mass between all groups. P3, P5 and P8% diets induced a decrease in essential amino acid concentrations in portal vein, whereas there was no significant difference between groups in veina cava. Severely protein restricted P3% and P5% diets induced an increase in hepatic gene expression involved in proteolysis as calpain 2 and ubiquitin, and an activation of ATF4-CHOP-TRB3 pathway. Conclusions These results suggested that under severe protein restriction, hepatic protein catabolism became a source of plasma amino acid that could partially compensate for the AA not provided by the diet. These observations confirm that liver plays a major role in the adaptation of the body to dietary protein restriction and highlight that severe dietary protein restriction induced liver protein catabolism by inducing an activation of ATF4-CHOP-TRB3 pathway in order to provide amino acids to body tissues. Funding Sources ABIES, AlimH-INRAE.

Amino acids are considered to be anabolic factors that affect protein turnover. The aim of this study was to test the effects of dietary L-arginine (Arg) levels on protein metabolism in the liver of laying hens and the expression of genes related to protein synthesis and proteolysis. Xinyang black commercial laying hens (n = 864, 31 wk of age) were randomly allotted to 6 treatments with 4 replicates of 36 birds. The dietary treatments were corn-corn gluten meal based diets containing 0.64, 0.86, 1.03, 1.27, 1.42, and 1.66% L-Arg, respectively. Serum concentrations of total protein and albumin were maximized in the 1.27% L-Arg group, and serum concentration of urea acid was the lowest in the 1.27% L-Arg group. The 1.27% L-Arg group had the highest fractional protein synthesis rate and fractional protein gain rate in the liver. Consistent with the data on protein turnover, mRNA abundances of target of rapamycin (TOR) and ribosomal protein S6 kinase 1 increased in the liver of layers fed 1.27% L-Arg, while mRNA abundances of cathepsin B and 20S proteasome decreased at the same dietary L-Arg level. In conclusion, the dietary level of L-Arg increased the liver fractional protein synthesis rate and fractional protein gain rate of laying hens, and the action of an appropriate level of dietary L-Arg involves upregulating the gene expression of the TOR signaling pathway accompanied by suppressing the mRNA expression of cathepsin B and 20S proteasome in the liver.

An important role in protein–energy metabolism has been attributed to leucine because of its long-term effects on body fat reduction and on the improvement of some indicators of protein status in rodents. The present study investigated the influence of leucine supplementation on the body composition and protein status of rats during the early phase of weight loss, which is characterized by a rapid loss of body weight. Thirty adult male Wistar rats were divided into 2 groups, a control and a leucine group (diet supplemented with 0.59% L-leucine), and were submitted to 1 week of 50% food restriction. The following parameters were evaluated: chemical carcass composition, protein and RNA content in liver and gastrocnemius muscle, and serum concentrations of insulin-like growth factor-1 and corticosterone. A higher liver weight and liver protein content were observed in the supplemented group (p < 0.05). However, no difference in body fat was found between groups (p > 0.05). The results indicate that low-dose leucine supplementation favors liver protein status but does not reduce body fat in rats during the early phase of rapid weight loss.

Peroxisome proliferators induce hepatic peroxisome proliferation and hepatocellular carcinomas in rodents. These chemicals increase the expression of the peroxisomal beta-oxidation pathway and the cytochrome P-450 4A family, which metabolizes lipids, including fatty acids. Mice lacking fatty acyl-CoA oxidase (AOX-/-), the first enzyme of the peroxisomal beta-oxidation system, exhibit extensive microvesicular steatohepatitis, leading to hepatocellular regeneration and massive peroxisome proliferation. To investigate proteins involved in peroxisome proliferation, we adopted a novel surface-enhanced laser desorption/ionization (SELDI) ProteinChip technology to compare the protein profiles of control (wild-type), AOX-/-, and wild-type mice treated with peroxisome proliferator, Wy-14,643. The results indicated that the protein profiles of AOX-/- mice were similar to the wild-type mice treated with Wy14,643, but significantly different from the nontreated wild-type mice. Using four different ProteinChip Arrays, a total of 40 protein peaks showed more than twofold changes. Among these differentially expressed peaks, a downregulated peak was identified as the major urinary protein in both AOX-/- and Wyl4,643-treated mice by SELDI. The identification of MUP was further confirmed by two-dimensional electrophoresis and liquid chromatography coupled tandem mass spectrometry (LC-MS-MS). This SELDI method offers several technical advantages for detection of differentially expressed proteins, including ease and speed of screening, no need for chromatographic processing, and small sample size.

International audience; Many rare monogenic diseases are treated by protein replacement therapy, in which the missing protein is repetitively administered to the patient. However, in several cases, the missing protein is required at a high and sustained level, which renders protein therapy far from being adequate. As an alternative, a gene therapy treatment ensuring a sustained effectiveness would be particularly valuable. Liver is an optimal organ for the secretion and systemic distribution of a therapeutic transgene product. Cutting edge non-viral gene therapy tools were tested in order to produce a high and sustained level of therapeutic protein secretion by the liver using the hydrodynamic delivery technique. The use of S/MAR matrix attachment region provided a slight, however not statistically significant, increase in the expression of a reporter gene in the liver. We have selected the von Willebrand Factor (vWF) gene as a particularly challenging large gene (8.4 kb) for liver delivery and expression, and also because a high vWF blood concentration is required for disease correction. By using the optimized miniplasmid pFAR free of antibiotic resistance gene together with the Sleeping Beauty transposon and the hyperactive SB100X transposase, we have obtained a sustainable level of vWFblood secretion by the liver, at 65% of physiological level. Our results point to the general use of this plasmid platform using the liver as a protein factory to treat numerous rare disorders by gene therapy.

Protein hydrolysates were prepared from chicken liver using fermentation and enzymatic hydrolysis. The lactic acid bacteria Pediococcus acidilactici NCIM5368 was employed in the fermentation process and a commercial protease (Alcalase® 2.5) was used in enzymatic hydrolysis. Chicken liver hydrolysates prepared by fermentation (FCLH) and enzymatic hydrolysis (ECLH) revealed appreciable amounts of protein [55.85 and 61.34 %; on dry weight basis, respectively]. Fermentation and enzymatic hydrolysis resulted in 14.3 and 26.12 % of degree of hydrolysis. Total antioxidant activity, reducing power, scavenging of superoxide, 2- diphenyl-1-picrylhydrazyl (DPPH) and 2, 2-azino-bis-3-ethyl-benzthiazoline-6-sulphonic acid (ABTS) radicals were determined for both FCLH & ECLH. FCLH & ECLH showed total antioxidant activity of 0.99 and 1.13 μg AAE mg(-1) proteins, respectively; while, they scavenged 96.14 and 92.76 % of DPPH radicals respectively. FCLH showed higher ABTS radical scavenging activity (32.16 %) than ECLH (19.29 %). Superoxide anion scavenging activity of FCLH & ECLH were found to be 95.02 & 88.94 %, respectively. Residues obtained after both treatments also exhibited antioxidant activities. FCLH reported highest antagonistic activity against Listeria monocytogenes (30 mm); while, ECLH showed antibacterial activity only against Micrococcus luteus (12 mm). Both hydrolysates have the potential to be a protein rich ingredient for use in formulated foods and possible help in reduction of oxidative stress.

At present, there is no accurate method for evaluating the quality of liver transplant from a brain-dead donor. Proteomics are used to investigate the mechanisms involved in brain death‑induced liver injury and to identify sensitive biomarkers. In the present study, age‑ and gender‑matched rabbits were randomly divided into the brain death and sham groups. The sham served as the control. A brain‑death model was established using an intracranial progressive pressurized method. The differentially expressed proteins extracted from the liver tissues of rabbits that were brain‑dead for 6 h in the two groups were determined by two‑dimensional gel electrophoresis and matrix‑assisted laser desorption ionization time of flight mass spectrometry. Although there was no obvious functional and morphological difference in 2, 4 and 6 h after brain death, results of the proteomics analysis revealed 973±34 and 987±38 protein spots in the control and brain death groups, respectively. Ten proteins exhibited a ≥2‑fold alteration. The downregulated proteins were: aldehyde dehydrogenase, runt‑related transcription factor 1 (RUNX1), inorganic pyrophosphatase, glutamate‑cysteine ligase regulatory subunit and microsomal cytochrome B5. By contrast, the expression of dihydropyrimidinase-related protein 4, peroxiredoxin‑6, 3‑phosphoinositide‑dependent protein kinase‑1, 3-mercaptopyruvate and alcohol dehydrogenase were clearly upregulated. Immunohistochemistry and western blot analysis results revealed that the expression of RUNX1 was gradually increased in a time‑dependent manner in 2, 4, and 6 h after brain death. In conclusion, alteration of the liver protein expression profile induced by brain death indicated the occurrence of complex pathological changes even if no functional or morphological difference was identified. Thus, RUNX1 may be a sensitive predict factor for evaluating the quality of brain death donated liver.

Seasonal variations in water temperature are a natural stressor of temperate fish that affect growth performance and metabolism globally. Gilthead sea bream is one of the most economically interesting species in the Mediterranean; but its liver metabolism is affected by the cold season. However, the effects of cold on protein turnover mechanisms have hardly been studied. Here, we study the relationship between liver oxidative status and protein homeostasis pathways during a 50-day low temperature period at 14 °C, and subsequent recovery at two times: 7 days (early recovery) and 30 days (late recovery). Liver redox status was determined by measuring oxidised lipids and proteins, the glutathione redox cycle and major antioxidant enzymes activities. Protein turnover was analysed via liver protein expression of HSP70 and HSP90; proteasome 26S subunits and polyubiquitination, as markers of the ubiquitin–proteasome system (UPS); and cathepsin D, as a lysosomal protease. Low temperature exposure depressed antioxidant enzyme activities, affecting the glutathione redox cycle and reducing total glutathione levels. Both the UPS and lysosomal pathways were also depressed and consequently, oxidised protein accumulated in liver. Interestingly, both protein oxidation and polyubiquitination tagging depended on protein molecular weight. Despite all these alterations, temperature recovery reverted most consequences of the cold at different rates: with delayed recovery of total glutathione levels and oxidised protein degradation with respect to enzyme activities recovery. All these findings demonstrate that protein liver homeostasis is compromised after chronic cold exposure and could be the cause of liver affectations reported in aquaculture of temperate fish.

Acetaminophen (APAP)-induced liver injury is the leading cause of acute liver failure in many countries. This study determined the extent of liver protein sulfhydryl depletion not only in whole liver homogenate but also in the zonal pattern of sulfhydryl depletion within the liver lobule. A single oral gavage dose of 150 or 300 mg/kg APAP in B6C3F1 mice produced increased serum alanine aminotransferase levels, liver necrosis, and glutathione depletion in a dose-dependent manner. Free protein sulfhydryls were measured in liver protein homogenates by labeling with maleimide linked to a near infrared fluorescent dye followed by SDS-polyacrylamide gel electrophoresis. Global protein sulfhydryl levels were decreased significantly (48.4%) starting at 1 hour after the APAP dose and maintained at this reduced level through 24 hours. To visualize the specific hepatocytes that had reduced protein sulfhydryl levels, frozen liver sections were labeled with maleimide linked to horseradish peroxidase. The centrilobular areas exhibited dramatic decreases in free protein sulfhydryls while the periportal regions were essentially spared. These protein sulfhydryl-depleted regions correlated with areas exhibiting histopathologic injury and APAP binding to protein. The majority of protein sulfhydryl depletion was due to reversible oxidation since the global- and lobule-specific effects were essentially reversed when the samples were reduced with tris(2-carboxyethy)phosphine before maleimide labeling. These temporal and zonal pattern changes in protein sulfhydryl oxidation shed new light on the importance that changes in protein redox status might play in the pathogenesis of APAP hepatotoxicity.

International audience; Epidemiologic and experimental studies showed that folate deficiency is associated with increased risk of degenerative diseases by enhancing abnormal one-carbon metabolism. We studied the changes in the proteome of liver, the main tissue of folate storage and metabolism, in a rat model of dietary folate depletion. Four-month-old rats were fed for 4 wk an amino acid-defined diet without folate and compared with pair-fed rats given the same diet adequately supplemented with folic acid. Folate deprivation decreased plasma and hepatic folate concentrations dramatically, while increasing homocysteinemia significantly. Using 2-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight MS, we identified 9 spots corresponding to differentially expressed proteins in the liver of folate-deficient rats compared with controls. Among those spots, 4 had a significantly increased volume, whereas the volume of the 5 other spots was decreased. Upregulated proteins included glutathione peroxidase (GPx) 1 and peroxiredoxin 6, 2 enzymes involved in the response to oxidative stress, and MAWD binding protein (MAWDBP), which has been associated with cancer. MAWDBP was simultaneously identified as a second spot with a lower isoelectric point (pI) that vanished almost completely after folate deficiency. Decreased abundance was also observed for cofilin 1, a protein linked to tumorigenesis, and for the GRP 75 precursor and preproalbumin, both of which are responsive to oxidative stress and/or inflammation. Moreover, an enzyme activity assay and/or Western blot analysis of GPx-1 and MAWDBP confirmed the proteomic findings. Our results show that folate deficiency modifies the abundance of several liver proteins consistently with adaptive tissue responses to oxidative and degenerative processes.

In eukaryotic cells, amino acid depletion reduces translation by a mechanism involving phosphorylation of eukaryotic initiation factor-2 (eIF2). Herein we describe that mice lacking the eIF2 kinase, general control nonderepressible 2 (GCN2) fail to alter the phosphorylation of this initiation factor in liver, and are moribund in response to dietary leucine restriction. Wild-type (GCN2(+/+)) and two strains of GCN2 null (GCN2(-/-)) mice were provided a nutritionally complete diet or a diet devoid of leucine or glycine for 1 h or 6 days. In wild-type mice, dietary leucine restriction resulted in loss of body weight and liver mass, yet mice remained healthy. In contrast, a significant proportion of GCN2(-/-) mice died within 6 days of the leucine-deficient diet. Protein synthesis in wild-type livers was decreased concomitant with increased phosphorylation of eIF2 and decreased phosphorylation of 4E-BP1 and S6K1, translation regulators controlled nutritionally by mammalian target of rapamycin. Whereas translation in the liver was decreased independent of GCN2 activity in mice fed a leucine-free diet for 1 h, protein synthesis in GCN2(-/-) mice at day 6 was enhanced to levels measured in mice fed the complete diet. Interestingly, in addition to a block in eIF2 phosphorylation, phosphorylation of 4E-BP1 and S6K1 was not decreased in GCN2(-/-) mice deprived of leucine for 6 days. This suggests that GCN2 activity can also contribute to nutritional regulation of the mammalian target of rapamycin pathway. As a result of the absence of these translation inhibitory signals, liver weights were preserved and instead, skeletal muscle mass was reduced in GCN2(-/-) mice fed a leucine-free diet. This study indicates that loss of GCN2 eIF2 kinase activity shifts the normal maintenance of protein mass away from skeletal muscle to provide substrate for continued hepatic translation.

Although the metabolism of liver-derived plasma proteins such as albumin has been extensively studied, human hepatic protein synthesis as a whole has not been well characterized, because a reproducible model for obtaining human liver tissue has not been available. In this study, the fractional synthesis rates of total liver protein and albumin in vivo were determined simultaneously in nine subjects undergoing elective laparoscopic cholecystectomy. L-[2H5]phenylalanine (45 mg/kg body wt) was administered for 10 minutes intravenously. Blood samples were collected at regular intervals for 90 minutes and a liver biopsy specimen was taken at 35 +/- 7 minutes. The enrichments of plasma free phenylalanine, plasma albumin, and total liver protein were measured with gas chromatography mass spectrometry (GC-MS). The fractional synthesis rate (FSR) of total liver protein was 24.7 +/- 3.1 %/d (mean +/- SD), and that of albumin was 5.9 +/-1.2%/d. The amount of albumin synthesized per day (absolute synthesis rate, ASR) was 109 +/- 21 mg/kg body wt. No correlation between FSR of total liver protein and ASR of albumin was found. It is concluded that the technique of obtaining liver tissue specimens during laparoscopic surgery may serve as a human in vivo model to study total liver protein synthesis. The fractional synthesis rate of total liver proteins (stationary and exported), equals approximately 25% of the liver protein content daily. Within the range of values of this study, the absolute synthesis rate of albumin was not correlated to the fractional synthesis rate of total liver protein.

Background: Protein tyrosine phosphatases (PTPases) appear to play an essential role in the regulation of steady-state phosphorylation of the insulin receptor and other proteins in the insulin-signaling pathway. Objectives: This study determined the role of PTPases in skeletal muscle and liver in an insulin-resistant model and the in vivo effect of troglitazone on PTPases. Methods: We used 26-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats as models of insulin resistance. PTPase activity was assessed with reduced carboxyamido-methylated and -maleylated lysozyme as substrates. The protein levels of 3 major candidate PTPases—leukocyte common antigen-related PTPase (LAR), PTPase 1B (PTP1B), and src homology 2-containing PTPase (SH-PTP2)—in skeletal muscle and liver of OLETF rats were analyzed using Western blot. The activities and amounts of PTPases in skeletal muscle and liver of OLETF rats treated with troglitazone for 7 days also were examined. Results: Particulate and cytosolic PTPase activities in skeletal muscle of OLETF rats were increased compared with those in control rats. Similar changes were observed in the liver. The protein levels of LAR, PTP1B, and SH-PTP2 in particulate fractions of skeletal muscle of OLETF rats were increased, but levels of PTP1B and SH-PTP2 in cytosolic fractions were unchanged. Similar findings were observed in the liver. Troglitazone normalized the increased PTPase activity and each protein level except cytosolic fraction of the liver. Conclusions: The findings suggest that troglitazone decreases elevated activities and levels of PTPases in the particulate fraction of the skeletal muscle and liver of OLETF rats, and that PTPase plays important roles through regulating the insulin-signaling system in the pathogenesis of insulin resistance in OLETF rats.

Cachexia is a multifactorial syndrome characterized by the loss of peripheral tissue and hypertrophy of visceral organs. The ApcMin/+ mouse model develops an IL-6 dependent cachexia, leading to gradual loss of muscle and fat and hypertrophy of spleen and liver. The purpose of this study was to examine liver function related to protein synthesis and glycogen storage during the progression of cancer cachexia. Livers collected from non-cachectic and cachectic ApcMin/+ mice were analyzed for marker of protein synthesis (p38) and degradation/angiogenesis marker - MMP2 protein expression. Liver glycogen content was examined by PAS staining intensity. The role of cachexia related inflammation on liver metabolism was studied by 10mg/kg/day administration of PDTC, a STAT3 and NF-ΚB inhibitor, for 2 weeks. As cachexia progressed there was suppression of Akt and p38 phosphorylation, and a reduction in liver glycogen content. MMP2 expression was also decreased 97% with severe cachexia. PDTC administration during seve...

In this study we investigated erythropoiesis and fetal liver protein secretion during late embryonic (Day 24 and Day 30) and early fetal (Day 40) development in pigs from domestic white crossbred (WC) gilts with a normal (intact; INT) or crowded (unilateral hysterectomized/ovariectomized; UHO) uterine environment, or from prolific Chinese Meishan (MS) gilts. Increased fetal weight, fetal liver weight, placental weight, total red blood cells, hematocrit, blood hemoglobin content, and maternal plasma erythropoietin (EPO) levels were observed as gestation advanced. Cultured fetal liver secretion of transferrin and a protein of Mr 12500 and pI 7.5 also increased as gestation advanced. Fetal plasma EPO declined between Day 30 and Day 40. Differential counts of circulating erythroid precursors revealed a decline in basophilic erythroblasts and polychromatic erythroblasts between Day 24 and Day 40, an increase in orthochromatic erythroblasts on Day 30 followed by a drop on Day 40, and an increase in the percentage of reticulocytes/ erythrocytes from < 1.0% to approximately 90% of circulating red blood cells between Day 24 and Day 40. Differences among the treatment groups included a lower fetal survival percentage in UHO (vs. INT or MS) on Day 40, and higher maternal hematocrits, fetal weights, fetal hematocrits, fetal EPO levels, and liver transferrin secretion in WC vs. MS pigs. MS pigs had a lower percentage of polychromatic erythroblasts overall and a higher percentage of orthochromatic erythroblasts on Day 24 followed by a higher percentage of erythrocytes on Day 40 than WC pigs, suggesting a more mature erythron (circulating red blood cells plus erythropoietic tissue) in the MS pigs. Covariate analysis indicated that MS had larger placentae per unit of body weight than did WC. Conclusions were that 1) Days 24-40 of gestation is a critical time for fetal erythropoiesis in pigs as well as survival in a crowded uterine environment, 2) the MS breed may differ in the development of the fetal erythropoietic system because of altered fetal or uterine physiology, and 3) the UHO procedure did not significantly affect erythropoiesis in the fetuses studied but did alter fetal survival and the relationship between fetal weight and both hematocrit and hemoglobin on Day 40.

The current anti-hepatitis C virus (HCV) therapy, based on pegylated-interferon alpha and ribavirin, has limited success rate and is accompanied by several side effects. The aim of this study was to identify protein profiles in pretreatment liver biopsies of HCV patients correlating with the outcome of antiviral therapy. Cytosolic or membrane/organelle-enriched protein extracts from liver biopsies of eight HCV patients were analyzed by two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. Overall, this analysis identified 21 proteins whose expression levels correlate with therapy response. These factors are involved in interferon-mediated antiviral activity, stress response, and energy metabolism. Moreover, we found that post-translational modifications of dihydroxyacetone kinase were also associated with therapy outcome. Differential expression of the five best performing markers (STAT1, Mx1, DD4, DAK, and PD-ECGF) was confirmed by immunoblotting assays in an independent group of HCV patients. Finally, we showed that a prediction model based on the expression levels of these markers classifies responder and nonresponder patients with an accuracy of 85.7%. These results provide evidence that the analysis of pretreatment liver protein profiles is valuable for discriminating between responder and nonresponder HCV patients, and may contribute to reduce the number of nonresponder patients exposed to therapy-associated risks.

We have explored the innervation of the rainbow trout (O. mykiss) liver using immunohistochemical procedures and light microscopy to detect in situ protein gene product 9.5 and neuronal nitric oxide synthase immunoreactivities (PGP-IR and NOS-IR). The results showed PGP-IR nerve fibres running with the extralobular biliary duct (EBD), hepatic artery (EHA) and portal vein (EPV) that form the hepatic hilum, as well as following the spatial distribution of the intrahepatic blood vessel and biliary channels. These nerve fibres appear as single varicose processes, thin bundles, or thick bundles depending on their diameter and location in the wall of the blood vessel or biliary duct. No PGP-IR fibres were detected in the liver parenchyma. NOS-IR nerve fibres were located only in the vessels and ducts that form the hepatic hilum (EBD, EHA, EPV); in addition, NOS-IR nerve cell bodies were found isolated or forming ganglionated plexuses in the peribiliary fibromuscular tissue of the EBD. No PGP-IR ganglionated plexuses were detected in the EBD. The location of the general (PGP-IR) and nitrergic (nNOS-IR) intrinsic nerves of the trout liver suggest a conserved evolutionary role of the nervous control of hepatic blood flow and hepatobiliary activity.

Fatty liver is a frequent subclinical health disorder in dairy cows that may lead to disorders related to the liver function. However, the effect of triglyceride(TG) accumulation on liver metabolic pathways is still unclear. The objective was, therefore, to characterize quantitative differences in the liver proteome betweenearly lactation dairy cows with a low or high liver TG content. The liver proteome analysis indicated that a high liver TG content in early lactation dairy cows isassociated with increased oxidation of saturated fatty acids, oxidative stress, and urea synthesis and decreasedoxidation of unsaturated fatty acids. Furthermore,liver gluconeogenesis is apparently not impaired by an increased liver TG content. Based on correlations between liver proteins and plasma components, we suggest that future studies investigate the sensitivity and specificity of plasma aspartate aminotransferase, β-hydroxybutyrate, total bilirubin, total bile acids, and γ-glutamyltransferase for potential use as blood-basedbiomarkers for early detection of fatty liver in dairy cows. Our study is the first to study the proteome of dairy cows with naturally occurring fatty liver in earlylactation. Fatty liver is a frequent subclinical health disorder in dairy cows that may lead to disorders related to the liver function. However, the effect of triglyceride(TG) accumulation on liver metabolic pathways is still unclear. The objective was, therefore, to characterize quantitative differences in the liver proteome betweenearly lactation dairy cows with a low or high liver TG content. The liver proteome analysis indicated that a high liver TG content in early lactation dairy cows isassociated with increased oxidation of saturated fatty acids, oxidative stress, and urea synthesis and decreasedoxidation of unsaturated fatty acids. Furthermore,liver gluconeogenesis is apparently not impaired by an increased liver TG content. Based on correlations between liver proteins and plasma components, we suggest that future studies investigate the sensitivity and specificity of plasma aspartate aminotransferase, β-hydroxybutyrate, total bilirubin, total bile acids, and γ-glutamyltransferase for potential use as blood-basedbiomarkers for early detection of fatty liver in dairy cows. Our study is the first to study the proteome of dairy cows with naturally occurring fatty liver in earlylactation.

We attempted to produce a model mouse with a liver injury resulting from an immunological mechanism in C57BL/6J mice, and the effect of hepatitis on the hepatic microsomal mixed-function oxidase system was studied. An experimental immunological liver injury model was caused by the intravenous injection of an anti-basic liver protein (BLP) antibody in mice which had been previously immunized with normal rabbit IgG (RGG) and complete Freund's adjuvant. C57BL/6J strain mice showed the highest susceptibility to the immunological liver injury. Typical histopathological changes in the liver included submassive hepatocellular necrosis and infiltration of lymphocytes into the portal tract and sinusoid area in a necrotic lesion. The liver injury in this model was markedly inhibited by the administration of prednisolone (20 mg/kg, p.o.), cyclophosphamide (15 mg/kg, i.p.), levamisole (10 mg/kg, p.o.), glycyrrhizin (50 mg/kg, i.p.) and cepharanthine (10 mg/kg, i.p.), which act on the immune system. Twenty-four hours after the injection of anti-BLP antibody, the activities of aminopyrine N-demethylase, aniline hydroxylase and NADPH-cytochrome c reductase and the content of cytochrome P-450 were mostly reduced, whereas cytochrome b5 and NADH-ferricyanide reductase were not. These results suggest that the experimental liver injury model in C57BL/6J mice is useful as a model of liver injury model, and its hepatitis was shown to inhibit the cytochrome P-450-dependent biotransformation of drugs in the mouse.

Portal vein glucose delivery (the portal glucose signal) stimulates glucose uptake and glycogen storage by the liver, whereas portal amino acid (AA) delivery (the portal AA signal) induces an increase in protein synthesis by the liver. During a meal, both signals coexist and may interact. In this study, we compared the protein synthesis rates in the liver and muscle in response to portal or peripheral glucose infusion during intraportal infusion of a complete AA mixture. Dogs were surgically prepared with hepatic sampling catheters and flow probes. After a 42-h fast, they underwent a 3-h hyperinsulinemic (4× basal) hyperglucagonemic (3× basal) hyperglycemic (≈160 mg/dl) hyperaminoacidemic (hepatic load 1.5× basal; delivered intraportally) clamp (postprandial conditions). Glucose was infused either via a peripheral (PeG; n = 7) or the portal vein (PoG; n = 8). Protein synthesis was assessed with a primed, continuous [14C]leucine infusion. Net hepatic glucose uptake was stimulated by portal glucose infusion (+1 mg·kg−1·min−1, P < 0.05) as expected, but hepatic fractional AA extraction and hepatic protein synthesis did not differ between groups. There was a lower arterial AA concentration in the PoG group (−19%, P < 0.05) and a significant stimulation (+30%) of muscle protein synthesis associated with increased expression of LAT1 and ASCT2 AA transporters and p70S6 phosphorylation. Concomitant portal glucose and AA delivery enhances skeletal muscle protein synthesis compared with peripheral glucose and portal AA delivery. These data suggest that enteral nutrition support may have an advantage over parenteral nutrition in stimulating muscle protein synthesis.

The role of protein glutathionylation in acetaminophen (APAP)-induced liver injury was investigated in this study. A single oral gavage dose of 150 or 300 mg/kg APAP in B6C3F1 mice produced increased serum alanine aminotransferase and aspartate aminotransferase levels and liver necrosis in a dose-dependent manner. The ratio of GSH to GSSG was decreased in a dose-dependent manner, suggesting that APAP produced a more oxidizing environment within the liver. Despite the increased oxidation state, the level of global protein glutathionylation was decreased at 1 h and continued to decline through 24 h. Immunohistochemical localization of glutathionylated proteins showed a complex dynamic change in the lobule zonation of glutathionylated proteins. At 1 h after APAP exposure, the level of glutathionylation decreased in the single layer of hepatocytes around the central veins but increased mildly in the remaining centrilobular hepatocytes. This increase correlated with the immunohistochemical localization of APAP covalently bound to protein. Thereafter, the level of glutathionylation decreased dramatically over time in the centrilobular regions with major decreases observed at 6 and 24 h. Despite the overall decreased glutathionylation, a layer of cells lying between the undamaged periportal region and the damaged centrilobular hepatocytes exhibited high levels of glutathionylation at 3 and 6 h in all samples and in some 24-h samples that had milder injury. These temporal and zonal pattern changes in protein glutathionylation after APAP exposure indicate that protein glutathionylation may play a role in protein homeostasis during APAP-induced hepatocellular injury.

This study was performed to examine the effect of consumption of pork-liver protein hydrolysate (PLH) on body fat accumulation in Otsuka Long-Evans Tokushima Fatty (OLETF) rats as a non-insulin-dependent diabetes mellitus model and in Long-Evans Tokushima Otsuka (LETO) rats as a control. Male 20-week-old OLETF and LETO rats were pair-fed either PLH or casein containing diet for 14 weeks. In the OLETF rats, dietary PLH significantly reduced the growth and weight of fat pad including perirenal and epididymal adipose tissues. Consumption of PLH markedly suppressed hepatic activities of lipogenesis enzymes such as glucose-6-phosphate dehydrogenase and fatty acid synthase and slightly elevated fecal excretion of total fat. In the LETO rats, growth and adipose tissue weight were unaffected by dietary treatment. The results suggest that PLH is a novel ingredient suppressing body fat in genetically obese rats by reducing lipogenesis.

A monospecific antibody (anti-CF3CO antibody) was obtained by affinity chromatography on a N epsilon-trifluoroacetyl-L-lysine (CF3CO-Lys) matrix of a rabbit polyclonal antiserum, directed against trifluoroacetylated protein adducts (CF3CO-proteins). The anti-CF3CO antibody recognized distinct CF3CO-proteins on immunoblots of a liver biopsy obtained from a human individual 10 h after halothane anaesthesia. Cross-reactive proteins of 52 kDa and 64 kDa were recognized on immunoblots of livers obtained from human individuals not exposed to halothane. Recognition of both CF3CO-proteins and the 52-kDa and 64-kDa cross-reactive proteins was abolished in the presence of 1 mM CF3CO-Lys. Anti-CF3CO antibody, affinity-adsorbed to the 52-kDa or the 64-kDa cross-reactive proteins of human liver, recognized the majority of target CF3CO-proteins on immunoblots of the human liver biopsy of an individual exposed to halothane. Liver biopsies of 5 out of 7 (71%) patients with halothane hepatitis exhibited an absence or low amounts of immunorecognizable 52-kDa and/or 64-kDa cross-reactive proteins. In contrast, of 22 control human individuals tested, all liver tissue samples were positive for the 52-kDa and/or the 64-kDa cross-reactive proteins. These data indicate that epitopes on the cross-reactive proteins of 52 kDa and 64 kDa of human liver bear strong immunochemical resemblance to epitopes on human liver CF3CO-proteins. Low-level expression of the cross-reactive proteins of 52 kDa and 64 kDa is discussed as one possible factor in human susceptibility to halothane hepatitis.

International audience; BACKGROUND: Hepatic glucose uptake is enhanced by the portal delivery of glucose, which creates a negative arterioportal substrate gradient. Hepatic amino acid (AA) utilization may be regulated by the same phenomenon, but this has not been proven. OBJECTIVE: We aimed to assess hepatic AA balance and protein synthesis with or without a negative arterioportal AA gradient. DESIGN: Somatostatin was infused intravenously, and insulin and glucagon were replaced intraportally at 4- and 3-fold basal rates, respectively, in 3 groups (n = 9 each) of conscious dogs with catheters for hepatic balance measurement. Arterial glucose concentrations were clamped at 9 mmol/L. An AA mixture was infused intravenously to maintain basal concentrations (EuAA), intraportally to mimic the postmeal AA increase (PoAA), or intravenously (PeAA) to match the hepatic AA load in PoAA. Protein synthesis was assessed with a primed, continuous [(14)C]leucine infusion. RESULTS: Net hepatic glucose uptake in the PoAA condition was < or =50% of that in the EuAA and PeAA conditions (P < 0.05). In the PoAA and PeAA conditions, hepatic intracellular leucine concentrations were 2- to 2.5-fold those in the EuAA condition (P < 0.05); net hepatic leucine uptake and [(14)C]leucine utilization were approximately 2-fold greater (P < 0.05) and albumin synthesis was 30% greater (P < 0.05) in the PoAA condition than in the EuAA and PeAA conditions. Phosphorylation of ribosomal protein S6 [downstream of the mammalian target of Rapamycin complex 1 (mTORC1)] was significantly higher in the PoAA, but not PeAA, condition than in the EuAA condition. CONCLUSIONS: Portal, but not peripheral, AA delivery significantly enhanced hepatic protein synthesis under conditions in which AAs, glucose, insulin, and glucagon did not differ at the liver, an effect apparently mediated by mTORC1 signaling.

International audience; A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.

The arrival of eggs in the liver during Schistosoma mansoni infection initiates a protective granulomatous response; however, as the infection progresses, this response results in chronic liver fibrosis. To better understand the impact of schistosomiasis on liver function, we used a proteomic approach to identify proteins whose expression was significantly altered in schistosome-infected mice 8 weeks postinfection. Identification of differentially expressed proteins by mass fingerprinting revealed that schistosome infection markedly reduced the abundance of proteins associated with several normal liver functions (i.e., citric acid cycle, fatty acid cycle, and urea cycle), while proteins associated with stress responses, acute phase reactants, and structural components were all significantly more abundant. The expression patterns of several immunity-related proteins (peroxiredoxin 1, arginase 1, and galectin 1) suggested that different protein forms are associated with schistosome infection. These findings indicate that acute schistosomiasis has a significant impact on specific liver functions and, moreover, that the alterations in specific protein isoforms and upregulation of unique proteins may be valuable as new markers of disease.

L-arginine supplementation in pigs decreases liver protein turnover and increases hindquarter protein turnover both during and after endotoxemia.Bruins MJ, Soeters PB, Lamers WH, Deutz NE.Department of Surgery, Maastricht University, Maastricht, The Netherlands.BACKGROUND: Accumulating evidence suggests that L-arginine, under conditions of septicemia, not only enhances immune function but also improves protein metabolism. OBJECTIVE: Because the effect of L-arginine administration on the protein metabolism of different organs is unknown, the aim of the study was to elucidate the effects of exogenous supplementation of L-arginine during endotoxemia on the in vivo protein metabolism of individual organs and at the whole-body level. DESIGN: Female pigs were cannulated with catheters in the aorta and the splenic, caval, portal, hepatic, and renal veins, enabling measurements across the hindquarter, portal-drained viscera, liver, and kidneys. Endotoxemia was induced by a 24-h continuous intravenous infusion of endotoxin (3 microg x kg body wt(-1) x h(-1)). At 8 h, an intravenous infusion of L-arginine was started (n = 8). Control pigs (n = 6) received L-alanine. At 24 h, blood was sampled. After cessation of the endotoxin infusion, L-arginine and L-alanine infusions were continued as a supplement in the enterally infused diet. At 48 h, blood samples were obtained during the postendotoxemic and nutritionally supported conditions. Stable isotopes were used to assess protein metabolism and phenylalanine hydroxylation. RESULTS: Both during and after the endotoxin challenge, L-arginine administration enhanced protein synthesis and degradation across the hindquarter and simultaneously reduced protein synthesis and degradation in the liver at equal rates. Protein turnover across the kidneys and portal-drained viscera remained unaffected. After endotoxemia, L-arginine infusion decreased whole-body protein turnover without affecting the net protein balance. CONCLUSION: L-Arginine administration affects protein turnover of the muscle area and the liver oppositely

At 6 wk of age, male Fischer F344 rats were fed a purified, casein-based diet either ad libitum or in the amount of 60% of the diet consumed by the rats fed ad libitum (restricted diet). Hepatocytes were isolated from the rats between 2.5 and 19 mo of age. The protein content of the hepatocytes isolated from the rats fed the restricted amount of diet was significantly lower than that of hepatocytes isolated from rats fed ad libitum. The DNA and RNA content of the hepatocytes were similar for the rats fed the two dietary regimens. The absolute rate of protein synthesis for hepatocytes isolated from rats fed ad libitum decreased 55 % between 2.5 and 19 mo of age. However, the rate of protein synthesis by hepatocytes from rats fed the restricted amount of diet decreased only slightly with increasing age. At 19 mo of age, the rate of protein synthesis by hepatocytes from the rats fed the restricted amount of diet was significantly higher than the rate of protein synthesis for hepatocytes from rats fed ad libitum. Therefore, dietary restriction retards the age-related decline in liver protein synthesis. Reproduced by permission. Maria C. Birchenall-Sparks, Michael S. Roberts, Jeffrey Staeker, James E. Hardwick, Arlan Richardson, Effect of Dietary Restriction on Liver Protein Synthesis in Rats. J. Nutr. 115 , 944-950 (1985).

Our aim was to analyze mechanisms involved in the adaptation of protein metabolism to food deprivation and refeeding in adult rats. Twelve-month-old rats, which had been food-deprived for 113 h and refed for 6 h, were injected subcutaneously with a flooding dose of valine (with 50% [1-13C]-L-valine) to measure in vivo protein synthesis in tibialis anterior, soleus and liver. Protein and RNA contents were also measured. In both muscles, protein mass was maintained during food deprivation. Due to a drop in protein synthetic capacity (Cs), total and myofibrillar protein synthesis rates were reduced in food-deprived rats and were not stimulated by a 6-h refeeding. In contrast, protein levels were maintained lower than RNA levels in liver during food deprivation, and Cs was higher than in fed rats. Protein synthesis rates and ribosomal efficiency were reduced in food-deprived rats. Due to maintenance of protein synthetic capacity, there was a rapid stimulation of liver protein synthesis with refeeding, which induced a significant rise in protein mass (also related to an inhibition of protein degradation). In conclusion, coordinated responses of liver and muscles allowed a sparing of muscle proteins during food deprivation and a rapid recovery of liver proteins during refeeding. Control of ribosome quantity could play a critical role in these adaptations in tissue protein synthesis in adult rats.

By means of our electrophoretic method applied to analyse the liver protein, as reported in Part I of this series, we studied the liver protein fractions in eight cases of chronic gastritis and 50 cases each of gastric or duodenal ulcer and of gastric cancer. The results obtained were as follows: 1. The liver protein in two cases of chronic gastritis and six of gastric ulcer, but only in mild grade as diagnosed clinically, was found to contain only a very small quantity of albumin (5.8% on the average), the remainder being all globulins, β-globulin being particularly abundant (47.1% on the average). 2. Of the severe cases of gastric diseases, the cases showing the liver protein fraction values within the range of the above mild cases were generally of good general conditions, but those with lowered β-globulin, raised γ globulin and/or albumin value were usually cases with complications or advanced disease. 3. The liver protein fraction values were more frequently within the values of mild cases in gastritis-ulcer cases than in gastric cancer cases, and the β-globulin value was lowered, the γ-globulin and/or the albumin value raised more frequently among the gastric cancer cases.

Chemical, physical, or mechanical insults to a cellular system cause changes in the rates of protein synthesis or degradation. To define patterns of quantitative protein changes and identify the biochemical basis of those changes, we are studying fluctuations in mouse liver protein abundance in response to factors such as stages of maturation, changes in diet, and exposure to chemicals. Two-dimensional gel electrophoresis (2DE) together with computer-assisted data analysis is used to collect quantitative data from groups of 2DE patterns. Our analysis of liver protein expression in mice treated with four different chemicals known to cause peroxisome proliferation serves as an example of the changes in protein expression that can be monitored by using this approach. The database we are building indicates that changes specific to a particular treatment as well as generalized effects can be identified, providing the opportunity to identify patterns of protein expression that are indicators of biological response to different types of cellular insults.

Changes in protein kinase A (PKA, or cAMP-dependent protein kinase) activity in the rat liver during different metabolic phases of sepsis were investigated. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into 3 groups: control, early sepsis, and late sepsis. Early and late sepsis refer to those animals killed at 9 and 18 h, respectively, after CLP. Hepatic PKA was extracted and partially purified by acid precipitation, ammonium sulfate fractionation, and diethylaminoethyl (DEAE)-cellulose chromatography. PKA was eluted from DEAE-cellulose column with a linear NaCl gradient. Two peaks of PKA, type I (eluted at low ionic strength) and type II (eluted at high ionic strength), were collected and their activities were determined on the basis of the rate of incorporation of [gamma-32-P]ATP into histone. The results show that during early sepsis, both type I and type II PKA activities remained unchanged. During late sepsis, type I PKA activity was decreased by 40.7-53.6%, whereas type II PKA activity was unaffected. Kinetic analysis of the data on type I PKA during the late phase of sepsis reveals that the Vmax (maximal velocity) values for ATP, cAMP, and histone were decreased by 40.7, 53.6, and 47.3%, respectively whereas the Km (substrate concentration required for half-maximal enzymatic activity) values for ATP, cAMP, and histone were unaltered. These data indicate that type I PKA was inactivated during the late hypoglycemic phase of sepsis in the rat liver. Because PKA-mediated phosphorylation plays an important role in the regulation of hepatic glucose metabolism, an inactivation of PKA may contribute to the development of hypoglycemia during the late phase of sepsis.

We analysed the respective influences of age and lysine deficiency on skeletal muscle and liver protein turnover. Growing male broilers were fed ad libirum on isoenergetic diets containing 2OO g crude protein/kg which varied in their lysine content (7·7 or 10·1 g/kg). Fractional rates of protein synthesis (FSR) were measured in vivo in the liver and the pectoralis major muscle of 2-, 3- and 4-week-old chickens (flooding dose of l-[143H]phenylalanine). Fractional rates of proteolysis (FBR) were estimated for the same tissues as the difference between synthesis and growth. Over the 2-week period liver FSR and FBR were unchanged, whereas muscle FSR decreased with age. This developmental decline was related to the lower capacity for protein synthesis (Cs) without any modifications of the translational efficiency. Whatever the age, lysine deficiency resulted in significant decreases in body weight, tissue protein content and tissue protein deposition, apparently because of reduced amounts of proteins synthesized. We recorded a difference in the response of the two tissues to lysine deficiency, the pectoralis major being more sensitive than the liver. When comparing birds of the same age, liver FSR and FBR were not modified by the diet, where as muscle FSR, Cs and FBR were higher in chicks fed on a lysinc-deficient diet than in the controls. Conversely, when chicks of similar weights were compared, the main effect of the dietary deficiency was an increase in muscle FBR. The results suggest that lysine deficiency not only delayed chick development so that protein turnover was affected, but also induced greater changes in metabolism. Thus, the principal mechanism whereby muscle mass decreased appeared to be a change in FBR.