Livers Of Sham-operated Rats Research Articles

Fam20C-mediated phosphorylation of osteopontin is critical for its secretion but dispensable for its action as a cytokine in the activation of hepatic stellate cells in liver fibrogenesis.

Osteopontin (OPN) is a phosphoglycoprotein secreted into the extracellular matrix upon liver injury, acting as a cytokine stimulates the deposition of fibrillary collagen in liver fibrogenesis. In livers of mice subjected to bile duct ligation (BDL) and in cultured activated hepatic stellate cells (HSCs), we show that OPN, besides being overexpressed, is substantially phosphorylated by family with sequence similarity 20, member C (Fam20C), formerly known as Golgi casein kinase (G-CK), which is exclusively resident in the Golgi apparatus. In both experimental models, Fam20C becomes overactive when associated with a 500-kDa multiprotein complex, as compared with the negligible activity in livers of sham-operated rats and in quiescent HSCs. Fam20C knockdown not only confirmed the role of Fam20C itself in OPN phosphorylation, but also revealed that phosphorylation was essential for OPN secretion. However, OPN acts as a fibrogenic factor independently of its phosphorylation state, as demonstrated by the increased expression of Collagen-I by HSCs incubated with either a phosphorylated or nonphosphorylated form of recombinant OPN. Collectively, our results confirm that OPN promotes liver fibrosis and highlight Fam20C as a novel factor driving this process by favoring OPN secretion from HSCs, opening new avenues for deciphering yet unidentified mechanisms underlying liver fibrogenesis.

Autofluorescence of liver tissue and bile: Organ functionality monitoring during ischemia and reoxygenation

Autofluorescence (AF) based optical biopsy of liver tissue is a powerful approach for the real-time diagnosis of its functionality. Since increasing attention is given to the bile production and composition to monitor the liver metabolic engagement in surgery and transplantation, we have investigated the bile AF properties as a potential, additional diagnostic parameter. Spectrofluorometric analysis has been performed in real time on a rat liver model of warm ischemia and reperfusion-60 minutes partial portal vein and hepatic artery clamping and subsequent restoration of blood circulation-in comparison with sham operated rats. The AF spectra have been recorded through a single fiber optic probe (366 nm excitation) from both liver tissue and bile, collected from the cannulated bile duct, and analyzed by means of curve fitting procedures. Bile composition has been also analyzed through biochemical assays of bilirubin, total bile acids (TBA) and proteins. Both liver and bile AF signal amplitude and spectral shape undergo changes during induction of ischemia and subsequent reperfusion. The liver tissue response is mainly ascribable to changes in NAD(P)H and flavins and their redox state, largely dependent on oxygen supply, and to the decrease of both vitamin A and fatty acid AF contributions. During comparable times, sham operated rat livers undergo smaller alterations in AF spectral shape, indicating a continuous, slight increase in the oxidized state. Bile AF emission shows a region in the 510-600 nm range ascribable to bilirubin, and resulting from the contribution of two bands, centered at about 515-523 and 570 nm, consistently with its bichromophore nature. Variations in the balance between these two bands depend on the influence of microenvironment on bilirubin intramolecular interchromophore energy transfer efficiency and are likely indicating alteration in a bile composition. This event is supported also by changes observed in the 400-500 nm emission region, ascribable to other bile components. In parallel with the intratissue AF properties, mainly reflecting redox metabolic activities, the bile AF analysis can provide additional information to assess alterations and recovery in the balance of liver metabolic activities.

The Vagal Nerve Stimulates Activation of the Hepatic Progenitor Cell Compartment via Muscarinic Acetylcholine Receptor Type 3

In the rat the hepatic branch of the nervus vagus stimulates proliferation of hepatocytes after partial hepatectomy and growth of bile duct epithelial cells after bile duct ligation. We studied the effect of hepatic vagotomy on the activation of the hepatic progenitor cell compartment in human and rat liver. The number of hepatic progenitor cells and atypical reactive ductular cells in transplanted (denervated) human livers with hepatitis was significantly lower than in innervated matched control livers and the number of oval cells in vagotomized rat livers with galactosamine hepatitis was significantly lower than in livers of sham-operated rats with galactosamine hepatitis. The expression of muscarinic acetylcholine receptors (M1-M5 receptor) was studied by immunohistochemistry and reverse transcriptase-polymerase chain reaction. In human liver, immunoreactivity for M3 receptor was observed in hepatic progenitor cells, atypical reactive ductules, intermediate hepatocyte-like cells, and bile duct epithelial cells. mRNA for the M1-M3 and the M5 receptor, but not the M4 receptor, was detected in human liver homogenates. In conclusion, the hepatic vagus branch stimulates activation of the hepatic progenitor cell compartment in diseased liver, most likely through binding of acetylcholine to the M3 receptor expressed on these cells. These findings may be of clinical importance for patients with a transplant liver.

Expression of basic fibroblast growth factor-like immunoreactivity in the nuclei of regenerating hepatocytes.

This study, utilizing rats subjected to two-thirds partial hepatectomy or sham operation, was designed (1) to investigate the content of basic fibroblast growth factor (bFGF) in the subcellular fractions of regenerating and sham-operated rat livers by immunoblot experiments and enzyme-linked immunosorbent assay (ELISA), (2) to show that bFGF immunoreactivity and proliferating cell nuclear antigen (PCNA) immunoreactivity are markers for hepatocellular mitosis before and after partial hepatectomy, and (3) to observe the location and fine structure of the bFGF immunoreaction within the regenerating liver with special attention to bFGF immunoreactivity in the nuclei of regenerating hepatocytes. Immunoblot experiments and ELISA showed a transient increase in high-molecular-weight forms of bFGF in the nuclear subcellular fraction of regenerating liver 48 h after partial hepatectomy. By light microscopy, bFGF and PCNA immunoreactivities were detected in the nuclei of regenerating hepatocytes. Electron microscopy demonstrated bFGF-like immunoreactivity mainly in the nuclear euchromatin and rarely in the heterochromatin or nucleoli of regenerating hepatocytes. The transient increase in high-molecular-weight forms of bFGF in the nuclear euchromatin of regenerating hepatocytes, together with the concomitant expression of PCNA in the regenerating liver, suggests an important role of the high-molecular-weight forms of bFGF in hepatocyte proliferation and/or mitosis, although authentic bFGF with a molecular form of 18 kDa is not considered to be involved in hepatic regeneration.

Increase in the number of atrial natriuretic hormone receptors in regenerating rat liver.

Forty-eight hours after partial (approximately 67%) hepatectomy the activity of the particulate guanylate cyclase was increased by 2-fold in the regenerating rat liver. This increase was not an artifact of membrane isolation procedures, and as determined by 125I-labeled Tyr-28 atrial natriuretic hormone-(1-28) ANF binding, was accompanied by a 2-fold increase in the number of ANF receptors. The Kd of the receptors in membranes of regenerating livers was not significantly different from the Kd of the receptors in livers of sham-operated rats. The linear synthetic descysteine analog of ANF, analog I, which binds only to the 66-kDa receptors, displaced approximately 40% of the specifically bound 125I-ANF in liver membranes from both hepatectomized and sham-operated (control) animals. Affinity cross-linking studies with 125I-ANF confirmed the increase in the 116-kDa ANF receptor in membranes of regenerating livers. In perfused livers derived from control and hepatectomized animals, the basal rates of cGMP production were not significantly different. However, atriopeptin II-stimulated cGMP production was twice as great in regenerating livers as compared with controls. These data demonstrate that the increase in particulate guanylate cyclase activity observed during liver regeneration is due to an increase in the 116-kDa ANF receptor-associated activity. Additionally, our data demonstrate that the regenerating rat liver may be a valuable model with which to study the role of the hepatic ANF receptor/particulate guanylate cyclase.

Activation of the glucose-regulated gene ( grp78) in regenerating rat liver is nonspecific and is related to acute phase response

The expression pattern of the hsp70 gene family during regeneration of rat liver has been investigated. Northern blots were prepared from total RNA isolated from livers at 0 h (control), 12 h (end of prereplication phase), 24 h (maximum of DNA synthesis) and 36 h (postmitotic phase) after partial hepatectomy. Blots were hybridized with probes specific for the hsp70 (heat-inducible), hsc70 (constitutively expressed), hst70 (testis-specific) and grp78 (glucose-regulated) gene. No hsp70 and hst70 gene transcripts have been detected at any time point investigated, and only a low increase of the hsc70 mRNA level has been observed 24 h after surgery. In contrast, a significant accumulation of the transcript coded by the grp78 gene has been detected in liver remnant 12 and 24 h after partial hepatectomy. however, we observed a comparable activation of this gene in livers of sham-operated rats or in rats injected with turpentine to cause sterile inflammation. Our results indicate that the activation of the grp78 gene in liver of wounded rats (partial hepatectomy or sham operation) is presumably a part of acute-phase response.

Effects of mitotic activity and water content on the proton relaxation times in partially hepatectomized and sham-operated rat livers.

Proton relaxation times, T1 and T2, water content, and mitotic activity in control, sham-operated and partially hepatectomized rat livers were measured before surgery, and at 28 hours, 48 hours and six days after surgery. T1, water content, and mitotic activity increased after partial hepatectomy, but T1 was also elevated after sham operations, whereas neither water content nor mitotic activity changed. T2 was unaffected by either procedure. The T1 changed is more likely to be the result of surgery related stress than of changes in water content or growth rate.

Liver carnitine metabolism after partial hepatectomy in the rat: Effects of nutritional status and inhibition of carnitine palmitoyltransferase

This study examined the effects of partial hepatectony on hepatic carnitine and acylcarnitine concentrations in fed or 24 h-starved partially hepatectonized (PH) or sham-operated (SO) rats at 1 or 4 days after surgery. The ratio of free to esterified carnitine was low in fed PH rats at day 1 : the low ratio was increased to the SO value when mitochondrial fat oxidation was inhibited by 2-tetradecylglycidate. Starvation (24 h) increased plasma [non-esterified fatty acid] in PH or SO rats, the increases being greater at day 1 than at day 4. Hepatic [long-chain acylcarnitine] were also increased. These latter increases were a consequence of increased mitochondrial fat oxidation since they were not observed in PH or SO rats treated with 2-tetradecylglycidate. Whereas the starvation-induced increase in long-chain acylcarnitine was associated with increased [ketone body] in livers of SO rats at both day 1 and day 4 after surgery, [ketone body] was inappropriately low for the steady-state long-chain [acylcarnitine] in livers of PH rats at the first post-operative day. This was not a consequence of a decrease in [total carnitine] in the liver. The results are discussed with reference to the role of the liver in determining the relative proportions of the fat fuels available for extrahepatic tissues and the effects of liver cell proliferation on hepatic triacylclycerol metabolism.

Blood flow of experimental liver metastases in rat as evaluated by the locally injected 133-Xenon washout.

The investigations on blood flow in liver metastases are interesting from both a pathophysiological and a therapeutic point of view. Available data, however, are few and not definitive, as these studies are complex and difficult to perform. In a group of 25 Sprague-Dawley rats, in which liver metastases of Walker-256 carcinoma had been implanted, the blood flows in a metastasis and in the normal liver surrounding it were determined by means of the locally injected 133-Xenon washout. Thirteen sham-operated rats were a control group. Blood flow in the metastases was decreased as a group compared to that in the normal liver surrounding metastases and to that in liver of sham-operated rats. Small metastases showed normal or increased blood flow, large ones decreased or stagnant blood flow. Moreover, a significant inverse correlation was found between blood flow and diameter of metastasis. It is concluded that liver metastases of Walker-256 carcinoma show a decrease in blood flow which is related to the size of metastasis.

Endogenous hepatic growth-modulating factors and effects of a choline-devoid diet and of phenobarbital on hepatocarcinogenesis in the rat.

The activities of an endogenous inhibitor and of a stimulator of cell proliferation were assayed in the livers of sham-operated (SO) or partially hepatectomized (PH) adult rats; rats fed a choline-supplemented (CS) or a choline-devoid (CD) diet; the same diets followed by acute CCl4 intoxication; the same diets supplemented with phenobarbital (PHB); or a CD diet containing DL-ethionine (ETH). The inhibitor and the stimulator were semipurified by fractional ethanol precipitation of a liver cytosolic fraction, and their activities were assessed by means of bioassays in vitro. The livers of SO rats and of rats fed the CS diet contained only inhibitor activity. Following PH, a CD diet, or CCl4 intoxication the inhibitor activity was suppressed, and there was a simultaneous appearance of a stimulator activity. Thus, PH, a CD diet, and CCl4 intoxication cause similar cellular (loss and regeneration) and humoral-homeostatic changes in adult rat livers. We propose that these changes constitute a basic attribute of the mechanism whereby the three conditions affect similarly hepatocarcinogenesis in the rat, especially in the case of a CD diet, because the changes it induces are chronic rather than acute. PHB, another promoter of chemical hepatocarcinogenesis, affected neither the inhibitor nor the stimulator activity. Thus, PHB seems to be acting by a different mechanism than that of the other three agents. ETH did not modify the shift in the balance of the growth-modulating factors induced by a plain CD diet. This shift may account for the marked stimulation of carcinogen-induced oval cell proliferation exerted by a CD diet. The significance of these results is discussed in the context of known effects of a CD diet and of PHB on hepatocarcinogenesis in rats.

Taurine metabolism in the rat: Effect of partial hepatectomy

1. 1. The biosynthesis of taurine in rat liver regenerating after partial hepatectomy is altered such that hypotaurine accumulates and the concentration of taurine is reduced. 2. 2. [ 35S]Cystine is converted to both [ 35S]hypotaurine and [ 35S]taurine in the liver regenerating after partial hepatectomy, whereas only [ 35S]taurine could be detected in the liver of sham-operated rats. 3. 3. The changes in activities of enzymes involved in this pathway, cystathionase (which increases), cysteinesulfinic acid decarboxylase (which decreases) and hypotaurine aminotransferase (which is unchanged), do not provide an adequate explanation of these observations. 4. 4. We conclude that the oxidation step in which hypotaurine is converted to taurine is altered in this. condition, although a convincing demonstration of this enzymatic reaction in vitro has not been accomplished to date.

Sialic acid metabolism in regenerating rat liver

Sialic acid metabolism was investigated in control rat liver, in regenerating liver at 24 h and 48 h after partial hepatectomy and in the liver of sham-operated animals. High levels of membrane-bound neuramindase, with no detectable changes in the soluble enzyme, were observed in regenerating rat liver. The neuraminidase activities in the liver of sham-operated rats were identical to those present in control liver. High levels of CMP- N-acetylneuraminic acid synthetase and sialyltransferase were observed both in regenerating liver as well as in the liver of sham-operated rats. The sialic acid content of regenerating rat liver, which was lower than that found in the liver of control and sham-operated rats at 24 h, returned to normal values 48 h after surgery.

The site of insulin resistance in acute uremia.

In order to define the mechanism of glucose intolerance in acutely uremic rats, various studies were carried out 24 hours after bilateral nephrectomy. Glucose removal following intravenous glucose was significantly (p is less than 0.001) decreased in uremic rats compared with sham-operated rats (k = 2.1 +/- 0.03 per cent vs. 5.1 +/- 0.2 per cent). This deterioration in glucose tolerance was associated with higher insulin levels in uremic rats from one to 40 minutes after glucose administration, suggesting that insulin resistance accounted for the decrease in glucose removal by uremic rats. To identify the site of the insulin resistance, we compared the ability of insulin to enhance net glucose uptake by isolated perfused liver and muscle (hindlimb) preparations obtained from uremic and sham-operated rats. Insulin suppressed glucose outflow from perfused livers of uremic rats at least as well as it did from livers of sham-operated rats, and suppression occurred at both maximal ( greater than 600 micromicron./ml.) and threshold (75 micromicron./ml.) perfusate insulin levels. In contrast, there was a significant decrease in the ability of insulin (mean perfusate level = 225 micromicron./ml.) to enhance glucose uptake of perfused hindlimbs of uremic as compared with sham-operated rats. These results suggest that the insulin resistance of acute uremia may be due primarily to decreased insulin-mediated uptake of glucose by skeletal muscle without any decrease in sensitivity of the liver to insulin.

Diversity of polyadenylated messenger RNA sequences in normal and 12-hr regenerating liver

Molecular hybridization techniques were used to examine the structural organization of the rat genome and gene expression during liver regeneration. The nonrepetitive fraction of rat DNA comprises approximately 69% of the total and has a complexity of 1.9 × 10 9 nucleotide pairs. Complementary DNAs (cDNA) were synthesized using polyadenylated polysomal mRNA templates from normal and 12-hr regenerating livers. Hybridization of cDNA with a large excess of total rat DNA indicates that the proportion of polyadenylated polysomal mRNA homologous to repetitive and nonrepetitive DNA is very similar in normal and regenerating livers. Hybridization of cDNAs with excess homologous or heterologous mRNA revealed that: (a) The sequence complexity of polyadenylated polysomal mRNA is essentially the same in normal and 12-hr regenerating liver. Using these data we estimate that the maximum proportion of single-copy DNA represented in polyadenylated polysomal mRNA in livers of sham-operated rats or in 12-hr regenerating liver is 0.85–0.90%; (b) 85–90% of polysomal polyadenylated mRNA is common to normal and regenerating livers, while 11–14% is unique either to normal or 12-hr regenerating liver polysomes. We cannot discern as yet whether the polysomal polyadenylated mRNA sequences unique to normal or regenerating livers are the result of differential gene transcription and/or selective transport from the nucleus to polysomes.

Studies on the structure and function of ribosomes from regenerating rat liver

Electrophoresis of ribosomal proteins from 1-day regenerating and sham-operated rat livers, doubly-labeled with two iodine isotopes, revealed that the relative stoichiometric ratios of proteins S6 from the 40S subunit and L5, L18 and L27 from the 60S subunit, vary significantly from those obtained with the other proteins. The ratios of the altered proteins return to normal within three days after partial hepatectomy. Polypeptide chain elongation and binding of aminoacyl-tRNA were slightly greater with regenerating preparations than with control preparation, when 80S ribosomes were used, but differences were not observed when the experiments were carried out with purified subunits and exogenous template.