Protein synthesis in mitochondrial and microsomal fractions from rat brain and liver after acute or chronic ethanol administration

Incorporation of C¹⁴ Leucine was determined $\text{in vitro}$ or $\text{in vivo}$ in isolated mitochondria and microsomes of rat brain and liver after acute or chronic ethanol administration $\text{in vivo}$.The protein synthesis in mitochondrial and microsomal preparation was inhibited respectively by chloramphenicol and cycloeximide, specific inhibitors for the two systems tested. The experimental data demonstrate that the $\text{in vitro}$ protein synthesis in both systems, mitochondrial and microsomal, is strongly affected only after chronic treatment which produces significant activation at the mitochondrial and microsomal level in the liver and an inhibition on the same systems of the brain.The data for $\text{in vivo}$ protein synthesis instead show strong inhibition after acute administration, except for brain mitochondria, which are practically unaffected, while after chronic treatment no significant alterations are observed.     

Endotoxin stimulation of liver parenchymal cell phosphofructokinase activity requires nonparenchymal cells.

The rate of carbohydrate flux through phosphofructokinase (measured as the rate of [3-3H]glucose detritiation) was increased fourfold in rat liver parenchymal cells incubated with conditioned medium from lipopolysaccharide-stimulated adherent liver non-parenchymal cells. The rate was not affected in parenchymal cells incubated either with lipopolysaccharide directly or with conditioned medium from non-stimulated non-parenchymal cells. The stimulation of carbohydrate flux through phosphofructokinase by conditioned medium was not duplicated by peptide cytokines known to be released by lipopolysaccharide-activated liver non-parenchymal cells (interleukin-1, interleukin-6, tumor necrosis factor-alpha, and transforming growth factor-beta) or platelet activating factor. Furthermore, formation of the active conditioned medium was not prevented by inclusion of cycloheximide or dexamethasone to inhibit cytokine synthesis, or indomethacin or BW755c to inhibit arachidonic acid metabolism, during lipopolysaccharide-stimulation of the non-parenchymal cells. The results indicate that intercellular communication between lipopolysaccharide-stimulated liver non-parenchymal cells and parenchymal cells by soluble mediators is responsible for the stimulation of liver phosphofructokinase activity during endotoxin-induced shock. Studies to isolate and identify the factor(s) in the conditioned medium are currently in progress.     

Uptake and degradation of bovine testes beta-galactosidase by parenchymal and nonparenchymal rat liver cells

The plasma half-life of beta-galactosidase in rat was about 1.5 min. Ten minutes after in vivo injection, 45% of the enzyme was recovered in liver, with hepatocytes and endothelial cells as the predominant cell types responsible for uptake. In vitro uptake of beta-galactosidase in hepatocytes and nonparenchymal liver cells was saturable, Ca2+-dependent and it could be partly inhibited by mannose or alpha-methyl-mannoside.     

Incorporation of labelled amino acids into proteins of isolated parenchymal and nonparenchymal rat liver cells in the absence and presence of ethanol.

Parenchymal and nonparenchymal cells were isolated from perfused rat livers and incubated at 37 degrees C in the absence and presence of ethanol (50 mM). 1. Nonparenchymal cells prepared by means of centrifugation showed a higher rate of incorporation of L-[U-14C]valine into protein than nonparenchymal cells prepared by means of pronase. Cells prepared by the former method were used for further studies. 2. Protein degradation was present in suspensions of both parenchymal and nonparenchymal cells evidenced by increasing levels of branched amino acids in the intracellular and extracellular compartment during cell incubation. 3. The rate of cellular protein synthesis (corrected for precursor pool specific radioactivity) was of the same order of magnitude in nonparenchymal and parenchymal cells when expressed as nmol valine incorporated per mg protein. This rate was also close to the value found in intact liver by other workers. 4. Approximately 25% of the total radioactivity incorporated during incubation for 2 h was found in proteins released to the medium from parenchymal cells, while the corresponding figure for nonparenchymal cells was 3.5%. 5. Ethanol inhibited incorporation of labelled valine into stationary and medium proteins of parenchymal cells. No such effects were found in nonparenchymal cells. 6. Nonparenchymal cells did not metabolize ethanol while parenchymal cells did, shown by changes in lactate/pyruvate ratio and medium pH. It was concluded that nonparenchymal cells are capable of synthesizing proteins at a rate comparable to that found in parenchymal cells. Protein synthesis in parenchymal cells was inhibited by ethanol, but nonparenchymal protein synthesis was unaffected. This difference may be linked to the ability of the former cell type to metabolize ethanol.     

Hydrolysis of retinyl esters by non-specific carboxylesterases from rat liver endoplasmic reticulum.

The four most important non-specific carboxylesterases from rat liver were assayed for their ability to hydrolyse retinyl esters. Only the esterases with pI 6.2 and 6.4 (= esterase ES-4) are able to hydrolyse retinyl palmitate. Their specific activities strongly depend on the emulsifier used (maximum rate: 440 nmol of retinol liberated/h per mg of esterase). Beside retinyl palmitate, these esterases cleave palmitoyl-CoA and monoacylglycerols with much higher rates, as well as certain drugs (e.g. aspirin and propanidid). However, no transacylation between palmitoyl-CoA and retinol occurs. Retinyl acetate also is a substrate for the above esterases and for another one with pI 5.6 (= esterase ES-3). Again the emulsifier influences the hydrolysis by these esterases (maximum rates: 475 nmol/h per mg for ES-4 and 200 nmol/h per mg for ES-3). Differential centrifugation of rat liver homogenate reveals that retinyl palmitate hydrolase activity is highly enriched in the plasma membranes, but only moderately so in the endoplasmic reticulum, where the investigated esterases are located. Since the latter activity can be largely inhibited with the selective esterase inhibitor bis-(4-nitrophenyl) phosphate, it is concluded that the esterases with pI 6.2 and 6.4 (ES-4) represent the main retinyl palmitate hydrolase of rat liver endoplasmic reticulum. In view of this cellular localization, the enzyme could possibly be involved in the mobilization of retinol from the vitamin A esters stored in the liver. However, preliminary experiments in vivo have failed to demonstrate such a biological function.     

Effects of long-term ethanol administration in a rat total enteral nutrition model of alcoholic liver disease

Male Sprague-Dawley rats were chronically fed a high-unsaturated-fat diet for 130 days by using total enteral nutrition (TEN), or the same diet in which ethanol (EtOH) isocalorically replaced carbohydrate calories. Additional groups were supplemented with the antioxidant N-acetylcysteine (NAC) at 1.7 g·kg(-1)·day(-1). Relative to an ad libitum chow-fed group, the high-fat-fed controls had three- to fourfold greater expression of fatty acid transporter CD36 mRNA and developed mild steatosis but little other hepatic pathology. NAC treatment resulted in increased somatic growth relative to controls (4.0 ± 0.1 vs. 3.1 ± 0.1 g/day) and increased hepatic steatosis score (3.5 ± 0.6 vs. 2.7 ± 1.2), associated with suppression of the triglyceride hydrolyzing protein adiponutrin, but produced no elevation in serum alanine aminotransferase (ALT). Chronic EtOH treatment increased expression of fatty acid transport protein FATP-2 mRNA twofold, resulting in marked hepatic steatosis, oxidative stress, and a twofold elevation in serum ALT. However, no changes in tumor necrosis factor-α or transforming growth factor-β expression were observed. Fibrosis, as measured by Masson's trichrome and picrosirius red staining, and a twofold increase in expression of type I and type III collagen mRNA, was only observed after EtOH treatment. Long-term EtOH treatment increased hepatocyte proliferation but did not modify the hepatic mRNAs for hedgehog pathway ligands or target genes or genes regulating epithelial-to-mesenchymal transition. Although the effects of NAC on EtOH-induced fibrosis could not be fully evaluated, NAC had additive effects on hepatocyte proliferation and prevented EtOH-induced oxidative stress and necrosis, despite a failure to reverse hepatic steatosis.     

Distribution and some properties of NADPH and NADH oxidase in parenchymal and nonparenchymal liver cells

The activities of NADPH and NADH oxidase were determined in homogenates of isolated pure parenchymal and nonparenchymal rat liver cells at neutral (7.4) and acid (5.5) pH. The NADPH oxidase at pH 7.4 is about equally active in parenchymal and nonparenchymal cells and in both cell types is rather insensitive to KCN (1 mm) inhibition. By lowering the pH to 5.5, the NADPH oxidase of the nonparenchymal cells is stimulated (twofold) while the activity in parenchymal cells is decreased. The NADH consumption at neutral pH in parenchymal cells is 75% inhibited by KCN, while this activity in nonparenchymal cells is relatively insensitive to KCN. The NADH oxidase in both parenchymal and nonparenchymal liver cells is less active when the pH is lowered from 7.4 to 5.5. The distribution of NAD(P)H oxidases between parenchymal and nonparenchymal liver cells and the effect of pH on their activities suggest that in the nonparenchymal cells, the NADPH oxidase might play a role in the synthesis of H₂O₂ within the phagocytic vacuole. A scheme is proposed which describes the metabolic events involved in H₂O₂ formation and catabolism of endo(phago)cytosed particles in nonparenchymal liver cells.     

In vivo uptake of human and rat low density and high density lipoprotein by parenchymal and nonparenchymal cells from rat liver.

The relative contribution of the parenchymal and nonparenchymal rat liver cells to the hepatic uptake of human and rat high density lipoprotein (HDL) and low density lipoprotein (LDL) was determined in vivo. Nonparenchymal cells, isolated 6 h after intravenous injection of iodinated human HDL and LDL, contained respectively 4.2 and 6.3 times the amount of trichloroacetic acid-precipitable radioactivity per mg cell protein as compared to parenchymal cells. For rat iodinated HDL and LDL these factors were 3.4 and 4.1, respectively. These results indicate that nonparenchymal liver cells play a substantial role in the hepatic uptake of human and rat HDL and LDL in vivo.     

Inhibition of rat liver retinyl palmitate hydrolase activity by ether analogs of cholesteryl esters and acylglycerides

In the present study the tissue distribution of [3H]methotrexate was studied after intravenous injection of [3H]methotrexate-containing liposomes in normal and macrophage-depleted mice. Elimination of macrophages was performed by treatment with dichloromethylene diphosphonate- (DMDP)-containing liposomes. After thorough elimination of the macrophages from spleen and liver, by two intravenous injections of DMDP liposomes 6 and 4 days before tissue distribution studies, we found dramatic changes in the localization pattern of [3H]methotrexate liposomes in the blood, due to a decreased uptake of [3H]methotrexate liposomes by the DMDP liposome-treated liver. Because of the absence of these macrophages that are able to clear the blood of liposomes, and because of the resulting higher blood level of liposomes, we found an enhanced uptake of [3H]methotrexate liposomes by the spleen. It may be concluded that, in the spleen, apart from uptake of liposomes by macrophages, at least one other mechanism is responsible for the clearance of liposomes from the circulation. When comparing cholesterol-rich with cholesterol-poor liposomes, we found basically the same results, although uptake of cholesterol-rich liposomes by macrophages was smaller than that of cholesterol-poor liposomes, as found in several other studies. We suggest that pretreatment with DMDP liposomes can help to maintain a high level of intravenous-injected liposome-entrapped material in the blood, which otherwise would be removed by macrophages.     

Bicarbonate ion-ATPase in rat liver cell fractions.

The distribution of HCO3MINUS-ATPase activity was studied in cell fractions prepared from homogenates of rat liver. The level of mitochondrial contamination in the microsomal fraction depended on the fractionation procedure and on the method of homogenization. With proper care, microsomes with undetectable mitochondrial contamination could be prepared. These microsomes had no detectable HCO3MINUS-ATPase activity. Approximately 85% of the total HCO3minus-ATPase activity of the post 6000 times g-min supernatant was recovered in the mitochondrial fraction. The properties of this mitochondrial HCO3minus-ATPase were not distinguishable from those of the various microsomal HCO3minus-ATPases previously described by other investigators.     

Growth and liver morphology after long-term ethanol consumption of rats

Ethanol was administered to female and male Wistar rats by mixing it with their drinking water. Ethanol concentrations were gradually increased up to either 8% or 15%. Female rats receiving 8% ethanol in their drinking water consumed 5-13 g, males 4-10 g daily. The ethanol/total food caloric intake percentages were 13 to 20% and 9 to 15% for female and male rats, respectively. There was no difference in body weight and relative liver weight between treated rats and their controls. Female and male rats receiving 15% of ethanol in their drinking water consumed 8-14 g ethanol per kg body weight per day. The percentages of ethanol/total food caloric intake were stabilized at about 25% for both sexes. Growth of the rats differed only slightly from controls; a tendency for a higher increase of body weight of the control rats was found. No difference in relative liver weight between ethanol-treated and control rats was observed. Microscopic examinations revealed that the ethanol treatment resulted in fat accumulation in the liver cells. A proliferation of the Smooth Endoplasmic Reticulum (SER) was more marked in the 15% dosed rats than in the 8% dosed rats and more distinct in female rats than in male rats in both dosage groups.     

Morphometrical analysis of the gap-junctional area in parenchymal cells of the rat liver after administration of dibutyryl cAMP and aminophylline

Summary In view of the presumed involvement of gap junctions in the coordination of metabolic activities, the influence of cAMP as a regulatory signal of cell metabolism on gap junctions of hepatocytes has been examined. Male rats received two intraperitoneal doses of 10 mg dibutyryl cAMP/100 g body weight with a time interval of 2.5 h and were decapitated 2.5 h later. After this 5-h interval, analysis of freeze-fracture replicas of fixed liver tissue revealed an increase in the mean (± SEM) gap-junctional membrane portion on the lateral hepatocyte membranes from 0.049 + 0.003 (n = 66) in controls to 0.061 ± 0.003 (n = 70) in treated rats, while the configuration of the connexons appeared unaltered. This effect could not be reinforced by prior administration of aminophylline: the relative gapjunctional area is similarly extended from 0.054 ± 0.003 (n = 126) in the control group to 0.065 ± 0.004 (n = 105) in the experimental animals. Probing for the time course of the junctional response, a group of rats was sacrificed 3 h after the onset of treatment. Already within this time, the gapjunctional area is augmented from 0.042 ± 0.004 (n = 63) in the concurrent controls to 0.069 ± 0.006 (n = 42) in the treated rats. These statistically significant increases in area may suggest a stimulating effect of cAMP on gap junctions of hepatocytes in vivo.This investigation was supported by grant No. 3.0059.81 (to D.W.S.) from the Fund for Medical Scientific Research (Belgium)     

Glycogen metabolism in adult rat liver parenchymal cell primary cultures.

Parenchymal cells were isolated from adult rat liver with an enzyme perfusion technique. The single-cell suspension, representing 40-50% of the liver's hepatocytes was suspended in medium and maintained in primary culture for up to four days. The cells were found to carry out glycogen synthesis for the first eight hours in culture after which time the accumulated glycogen was gradually degraded. The ability of the liver cell cultures to accumulate glycogen was found to be dependent upon the metabolic state of the animal prior to cell isolation. Cells prepared during the feeding period from animals on the 8+16 feeding schedule had markedly different capacities for glycogen accumulation. Changes in glycogen metabolism were found to be due, in part, to changes in the fraction of cells involved in metabolism at any given time. High concentrations of glucose stimulated the cells to deposit glycogen but the response was reduced the longer the cells were in culture over a 3-day period. This loss of glycogen synthesizing capacity appears to be due to a decrease in glycogen synthetase activity. The activities of pyruvate kinase, hexokinase and aldolase also decrease during the culture period.     

Retinol esterification in cultured rat liver cells.

Retinol esterification was examined in cultured hepatocytes and stellate cells from the rat. Esterification of [3H]retinol was linear for 2 h in both cell types. By increasing the concentration of retinol in the medium, there was a marked increase in retinol esterification in both cell types. The capacity for esterification of retinol was in the same order of magnitude in the two cell types at 3.5 microM-retinol in the medium. This represents a rate of retinol esterification which far exceeds that required to esterify the amount of retinol absorbed in the intestine. It was demonstrated in particulate homogenates from cultured hepatocytes that the esterification of retinol was dependent on acyl-CoA. Addition of 25-hydroxycholesterol or mevalonolactone promoted an increase in cholesterol esterification, whereas retinol esterification was unaffected, suggesting that cholesterol and retinol are esterified by two different enzymes. Some 80% of vitamin A in cultured hepatocytes is retinyl esters, mostly retinyl palmitate. By adding 87 microM-retinol in the medium the cells accumulated 100-fold free retinol and 2.5-3.0-fold retinyl esters within 1 h. When retinol-loaded cells were incubated without retinol, there was a marked decrease especially in free but also in esterified retinol. In the presence of 1 mM-oleic acid in the medium the amount of retinyl oleate was twice that in control cells.     

Changes in activities of some ammonia-metabolizing enzymes in the rat liver and the brain after chronic ethanol administration

The changes in the activities of ammonia-metabolizing enzymes in liver and brain after ethanol intoxication has been investigated in rats. After administration of ethanol 30% (w/v) 6g kg-1 for 4 weeks we found an increase in liver glutamate dehydrogenase and glutaminase activity. In brain tissue the glutaminase activity was significantly higher and glutamate dehydrogenase was significantly lower. Glutamine synthetase activity in liver and brain was practically unchanged. The reasons for these changes in the activities of some ammonia-metabolizing enzymes in liver and brain after ethanol ingestion have been discussed.     

Reduced hepatic alpha-tocopherol content after long-term administration of ethanol to rats

We have studied the effects of long-term administration of ethanol on the distribution and pharmacokinetics of alpha-tocopherol. In rats fed ethanol (35% of total energy) for 5-6 weeks concentration of alpha-tocopherol in whole liver was reduced by 25% as compared to the pair-fed controls (P less than 0.003). This reduction was significant in the parenchymal cells (28%, P less than 0.004), whereas no significant difference was observed for the nonparenchymal cells. Mitochondrial alpha-tocopherol content was reduced by 55% in the ethanol-treated rats as compared to the controls (P less than 0.002), whereas no significant difference was observed in microsomes, light mitochondria or cytosol. The serum levels of alpha-tocopherol showed no significant difference between the groups. When in vivo labeled chylomicron alpha-[3H]tocopherol was injected intravenously to anesthetized rats, we found a significant increase in serum half-life of alpha-tocopherol in the ethanol-treated group as compared to the controls (P less than 0.025). Hepatic alpha-[3H]tocopherol content was similar in the two groups 24 h after injection.     

Gap junction ultrastructure in rat liver parenchymal cells after in vivo ischemia

The ultrastructure of gap junctions between rat liver parenchymal cells has been studied after in vivo ischemia, with and without subsequent blood reflow. Freeze fracture replicas were analysed by electron microscopic observation, optical diffraction and morphometric analysis. In control specimens gap junction connexons were widely dispersed and arranged in nearly random fashion over nearly the whole junctional area, with only minute spots of hexagonal connexon arrangement. An ischemic period of 30 min, from which the vast majority of cells are capable of recovery after restoration of the blood supply, usually entails only a slight enlargement of the areas of hexagonally arranged connexons. After 120 min of ischemia without reflow, which results in necrosis of most parenchymal cells, all gap junctions showed a completely hexagonal arrangement of connexons. The numerical density of connexons after 30 and 120 min of ischemia without reflow was significantly higher than in controls, whereas after 30 min of ischemia followed by 2 h of reflow the numerical density had returned to control levels. A fully hexagonal arrangement of gap junction connexons, as occurs after longer periods of ischemia, seems to be related to irreversible cell damage and presumably to metabolic uncoupling of cells. This was preceded by an increase in the numerical density of connexons, which is probably a reversible phenomenon.     

Growth factors and nonparenchymal cell conditioned media induce mitogenic responses in stable long-term adult rat hepatocyte cultures

Most prior studies have characterized hepatocyte proliferative responses in culture systems that do not express a stable differentiated phenotype. We investigated the DNA synthetic response of long-term stable hepatocyte cultures to growth factor stimulation as well as conditioning with nonparenchymal cells (NPCs). Primary rat hepatocytes were cultured on a single layer of collagen (h/C) or Matrigel (h/M), or in a collagen sandwich (C/h/C) or collagen-Matrigel sandwich (M/h/C). Hepatocytes were cultured for 7 days to allow phenotypic stabilization before growth factor addition, except for h/C cultures, which are unstable, where growth factors were added 1 day after seeding. Culture medium was supplemented with a mixture of hepatocyte, epidermal, and vascular endothelial growth factors and interleukin-6, either directly or after conditioning with NPCs for 24 h. Growth factors alone induced hepatocyte DNA synthesis, as measured via [3H]thymidine uptake, in the h/C, C/h/C, and M/h/C configurations. h/M exhibited very low levels of DNA synthesis. In the C/h/C and M/h/C configurations, the greatest stimulation was obtained using NPC-conditioned growth factors. This response was sustained for several days and without decreasing albumin or urea synthesis. These results suggest that hepatocyte mitogens and NPC-derived factors can stimulate DNA synthesis in stable and differentiated hepatocyte cultures.