Liver Perfusion: an in Vitro Technique for the Study of Intracellular Protein Turnover and Its Regulation in Vivo

Mortimore, Glenn E.; Surmacz, C A

doi:10.1079/pns19840039

Cited by 17 publications

(11 citation statements)

References 44 publications

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“…The method used in this study has been shown to give a valid estimate of the rate of protein degradation prevailing in the liver at the time when the perfusion is started [11]. Also in this study, the fractional degradation rates in the livers of the control rats were well in accord with previously reported values [21].…”

Section: Discussionsupporting

confidence: 88%

“…The rate of protein degradation was measured as accumulation of valine into the perfusate, which contained 18 /aM-cycloheximide to prevent the re-incorporation of valine. It has been shown previously [11] that during the 15 min period the release of valine into the perfusate is linear and the valine originates from the lysosomes formed in the liver before the perfusion was started. Because valine is not appreciably metabolized in liver [11], its accumulation into the perfusate gives a valid estimate of the rate of protein degradation prevailing in vivo at the moment the perfusion was started.…”

Section: Protein Degradationmentioning

confidence: 96%

“…It has been shown previously [11] that during the 15 min period the release of valine into the perfusate is linear and the valine originates from the lysosomes formed in the liver before the perfusion was started. Because valine is not appreciably metabolized in liver [11], its accumulation into the perfusate gives a valid estimate of the rate of protein degradation prevailing in vivo at the moment the perfusion was started. The rate of protein degradation was measured at four time points (07:00, 11:00, 17:00 and 23:00 h).…”

Section: Protein Degradationmentioning

confidence: 96%

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Inhibition of proteolysis in the liver by chronic ethanol feeding

Pösö

Hirsimäki²

1991

Biochemical Journal

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Effects of chronic ethanol feeding on the volume density of lysosomes, the rate of protein degradation and the amount of protein were studied in livers perfused in situ at 07:00, 11:00, 17:00 and 23:00 h. Ethanol was given to the rats in drinking water for either 3 or 8-10 weeks. During week 3 of treatment and onwards, the average daily consumption of ethanol was 12.3 +0.3 g/kg body wt. Morphometric analysis revealed that the volume density of autophagosomes and autolysosomes was lower in the ethanol-fed rats than in the controls. When compared with age-matched controls, the rate of proteolysis, measured as release of valine, was 33 % and 26 % less in the ethanol-fed rats after treatment for 3 and 8-10 weeks respectively. The difference between the two groups was most pronounced at 07:00 and 11:00 h. Protein content of the liver increased significantly after the longer ethanol treatment. According to these results, chronic ethanol feeding inhibits proteolysis in the liver by preventing the sequestration of protein into lysosomes. INTRODUCTIONChronic ethanol ingestion disturbs the finely adjusted balance of protein turnover in liver and leads to accumulation of hepatocellular protein (for review, see [1]). Part of this accumulation can be explained by an increase in the amount of fatty-acid-binding protein [2] and by plasma proteins which in the presence of ethanol are retained in the liver [3]. It can, however, be calculated that the amount of plasma proteins together with the fatty-acid-binding protein account for less than 30 % of the total protein accumulation during chronic ethanol feeding [2,3], which leaves the nature of the major contributor(s) unknown.The ethanol-induced increase in hepatic protein mass can be caused by a change in the rate of either protein synthesis or degradation. Despite extensive studies, effects of chronic ethanol feeding on protein synthesis in vivo are far from clear, and the reported results range from stimulation to inhibition (for review, see [1]). Ethanol has been shown to inhibit the rate of intracellular protein degradation in perfused rat liver [4], whereas the chronic effects of ethanol have only been calculated from the changes in the amount of protein and the rate of protein synthesis [5,6].The bulk of long-lived protein in the liver is degraded intralysosomally [7]. The process has been shown to be more sensitive to nutritional factors than is protein synthesis, and there are several lines of evidence which indicate that protein degradation rather than synthesis is the dominant site for regulating the cytoplasmic protein content of the hepatocyte [7,8]. It was shown previously that in perfused rat liver ethanol inhibits the formation of autophagosomes [4], the first step of lysosomal proteolysis, whereas the effect of chronic ethanol feeding on autophagocytosis is not known. The purpose of the present study was to measure the rate of protein degradation and volume densities of lysosomal components in livers of rats that were chronically fed on an ethanol-containing die...

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Section: Discussionsupporting

confidence: 88%

Section: Protein Degradationmentioning

confidence: 96%

Section: Protein Degradationmentioning

confidence: 96%

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Inhibition of proteolysis in the liver by chronic ethanol feeding

Pösö

Hirsimäki²

1991

Biochemical Journal

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“…These results differ from those of M0rland & Bessesen (1977), who obtained no effect of ethanol in isolated hepatocytes incubated in the absence of amino acids. The reason for this discrepancy is not clear, but it should be pointed out that protein degradation in isolated hepatocytes (Seglen et al, 1980;Ballard & Gunn, 1982) is not as responsive to physiological regulation as it is in the perfused liver (Mortimore & Pbso, 1984;Mortimore & Surmacz, 1984 the liver and leads to the accumulation of amino acids (Krebs et al, 1973), which are recognized as the primary inhibitors of hepatic protein degradation (Poso et al, 1982b). Fig.…”

Section: Results and Discussion Protein Degradationmentioning

confidence: 99%

“…As an experimental model we chose isolated perfused rat liver, which has been shown to be especially well suited for studies on protein metabolism (Mortimore & Surmacz, 1984).…”

Section: Introductionmentioning

confidence: 99%

Inhibition of intracellular protein degradation by ethanol in perfused rat liver

Pösö¹,

Surmacz²,

Mortimore³

1987

Biochemical Journal

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Ethanol (50 mM) inhibited proteolysis in the perfused rat liver during stringent amino acid deprivation and also in the presence of normal and 10 times normal concentrations of plasma amino acids. The concentration-response curve of ethanol reached a plateau after 5 mm in both the presence and the absence of normal plasma amino acids, suggesting inhibition by oxidation products of ethanol. Intracellular glutamine, tyrosine and proline increased in concentration with ethanol, but the increases were too small to explain the observed inhibition of proteolysis. The uptake of 1251-asialofetuin was slightly decreased and the output of ammonia increased in the presence of ethanol. These, together with a significant suppression of basal proteolysis in the presence of amino acids, suggest that lysosomal function was directly affected. Electron-microscopic examination of lysosomal components showed that the aggregate volume of autophagosomes (initial vacuoles) were significantly smaller in livers perfused with ethanol than in controls. However, the equivalent volume of autolysosomes (degradative vacuoles) was the same in both groups. According to these results, ethanol inhibits protein degradation in the liver by two discrete mechanisms: one decreasing the formation of autophagic vacuoles and the other involving lysosomotropic inhibition, possibly via ammonia.

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Contribution of gene expression to metabolic fluxes in hypermetabolic livers induced through burn injury and cecal ligation and puncture in rats

Banta

Vemula

Yokoyama

et al. 2006

Biotech & Bioengineering

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Severe injury activates many stress-related and inflammatory pathways that can lead to a systemic hypermetabolic state. Prior studies using perfused hypermetabolic rat livers have identified intrinsic metabolic flux changes that were not dependent upon the continual presence of elevated stress hormones and substrate loads. We investigated the hypothesis that such changes may be due to persistent alterations in gene expression. A systemic hypermetabolic response was induced in rats by applying a moderate burn injury followed 2 days later by cecum ligation and puncture (CLP) to produce sepsis. Control animals received a sham-burn followed by CLP, or a sham-burn followed by sham-CLP. Two days after CLP, livers were analyzed for gene expression changes using DNA microarrays and for metabolism alterations by ex vivo perfusion coupled with Metabolic Flux Analysis. Burn injury prior to CLP increased fluxes while decreases in gene expression levels were observed. Conversely, CLP alone significantly increased metabolic gene expression, but decreased many of the corresponding metabolic fluxes. Burn injury combined with CLP led to the most dramatic changes, where concurrent changes in fluxes and gene expression levels occurred in about 1/3 of the reactions. The data are consistent with the notion that in this model, burn injury prior to CLP increased fluxes through post-translational mechanisms with little contribution of gene expression, while CLP treatment up-regulated the metabolic machinery by transcriptional mechanisms. Overall, these data show that mRNA changes measured at a single time point by DNA microarray analysis do not reliably predict metabolic flux changes in perfused livers.

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Liver Perfusion: an in Vitro Technique for the Study of Intracellular Protein Turnover and Its Regulation in Vivo

Cited by 17 publications

References 44 publications

Inhibition of proteolysis in the liver by chronic ethanol feeding

Inhibition of proteolysis in the liver by chronic ethanol feeding

Inhibition of intracellular protein degradation by ethanol in perfused rat liver

Contribution of gene expression to metabolic fluxes in hypermetabolic livers induced through burn injury and cecal ligation and puncture in rats

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