Secretion into the circulating medium of lipids synthetized by isolated Wistar rat liver perfused with high concentrations of oleic acid and glycerol

In previously 18-h fasting Wistar rats, the liver is isolated and perfused with [9, 10(-3)H2] oleic acid (346 mumol and [1-14C] glycerol (115 mumol). Then, in a circulating medium, the secretion of triacylglycerols -- synthetized de novo and by esterification of exogenous oleic acid -- and VLDL is inhibited. On the other hand, the secretion of phospholipids is getting away that regulatory process.     

Comparison in genetically hyperlipoproteinemic and normal rats of the secretion of lipids synthesized by isolated livers perfused with isotopic oleic acid and glycerol at high concentrations

After perfusing isolated livers of Zucker fa/fa ad Fa/- rats with loads of [9,10-3H2] oleic acid (346 mumol) and [1-14C] glycerol (115 mumol), glycerol inhibited the hepatic secretion of triacylglycerol and phospholipids in the two groups of rats. However, the amount of acylglycerols synthetized from these exogenous substrates is slightly higher in the obese rats than in the normal rats. These results suggest that glycerol, present in high amounts in blood of fa/fa rats, failed to regulate triacylglycerols and phospholipids secretion.     

Serum antitrypsin synthesis by the isolated perfused rat liver.

The isolated perfused rat liver synthesizes antitrypsin activity in a linear fashion as long as 24 hr. The rate of synthesis may be ten times the rate necessary for physiologic levels in vivo. These data are compatible with the liver as the principal site of synthesis of serum antitryptic activity in the rat. The antitrypsin activity of the rat perfusate resembles that of normal human plasma in liability to heat (56 degrees) and to acid (below pH 6) and in apparent molecular size (elution from Sephadex G-200).     

Uptake and incorporation of 3H-orotic acid in isolated perfused rat liver

3H-orotic acid incorporation into RNA and the level of RNA polymerase activity in isolated rat liver perfused for 5 hrs were investigated. In spite of a dramatic decrease in 3H-orotic acid uptake by liver cells during perfusion, a constant rate of RNA synthesis was observed. Moreover, RNA polymerase I and II activities were not affected by a 5-hr perfusion. It is suggested that isolated perfused rat liver can be used to study direct effects of hormones and drugs on RNA synthesis.     

Contributions of fatty acid and sterol synthesis to triglyceride and cholesterol secretion by the perfused rat liver in genetic hyperlipemia and obesity

The relative importance of fatty acid synthesis in triglyceride secretion by perfused livers from lean (normal control) and obese Zucker rats was investigated. Livers from fed animals were perfused in a recirculating system with tritiated water and a constant infusion of oleic acid. Triglyceride secretion was 5 times greater and cholesterol secretion was 35% greater in the obese rat livers. The very-low-density lipoprotein hypersecreted by perfused livers from obese rats contained more apolipoprotein B and exhibited an increased B-48/B-100 ratio. Apo-B was also elevated in the hypertriglyceridemic plasma of obese rats in both fed and fasting states. The very-low-density lipoprotein isolated therefrom was likewise characterized by an increased B-48/B-100 ratio. Ketogenesis was depressed 40% in the obese rat livers and increased hepatic malonyl-CoA was implicated in this alteration. The de novo synthesis and secretion of newly synthesized cholesterol was moderately increased in the perfused livers from obese rats. Tritium incorporation into fatty acids was 15 times greater in the obese genotype. Most of the synthesized fatty acids remained in the liver and were recovered after perfusion in triglyceride and phospholipids. Newly synthesized fatty acids accounted for only 3 and 15% of the triglyceride secreted by the lean and obese rat livers, respectively. A large portion of the secreted triglyceride fatty acids was derived from endogenous liver lipids. When the turnover of newly synthesized fatty acids in these pools was considered, the contribution of de novo fatty acid synthesis to triglyceride secretion was estimated to be 9% in the lean and 44% in the obese rat livers. Therefore, the altered partition of free fatty acids (Fukuda, N., Azain, M. J., and Ontko, J. A. (1982) J. Biol. Chem. 257, 14066-14072) and increased fatty acid synthesis are both major determinants of the hypersecretion of triglyceride-rich lipoproteins by the liver in the genetically obese Zucker rat.     

Futile cycles in isolated perfused rat liver and in isolated rat liver parenchymal cells.

Isolated livers from fed rats were perfused with a medium containing glucose labeled uniformly with ¹⁴C and specifically with ³H. There was considerable formation of glucose from endogenous sources but simultaneously uptake of about half of the ¹⁴C in glucose. After 2 hours the ${}^3\text{H}{}^{14}\text{C}$ ratios in perfusate glucose decreased by 55–60% with (2-³H, U-¹⁴C), 40–50% with (5-³H, U-¹⁴C), 25–30% with (3-³H or 4-³H, U-¹⁴C) and by 10–15% with (6-³H, U-¹⁴C) glucose. Qualitatively comparable patterns were obtained with rat hepatocytes. These results demonstrate recycling of carbon between glucose and pyruvate. Superimposed upon this there is an extensive futile cycle between glucose and glucose 6-P. There is also futile cycling between fructose 6-P and fructose 1,6 P₂ and to a small extent between phosphoenol pyruvate and pyruvate.     

Electron microscopic and biochemical study of lipoprotein synthesis in the isolated perfused rat liver

The isolated perfused rat liver was used to study the 300-800 A electron-opaque bodies which had previously been described in the liver cell Golgi apparatus, smooth endoplasmic reticulum, and space of Disse. When the perfusion medium was enriched with linoleate, the number and electron opacity of these particles increased markedly. Sequential biopsies showed that they appeared first in the smooth surfaced terminal ends of the rough reticulum, the smooth endoplasmic reticulum proper, and the Golgi apparatus and later in the space of Disse. After 60 min of perfusion, particles of the same size and shape as those in the liver cells could be isolated in large numbers from the d < 1.006 fraction of the perfusate. Control livers perfused with an identical medium but without linoleate did not show these changes. Puromycin markedly depressed the production of 300-800 A particles by livers perfused with an oleate-rich medium; however, it did not interfere with the formation of large cytoplasmic droplets of neutral fat. In keeping with these findings, puromycin blocked the incorporation of oleate-(14)C into lipoprotein triglyceride isolated from the perfusate, but did not interfere with the appearance of the labeled fatty acid in tissue triglyceride. Puromycin also blocked the incorporation of leucine-(3)H into both tissue protein and perfusate lipoprotein. We concluded that the 300-800 A particles observed are, in all likelihood, very low density lipoproteins and that their formation is blocked by puromycin, presumably through interference with the synthesis of their apoprotein.     

Metabolism of triglyceride fatty acid by the perfused rat heart

1. Chyle lipids, labelled with (14)C, are taken up and oxidized by the isolated perfused rat heart. 2. In recirculatory perfusions, when chyle lipids are the sole exogenous energy source, about 24% of the total oxygen uptake is accounted for by their oxidation. This proportion is not changed by starvation of the rats for 48hr. and falls when an external work load is imposed on the left ventricle. 3. With albumin in the perfusion medium, the rate of (14)CO(2) output is reduced by half and there is a rise in the proportion of (14)C-labelled free fatty acids in the medium. 4. Clearing-factor lipase appears in the perfusion medium when chyle lipids are perfused through the heart. In the absence of albumin, the activity of the medium enzyme is low and only a small proportion of the (14)CO(2) output can be accounted for by the oxidation of free fatty acids released by it. In the presence of albumin, the enzyme is more active in the medium. 5. When a substantial proportion of the total clearing-factor lipase is removed from the heart by a prior perfusion with heparin, (14)C-labelled chyle lipid perfused subsequently is oxidized at only half the normal rate.     

Comparison of isolated rat hepatocytes prepared from liver slices and perfused liver with special reference to protein metabolism

Two different methods were used to prepare isolated hepatocytes from the same rat livers. The cells prepared by a slice technique (HS cells) were compared with those prepared by a collagenase perfusion technique (HP cells). The cell yield was higher by the latter technique, HP cells had higher viability, content of RNA and protein, and initial oxygen consumption than HS cells. The rate of protein degradation and protein synthesis as well as the fractional incorporation of labeled valine into medium protein was also higher in HP cells. HP cells had a lower leakage of ALAT and LDH than HS cells. Some differences, such as leakage of ASAT and oxygen consumption, became reduced or were abolished with time during subsequent cell incubation. On the other hand differences with respect to cell viability, leakage of ALAT and LDH, fractional incorporation of labeled valine into medium proteins, and protein synthetic rate all became more marked with time.