Comparison in genetically hyperlipoproteinemic and normal rats of the captation and incorporation of isotopic oleic acid and glycerol at high concentration by the perfused liver

Perfusions of isolated livers from genetically hyperlipoproteinemic Zucker fa/fa and normolipemic Zucker Fa/- rats are performed with loads of ]9,10-3H2] oleic acid and [1-14C] glycerol. The hepatic acylglycerols anabolism from these precursors is higher in the fa/fa rat than in the control Fa/- rats. Synthesis by esterification (of oleic acid) is more increased than de novo synthesis (from glycerol). The increase in lipid anabolism is due to an augmentation of the hepatic cellular mass, but this anabolism is not regulated in the same way than in the normal rat.     

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 of triglyceride and phospholipid synthesis in isolated Wistar rat liver perfused with high quantities of oleic acid and glycerol

In a recirculating system, [9,10(-3)H2] oleic acid (346 mumol) and [1-14C] glycerol (115 mumol) are perfused into livers of 18-h fasting Wistar rats. These precursors are incorporated in same amounts into triacylglycerols, and in amounts growing up with the duration of the experiment (5 to 120 min). Their incorporation is slight into phospholipids. However, during the experiment, the increase of 3H/14C ratio of every acylglycerols shows that more lipids are synthetized in the acylation way than in the de novo way. The only synthesis of phospholipids, studied in the two ways, seems to be regulated, unlike the one of triacylglycerols in those experimental conditions.     

Comparison of metabolism of free fatty acid by isolated perfused livers from male and female rats.

Livers from normal, fed male and female rats were perfused with different amounts of [1-14C]oleate under steady state conditions, and the rates of uptake and utilization of free fatty acid (FFA) were measured. The uptake of FFA by livers from either male or female rats was proportional to the concentration of FFA in the medium. The rate of uptake of FFA, per g of liver, by livers from female rats exceeded that of the males for the same amount of FFA infused. The incorporation by the liver of exogenous oleic acid into triglyceride, phospholipid, and oxidation products was proportional to the uptake of FFA. Livers from female rats incorporated more oleate into triglyceride (TG) and less into phospholipid (PL) and oxidation products than did livers from male animals. Livers from female rats secreted more TG than did livers from male animals when infused with equal quantities of oleate. The incorporation of endogenous fatty acid into TG of the perfusate was inhibite) by exogenous oleate. At low concentrations of perfusate FFA, however, endogenous fatty acids contributed substantially to the increased output of TG by livers from female animals. Production of 14CO2 and radioactive ketone bodies increased with increasing uptake of FFA. The partition of oleate between oxidative pathways (CO2 production and ketogenesis) was modified by the availability of the fatty acid substrate with livers from either sex. The percent incorporation of radioactivity into CO2 reached a maximum, whereas incorporation into ketone bodies continued to increase. The output of ketone bodies was dependent on the uptake of FFA, and output by livers from female animals was less than by livers from male rats. The increase in rate of ketogenesis was dependent on the influx of exogenous FFA, while ketogenesis from endogenous sources remained relatively stable. The output of glucose by the liver increased with the uptake of FFA, but no difference due to sex was observed. The output of urea by livers from male rats was unaffected by oleate, while the output of urea by livers from females decreased as the uptake of FFA increased. A major conclusion to be derived from this work is that oleate is not metabolized identically by livers from the two sexes, but rather, per gram of liver, livers from female rats take up and esterify more fatty acid to TG and oxidize less than do livers from male animals; livers from female animals synthesize and secrete more triglyceride than do livers from male animals when provided with equal quantities of free fatty acid.     

Formation of free acetate by isolated perfused livers from normal, starved and diabetic rats

Isolated rat livers perfused in an open system exhibited a continous net release of free acetate. Upon intraportal infusion of hexanoate the net release of total ketone bodies and of free acetate increased significantly in livers from fed and 48 hours starved rats. The ratio ketone body production/acetate production during infusion of hexanoate was similar with livers from fed and starved rats. Livers from diabetic rats, however, did not only exhibit a higher rate of ketone body and acetate production, but also a significant decrease of the ratio ketone body production/acetate production. Intraportal infusion of oleate led also to an enhanced release of free acetate. An examination of the activities of 5 enzymes involved in ketone body and acetate metabolism showed no correlation with the higher rate of acetate production by diabetic livers.     

Fatty acid metabolism and lipid secretion by perfused livers from rats fed laboratory stock and sucrose-rich diets

To assess the possible role of altered hepatic processing of free fatty acids in dietary sucrose-induced accumulation of triglyceride in the liver and blood plasma, livers from rats fed commercial laboratory stock and high sucrose diets were perfused both with and without oleic acid substrate. Consumption of the sucrose diet exerted a multiplicity of effects on oleic acid metabolism, characterized by decreased conversion to both ketone bodies and carbon dioxide, increased esterification into liver triglyceride, and increased secretion in triglyceride-rich lipoproteins. During the infusion of oleic acid, livers from sucrose-fed rats also exhibited decreased ketogenesis, and increased secretion of triglyceride from endogenous sources. Since oleic acid uptake from the perfusion medium was identical in both groups, the observed effects of sucrose feeding are ascribed to altered rates of intracellular metabolic processes. Mass and radiochemical analyses of perfusate ketone bodies and triglycerides were indicative of greater mobilization of triglycerides from hepatocellular lipid droplets in the livers from sucrose-fed rats. These livers contained more triglyceride and secreted more triglyceride even in the absence of infused oleic acid. In summary, the sucrose-rich diet increased the esterification:oxidation ratio of intracellular free fatty acids derived from both the circulation and endogenous sources within the hepatocyte. In response, secretion of triglyceride-rich lipoproteins by the liver and deposition of triglyceride within the liver were promoted. It is concluded that alterations in the processing of free fatty acids by the liver contribute significantly to the liver and plasma triglyceride accumulation following sucrose consumption.     

Metabolism of glutamine and glutamic acid by isolated perfused kidneys of normal and acidotic rats

1. When isolated kidneys from fed rats were perfused with glutamine the rate of ammonia release at pH7.4 (110–360μmol/h per g dry wt.) was one to two times that of glutamine removal. Glucose formation from 5mm-glutamine was 16μmol/h per g. If kidneys were perfused with glutamine at pH7.1 (10–13mm-sodium bicarbonate) there was no increase in glutamine removal or in the formation of ammonia or glucose. 2. When isolated kidneys from fed rats were perfused with glutamate at pH7.4, glucose formation was 59μmol/h per g, glutamine formation was 182μmol/h per g and ammonia release was negligible. At pH7.1 glutamine synthesis was inhibited and formation of ammonia and glucose were increased. 3. In perfused kidneys from acidotic rats, which had received 1.5% (w/v) NH₄Cl to drink for 7–10 days, gluconeogenesis from glutamine was enhanced (101μmol/h per g). Glutamine removal and ammonia formation were also increased, compared with the rates in perfused kidney from normal rats. The extra glutamine consumed was equivalent to the extra glucose formed. 4. When the kidney from the 7–10-day-acidotic rat was perfused with glutamate gluconeogenesis was increased (113μmol/h per g). Synthesis of glutamine was decreased, and ammonia release was approximately equal to the rate of glutamate removal. 5. The time-course of these metabolic alterations was investigated after the rapid induction of acidosis by infusion of 0.25m-HCl into the right side of the heart. The increase in gluconeogenesis from glutamine developed gradually over several hours. When kidneys from 6h-acidotic rats were perfused with glutamate, formation of glucose and glutamine were both rapid. 6. In acidotic rat kidneys perfused with glutamine, tissue concentrations of glutamate and glucose 6-phosphate were increased compared with those in control perfused kidneys from non-acidotic rats. 7. The results are discussed in terms of control of the renal metabolism of glutamine. In particular, it is suggested that in acidotic rats glucose formation is the major fate of the carbon of the extra glutamine utilized by the kidney, and that inhibition of glutamine synthetase could contribute to the increase in intracellular ammonia concentration in the kidney.     

Hormone secretion and glucose metabolism in islets of Langerhans of the isolated perfused pancreas from normal and streptozotocin diabetic rats.

The glucose responsiveness of alpha- and beta-cells of normal as well as untreated and insulin-treated streptozotocin diabetic rats was tested in the extracorporeal perfusion system. Also assessed was the possible in vitro effect of added insulin on the glucose sensitivity of islets from untreated diabetic animals. Insulin and glucose responsiveness of the two cell types. The rate of glucose entry islet tissue was estimated, and the effect of glucose on the tissue supply of ATP and lactate and the cyclic 3':5'-AMP level of islets was measured under the above in vitro conditions. It was demonstrated that beta-cells are more accessible to glucose than alpha-cells, that glucose entry into islet cells is not significantly modified by insulin and that glucose had no effect on ATP, lactate and cyclic 3':5'-AMP levels of islet tissue under any of the conditions investigated. High insulin in vitro elevated ATP levels of alpha-cell islets independent of extracellular glucose. Glucose caused insulin release from normal but not from diabetic islets and rapidly and efficiently suppressed stimulated glucagon secretion of the pancreas from normal and insulin treated diabetic rats. Glucose was less effective in inhibiting stimulated glucagon secretion by the pancreas from untreated diabetic rats whether insulin was added to the perfusion media or not. Therefore, profound differences of glucose responsiveness of alpha-cells fail to manifest themselves in alterations of basic parameters of glucose and energy metabolism in contrast to what had been postulated in the literature. It is however, apparent that the glucose responsiveness of alpha-cells is modified by insuling by an as yet undefined mechanism.     

Regulation of 3-hydroxybutyrate formation and secretion of very-low-density-lipoprotein triacylglycerol by perfused livers from fed and starved rats.

Acetoacetate was the sole ketone body formed when livers from starved rats were perfused with minimal concentrations of non-esterified fatty acid. Absence of 3-hydroxybutyrate was related to a low substrate potential, which caused a more oxidized redox state and a decreased [ATP]/[ADP] ratio. Only under conditions of comparable non-esterified fatty acid uptake was lipoprotein triacylglycerol production inversely related to ketogenesis.     

Correlation between lactate output and insulin secretion of the isolated perfused rat pancreas under the influence of high concentrations of phenformin]

On the isolated perfused rat pancreas phenformin at high concentrations (10 mg/1, 50 mg/1 and 100 mg/1) provokes an increase of the insulin and lactate output in the effluent liquid. In no case is glucagon secretion modified by this substance. There exists a statistically significant correlations between the increase in insulin output and the increase in lactate output induced by phenformin.     

Insulin stimulates triacylglycerol secretion by perfused livers from fed rats but inhibits it in livers from fasted or insulin-deficient rats implications for the relationship between hyperinsulinaemia and hypertriglyceridaemia.

We determined whether the direction of the acute effect of insulin on hepatic triacylglycerol secretion is dependent on the prior physiological state or on the in vitro experimental system used. The effect of insulin on triacylglycerol secretion was studied using perfused livers isolated from rats under three metabolic conditions: fed normo-insulinaemic, 24-h fasted and fed, streptozotocin-diabetic (insulin-deficient). Insulin acutely activated triacylglycerol secretion (by 43%) in organs from fed, normo-insulinaemic animals, whereas it inhibited triacylglycerol secretion in livers isolated from fasted or insulin-deficient rats (by 30 and 33%, respectively). By contrast, in 24-h-cultured hepatocytes insulin invariably acutely inhibited triacylglycerol secretion irrespective of the metabolic state of the donor animals. It is concluded that the use of perfused livers enables the observation of a switch in the direction of insulin action on hepatic triacylglycerol secretion from stimulatory, in the normo-insulinaemic state, to inhibitory in the fasting or insulin-deficient state. The possible implications of this switch for the relationship between hyperinsulinaemia, increased hepatic very-low-density lipoprotein-triacylglycerol secretion and hypertriglyceridaemia observed in vivo are discussed.     

Characteristics of synthesized RNA in the isolated and perfused rat liver

The incorporation of 3H-orotic acid into nuclear and microsomal RNA from isolated perfused rat liver has been studied. The specific radioactivity of nuclear RNA indicates that the efficiency for RNA synthesis in the perfused liver is similar to that of the liver 'in vivo'. In contrast, the microsomal RNA specific radioactivity is well below that observed 'in vivo'. This may indicate a slower transport of the labelled RNA from the nucleus to the cytoplasm. Labelling pattern of total nuclear RNA, nuclear poly(A) containing RNA and microsomal RNA appear to be in line with these assumptions.     

Metabolism of 5-hydroxytryptophan in isolated perfused liver from normal and x-irradiated rats

Summary Metabolism of¹⁴C-5-OH-tryptophan was studied in isolated perfused liver from normal and whole body irradiated rats (7 to 8 days after 1000 R). Metabolism of 5-OH-tryptophan is slow compared to that of 5-OH-tryptamine, and the metabolites isolated were mainly conjugates of 5-OH-tryptamine and 5-OH-indole acetic acid. Irradiation does not diminish significantly the conversion of 5-OH-tryptophan to 5-OH-tryptamine, rather an increased formation of 5-OH-indole acetic acid and of conjugates could be observed in irradiated liver.

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Zusammenfassung Der Stoffwechsel von¹⁴-C-5-OH-Tryptophan wurde in isoliert perfundierter Leber normaler und bestrahlter Ratten untersucht (7 bis 8 Tage nach 1000 R). Der Abbau von 5-OH-Tryptophan ist gering im Vergleich zu dem von 5-OH-Tryptamin. Die beobachteten Metaboliten waren im wesentlichen die Konjugate von 5-OH-Tryptamin und 5-OH-Indolacetat. Bestrahlung vermindert nicht die Umwandlung von 5-OH-Tryptophan zu 5-OH-Tryptamin und führt sogar zu einer vermehrten Bildung von 5-OH-Indolessigsäure und ihrer Konjugate.

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This is publication No. 706 of the Euratom Biology Division Contract Euratom-C.E.N./ S.C.K. No. 078-69-1 BIAC.     

Effects of oleic acid on the biosynthesis of lipoprotein apoproteins and distribution into the very-low-density lipoprotein by the isolated perfused rat liver.

The effects of oleic acid on the biosynthesis and secretion of VLDL (very-low-density-lipoprotein) apoproteins and lipids were investigated in isolated perfused rat liver. Protein synthesis was measured by the incorporation of L-[4,5-3H]leucine into the VLDL apoproteins (d less than 1.006) and into apolipoproteins of the whole perfusate (d less than 1.21). Oleate did not affect incorporation of [3H]leucine into total-perfusate or hepatic protein. The infusion of oleate, however, increased the mass and radioactivity of the VLDL apoprotein in proportion to the concentration of oleate infused. Uptake of oleate was similar with livers from fed or fasted animals. Fasting itself (24 h) decreased the net secretion and incorporation of [3H]leucine into total VLDL apoprotein and decreased the output of VLDL protein by the liver. A linear relationship existed between the output of VLDL triacylglycerol (mumol/h per g of liver) and secretion and/or synthesis of VLDL protein. Net output of VLDL cholesterol and phospholipid also increased linearly with VLDL-triacylglycerol output. Oleate stimulated incorporation of [3H]leucine into VLDL apo (apolipoprotein) E and apo C by livers from fed animals, and into VLDL apo Bh, B1, E and C by livers from fasted rats. The incorporation of [3H]leucine into individual apolipoproteins of the total perfusate lipoprotein (d less than 1.210 ultracentrifugal fraction) was not changed significantly by oleate during perfusion of livers from fed rats, suggesting that the synthesis de novo of each apolipoprotein was not stimulated by oleate. This is in contrast with that observed with livers from fasted rats, in which the synthesis of the total-perfusate lipoprotein (d less than 1.210 fraction) apo B, E and C was apparently stimulated by oleate. The observations with livers from fed rats suggest redistribution of radioactive apolipoproteins to the VLDL during or after the process of secretion, rather than an increase of apoprotein synthesis de novo. It appears, however, that the biosynthesis of apo B1, Bh, E and C was stimulated by oleic acid in livers from fasted rats. Since the incorporations of [3H]leucine into the VLDL and total-perfusate apolipoproteins were increased in fasted-rat liver when the fatty acid was infused, part of the apparent stimulated synthesis of the VLDL apoprotein may be in response to the increased formation and secretion of VLDL lipid.