Lactate production in the perfused rat liver

1. In aerobic conditions the isolated perfused liver from well-fed rats rapidly formed lactate from endogenous glycogen until the lactate concentration in the perfusion medium reached about 2mm (i.e. the concentration of lactate in blood in vivo) and then production ceased. Pyruvate was formed in proportion to the lactate, the [lactate]/[pyruvate] ratio remaining between 8 and 15. 2. The addition of 5mm- or 10mm-glucose did not affect lactate production, but 20mm- and 40mm-glucose greatly increased lactate production. This effect of high glucose concentration can be accounted for by the activity of glucokinase. 3. The perfused liver released glucose into the medium until the concentration was about 6mm. When 5mm- or 10mm-glucose was added to the medium much less glucose was released. 4. At high glucose concentrations (40mm) more glucose was taken up than lactate and pyruvate were produced; the excess of glucose was probably converted into glycogen. 5. In anaerobic conditions, livers of well-fed rats produced lactate at relatively high rates (2.5mumol/min per g wet wt.). Glucose was also rapidly released, at an initial rate of 3.2mumol/min per g wet wt. Both lactate and glucose production ceased when the liver glycogen was depleted. 6. Addition of 20mm-glucose increased the rate of anaerobic production of lactate. 7. d-Fructose also increased anaerobic production of lactate. In the presence of 20mm-fructose some glucose was formed anaerobically from fructose. 8. In the perfused liver from starved rats the rate of lactate formation was very low and the increase after addition of glucose and fructose was slight. 9. The glycolytic capacity of the liver from well-fed rats is equivalent to its capacity for fatty acid synthesis and it is pointed out that hepatic glycolysis (producing acetyl-CoA in aerobic conditions) is not primarily an energy-providing process but part of the mechanism converting carbohydrate into fat.     

A rise in cyclic AMP with a lack of glycogenolysis by secretin in the isolated perfused rat liver and its inhibition by epinephrine

The effects of secretin on glucose output and cyclic AMP from the isolated perfused rat liver were investigated. Secretin 0.1 U/ml increased cyclic AMP in the effluent without an increase in glucose output. Glucose output induced by epinephrine 10(-8)M was not affected by secretin 0.1 U/ml administered simultaneously, whereas the increase in cyclic AMP produced by secretin 0.1 U/ml was inhibited by epinephrine 10(-8)M. The increase in cyclic AMP produced by glucagon 10(-10)M was not affected by epinephrine 10(-8)M. These results suggest that secretin does not affect glycogenolysis in the liver and secretin activates adenylate cyclase through a different receptor from glucagon in the liver.     

Regulation of production and release of lipoprotein by the perfused rat liver

The relationship between protein and triglyceride release into d < 1.007 lipoprotein was studied in the isolated perfused rat liver. Livers were perfused with a medium either high or low in linoleate content. Perfusion with the linoleate-rich medium resulted in a marked increase in the net release of both d < 1.007 lipoprotein triglyceride and lipoprotein protein, and caused a significant increase in amino acid incorporation into the protein moiety. Amino acid incorporation into d 1.008-1.21 protein was not affected by fatty acid concentration, while incorporation into whole perfusate and tissue proteins was depressed by a perfusate high in fatty acid content. Electron microscopic studies demonstrated that the livers with the higher rate of triglyceride release also produced a greater number of lipoprotein particles. The particles they released were also somewhat larger. These studies suggest that the intracellular concentration of newly esterified triglyceride and (or) some other lipid metabolite can specifically influence the release and perhaps the synthesis of d < 1.007 lipoprotein protein. They also suggest that the liver increases its rate of triglyceride release primarily by producing more lipoprotein particles.     

The transfer of estrone glucuronide to bile in an isolated perfused rat liver system.

An isolated rat liver perfusion system has been utilized in a study of the biliary excretion of estrone glucuronide. The estrogen was initially shown to be excreted without prior metabolism. Disappearance from the medium was rapid and biliary concentrations exceeded that in the medium by more than a thousand-fold. Disappearance rates were decreased when medium estrone glucuronide concentrations exceeded 0.29 mM. Inhibition by other steroidal conjugates, testosterone glucuronide, 2-methoxyestrone (3-hydroxy-2-methoxy-estra-1,3,5(10)-trien-17-one glucuronide and 2-hydroxyestrone (2,3-dihydroxyestra-1,3,5(10)-trien-17-one) glutathione, was also demonstrated. Phenolphthalein glucuronide, at 10 times the molar concentration of estrone glucuronide, did not affect the medium clearance of the latter compound. These findings indicate the possibility of utilizing this system for further studies of possible interactions by other organic compounds for excretion via the biliary route.     

Influence of ischaemic time on the production of bile by perfused rat liver

Production of bile has been studied in rat livers in situ and in livers perfused with rat blood or with bovine erythrocytes in Krebs Ringer buffer containing bovine albumin. The mean rate bile flow in four in situ livers remained almost constant for 3 hr following cannulation while bile flow in vitro decreased gradually throughout the 3-hr perfusion period. The total amount of bile produced in vitro decreased linearly with increase in ischaemic time. This decrease was greater in livers perfused with rat blood than with bovine erythrocytes. The length of the ischaemic time had no effect on other indices of liver function which were measured, i.e., urea synthesis, the ability to maintain a low concentration of ammonia in the perfusate, and the ability to retain potassium within the cells.     

The inhibition of oxalate biosynthesis in isolated perfused rat liver by DL-phenyllactate and n-heptanoate

Glycolate oxidase was isolated and partially purified from human and rat liver. The enzyme preparation readily catalyzed the oxidation of glycolate, glyoxylate, lactate, hydroxyisocaproate and α-hydroxybutyrate. The oxidation of glycolate and glyoxylate by glycolate oxidase was completely inhibited by 0.02 m dl-phenyllactate or n-heptanoate. The oxidation of glyoxylate by lactic dehydrogenase or xanthine oxidase was not inhibited by 0.067 m dl-phenyllactate or n-heptanoate. The conversion of [U-¹⁴C] glyoxylate to [¹⁴C] oxalate by isolated perfused rat liver was completely inhibited by dl-phenyllactate and n-heptanoate confirming the major contribution of glycolate oxidase in oxalate synthesis. Since the inhibition of oxalate was 100%, lactic dehydrogenase and xanthine oxidase do not contribute to oxalate biosynthesis in isolated perfused rat liver. dl-Phenyllactate also inhibited [¹⁴C] oxalate synthesis from [1-¹⁴C] glycolate, [U-¹⁴C] ethylene glycol, [U-¹⁴C] glycine, [3-¹⁴C] serine, and [U-¹⁴C] ethanolamine in isolated perfused rat liver. Oxalate synthesis from ethylene glycol was inhibited by dl-phenyllactate in the intact male rat confirming the role of glycolate oxidase in oxalate synthesis in vivo and indicating the feasibility of regulating oxalate metabolism in primary hyperoxaluria, ethylene glycol poisoning, and kidney stone formation by enzyme inhibitors.     

Stimulation of glycogenolysis by epinephrine and glucagon and its inhibition by insulin in isolated rat liver hepatocytes

Rat liver hepatocytes were isolated by collagenase $\text{in vitro}$ perfusion technique and the effect of epinephrine, glucagon and insulin on glycogenolysis was studied. Both glucagon and epinephrine at the concentration of 10^?6M, stimulated gluconeogenesis by 80–100%. Addition of insulin (33 μUnits/ml) completely abolished the epinephrine-stimulated glycogenolysis whereas only 50% inhibition was observed with insulin in glucagon stimulated glycogenolysis. This stimulation was observed within 2–5 min after the addition of the hormones. These results suggest that hepatocytes isolated with low concentrations of collagenase retain glucagon, epinephrine and insulin receptor sites.     

Uptake of asialoglycophorin-liposomes by the perfused rat liver

Asialoglycophorin-containing liposomes and their contents (¹²⁵I-labeled bovine serum albumin) were taken up by a perfused rat liver with subsequent digestion of their protein components. The uptake of these liposomes required Ca²⁺ as well as desialylation. The process was inhibited partially by asialofetuin and completely by further addition of asialoglycophorin to the perfusate.     

PGE1 metabolism by the perfused rat liver

The hepatic and biliary metabolites of PGE1 have been isolated and identified after infusions of PGE1 into isolated rat liver preparations. The results demonstrate that in general PGE1 undergoes metabolism similar to that of PGE2 in the rat and reveals the possibility of a selective PG metabolite transport system across the biliary canalicular membrane.     

Dopamine production by the isolated perfused rat kidney

We used isolated perfused rat kidneys to examine dopamine (DA) production and its relation to renal function. Both innervated and chronically surgically denervated kidneys perfused with a solution containing neither albumin nor tyrosine, excreted 0.2 +/- 0.1 ng DA X min-1 X g wet weight-1 during the 10-min collection period between 30 and 40 min after starting perfusion. When perfused with 6.7% albumin, without tyrosine, innervated kidneys excreted 1.0 +/- 0.06 ng DA X min-1 X g-1 and denervated kidneys excreted 1.0 +/- 0.07 DA X min-1 X g-1. When 0.03 mM tyrosine was included in the albumin perfusate, innervated kidneys excreted 1.2 +/- 0.1 ng DA X min-1 X g-1 (p less than 0.1). Under these conditions DA excretion continued for at least 100 min at which time it was 0.6 ng X min-1 X g-1 and 86 ng/g kidney weight had been excreted. Denervated kidneys perfused with albumin + tyrosine excreted 0.9 +/- 0.13 ng DA X min-1 X g-1. Renal stores of free DA, conjugated DA, and dihydroxyphenylalanine (DOPA) could have provided at the most 30 ng/g of DA. Carbidopa inhibited DA excretion completely. DA excretion did not correlate with renal vascular resistance, inulin clearance, or fractional sodium excretion. In summary, nonneural tissue in isolated perfused kidneys produced DA at the same rate as denervated kidneys in vivo. Less than one-third of the DA produced by isolated kidneys could have come from intrarenal stores of DOPA, free DA, and conjugated DA; the rest was synthesized from unknown precursors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gluconeogenesis in the perfused rat liver

1. A modification of the methods of Miller and of Schimassek for the perfusion of the isolated rat liver, suitable for the study of gluconeogenesis, is described. 2. The main modifications concern the operative technique (reducing the period of anoxia during the operation to 3min.) and the use of aged (non-glycolysing) red cells in the semi-synthetic perfusion medium. 3. The performance of the perfused liver was tested by measuring the rate of gluconeogenesis, of urea synthesis and the stability of adenine nucleotides. Higher rates of gluconeogenesis (1mumole/min./g.) from excess of lactate and of urea synthesis from excess of ammonia (4mumoles/min./g. in the presence of ornithine) were observed than are likely to occur in vivo where rates are limited by the rate of supply of precursor. The concentrations of the three adenine nucleotides in the liver tissue were maintained within 15% over a perfusion period of 135min. 4. Ca(2+), Na(+), K(+), Mg(2+) and phosphate were found to be required at physiological concentrations for optimum gluconeogenesis but bicarbonate and carbon dioxide could be largely replaced by phosphate buffer without affecting the rate of gluconeogenesis. 5. Maximal gluconeogenesis did not decrease maximal urea synthesis in the presence of ornithine and ammonia and vice versa. This indicates that the energy requirements were not limiting the rates of gluconeogenesis or of urea synthesis. 6. Addition of lactate, and especially ammonium salts, increased the uptake of oxygen more than expected on the basis of the ATP requirements of the gluconeogenesis and urea synthesis.     

Effect of DG5128 on epinephrine and glucagon induced glucose output from the isolated perfused rat liver

The effect of a specific alpha 2-adrenergic antagonist 2-[2-(4,5-dihydro-1.H-imidazol-2-yl)-1-phenyl-ethyl] pyridine dihydrochloride sesquihydrate (DG5128), on the glucose output by epinephrine and/or glucagon was studied using the perfused rat liver. The administration of DG5128 alone did not affect the glucose output. However, DG5128 produced a significant inhibition of the increased glucose output when induced by 10(-6) M epinephrine alone or 10(-6) M epinephrine plus 1.4 x 10(-10) M glucagon. There were no significant changes of the glucose output by 1.4 x 10(-10) M or 7.0 x 10(-11) M glucagon alone. On the other hand, addition of 1 mU/ml insulin to the perfusate suppressed the 7.0 x 10(-11) M glucagon-induced glucose output, but failed to decrease the 1.4 x 10(-10) M glucagon effect. DG5128 suppressed further the glucagon (7.0 x 10(-11) M)-induced increase of glucose output in the presence of insulin. These results suggest that DG5128 produces a hypoglycemic effect partly through an inhibition of the increased hepatic glucose output elicited by epinephrine and glucagon.     

Heparin is not metabolized by the perfused rat liver

The role of the liver in metabolism of heparin was studied using the isolated rat liver perfused in vitro for 10 hr. Porcine intestinal heparin (1000 u) was added to the recirculating liver perfusate, and serial heparin measurements were performed on the liver perfusate every 2 hr, as well as on bile samples secreted by the perfused liver. Heparin concentration remained at a constant level throughout the 10 hr of perfusion, and there was no detectable heparin secreted into bile samples. The findings suggest that hepatic metabolism/clearance plays a minimal role in heparin kinetics in plasma.     

Apolipoprotein A-I isoprotein synthesis by the perfused rat liver

The hepatic pattern of synthesis of the apolipoprotein A-I isoforms has been analyzed in the rat. After isolated livers were perfused with defibrinated rat blood and [³H]leucine, the radioactivity associated with apolipoprotein A-I and other apolipoproteins was determined following two-dimensional gel electrophoresis of the perfusate d < 1.21 g/ml lipoprotein fraction. In rat serum, apolipoprotein A-I is a polymorphic system consisting of two major isoproteins and a series of minor species. Following liver perfusion, 72% of the radioactivity associated with apolipoprotein A-I isoproteins was recovered in the more acidic and quantitatively less abundant of the two major isoforms. Only 8% was associated with the major apolipoprotein A-I isoform, and similar or lower amounts were found in the other minor isoproteins. These results are consistent with the concept that, in the rat, the major apolipoprotein A-I isoforms differ in their pattern of biosynthesis and/or metabolism.