The effect of fatty acids on the rate of pyruvate decarboxylation was studied in perfused livers from fed rats. The production of 14C02 from infused [l-14C]pyruvate was employed as a monitor of the flux through the pyruvate dehydrogenase reaction. A correction for other decarboxylation reactions was made using kinetic analyses. Fatty acid (octanoate or oleate) infusion caused a stimulation of pyruvate decarboxylation at pyruvate concentrations in the perfusate below 1 mM (up to 3-fold at 0.05 mM pyruvate) but decreased the rate to one-third of control rates at pyruvate concentrations near 5 mM. These effects were half-maximal at fatty acid concentrations below 0.1 mM. Infusion of 3-hydroxybutyrate also caused a marked stimulation of pyruvate decarboxylation at low pyruvate concentrations.The data suggest that the mechanism by which fatty acids stimulate the flux through the pyruvate dehydrogenase reaction in perfused liver at low (limiting) pyruvate concentrations involves an acceleration of pyruvate transport into the mitochondrial compartment due to an exchange with acetoacetate. Furthermore, it is proposed that a relationship exists between ketogenesis and the regulation of pyruvate oxidation at pyruvate concentrations approximating conditions in vivo.The pyruvate dehydrogenase multienzyme complex from mammalian tissues is regulated by two different mechanisms. Early studies indicated that this enzyme is inhibited by products of the reaction, NADH and acetyl-CoA, each of which is a competitive inhibitor with respect to the corresponding substrate, i.e. NAD' and coenzyme A [4-61. Later it was found that pyruvate dehydrogenase is inactivated upon phosphorylation by a specific protein kinase, whereas the phosphorylated enzyme complex may be reactivated by a phosphoprotein phosphatase [7 - explanation for this effect was based upon the fact that the inhibitory products of the pyruvate dehydrogenase reaction, i.e. NADH and acetyl-CoA, are also produced during P-oxidation of fatty acids [4 -61. However, the observation that addition of fatty acids to intact organs [25 -271 or mitochondrial preparations [ll, 17,19,20] resulted in a conversion of active pyruvate dehydrogenase into its inactive form indicated that the fatty acid effect was more than a simple, direct feedback inhibition of the enzyme by NADH and acetyl-CoA. Initial proposals for the mechanism of the fatty-acid-mediated inactivation of pyruvate dehydrogenase have centered around the suggestion that the addition of long-chain fatty acids to tissues may cause an increased ATP/ADP ratio in the mitochondrial compartment [20] which would accentuate the kinase-mediated inactivation, i.e. ATP is a substrate of the kinase while ADP is a competitive inhibitor [23]. This hypothesis was supported by experiments with isolated mitochondria in which the interconversion of pyruvate dehydrogenase between its active and inactive forms is sensitive to changes in the intramitochondrial ATP/ADP ratio [13,19,20,221. On the other hand, several experiments suggested that the...

The hepatic removal of the glutathione conjugate of bromosulfophthalein (BSPGSH) was studied in the single-pass perfused rat liver with the multiple indicator dilution (MID) technique against various background concentrations of BSPGSH (20 to 214 mumol/L) over which nonlinear binding to both plasma (albumin) and tissue proteins with two classes of binding sites was found. A bolus containing 51Cr-labeled red blood cell (a vascular reference), [125I]albumin and [14C]sucrose (large and small molecular weight interstitial references, respectively), D2O (a cellular space reference), and [3H]BSPGSH was injected into the portal vein during steady-state. The eliminated fraction of dose, obtained by subtracting the survival fraction of [3H]BSPGSH in plasma from one, corresponded to the steady state extraction ratio (E) with bulk data, which declined from 0.74 +/- 0.04 to 0.27 +/- 0.01 with concentration. The major portion of the tracer outflow profile was a throughput component, which is the proportion of tracer that did not enter liver cells during its transit through the liver. The influx, efflux, and sequestration coefficients, evaluated with previously developed barrier-limited models, provided the corresponding influx (k1), efflux (k-1) and excretion (kseq) rate constants. Concentration-dependent influx (Vmax = 83 nmol min-1 g-1 and Km = 3.7 mumol/L), efflux (Vmax = 15 nmol min-1 g-1 and Km = 1.8 mumol/L), and excretion (Vmax = 94 nmol min-1 g-1 and Km = 1.8 mumol/L) were obtained for BSPGSH, when Km values are expressed in terms of the unbound concentrations. In these calculations, the observed unbound tissue concentration was not used for estimation of the Vmax and Km for efflux and excretion because of overestimation, because of the presence of highly concentrated BSPGSH in ductular elements present in liver homogenates; rather, the unbound tissue concentration was calculated from the influx, efflux, and removal rate coefficients. Because of carrier-mediated entry, the unbound tissue concentration does not equal the unbound plasma concentration, and kinetic parameters for BSPGSH excretion could be alternately estimated when the rate of excretion or net rate of loss of BSPGSH from plasma was regressed against the estimated tissue unbound concentration. This yielded a Vmax of 97 nmol min-1 g-1 and a Km of 3.6 mumol/L, values similar to those obtained from MID.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple, noneliminated references ((51)Cr-labeled erythrocytes, (125)I-albumin, [(14)C]- or [(3)H]sucrose, and [(2)H](2)O), together with [(3)H]hippurate or [(14)C]benzoate, were injected simultaneously into the portal vein of the perfused rat liver during single-pass delivery of benzoate (5-1,000 microM) and hippurate (5 microM) to investigate hippurate formation kinetics and transport. The outflow dilution data best fit a space-distributed model comprising vascular and cellular pools for benzoate and hippurate; there was further need to segregate the cellular pool of benzoate into shallow (cytosolic) and deep (mitochondrial) pools. Fitted values of the membrane permeability-surface area products for sinusoidal entry of unbound benzoate were fast and concentration independent (0.89 +/- 0.17 ml. s(-1). g(-1)) and greatly exceeded the plasma flow rate (0.0169 +/- 0.0018 ml. s(-1). g(-1)), whereas both the influx of benzoate into the deep pool and the formation of hippurate occurring therein appeared to be saturable. Results of the fit to the dilution data suggest rapid uptake of benzoate, with glycination occurring within the deep and not the shallow pool as the rate-determining step.