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Despite being first published over 40 years ago, the combination of 13C nuclear magnetic resonance spectroscopy (NMR) and the isolated perfused liver preparation remains a unique and relevant approach in investigating the effects of pharmacological interventions on hepatic metabolism. The use of intact, perfused livers maintains many metabolic reactions at their respective rates in vivo, while the use of 13C-labelled substrates in combination with 13C NMR allows for a detailed study of specific pathways, as well as the design of robust assays which can be used to evaluate novel pharmacological agents. In this review article, we share some of the methods used to probe glucose metabolism, and highlight key findings and successes derived from the application of this specialized technique to the area of drug development for diabetes and related metabolic disorders.

Chlorhexidine (CHX) is an over-the-counter antiseptic amply used by the population. There are reports that CHX acts in mitochondria as an uncoupler and inhibitor. The purpose of this study was to investigate the short-term effects of CHX on hepatic metabolic pathways linked to energy metabolism in the perfused rat liver. The compound inhibited both glucose synthesis and the urea cycle. Oxygen consumption was raised at low concentrations (up to 10 μM) and diminished at higher ones. A pronounced diminution in the cellular ATP content was observed. Conversely, CHX stimulated glycolysis and enhanced leakage of cellular enzymes (lactate dehydrogenase and fumarase). In isolated mitochondria, this antiseptic inhibited pyruvate carboxylation, oxidases, and oxygen uptake at very low concentrations (2 μM) and promoted uncoupling. The results described herein raise great concerns about the safety of CHX, as the observed effects can induce hypoglycemia, lactic acidosis, ammonemia as well as cell membrane disruption. [Display omitted] • CHX reduced the cellular ATP content and the ATP/AMP ratio. • Hepatic anabolic pathways rate are inhibited and catabolic ones are increased. • Oxygen uptake was stimulated at slow concentrations and inhibited at higher ones in the liver. • CHX inhibited pyruvate carboxylation in isolated mitochondria. • CHX compromised mitochondrial energetic efficiency and disrupted the cell membrane. [ABSTRACT FROM AUTHOR]

Isolated hepatocytes and liver S9 fractions have been used to collect in vitro biotransformation data for fish as a means of improving modeled estimates of chemical bioaccumulation. To date, however, there have been few direct comparisons of these 2 methods. In the present study, cryopreserved trout hepatocytes were used to measure in vitro intrinsic clearance rates for 6 polycyclic aromatic hydrocarbons (PAHs). These rates were extrapolated to estimates of in vivo intrinsic clearance and used as inputs to a well stirred liver model to predict hepatic clearance. Predicted rates of hepatic clearance were then evaluated by comparison with measured rates determined previously using isolated perfused livers. Hepatic clearance rates predicted using hepatocytes were in good agreement with measured values (<2.1-fold difference for 5 of 6 compounds) under 2 competing binding assumptions. These findings, which may be attributed in part to high rates of PAH metabolism, are similar to those obtained previously using data from liver S9 fractions. For 1 compound (benzo[ a]pyrene), the in vivo intrinsic clearance rate calculated using S9 data was 10-fold higher than that determined using hepatocytes, possibly due to a diffusion limitation on cellular uptake. Generally, however, there was good agreement between calculated in vivo intrinsic clearance rates obtained using either in vitro test system. These results suggest that both systems can be used to improve bioaccumulation assessments for fish, particularly when vitro rates of activity are relatively high, although additional work is needed to determine if the chemical domain of applicability for each system differs. Environ Toxicol Chem 2017;36:463-471. Published 2016 SETAC. This article is a US government work and, as such, is in the public domain in the United States of America. [ABSTRACT FROM AUTHOR]

Isolated perfused trout livers were used to evaluate in vitro-in vivo metabolism extrapolation procedures for fish. In vitro depletion rates for 6 polycyclic aromatic hydrocarbons (PAHs) were measured using liver S9 fractions and extrapolated to the intact tissue. Predicted hepatic clearance (CLH) values were then compared with values exhibited by intact livers. Binding in liver perfusates was manipulated using bovine serum albumin (BSA) and was characterized by solid-phase microextraction. Additional studies were conducted to develop binding terms (fU; calculated as the ratio of unbound fractions in liver perfusate [fU,PERF] and the S9 system [fU,S9]) used as inputs to a well-stirred liver model. Hepatic clearance values for pyrene and benzo[a]pyrene, predicted by extrapolating in vitro data to the intact tissue, were in good agreement with measured values (< 2-fold difference). This can be partly attributed to the rapid rate at which both compounds were metabolized by S9 fractions, resulting in perfusion-limited clearance. Predicted levels of CLH for the other PAHs underestimated observed values although these differences were generally small (< 3-fold, except for naphthalene). Setting fU = 1.0 improved clearance predictions at the highest tested BSA concentration (10mg/ml), suggesting that trout S9 fractions exhibit lower levels of intrinsic activity than the intact tissue or that the full binding assumption (ie, fU = fU,PERF/fU,S9) underestimates the availability of hydrophobic substrates to hepatic metabolizing enzymes. These findings provide qualified support for procedures currently being used to predict metabolism impacts on chemical accumulation by fish based on measured rates of in vitro activity. [ABSTRACT FROM PUBLISHER]

Background: Submandibular salivary glands (SMGs) synthesize, accumulate and secrete a large amount of epidermal growth factor (EGF) in mice. It is known that surgical removal of SMG (sialoadenectomy) alters cell turnover in the liver and exacerbates liver injury induced by lipopolysaccharide/galactosamine (LPS/GalN). Results: Here we show that such increased hepatotoxicity is not the consequence of the lack of EGF production from SMG. On the contrary, it appears to be the consequence of an inadequate cytokine production by the liver of sialoadenectomized mice. Thus, we found that the increase of plasma tumour necrosis factor-α and interleukin-6 was slower in sialoadenectomized than in sham-operated mice. This is because of a decreased rate of production of both cytokines by the liver. We found that the increase of plasma corticosterone (CS) concentration is lower in sialoadenectomized than that in sham-operated mice. Adrenalectomy exacerbated liver injury induced by LPS/GalN. In these animals, sialoadenectomy did not further increase the effect of LPS/GalN. Conclusions: Our results suggest that the effect of sialoadenectomy on LPS/GalN-induced liver toxicity may be the consequence of an altered cytokine production by the liver and a reduced CS release from adrenal glands. [ABSTRACT FROM AUTHOR]

Abstract: The demonstration of pharmacodynamic efficacy of novel chemical entities represents a formidable challenge in the early exploration of synthetic lead classes. Here, we demonstrate a technique to validate the biological efficacy of novel antagonists of the human glucagon receptor (hGCGR) in the surgically removed perfused liver prior to the optimization of the pharmacokinetic properties of the compounds. The technique involves the direct observation by 13C NMR of the biosynthesis of [13C]glycogen from [13C]pyruvate via the gluconeogenic pathway. The rapid breakdown of [13C]glycogen (glycogenolysis) following the addition of 50pM exogenous glucagon is then monitored in real time in the perfused liver by 13C NMR. The concentration-dependent inhibition of glucagon-mediated glycogenolysis is demonstrated for both the peptidyl glucagon receptor antagonist 1 and structurally diverse synthetic antagonists 2–7. Perfused livers were obtained from a transgenic mouse strain that exclusively expresses the functional human glucagon receptor, conferring human relevance to the activity observed with glucagon receptor antagonists. This technique does not provide adequate quantitative precision for the comparative ranking of active compounds, but does afford physiological evidence of efficacy in the early development of a chemical series of antagonists. [Copyright &y& Elsevier]

Abstract: NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD–FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH /FMNH2 couple donates electrons to cytochrome P450 at constant oxidation–reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form can function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed. [Copyright &y& Elsevier]

Abstract: A clearer picture of the hepatic metabolic pathways affected by fulminant hepatic failure (FHF) would help develop nutritional support and nonsurgical therapies for FHF. We characterized the evolution of hepatic metabolism in a rat model of FHF using an isolated perfused liver system together with a mass-balance model of intermediary metabolism. Principal component analysis (PCA) was used to identify potential new sensitive markers for FHF. To induce FHF, rats were given two D-galactosamine injections under fasting conditions. Controls were fasted only. Livers were harvested 1, 4, 8, and 12h later and perfused with Eagle minimal essential medium supplemented with amino acids and bovine serum albumin, and equilibrated with 95% O2/5% CO2. At the 1h time point, lactate release increased concomitant with a decrease in gluconeogenesis, TCA cycle and mitochondrial electron transport fluxes. At 4h, amino acid metabolism and urea cycle fluxes were significantly depressed. By 8h, gluconeogenesis had switched to glycolysis. By 12h, amino acid metabolism was broadly inhibited, and there was a net release of many amino acids. Mass-balance analysis shows that the main source of ATP production in the FHF liver gradually changed from mitochondrial oxidative phosphorylation to glycolysis. PCA suggests that a linear combination of glucose, lactate, and glutamine concentrations in arterial plasma is a sensitive marker for FHF. We conclude that D-galactosamine causes early mitochondrial dysfunction while glycolytic ATP synthesis remains functional. Markers that are indirectly linked to these pathways may be used to evaluate the progression of FHF. [Copyright &y& Elsevier]

Abstract: The swift increase in alcohol metabolism (SIAM) is a phenomenon defined as a rapid increase in hepatic respiration and alcohol metabolism after administration of a bolus dose of alcohol. Continuous exposure to alcohol is known to produce adaptive changes in liver alcohol and oxygen metabolism. A considerable burst of hepatic respiration can also occur after administration of a single large dose of alcohol and results in a near doubling of alcohol metabolism, a high demand for oxygen, and downstream or pericentral hypoxia. These dramatic changes in rates of alcohol metabolism and tissue concentrations of oxygen are not due to induced enzyme activity in liver. This phenomenon depends on activation of mitochondrial function, an increase in co-factor supply for nicotinamide adenine dinucleotide–dependent alcohol metabolism, depletion of glycogen reserves, liberation of fatty acids through activation of an adrenergic response to alcohol providing substrate for catalase, and activation of Kupffer cells, the hepatic resident macrophages responsible for production of cytokines and prostaglandins. An understanding of the mechanisms of hypermetabolism in liver can have vital ramifications for knowledge of both alcohol-related and alcohol-unrelated liver injury because hypoxia that is a result of hypermetabolism can compound effects of pharmaceuticals and environmental agents on the liver. Swift increase in alcohol metabolism is an excellent example of the complexity of cell–cell interactions in liver and extrahepatic regulation of biochemical and molecular events in this organ, and this important phenomenon shall be considered in studies of liver disease and biochemistry. [Copyright &y& Elsevier]

The effect of oral taurine supplementation on endotoxin-induced cholestasis was investigated in rat liver. At 12 h following lipopolysaccharide (LPS) injection (4 mg/kg body weight i.p.) bile flow and bromosulfophthalein (BSP) and taurocholate (TC) excretion were determined in the perfused liver and the expression of the canalicular transporters multidrug resistance protein 2 (Mrp2) and bile salt export pump (Bsep) was analyzed. Injection of LPS induced a significant decrease of bile flow ( 2.2±0.2 μl/g liver wet weight/min vs 3.3±0.1 μl/g liver wet weight in controls), biliary BSP excretion (10.8±2.2 nmol/g/min vs 21.0±3.8 nmol/g/min), and biliary TC excretion (114±23 nmol/g/min vs 228±8 nmol/g/min). These effects were due to transporter retrieval from the canalicular membrane and downregulation of Mrp2 and Bsep expression. In taurine-supplemented rats bile flow was 30% higher than that in untreated rats and the expression of Mrp2 and Bsep protein was increased two- to threefold. In taurine-supplemented rats there was no significant reduction of bile flow or of BSP and TC excretion at 12 h following LPS injection. This protective effect of taurine was due to higher Mrp2 and Bsep protein levels compared to nonsupplemented LPS-treated rats, whereas relative Mrp2 retrieval from the canalicular membrane induced by LPS was not significantly different. LPS-induced tumor necrosis factor α and interleukin-1β release were lower in taurine-fed rats; however, downregulation of Mrp2 and Bsep expression by LPS was delayed but not prevented. The data show that oral supplementation of taurine induces Mrp2 and Bsep expression and may prevent LPS-induced cholestasis. [Copyright &y& Elsevier]

Multidrug resistance-associated protein 3 (Mrp3/ABCC3), which can mediate the cellular extrusion of bile acids, is induced on the hepatic sinusoidal membrane of Mrp2/ABCC2-deficient rats (Eisai hyperbilirubinemic rats; EHBRs) and phenobarbital-treated Sprague–Dawley rats. In the present study, the correlation between the sinusoidal efflux clearance (PSeff) of [3H]taurocholate (TC) and the hepatic expression of Mrp3 was investigated using perfused liver from these rats. A significant correlation was observed between the PSeff and the hepatic expression level of Mrp3, suggesting a contribution by Mrp3 to the sinusoidal efflux of TC. The results of the kinetic analysis also suggested that other transporter(s) on the sinusoidal plasma membrane may participate in the efflux of TC under physiological conditions. The contribution of Mrp3 to the sinusoidal efflux of TC in EHBRs and phenobarbital (80 and 40 mg/kg)-treated rats was revealed to be 58%, 48%, and 31%, respectively. [Copyright &y& Elsevier]

Purpose. It has been shown that plasma concentration and urinary excretion of bile acids is elevated under the cholestatic/hyperbilirubinemic conditions. Previously, it was demonstrated that the plasma concentration of bile acids was elevated in the multidrug resistance-associated protein 2 (Mrp2)-deficient rats. The purpose of the present study was to compare the sinusoidal efflux clearance of taurocholate (TC) between Mrp2-deficient Eisai hyperbilirubinemic rats (EHBR) and normal rats. Method. Hepatic disposition of the [3H]TC was examined in the perfused liver. Apparent efflux clearance (PSnet, eff) of [3H]TC from hepatocytes to outflow across the sinusoidal membrane was defined as the amount of [3H]TC excreted into the outflow from the liver divided by hepatic AUC of [3H]TC. Additionally, influx clearance (PSinf) was also determined by multiple indicator dilution method because PSnet, eff is also affected by PSinf. Results. PSnet, eff was significantly higher in EHBR than that in Sprague-Dawley (SD) rats (16.6 ±1.7 vs. 6.1 ± 1.3 μL/min/g liver, P <0.01). In contrast, PSinf was comparable between SD rats and EHBR. Kinetic analysis suggested that the intrinsic clearance for the efflux of [3H]TC across the sinusoidal membrane in EHBR was higher than that in SD rats (10.4 ± 1.0 v.s. 23.3 ± 1.7 μL/min/g liver, P <0.01). Conclusions. Enhanced sinusoidal efflux of TC in EHBR may be related to the altered disposition of bile acids in the mutant rats. Because Mrp3 transports TC and its expression is induced on the basolateral membrane of Mrp2-deficient rats, the enhanced sinusoidal efflux of TC in EHBR may be accounted for, at least partially, by the increased expression of Mrp3. [ABSTRACT FROM AUTHOR]

The effects of glucagon and insulin on glucose production were explored directly using the isolated perfused liver of the Goto-Kakizaki (GK) rat, an animal model of type-2 diabetes. In the perfused liver of control rats, infusion of glucagon (0.06–1.0 nM) into the portal vein dose-dependently increased glucose output. In the GK rat liver, in which the intracellular distribution of glycogen was heterogeneous, basal glucose output during perfusion was significantly higher than in control, whereas the effect of glucagon on the maximum glucose output was not different. Infusion of insulin inhibited the glucagon-induced hepatic glucose output by 30–40% in control livers, but had no effect on that from the GK rat liver. The increase in hepatic cAMP content after glucagon infusion was antagonized by insulin in control livers, but not in the livers of GK rats. These results indicate that the antagonistic effect of insulin on glucagon-induced hepatic glucose production was attenuated in the isolated liver of the GK rat and suggest that this insulin resistance appeared in the signal transduction process of glucagon upstream from cAMP production. [ABSTRACT FROM AUTHOR]

Despite being first published over 40 years ago, the combination of 13C nuclear magnetic resonance spectroscopy (NMR) and the isolated perfused liver preparation remains a unique and relevant approach in investigating the effects of pharmacological interventions on hepatic metabolism. The use of intact, perfused livers maintains many metabolic reactions at their respective rates in vivo, while the use of 13C-labelled substrates in combination with 13C NMR allows for a detailed study of specific pathways, as well as the design of robust assays which can be used to evaluate novel pharmacological agents. In this review article, we share some of the methods used to probe glucose metabolism, and highlight key findings and successes derived from the application of this specialized technique to the area of drug development for diabetes and related metabolic disorders. [ABSTRACT FROM AUTHOR]

The regulatory mechanism of ketone-body output by the hepatic sympathetic nerves was studied in rat liver perfused in situ. Enrichment of the perfusion medium with 1 mM octanoate increased the basal ketone-body output from the liver up to 1.5 µmol · min-1 · g liver-1. Under these conditions, electrical stimulation of the hepatic nerves (20 V, 20 Hz, 2 ms) decreased the output of both acetoacetate and β-hydroxybutyrate, and was accompanied by an accumulation of β-hydroxybutyrate in the liver. The effects of nerve stimulation were inhibited by the α1-antagonist bunazosin (10 µM). However, noradrenaline, a typical sympathetic neurotransmitter, at a concentration of 1 µM decreased the output of acetoacetate but did not affect β-hydroxybutyrate output. Prostaglandin F2α at a concentration of 10 µM produced an effect similar to treatment with noradrenaline, without a decrease in β-hydroxybutyrate output. ATP at 50 µM, however, decrease the output of both acetoacetate and β-hydroxybutyrate and increased the tissue concentration of β-hydroxybutyrate, mimicking the effect of nerve stimulation. Moreover, in the presence of 0.2 µM ATP, a concentration that produced neither metabolic nor hemodynamic changes, noradrenaline (1 µM) was shown to decrease the β-hydroxybutyrate output. These results indicate the possible involvement of ATP in the action of hepatic sympathetic nerves on β-hydroxybutyrate output from the liver, presumably through its interaction with noradrenaline. [ABSTRACT FROM AUTHOR]

Male Wistar rats show typical age-related variations in the distribution of high-density lipoprotein subfractions that include an increase in HDL1 and a decrease in HDL2 proportion. The role of liver in these variations was evaluated by studying the lipoprotein and bile secretions from perfused livers of 14±1 and 3.5±0.5 month old Wistar rats (adult and young animals, respectively). The lipid content of lipoproteins secreted from adult livers was higher in HDL2 fraction and lower in VLDL fraction. The lipid output did not show significant age-related variations in the case of HDL1 fraction. However, the lipoproteins secreted from adult livers contained a higher proportion of phospholipids, and a lower proportion of triacylglycerols in comparison with lipoproteins secreted by young livers. Therefore, the molar ratio of core to surface lipids was lower in lipoproteins secreted by adult livers. Adult livers showed a reduction in bile flow by about 37% with a significantly higher phospholipid secretion. These findings suggest that both the hepatic metabolism of glycerophospholipids and their repartition between plasma and bile compartments are affected by aging process. In conclusion, present data show that the age-related increase in plasma HDL1 proportion, previously observed in this rat strain in vivo, are not due to a higher liver secretion of these particles. Conversely, liver appears to have a major role in the age-related VLDL increase and in the variations of phospholipid lipoprotein secretion. [ABSTRACT FROM AUTHOR]

The hepatic metabolism and biliary and pancreatic excretion of the serine protease inhibitor camostat mesilate and its metabolites FOY-251 and GBA were studied in rats in vivo and in in situ liver-perfusion experiments. After oral feeding (100 mg/kg) and iv infusion (5 mg/kg-h) of camostat mesilate, the original compound and both metabolites appeared in bile, but could not be detected in pancreatic juice. In plasma, only FOY-251 and GBA were detected. In the perfused rat liver, camostat mesilate (10 μM) was eliminated by 33.8% and its molar rate of degradation to FOY-251 was 25.1%. During the study period about 0.1% of camostat mesilate and FOY-251 appeared in bile. The liver perfusion of FOY-251 or GBA revealed very low hepatic extraction rates of 10.3 and 2.4%, respectively. In conclusion, the low hepatic extraction rate of camostat mesilate and its metabolites leads to high concentrations of the active metabolite FOY-251 in plasma. Camostat mesilate and its metabolites are effectively excreted into bile, but not in rat pancreatic juice. [ABSTRACT FROM AUTHOR]

The t of insulin removal by perfused liver from hyperinsulinaemic ob/ob mice has previously been shown to be prolonged as compared to livers from nonobese mice. Since this defect improved when insulin levels were lowered in vivo, it appeared that the abnormality might be secondary to hyperinsulinaemia. To test this hypothesis further, insulin removal by perfused liver from one year and 6 week old Sprague-Dawley rats has been compared. Older rats were both obese and hyperinsulinaemic, and their t of insulin removal (mean ± SEM) was significantly prolonged (11.0±0.56 min vs 7.6±0.29 min). In addition, the induction of severe streptozotocin-induced hypoinsulinaemia in young rats led to a shortening of the t (5.56±0.72 min). However, the t also fell comparably when weight gain of young control rats was limited to that of young hypoinsulinaemic rats by caloric restriction (5.89±0.81 min). Furthermore, t was not increased when young rats were made hyperinsulinaemic by administration of NPH insulin for 12-14 days, nor was it decreased when older rats were made moderately hypoinsulinaemic by the administration of streptozotocin. These results demonstrate that the defect in hepatic insulin removal observed in the ob/ob mouse is not unique to this genetic variant, but is also observed in normal obese rats. Although the mechanism responsible for this phenomenon remains to be defined, it does not seem to be a simple function of relatively short-term (1-2 weeks) changes in plasma insulin concentration. On the other hand, it is still possible that the prolongation of insulin removal in both the ob/ob mouse and the obese rat is secondary to more chronic elevations in plasma insulin, or, alternatively is related to the obese state itself. In either event, the defect in hepatic insulin removal may be a general feature of obesity and, as such, contribute to the maintenance of hyperinsulinaemia in a variety of states characterized by obesity. [ABSTRACT FROM AUTHOR]

Carbohydrate and lipid metabolism has been studied in perfused livers of lean and obese-hyper-glycaemic (ob/ob) mice. The capacity for gluconeogenesis from lactate or pyruvate was similar in livers of both groups of mice. Incorporation of carbon from labelled pyruvate into hepatic lipids was much higher in livers of ob/ob mice than in those of lean controls. Total lipogenesis, as well as newly synthesized triglyceride secretion by perfused livers, was also estimated, by measuringH (fromHO) incorporation into total (i.e. liver + perfusate) and perfusate triglyceride fatty acids. Lipogenesis, both in the absence or in the presence of substrates, was greater in livers of ob/ob mice than in those of lean controls, as was newly synthesized triglyceride secretion. In the absence of oleate in the perfusate, the secretion of unlabelled triglyceride by livers of 06/06 mice was much higher than that of non-obese mice, but it did not increase further upon addition of oleate, as it did in livers of lean controls. Ketone body production by livers of ob/ob mice, perfused with albumin bound oleate, was considerably lower than that observed in control livers. When ob/ob mice were made relatively insulin deficient by streptozotocin treatment, all these anomalies of lipid metabolism were restored towards normal. It is proposed that hyperinsulinaemia is responsible, at least in part, for the abnormalities in lipogenesis, triglyceride secretion and fatty acid oxidation to ketone bodies observed in livers of the obese-hyperglycaemic mice. [ABSTRACT FROM AUTHOR]

The addition of L-alanine as substrate to a perfused rat liver preparation produced a five-fold increase in the rate of glucose production. This enhancement of the gluconeogenic flux seems to be a consequence of a rise in the steady-state levels of pyruvate and oxaloacetate subsequent to the rise in alanine concentration. Glucagon (2×10 M) increased the gluconeogenic flux from alanine (10 mM) by 50 percent, even though the concentration of the substrate in the perfusion fluid was at saturation. This effect was accompanied by a rise in the intracellular concentrations of alanine. However, the steady-state concentrations of pyruvate and oxaloacetate were decreased, probably as a consequence of a more reduced state of the nicotinamide-nucleotide system. In vivo, the intraperitoneal administration of glucagon to starved rats was accompanied by a decrease in the hepatic alanine and pyruvate concentrations despite the striking effects raising the plasma glucose levels. These observations seem to indicate that the effect of the hormone increasing the hepatic glucose output must be mediated through some other mechanism(s) independent of the intracellular variations in the hepatic amino acids levels. [ABSTRACT FROM AUTHOR]

The carbon balances in isolated perfused rat liver during gluconeogenesis from l-alanine and sodium l-lactate indicate that assuming the substrate unaccounted for were fully oxidized the energy yielded was not sufficient to support the observed rates of glucose synthesis. This observation indicates that endogenous substrates must also be oxidized. The possibility that endogenous fatty acid oxidation was the source of the energy needed to support glucose synthesis was investigated by measuring the rate ofCO formation from tracer quantities of added [U-C] palmitate. Short pulses of l-alanine or sodium l-lactate infusion produced an increased rate ofCO production paralleled by increases in oxygen uptake indicating that more endogenous fuel is being mobilized. That the rate ofCO output is an expression of fatty acid mobilization was supported by experiments demonstrating that the addition of octanoate to dilute the fatty acid pool produced an immediate fall in the rate ofCO output. On the other hand, the administration of glucose produced no changes in oxygen uptake orCO output. However, lactate even in the presence of glucose induced a rise inCO production which occurred in parallel with the enhancement in oxygen uptake. It is concluded that mobilization of hepatic endogenous fatty acid is a metabolic event intimately associated with enhancement of gluconeogenesis. Consequently the control of the different steps of this process may indirectly control gluconeogenesis. [ABSTRACT FROM AUTHOR]

Glucose production was measured in isolated rat livers perfused with 100 ml of blood-free recirculating medium. The gluconeogenic rate using l-alanine as substrate was only 55% of that obtained with l-lactate. The steady-state concentration of gluconeogenic and tricarboxylic acid cycle intermediates were measured in freeze clamped biopsies. Livers perfused with l-lactate displayed higher concentrations of malate, α-glycerophosphate and β-hydroxybutyrate probably as a result of a higher state of reduction of the nicotinamide system. Hexose-phosphate intermediates were also increased when l-lactate was the substrate. Phosphoenolpyruvate and 3-phosphoglycerate were considerably elevated when l-alanine was the glucose precursor. Livers perfused with l-lactate displayed higher cytosolic concentration of all the tricarboxylic acid cycle intermediates except oxaloacetate while glutamate was slightly and aspartare considerably higher when alanine was the substrate. In the mitochondrial compartment the pattern of distribution tended to be the opposite; that is, livers perfused with l-lactate showed lower concentrations of all the intermediates except α-ketoglutarate. The mitochondrial: cytosolic metabolite gradients of all the intermediates whose distribution was studied were higher in livers perfused with l-alanine. The relevance of these findings to the observed differences in the gluconeogenic fluxes are discussed. [ABSTRACT FROM AUTHOR]

Rabbit livers were perfused with Krebs solution via the portal vein and small bundles of the hepatic nerve were monitored for spontaneous afferent impulses arising in the liver. The concentration of the Krebs solution was varied by altering the amount of water in which the salts were dissolved. An alteration from 5% more concentrated to 5% more dilute and vice versa did not affect this spontantous discharge. Addition to the Krebs solution of up to 60 mM sucrose, of up to 22 mM mannitol or of 0.8 mM polyethylenglycol 6000 mol. wt. did not alter the frequency of impulses. Perfusion with bovine serum, or Krebs solution containing approx. 0.6 mM polyvinylpyrrolidone, mol. wt. 44000, or dextran mol. wt. 110 000 increased the frequency of discharge in some nerve bundles. An alteration of 0.06 mM in the concentration of dextran produced a significant change of frequency. It is concluded that spontaneous discharge in some afferent hepatic nerves is influenced by colloidal osmotic pressure but not by changes of ionic osmotic pressure. The nerves affected differ from 'sodium-sensitive' nerves and from the 'osmoreceptors' of Niijima. [ABSTRACT FROM AUTHOR]

Exposure of the perfused rat liver to a perfusate made hyperosmotic by the presence of 200 mmol lglucose led, as expected, to marked, transient hepatocellular shrinkage followed by volume-regulatory net K uptake. However, even after this volume-regulatory K uptake had ceased, the liver cells are still slightly shrunken. Withdrawal of glucose from the perfusate resulted in marked transient cell swelling, net K release from the liver and restoration of cell volume. However, when the Krebs-Henseleit perfusate was made hyperosmotic by the presence of urea (20-300 mM), there was no immediate decrease in liver mass, yet a slight and persistent cell shrinkage developing 2 min after the onset of exposure to urea. Surprisingly, urea induced concentration-dependent net K efflux from the liver and removal of urea net K reuptake from the inflowing perfusate. The urea (200 mM)-induced net K release resembled that observed following a lowering of the influent [NaCl]: making the perfusate hypoosmotic (245 mosmol l, by reducing influent [NaCl] by 30 mM) gave roughly the same K response as hyperosmotic exposure (505 mosmol/l) resulting from the presence of 200 mM urea. The urea-induced K efflux was not inhibited in the presence of ouabain (1 mM), or in Ca-free perfusion, but was modified in the presence of quinidine (1 mM) or Ba (1 mM). The direction in which the liver was perfused had no effect on the urea-induced net K release. Electrophysiological studies showed that urea led to quinidine-sensitive hyperpolarization and increase in K selectivity of plasma membranes, suggesting opening of K channels in the hepatocyte plasma membrane in response to urea. The data suggest that urea, but not glucose, enters the hepatocyte as quickly as water. Furthermore, urea at high concentrations apparently leads to K efflux from the hepatocyte and cell shrinkage, possibly due to opening of K channels in the hepatocyte plasma membrane. [ABSTRACT FROM AUTHOR]

The metabolism of 3α-[(10,11-dihydro-5 H-dibenzo-[ a,d]cyclohepten-5-yl)oxy]-8-methyltropanium iodide (deptropine methiodide; N-methyldeptropine), a potential agent for the treatment of peptic ulcers and gastrointestinal spasms, was studied by passing the N-CH-labelled compound through the isolated perfused rat liver. The substance was rapidly taken up by the liver. This process followed first-order kinetics with a half-life of 5 min. It was calculated that 94% of the drug was removed by the liver during one passage. About 70% of the initial radioactivity was excreted in the bile during a perfusion period of 2 h. This elimination process exhibited first-order kinetics with a half-life of about 25 min. N-Methyldeptropine was found to be broken down in the liver into N-methyltropine, and a non radioactive fragment. N-methyltropine and N-methyldeptropine were eliminated in the bile. The former also diffused back into the perfusion fluid. In addition, the bile contained an uncharacterised radioactive product related to N-methyltropine, possibly a complex of N-methyltropine with bile components. Both the rapid uptake of N-methyldeptropine in the liver and the biotransformation into products with a much lower pharmacological and toxicological potency, should lessen the likelihood of side effects outside the gastrointestinal tract after oral dosage. [ABSTRACT FROM AUTHOR]

Livers from fed and starved rats were perfused with either rat blood, washed bovine erythrocytes suspended in Eagle minimal essential medium, or the same medium without erythrocytes. The activity of microsomal mixed function oxidase under different perfusion conditions was estimated in the intact liver by following the disappearance of hexobarbital from the perfusion medium. Half-lives of 9 and 29 min were determined in perfusion systems with and without erythrocytes, respectively. They were 70% greater in livers from rats starved for 20 h. Identical half-lives were determined in perfusions with rat blood or washed erythrocytes suspended in the synthetic medium. Testosterone inhibited hexobarbital metabolism when added at an equimolar concentration of 0.5 mM. However, no significant inhibition could be detected with 1 mM hydrocortisone. Furthermore, the porphyria-inducing agent allylisopropyl-acetamide markedly inhibited hexobarbital metabolism prior to decreasing the level of cytochrome P 450. Microsomal cytochrome b, cytochrome P 450 and NADPH cytochrome c reductase were stable during 4 h of perfusion in media containing erythrocytes but their levels were lower by 20, 40 and 45%, respectively, after perfusion with the erythrocyte-free medium. However, in the latter medium the specific activity of NAD glycohydrolase in microsomes was 35% greater. The stability of these proteins was compared with their turnover in vivo and with the morphology of the perfused livers at the light and electron microscopic levels. [ABSTRACT FROM AUTHOR]

Livers from fed and starved rats were perfused with either washed bovine erythrocytes suspended in Eagle minimal essential medium or the same medium without erythrocytes. Albumin synthesis was studied under these perfusion conditions by following the appearance of the secreted protein in the perfusion medium with specific antibodies against rat albumin. Albumin was synthesized linearly between 1 and 4 h after the start of perfusion at a rate of 2.8 mg albumin/h x whole liver. In perfusions without erythrocytes and in perfused livers from rats starved for 20 h the synthesis rate was reduced by 50%. Albumin synthesis was not changed in livers from rats which had been pretreated with phenobarbital for 4 h and 8 days despite marked liver growth after 8 days of treatment. [ABSTRACT FROM AUTHOR]

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Metformin has been proposed as a potential drug treatment to reduce liver steatosis. Therefore, the study of the effects of in vivo metformin administration, on hepatic fat metabolism in the isolated perfused liver is of great interest. We have studied the effects of in vivo metformin treatment of rats with experimentally induced overweight, hyperglycemia and hypertriglyceridemia, on plasma hormone and metabolite concentrations, as well as on triglyceride and ketone body output by the isolated perfused livers. Sprague–Dawley rats were fed ad libitum with a high lipid diet for 10 weeks. Then, one rat group was treated for 7 days with 350 μg/kg of BW of metformin per day, whereas the control group received only water. Thereafter, the livers were excised and perfused in vitro under controlled conditions. The hypertriglyceridemic rats had a greater body weight, as well as greater plasma glucose, insulin and triglyceride concentrations, than the control rats fed ordinary chow. In vivo metformin treatment decreased plasma glucose, insulin and triglyceride concentrations. With respect to the overweight, hyperglycemic, hypertriglyceridemic, untreated control rats, the cumulative (i.e. over 3 h of perfusion) triglyceride output by the perfused livers was decreased by >60%, whereas total ketone body (i.e. the sum of 3-hydroxybutyrate and acetoacetate) output was increased by >100% ( p < 0.01 for both). In conclusion, the in vivo treatment with metformin of rats with diet-induced overweight and hypertriglyceridemia is capable to re-address hepatic fatty acid metabolism from lipogenesis toward fat oxidation and ketone body production, either directly or through a reduction of insulin concentrations. [ABSTRACT FROM AUTHOR]

Cell volume plays a decisive role in the regulation of hepatic metabolism. The present study has been performed to test for an effect of insulin and glucagon on liver cell volume. To this end, the effect of these hormones has been studied in isolated perfused rat livers and isolated rat hepatocytes. Insulin leads to rapid stimulation of cellular K+ uptake and increase of cell volume, effects reversed by glucagon or cAMP. The insulin stimulated cellular K+ uptake is significantly decreased in the presence of either loop diuretics (furosemide or bumetanide) or amiloride and is completely inhibited in the presence of both, bumetanide and amiloride. The glucagon stimulated cellular K+ release in the presence of insulin is blunted by K+ channel blocker quinidine. The effects of insulin and glucagon on liver cell volume could participate in the regulation of hepatic metabolism by these hormones. [ABSTRACT FROM AUTHOR]