Harmful effects of usnic acid on hepatic metabolism.

Usnic acid is a naturally occurring dibenzofuran derivative found in several lichen species. The compound has been marketed as an ingredient of food supplements for weight reduction. There is evidence that the compound acts as an uncoupler of mitochondrial oxidative phosphorylation and it is also clear that consumption of the drug can lead to severe hepatotoxicity depending on the doses. Based on these and other ideas the objective of the present work was to investigate the possible effects of usnic acid on liver metabolism. Livers of male Wistar rats were perfused in a non-recirculating system. Usnic acid stimulated oxygen consumption at low concentrations, diminished the cellular ATP levels, increased the cytosolic but diminished the mitochondrial NADH/NAD(+) ratio, strongly inhibited gluconeogenesis from three different substrates (IC(50) between 1.33 and 3.61 µM), stimulated glycolysis, fructolysis, glycogenolysis and ammoniagenesis and inhibited ureogenesis. The (14)CO(2) production from [1-(14)C]octanoate and [1-(14)C]oleate was increased by usnic acid, but ketogenesis from octanoate was diminished and that from oleate was not affected. It may be concluded that the effects of usnic acid up to 2.5 µM reflect predominantly its activity as an uncoupler. At higher concentrations, however, several other effects may become significant, including inhibition of mitochondrial electron flow and inhibition of medium-chain fatty acid oxidation. In metabolic terms, toxicity of usnic acid can be predicted to be especially dangerous in the fasted state due to the combination of several deleterius events such as diminished hepatic glucose and ketone bodies output to the brain and increased ammonia production.

Raloxifene affects fatty acid oxidation in livers from ovariectomized rats by acting as a pro-oxidant agent.

Estrogen deficiency accelerates the development of several disorders including visceral obesity and hepatic steatosis. The predisposing factors can be exacerbated by drugs that affect hepatic lipid metabolism. The aim of the present work was to determine if raloxifene, a selective estrogen receptor modulator (SERM) used extensively by postmenopausal women, affects hepatic fatty acid oxidation pathways. Fatty acids oxidation was measured in the livers, mitochondria and peroxisomes of ovariectomized (OVX) rats. Mitochondrial and peroxisomal ß-oxidation was inhibited by raloxifene at a concentration range of 2.5-25 µM. In perfused livers, raloxifene reduced the ketogenesis from endogenous and exogenous fatty acids and increased the ß-hydroxybutyrate/acetoacetate ratio. An increase in ¹4CO2 production without a parallel increase in the oxygen consumption indicated that raloxifene caused a diversion of NADH from the mitochondrial respiratory chain to another oxidative reaction. It was found that raloxifene has a strong ability to react with H2O2 in the presence of peroxidase. It is likely that the generation of phenoxyl radical derivatives of raloxifene in intact livers led to the co-oxidation of NADH and a shift of the cellular redox state to an oxidised condition. This change can perturb other important liver metabolic processes dependent on cellular NADH/NAD⁺ ratio.

Foot-and-mouth disease in feral swine: susceptibility and transmission.

Experimental studies of foot-and-mouth disease (FMD) in feral swine are limited, and data for clinical manifestations and disease transmissibility are lacking. In this report, feral and domestic swine were experimentally infected with FMDV (A24-Cruzeiro), and susceptibility and virus transmission were studied. Feral swine were proved to be highly susceptible to A-24 Cruzeiro FMD virus by intradermal inoculation and by contact with infected domestic and feral swine. Typical clinical signs in feral swine included transient fever, lameness and vesicular lesions in the coronary bands, heel bulbs, tip of the tongue and snout. Domestic swine exhibited clinical signs of the disease within 24 h after contact with feral swine, whereas feral swine did not show clinical signs of FMD until 48 h after contact with infected domestic and feral swine. Clinical scores of feral and domestic swine were comparable. However, feral swine exhibited a higher tolerance for the disease, and their thicker, darker skin made vesicular lesions difficult to detect. Virus titration of oral swabs showed that both feral and domestic swine shed similar amounts of virus, with levels peaking between 2 to 4 dpi/dpc (days post-inoculation/days post-contact). FMDV RNA was intermittently detectable in the oral swabs by real-time RT-PCR of both feral and domestic swine between 1 and 8 dpi/dpc and in some instances until 14 dpi/12 dpc. Both feral and domestic swine seroconverted 6-8 dpi/dpc as measured by 3ABC antibody ELISA and VIAA assays. FMDV RNA levels in animal room air filters were similar in feral and domestic swine animal rooms, and were last detected at 22 dpi, while none were detectable at 28 or 35 dpi. The FMDV RNA persisted in domestic and feral swine tonsils up to 33-36 dpi/dpc, whereas virus isolation was negative. Results from this study will help understand the role feral swine may play in sustaining an FMD outbreak, and may be utilized in guiding surveillance, epidemiologic and economic models.

Pathogenicity and molecular characterization of emerging porcine reproductive and respiratory syndrome virus in Vietnam in 2007.

In 2007, Vietnam experienced swine disease outbreaks causing clinical signs similar to the 'porcine high fever disease' that occurred in China during 2006. Analysis of diagnostic samples from the disease outbreaks in Vietnam identified porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV-2). Additionally, Escherichia coli and Streptococcus equi subspecies zooepidemicus were cultured from lung and spleen, and Streptococcus suis from one spleen sample. Genetic characterization of the Vietnamese PRRSV isolates revealed that this virus belongs to the North American genotype (type 2) with a high nucleotide identity to the recently reported Chinese strains. Amino acid sequence in the nsp2 region revealed 95.7-99.4% identity to Chinese strain HUN4, 68-69% identity to strain VR-2332 and 58-59% identity to strain MN184. A partial deletion in the nsp2 gene was detected; however, this deletion did not appear to enhance the virus pathogenicity in the inoculated pigs. Animal inoculation studies were conducted to determine the pathogenicity of PRRSV and to identify other possible agents present in the original specimens. Pigs inoculated with PRRSV alone and their contacts showed persistent fever, and two of five pigs developed cough, neurological signs and swollen joints. Necropsy examination showed mild to moderate bronchopneumonia, enlarged lymph nodes, fibrinous pericarditis and polyarthritis. PRRSV was re-isolated from blood and tissues of the inoculated and contact pigs. Pigs inoculated with lung and spleen tissue homogenates from sick pigs from Vietnam developed high fever, septicaemia, and died acutely within 72 h, while their contact pigs showed no clinical signs throughout the experiment. Streptococcus equi subspecies zooepidemicus was cultured, and PRRSV was re-isolated only from the inoculated pigs. Results suggest that the cause of the swine deaths in Vietnam is a multifactorial syndrome with PRRSV as a major factor.

Genetic identification of novel poxviruses of cetaceans and pinnipeds.

Novel poxviruses were identified in skin lesions of several species of cetaceans and pinnipeds using polymerase chain reaction targeting DNA polymerase and DNA topoisomerase I genes of members of the subfamily Chordopoxvirinae. With the exception of parapoxviruses, no molecular data of marine mammal poxviruses were available to infer genetic and evolutionary relatedness to terrestrial vertebrate poxviruses. Viruses were assigned to a cetacean poxvirus 1 (CPV-1) group based on nucleotide and amino acid identities of gene fragments amplified from skin lesions of Asian bottlenose (Tursiops aduncus), Atlantic bottlenose (Tursiops truncatus), rough-toothed (Steno bredanensis), and striped (Stenella coeruleoalba) dolphins. A different poxvirus was detected in skin lesions of a bowhead whale (Balaena mysticetus) and provisionally assigned to a CPV-2 group. These viruses showed highest identity to terrestrial poxviruses of the genera Orthopoxvirus and Suipoxvirus. A novel species-specific poxvirus was also identified in skin lesions of Steller sea lions (Eumetopias jubatus). None of these poxviruses were found to have amplifiable hemagglutinin gene sequences. Novel parapoxviruses were also identified in skin lesions of Steller sea lions and spotted seals (Phoca largha). A significant degree of divergence was observed in sequences of Steller sea lion parapoxviruses, while those of spotted seals and harbor seals (Phoca vitulina) were highly conserved.

Efficiency of combined methotrexate/chloroquine therapy in adjuvant-induced arthritis.

The present study evaluates the effects of methotrexate (MTX) and chloroquine (CQ), and of combined MTX + CQ treatment, on the inflammatory response and on plasma and liver phosphatase and transaminase activities, employing an adjuvant-induced arthritis model in rats. Arthritis was induced by the intradermal injection of a suspension of Mycobacterium tuberculosis in mineral oil into the plantar surface of the hind paws. Development of the inflammatory response was assessed over a 21-day period. Animal groups received either: (i) MTX, administered i.p., weekly, in 0.15, 1.5, 3, 6 or 12 mg/kg doses; (ii) CQ, given intragastrically, in daily 25 or 50 mg/kg doses; or (iii) MTX + CQ, administered in two combinations (MTX1.5 mg/kg + CQ50 mg/kg, or MTX6 mg/kg + CQ50 mg/kg). At the end of the experimental period, the animals were anesthetized and killed, blood and liver samples were collected and prepared for measurement of acid and alkaline phosphatase (AP, ALP), and aspartate (AST) and alanine aminotransferase (ALT) activities. MTX at 6 and 12 mg/kg reduced the inflammatory response while CQ had no effect. MTX6 mg/kg + CQ50 mg/kg reduced the inflammatory response similar to MTX12 mg/kg, without affecting the bone marrow. Plasma AP and liver ALP activities were very elevated in the arthritic rats. While MTX treatment partially reduced both plasma AP and liver ALP activities at all doses used in the arthritic rats, CQ treatment reduced plasma AP, but increased liver AP activity. MTX + CQ treatment decreased plasma AP and liver ALP activities in the arthritic rats to control values. Plasma and liver AST activities were unaltered in the arthritic rats, and were unaffected by treatment. However, plasma and liver ALT activities were significantly reduced in the arthritic rats. While MTX or CQ treatment did not alter plasma transaminase activity in the arthritic rats, after MTX + CQ treatment, plasma ALT activity returned to normal values. In conclusion, the present data suggest that MTX + CQ treatment provides more effective anti-inflammatory protection against adjuvant-induced arthritis than does MTX alone, reverting the alterations in enzyme activities induced by this inflammatory disease in rats.

DNA strand breaks in the lymphocytes of workers exposed to diisocyanates: indications of individual differences in susceptibility after low-dose and short-term exposure.

Diisocyanates are chemically reactive and induce asthma, but data on genotoxic effects of diisocyanates in humans are limited. The investigation presented here used short term diisocyanate chamber exposure to study DNA strand breaks in lymphocytes of 10 healthy individuals and of 42 workers, with airway symptoms, who had previously been exposed to diisocyanates. The alkaline version of the Comet assay was used to analyse DNA strand breaks in lymphocytes. In addition, blood samples of 10 further control individuals without any exposure to diisocyanates were studied. Substances studied were 4,4'-methylenediphenyldiisocyanate (MDI, n=25), 2,4-toluenediisocynate and 2,6-toluenediisocyanate (TDI, n=5), and 1,6-hexamethylenediisocyanate (HDI, n=12), at concentrations between 5 and 30 ppb for 2 h. Lymphocytes isolated from the subjects before exposure and 30 min and 19 h after were used to evaluate DNA damage. No significant changes in DNA strand-break frequencies were measured, as Olive tail moment (OTM), either between groups or before and after diisocyanate exposure. OTM was similar in subjects with an asthmatic reaction (MDI, n=5; TDI, n=1; HDI, n=1) and in subjects without such a reaction. However, a small and susceptible group (about 10% of the individuals studied) could be identified with higher frequencies of DNA strand breaks in lymphocytes after chamber exposure. The occurrence of DNA damage in this group may be based on indirect mechanisms such as oxidative stress or apoptosis.

Transport, transformation and distribution space of propofol in the rat liver studied by means of the indicator-dilution technique.

1. The transport, transformation and distribution space of the endovenous anaesthetic propofol in the isolated perfused rat liver were investigated by using the multiple-indicator dilution technique with constant infusion (step input). 2. The behaviour of propofol in the liver was described by a space-distributed variable transit-time model. The drug permeated the cell membrane at very high rates and its distribution into the cellular space was flow-limited. The apparent distribution space of propofol varied between 284 and 125 times the water space, and was inversely related to the tested portal concentrations (33-250 microM). 3. The corresponding ratios of intra- to extracellular concentration varied between 319 and 187, revealing a very high affinity of the liver for propofol. They most probably reflect binding to several cellular structures, including membranes and proteins. 3. The single-pass rate coefficients for biotransformation decreased with increases in the portal concentration of propofol. The liver released significant amounts of 4-hydroxypropofol, reaching 41.7 % of the total single pass of 67.2 microM propofol biotransformation. These results disprove previous notions that hydroxylation is rate limiting for conjugation and suggest that the liver might function as a 4-hydroxypropofol source for conjugation to glucuronic acid or sulfate in other tissues.

Actions of quercetin on gluconeogenesis and glycolysis in rat liver.

1. The action of quercetin on glucose catabolism and production was investigated in the perfused rat liver. 2. Quercetin inhibited lactate production from glucose: 80% inhibition was found at a quercetin concentration of 100 micro M, and at higher concentrations inhibition was complete. 3. Pyruvate production from glucose presented a complex pattern, but stimulation was evident at 100 and 300 micro M quercetin. Oxygen uptake tended to be increased. 4. Glucose synthesis from lactate and pyruvate was inhibited. Inhibition was already evident at 50 micro M quercetin and almost complete at 300 micro M. Concomitantly, the increment in oxygen uptake caused by lactate plus pyruvate was stimulated by 50 micro M quercetin, but clearly inhibited by higher concentrations (100-500 micro M). 5. Glucose phosphorylation in the high-speed supernatant fractions of liver homogenates was inhibited by quercetin, but only at concentrations above 150 micro M. 6. It is concluded that quercetin can inhibit both glucose degradation and production and increase the cytosolic NAD(+)/NADH ratio. 7. These effects are likely to arise from many causes. Reduction of oxidative phosphorylation, inhibition of Na(+)-K(+)-ATPase, inhibition of glucokinase and inhibition of glucose 6-phosphatase could all contribute to the overall action of quercetin.

Action of quercetin on glycogen catabolism in the rat liver.

1. The influence of quercetin on glycogen catabolism and related parameters was investigated in the isolated perfused rat liver and subcellular systems. 2. Quercetin stimulated glycogenolysis (glucose release). This effect was already evident at a concentration of 50 microM maximal at 300 microM and declined at higher concentrations. Quercetin also stimulated oxygen consumption, with a similar concentration dependence. 3. Lactate production from endogenous glycogen (glycolysis) was diminished by quercetin without significant changes in pyruvate production. 4. Quercetin did not inhibit glucose transport into cells but decreased intracellular sequestration of [5-(3)H]glucose under conditions of net glucose release. 5. In isolated mitochondria, quercetin diminished the energy transduction efficiency. It also inhibited several enzymatic activities, e.g. the K(+)-ATPase/Na(+)-ATPase of plasma membrane vesicles and the glucose 6-phosphatase of isolated microsomes. 6. No significant changes of the cellular contents of AMP, ADP and ATP were found. The cellular content of glucose 6-phosphate, however, was increased (3.12-fold). 7. Some of the effects of quercetin (glycogenolysis stimulation) can be attributed to its action on mitochondrial energy metabolism, as, for example, uncoupling of oxidative phosphorylation. However, the multiplicity of the effects on several enzymatic systems certainly produces an intricate interplay that also generates complex and apparently contradictory effects.

Glycogen levels and glycogen catabolism in livers from arthritic rats.

Hepatic glycogen catabolism and glycogen levels in rats with chronic arthritis were investigated. At 9:00 a.m., the hepatic glycogen contents of ad libitum fed arthritic and normal rats were 225.5+/-17.7 and 332.1+/-28.6 micromol glucosyl units x (g liver)(-1), respectively. Food intake of arthritic and normal rats was equal to 100.1+/-6.7 and 105.0+/-3.1 mg x (g body w)(-1) x (per 24 h)(-1), respectively. In isolated perfused livers from normal and arthritic rats the rates of glucose, lactate and pyruvate release were the same when substrate- and hormone-free perfusion was performed. During an infusion period of 20 min glucagon caused an increment in glucose release of 35.3+/-4.7 micromol x (g liver)(-1) in livers from arthritic rats; in the normal condition the corresponding increment was 69.6+/-5.7 micromol x (g liver)(-1). Lactate and pyruvate productions (indicators of glycolysis) were diminished by glucagon in livers from normal rats; in the arthritic condition an initial stimulation was found, followed by a slow decay, which did not result in significant inhibition at the end of the glucagon infusion period (20 min). The actions of cAMP and dibutyryl-cAMP were similar to those of glucagon. It was concluded that livers from arthritic rats show an impaired capacity of releasing glucose under the stimulus of glucagon. This can be partly due to the lower glycogen levels and partly to a smaller capacity of inhibiting glycolysis. Reduction in glycogen levels was not associated with reduction in food intake or failure in the energetic state of the hepatic cells. These changes in glycogen metabolism may be related to reduced gluconeogenic capacity of the livers and/or to production of inflammatory mediators observed in the arthritis disease.

Changes in distribution spaces and cell permeability caused by ATP in the rat liver.

AIMS/BACKGROUND: Cellular and extracellular volume changes caused by ATP were investigated in the liver as well as the possible formation of diffusion barriers, which could be responsible for some of its metabolic effects. METHODS: The experimental system was the bivascularly perfused rat liver. [(14)C]Sucrose and [(3)H]water were simultaneously injected into either the portal vein or the hepatic artery. Mean transit times, distribution spaces, variances and linear superimpositions were calculated. RESULTS: In the portal system, ATP reduced the transit time in the great vessels, had little or no effect on sinusoidal and cellular spaces, but impaired the flow-limited distribution of both [(14)C]sucrose and [(3)H]water. In the arterial bed ATP infused into either the portal vein or the hepatic artery produced vasodilation and increased the aqueous extra-sucrose space. These effects were inhibited by Nomega-nitro-L-arginine methyl ester infused into the hepatic artery. CONCLUSIONS: Sucrose and extra-sucrose space changes caused in the arterial bed by portally infused ATP are most probably analogous to the transhepatic vasodilation effect already described for the rabbit liver. Impairment of flow-limited distribution of tracers in the sinusoidal bed indicates that ATP induces the formation of permeability barriers, which could be responsible for some of its metabolic effects.

The uncoupling effect of the nonsteroidal anti-inflammatory drug nimesulide in liver mitochondria from adjuvant-induced arthritic rats.

The aim of the present study was to evaluate the changes caused by adjuvant-induced arthritis in liver mitochondria and to investigate the effects of the nonsteroidal anti-inflammatory drug nimesulide. The main alterations observed in liver mitochondria from arthritic rats were: higher rates of state IV and state III respiration with beta-hydroxybutyrate as substrate; reduced respiratory control ratio and impaired capacity for swelling dependent on beta-hydroxybutyrate oxidation. No alterations were found in the activities of NADH oxidase and ATPase. Nimesulide produced: (1) stimulation of state IV respiration; (2) decrease in the ADP/O ratio and in the respiratory control ratio; (3) stimulation of ATPase activity of intact mitochondria; (4) inhibition of swelling driven by the oxidation of beta-hydroxybutyrate; (5) induction of passive swelling due to NH(3)/NH(4)+ redistribution. The activity of NADH oxidase was insensitive to nimesulide. Mitochondria from arthritic rats showed higher sensitivity to nimesulide regarding respiratory activity. The results of this work allow us to conclude that adjuvant-induced arthritis leads to quantitative changes in some mitochondrial functions and in the sensitivity to nimesulide. Direct evidence that nimesulide acts as an uncoupler was also presented. Since nimesulide was active in liver mitochondria at therapeutic levels, the impairment of energy metabolism could lead to disturbances in the liver responses to inflammation, a fact that should be considered in therapeutic intervention.

Zymosan-induced changes in glucose release and fatty acid oxidation in the perfused rat liver.

The aim of the present study was to investigate the actions of zymosan on glucose release and fatty acid oxidation in perfused rat livers and to determine if Kupffer cells and Ca2+ ions are implicated in these actions. Zymosan caused stimulation of glycogenolysis in livers from fed rats. In livers from fasted rats zymosan caused gradual inhibition of glucose production and oxygen consumption from lactate plus pyruvate. Ketogenesis, oxygen consumption, and [14C-]-CO2 production were inhibited by zymosan when the [1-14C]-palmitate was supplied exogenously. However, ketogenesis and oxygen consumption from endogenous sources were not inhibited. An interference with substrate-uptake by the liver may be the cause of the changes in gluconeogenesis and oxidation of fatty acids from exogenous sources. The pretreatment of the rats with gadolinium chloride and the removal of Ca2+ ions did not suppress the effects of zymosan on glucose release, a finding that argues against the participation of Kupffer cells or Ca2+ ions in the liver responses. The hepatic metabolic changes caused by zymosan could play a role in the systemic metabolic alterations reported to occur after in vivo zymosan administration.

Effects of norepinephrine on the metabolism of fatty acids with different chain lengths in the perfused rat liver.

The effects of norepinephrine on ketogenesis in isolated hepatocytes have been reported as ranging from stimulation to inhibition. The present work was planned with the aim of clarifying these discrepancies. The experimental system was the once-through perfused liver from fasted and fed rats. Fatty acids with chain lengths varying from 8-18 were infused. The effects of norepinephrine depended on the metabolic state of the rat and on the nature of the fatty acid. Norepinephrine clearly inhibited ketogenesis from long-chain fatty acids (stearate > palmitate > oleate), but had little effect on ketogenesis from medium-chain fatty acids (octanoate and laureate). With palmitate the decrease in oxygen uptake was restricted to the substrate stimulated portion; with stearate, the decrease exceeded the substrate stimulated portion; with oleate, oxygen uptake was transiently inhibited. Withdrawal of Ca2+ attenuated the inhibitory effects. 14CO2 production from [1-14C]oleate was inhibited. Net uptake of the fatty acids was not affected by norepinephrine. In livers from fed rats, oxygen uptake and ketogenesis from stearate were only transiently inhibited. The conclusions are: (a) in the fasted state norepinephrine reduces ketogenesis and respiration by means of a Ca2+-dependent mechanism; (b) the degree of inhibition varies with the chain length and the degree of saturation of the fatty acids; (c) norepinephrine favours esterification of the activated long-chain fatty acids in detriment to oxidation; (d) in the fed state the stimulatory action of norepinephrine on glycogen catabolism induces conditions which are able to reverse inhibition of ketogenesis and oxygen uptake.

Uptake of lactate by the liver: effect of red blood cell carriage.

Multiple-indicator dilution experiments with labeled lactate were performed in the livers of anesthetized dogs. A mixture of (51)Cr-labeled erythrocytes, [(3)H]sucrose, and L-[1-(14)C]lactate or a mixture of (51)Cr-labeled erythrocytes, [(14)C]sucrose, and L-[2-(3)H]lactate was injected into the portal vein, and samples were obtained from the hepatic vein. Data were evaluated using a model comprising flow along sinusoids, exchange of lactate between plasma and erythrocytes and between plasma and hepatocytes, and, in the case of L-[1-(14)C]lactate, metabolism to H[(14)C]O(-)(3) within hepatocytes. The coefficient for lactate efflux from erythrocytes was 0.62 +/- 0.24 s(-1), and those for influx into and efflux from hepatocytes were 0.44 +/- 0.13 and 0.14 +/- 0.07 s(-1), respectively. The influx permeability-surface area product of the hepatocyte membrane for lactate (P(in)S, in ml x s(-1) x g(-1)) varied with total flow rate (F, in ml s(-1) x g(-1)) according to P(in)S = (3.1 +/- 0.5)F + (0.021 +/- 0.014). Lactate in plasma, erythrocytes, and hepatocytes was close to equilibrium, whereas lactate metabolism was rate limiting.

Glucose phosphorylation capacity and glycolysis in the liver of arthritic rats.

OBJECTIVE AND DESIGN: Glycolysis and the glucose phosphorylation capacity of livers from arthritic rats were studied because alterations in these parameters are suggested by some studies. SUBJECTS: Arthritis was induced in male albino rats (Wistar; 180-220 g). TREATMENT: The animals were injected with 100 microl heat inactivated Mycobacterium tuberculosis suspended in mineral oil at a concentration of 0.5% (w/v). Animals showing lesions after 14 to 21 days were selected. METHODS: Glucose phosphorylation was measured in the high speed supernatant fraction of liver homogenates and glycolysis in the isolated perfused liver. RESULTS: The glucose concentration for half-maximal rates was reduced from 18.32+/-5.69 in normal to 9.84+/-3.15 mM in arthritic rats (p = 0.024). Vmax was increased from 8.77+/-0.27 in normal to 11.49+/-0.29 nmol min(-1) mg protein(-1) in arthritic rats (p = 0.001). Perfused livers from arthritic rats showed a 2.43-fold higher rate of glycolysis. CONCLUSIONS: Livers from arthritic rats present a higher glucose phosphorylation capacity. Possibly this phenomenon is caused by circulating inflammatory mediators produced during adjuvant-induced arthritis.

Transport of cyclic AMP and synthetic analogs in the perfused rat liver.

The purpose of the present work was to investigate the transport of cyclic AMP (cAMP) and analogs in the rat liver. The experimental system was the isolated once-through perfused liver. Transport was measured by employing the multiple-indicator dilution technique. The single-pass recovery of tracer [(32)P]cAMP was equal to 94.4 +/- 1. 4%; no significant extracellular transformation of cAMP occurred during a single passage. The unidirectional influx rates of dibutyryl-cAMP were a saturable function of its concentration, with K(m) = 72.75 +/- 9.24 microM and V(max) = 0.464 +/- 0.026 micromol min(-1) (mL cellular space)(-1). The unidirectional influx rates of cAMP were much lower than those of dibutyryl-cAMP and were a linear function of the concentration (up to 100 microM). The transfer coefficient for influx (k(in)) was equal to 0.860 +/- 0.058 mL min(-1) (mL extracellular space)(-1). cAMP inhibited the influx of dibutyryl-cAMP; the IC(50) was 0.83 mM. The following series of increasing unidirectional influx rates was found: cAMP < monobutyryl-cAMP approximately 2-aza-epsilon-cAMP < rp-cAMPS approximately sp-cAMPS < 8-Br-cAMP approximately dibutyryl-cGMP approximately 8-Cl-cAMP < O-dibutyryl-cAMP. There was no precise correlation between the rates of influx of the various cyclic nucleotides and their lipophilicity. It was concluded that the penetration of cAMP and its analogs into the liver cells was a facilitated process. Lipophilicity was not the only factor determining the rate of transport. The transformation of dibutyryl-cAMP was limited by both transport and activity of the intracellular enzymic systems. The intracellular transformation of exogenous cAMP, however, was limited by the transport process.

Modeling the transformation of exogenously supplied cAMP in the perfused rat liver.

A kinetic model describing the behavior of extracellularly supplied cAMP in the perfused rat liver was derived and compared with experimental data. The model was based on the following conditions and assumptions: a) labeled cAMP is being constantly infused (step input); b) permeation of the cell membrane is an essentially irreversible step (k(in) as transfer coefficient); c) the adenine moiety of cAMP incorporates into a nucleotide pool (km1 as transformation coefficient), which cannot permeate the cell membrane; d) the adenine moiety of cAMP can be transferred from the nucleotide pool to a nucleoside + free base pool (km2 as transformation coefficient), which is able to permeate the cell membrane (k(ef) as transfer coefficient). These events were described by a series of differential equations for which an analytical solution was obtained. Total cellular incorporation of label derived from [3H]cAMP was measured in the isolated perfused rat liver. The equations of the model were fitted to these experimental data by means of a least-squares procedure. In the fitting procedure the previously determined k(in) value (0.55 ml min(-1) ml cellular space(-1)) was used. The model is able to describe the experimental data (correlation coefficient = 0.993 +/- 0.008) with km1, km2 and k(ef) values of 17.11, 0.0948 and 1.385 min(-1), respectively. Simulations revealed the following sequence of decreasing intracellular pool sizes: nucleotide pool > nucleoside + free base pool > intracellular cAMP. The intracellular cAMP concentrations correspond to only 3.2% of the extracellular ones. This low proportion explains why it was generally difficult to detect cAMP in the cell space when this compound was added to an isolated cell system. The model and the parameters determined in the present work can be used to predict intracellular cAMP concentrations in the perfused liver for specific extracellular concentrations.

Gluconeogenesis in the liver of arthritic rats.

The gluconeogenic response in the liver from rats with chronic arthritis to various substrates and the effects of glucagon were investigated. The experimental technique used was the isolated liver perfusion. Hepatic gluconeogenesis in arthritic rats was generally lower than in normal rats. The difference between normal and arthritic rats depended on the gluconeogenic substrate. In the absence of glucagon the following sequence of decreasing differences was found: alanine (-71.8 per cent) reverse similarglutamine (-71.7 per cent)>pyruvate (-60 per cent)>lactate+pyruvate (-44.9 per cent)>xylitol (n.s.=non-significant) reverse similarglycerol (n.s.). For most substrates glucagon increased hepatic gluconeogenesis in both normal and arthritic rats. The difference between normal and arthritic rats, however, tended to diminish, as revealed by the data of the following sequence: alanine (-48.9 per cent) reverse similarpyruvate (-47.6 per cent)>glutamine (-33.8 per cent)>glycerol (n.s.) reverse similarlactate+pyruvate (n.s.) reverse similarxylitol (n.s.). The causes for the reduced hepatic gluconeogenesis in arthritic rats are probably related to: (a) lower activities of key enzymes catalyzing most probably steps preceding phosphoenolpyruvate (e.g. phosphoenolpyruvate carboxykinase, pyruvate carboxylase, etc. ); (b) a reduced availability of reducing equivalents in the cytosol; (c) specific differences in the situations induced by hormones or by the individual substrates. Since glycaemia is almost normal in chronically arthritic rats, it seems that lower gluconeogenesis is actually adapted to the specific needs of these animals.