Lactate signaling and fuel selection in rainbow trout: mobilization of energy reserves.

Lactate is now recognized as a regulator of fuel selection in mammals because it inhibits lipolysis by binding to the hydroxycarboxylic acid receptor 1 (HCAR1). The goals of this study were to quantify the effects of exogenous lactate on: 1) lipolytic rate or rate of appearance of glycerol in the circulation (Ra glycerol) and hepatic glucose production (Ra glucose), and 2) key tissue proteins involved in lactate signaling, glucose transport, glycolysis, gluconeogenesis, lipolysis, and ß-oxidation in rainbow trout. Measurements of fuel mobilization kinetics show that lactate does not affect lipolysis as it does in mammals (Ra glycerol remains at 7.3 ± 0.5 µmol·kg-1·min-1), but strongly reduces hepatic glucose production (16.4 ± 2.0 to 8.9 ± 1.2 µmol·kg-1·min-1). This reduction is likely induced by decreasing gluconeogenic flux through the inhibition of cytosolic phosphoenolpyruvate carboxykinase (Pck1, alternatively called Pepck1; 60% and 24% declines in gene expression and protein level, respectively). It is also caused by lactate substituting for glucose as a fuel in all tissues except white muscle that increases glut4a expression and has limited capacity for monocarboxylate transporter (Mct)-mediated lactate import. We conclude that lipolysis is not affected by hyperlactatemia because trout show no activation of autocrine Hcar1 signaling (gene expression of the receptor is unchanged or even repressed in red muscle). Lactate regulates fuel mobilization via Pck1-mediated suppression of gluconeogenesis and by replacing glucose as a fuel. This study highlights important functional differences in the Hcar1 signaling system between fish and mammals for the regulation of fuel selection.

The mammalian insulin antagonist S961 does not exhibit insulin receptor antagonism in rainbow trout in vivo.

Due to their reported 'glucose-intolerant' phenotype, rainbow trout have been the focus of comparative studies probing underlying endocrine mechanisms at the organismal, tissue and molecular level. A particular focus has been placed on the investigation of the comparative role of insulin, an important glucoregulatory hormone, and its interaction with macronutrients. A limiting factor in the comparative investigation of insulin is the current lack of reliable assays to quantify circulating mature and thus bioactive insulin. To circumvent this limitation, tissue-specific responsiveness to postprandial or exogenous insulin has been quantified at the level of post-translational modifications of cell signalling proteins. These studies revealed that the insulin responsiveness of these proteins and their post-translational modifications are evolutionarily highly conserved and thus provide useful and quantifiable proxy indices to investigate insulin function in rainbow trout. While the involvement of specific branches of the intracellular insulin signalling pathway (e.g., mTor) in rainbow trout glucoregulation have been successfully probed through pharmacological approaches, it would be useful to have a functionally validated insulin receptor antagonist to characterize the glucoregulatory role of the insulin receptor pathway in its entirety for this species. Here, we report two separate in vivo experiments to test the ability of the mammalian insulin receptor antagonist, S961, to efficiently block insulin signalling in liver and muscle in response to endogenously released insulin and to exogenously infused bovine insulin. We found that, irrespective of the experimental treatment or dose, activation of the insulin pathway in liver and muscle was not inhibited by S961, showing that its antagonistic effect does not extend to rainbow trout.

Epigenetic and post-transcriptional repression support metabolic suppression in chronically hypoxic goldfish.

Goldfish enter a hypometabolic state to survive chronic hypoxia. We recently described tissue-specific contributions of membrane lipid composition remodeling and mitochondrial function to metabolic suppression across different goldfish tissues. However, the molecular and especially epigenetic foundations of hypoxia tolerance in goldfish under metabolic suppression are not well understood. Here we show that components of the molecular oxygen-sensing machinery are robustly activated across tissues irrespective of hypoxia duration. Induction of gene expression of enzymes involved in DNA methylation turnover and microRNA biogenesis suggest a role for epigenetic transcriptional and post-transcriptional suppression of gene expression in the hypoxia-acclimated brain. Conversely, mechanistic target of rapamycin-dependent translational machinery activity is not reduced in liver and white muscle, suggesting this pathway does not contribute to lowering cellular energy expenditure. Finally, molecular evidence supports previously reported chronic hypoxia-dependent changes in membrane cholesterol, lipid metabolism and mitochondrial function via changes in transcripts involved in cholesterol biosynthesis, ß-oxidation, and mitochondrial fusion in multiple tissues. Overall, this study shows that chronic hypoxia robustly induces expression of oxygen-sensing machinery across tissues, induces repressive transcriptional and post-transcriptional epigenetic marks especially in the chronic hypoxia-acclimated brain and supports a role for membrane remodeling and mitochondrial function and dynamics in promoting metabolic suppression.

Alanine alters the carbohydrate metabolism of rainbow trout: glucose flux and cell signaling.

In rainbow trout, dietary carbohydrates are poorly metabolized compared with other macronutrients. One prevalent hypothesis suggests that high dietary amino acid levels could contribute to the poor utilization of carbohydrates in trout. In mammals, alanine is considered an important gluconeogenic precursor, but has recently been found to stimulate AMP-activated protein kinase (AMPK) to reduce glucose levels. In trout, the effect of alanine on glucose flux is unknown. The goal of this study was to determine the effects of 4 h exogenous alanine infusion on glucose metabolism in rainbow trout. Glucose flux, and the rate of glucose appearance (Ra) and disposal (Rd) were measured in vivo. Key glycolytic and gluconeogenic enzyme expression and activity, and cell signaling molecules relevant to glucose metabolism were assessed in the liver and muscle. The results show that alanine inhibits glucose Ra (from 13.2±2.5 to 7.3±1.6 µmol kg-1 min-1) and Rd (from 13.2±2.5 to 7.4±1.5 µmol kg-1 min-1) and the slight mismatch between Ra and Rd caused a reduction in glycemia, similar to the effects of insulin in trout. The reduction in glucose Rd can be partially explained by a reduction in glut4b expression in red muscle. In contrast to mammals, trout alanine-dependent glucose-lowering effects did not involve hepatic AMPK activation, suggesting a different mechanistic basis. Interestingly, protein kinase B (AKT) activation increased only in muscle, similar to effects observed in insulin-infused trout. We speculate that alanine-dependent effects were probably mediated through stimulation of insulin secretion, which could indirectly promote alanine oxidation to provide the needed energy.

Escitalopram (S-citalopram) and its metabolites in vitro: cytochromes mediating biotransformation, inhibitory effects, and comparison to R-citalopram.

Transformation of escitalopram (S-CT), the pharmacologically active S-enantiometer of citalopram, to S-desmethyl-CT (S-DCT), and of S-DCT to S-didesmethyl-CT (S-DDCT), was studied in human liver microsomes and in expressed cytochromes (CYPs). Biotransformation of the R-enantiomer (R-CT) was studied in parallel. S-CT was transformed to S-DCT by CYP2C19 (K(m) = 69 microM), CYP2D6 (K(m) = 29 microM), and CYP3A4 (K(m) = 588 microM). After normalization for hepatic abundance, relative contributions to net intrinsic clearance were 37% for CYP2C19, 28% for CYP2D6, and 35% for CYP3A4. At 10 microM S-CT in liver microsomes, S-DCT formation was reduced to 60% of control by 1 microM ketoconazole, and to 80 to 85% of control by 5 microM quinidine or 25 microM omeprazole. S-DDCT was formed from S-DCT only by CYP2D6; incomplete inhibition by quinidine in liver microsomes indicated participation of a non-CYP pathway. Based on established index reactions, S-CT and S-DCT were negligible inhibitors (IC(50) > 100 microM) of CYP1A2, -2C9, -2C19, -2E1, and -3A, and weakly inhibited CYP2D6 (IC(50) = 70-80 microM). R-CT and its metabolites, studied using the same procedures, had properties very similar to those of the corresponding S-enantiomers. Thus S-CT, biotransformed by three CYP isoforms in parallel, is unlikely to be affected by drug interactions or genetic polymorphisms. S-CT and S-DCT are also unlikely to cause clinically important drug interactions via CYP inhibition.

Relative contributions of CYP2C9 and 2C19 to phenytoin 4-hydroxylation in vitro: inhibition by sulfaphenazole, omeprazole, and ticlopidine.

OBJECTIVES: To determine the relative contribution of cytochromes P450 (CYP) 2C9 and 2C19 to the formation of 5-(-4-hydroxyphenyl)-5-phenylhydantion (HPPH) from phenytoin (PPH). DESIGN: Hydroxylation of PPH to form HPPH was studied in vitro using human liver microsomes and microsomes from cDNA-transfected human lymphoblastoid cells. RESULTS: Formation of HPPH from PPH in liver microsomes had a mean (+/- SEM) apparent Km [substrate concentration corresponding to 50% of maximal reaction velocity (Vmax)] of 23.6 +/- 1.8 mumol/l. Coincubation with the CYP2C9 inhibitor, sulfaphenazole (SPA), at 5 mumol/l reduced reaction velocity to less than 15% of control values. The mean inhibitor concentration at which 50% inhibition is achieved (IC50 value) for SPA versus PPH hydroxylation (0.49 microM) was similar to the SPA IC50 versus flurbiprofen hydroxylation (0.46 microM) and tolbutamide hydroxylation (0.7-1.5 microM). In contrast, the CYP2C19 inhibitor omeprazole (OME) at 10 mumol/l produced only a small degree of inhibition. Incubation of PPH with microsomes from cDNA-transfected human lymphoblastoid cells containing CYP1A2, 2A6, 2B6, 2C8, 2D6, 2E1, or 3A4 yielded no detectable formation of HPPH. Only CYP2C9 and 2C19 had PPH hydroxylation activity, with apparent Km values for the high-affinity component of 14.6 mumol/l and 24.1 mumol/l, respectively. Based on Vmax values in liver microsomes, the Vmax and Km values in expressed CYPs and the relative abundance of the two isoforms in human liver, CYP2C9, and 2C19 were estimated to have relative contributions of 90% and 10%, respectively, to net intrinsic clearance. CONCLUSIONS: Formation of HPPH from PPH is mediated exclusively by CYP2C9 and 2C19, with CYP2C9 playing the major role.

Inhibition of human cytochrome P450 isoforms by nonnucleoside reverse transcriptase inhibitors.

The capacity of three clinically available nonnucleoside reverse transcriptase inhibitors (NNRTIs) to inhibit the activity of human cytochromes P450 (CYPs) was studied in vitro using human liver microsomes. Delavirdine, nevirapine, and efavirenz produced negligible inhibition of phenacetin O-deethylation (CYP1A2) or dextromethorphan O-demethylation (CYP2D6). Nevirapine did not inhibit hydroxylation of tolbutamide (CYP2C9) or S-mephenytoin (CYP2C19), but these CYP isoforms were importantly inhibited by delavirdine and efavirenz. This indicates the likelihood of significantly impaired clearance of CYP2C substrate drugs (such as phenytoin, tolbutamide, and warfarin) upon initial exposure to these two NNRTIs. Delavirdine and efavirenz (but not nevirapine) also were strong inhibitors of CYP3A, consistent with clinical hazards of initial cotreatment with either of these drugs and substrates of CYP3A. The in vitro microsomal model provides relevant predictive data on probable drug interactions with NNRTIs when the mechanism is inhibition of CYP-mediated drug biotransformation. However, the model does not incorporate interactions attributable to enzyme induction.

Inhibition of human cytochrome P450 isoforms in vitro by zafirlukast.

Zafirlukast is a cysteinyl leukotriene antagonist used to treat allergic and exercise-induced asthma. This in vitro study used human liver microsomes to evaluate the inhibitory activity of zafirlukast versus six human cytochrome P450 (CYP) isoforms. Zafirlukast (0-250 microM) was co-incubated with fixed concentrations of index substrates. Zafirlukast inhibited the hydroxylation of tolbutamide (CYP2C9; mean IC(50)=7.0 microM), triazolam (CYP3A; IC(50)=20.9 microM) and S-mephenytoin (CYP2C19; IC(50)=32.7 microM), and was a less potent inhibitor of phenacetin O-deethylation (CYP1A2; IC(50)=56 microM) and dextromethorphan O-demethylation (CYP2D6; IC(50)=116 microM). Zafirlukast produced negligible inhibition of CYP2E1. In vitro inhibition of CYP2C9 by zafirlukast is consistent with clinical studies showing impaired clearance of S-warfarin and enhanced anti-thrombotic effects, although the in vitro IC(50) value is higher than the usual range of clinically relevant plasma concentrations. Zafirlukast deserves further clinical study as an inhibitor of other CYP2C9 substrates such as nonsteroidal anti-inflammatory agents, tolbutamide, phenytoin and mestranol. Clinically important inhibition by zafirlukast of other CYP isoforms is not established.

Analysis of ritonavir in plasma/serum and tissues by high-performance liquid chromatography.

A method has been developed to quantify ritonavir concentrations in human plasma and in mouse serum, liver, and brain using high-performance liquid chromatography. Extraction recoveries for ritonavir and its internal standard averaged greater than 95%. Within-day variability, expressed as a coefficient of variation, averaged 6% over the concentration range 0.5 microg/mL to 15 microg/mL ritonavir, and between-day variability averaged 5.6% over 5 microg/mL to 15 microg/mL ritonavir. The method was applied to quantitation of ritonavir in mouse serum and tissue. Measured values deviated less than 5% from the actual values in mouse serum, liver, and brain samples containing 5 microg/mL ritonavir. The slopes of calibration curves for extracted calf serum, mouse serum, mouse liver and mouse brain standards were nearly identical to the calibration slope of standards which were not extracted. All curves were linear through zero, and r2 was no less than 0.998 for any form of calibration. In addition, there was no chromatographic interference from commonly prescribed medications.