In Vivo Pharmacodynamic Target Investigation of Two Bacterial Topoisomerase Inhibitors, ACT-387042 and ACT-292706, in the Neutropenic Murine Thigh Model against Streptococcus pneumoniae and Staphylococcus aureus.

ACT-387042 and ACT-292706 are two novel bacterial topoisomerase inhibitors with broad-spectrum activity against Gram-positive and -negative bacteria, including methicillin-resistant Staphylococcus aureus and penicillin- and fluoroquinolone-resistant Streptococcus pneumoniae We used the neutropenic murine thigh infection model to characterize the pharmacokinetics (PK)/pharmacodynamics (PD) of these investigational compounds against a group of 10 S. aureus and S. pneumoniae isolates with phenotypic resistance to beta-lactams and fluoroquinolones. The in vitro activities of the two compounds were very similar (MIC range, 0.03 to 0.125 mg/liter). Plasma pharmacokinetics were determined for each compound by using four escalating doses administered by the subcutaneous route. In treatment studies, mice had 10(7.4) to 10(8) CFU/thigh at the start of therapy with ACT-387042 and 10(6.7) to 10(8.3) CFU/thigh at the start of therapy with ACT-292706. A dose-response relationship was observed with all isolates over the dose range. Maximal kill approached 3 to 4 log10 CFU/thigh compared to the burden at the start of therapy for the highest doses examined. There was a strong relationship between the PK/PD index AUC/MIC ratio (area under the concentration-time curve over 24 h in the steady state divided by the MIC) and therapeutic efficacy in the model (R(2), 0.63 to 0.82). The 24-h free-drug AUC/MIC ratios associated with net stasis for ACT-387042 against S. aureus and S. pneumoniae were 43 and 10, respectively. The 24-h free-drug AUC/MIC ratios associated with net stasis for ACT-292706 against S. aureus and S. pneumoniae were 69 and 25, respectively. The stasis PD targets were significantly lower for S. pneumoniae (P < 0.05) for both compounds. The 1-log-kill AUC/MIC ratio targets were ∼2- to 4-fold higher than stasis targets. Methicillin, penicillin, or ciprofloxacin resistance did not alter the magnitude of the AUC/MIC ratio required for efficacy. These results should be helpful in the design of clinical trials for topoisomerase inhibitors.

Levels in neurotransmitter precursor amino acids correlate with mental health in patients with breast cancer.

Breast cancer is the most common cancer among females. Approximately 30% of cancer patients develop depression or depressive adaptation disorder within 5 years post diagnosis. Low grade inflammation and subsequent changes in neurotransmitter levels could be the pathophysiological link. In the current study we investigated the association of neurotransmitter precursor amino acids with a diagnosis of depression or state anxiety in 154 subjects suffering from breast cancer (BCA(+)), depression (DPR(+)), both or neither. Sociodemographic parameters, severity of depressive symptoms, and state anxiety (ANX) were recorded. Neopterin, kynurenine/tryptophan and phenylalanine/tyrosine were analysed by HPLC or ELISA. Significantly higher serum neopterin values were found in DPR(+) patients (p = 0.034) and in ANX(+) subjects (p = 0.008), as a marker of Th1-related inflammation. The phenylalanine/tyrosine ratio (index of the catecholamine pathway) was associated with the factors "breast cancer" and "depression" and their interaction (all p < 0.001); it was highest in the DPR(+)BCA(+) group. The kynurenine/tryptophan ratio (index of the serotonin pathway) was significantly associated with the factors "breast cancer" and "state anxiety" and their interaction (p < 0.001, p = 0.026, p = 0.02, respectively); it was highest in the ANX(+)BCA(+) group. In BCA(+) patients kynurenine/tryptophan ratios correlated with severity of state anxiety (r = 0.226, p = 0.048, uncorrected) and phenylalanine/tyrosine ratios with severity of depressive symptoms (r = 0.376, p < 0.05, corrected). In conclusion, levels of neurotransmitter precursor amino acids correlate with mental health, an effect which was much more pronounced in BCA(+) patients than in BCA(-) subjects. Aside from identifying underlying pathophysiological mechanisms, these results could be the basis for future treatment studies: in BCA(+) patients with depression the use of serotonin-noradrenaline reuptake inhibitors might be recommended while in those with predominant anxiety selective serotonin reuptake inhibitors might be the treatment of choice.

[Difference and distance between the central and thinnest points of the cornea: impact of refractive state, age and ocular side]. / Differenz und Entfernung zwischen zentraler und dünnster Stelle der Hornhaut: Einfluss von Refraktion, Alter und Augenseite.

BACKGROUND: The aim of the study was to quantify the difference in corneal thickness between the central and thinnest points (∆PachyZ-PachyD), the distance between the center of the cornea and its thinnest point (vector length PachyD) and to explore the impact of refractive state, age and ocular side. PATIENTS AND METHODS: This was a multicenter, retrospective, cross-sectional study and medical records of 16,872 eyes were reviewed. The Orbscan® (Bausch and Lomb) procedure was used for pachymetry and keratometry. RESULTS: The results showed that ∆PachyZ-PachyD and vector length PachyD were higher in hyperopic eyes (∆PachyZ-PachyD: 11.99 ± 12.08 µm, vector length PachyD: 0.85 ± 0.44 mm) compared to myopic eyes (∆PachyZ-PachyD: 9.2 ± 7.86 µm, vector length PachyD: 0.7 ± 0.37 mm; p < 0.001). Refractive state, age and ocular side demonstrated an independent, statistically significant impact on ∆PachyZ-PachyD and vector length PachyD. CONCLUSIONS: As a result of the significant impact of refractive state, age and ocular side on ∆PachyZ-PachyD and vector length PachyD, these variables should be considered in a normative data collection.

The CERTAIN Registry: a novel, web-based registry and research platform for pediatric renal transplantation in Europe.

BACKGROUND: The results of pediatric renal transplantation have improved markedly in the last decade. However, a number of relevant clinical problems remain, such as organ damage caused by chronic rejection, long-term toxicity of immunosuppressive therapy, difficulty in developing tolerance-inducing protocols, secondary cardiovascular comorbidity, post-transplantation lymphoproliferative disease, suboptimal longitudinal growth, quality of life, adherence to immunosuppressive medication, and structured transition programs to adult care. These unmet clinical needs require intense collaborative and interdisciplinary clinical research. We recently founded the Cooperative European Paediatric Renal TransplAnt INitiative (CERTAIN; www.certain-registry.eu) as a research network and platform built on a novel, web-based registry. RESULTS: The registry's dataset provides essential information on generic kidney transplantation-related topics and also captures pediatric-specific topics, such as growth, physical and psychosocial development, and adherence. Due to its flexibility the system can be used as follows: (1) as a registry capturing a minimal or an extended dataset; (2) as a center and/or country-specific transplantation database; or (3) as a patient-specific electronic transplantation chart. The data can be exported directly from the CERTAIN web application into statistical software packages for scientific analyses. The rights regarding data ownership, evaluation, and publications are regulated in the registry's rules of procedure. Data quality is ensured by automatic software validation and a manual data review process. To avoid redundant data entry, CERTAIN has established interfaces for data change with Eurotransplant, the Collaborative Transplant Study (CTS), and the registry of the European Society of Pediatric Nephrology (ESPN) and European Renal Association - European Dialysis and Transplant Association (ERA-EDTA) (ESPN/ERA-EDTA registry). CERTAIN fulfils all regulatory and ethical requirements of the European Union and Germany, in particular, regarding patients' data privacy and security. CONCLUSION: Using modern information technology, the recently established multinational CERTAIN Registry fills a gap in Europe for collaborative 5 research and quality assurance in the field of pediatric renal transplantation.

In vivo and in vitro investigations of heterozygous nebulin knock-out mice disclose a mild skeletal muscle phenotype.

Nemaline myopathy is the most common congenital skeletal muscle disease, and mutations in the nebulin gene account for 50% of all cases. Recent studies suggest that the disease severity might be related to the nebulin expression levels. Considering that mutations in the nebulin gene are typically recessive, one would expect that a single functional nebulin allele would maintain nebulin protein expression which would result in preserved skeletal muscle function. We investigated skeletal muscle function of heterozygous nebulin knock-out (i.e., nebulin(+/-)) mice using a multidisciplinary approach including protein and gene expression analysis and combined in vivo and in vitro force measurements. Skeletal muscle anatomy and energy metabolism were studied strictly non-invasively using magnetic resonance imaging and 31P-magnetic resonance spectroscopy. Maximal force production was reduced by around 16% in isolated muscle of nebulin(+/-) mice while in vivo force generating capacity was preserved. Muscle weakness was associated with a shift toward a slower proteomic phenotype, but was not related to nebulin protein deficiency or to an impaired energy metabolism. Further studies would be warranted in order to determine the mechanisms leading to a mild skeletal muscle phenotype resulting from the expression of a single nebulin allele.

S-15261, a new anti-hyperglycemic agent, reduces hepatic glucose production through direct and insulin-sensitizing effects.

S-15261 is a new oral anti-hyperglycemic agent that increases insulin sensitivity in various insulin-resistant animal models. The aim of this study was to determine the short- and long-term effects of S-15261 and its metabolites (S-15511 and Y-415) on fatty acid and glucose metabolism in hepatocytes isolated from 24-h starved rats. During short-term exposure (1h) neither S-15261 nor its metabolites affected fatty acid oxidation whatever the concentration used. By contrast, S-15261 and its two metabolites reduced the rates of glucose production from lactate/pyruvate and dihydroxyacetone. Using crossover plot analysis, it was shown that Y-415 reduced hepatic gluconeogenesis upstream the formation of dihydroxyacetone phosphate. After 48 h in culture, S-15261 and its two metabolites reduced the rates of glucose production from lactate/pyruvate secondarily to a decrease in PEPCK and Glc-6-Pase mRNA levels. A part of these effects on gene expression could be due to a drug-induced reduction in PGC-1 gene expression. When hepatocytes were cultured in the presence of a submaximal concentration of insulin (10(-9)M), S-15261, through its metabolite S-15511, enhanced insulin sensitivity both on gene expression (PEPCK, Glc-6-Pase, PGC-1) and on gluconeogenesis. Furthermore, S-15261 and S-15511 induced the expression of GK and FAS genes as the result of an increased in SREBP-1c mRNA levels. Finally, S-15511 enhanced the stimulatory effect of insulin on GK mRNA level through an additional increase in SREBP-1c gene expression. In conclusion, this work reveals that S-15261 via its metabolites reduces hepatic glucose production through direct and insulin-sensitizing effects on genes encoding regulatory proteins of hepatic glucose metabolism.

Effect of metformin on fatty acid and glucose metabolism in freshly isolated hepatocytes and on specific gene expression in cultured hepatocytes.

The short-term effect of metformin on fatty acid and glucose metabolism was studied in freshly incubated hepatocytes from 24-hr starved rats. Metformin (5 or 50 mM) had no effect on oleate or octanoate oxidation rates (CO(2)+ acid-soluble products), whatever the concentration used. Similarly, metformin had no effect on oleate esterification (triglycerides and phospholipid synthesis) regardless of whether the hepatocytes were isolated from starved (low esterification rates) or fed rats (high esterification rates). In contrast, metformin markedly reduced the rates of glucose production from lactate/pyruvate, alanine, dihydroxyacetone, and galactose. Using crossover plot experiments, it was shown that the main effect of metformin on hepatic gluconeogenesis was located upstream of the formation of dihydroxyacetone phosphate. Increasing the time of exposure to metformin (24 hr instead of 1 hr) led to significant changes in the expression of genes involved in glucose and fatty acid metabolism. Indeed, when hepatocytes were cultured in the presence of 50 to 500 microM metformin, the expression of genes encoding regulatory proteins of fatty acid oxidation (carnitine palmitoyltransferase I), ketogenesis (mitochondrial hydroxymethylgltaryl-CoA synthase), and gluconeogenesis (glucose 6-phosphatase, phosphoenolpyruvate carboxykinase) was decreased by 30 to 60%, whereas expression of genes encoding regulatory proteins involved in glycolysis (glucokinase and liver-type pyruvate kinase) was increased by 250%. In conclusion, this work suggests that metformin could reduce hepatic glucose production through short-term (metabolic) and long-term (genic) effects.

Long-chain fatty acids regulate liver carnitine palmitoyltransferase I gene (L-CPT I) expression through a peroxisome-proliferator-activated receptor alpha (PPARalpha)-independent pathway.

Liver carnitine palmitoyltransferase I (L-CPT I) catalyses the transfer of long-chain fatty acid (LCFA) for translocation across the mitochondrial membrane. Expression of the L-CPT I gene is induced by LCFAs as well as by lipid-lowering compounds such as clofibrate. Previous studies have suggested that the peroxisome-proliferator-activated receptor alpha (PPARalpha) is a common mediator of the transcriptional effects of LCFA and clofibrate. We found that free LCFAs rather than acyl-CoA esters are the signal metabolites responsible for the stimulation of L-CPT I gene expression. Using primary culture of hepatocytes we found that LCFAs failed to stimulate L-CPT I gene expression both in wild-type and PPARalpha-null mice. These results suggest that the PPARalpha-knockout mouse does not represent a suitable model for the regulation of L-CPT I gene expression by LCFAs in the liver. Finally, we determined that clofibrate stimulates L-CPT I through a classical direct repeat 1 (DR1) motif in the promoter of the L-CPT I gene while LCFAs induce L-CPT I via elements in the first intron of the gene. Our results demonstrate that LCFAs can regulate gene expression through PPARalpha-independent pathways and suggest that the regulation of gene expression by dietary lipids is more complex than previously proposed.

Reduced hepatic fatty acid oxidation in fasting PPARalpha null mice is due to impaired mitochondrial hydroxymethylglutaryl-CoA synthase gene expression.

Glucose and fatty acid metabolism (oxidation versus esterification) has been measured in hepatocytes isolated from 24 h starved peroxisome proliferator-activated receptor-alpha (PPARalpha) null and wild-type mice. In PPARalpha null mice, the development of hypoglycemia during starvation was due to a reduced capacity for hepatic gluconeogenesis secondary to a 70% lower rate of fatty acid oxidation. This was not due to inappropriate expression of the hepatic CPT I gene, which was similar in both genotypes, but to impaired mitochondrial hydroxymethylglutaryl-CoA synthase gene expression in the PPARalpha null mouse liver. We also demonstrate that hepatic steatosis of fasting PPARalpha null mice was not due to enhanced triglyceride synthesis.

Prediction of pharmacokinetic drug/drug interactions from In vitro data: interactions of the nonsteroidal anti-inflammatory drug lornoxicam with oral anticoagulants.

CYP2C9 is involved in the metabolism of the oral anticoagulants warfarin, phenprocoumon, and acenocoumarol. It is also responsible for the 5'-hydroxylation of the nonsteroidal anti-inflammatory drug lornoxicam. Therefore, lornoxicam and the oral anticoagulants are potential inhibitors of their metabolism. Their inhibitory potency was investigated in microsomes from six human livers. An approach to predict pharmacokinetic interactions of lornoxicam from in vitro inhibition data was developed. Where possible, the forecasts were verified by comparison with data from clinical interaction studies. The following increases in steady-state plasma concentrations or areas under the plasma concentration-time curve of the oral anticoagulants by concomitant lornoxicam medication were predicted (values in parentheses are for healthy volunteers): (S)-warfarin, 1. 58-fold (1.32-fold for racemate); racemic-acenocoumarol, 1.28-fold (1.09-fold); (R)-acenocoumarol, 1.10-fold (1.0-fold); racemic-phenprocoumon, 1.11-fold (1.18-fold); and (S)-phenprocoumon, 1.13-fold (1.24-fold). Lornoxicam 5'-hydroxylation was competitively inhibited in vitro by both phenprocoumon (K(i) = 1.2 +/- 0.4 microM) and acenocoumarol (K(i) = 5.5 +/- 3.5 microM). The present results indicate that relatively close predictions of the interactions of lornoxicam with oral anticoagulants from in vitro data are possible under the assumption that hepatic lornoxicam concentrations are similar to its total plasma concentrations. The degree of pharmacokinetic interactions exhibited by oral anticoagulants and lornoxicam is dependent on the respective contribution of CYP2C9 to their total clearance.

Atypical expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in subcutaneous adipose tissue of male rats.

The mRNAs encoding mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mtHMG-CoA synthase), the rate limiting enzyme in ketone body production, are highly expressed in subcutaneous (SC) and, to a lesser extent, in peri-epididymal (PE) rat adipose tissues. This atypical mtHMG-CoA synthase gene expression is dependent on the age (from 9 weeks of age) and sex (higher in male than in female) of the rats. In contrast, the expression of mtHMG-CoA synthase in SC adipose deposit is independent of the nutritional state (fed versus starved) or of the thermic environment (24 degrees C versus 4 degrees C). The expression of mtHMG-CoA synthase is suppressed in SC fat pads of castrated male rats whereas treatment of castrated rats with testosterone restores a normal level of expression. Moreover, testosterone injection induces the expression mtHMG-CoA synthase in SC adipose tissue of age-matched females. The presence of the mtHMG-CoA synthase immunoreactive protein confers to mitochondria isolated from SC adipose deposits, the capacity to produce ketone bodies at a rate similar to that found in liver mitochondria (SC = 13.7 +/- 0.7, liver = 16.4 +/- 1.4 nmol/min/mg prot). mtHMG-CoA synthase is expressed in the stromal vascular fraction (SVF) whatever the adipose deposit considered. While acetyl-CoA carboxylase (ACC) is only expressed in mature adipocytes, the other lipogenic enzymes, fatty acid synthase (FAS) and citrate cleavage enzyme (CCE), are expressed both in SVF cells and mature adipocytes. The expression of lipogenic enzyme genes is markedly reduced in adipocytes but not in SVF cells isolated from 48-h starved male rats. When SVF is subfractionated, mtHMG-CoA synthase mRNAs are mainly recovered in two fractions containing poorly digested structures such as microcapillaries whereas the lowest expression is found in the pre-adipocyte fraction. Interestingly, FAS and CCE mRNAs co-segregate with mtHMG-CoA synthase mRNA. The possible physiological relevance of such atypical expression of mtHMG-CoA synthase is discussed.

The N-terminal domain of rat liver carnitine palmitoyltransferase 1 mediates import into the outer mitochondrial membrane and is essential for activity and malonyl-CoA sensitivity.

The rat liver carnitine palmitoyltransferase 1 (L-CPT1), an integral outer mitochondrial membrane (OMM) protein, is the key regulatory enzyme of fatty acid oxidation and is inhibited by malonyl-CoA. In vitro import of L-CPT1 into the OMM requires the presence of mitochondrial receptors and is stimulated by ATP but is membrane potential-independent. Its N-terminal domain (residues 1-150), which contains two transmembrane segments, possesses all of the information for mitochondrial targeting and OMM insertion. Deletion of this domain abrogates protein targeting, whereas its fusion to non-OMM-related proteins results in their mitochondrial targeting and OMM insertion in a manner similar to L-CPT1. Functional analysis of chimeric CPTs expressed in Saccharomyces cerevisiae shows that this domain also mediates in vivo protein insertion into the OMM. When the malonyl-CoA-insensitive CPT2 was anchored at the OMM either by a specific OMM signal anchor sequence (pOM29) or by the N-terminal domain of L-CPT1, its activity remains insensitive to malonyl-CoA inhibition. This indicates that malonyl-CoA sensitivity is an intrinsic property of L-CPT1 and that its N-terminal domain cannot confer malonyl-CoA sensitivity to CPT2. Replacement of the N-terminal domain by pOM29 results in a less folded and less active protein, which is also malonyl-CoA-insensitive. Thus, in addition to its role in mitochondrial targeting and OMM insertion, the N-terminal domain of L-CPT1 is essential to maintain an optimal conformation for both catalytic function and malonyl-CoA sensitivity.

Topological and functional analysis of the rat liver carnitine palmitoyltransferase 1 expressed in Saccharomyces cerevisiae.

The rat liver carnitine palmitoyltransferase 1 (L-CPT 1) expressed in Saccharomyces cerevisiae was correctly inserted into the outer mitochondrial membrane and shared the same folded conformation as the native enzyme found in rat liver mitochondria. Comparison of the biochemical properties of the yeast-expressed L-CPT 1 with those of the native protein revealed the same detergent lability and similar sensitivity to malonyl-CoA inhibition and affinity for carnitine. Normal Michaelis-Menten kinetics towards palmitoyl-CoA were observed when careful experimental conditions were used for the CPT assay. Thus, the expression in S. cerevisiae is a valid model to study the structure-function relationships of L-CPT 1.

Denitrification of the genotoxicant 2-nitropropane: relationship to its mechanism of toxicity.

1. The stabilities of the industrial chemical and constituent of cigarette smoke 2-nitropropane (2-NP) and its aci tautomer propane 2-nitronate (P2N) towards hepatic enzymes and proteins such as serum proteins and oxyhaemoglobin were investigated in vitro in biological (hepatocytes and subcellular liver fractions) and model systems (serum proteins, oxyhaemoglobin, methylene blue). 2. Denitrification of 2-NP, P2N and 2-deutero 2-nitropropane (2H-NP) occurred in murine hepatocytes significantly faster than in rat cells. For 2-NP the rates were 1271 +/- 167 versus 820 +/- 125 pmol nitrite x min-1 x 10(6) cells-1. 3. A similar observation was made in microsomes, where 2-NP denitrification was 1460 +/- 110 (mouse) versus 480 +/- 80 pmol nitrite x min-1 x mg protein-1 (rat). 4. The major NO2(-)-forming activity was found to be localized in the microsomal fraction. 5. Conversion of 2-NP into P2N, either chemically or enzymatically, was a prerequisite for rapid denitrification. 6. Serum proteins and oxyhaemoglobin proved to be capable of denitrifying P2N (198 +/- 24 pmol nitrite x min-1 x mg protein-1 and 7.1 +/- 1.0 nmol nitrite x min-1 x nmol HbO2(-1) respectively), but were much less active towards 2-NP (24 +/- 2 pmol nitrite x min-1 x mg protein-1 and none respectively). 7. Methylene blue decomposed 2-NP and P2N at rates of 11 +/- 3 and 192 +/- 4 pmol nitrite x min-1 x nmol, methylene blue-1 respectively. The dye also enhanced NO2- formation from P2N and 2-NP in the presence of hepatocytes or serum proteins, with a concomitant enhancement of both 2-NP and P2N toxicity. 8. The results presented report species differences in the denitrification rate of 2-NP and highlight the crucial nitro-aci tautomerism of 2-NP as a pivotal determinant of 2-NP toxicity.

Secretoneurin: a market in rat hippocampal pathways.

Secretoneurin is a 33-amino acid peptide, generated in brain by proteolytic processing of secretogranin II. The distribution of secretoneurin-like immunoreactivity and secretogranin II mRNA was investigated in the hippocampus of the rat. Secretogranin II mRNA was found in high concentrations throughout the granule cell and pyramidal cell layers and in many local neurons, notably in the hilus of the dentate gyrus. The general distributional pattern of secretoneurin-like immunoreactivity was characterized by a prominent staining in the area of the terminal field of mossy fibers with an obvious staining in the infrapyramidal area of CA3 and a strongly immunopositive band in the inner third of the molecular layer of the dentate gyrus. Lesions of the granule cells by local injection of colchicine significantly reduced secretoneurin-like immunoreactivity in the terminal field of mossy fibers, but not in the inner molecular layer of the dentate gyrus. On the other hand, destruction of interneurons of the dentate gyrus (mossy cells and certain gamma-aminobutyricacid-ergic interneurons) by kainic acid-induced seizures was associated with a reduction of secretoneurin-like immunoreactivity in the inner molecular layer of the dentate gyrus. However, 30 days after kainic acid-induced seizures, a strongly secretoneurin-immunoreactive band reappeared in this area, which at this late time point is due to sprouting of mossy fibers collaterals. Our experiments suggest a widespread distribution of secretoneurin-like immunoreactivity in neurons of the hippocampal formation with a preferential localization in excitatory pathways including associational/commissural fibers originating from secretoneurin-containing mossy cells.

Troglitazone inhibits fatty acid oxidation and esterification, and gluconeogenesis in isolated hepatocytes from starved rats.

The effects of troglitazone and pioglitazone on glucose and fatty acid metabolism were studied in hepatocytes isolated from 24-h-starved rats. These thiazolidinediones inhibited long-chain fatty acid (oleate) oxidation and produced a very oxidized mitochondrial redox state. By contrast, thiazolidinediones did not affect the rate of medium-chain fatty acid (octanoate) oxidation or the activity of mitochondrial carnitine palmitoyltransferase (CPT) I. Thiazolidinediones inhibited selectively triglyceride synthesis but not phospholipid synthesis. The combined inhibition of oleate oxidation and esterification by troglitazone was due to a noncompetitive inhibition of mitochondrial and microsomal long-chain acyl-CoA synthetase (ACS) activities. It was suggested that troglitazone must be metabolized into its sulfo-conjugate derivative in liver cells to inhibit mitochondrial and microsomal ACS activities. Thiazolidinediones inhibited glucose production from lactate/pyruvate or from alanine. Analysis of gluconeogenic metabolite concentrations suggested that troglitazone would inhibit gluconeogenesis at the level of pyruvate carboxylase and glyceraldehyde-3-phosphate dehydrogenase reactions. It was concluded that 1) at a similar concentration, troglitazone was more efficient than pioglitazone to inhibit fatty acid metabolism and gluconeogenesis and 2) the inhibition of gluconeogenesis by troglitazone could be the result of the inhibition of long-chain fatty acid oxidation (decrease in acetyl-CoA, NADH-to-NAD+, and ATP-to-ADP ratios).

Spinal cord injury increases the risk of abdominal aortic aneurysm.

We have observed apparently disproportionate numbers of abdominal aortic aneurysms (AAAs) in chronic spinal cord injury (SCI) patients. To test whether aortic enlargement is more frequent in SCI, we measured maximum infrarenal aortic diameters (AoDmax) by B mode ultrasound in 89 SCI subjects and 223 age and sex matched controls. The average AoDmax in SCI subjects (mean age 60.3 years) was 2.27+/-0.80 cm compared to 2.07+/-0.69cm in the controls. This difference was significant (P = 0.023), as were the proportions of subjects with AoDmax >/- 3cm. A total of 20.2 per cent of the SCI group had AoDmax >/- 3cm compared to 8.9 per cent for the controls (P < 0.0001, chi-square). Race, height, and weight distributions were similar. SCI patients had lower levels of hypertension and cigarette smoking than controls. Within the SCI and control groups, subjects with AoDmax >/- 3cm had increased cigarette consumption compared to /- 3cm group than in the >/- 3cm group, T6 versus T8, but not significantly (P = 0.23)> Based on these results, SCI patients have over a two-fold risk of aortic enlargement as a consequence of spinal cord injury.

Cyclic AMP and fatty acids increase carnitine palmitoyltransferase I gene transcription in cultured fetal rat hepatocytes.

In the rat, the gene for liver mitochondrial carnitine palmitoyltransferase I (CPT I), though dormant prior to birth, is rapidly activated postnatally. We sought to elucidate which hormonal and/or nutritional factors might be responsible for this induction. In cultured hepatocytes from 20-day-old rat fetus, the concentration of CPT I mRNA, which initially was very low, increased dramatically in a dose-dependent manner after exposure of the cells to dibutyryl cAMP (Bt2cAMP). Similar results were obtained when long-chain fatty acids (LCFA), but not medium-chain fatty acids, were added to the culture medium. The effects of Bt2cAMP and LCFA were antagonized by insulin, also dose dependently. In contrast, CPT II gene expression, which was already high in fetal hepatocytes, was unaffected by any of the above manipulations. Bt2cAMP stimulated CPT I gene expression even when endogenous triacylglycerol breakdown was suppressed by lysosomotropic agents suggesting that the actions of cAMP and LCFA were distinct. Moreover, half-maximal concentrations of Bt2cAMP and linoleate produced an additive effect CPT I mRNA accumulation. While linoleate and Bt2cAMP stimulated CPT I gene transcription by twofold and fourfold, respectively, the fatty acid also increased the half-life of CPT I mRNA (50%). When hepatocytes were cultured in the presence of 2-bromopalmitate, (which is readily converted by cells into its non-metabolizable CoA ester) CPT I mRNA accumulation was higher than that observed with oleate or linoleate. Similarly, the CPT I inhibitor, tetradecylglycidate, which at a concentration of 20 microM did not itself influence the CPT I mRNA level, enhanced the stimulatory effect of linoleate. The implication is that induction of the CPT I message by LCFA does not require mitochondrial metabolism of these substrates; however, formation of their CoA esters is a necessary step. Unlike linoleate, the peroxisome proliferator, clofibrate, increased both CPT I and CPT II mRNA levels and neither effect was offset by insulin. It thus appears that the mechanism of action of LCFA differs from that utilized by clofibrate, which presumably works through the peroxisome proliferator activated receptor. We conclude that the rapid increase in hepatic CPT I mRNA level that accompanies the fetal to neonatal transition in the rat is triggered by the reciprocal change in circulating insulin and LCFA concentrations, coupled with elevation of the liver content of cAMP.

Catalysis of nitro-aci tautomerism of the genotoxicant 2-nitropropane by cytosol from rodent and human liver.

2-Nitropropane (2-NP) is a genotoxicant and hepatocarcinogen in rodents. Conversion to propane 2-nitronate (P2N), the anion of the tautomeric aci form of 2-NP, seems to be a pivotal part of the mechanism by which 2-NP causes its toxicity. We tested the hypothesis that the tautomeric equilibrium is influenced by enzymes in the liver, the target organ of 2-NP toxicity. Rat or mouse hepatocytes were incubated with 2-NP, P2N or the 2-NP isotopomer 2-deutero 2-nitropropane (2H-2-NP), which equilibrates with P2N much more slowly than 2-NP. Tautomers were analyzed by HPLC. The rates of conversion of 2-NP to P2N expressed as nmol P2N x (10(6) cells/ml)-1 x min-1 were 4.0 and 4.2 in the presence of hepatocytes from rats or mice, respectively, and 2.6 in the absence of cells. Production of 2-NP to P2N expressed as nmol 2-NP x (10(6) cells/ml)-1 x min-1 was increased from 6.1 in the absence of cells to 11.9 or 9.9 in the presence of hepatocytes from rats or mice, respectively. The rate of formation of P2N from 2H-2-NP as compared to 2-NP was characterised by a primary isotope effect of 3.4 and 3.8 in hepatocytes from rats and mice, respectively, contrasting with a value of 9.6 measured in medium omitting cells. When 2-NP was incubated with subfractions of rodent or human liver homogenate, production of P2N by cytosol was between 7.3 (mouse liver) and 28.1 times (human liver) higher than that observed in microsomes. Similarly generation of 2-NP from P2N by cytosol exceeded that in microsomes by a factor of two. Tautomerism in heat-activated cytosol, mitochondria or microsomes was not different from that in buffer only. The results suggest that the nitro-aci tautomerism of secondary nitroalkanes is catalysed by a hepatic enzyme which resides predominantly in the cytosol and may thus contribute to the generation of the toxic species via which 2-NP exerts its toxicity.