Metabolism Of Several Substrates Research Articles

Role of natriuretic peptides in the cardiovascular-adipose communication: a tale of two organs.

Natriuretic peptides have long been known for their cardiovascular function. However, a growing body of evidence emphasizes the role of natriuretic peptides in the energy metabolism of several substrates in humans and animals, thus interrelating the heart, as an endocrine organ, with various insulin-sensitive tissues and organs such as adipose tissue, muscle skeletal, and liver. Adipose tissue dysfunction is associated with altered regulation of the natriuretic peptide system, also indicated as a natriuretic disability. Evidence points to a contribution of this natriuretic disability to the development of obesity, type 2 diabetes mellitus, and cardiometabolic complications; although the causal relationship is not fully understood at present. However, targeting the natriuretic peptide pathway may improve metabolic health in obesity and type 2 diabetes mellitus. This review will focus on the current literature on the metabolic functions of natriuretic peptides with emphasis on lipid metabolism and insulin sensitivity. Natriuretic peptide system alterations could be proposed as one of the linking mechanisms between adipose tissue dysfunction and cardiovascular disease.

Functional characterization of genetic variants of human FMO3 associated with trimethylaminuria

Impaired conversion of trimethylamine to trimethylamine N-oxide by human flavin containing monooxygenase 3 (FMO3) is strongly associated with primary trimethylaminuria, also known as ‘fish-odor’ syndrome. Numerous non-synonymous mutations in FMO3 have been identified in patients suffering from this metabolic disorder (e.g., N61S, M66I, P153L, and R492W), but the molecular mechanism(s) underlying the functional deficit attributed to these alleles has not been elucidated. The purpose of the present study was to determine the impact of these disease-associated genetic variants on FMO3 holoenzyme formation and on steady-state kinetic parameters for metabolism of several substrates, including trimethylamine. For comparative purposes, several common allelic variants not associated with primary trimethylaminuria (i.e., E158K, V257M, E308G, and the E158K/E308G haplotype) were also analyzed. When recombinantly expressed in insect cells, only the M66I and R492W mutants failed to incorporate/retain the FAD cofactor. Of the remaining mutant proteins P153L and N61S displayed substantially reduced (<10%) catalytic efficiencies for trimethylamine N-oxygenation relative to the wild-type enzyme. For N61S, reduced catalytic efficiency was solely a consequence of an increased Km, whereas for P153L, both Km and kcat were altered. Similar results were obtained when benzydamine N-oxygenation was monitored. A homology model for FMO3 was constructed based on the crystal structure for yeast FMO which places the N61 residue alone, of the mutants analyzed here, in close proximity to the FAD catalytic center. These data demonstrate that primary trimethylaminuria is multifactorial in origin in that enzyme dysfunction can result from kinetic incompetencies as well as impaired assembly of holoprotein.

Re-engineering cytochrome P450 2B11dH for enhanced metabolism of several substrates including the anti-cancer prodrugs cyclophosphamide and ifosfamide

Based on recent directed evolution of P450 2B1, six P450 2B11 mutants at three positions were created in an N-terminal modified construct termed P450 2B11dH and characterized for enzyme catalysis using five substrates. Mutant I209A demonstrated a 3.2-fold enhanced k cat/ K m for 7-ethoxy-4-trifluoromethylcourmarin O-deethylation, largely due to a dramatic decrease in K m (0.72 μM vs. 18 μM). I209A also demonstrated enhanced selectivity for testosterone 16β-hydroxylation over 16α-hydroxylation. In contrast, V183L showed a 4-fold increased k cat for 7-benzyloxyresorufin debenzylation and a 4.7-fold increased k cat/ K m for testosterone 16α-hydroxylation. V183L also displayed a 1.7-fold higher k cat/ K m than P450 2B11dH with the anti-cancer prodrugs cyclophosphamide and ifosfamide, resulting from a ∼4-fold decrease in K m. Introduction of the V183L mutation into full-length P450 2B11 did not enhance the k cat/ K m. Overall, the re-engineered P450 2B11dH enzymes exhibited enhanced catalytic efficiency with several substrates including the anti-cancer prodrugs.

Engineering of Cytochrome P450 3A4 for Enhanced Peroxide-Mediated Substrate Oxidation Using Directed Evolution and Site-Directed Mutagenesis

CYP3A4 has been subjected to random and site-directed mutagenesis to enhance peroxide-supported metabolism of several substrates. Initially, a high-throughput screening method using whole cell suspensions was developed for H2O2-supported oxidation of 7-benzyloxyquinoline. Random mutagenesis by error-prone polymerase chain reaction and activity screening yielded several CYP3A4 mutants with enhanced activity. L216W and F228I showed a 3-fold decrease in Km, HOOH and a 2.5-fold increase in kcat/Km, HOOH compared with CYP3A4. Subsequently, T309V and T309A were created based on the observation that T309V in CYP2D6 has enhanced cumene hydroperoxide (CuOOH)-supported activity. T309V and T309A showed a > 6- and 5-fold higher kcat/Km, CuOOH than CYP3A4, respectively. Interestingly, L216W and F228I also exhibited, respectively, a > 4- and a > 3-fold higher kcat/Km, CuOOH than CYP3A4. Therefore, several multiple mutants were constructed from rationally designed and randomly isolated mutants; among them, F228I/T309A showed an 11-fold higher kcat/Km, CuOOH than CYP3A4. Addition of cytochrome b5, which is known to stimulate peroxide-supported activity, enhanced the kcat/Km, CuOOH of CYP3A4 by 4- to 7-fold. When the mutants were tested with other substrates, T309V and T433S showed enhanced kcat/Km, CuOOH with 7-benzyloxy-4-(trifluoromethyl)coumarin and testosterone, respectively, compared with CYP3A4. In addition, in the presence of cytochrome b5, T433S has the potential to produce milligram quantities of 6beta-hydroxytestosterone through peroxide-supported oxidation. In conclusion, a combination of random and site-directed mutagenesis approaches yielded CYP3A4 enzymes with enhanced peroxide-supported metabolism of several substrates.

The Isolation and Comparison of Multiple Forms of CYP2B from Untreated and Phenobarbital-Treated Rabbit Liver Microsomes

At low levels of phenobarbital induction two forms of isoenzyme 2 (LM2; CYP2B4) were obtained during purification of cytochrome P450 from rabbit liver microsomes. At high levels of induction only one form (LM2A) was present. Although the two purified forms (LM2A and LM2B) were very similar they differed in: (a) peak elution on CM-Sepharose, (b) wavelength maximum of the reduced P450-CO spectrum, and (c) metabolism of several substrates, where the activities of LM2B ranged from 0.6 to 2.65 times that of LM2A. A third LM2 fraction (2C) was isolated from untreated rabbit liver and, although homogenous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, appeared to be a mixture of LM2B and a form of P450 LM2 other than LM2A. LM2A was not found in the untreated rabbit liver microsomes. On CM-Sepharose the elution of fraction 2C overlapped that of LM2B. The apparent molecular weight and immunoresponse to anti-LM2A IgG were the same for fraction 2C as for LM2A and LM2B. Peptide mapping using trypsin showed no difference between LM2A and LM2B, but consistently revealed at least one extra band with fraction 2C. After CNBr cleavage and high-pressure liquid chromatography separation of the LM2A and LM2B fragments the peptide beginning with Pro(347) of LM2A (peak 4A) eluted 12 min later than that of LM2B (peak 4B) indicating a difference in the fragments, although partial NH2-terminal amino acid sequences and molecular masses were the same. The corresponding CNBr fragment of fraction 2C splits into two peaks (4C:1 and 4C:2) with retention times corresponding to 4B and 4A, respectively. The mass of 4C:1. was the same as that of 4B, while the mass of 4C:2 markedly differed from that of 4A and 4B. Both fragments had the same partial NH2-terminal amino acid sequence as 4A and 4B. After comparing the physicochemical properties as well as catalytic activities of these isolated and purified LM2 forms with the cDNA-expressed forms 2B-B0, 2B-B1, 2B-B2, and 2B-Bx [see R. Ryan et al. (1993) Arch. Biochem. Biophys. 304, 454-463], the data suggest that LM2A is the form designated as 2B-B0 (LM2), LM2B is 2B-Bx, and fraction 2C is a mixture containing 2B-B1 and 2B-Bx. This is the first isolation and identification of the three isozymic LM2 proteins from rabbit liver.

Metabolic oxidation phenotypes as markers for susceptibility to lung cancer.

That bronchial carcinoma is not an inevitable consequence of cigarette smoking has stimulated the search for host factors that might influence the susceptibility of the individual smoker. One plausible host factor would be a polymorphic gene controlling the metabolic oxidative activation of chemical carcinogens, giving rise to wide inter-subject variation in the generation of cancer-inducing and/or promoting species. Recently, three genetic polymorphisms of human metabolic oxidation have been demonstrated (as characterized by debrisoquine, mephenytoin and carbocysteine), with the metabolism of several substrates exhibiting the phenomenon. Debrisoquine 4-hydroxylation segregates into two human phenotypes, each comprising characteristic metabolic capability. We report here the frequency of debrisoquine 4-hydroxylation phenotypes in age-, sex- and smoking history-matched bronchial carcinoma and control patients. Cancer patients showed a preponderance of probable homozygous dominant extensive metabolizers (78.8%) with few recessive poor metabolizers (1.6%) compared with smoking controls (27.8% and 9.0% respectively). We conclude that the gene controlling debrisoquine 4-hydroxylation may be a host genetic determinant of susceptibility to lung cancer in smokers and that it represents a marker to assist in assessing individual risk.

Alveolar type II and Clara cells: isolation and xenobiotic metabolism.

This paper describes one isolation procedure for two pulmonary cell types and discusses how these cells are being used for toxicological studies. Alveolar Type II cells and Clara cells have been isolated from rabbits and rats and separated into highly enriched fractions. The lungs were digested with protease, and the pulmonary cell digests were separated into discrete fractions on the basis of cellular size and density differences. Several studies have been conducted to compare the metabolism of xenobiotics in these cell types and three examples are discussed. The metabolic activation and covalent binding of the pulmonary toxin, 4-ipomeanol was found to occur in both Clara and Type II cells in vitro, although to a much greater extent in the Clara cells. Also, the metabolism of several substrates, including 7-ethoxycoumarin, coumarin and benzo(a)pyrene, was compared in the isolated cell fractions and found to be much greater in the Clara cells than in the Type II cells. Immunohistochemical analysis and gel electrophoresis have also been used to demonstrate the presence of the two major rabbit pulmonary cytochrome P-450 isozymes in both the isolated Clara cells and alveolar Type II cells.

Protective effect of diethyldithiocarbamate and carbon disulfide against liver injury induced by various hepatotoxic agents

Diethyldithiocarbamate (DTC) and carbon disulfide (CS 2), at nearly equimolar oral dose levels, protected mice against liver damage induced by carbon tetrachloride, chloroform, bromotri-chloromethane, thioacetamide, bromobenzene, furosemide, acetaminophen, dimethylnitrosamine and trichloroethylene, as evidenced by the suppression of elevations in plasma GPT activity and liver calcium content, and of histopathological alterations. Both agents also prolonged hexobarbital sleeping time and zoxazolamine paralysis time in mice. DTC and CS 2 alone, given orally, decreased microsomal metabolism of several substrates (aniline, p-nitroanisole, hexobarbital. zoxazolamine, aminopyrine and 3,4-benzpyrene), CCl 4-induced lipid peroxidation, and cytochrome P-450 content. The loss of microsomal drug-metabolizing enzyme activity was also observed in the experiments in vitro using liver slices and isolated microsomes. Since a characteristic common to such diverse hepatotoxins is that they require metabolic activation before exhibiting hepatotoxicity, the protective mechanisms of DTC and CS 2 may involve their interference with the process of metabolic activation of these hepatotoxins. The protective action of DTC may be mediated almost entirely through CS 2 when administered orally and at least partly with parenteral administration, since, in CCl 4-induced liver injury, DTC was most effective when given orally, while the action of CS 2 was less dependent on the route of administration. Thus, CS 2 and CS 2-producing agents in vivo such as dithiocarhamate derivatives and disulfiram may modify toxicological and pharmacological effects of foreign compounds by inhibiting microsomal drug-metabolizing enzyme activity in the liver.

Purification and characterization of a minor form of hepatic microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls

A minor form of hepatic microsomal cytochrome P-450 has been purified to apparent homogeneity from rats treated with the polychlorinated biphenyl mixture, Aroclor 1254. This newly isolated hemoprotein, cytochrome P-450 e, is inducible in rat liver by Aroclor 1254 and phenobarbital, but not by 3-methylcholanthrene. Two other hemoproteins, cytochromes P-450 b and P-450 c, have also been highly purified during the isolation of cytochrome P-450 e based on chromatographic differences among these proteins. By Ouchterlony double-diffusion analysis with antibody to cytochrome P-450 b, highly purified cytochrome P-450 e is immunochemically identical to cytochrome P-450 b but does not cross-react with antibodies prepared against other rat liver cytochromes P-450 ( P-450 a, P-450 c, P-450 d) or epoxide hydrolase. Purified cytochrome P-450 e is a single protein-staining band in sodium dodecyl sulfate-polyacrylamide gels with a minimum molecular weight (52,500) slightly greater than cytochromes P-450 b or P-450 d (52,000) but clearly distinct from cytochromes P-450 a (48,000) and P-450 c (56,000). The carbon monoxide-reduced difference spectral peak of cytochrome P-450 e is at 450.6 nm, whereas the peak of cytochrome P-450 b is at 450 nm. Ethyl isocyanide binds to ferrous cytochromes P-450 e and P-450 b to yield two spectral maxima at 455 and 430 nm. At pH 7.4, the 455:430 ratio is 0.7 and 1.4 for cytochromes P-450 b and P-450 e, respectively. Metyrapone binds to reduced cytochromes P-450 e and P-450 b (absorption maximum at 445–446 nm) but not cytochromes P-450 a, P-450 c, or P-450 d. Metabolism of several substrates catalyzed by cytochrome P-450 e or P-450 b reconstituted with NADPH-cytochrome c reductase and dilauroylphosphatidylcholine was compared. The substrate specificity of cytochrome P-450 e usually paralleled that of cytochrome P-450 b except that the rate of metabolism of benzphetamine, benzo[ a]pyrene, 7-ethoxycoumarin, hexobarbital, and testosterone at the 16α-position catalyzed by cytochrome P-450 e was only 15–25% that of cytochrome P-450 b. In contrast, cytochrome P-450 e catalyzed the 2-hydroxylation of estradiol-17β more efficiently (threefold) than cytochrome P-450 b. Cytochrome P-450 d, however, catalyzed the metabolism of estradiol-17β at the greatest rate compared to cytochromes P-450 a, P-450 b, P-450 c, or P-450 e. The peptide fragments of cytochromes P-450 e and P-450 b, generated by either proteolytic or chemical digestion of the hemoproteins, were very similar but not identical, indicating that these two proteins show minor structural differences.