In vivo effect of insulin to decrease matrix metalloproteinase-2 and -9 activity after arterial injury.

UNLABELLED: In vitro, insulin has both growth-promoting and vasculoprotective effects. In vivo, the effect of insulin is mainly protective. Insulin treatment (3 U/day) decreases smooth muscle cell (SMC) migration and neointimal growth after carotid angioplasty in normal rats maintained at normoglycemia by oral glucose. SMC migration requires limited proteolysis of the extracellular matrix, which is mediated by matrix metalloproteinases (MMPs). In this study, we investigated the effects of normoglycemic hyperinsulinemia on MMP activity after balloon angioplasty. Rats were divided into three groups: (1) control implants and tap water; (2) control implants and oral glucose, and (3) insulin implants (3 U/day) and oral glucose. RESULTS: Gelatin zymography revealed that insulin reduced the gelatinolytic activity of pro-MMP-2 by 46% (p < 0.05), MMP-2 by 44% (p < 0.05) and MMP-9 by 51% (p < 0.05) compared to controls after arterial injury. Insulin also reduced mRNA levels of MMP-2 (p < 0.05) and MMP-9 (p < 0.05) and protein levels of MMP-2 (p < 0.05). In contrast, there were no significant changes in membrane-type 1 MMP protein and tissue inhibitors of MMP activity after insulin treatment. Thus, these results suggest a mechanism by which insulin inhibits SMC migration and supports a vasculoprotective role for insulin in vivo.

Insulin increases reendothelialization and inhibits cell migration and neointimal growth after arterial injury.

OBJECTIVE: Insulin has both growth-promoting and protective vascular effects in vitro, however the predominant effect in vivo is unclear. We investigated the effects of insulin in vivo on neointimal growth after arterial injury. METHODS AND RESULTS: Rats were given subcutaneous control (C) or insulin implants (3U/d;I) 3 days before arterial (carotid or aortic) balloon catheter injury. Normoglycemia was maintained by oral glucose and, after surgery, by intraperitoneal glucose infusion (saline in C). Insulin decreased intimal area (P<0.01) but did not change intimal cell proliferation or apoptosis. However, insulin inhibited cell migration into the intima (P<0.01) and increased expression of smooth muscle cell (SMC) differentiation markers (P<0.05). Insulin also increased reendothelialization (P<0.01) and the number of circulating progenitor cells (P<0.05). CONCLUSIONS: These results are the first demonstration that insulin has a protective effect on both SMC and endothelium in vivo, resulting in inhibition of neointimal growth after vessel injury.

Hepatic CYP2A6 levels and nicotine metabolism: impact of genetic, physiological, environmental, and epigenetic factors.

PURPOSE: We investigated the role of genetic, physiological, environmental, and epigenetic factors in regulating CYP2A6 expression and nicotine metabolism. METHODS: Human livers (n = 67) were genotyped for CYP2A6 alleles and assessed for nicotine metabolism and CYP2A6 expression (mRNA and protein). In addition, a subset of livers (n = 18), human cryopreserved hepatocytes (n = 2), and HepG2 cells were used for DNA methylation analyses. RESULTS: Liver samples with variant CYP2A6 alleles had significantly lower CYP2A6 protein expression, nicotine C-oxidation activity, and affinity for nicotine. Female livers had significantly higher CYP2A6 protein and mRNA expression compared to male livers. Livers exposed to dexamethasone and phenobarbital had higher CYP2A6 expression and activity, however the difference was not statistically significant. Age and DNA methylation status of the CpG island and a regulatory site were not associated with altered CYP2A6. CONCLUSIONS: We identified genotype, gender, and exposure to inducers as sources of variation in CYP2A6 expression and activity, but much variation remains to be accounted for.

A novel CYP2A6 allele (CYP2A6*35) resulting in an amino-acid substitution (Asn438Tyr) is associated with lower CYP2A6 activity in vivo.

Cytochrome P450 2A6 (CYP2A6) is the primary human enzyme involved in nicotine metabolism. The objective of this study was to characterize two nonsynonymous single nucleotide polymorphisms in CYP2A6(*)24, 594G>C (Val110Leu) and 6458A>T (Asn438Tyr). We determined their haplotype, allele frequencies, effect on CYP2A6 activity in vivo, as well as their stability and ability to metabolize nicotine in vitro. CYP2A6(*)35 (6458A>T) occurred at a frequency of 2.5-2.9% among individuals of black African descent, 0.5-0.8% among Asians and was not found in Caucasians. In addition, we identified two novel alleles, CYP2A6(*)36 (6458A>T and 6558T>C (Ile471Thr)) and CYP2A6(*)37 (6458A>T, 6558T>C and 6600G>T (Arg485Leu)). In vivo, CYP2A6(*)35 was associated with lower CYP2A6 activity as measured by the 3HC/COT ratio. In vitro, CYP2A6.35 had decreased nicotine C-oxidation activity and thermal stability. In conclusion, we identified three novel CYP2A6 alleles (CYP2A6(*)35, (*)36 and (*)37); the higher allele frequency variant CYP2A6(*)35 was associated with lower CYP2A6 activity.

Novel and established CYP2A6 alleles impair in vivo nicotine metabolism in a population of Black African descent.

Cytochrome P450 2A6 (CYP2A6) is a human enzyme best known for metabolizing tobacco-related compounds, such as nicotine, cotinine (COT), and nitrosamine procarcinogens. CYP2A6 genetic variants have been associated with smoking status, cigarette consumption, and tobacco-related cancers. Our objective was to functionally characterize four nonsynonymous CYP2A6 sequence variants with respect to their haplotype, allele frequency, and association with in vivo CYP2A6 activity. In vivo, nicotine was administered orally to 281 volunteers of Black African descent. Blood samples were collected for kinetic phenotyping and CYP2A6 genotyping. In vitro, nicotine C-oxidation catalytic efficiencies of heterologously expressed variant enzymes were assessed. The four uncharacterized sequence variants were found in seven novel alleles CYP2A6(*)24A&B ; (*)25, (*)26, (*)27, and *28A&B, most were associated with impaired in vivo CYP2A6 activity. Nicotine metabolism groupings, based on the in vivo data of variant alleles, were created. Mean trans-3'-hydroxycotinine/cotinine (3HC/COT) differed (P<0.001) between normal (100%), intermediate (64%), and slow (40%) groups. Systemic exposure to nicotine following oral administration also differed (P<0.001) between normal (100%), intermediate (139%), and slow (162%) metabolism groups. In addition, alleles of individuals with unusual phenotype-genotype relationships were sequenced, resulting in the discovery of five novel uncharacterized alleles and at least one novel duplication allele. A total of 7% of this population of Black African descent had at least one of the eight novel characterized alleles and 29% had at least one previously established allele. These findings are important for increasing the accuracy of association studies between CYP2A6 genotype and behavioral, disease, or pharmacological phenotypes.

The role of CYP2A6 in the emergence of nicotine dependence in adolescents.

OBJECTIVES: The objectives of our study were to evaluate whether genetic variation in nicotine metabolic inactivation accounted for the emergence of nicotine dependence from mid- to late adolescence and whether initial smoking experiences mediated this effect. METHODS: Participants were 222 adolescents of European ancestry who participated in a longitudinal cohort study of the biobehavioral determinants of adolescent smoking. Survey data were collected annually from grade 9 to the end of grade 12. Self-report measures included nicotine dependence, smoking, age first smoked, initial smoking experiences, peer and household member smoking, and alcohol and marijuana use. DNA collected via buccal swabs was assessed for CYP2A6 alleles that are common in white people and are demonstrated to decrease enzymatic function (CYP2A6*2, *4, *9, *12). RESULTS: Latent growth-curve modeling indicated that normal metabolizers (individuals with no detected CYP2A6 variants) progressed in nicotine dependence at a faster rate and that these increases in nicotine dependence leveled off more slowly compared with slower metabolizers (individuals with CYP2A6 variants). Initial smoking experiences did not account for how CYP2A6 genetic variation impacts nicotine dependence. CONCLUSIONS: These findings may help to promote a better understanding of the biology of smoking behavior and the emergence of nicotine dependence in adolescents and inform future work aimed at understanding the complex interplay between genetic, social, and psychological factors in adolescent smoking behavior.

Characterization of the novel CYP2A6*21 allele using in vivo nicotine kinetics.

OBJECTIVE: The impact of CYP2A6*21 (K476R) on in vivo nicotine metabolism and disposition was investigated. METHODS: A two-step allele-specific PCR assay was developed to detect the 6573A>G single nucleotide polymorphism (SNP) in CYP2A6*21. Nicotine metabolism phenotypes from a previously described intravenous labeled nicotine and cotinine infusion study [1] was used to assess the impact of CYP2A6*21. Genomic DNA samples from 222 (111 monozygotic and dizygotic twin pairs) Caucasian subjects were genotyped for CYP2A6 alleles (CYP2A6*1X2, -*1B, -*2, -*4, -*7, -*9, -*10, -*12, and -*21). The pharmacokinetic parameters were compared between individuals with no detected CYP2A6 variants (CYP2A6*1/*1, n = 163) and individuals heterozygous for the CYP2A6*21 allele (CYP2A6*1/*21, n = 9). RESULTS: The frequency of the CYP2A6*21 allele was found to be 2.3% in Caucasians (n = 5/222 alleles, evaluated in one twin from each twin pair). In vivo pharmacokinetic parameters, such as nicotine clearance (1.32+/-0.37 vs. 1.18+/-0.20 L/min), fractional clearance of nicotine to cotinine (1.02+/-0.36 vs. 0.99+/-0.23 L/min), nicotine half-life (111+/-37 vs. 116+/-29 min), and the trans-3'-hydroxycotinine to cotinine ratio (1.92+/-1.0 vs. 1.55+/-0.58) indicated no substantial differences in nicotine metabolism between those without the variant (CYP2A6*1/*1, n = 163) and those with the variant (CYP2A6*1/*21, n = 9), respectively. CONCLUSIONS: CYP2A6*21 does not have a detectable impact on nicotine metabolism in vivo. Our data suggest that CYP2A6*21 may not be important for future studies of nicotine metabolism and the resulting impacts on smoking behaviors.

Genetic influences on smoking: a brief review.

Smoking is a complex behavior influenced by environ-mental and genetic factors. A number of twin studies demonstrate genetic influences on multiple aspects of smoking including initiation, dependence, continuation, amount consumed, and cessation. It is likely that this genetic risk involves a complex interaction between multiple genes in different biologic pathways. These may include genes involved in the general aspect of dependence (eg, genes within the dopaminergic system) and those more specific to nicotine dependence (eg, genes encoding nicotinic acetylcholine receptors and nicotine-metabolizing enzymes). Genome-wide linkage analyses and gene association studies provide approaches for identifying these genes. This review provides a brief highlight of data from some of the candidate gene association studies and discusses some of the discrepancies found in the literature. In addition, the authors have reviewed some recent studies examining how genetic variations in drug-metabolizing enzymes and drug targets can influence the response to treatment with drugs such as bupropion and nicotine.These latter studies provide the potential for maximizing treatment efficacy by individualizing the type, dosage, and duration of the treatment based on an individual's genotype/phenotype. Significant advances have been made in understanding the genetic contribution to smoking behaviors. These and future studies will provide potential directions for novel prevention and treatment strategies.