Biomedical subjects

Raymond F Novak

Publications and source records attributed to Raymond F Novak.

14 recordsLinked to original sources

PubMed2006

The mitogen-activated protein kinase kinase (mek) inhibitor PD98059 elevates primary cultured rat hepatocyte glutathione levels independent of inhibiting mek.

The antioxidant activity of flavonoids, directly through scavenging oxidizing species and indirectly through modulating drug-metabolizing enzyme activities, is associated with chemopreventive and chemotherapeutic effects. However, little published information is available concerning the effect of flavonoids on glutathione (GSH) homeostasis. We previously demonstrated that PD98059 (2'-amino-3'-methoxyflavone), a flavone derivative and selective mitogen-activated protein kinase kinase (MEK) 1 inhibitor, enhanced the insulin-mediated increase in GSH levels. To determine whether the PD98059-mediated increase in GSH levels was associated with MEK inhibition, primary cultured rat hepatocytes were treated with PD98059, the MEK inhibitor U0126, which is not a flavone derivative, or flavone. PD98059 increased GSH levels in a concentration-dependent manner in hepatocytes cultured in the presence or absence of insulin. In contrast, GSH levels were not affected by U0126 at concentrations sufficient to inhibit insulin-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Flavone, however, markedly increased GSH levels without inhibition of ERK1/2 phosphorylation. The concentration of GSH in the culture medium was also elevated by PD98059 or flavone, suggesting that the cellular GSH elevation could not be accounted for by the inhibition of GSH efflux into medium. Interestingly, PD98059 and flavone increased cellular cysteine levels, which may be responsible for the PD98059- and flavone-mediated elevation of GSH levels. These results provide evidence that PD98059 and flavone produce dramatic changes in GSH homeostasis in hepatocytes, through a mechanism(s) unrelated to MEK inhibition. Moreover, the current study implies that flavonoid-induced chemopreventive and chemotherapeutic effects may be mediated by regulation of redox state through the stimulation of GSH synthesis.

Animals↗

PubMed2005

Secretome analysis of microarray data reveals extracellular events associated with proliferative potential in a cell line model of breast disease.

It is widely believed that breast cancer develops in a multistep process with premalignant lesions preceding invasive carcinoma. The characterization of molecular events associated with premalignant progression would improve our understanding of carcinogenesis and greatly benefit the development of early detection methods and chemoprevention strategies. However, the molecular biology of precancerous breast disease is poorly understood. To better characterize extracellular events associated with disease progression, such as cell-cell and cell-extracellular matrix (ECM) signaling, we analyzed gene expression profiles for the set of genes coding for secreted proteins (the secretome) in a cell line model of human proliferative breast disease (PBD). PBD describes a series of preneoplastic changes in the inner lining of milk glands associated with a dramatic increase in the risk of breast cancer. We used a series of cell lines with increasing proliferative propensity, and cell cultures were grown on matrigel to emulate in vivo growth and ECM interactions. Microarray analysis identified two clusters of secretome genes with expression profiles correlating to PBD progression. Some of the identified genes have previously been associated with breast malignancies, and our results suggest that changes in expression for these genes begin in the premalignant stage, offering potential use for early detection and as chemotherapeutic targets. RT-PCR validation demonstrates the reliability of the microarray results.

Breast Neoplasms↗

PubMed2005

Identification of the insulin signaling cascade in the regulation of alpha-class glutathione S-transferase expression in primary cultured rat hepatocytes.

We reported previously that insulin elevated alpha-class glutathione S-transferase (GSTs) protein levels in primary cultured rat hepatocytes (Kim et al., 2003b). In contrast, glucagon down-regulated alpha- and pi-class GST expression, and mechanistic research implicated cAMP and protein kinase A in this process (Kim et al., 2003b). The present study examines the signaling pathways involved in the regulation of alpha-class GST in response to insulin in primary cultured rat hepatocytes. Protein levels of GSTA1/2 and GSTA3/5 and activity of GST toward 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) were increased in an insulin concentration-dependent manner. Treatment of cells with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] or rapamycin, an inhibitor of mammalian target of rapamycin and ribosomal p70 S6 kinase (p70S6K) phosphorylation, or with an adenovirus containing green fluorescent protein and a dominant-negative and kinase-dead Akt, effectively inhibited the insulin-mediated increase in alpha-class GST expression and GST activity toward NBD. In contrast, PD98059 (2'-amino-3'-methoxyflavone), an inhibitor of mitogen-activated protein kinase kinase, SP600125 (1,9-pyrazoloanthrone), an inhibitor of c-Jun N-terminal kinase, SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imadazole], an inhibitor of p38 mitogen-activated protein kinase, or bisindolylmaleimide, a broad spectrum inhibitor of protein kinase C, did not inhibit the insulin-mediated increase in alpha-class GST protein levels in hepatocytes. These results show that PI3K/Akt/p70S6K signaling is active in the insulin-mediated up-regulation of the antioxidant defense system and that low insulin levels, as encountered in diabetes, potentially increase the susceptibility of hepatocytes to xenobiotic-mediated and/or oxidative stress-mediated damage.

Animals↗

PubMed2005

Role of mechanical and redox stress in activation of mitogen-activated protein kinases in primary cultured rat hepatocytes.

Mechanical stress is known to activate signaling cascades, including mitogen-activated protein kinase (MAPK) pathways. Although mechanical stress has been implicated in hepatic cirrhosis and liver regeneration following hepatectomy, the signaling pathway(s) that may be activated in hepatocytes in response to mechanical stress have not been determined. Using primary cultured rat hepatocytes to examine cellular signaling in response to mechanical stress associated with medium change, we observed that the phosphorylation status of extracellular signal-regulated kinase 1/2 (ERK1/2), Jun N-terminal kinase and p38 MAPK, but not Akt, was altered. MAPK activation, especially ERK1/2, was rapidly increased within 5 min, followed by a subsequent decrease to below basal levels between 30 min and 1 h following medium change. MAPK/ERK kinase (MEK1/2) phosphorylation followed the same pattern. The phosphorylation of Raf-1 in response to medium change was also consistent with Raf-1 serving as an upstream regulator of MEK1/2-ERK1/2 signaling. Phosphorylation of ERK1/2 was increased by mechanical stress alone, suggesting that mechanical stress may be primarily responsible for ERK1/2 activation in response to medium change. Medium change produced a marked decline in oxidized glutathione and malondialdehyde levels, and the antioxidant N-acetyl-L-cysteine decreased basal ERK1/2 phosphorylation, suggesting a role for oxidative stress in maintaining basal ERK1/2 phosphorylation in cultured hepatocytes. These data suggest that medium change results in immediate activation of the MAPK signaling pathway due to mechanical stress, followed by a subsequent inactivation of MAPK signaling due to a reduction in oxidative stress levels. These processes may be associated with alteration of hepatic hemodynamic circulation observed in hepatic diseases and in liver transplantation.

Animals↗

PubMed2005

Proceedings of a workshop on DNA adducts: biological significance and applications to risk assessment Washington, DC, April 13-14, 2004.

In April 2004, the Health and Environmental Sciences Institute, a branch of the International Life Sciences Institute, with support from the National Institute of Environmental Health Sciences, organized a workshop to discuss the biological significance of DNA adducts. Workshop speakers and attendees included leading international experts from government, academia, and industry in the field of adduct detection and interpretation. The workshop initially examined the relationship between measured adduct levels in the context of exposure and dose. This was followed by a discussion on the complex response of cells to deal with genotoxic insult in complex, interconnected, and interdependent repair pathways. One of the major objectives of the workshop was to address the recurring question about the mechanistic and toxicological relevance of low-concentration measured adducts and the presentations in the session entitled "Can low levels of DNA adducts predict adverse outcomes?" served as catalysts for further discussions on this subject during the course of the workshop. Speakers representing the regulatory community and industry reviewed the value, current practices, and limitations of utilizing DNA adduct data in risk assessment and addressed a number of practical questions pertaining to these issues. While no consensus statement emerged on the biological significance of low levels of DNA adducts, the workshop concluded by identifying the need for more experimental data to address this important question. One of the recommendations stemming from this workshop was the need to develop an interim "decision-logic" or framework to guide the integration of DNA adduct data in the risk assessment process. HESI has recently formed a subcommittee consisting of experts in the field and other key stakeholders to address this recommendation as well as to identify specific research projects that could help advance the understanding of the biological significance of low levels of DNA adducts.

Animals↗

PubMed2005

Acetoacetate induces CYP2E1 protein and suppresses CYP2E1 mRNA in primary cultured rat hepatocytes.

The ketone body acetoacetate (AA) in the absence of insulin or in the presence of diabetic insulin levels decreases CYP2E1 mRNA expression in a concentration- and time-dependent manner in primary cultured rat hepatocytes. AA activates p70 ribosomal S6 kinase (p70S6K) and protein kinase C (PKC) by approximately 2- to 2.5-fold, respectively, following 6-h treatment. The AA-mediated activation of p70S6K, but not PKC, was abolished by inhibition of PI 3-K with LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] or wortmannin, in agreement with p70S6K being downstream of phosphatidylinositol 3-kinase (PI 3-K). Inhibition of PI 3-K, mTOR with rapamycin, or PKC with bisindolylmaleimide ameliorated the AA-mediated down-regulation of CYP2E1 mRNA expression. Neither the mitogen-activated protein kinase kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone) nor the p38 mitogen-activated protein kinase inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] ameliorated the AA-mediated suppression of CYP2E1 mRNA expression. Heterogeneous nuclear RNA analysis revealed that AA suppressed CYP2E1 gene transcription by approximately 50% and that inhibition of PI 3-K and PKC diminished this AA-mediated effect on transcription. CYP2E1 mRNA half-life slightly increased from approximately 24 h in untreated hepatocytes to approximately 32 h in AA-treated cells. Interestingly, AA increased CYP2E1 protein levels by approximately 2- and 2.5-fold at 24 and 48 h, respectively. DL-beta-hydroxybutyrate was without effect. Polysomal distribution studies revealed that AA increased the proportion of RNA associated with the actively translated polysomal fractions versus the 40S to 60S untranslated fractions by approximately 40%. CYP2E1 protein half-life increased from approximately 8 h in untreated hepatocytes to approximately 24 in AA-treated cells. These data show that AA decreases CYP2E1 mRNA expression through inhibition of gene transcription while simultaneously elevating CYP2E1 protein levels through increased translation and decreased protein degradation.

Acetoacetates↗

PubMed2004

Insulin signaling regulates gamma-glutamylcysteine ligase catalytic subunit expression in primary cultured rat hepatocytes.

Decreased glutathione (GSH) levels and gamma-glutamylcysteine ligase (GCL) activity have been observed in diabetic patients, and insulin reportedly increases GSH synthesis via increased GCL catalytic subunit (GCLC) gene expression. The signaling pathways responsible for mediating insulin effects on GCLC expression and GSH levels, however, are unknown. The signaling pathways involved in the regulation of GSH synthesis in response to insulin were examined in primary cultured rat hepatocytes. GSH levels, GCL activity, GCLC protein, and mRNA levels were increased to 140, 160, 600, and 340% of that monitored in untreated cells, respectively, in hepatocytes cultured with 100 nM insulin. The phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002 [2-(4-morpholinyl)-9-phenyl-4H-1-benzopyran-4-one], dominant-negative Akt, or rapamycin, an inhibitor of mTOR (mammalian target of rapamycin) and ribosomal p70 S6 kinase (p70S6K) phosphorylation, inhibited the insulin-mediated increase in GCLC protein and GSH levels. Although the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase, p38 MAPK, and JNK (c-Jun N-terminal kinase) were activated in response to insulin, PD98059 (2'-amino-3'-methoxyflavone), an inhibitor of mitogen-activated protein kinase kinase, SP600125 (1,9-pyrazoloanthrone), an inhibitor of JNK, and SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], an inhibitor of p38 MAPK, failed to inhibit the insulin-mediated increase in GCLC protein levels. In conclusion, these data show that insulin signaling pathways involving PI3K/Akt/p70S6K, but not MAPKs, are active in the insulin-mediated regulation of GSH synthesis via increased GCLC expression.

Animals↗

PubMed2004

Acetoacetate activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase in primary cultured rat hepatocytes: role of oxidative stress.

Diabetes is characterized by elevated levels of ketone bodies acetoacetate (AA) and 3-hydroxybutyrate (3HB). High levels of ketone bodies have been implicated in generation of cellular oxidative stress. Ketone body activation of cellular signaling pathways associated with oxidative stress, however, has not been established. Thus, ketone body effects on kinase activation in primary cultured rat hepatocytes have been examined. Treatment with AA increased the phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 mitogen-activated protein kinase (MAPK), maximally by approximately 2.5- and 4-fold, respectively. AA failed to activate c-Jun NH(2)-terminal kinase. AA-mediated Erk1/2 and p38 MAPK activation was detectable at 3 h post-treatment with maximal activation occurring at 12 h. In contrast, 3HB failed to activate any of these kinases. Elevated phosphorylation of Raf and MKK3/6 also occurred in response to AA. Bisindolylmaleimide, a generalized protein kinase C (PKC) inhibitor, and B581, a Ras farnesylation inhibitor, inhibited AA-mediated activation of Erk1/2 and p38 MAPK, suggesting a role for PKC and Ras in mediating such activation. Interestingly, the tyrosine kinase inhibitor genistein prevented the AA-mediated phosphorylation of Erk1/2, but not p38 MAPK. AA treatment resulted in the generation of reactive oxygen species (ROS) and the depletion of cellular glutathione levels, which was ameliorated by the antioxidants N-Acetyl-l-cysteine (NAC) and Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid). NAC and Trolox also ameliorated AA-mediated Erk1/2 and p38 MAPK activation, suggesting that this activation is associated with ROS and oxidative stress.

Acetoacetates↗

PubMed2004

Gene-specific dye bias in microarray reference designs.

The most widely used microarray experiment design includes the use of a reference standard. Comparisons of gene expression between samples are facilitated because each sample is directly measured against the reference standard, using two fluorescent dyes. Numerous reports indicate that some genes incorporate the two commonly used dyes with different efficiencies, contributing to inaccurate data. However, it is widely assumed that these effects will not corrupt results if the reference standard is labeled with the same dye on each microarray. We demonstrate that this assumption is not reliable and that dye orientation can significantly influence measured changes in gene expression.

Animals↗

PubMed2003

Oxidative DNA damage and repair in a cell lineage model of human proliferative breast disease (PBD).

Oxidative damage to DNA is thought to play a significant role in mutagenesis, aging, and cancer. Sensitivity to oxidative DNA damage and DNA repair efficiency were examined using a series of human breast epithelial cell lines-MCF-10A, MCF-10AT, and MCF-10ATG3B-with progressively elevated Ras protein. Breast epithelial cells were treated with H2O2, in the absence and presence of the DNA-repair inhibitors hydroxyurea (HU) and cytosine arabinoside (Ara-C). DNA strand breaks were assessed by the mean olive tail moment (microm) using the alkaline single-cell gel electrophoresis (Comet) assay. In untreated cells, the mean olive tail moment values were 4.3 +/- 0.7, 8.3 +/- 1.1, and 7.1 +/- 0.6 microm in the MCF-10A, MCF-10AT, and MCF-10ATG3B cells, respectively. Five min H2O2 treatment produced concentration-dependent DNA damage, with the MCF-10A cells most susceptible and the tumorigenic MCF-10ATG3B cells the least susceptible. Treatment with 100 microM H2O2 resulted in approximately 17-, 6-, and 4.5-fold increases in mean olive tail moment values in the MCF-10A, MCF-10AT, and MCF-10ATG3B cells, respectively, compared to untreated cells. The HCC1937 tumor cell line responded in a manner comparable to the MCF-10ATG3B cells treated with H2O2, HU/Ara-C pre-treatment resulted in a approximately 1.5-fold increase in olive tail moment values in all three cell lines. Protein levels of antioxidant enzymes, including catalase, copper/zinc superoxide dismutase (Cu/Zn SOD), and manganese SOD (MnSOD) were determined in order to examine a potential mechanism for increased resistance to H2O2-mediated DNA damage. Levels of these enzymes increased progressively, with highest expression in MCF-10ATG3B cells. Increased cellular resistance also coincided with marked decreases in p53 protein levels. These results demonstrate that, in this cell lineage, sensitivity to oxidative DNA damage by H2O2 decreases with tumorigenicity (i.e., MCF-10A vs. MCF-10ATG3B), and show that DNA repair, altered Ras, and p53 expression, or compensatory mechanisms involving elevated antioxidant enzymes are involved in mediating these effects.

Animals↗

PubMed2003

Insulin and glucagon signaling in regulation of microsomal epoxide hydrolase expression in primary cultured rat hepatocytes.

Microsomal epoxide hydrolase (mEH) plays an important role in the detoxification of a broad range of epoxide intermediates and has been reported to be decreased during diabetes and fasting. The signaling pathways involved in the regulation of mEH expression in response to insulin and glucagon were examined in primary cultured rat hepatocytes. mEH protein levels were increased 2- to 6-fold in hepatocytes cultured for 1 to 4 days, respectively, in the presence of insulin. Concentration-response studies revealed that insulin concentrations >or=1 nM resulted in increased mEH protein levels. The phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin or LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], and rapamycin, an inhibitor of p70 S6 kinase phosphorylation, ameliorated the insulin-mediated increase in mEH protein levels. The p38 mitogen-activated protein (MAP) kinase inhibitors SB203580 and SB202190 also abrogated the insulin-mediated increase in mEH protein. Treatment of cells with glucagon, 8-bromo-cAMP, or dibutyryl-cAMP for 3 days resulted in decreased mEH protein levels. Pretreatment with the protein kinase A (PKA) inhibitor H89 (N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline) prior to glucagon addition markedly attenuated the glucagon effect, implicating PKA signaling in the regulation of mEH expression. These data demonstrate that insulin and glucagon regulate, in an opposing manner, the expression of mEH in primary cultured rat hepatocytes. Furthermore, these data suggest that PI3K and p70 S6 kinase are active in the regulation of insulin-mediated mEH expression. We also provide data implicating p38 MAP kinase in the insulin-mediated increase in mEH levels. Moreover, cAMP and PKA are implicated in mediating the inhibitory effect of glucagon on mEH expression.

Animals↗

PubMed2003

Insulin and glucagon regulation of glutathione S-transferase expression in primary cultured rat hepatocytes.

Diabetes is a major cause of morbidity and mortality, and complications resulting from diabetes have been attributed in part to increased oxidative stress. Glutathione S-transferases (GSTs) constitute a major protective mechanism against oxidative stress. Studies of the expression and activity of GSTs during diabetes are inconclusive, with both increased and decreased GST expression being reported in vivo. Insulin and glucagon effects on GST expression and the signaling pathway involved in the glucagon regulation of GST expression were examined in primary cultured rat hepatocytes. The addition of insulin resulted in the elevation of alpha-class GST protein levels, whereas alpha- and pi-class GST protein levels were markedly decreased in hepatocytes cultured with glucagon. In contrast, mu-class GST protein expression was unaffected by insulin or glucagon treatment. Insulin concentrations >/=1 nM resulted in increased GST activities and alpha-class GST protein levels, whereas glucagon concentrations >/=20 nM decreased alpha- and pi-class protein levels and activity. Treatment of cells with 8-bromo-cAMP or dibutyryl-cAMP also resulted in decreased alpha- and pi-class GST protein levels. Pretreatment with N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H89), a selective inhibitor of protein kinase A, before glucagon addition markedly attenuated the glucagon effect. This study demonstrates that insulin and glucagon regulate, in an opposing manner, the expression of alpha-class GSTs and that glucagon completely inhibits pi-class GST expression in vitro, suggesting that hepatic GST expression may be decreased during diabetes. Furthermore, the present study implicates cAMP and protein kinase A in mediating the inhibitory effect of glucagon on GST expression.

Animals↗

PubMed2002

Induction of microsomal epoxide hydrolase by sulfur amino acid deprivation via the pathway of C-Jun N-terminal kinase and its extracellular exposure during cell death.

Microsomal epoxide hydrolase (mEH), an epoxide detoxifying enzyme and putative cell surface autoantigen, is inducible by xenobiotics and by certain pathophysiological conditions (e.g., tumorigenesis and protein-calorie malnutrition). The present study was designed to determine mEH expression in H4IIE cells during cell death initiated by sulfur amino acid deprivation (SAAD) and to identify the signaling pathway for the enzyme induction. SAAD induced cell death at 48-72 h with translocation of Bax to mitochondria and increased mitochondrial permeability with cytochrome c release, both of which were prevented by SB203580 or by dominant-negative JNK1 [JNK1(-)] stable transfection. Caspase-3 activity was only marginally increased by SAAD. Neither genomic DNA fragmentation nor poly(ADP-ribose) polymerase cleavage was observed during SAAD-induced cell death. Thus, SAAD induced cell death independent of caspase activation. This was supported by the observation that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a general caspase inhibitor, did not prevent cell death. The levels of mEH mRNA and protein were notably increased in cells under SAAD for 48-72 h. The induction of mEH occurred in parallel with cell death. Whereas SAAD-induced cell death resulted from both JNK1 and p38 kinase activation, mEH induction was decreased only by JNK1(-) transfection. Immunocytochemistry revealed that mEH protein was intensely stained in dying cells, cellular fragments and cell debris. Furthermore, the number of cells positive for surface mEH substantially increased by SAAD, as evidenced by flow cytometry analysis. These results demonstrated that SAAD induced nonapoptotic cell death with Bax translocation to mitochondria and mitochondrial cytochrome c release, but not through caspase-3 activation, and that mEH was induced by SAAD via the pathway of JNK1, but not ERK1/2 or p38 kinase, in parallel with cell death.

Amino Acids, Sulfur↗

PubMed2002

Insulin signaling in the transcriptional and posttranscriptional regulation of CYP2E1 expression.

Diabetes has been reported to increase the expression of cytochrome P450 (CYP) 2E1 messenger RNA (mRNA) and protein several-fold, and enhanced expression has been associated with elevated ketone bodies. Primary cultured rat hepatocytes were used to explore ketone body and insulin regulation of CYP2E1 expression. Hydroxybutyrate and acetoacetate (AC), alone or in combination, either failed to affect or decreased CYP2E1 mRNA levels by up to 90% relative to untreated hepatocytes. Insulin produced a concentration-dependent decrease in CYP2E1 mRNA levels, and insulin receptor immunoprecipitation showed a correspondence between receptor phosphorylation and the decrease in CYP2E1 mRNA levels at physiologic levels of insulin. Phosphatase inhibitors decreased CYP2E1 mRNA levels by greater than 95%. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitors wortmannin or LY294002 and rapamycin, an inhibitor of p70 S6 kinase phosphorylation, ameliorated the insulin-mediated decrease in CYP2E1 mRNA levels. Geldanamycin, which inhibits Src kinase, also abrogated the insulin-mediated decrease in CYP2E1 mRNA levels. In contrast, the protein kinase C (PKC) inhibitor bisindolylmaleimide, the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, and the p38 mitogen-activated protein (MAP) kinase inhibitor SB202190 did not affect the insulin-mediated decrease in CYP2E1. CYP2E1 mRNA half-life decreased from approximately 48 hours in the absence of insulin to approximately 15 hours at 10 nmol/L insulin, and this decrease was prevented by wortmannin. The half-life of CYP2B mRNA was increased by insulin, whereas that of CYP3A was unaffected. Analysis of CYP2E1 gene transcription using heterogeneous nuclear RNA (hnRNA) showed that insulin suppressed CYP2E1 transcription. In conclusion, these data show involvement of transcriptional and posttranscriptional mechanisms in the insulin-mediated regulation of CYP2E1 and implicate PI3-kinase, p70 S6 kinase, and Src kinase in mediating these effects.

Animals↗