![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 289, Issue 2, 1121-1127, May 1999
Institute of Chemical Toxicology, Wayne State University, Detroit, Michigan
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Uncontrolled diabetes results in enhanced expression of cytochrome P-450 (CYP)2E1, CYP2B, CYP3A, and CYP4A. Because of the simultaneous and confounding metabolic and hormonal changes that occur in vivo as a consequence of diabetes, primary cultured rat hepatocytes provide an excellent model system for examination of the effects of insulin on P-450 expression and on xenobiotic-mediated P-450 expression. In the present study, we examined the effects of insulin on pyridine-, phenobarbital-, and ciprofibrate-mediated expression of CYP2E1, CYP2B, CYP3A, and CYP4A in primary cultured rat hepatocytes. Pyridine addition to primary rat hepatocytes cultured in the presence of 1 nM insulin or in the absence of insulin resulted in a 3.5-fold and 3-fold enhancement in CYP2E1 protein expression, respectively, in the absence of any pyridine-mediated increase in mRNA expression. In contrast, hepatocytes cultured in the standard concentration of 1 µM insulin resulted in only a 2-fold increase in protein expression. Thus, the fold-induction of CYP2E1 protein in response to pyridine was 1.5- to 1.8-fold greater in either the absence of insulin or in the presence of 1 nM insulin, respectively, than that monitored in the presence of 1 µM insulin. To examine whether insulin effects on xenobiotic-mediated CYP2E1 expression were selective, insulin effects on xenobiotic-mediated expression of transcriptionally regulated CYP2B, CYP3A, and CYP4A were examined. Pyridine- or phenobarbital-mediated induction of CYP2B mRNA and protein expression in hepatocytes was suppressed by as much as 80% at lower insulin levels (0 and 1 nM), relative to the level monitored in the presence of 1 µM insulin. Omitting insulin from the medium resulted in a 50% decrease in CYP3A mRNA levels in response to phenobarbital treatment and a 30% decrease in CYP4A mRNA levels in response to ciprofibrate treatment, relative to the level obtained in response to these treatments in the presence of 1 µM insulin. The results of this study demonstrate that decreasing the insulin level in the primary hepatocyte culture medium enhanced xenobiotic-mediated CYP2E1 expression, whereas lower insulin levels suppressed xenobiotic-mediated CYP2B, CYP3A, and CYP4A expression in this cell culture system.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Pathophysiologic
alterations such as diabetes, fasting, and high-fat diet increase
cytochrome P-450 (CYP)2E1 expression by ~3- to 8-fold at both the
mRNA and protein levels (Hong et al., 1987
; Song et al., 1987; Bellward
et al., 1988; Dong et al., 1988; Favreau and Schenkman, 1988; Johansson
et al., 1988; Shimojo et al., 1993). CYP2E1 mRNA and protein are
elevated in both chemically induced (Dong et al., 1988; Favreau and
Schenkman, 1988; Thummel and Schenkman, 1990; Shimojo et al., 1993) and
spontaneous (Bellward et al., 1988; Dong et al., 1988) diabetic rats.
Elevation of CYP2E1 mRNA levels in the diabetic state in vivo has been
attributed to mRNA stabilization (Song et al., 1987). CYP2B1 (Yamazoe
et al., 1989b; Barnett et al., 1990a; Donahue and Morgan, 1990), CYP3A (Barnett et al., 1990b; Shimojo et al., 1993), and CYP4A (Barnett
et al., 1990b; Shimojo et al., 1993) protein and activity levels have
also been reported to be increased ~2- to 5-fold in rats made
diabetic by alloxan or streptozotocin treatment. The elevated
expression of these P-450s has been largely attributed to
diabetes-induced alterations in metabolism (elevated ketone body
levels; Bellward et al., 1988; Dong et al., 1988; Barnett et al.,
1990a; Barnett et al., 1990b) or hormone secretion (decreased growth
hormone and testosterone levels; Yamazoe et al., 1989a,b; Thummel and
Schenkman, 1990; Richardson et al., 1992). Although insulin
administration to diabetic rats has been shown to lower CYP2E1, CYP2B,
CYP3A, and CYP4A to control levels (Dong et al., 1988; Favreau and
Schenkman, 1988; Yamazoe et al., 1989b; Barnett et al., 1990b; Donahue
and Morgan, 1990; Shimojo et al., 1993), these effects have been
attributed to the normalization of ketone body and/or growth hormone
levels. We have demonstrated, using primary cultured rat hepatocytes,
that CYP2E1 mRNA and protein expression are primarily regulated by
insulin, with lower insulin concentrations (which reflect the diabetic
state) elevating CYP2E1 mRNA and protein levels ~4- to 11-fold
(Woodcroft and Novak, 1997). In contrast, lower insulin levels were
found to have minimal effect on basal CYP2B, CYP3A, or CYP4A expression
in primary cultured rat hepatocytes (Woodcroft and Novak, 1997).
Treatment of rats with xenobiotics such as pyridine, acetone, or
alcohols (ethanol and isopropanol) results in increased hepatic CYP2E1
protein levels in the absence of a concomitant increase in CYP2E1 mRNA
levels, indicating that posttranscriptional mechanisms are involved in
the regulation of this P-450 (Johansson et al., 1988; Kim et al.,
1988). Mechanistic studies in vivo have implicated both translational
efficiency (Kim and Novak, 1990; Kim et al., 1990) and protein
stabilization (Song et al., 1989; Roberts et al., 1995) in
xenobiotic-mediated elevation of CYP2E1 protein levels.
It has been reported that administration of the CYP2E1 inducer
4-methylpyrazole to rats made diabetic by streptozotocin treatment results in an additive increase in hepatic CYP2E1 protein levels relative to the increases obtained by either 4-methylpyrazole or
streptozotocin treatment alone, indicating that xenobiotic-mediated CYP2E1 protein expression is enhanced during diabetes (Wu and Cederbaum, 1993). Thus, the mechanisms governing regulation of CYP2E1
expression are complex and involve transcriptional,
post-transcriptional, translational, and post-translational events.
Moreover, the superimposition of xenobiotic effects upon metabolic
and/or hormonal changes further complicates delineation of the factors
and/or mechanisms regulating CYP2E1 expression.
In the present study, the effect of insulin on xenobiotic-mediated
CYP2E1 mRNA and protein expression in primary cultured rat hepatocytes
was examined for the purpose of further delineating the contribution of
insulin to CYP2E1 expression. Moreover, the effects of insulin (or
diabetes) on xenobiotic-mediated expression of CYP2B, CYP3A, or CYP4A
have not been examined either in vivo or in cultured hepatocytes,
despite reports that these P-450s are elevated during diabetes (Yamazoe
et al., 1989b; Barnett et al., 1990a, 1990b; Donahue and Morgan, 1990;
Shimojo et al., 1993). Thus, xenobiotics [e.g., pyridine (PYR),
phenobarbital (PB), and ciprofibrate (CIPRO)] known to enhance the
expression of CYP2E1, CYP2B, CYP3A, and CYP4A were used to assess the
effect of insulin on xenobiotic-enhanced expression of these P-450s in
primary cultured rat hepatocytes. Treatment of primary rat hepatocytes
with a CYP2E1-inducing agent, in combination with lower insulin
concentrations in the medium (to mimic the diabetic state), results in
greater CYP2E1 protein levels than occurs with either of these
treatments alone. CYP2E1 protein levels are elevated in response to the
inducing agent by 1.5- to 1.8-fold in the presence of lower insulin
concentrations over the levels monitored in the presence of the
standard concentration of 1 µM insulin. In contrast,
xenobiotic-mediated CYP2B, CYP3A, and CYP4A induction, which occurs
primarily through transcriptional activation, was suppressed by 30 to
80% by lowering the insulin concentration. Thus, insulin
differentially regulates xenobiotic-mediated expression of CYP2E1
relative to CYP2B, CYP3A, and CYP4A.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Chemicals. Modified Chee's medium, L-glutamine, tricine, and gentamicin were obtained from Life Technologies, Inc. (Gaithersburg, MD). Insulin (NovolinR) was purchased from Novo-Nordisk (Princeton, NJ). Collagenase (type I) was purchased from Worthington Biochemicals (Freehold, NJ). Culture dishes (Falcon) or flasks (Corning) were obtained from Baxter (McGaw Park, IL). Vitrogen (95-98% type I collagen, 2-5% type III collagen) was obtained from Celtrix Pharmaceuticals (Santa Clara, CA). Anti-rat CYP2B1 was purchased from XenoTech (Kansas City, KS). Horseradish peroxidase-conjugated goat anti-rabbit antibody was obtained from BioRad (Melville, NY). Enhanced chemiluminescence reagents were purchased from Amersham (Arlington Heights, IL). PB was obtained from Mallinckrodt (Chesterfield, MO). PYR (technical grade) was obtained from Fisher (Pittsburgh, PA). CIPRO was a gift from Sterling-Winthrop Pharmaceuticals (Rensselaer, NY). All other reagents were from Sigma Chemical Co. (St. Louis, MO).
Primary Rat Hepatocyte Cultures.
Hepatocytes were isolated
from the livers of male Sprague-Dawley rats (200-300 g) using
collagenase perfusion as described previously (Seglen, 1982; Zangar et
al., 1995). Hepatocytes were plated onto dishes or flasks covalently
coated with Vitrogen as described previously (Waxman et al., 1990;
Zangar et al., 1995). Cells were plated at densities of 12 × 106 cells/75 cm2 flask or
4 × 106 cells/25 cm2
flask. Modified Chee's medium was fortified as described (Waxman et
al., 1990), except that it was supplemented with 0.1 µM dexamethasone and 1 µM insulin, the standard concentration of insulin used in primary hepatocyte culture. Four hours after plating, medium was replaced with medium containing various concentrations of insulin (no
insulin, 1 nM, and 1 µM). The medium was changed every 24 h thereafter.
; Zangar and Novak, 1997; Woodcroft
and Novak, 1998). All cells were harvested 96 h after initiation
of culture. When treatment involved the volatile chemical PYR, the
tissue culture flasks were sealed to prevent loss of the chemical.
Thus, after media change, loosely capped flasks were returned to the
incubator for 1 to 2 h to allow equilibration of media with the
incubator chamber atmosphere. Chemicals were then added, and flasks
were sealed. We have reported previously that sealing of the flasks for
the duration of the xenobiotic treatment period did not affect CYP2E1
or 2B expression (Woodcroft and Novak, 1998). PYR was prepared as a 5 M
stock solution in sterile water. PB was prepared as a 100 mM stock
solution in sterile water. CIPRO was prepared as a 30 mM stock solution
in 100 mM HEPES (pH 7.2). Data for statistical analysis were obtained
from three separate microsome or total RNA preparations from a single hepatocyte preparation, and reproducibility of results were confirmed in at least two hepatocyte preparations (data not shown).
The xenobiotic and insulin concentrations used in this study have
previously been demonstrated by our laboratory to be nontoxic to
primary cultured rat hepatocytes, as measured by lactate dehydrogenase release (Zangar et al., 1995; Woodcroft and Novak, 1997).
Immunoblot Analyses.
Hepatocyte microsomes were prepared
essentially as described previously (Zangar et al., 1995; Woodcroft and
Novak, 1998). Cells were washed twice with 5 ml of 4°C PBS (pH 7.4),
suspended in 6 ml of PBS/flask using three flasks/treatment, and
pelleted by centrifugation at 200g for 3 min. To minimize
the possibility of proteolytic degradation of CYP2E1 protein during
isolation, cells were suspended in 1 ml of buffer containing protease
inhibitors (20 mM Tris-Cl, pH 7.4, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 5 µg/ml pepstatin A, 50 mM spermine, 50 mM spermidine, 250 mM sucrose, 10 mM 
-mercaptoethanol, 2 mM EDTA, and 2 mM EGTA) and repelleted. Cells were then suspended in 250 µl of this buffer, homogenized for 30 s using an Omni 1000 homogenizer (Waterbury, CT) set on high, and centrifuged for 10 min at 10,000g,
4°C. The resulting supernatant was centrifuged for 1.5 h at
105,000g, 4°C. The pelleted microsomes were washed with 1 ml of microsome storage buffer (50 mM Tris-acetate, 20% glycerol, 1 mM
EDTA, pH 7.4) and then suspended in 100 µl of this buffer by very
gentle sonication with a probe sonicator (setting 2 on a Tekmar Sonic
Disruptor for 20 to 30 s). Microsomal proteins (20 µg/lane) were
separated on 10% polyacrylamide gels and transferred to nitrocellulose
for immunoblot analysis as described previously (Laemmli, 1970).
). Detection of the bound primary antibodies was performed using
horseradish peroxidase-conjugated secondary antibody followed by
detection by chemiluminescence. Densitometry was performed using a
Molecular Dynamics (Sunnyvale, CA) laser densitometer and the
ImageQuant analysis program.
Northern Blot Analysis.
Total RNA from hepatocytes was
isolated and separated (10 µg/lane) on formaldehyde-agarose gels and
capillary blotted as described previously (Sambrook et al., 1989; Xie
and Rothblum, 1991). A CYP2E1 cDNA probe was prepared as described
previously (Zangar et al., 1995). cDNAs for CYP2B1 (Doehmer et al.,
1988), CYP3A1 (pDex12) (Wrighton et al., 1985), and CYP4A1 (Hardwick et
al., 1987) were generously provided by Drs. Milton Adesnik (New
York University Medical Center, NY), Philip Guzelian (University of Colorado, Denver, CO), and Frank Gonzalez (National Cancer Institute, Bethesda, MD), respectively, and the cytoplasmic 7S cDNA (Balmain et
al., 1982) was generously provided by Dr. Allan Balmain (Beatson Institute for Cancer Research, Glasgow, UK). The cDNAs were labeled using a random primer kit (Life Technologies, Inc.) according to the
manufacturer's instructions.
). The blots were then exposed
to X-ray film (Kodak XAR) for 2 h to 4 days, and bands were
quantified using a Molecular Dynamics (Sunnyvale, CA) laser
densitometer and the ImageQuant analysis program.
Statistics. Significant differences between treatment groups were determined by ANOVA, followed by Tukey-Kramer, p < .05.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Insulin Effects on CYP2E1 mRNA and Protein Expression.
Basal
expression of CYP2E1 mRNA was monitored in primary hepatocytes cultured
for 96 h in the presence of the standard concentration of 1 µM
insulin or 1 nM insulin or in the absence of insulin. Relative to the
level of CYP2E1 mRNA in hepatocytes cultured in the presence of 1 µM
insulin, hepatocytes maintained in culture in the presence of 1 nM
insulin exhibited an ~4-fold increase in the basal expression of
CYP2E1 mRNA, and those cultured in the absence of insulin exhibited an
~12-fold increase in CYP2E1 mRNA expression (Fig.
1).
|
|
Insulin Effects on CYP2B mRNA and Protein Expression.
Basal
CYP2B mRNA expression was marginally increased (maximally 3.5-fold) by
maintaining hepatocytes in the presence of 1 nM insulin or in the
absence of insulin, relative to levels monitored in hepatocytes
cultured in the presence of 1 µM insulin (Fig. 3).
|
|
|
Insulin Effects on CYP3A and CYP4A mRNA Expression. Because the expression and activities of CYP3A and CYP4A have also been reported to be elevated in diabetic rats, and these P-450s are regulated primarily at the level of transcription, we examined the effect of insulin on xenobiotic-mediated CYP3A and CYP4A mRNA expression.
CYP3A mRNA levels were decreased slightly (~35%) in primary hepatocytes cultured in the absence of insulin compared with hepatocytes cultured in the presence of 1 µM insulin (Fig. 6). PB-induced CYP3A mRNA levels were significantly (~50%) lower in hepatocytes maintained in the absence of insulin relative to those maintained in the presence of 1 µM insulin (Fig. 6). The induction of CYP3A mRNA by PB decreased from ~6-fold in the presence of 1 µM insulin to ~4.5-fold in the absence of insulin. Thus, the absence of insulin significantly decreased PB-mediated expression of CYP3A mRNA.
|
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Primary rat hepatocyte culture offers an excellent system with
which to examine the effects of insulin on xenobiotic-mediated expression of these P-450s, because it permits control of the insulin
levels in the absence of simultaneous changes in metabolic or hormonal
factors (e.g., ketone bodies, growth hormone, and testosterone), which
occur during diabetes in vivo. We have shown that a primary rat
hepatocyte culture system that provides detectable basal expression of
CYP2E1, CYP2B, CYP3A, and CYP4A mRNA and protein is also responsive to
xenobiotic-mediated increases in these P-450s in a manner that
parallels that monitored in vivo (Zangar et al., 1995; Zangar and
Novak, 1997; Zangar and Novak, 1998; Woodcroft and Novak, 1998). We
have also used this primary rat hepatocyte culture system to examine
insulin effects on basal expression of CYP2E1, CYP2B, CYP3A, and CYP4A
(Woodcroft and Novak, 1997).
The basal expression of CYP2E1, and to a lesser extent CYP2B, mRNA and
protein was enhanced in primary cultured rat hepatocytes by either
lowering the insulin concentration or completely excluding insulin from
the medium (Figs. 1-4; Woodcroft and Novak, 1997). In contrast,
exclusion of insulin from the culture medium resulted in a slight
decrease in the basal levels of CYP3A and CYP4A mRNA (Figs. 6 and 7;
Woodcroft and Novak, 1997).
The substantial increase in CYP2E1 mRNA and protein levels in
hepatocytes cultured in lower concentrations of insulin observed in the
present study and in previous studies by our laboratory (Woodcroft and
Novak, 1997) demonstrate that insulin is primarily responsible for the
increase in CYP2E1 expression. In more detailed concentration-response
experiments performed in primary cultured rat hepatocytes in our
laboratory, the level of insulin monitored in the diabetic
Sprague-Dawley rat (<0.02 nM; Donahue and Morgan, 1990) corresponds to
the insulin concentration at which we begin to observe a positive
effect on CYP2E1 expression (0.01 nM; Woodcroft and Novak, unpublished
observations). That insulin appears to be the primary regulator of the
increase in CYP2E1 expression is supported further by additional
studies from our laboratory, which demonstrated that ketone bodies
(3-hydroxybutyrate and acetoacetate) did not increase the expression of
CYP2E1 mRNA or protein in primary cultured rat hepatocytes (Zangar and
Novak, 1997).
Whereas insulin plays a primary role in regulating CYP2E1 expression in
primary cultured rat hepatocytes, it does not appear to be a primary
mediator of CYP2B expression, because minimal increases in basal
expression were observed in this study in the presence of lowered
insulin levels (Figs. 3-5). Concomitant research, however, has
demonstrated that 3-hydroxybutyrate and acetoacetate do cause
substantial increases in CYP2B expression (Zangar and Novak, 1997). Our
studies also indicate that insulin is not a major factor in regulating
CYP3A or CYP4A expression, because maintaining hepatocytes in the
absence of insulin resulted in minimal decreases in the basal levels of
these two P-450s (Figs. 6 and 7).
The effects of insulin on xenobiotic-enhanced CYP2E1 expression
observed in this study suggest that insulin can also affect protein
expression independent of effects on mRNA expression. Maintaining
hepatocytes in the presence of 1 nM insulin or in the absence of
insulin, compared with in the presence of the standard concentration of
1 µM insulin, resulted in an increase in basal expression of CYP2E1
mRNA (Fig. 1) and CYP2E1 protein (Fig. 2). However, lowering the
insulin levels resulted in a greater increase in the expression of
CYP2E1 protein after PYR treatment than that monitored in the presence
of 1 µM insulin (Fig. 2), with no concomitant xenobiotic-mediated
increase in CYP2E1 mRNA (Fig. 1) (i.e., lowering the insulin
concentration enhanced the degree of CYP2E1 protein induction mediated
by PYR). These findings support similar observations in rats by Wu and
Cederbaum (1993), wherein hepatic CYP2E1 protein levels were reported
to be increased to a greater degree by 4-methylpyrazole treatment of
streptozotocin-induced diabetic rats than by either 4-methylpyrazole or
streptozotocin treatment alone without additional effect on CYP2E1 mRNA
levels other than that produced by streptozotocin alone.
In contrast to the elevation of xenobiotic-mediated CYP2E1 protein
levels produced by lower insulin concentrations, CYP2B mRNA and protein
expression in response to PYR or PB treatment were suppressed by lower
insulin levels (Figs. 3-5). Interestingly, lower insulin
concentrations exerted opposing effects on basal and
xenobiotic-mediated CYP2B expression, enhancing basal levels while
suppressing xenobiotic-mediated CYP2B expression. The insulin effect on
CYP2B is similar to that reported with dexamethasone, which at low
concentrations suppresses basal expression of CYP2B mRNA in primary
cultured rat hepatocytes while enhancing PB-mediated induction (Kocarek
et al., 1994). This may be a result of xenobiotic inducers of CYP2B
acting on intermediary components of the insulin receptor-mediated
signal transduction pathway, leading to an enhancement of the insulin
effect on CYP2B gene expression.
The absence of insulin decreased expression of CYP3A and CYP4A mRNA in
both untreated and xenobiotic-treated hepatocytes, a result that
differs from that of CYP2E1 or CYP2B, illustrating that insulin can
have multiple and varied effects on gene expression in primary cultured
hepatocytes. This is consistent with the effects of insulin on other
genes. For example, insulin has been reported to inhibit the expression
of cholesterol 7
-hydroxylase, sterol 27-hydroxylase, aspartate
aminotransferase, phosphoenolpyruvate carboxykinase, growth
hormone, and glucagon while stimulating the expression of
gylceraldehyde-3-phosphate dehydrogenase, c-Fos, -amylase, and
glucokinase (O'Brien and Granner, 1991; Twisk et al., 1995; and
references therein).
Mechanistic studies on the regulation of expression of CYP2E1 have been
limited by the lack of a cell culture system that expresses detectable
basal CYP2E1 mRNA and protein and that mimics the in vivo response of
CYP2E1 to xenobiotics. Our laboratory has used primary rat hepatocytes
cultured in modified Chee's medium on Vitrogen substratum to achieve
both measurable basal expression of CYP2E1 and a significant induction
response to xenobiotics known to enhance the hepatic expression of
CYP2E1 in vivo (Zangar et al., 1995; Woodcroft and Novak, 1998). The
results of the present study using this cell culture model indicate
that insulin differentially regulates the xenobiotic-enhanced
expression of CYP2E1, CYP2B, CYP3A, and CYP4A, enhancing that of CYP2E1
while suppressing the others. We have also demonstrated that insulin
can affect the level of xenobiotic-mediated expression of CYP2E1
protein exclusive of any effect of insulin on CYP2E1 mRNA. Thus, this
cell culture system provides an excellent model for additional
mechanistic investigations into the insulin-mediated regulation of
P-450 expression.
| |
Footnotes |
|---|
Accepted for publication January 11, 1999.
Received for publication August 21, 1998.
1 This work was supported by Grant ES03656 and the Environmental Health Sciences Center Grant P30 ES06639 from the National Institute of Environmental Health Sciences.
Send reprint requests to: Dr. Raymond F. Novak, Institute of Chemical Toxicology, Wayne State University, 2727 Second Avenue, Room 4000, Detroit, MI 48201. E-mail: r.novak{at}wayne.edu
| |
Abbreviations |
|---|
CYP, cytochrome P-450; PYR, pyridine; PB, sodium phenobarbital; CIPRO, ciprofibrate.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
-hydroxylase (cytochrome P450LA): Identification of a new cytochrome P450 gene family.
J Biol Chem
262:
801-810-hydroxylase and sterol 27-hydroxylase gene transcription.
Hepatology
21:
501-510[Medline].This article has been cited by other articles:
|
|
 |
|
  H.-N. Chen, S. Fan, and C.-F. Weng Down-regulation of TGF{beta}1 and leptin ameliorates thioacetamide-induced liver injury in lipopolysaccharide-primed rats Innate Immunity, June 1, 2007; 13(3): 176 - 188. [Abstract] [PDF]
|
|
|
|
 |
|
 J. Sugatani, T. Wada, M. Osabe, K. Yamakawa, K. Yoshinari, and M. Miwa Dietary Inulin Alleviates Hepatic Steatosis and Xenobiotics-Induced Liver Injury in Rats Fed a High-Fat and High-Sucrose Diet: Association with the Suppression of Hepatic Cytochrome P450 and Hepatocyte Nuclear Factor 4{alpha} Expression Drug Metab. Dispos., October 1, 2006; 34(10): 1677 - 1687. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. K. Kim, K. J. Woodcroft, S. S. Khodadadeh, and R. F. Novak Insulin Signaling Regulates {gamma}-Glutamylcysteine Ligase Catalytic Subunit Expression in Primary Cultured Rat Hepatocytes J. Pharmacol. Exp. Ther., October 1, 2004; 311(1): 99 - 108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Abdelmegeed, S. K. Kim, K. J. Woodcroft, and R. F. Novak Acetoacetate Activation of Extracellular Signal-Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase in Primary Cultured Rat Hepatocytes: Role of Oxidative Stress J. Pharmacol. Exp. Ther., August 1, 2004; 310(2): 728 - 736. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Raucy, J. Lasker, K. Ozaki, and V. Zoleta Regulation of CYP2E1 by Ethanol and Palmitic Acid and CYP4A11 by Clofibrate in Primary Cultures of Human Hepatocytes Toxicol. Sci., June 1, 2004; 79(2): 233 - 241. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Kim, K. J. Woodcroft, S. G. Kim, and R. F. Novak INSULIN AND GLUCAGON SIGNALING IN REGULATION OF MICROSOMAL EPOXIDE HYDROLASE EXPRESSION IN PRIMARY CULTURED RAT HEPATOCYTES Drug Metab. Dispos., October 1, 2003; 31(10): 1260 - 1268. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. A. Jessen, J. S. Mullins, A. de Peyster, and G. J. Stevens Assessment of Hepatocytes and Liver Slices as in Vitro Test Systems to Predict in Vivo Gene Expression Toxicol. Sci., September 1, 2003; 75(1): 208 - 222. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Kim, K. J. Woodcroft, and R. F. Novak Insulin and Glucagon Regulation of Glutathione S-Transferase Expression in Primary Cultured Rat Hepatocytes J. Pharmacol. Exp. Ther., April 1, 2003; 305(1): 353 - 361. [Abstract] [Full Text]
|
|
|
|
 |
|
 A. Moncion, N. T. Truong, A. Garrone, P. Beaune, R. Barouki, and I. de Waziers Identification of a 16-Nucleotide Sequence That Mediates Post-transcriptional Regulation of Rat CYP2E1 by Insulin J. Biol. Chem., November 22, 2002; 277(48): 45904 - 45910. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Sidhu, F. Liu, S. M. Boyle, and C. J. Omiecinski PI3K Inhibitors Reverse the Suppressive Actions of Insulin on CYP2E1 Expression by Activating Stress-Response Pathways in Primary Rat Hepatocytes Mol. Pharmacol., April 16, 2001; 59(5): 1138 - 1146. [Abstract] [Full Text]
|
|
|
|
 |
|
 E. R. Jacobs and D. C. Zeldin The lung HETEs (and EETs) up Am J Physiol Heart Circ Physiol, January 1, 2001; 280(1): H1 - H10. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
) of insulin or in the presence of 1 nM or 1 µM
insulin. PYR was added 24 h before harvesting cells at 96 h.
Columns and cross bars represent means ± S.E.M. of Northern blot
band densities of three preparations of total RNA. All values are shown
as a percentage of the CYP2E1 mRNA level monitored in untreated
hepatocytes cultured in the presence of 1 µM insulin. #,
significantly different than corresponding treatment in the presence of
1 µM insulin, p < .05.
