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Hepatic Cytochrome P450 4A Expression Level in a Rat
Model of Microvesicular Steatosis
AMS Abdul Majid1,2
1. School of Pharmaceutical Sciences, University Science Malaysia, Penang, Malaysia,
2. School of Medical Sciences, University of New South Wales, Sydney, Australia
Keywords: Cytochrome P450; Orotic acid; Peroxisome proliferator-activated receptor
ABSTRACT
Microvesicular steatosis in general plays an important role in the pathogenesis
of drug, disease and alcohol induced liver damage. Such an event is manifested by the
accumulation of lipid within the hepatocytes. Initial experiments conducted previously
showed that short-term intake of diets containing Orotic acid (OA) produced rapid and
extensive steatosis that follows a microvesicular distribution (Su, Sefton et al. 1999).
The present study evaluated the hepatic cytochrome P450 4A expression level in a rat
model of microvesicular steatosis that was marked by the intake of diets containing 1%
of the Orotic acid for a time period of 21 days. The animals were sacrificed on the 22 nd
day and the hepatic microsomal P450 A4 protein level was quantitated using
immunoblot analysis. The result of the investigation showed a significant induction of
CYP 4A expression level in groups that were fed with diets containing the 1% OA. The
CYP4A in the test group livers was found to have been up regulated by 53% when
compared to the control group.
1
INTRODUCTION
Steatosis is a disease state that occurs due to accumulation of lipid within
hepatocytes. This is often due to the intake of alcohol and drugs such as corticosteroids,
tetracycline and some non-steroidal anti-inflammatory agents. Factors such as inherited
metabolic disorders and diseases with hepatic involvement can also cause such a state
(Farrell 1994; Burt, Mutton et al. 1998). The advent of steatosis by OA is thought to be
mediated via mitochondrial injury, resulting in the impaired β-oxidation of fatty acids
(Berson, De Beco et al. 1998; Burt, Mutton et al. 1998), however the detailed effects of
lipid accumulation on hepatic gene expression are still unclear.
In the rat, OA has been shown to cause severe fatty infiltration of the liver when
administered in concentrations of 0.2% or higher in a purine-deficient diet (Standerfer
and Handler 1955). The accumulation of lipids (mainly triglycerides) is due to
inhibition of lipoprotein synthesis. The lipoprotein precursors, i.e. the apoprotein and
lipid moieties, are synthesised, but the apoprotein is deficient in N-acetylglucosamine,
D galactose and N-acetyl neuraminic acid (Martin, Biol et al. 1982) and the conjugation
of the two moieties in the liver is impeded (Roheim, Switzer et al. 1965), resulting in a
progressive increase in liver fat with a concomitant reduction in serum concentration of
very low density lipoproteins and triglycerides (Hay, Fleming et al. 1988). The
mechanism however, is not yet fully understood.
Previous study showed a significant increase in cholesterol, triglycerides and
phospholipids levels in rat liver after feeding of experimental diets containing OA for
21 days (Su, Sefton et al. 1999). Fatty acid overload is accompanied by an increased
2
capacity for oxidation of fatty acids by the mitochondrial peroxisomal and microsomal
pathways of liver and heart (Mannaerts and Van Veldhoven 1992). The peroxisomal βoxidation is only a minor pathway for fatty acid oxidation relative to the mitochondrial
counterpart, its role is more important for the β-oxidation of very long-chain fatty acids
(Osmundsen, Bremer et al. 1991). Diets containing long chain fatty acids have been
shown to induce peroxisomal and microsomal oxidation enzymes and also the
expression of the cytoplasmic liver-type fatty acid-binding protein (L-FAB) (Issemann,
Prince et al. 1992; Reddy and Mannaerts 1994).
The L-FAB has been shown to have marked affinity for long-chain fatty acids
and is considered to have a significant role in mediating the cellular uptake and
intracellular targeting of long-chain fatty acids (Glatz and van der Vusse 1996).
Therefore, in the metabolism and transport of long-chain fatty acids during fatty acid
overload, the activity of peroxisomal, microsomal enzymes and the expression of LFAB are of great importance.
The peroxisome proliferator activated receptors (PPARs; NR1C) belong to the
steroid/thyroid/retinoid receptor superfamily. They are nuclear lipid-activatable
receptors that control a variety of genes in several pathways of lipid metabolism,
including fatty acid transport, uptake by the cells, intracellular binding and activation,
as well as catabolism (β -oxidation and ω-oxidation) or storage.
Three related isotypes of PPAR have been identified in rat and human, they are
PPAR α (NR1C1), PPAR β (NR1C2) and PPAR γ (NR1C3) (Issemann and Green 1990;
Dreyer, Krey et al. 1992; Gottlicher, Widmark et al. 1992; Schmidt, Endo et al. 1992;
Chen, Law et al. 1993; Sher, Yi et al. 1993; Zhu, Alvares et al. 1993; Kliewer, Forman
et al. 1994; Amri, Bonino et al. 1995; Aperlo, Pognonec et al. 1995; Greene, Blumberg
3
et al. 1995; Xing, Zhang et al. 1995). PPARs belong to the TR/RAR subfamily that
recognize preferentially the core hexanucleotide motif AGGTCA in the up stream
region of the target genes and are also characterized by the ability to form heterodimers
with the 9-cis-retinoic acid receptor, RXR (NR2B). PPARs bind to DNAs
heterodimerically with RXR as their binding partner. The PPAR: RXR heterodimeric
protein binds to the PPAR response elements (PPRE), a direct repeat of two core
recognition motifs AGGTCA spaced by one nucleotide (also known as DR1)(Kliewer,
Umesono et al. 1992). The PPRE was first located in the promoter region of the acylCoA oxidase gene (Dreyer, Krey et al. 1992; Tugwood, Issemann et al. 1992). PPAR
interacts with the upstream-extended core hexamer of the DR1, whereas RXR occupies
the downstream motif (Ijpenberg, Jeannin et al. 1997). It has been demonstrated that the
carboxy-terminal extension (CTE) regions of both receptors is indeed responsible for
the recognition of the 5'-flank of the DR1 in PPREs (Hsu, Palmer et al. 1998).
Interestingly, it was observed that the expression of RXR abolished PPARα
stimulation of the PRL promoter in pituitary GH4C1 cells (Tolon, Castillo et al. 1998)
which suggests that stimulation of the PRL promoter by PPARα was mediated by
protein-protein interaction rather than binding of PPAR:RXR to the promoter. It has
been proposed that the mechanism of this phenomenon is a ligand-dependent
association of PPARα with the transcription factor GHF-1 that stimulates transcription
(Tolon, Castillo et al. 1998). This also implies that PPARα would act similarly to a coactivator. It is postulated that in the event of over-expression of RXR, the stimulatory
effect of PPARα can be suppressed as RXR might compete for its association with
GHF-1.
4
EXPERIMENTAL PROCEDURE
Materials
The Hyperfilm-MP and reagents for enhanced chemiluminescence were acquired from
Amersham Australia (North Ryde, NSW, Australia). Retinal, retinyl acetate and αtocopheryl acetate were purchased from Sigma
Chemical Co. (St. Louis, MO).
Chemicals used for SDS-polyacrylamide gel electrophoresis were obtained from BioRad laboratories (Richmond, CA). Constituents for the rat experimental diets were
purchased from ICN Biochemicals (Seven Hills, NSW, Australia). Other biochemicals
were procured from Sigma Chemical Co. (Castle Hill, NSW, Australia) The rabbit antiCYP4A1 IgG was a gift from Prof. G.G. Gibson, University of Surrey.
Animal Treatments
The research work was carried out according to the guidelines endorsed by the
Australian National Health and Medical Research Council and was approved by the
University of New South Wales and Central Area Health Service Animal Care and
Ethics Committees. 12 male Wistar rats of approximately 3 weeks old with weight
range of 237-282 g were divided equally into 2 groups with 3 rats per cage. Each group
received basal diets of a high sucrose-containing semi-purified (SP) diet with each
kilogram consisting of sucrose (600g), casein (200 g), cellulose (110 g), corn oil (40 g),
ICN salt mixture 4179 (40 g), ICN vitamin diet fortification mixture (10 g), αtocopherol (20 mg), and retinyl acetate (8.7 mg). The rat group that had 1% Orotic Acid
in their diet was labelled as OA+ group and the group without any Orotic Acid (the
control group) was labelled as OA- . The rats were held on the diet for 21 days and had
5
free access to water. All the rats were individually labelled and their weights recorded
throughout the 21 days and all the animals were sacrificed on the 22nd day under
anaesthesia.
Microsome preparation
The rat livers harvested were perfused with cold saline solution followed by snap
freezing in liquid N2 before storage at -70o C. The hepatic microsomes were prepared
according to methods established earlier (Murray, Zaluzny et al. 1986). The liver
sections from storage were thawed on ice in microsome preparation buffer (pH 7.4)
containing 0.01M K2HPO4, 1mM EDTA and 0.25 M sucrose. The samples were
homogenised and centrifuged at 10 000g for 25 minutes at 4o C under vacuum. The
pallets were discarded and the supernatant was subjected to ultracentrifugation under
vacuum at 35 000rpm for 1 hour 10 minutes at 4 oC. The supernatant was transferred
into another tube and stored at -70 oC and the remaining pellet was covered with
preparation buffer and homogenised for 5 passes followed by further ultracentrifugation
at 4 oC at 35 000rpm for 35 minutes under vacuum. All the supernatant was discarded
and the final microsomal pellet was resuspended in 50mM potassium phosphate buffer
(pH 7) containing 1 mM EDTA and 20% glycerol and homogenised for 5 passes before
being snapped frozen in liquid N2 and stored at -70ºC.
Immunoblotting for CYP 450 4A Apoproteins in Rat Hepatic Microsomes.
The rat hepatic microsomes protein concentration was determined and standardised
using Lowry Protein quantification assay prior to Western Blot analysis. 5 mg / lane of
each sample was subjected to electrophoresis on 7.5% acrylamide running gel and 3%
polyacrylamide stacking gel (Towbin, Staehelin et al. 1979; Murray, Zaluzny et al.
6
1986). Proteins were transferred to nitrocellulose paper via electrophoresis gel transfer
operation. The nitrocellulose paper was left to incubate in anti-rabbit IgG 4A antibody
followed by anti-sheep IgG antibody and the immunoreactive proteins were detected by
enhanced chemiluminescence on Hyperfilm-MP, and the resultant signals were
analyzed by densitometry (Bio-Rad, Richmond, CA).
RESULTS:
Table 1: Change in rat body mass in during 21 days of dietary conditioning.
RAT BODY MASS (g)
DAY
1
3
5
7
9
11 13 15 17 19 21
GROUP
OA-1
240 226 245 261 274 287 306 322 330 343 351
OA-2
264 253 273 289 299 307 322 332 342 350 359
OA-3
261 243 264 279 289 302 312 325 334 342 352
OA-4
278 269 284 298 308 319 331 342 354 365 375
OA-5
267 256 281 295 309 317 329 336 345 357 370
OA-6
253 241 258 272 284 294 300 316 320 330 332
OA+1
261 249 262 272 277 283 289 296 294 304 317
OA+2
283 271 276 285 289 290 291 301 310 305 311
OA+3
258 251 259 267 267 269 272 280 279 285 295
OA+4
252 241 257 254 261 261 265 267 271 274 278
OA+5
237 230 240 247 252 251 264 264 270 279 289
OA+6
252 243 255 262 265 274 277 282 284 284 287
Table 2 : Food consumption during the 21 days period
MASS OF DIET CONSUMED (g)
DAY
1
2
CAGE 1 (-OA 1,2,3)
CAGE 2 (-OA 4,5,6)
CAGE 3(+OA 1,2,3)
CAGE 4(+OA 4,5,6)
48
48
48
48
48
48
48
48
3
4
100 100
100 100
100 80
100 87
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
83
90
63
63
86
87
74
73
82
90
85
65
68
70
65
38
77
79
82
61
77
67
85
65
81
70
88
62
82
76
92
58
89
78
91
78
85
73
87
81
91
83
98
100
84
77
99
82
97
83
99
93
100
86
99
81
91
90
82
97
91
83
99
71
92
82
98
99
Total
(Kg)
1752
1660
1762
1546
7
Figure 1: Cyp4A immunoreactive protein in rat liver microsomes by Western analysis.
103
76
49
33
+1
Marker MW
(kD)
+2
+3
+4
+5
OA+ Group
+6
-1
-2
-3
-4
-5
-6
OA- Group
Figure 2: Cyp4A level between OA+ group and OA-group.
Chart showing the Cyp450 Protein
level in OA- and OA+ group
Densitometer unit
2
1.5
1
0.5
OA+
OA-
0
8
Effects of intake of Orotic Acid containing diets on rat body weight
Table 1 shows that the group of rats that were fed with diets containing Orotic
acid (OA+) had lower body weights when compared with control (OA-). Student's t-test
indicated that the mean values of the two population body mass were significantly
different from each other (P< 0.05). The rats also had a lower rate of weight gain when
compared to the control, but the total food consumed per cage over the 21 days period
was not different as shown by Table 2. The OA- group consumed 1706+65 g and the
OA+ group consumed 1655+152g of rat diet. On Day 21, the OA- group had a mean
body mass of 357+15 g and the OA+ group had a mean mass of 296+14g.
The chart in Figure 2 is a graphical representation of the results shown in Figure
1. The data represents the total amount of Cyp4A in liver microsomes from OA+ group
and OA- rats in densitometry units. The OA+ group had a value of 1.560+0.232 unit
and the OA- group had a value of 1.0173+0.2463 unit. The student's t-test showed a
significant difference between the values of the two groups (P<0.05). This showed that
the Cyp4A level was approximately 53+35 % in the OA+ group when compared to the
control.
DISCUSSION
The results of this experiment clearly indicated the up regulation of CyP450 4A
proteins in the rat group that was fed with diet containing Orotic acid. Thus, extensive
fatty infiltration of the liver was evident. The state of steatosis was observed in the OA+
group liver sample under microscope but no micrographs were taken. However similar
9
findings were made during previous studies (Su, Sefton et al. 1999). The Cyp4A level
in the test group was approximately 53+35 % higher than the control group.
Previous investigation showed no significant difference between the body
weights of the rat group that was fed with diet containing 1% OA and the control group
(Su, Sefton et al. 1999). The current investigation however showed otherwise. The body
weight of the control group was approximately 20 % greater than the test group
although the animals consumed the same amount of diet. Throughout the study, it was
observed that the rats which were fed with Orotic acid containing diet appeared to be
less physically active compared to the control group.
Previous experiments showed that the Orotic acid containing diet resulted in an
increase in cholesterol, triglyceride and phospholipids levels in rat liver after feeding of
the experimental diets for 21 days (Su, Sefton et al. 1999). It was proposed that lipid
deposition could influence CYP function (Murray, Cantrill et al. 1992). This was clearly
observed in the previous experiment where Orotic acid containing diet resulted in a
decrease in the testosterone hydroxylation activity of CYP2C11, CYP3A and CYP2A1.
The expression level of the isoenzymes was also significantly reduced (Su, Sefton et al.
1999).
In this experiment the expression level of the CYP4A was dramatically
increased in steatotic rats. The CYP4A expression level could be influenced by fatty
acids acting as ligands of the PPAR receptors which are known to influence CYP4A
regulation. PPARs are capable of specifically interacting with ligands including certain
natural compounds such as fatty acids. However the receptors have several peculiarities.
Firstly, in contrast to a number of related receptors such as the thyroid hormone
receptor, PPARs accommodate several types of ligands and most of the known
10
activators are bona fide ligands (Willson and Wahli 1997). Secondly most of the
molecules that specifically bind to PPAR do so with a rather low affinity as compared
with the affinity of classical hormones for their cognate receptors. Thirdly, it is found
that there is some overlap in ligand recognition by different PPAR isotypes (PPAR α, β
and γ).
The role of fatty acids has changed from a passive energy-providing molecule to
metabolic regulators. The discovery that fatty acids could act as PPAR ligands indicated
that at least part of the PPAR-dependent transcriptional activity of fatty acids results
from a direct interaction of the nuclear receptor with these molecules. These fatty acids
bind to all three PPARs α, β and γ with PPAR α exhibiting the highest affinity, at
concentrations that are in agreement with their circulating blood levels. Compared to
the unsaturated fatty acids, saturated fatty acids are poor PPAR ligands in general
(Forman, Chen et al. 1997; Kliewer, Sundseth et al. 1997; Krey, Braissant et al. 1997),
whereas phytanic acid, a dietary branched-chain, isoprenoid-derived fatty acids, binds
to PPAR α efficiently (Ellinghaus, Wolfrum et al. 1999). Other PPAR ligands include
dicarboxylic fatty acids, eicosanoids and a range of synthetic ligands such as
hypolipidermic agents, Prostaglandin 12 analogues and Leukotriene B4 analogues.
Alternative Pathways for PPAR activation
PPAR α and γ are phosphoproteins. Similar to several other nuclear hormone
receptors, PPARs can also be regulated by phophorylation in addition to liganddependent activation. The phophorylation of PPAR α and γ can be influenced by
insulin (Shalev, Siegrist-Kaiser et al. 1996). The effect of insulin occurs through the
11
phosphorylation of two microtubule-associated protein (MAP) kinase sites located in
the A/B domain of hPPAR α (Juge-Aubry, Hammar et al. 1999). Zhang et al (Zhang,
Berger et al. 1996) showed a synergistic effect between insulin treatment and PPAR γ
ligand dependent activation on the
expression of the target gene aP2. However
contrasting results were obtained when exploring the effects of epidermal growth factor
(EGF) and platelet-derived growth factor (PDGF) in modulating PPAR activity (Hu,
Kim et al. 1996; Camp and Tafuri 1997). These growth factors were found to decrease
the transcriptional activity of PPAR γ while increasing PPAR phosphorylation through
MAP kinase signalling.
It remains unclear how the nature of triggering signals such as insulin and
growth factors can have quite opposite effects on PPAR α signalling. It is possible that
their activity involves either specific pleiotropic actions or use of different kinase
pathways such as the use of Ras/MAP kinase pathway by insulin to mediate the upregulation of phosphophenol pyruvate carboxykinase gene expression and the inhibition
of PPAR α activity by growth hormone through the Janus kinase 2-signal transducer
and activator of transcription 5b (JAK2-STAT5b) pathway.
The PPAR:RXR heterodimer can alternatively be activated through ligand
binding to RXR. Either partner of the PPAR:RXR heterodimer can regulate the
transcriptional activity of the DNA-bound complex by interacting with their respective
cognate ligand activity of the DNA-bound complex by interacting with their cognate
ligand individually or simultaneously. This was observed when RXR ligands such as 9cis-RA and LG 1069 activated the PPRE-driven reporter gene in PPAR:RXR-dependent
manner (Kliewer, Umesono et al. 1992; Gearing, Gottlicher et al. 1993; Keller, Dreyer
et al. 1993).
12
PPAR-mediated transactivation properties
PPAR-mediated transactivation results from the combination of PPAR:RXR
binding to PPRE ligand activation of the complex formed by the two species. It is
possible that along with the complex is a repressor molecule(s) that renders the
PPAR:RXR heterodimer inactive (Horwitz, Jackson et al. 1996; Glass, Rose et al.
1997). Ligand binding (or other activation processes e.g. Phosphorylation) to the PPAR
or RXR molecule may change the conformational structure, thus resulting to the
dissociation of the repressor molecule(s) and activating the heterodimer complex. This
would form a new protein-protein interaction surface that would allow specific contacts
with co-activator(s). This subsequently transduces regulatory action to the basal
transcriptional machinery. The activated PPAR:RXR complex binds to the PPRE
resulting to a change in chromatin structure causing the release of histone molecules.
The PPRE-bound PPAR:RXR complex triggers the activation of a co-activator-acetyltransferase complex. This enzyme acetylates the histone tails of the promoter chromatin
at the transcription initiation region thereby producing a transcriptionally competent
structure. This permits additional transcription factors and the basal transcriptional
machinery, including the RNA Pol II initiation complex to assemble with the promoter
thus initiating transcription of the particular mRNA.
PPAR α tends to be expressed in late development stages in mouse and rat. High
levels of the PPAR α mRNA are in brown fat, liver, kidney and the mucosa of stomach
and duodenum of adult rat. Other organs that express significant amounts of PPAR α
mRNA include the retina, adrenal gland, skeletal muscle and pancreatic islets
(Braissant, Foufelle et al. 1996; Lemberger, Braissant et al. 1996). In summary, the
expression of PPAR α correlates with high mitochondrial and peroxisomal β-oxidation
13
activities in tissues that primarily use fatty acids as an energy source such as cells of
kidney proximal tubules and cardiomyocytes.
It has been shown by several investigators that PPAR α expression in rat liver is
subject to differential regulation by insulin and glucocorticoids and therefore situations
that can induce the levels of plasma glucocorticoids such as stress or fasting can have an
effect in the expression of PPAR α (Lemberger, Staels et al. 1994; Steineger, Sorensen
et al. 1994). Growth Hormone in contrast has been shown to decrease the PPAR α
mRNA levels by 50 % in primary culture of rat hepatocytes (Yamada, Sugiyama et al.
1995). Down-regulation of PPAR α gene expression was also observed in chronic
alcoholic liver disease in rat (Wan, Morimoto et al. 1995).
The CYP4A enzymes belong to the cytochrome mono-oxygenase that plays a
central role in the oxidation of a wide variety of endogenous as well as exogenous
compounds. The CYP4A enzymes have been shown to catalyse the ω-hydroxylation of
fatty acids and eicosanoids. The rat CYP4A gene family consists of four members that
are expressed in the kidney, liver, intestine, heart, colon, lungs, brain and prostate
(Kusunose, Ogita et al. 1981; Kimura, Hardwick et al. 1989; Stromstedt, Hayashi et al.
1990; Hardwick 1991). Three isoenzymes of CYP4A have been identified in rat livers
that include CYP4A1, CYP4A2 and CYP4A3. CYP4A1 has been found to contain
PPRE in their promoter sequence and was shown to respond to PPAR α activators in
vivo and cell culture (Muerhoff, Griffin et al. 1992; Krey, Keller et al. 1993; Aldridge,
Tugwood et al. 1995). The CYP4A are distinct from other CYP enzymes with less than
30 % homology (Aoyama, Hardwick et al. 1990). All of the three members of the rat
CYP4 enzymes have been shown to be induced in the liver by hypolipidemic drugs that
includes CYP4A1, CYP4A2 and CYP4A3 (Hardwick, Song et al. 1987; Kimura,
14
Hardwick et al. 1989). Their cDNA sequences show great homology of more than 95 %
(Kimura, Hanioka et al. 1989; Kimura, Hardwick et al. 1989). The Cyp4A1 is one of the
most active isoenzymes to hydroxylate palmitate, laurate and arachiodonate and this
occurs mainly at the ω position (Gibson 1989; Aoyama, Hardwick et al. 1990).
Peroxisome proliferators have been shown to induce the expression of several
P450 4A enzymes in rat (Kimura, Hardwick et al. 1989). P450 4A enzymes represent a
distinct evolutionary branch of the cytochrome P450 superfamily and are distinguished
from other mammalian cytochrome P450 enzymes by their ability to catalyse the ωhydroxylation of long and medium chain fatty acids and prostaglandins. The other
mammalian P450 iso-enzymes also oxidise fatty acids but their degree of specificity for
the terminal, primary C-H bonds of the fatty acid is low when compared to the CYP450
4A sub-type. The significance of ω-hydroxylation as a pathway for the metabolism of
saturated fatty acids is still poorly understood. It is however likely that it contributes to
the formation of dicarboxylic acids through the subsequent oxidation of the product ωalcohols by cytosolic enzymes. Dicarboxylic fatty acids are substrates for peroxisomal
or mitochondrial β-oxidation and it appears that the peroxisomal pathways to be more
efficient for the β-oxidation of dicarboxylic acids than the mitochondrial pathway
(Okita 1994; Tontonoz, Hu et al. 1994). This difference suggests that microsomal ωhydroxylation would shunt fatty acids to peroxisomes for degradation, which in turn
would affect energy conservation from fatty acid oxidation, as the initial step of the
peroxisomal pathway is not coupled to ATP production, as it is in the mitochondrial
pathways (Tontonoz, Hu et al. 1994). In certain situations, β-oxidation may be
incomplete leading to the production of dicarboxylic fatty acids of intermediate length,
C6-C10. The levels of C6-C10 dicarboxylic acids are generally elevated in tissues,
15
plasma and urine during fasting and diabetic states where the concentrations of
circulating fatty acids are increased due to lypolysis in adipocytes (Mortensen 1992). It
was shown that under such conditions, the hepatic concentrations of P450 4A enzymes
are also elevated (Imaoka, Shimojo et al. 1988; Barnett, Gibson et al. 1990; Ferguson,
Donahue et al. 1993). However, when peroxisome proliferator such as clofibrate was
administered (which induces P450 4A) no increase of such dicarboxylic acids were
observed (Mortensen 1992). This may reflect a more complete shortening of the
dicarboxylic acid by peroxisomal β-oxidation, which is also induced by clofibrate
(Okita 1994).
Other factors that could contribute to the increase of hepatic free fatty acids
level could also be due to competition for metabolic pathways and binding proteins by
peroxisome proliferators. This has led to the speculation that fatty acids mediate the
response of PPARα to peroxisome proliferators (Lock, Mitchell et al. 1989; Issemann,
Prince et al. 1992) and fatty acids have been shown to activate PPARα (Gottlicher,
Widmark et al. 1992; Issemann, Prince et al. 1992; Keller, Dreyer et al. 1993).
Furthermore, selective inactivation of P450 4A enzymes has been shown to decrease the
induction of the peroxisomal fatty acyl-CoA oxidase of rat hepatocytes, suggesting that
the production of dicarboxylic acids by P450 4A enzymes contributes to the response to
peroxisome proliferators (Kaikaus, Chan et al. 1993).
Studies conducted by Kaikaus et al (Kaikaus, Chan et al. 1993) demonstrated
that induction of peroxisomal β-oxidation and L-FABP by peroxisome proliferators is
mediated via the cytochrome P450 4A ω-hydroxylation pathways. It is suggested that
fatty acids metabolised by the enzyme forms into dicarboxylic acid products that may
activate the soluble PPAR receptor of the L-FAB and peroxisomal enzymes thus
16
induces them pretranslationally. Oxidised fatty acids may activate the receptors by
acting as ligands. The other possibility may include activation of PPAR by protein
kinase C-mediated phosphorylation. By comparing PPAR α-deficient and wild type
mice, it was shown that starvation and insulin dependent diabetes result in a strong
induction of the hepatic CYP4A genes and other lipid-metabolising enzymes such as
Acetyl-Coenzyme A through activation of PPAR α provides a negative feedback on the
intracellular levels of an endogenous ligand (Devchand, Keller et al. 1996). Secondly it
can also be concluded that a pathophysiological state can induce cellular changes that
could lead to the activation of PPAR α (Kroetz, Yook et al. 1998). Finally, it can also be
assumed that PPAR α may have an important role in the detoxification of some
xenobiotics.
A more detailed analysis conducted by this laboratory that evaluated the impact
of lipid-deficient OA-diet on CYP4A expression and PPARα activation in rodent liver
found that the CYP4A protein and laurate ω-hydroxylation activity were elevated in rat
hepatocytes after 21 days (Su et al. 2005). CYP4A1 and CYP4A2 mRNAs were found
to have been induced more than 2 fold of the control with no apparent effect in
corresponding mRNAs level of CYP4A3 and peroxisomal acyl-CoA oxidase.
Coadministration of clofibric acid and the OA-diet was found to have prevented hepatic
lipid accumulation and caused significant upregulation of CYP4A1-3 and acyl-CoA
oxidase mRNAs. Hepatic PPARα protein and retinoid X-receptor-α (RXRα) level was
also found to have decreased by the S/OA-diet. The administration of the OA
containing diet to control and PPARα-null mice led to hepatic lipid deposition and the
microsomal CYP4A protein upregulation in the wild-type but not PPARα-null mice.
The results of this study concluded PPARα involvement in the induction of CYP4A in
17
rodent liver by the lipid-devoid OA-diet and suggested that the decreased availability of
hepatic PPARα and RXRα after intake of the diet may add to the selective upregulation
of hepatic CYP4A1 and CYP4A2 in this model of steotosis.
Conclusion
The findings from the present study that CYP4A protein expression is up regulated in
hepatic steatosis indicate that pathophysiological accumulation of fatty acids may be
responsible. Activation of PPARα-mediated gene transcription in fatty liver may
contribute to the disordered homeostasis of lipid oxidation that occurs in genetic disease
and altered nutrition.
Acknowledgements
I would like to express my gratitude to Prof Michael Murray for his supervision during
the length of this research and to Dr Gloria Su for her kind assistance throughout.
18
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