Dietary lipids regulate b-oxidation enzyme
gene expression in the developing rat kidney
F. OUALI, F. DJOUADI, C. MERLET-BÉNICHOU, AND J. BASTIN
Institut National de la Santé et de la Recherche Médicale Unité 319,
Université Paris 7 Denis Diderot, 75251 Paris Cedex 05, France
energy metabolism; postnatal period; mitochondria; peroxisome; gene regulation; peroxisome proliferator-activated receptor
FATTY ACIDS HAVE BEEN identified for a long time as one
of the main energy substrates used by the kidney of rat
and other species (36). Mitochondrial fatty acid b-oxidation, which allows high yields of ATP production, plays
an essential role in supporting kidney reabsorptive
functions, as evidenced by the effects of mitochondrial
fatty acid utilization blockers, which induce marked
impairments in proximal tubule reabsorption (36).
Mitochondrial energy metabolism is not mature in
the newborn rat kidney (3) and undergoes profound
changes during the first weeks after birth (14, 35). In
particular, the postnatal development of energy metabolism involves an enhanced expression of nuclear genes
encoding mitochondrial malate dehydrogenase, a tricarboxylic acid cycle enzyme, and medium-chain acyl-CoA
dehydrogenase (MCAD), a mitochondrial fatty acid
b-oxidation enzyme, in the renal cortex and inner
stripe of the outer medulla (ISOM) (12). Gene expres-
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sion of these enzymes is controlled, during the suckling
period, by physiological factors, such as changes in
glucocorticoid (12) or thyroid hormone plasma levels
(13). During this period, lipids supplied by maternal
milk provide 60% of total energy, compared with only
15% in the standard adult diet (17). The present work
addresses the role of dietary lipids in regulating b-oxidation enzyme genes in the immature kidney.
The kidney is the only other organ in addition to the
liver in which fatty acid b-oxidation can occur both in
the mitochondria and in the peroxisomes (33, 37). The
renal peroxisomes, which are found exclusively in the
proximal tubule of the nephron, rank among the largest
peroxisomes known in different cell types (37). The
presence of peroxisomes in the fetal kidney is ascertained by morphological studies in rat and mouse and
in the human (37). After birth, there is a dramatic
increase in the number of peroxisomes in the proximal
tubule of rat and mouse kidney (37).
Peroxisomal b-oxidation, which involves a set of
specific enzymes (24), differs from its mitochondrial
counterpart in many aspects. In particular, peroxisomal fatty acid b-oxidation is not directly coupled to
the mitochondrial respiratory chain, does not perform
complete oxidation of fatty acids, and exhibits a different substrate specificity, compared with the mitochondrial pathway (33). Biochemical studies showed that
liver peroxisomes can shorten very-long-chain fatty
acids and other fatty acids that are poor substrates for
the mitochondrial b-oxidation machinery, allowing their
further use as energy substrates by mitochondria (33).
Feeding adult rats a high-fat diet leads to a stimulation of peroxisomal b-oxidation in the liver (32). Nevertheless, the effects of fat supply on kidney peroxisomal
or mitochondrial b-oxidation have not, so far, been
investigated. Peroxisomal metabolism is insufficiently
documented in the developing kidney (37). There are, in
particular, no data on peroxisomal fatty acid b-oxidation in the kidney of suckling pups.
The aim of this study was to evaluate the effectiveness of dietary lipid to exert long-term or short-term
control on b-oxidation enzyme mRNA abundance in the
immature kidney cortex and ISOM. We focused on the
long-chain-acyl-CoA dehydrogenase (LCAD) and MCAD,
which catalyze the first step of mitochondrial fatty acid
b-oxidation, with distinct carbon chain length specificities. Gene expression of acyl-CoA oxidase (ACO), which
catalyzes the initial step of peroxisomal b-oxidation,
was studied in parallel.
The mechanism(s) underlying the effects of dietary
fat on enzymes and protein of fatty acid metabolism
remain hypothetical. Recent data suggest that these
effects might involve a control of peroxisomal and
0363-6127/98 $5.00 Copyright r 1998 the American Physiological Society
F777
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Ouali, F., F. Djouadi, C. Merlet-Bénichou, and J.
Bastin. Dietary lipids regulate b-oxidation enzyme gene
expression in the developing rat kidney. Am. J. Physiol. 275
(Renal Physiol. 44): F777–F784, 1998.—This study examines
the ability of dietary lipids to regulate gene expression of
mitochondrial and peroxisomal fatty acid b-oxidation enzymes in the kidney cortex and medulla of 3-wk-old rats and
evaluates the role of glucagon or of the a-isoform of peroxisome proliferator-activated receptor (PPARa) in mediating
b-oxidation enzyme gene regulation in the immature kidney.
The long-chain (LCAD) and medium-chain acyl-CoA dehydrogenases (MCAD) and acyl-CoA oxidase (ACO) mRNA levels
were found coordinately upregulated in renal cortex, but not
in medulla, of pups weaned on a high-fat diet from day 16 to
21. Further results establish that switching pups from a lowto a high-fat diet for only 1 day was sufficient to induce large
increases in cortical LCAD, MCAD, and ACO mRNA levels,
and gavage experiments show that this upregulation of
b-oxidation gene expression is initiated within 6 h following
lipid ingestion. Treatment of pups with clofibrate, a PPARa
agonist, demonstrated that PPARa can mediate regulation of
cortical b-oxidation enzyme gene expression, whereas glucagon was found ineffective. Thus dietary lipids physiologically
regulate gene expression of mitochondrial and peroxisomal
b-oxidation enzymes in the renal cortex of suckling pups, and
this might involve PPARa-mediated mechanisms.
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REGULATION OF b-OXIDATION GENE BY DIETARY LIPIDS
MATERIALS AND METHODS
Animals and diets. Pregnant Wistar rats were bred and
mated in our laboratory and had free access to water and
standard food (UAR 113, containing, per 100 g food, 51 g
carbohydrate, 22 g protein, and 5 g lipid; Usine d’Alimentation
Rationnelle, Villemoisson-sur-Orge, France). Each litter was
reduced to 10 animals at birth. Pups were kept with their
mother until day 21 after birth, or precociously weaned on
postnatal day 16 and then put on a low- or a high-fat solid
food diet.
The low-fat diet was obtained commercially from Usine
d’Alimentation Rationnelle and provided, per 100 g, 58 g
carbohydrate, 19 g protein, and less than 1 g residual lipids;
under this diet, lipids account for less than 3% of the total
caloric supply. The high-fat diet, prepared by adding 25%
coconut oil (Sigma, St. Louis, MO) to the low-fat food,
provided, per 100 g, 41 g carbohydrate, 14 g protein, and 25 g
lipid. Accordingly, lipids accounted for 55% of the caloric
supply in the high-fat diet. By comparison, milk lipids provide
60% of the total caloric intake in suckling rats (17). Coconut
oil was chosen because it contains 86% saturated long-chain
and medium-chain fatty acids (25), which are physiological
substrates of LCAD, MCAD, and ACO and which represent
,65% of total fatty acids supplied by maternal milk (2, 19).
In a first set of experiments, litters were divided in two
groups on day 16, and from day 16 to 21, one-half of the litter
was kept on the low-fat diet, while the other half was put on
the high-fat regimen. Food intake in the various groups of
animals was determined daily by weighing the amount of food
consumed. Body weight measurements were performed daily
in parallel.
Further experiments were designed to evaluate short-term
regulation of gene expression by dietary lipids. In contrast to
adult rats, young rats aged less than 1 mo eat at any time of
the day (17), and cannot be conditioned to consume their food
within a few hours. We found that the shortest period of time
during which reproducible food intake values can be obtained
from 3-wk-old pups is 24 h (data not shown). To investigate
the effects of a high-fat diet over 24 h, litters of 16-day-old rats
were first weaned on the low-fat diet from day 16 to 21, to
avoid background effects due to milk feeding. Then, from day
21 to 22, one-half of the litter was switched on the high-fat
diet, whereas the other half was maintained on low-fat food. A
similar protocol was used to study the effects of a 1-day-long
supplementation by clofibrate, a PPARa ligand (15). Accordingly, pups weaned on low-fat diet from day 16 to 21 were
switched on low-fat food supplemented with 0.5% clofibrate
(Sigma) from day 21 to 22. Control pups were kept on low-fat
food during the same period.
Finally, the very short-term regulation of MCAD gene
expression was investigated in 21-day-old rats kept on a
low-fat diet from day 16 to 21. A first group of animals were
given 1 ml of coconut oil or vehicle (sterile water) by gavage;
another group received 1 ml of 50 mg/ml clofibrate solution or
vehicle (NaCl 0.9%, ethanol 7%), and a last group received a
single subcutaneous injection of glucagon (150 µg/100 g body
wt; Sigma); all these animals were killed 6 h later.
The kidneys and liver were removed on day 21 or 22, under
ketamine anesthesia (100 mg/kg body wt, Imalgène; RhôneMérieux, Lyon, France). The tissues were immediately frozen
in liquid nitrogen and stored at 280°C. Cortex and ISOM
were dissected by hand at 220°C. Blood samples were
collected at 9:30 AM, from axillary artery, in heparinized
glass tubes and immediately centrifuged. Plasma samples
were kept at 280°C until analysis. Nonesterified fatty acids
(NEFA) plasma levels were determined using the NEFA C
WAKO kit (Dardilly, France).
The values of growth, food, energy intake, lipid consumption, and NEFA plasma levels in high- and low-fat fed pups
are presented in Table 1. Body growth was slightly lower in
high-fat than in low-fat fed pups, but growth rates in both
groups remained in the physiological range for 3-wk-old rats
(26, 29). Pups exhibited lower appetence for high-fat than for
low-fat food, as reflected by the food intake values. However,
since the caloric content per gram of high-fat food (4 kcal/g)
was higher than that of low-fat food (2.2 kcal/g), the daily
dietary energy intakes were similar regardless of whether
rats were kept on a low- or a high-fat diet. Altogether, the
protocols and diets used in this study allowed us to induce a
marked increase in the dietary intake of saturated fat and in
circulating fatty acids in high-fat fed animals compared with
low-fat fed littermates (Table 1).
Northern blot analysis. Isolation of total RNA from frozen
liver and kidney cortex and ISOM, electrophoresis through a
formaldehyde-containing agarose gel (15 µg of RNA/lane),
and transfer to nylon membrane followed by ultraviolet
cross-linking were carried out as described elsewhere (12, 13).
The membranes were probed with cDNAs labeled [a-32P]dCTP
using the random primer technique. The mitochondrial enzyme cDNA probes used in this study were rat MCAD EcoR I
fragment of 871 bp (21) and rat LCAD EcoR I fragment of
1,200 bp (20). A 559-bp ACO cDNA was synthesized from total
liver RNA by RT-PCR for 25 cycles using primers 58-CAATCACGCAATAGTTCTGGCTC-38 (upstream) and 58-AAGCTCAGGCAGTTCACTCAGG-38 (downstream) chosen from the
Table 1. Body growth, food, energy, and lipid intakes
and fatty acid plasma levels of animals fed the lowor the high-fat diet during the 16- to 21-day period
Diet
Body
Growth,
g/day
Food
Intake,
g/day
Energy
Intake,
kcal/day
Lipid
Intake,
g/day
Fatty
Acid
Plasma
Levels,
mmol/l
Low fat 4.0 6 0.1 10.2 6 0.2 23.0 6 0.7 0.07 6 0.01 0.20 6 0.03
High fat 2.9 6 0.3* 6.3 6 0.4† 25.0 6 1.6 1.60 6 0.12† 0.60 6 0.07†
Values are means 6 SE of 4–10 animals. * P , 0.05 and † P , 0.001
compared with the low-fat group value.
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mitochondrial b-oxidation gene expression mediated
through binding of fatty acids to a nuclear receptor, the
a-isoform of peroxisome proliferator-activated receptor
(PPARa) (27). Other data suggest that glucagon, the
plasma level of which increases with a high-fat diet,
could trigger gene expression of liver b-oxidation enzymes (34). This led us to study PPARa mRNA levels in
the kidney of pups fed high- and low-fat diets, and to
determine whether clofibrate, a PPARa agonist, can
stimulate gene expression of the b-oxidation enzymes
studied in the immature kidney. The effects of glucagon
in regulating gene expression of these enzymes were
investigated in parallel.
Our data indicate that changes in the dietary fat
supply can induce large changes in gene expression of
mitochondrial and peroxisomal b-oxidation enzymes in
the kidney cortex, but not in the ISOM. These changes
in b-oxidation enzyme mRNA levels appear unrelated
to the plasma levels of glucagon. In contrast, PPARadependent gene regulation of mitochondrial and peroxisomal b-oxidation enzymes can clearly operate in the
developing kidney during the suckling period.
REGULATION OF b-OXIDATION GENE BY DIETARY LIPIDS
500 µl of reaction mixture containing 100 mM HEPES, pH
7.6, 0.1 mM EDTA, 200 µM ferricenium hexafluorophosphate
(FcPF6 ), 0.5 mM sodium tetrathionate, and 100 µM octanoylCoA. MCAD activity was calculated from the decrease in
FcPF6 absorbance observed during the first minute of reaction. The results were corrected for absorbance decrease
measured in the absence of octanoyl-CoA.
Expression of results and statistical analysis. The b-oxidation enzyme mRNA abundance was expressed on a relative
percentage basis; the results were obtained from at least two
different Northern blots. MCAD enzyme activity is expressed
as micromoles of octanoyl-CoA oxidized per minute per gram
wet weight. All the data are expressed as means 6 SE. The
means from 4–10 rats in each experimental group were
subjected to ANOVA and Fishers test.
RESULTS
Mitochondrial and peroxisomal b-oxidation enzyme
gene expression in immature kidney cortex and ISOM:
Effects of dietary lipid content. In the renal cortex, the
mRNA levels of LCAD, MCAD, and ACO were markedly higher (1175%, 150%, and 171%, respectively) in
rats fed a high-fat diet from day 16 to 21, than in
littermates kept on a low-fat diet during the same
period (Fig. 1A). Upregulation of MCAD gene expression in response to a high-fat diet went together with a
Fig. 1. Effects of change in lipid supply over the period
from day 16 to 21 on mRNA abundance of long-chain
(LCAD) and medium-chain acyl-CoA dehydrogenases
(MCAD) and acyl-CoA oxidase (ACO) in kidney cortex
and inner stripe of outer medulla (ISOM). Rats were fed
a low-fat (LF) or a high-fat (HF) diet from day 16 to 21,
and Northern blot analyses of total RNA were performed from kidney cortex (A) and ISOM (B) on postnatal day 21. Representative Northern blots are shown in
the top of A and B, and bars represent mean mRNA
values 6 SE obtained from 4–10 animals. Values found
in the low-fat group were taken as reference. * P , 0.05,
** P , 0.01, and *** P , 0.001 compared with LF values.
Absence of error bars indicates that the SE is smaller
than the symbol.
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rat ACO cDNA sequence published by Miyazawa et al. (30).
The ACO cDNA was then directly cloned into pCR II TA
cloning vector according to the manufacturer’s protocol (Invitrogen). A cDNA comprising part of the D and E domains of the
rat PPARa (28) was obtained by RT-PCR (30 cycles). The
primers used were 58-CCCGGGTCATACTCGCAGG-38 (upstream) and 58-TCAGTACATGTCTCTGTAG-38 (downstream).
The resulting cDNA (717 nucleotides long) was purified on
agarose gel and directly used for labeling. Prehybridization
and hybridization were performed in an hybridization oven at
68°C, using the QuickHyb solution from Stratagene following
the manufacturer’s instructions. The membranes were washed
twice with 23 SSC (13 SSC is 0.15 M NaCl/0.015 M sodium
citrate) for 15 min at room temperature, once with 13 SSC
and 1% SDS for 10 min at room temperature, and with 13
SSC and 1% SDS for 30 min at 60°C. Signal densities for each
mRNA were quantified by computerized densitometric analysis of the autoradiograms. The blots were also hybridized
with an 18S cDNA probe to correct variations in the amount
of RNA loaded.
Measurement of MCAD activity. MCAD activity was determined spectrophotometrically at 37°C by following the decrease in ferricenium ion absorbance at 300 nm as previously
described (12). Briefly, kidney cortex or ISOM were weighed
and homogenized (1:5 wt/vol) in ice-cold 100 mM HEPES
(pH 7.6)/0.1 mM EDTA, using a motor-driven Teflon/glass
homogenizer. The homogenates were centrifuged at 7,200 g
for 1 min. For enzyme assay, 5 µl of supernatant was added to
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REGULATION OF b-OXIDATION GENE BY DIETARY LIPIDS
Fig. 2. Effects of change in lipid supply over the period from day 21 to
22 on LCAD, MCAD, and ACO mRNA abundance in kidney cortex.
Rats were fed a low-fat diet from day 16 to 21 and then switched onto
a high-fat diet (HF), or kept on low-fat food (LF), until day 22. Total
RNA extracted from kidney cortex was analyzed by Northern blot.
Bars are means 6 SE of 4–6 animals. Values found in the low-fat
group were taken as reference. ** P , 0.01. *** P ,0.001. Absence of
error bars indicates that the SE is smaller than the symbol.
parallel increase in MCAD enzyme activity (in
µmol · min21 · g wet wt21 ) in the cortex (6.37 6 0.25 in
the low-fat vs. 8.29 6 0.28 in the high-fat group; n 5 6,
P , 0.001). This was in contrast to the data obtained
from the renal ISOM, since similar b-oxidation enzyme
mRNA levels (Fig. 1B) and MCAD enzyme activities
(low fat, 2.54 6 0.15; high fat, 2.21 6 0.22 µmol · min21 · g
wet wt21; n 5 6) were found in this part of the kidney, in
both groups of animals.
Further experiments were performed to determine
whether upregulation of b-oxidation enzyme gene expression can occur in the immature cortex in response
to a shorter exposure to a high-fat diet. Rats were
conditioned to eat low-fat food from day 16 to 21 and
then kept on the same diet, or switched onto the
high-fat food for only 1 day, i.e., from day 21 to 22. As
Fig. 3. Compared PPARa mRNA levels in kidney cortex and ISOM of
21-day-old suckling rat pups. Representative Northern blots are
shown at top, and bars represent mean mRNA values 6 SE obtained
from 5 animals. *** P , 0.001.
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can be seen in Fig. 2, the animals put on a high-fat diet
for 1 day exhibited a large and coordinated increase in
LCAD (1100%), MCAD (184%), and ACO (1350%)
gene expression in the kidney cortex, compared with
those kept on a low-fat diet.
PPARa gene expression in 3-wk-old rat kidney: Effects
of clofibrate on b-oxidation enzyme gene expression. In
Northern blots run from cortex and ISOM of 3-wk-old
rats, the PPARa cDNA probe generated a single band of
,8.5 kb, similar to that reported in studies of adult rat
kidney or liver (22). PPARa mRNA signals were fourfold higher in the cortex than in the ISOM (Fig. 3).
Comparable amounts of PPARa transcripts were found
in the immature cortex of 22-day-old rats fed low- or
high-fat diet for 24 h (Fig. 4). Addition of 0.5% clofibrate
to low-fat food for 1 day did not lead to significant
change in PPARa mRNA, compared with the levels
found in littermates on the low-fat diet only (Fig. 4).
Switching pups onto a clofibrate-containing diet resulted in a marked and coordinated increase of LCAD
(156%), MCAD (1110%), and ACO (1700%) mRNA
steady-state levels (Fig. 5). In contrast, no changes in
MCAD gene expression occurred in response to clofibrate
in the ISOM of the same animals (data not shown).
Short-term regulation of MCAD gene expression: Effects of lipids, clofibrate, and glucagon. To determine
whether upregulation of b-oxidation enzyme gene can
REGULATION OF b-OXIDATION GENE BY DIETARY LIPIDS
F781
Fig. 4. PPARa mRNA abundance in kidney cortex of low-fat-fed,
high-fat-fed, and clofibrate-treated rats. Rats fed a low-fat diet from
day 16 to 21 were put on a high-fat diet from day 21 to 22 (HF) or fed a
low-fat diet containing 0.5% clofibrate during the same period (LF 1
clofibrate). Controls were kept on low-fat food until day 22 (LF).
PPARa mRNA steady-state levels were studied by Northern blots
from cortex total RNA (top). Bars are means 6 SE of 4 animals.
Values found in low-fat group were taken as reference.
occur within hours, MCAD mRNA levels were studied
in kidney cortex 6 h after gavage with coconut oil or
clofibrate. Figure 6A shows that a 30% increase in
MCAD mRNA occurred in both groups of animals,
compared with vehicle-treated pups.
To test a possible effect of glucagon in mediating
short-term regulation of MCAD gene expression, 21-dayold rats kept on a low-fat diet from day 16 to 21 received
a bolus dose of glucagon, and the liver and kidneys were
removed 6 h later. Liver was used as a control, since
glucagon was previously reported to increase MCAD
gene expression in this tissue (31). Accordingly, Northern blot analysis revealed a 38% (P , 0.05) increase in
hepatic levels of MCAD mRNA (Fig. 6B). In contrast,
MCAD mRNA abundance was unchanged in the kidney
cortex of pups receiving glucagon (Fig. 6B).
DISCUSSION
The present data clearly indicate that changes in the
dietary fat supply can induce marked changes in fatty
acid oxidation enzyme gene expression in the immature
rat kidney cortex. Upregulation of mitochondrial and
peroxisomal b-oxidation enzyme mRNA, together with
a parallel increase in MCAD enzyme activity, was first
shown to occur in the kidney cortex of pups fed a
high-fat diet over the period from day 16 to 21. In
contrast, in the renal medulla gene, expression of these
enzymes was unaffected by changes in lipid supply.
This cortex-specific response is consistent with previous data showing that, after 5 days on a high-fat diet,
the activity of b-hydroxyacyl-CoA dehydrogenase, a
Fig. 5. Effects of clofibrate on b-oxidation enzyme gene expression in
kidney cortex over the period from day 21 to 22. Rats were fed low-fat
food 1 0.5% clofibrate as mentioned in Fig. 4. Bars are means 6 SE of
4–6 animals. * P , 0.05 and *** P , 0.001 compared with the low-fat
value (100%). Absence of error bars indicates that the SE is smaller
than the symbol.
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mitochondrial b-oxidation enzyme, is upregulated in
the proximal convoluted tubule, but not in the medullary thick ascending limb, of 21-day-old rat kidney (4).
The present results also show that lipid supply can
exert short-term regulatory effects on b-oxidation enzyme gene expression. Thus increasing lipid supply for
only 24 h was sufficient to induce large and coordinated
increases in the mRNA levels of LCAD, MCAD, and
ACO in the immature kidney cortex. In addition,
experiments run in 21-day-old rats receiving coconut
oil by gavage showed that upregulation of MCAD gene
expression was already initiated within 6 h after lipid
ingestion.
Regulation by lipid supply of enzymes and proteins of
fatty acid metabolism has been very little studied in the
rat kidney (4, 6) and is documented more thoroughly in
F782
REGULATION OF b-OXIDATION GENE BY DIETARY LIPIDS
other organs (10, 18, 33). ACO activity increases markedly in the liver of adult rats chronically fed a high-fat
diet (32, 33). In weanling rats, the activity of liver
carnitine-palmitoyl-transferase 1, which catalyzes the
import of long-chain fatty acid into the mitochondria, is
stimulated after 10 days on a high-fat diet (34). In the
long-term range, these changes in enzyme activities or
gene expression are probably mediated by multiple
regulatory pathways. Indeed, chronic changes in fat
supply modify pancreatic hormone plasma levels (17)
but also induce progressive changes in cell membrane
phospholipid composition, and these latter changes
might in turn result in changes in a number of signal
transduction pathways (10).
The hypothesis of short-term regulatory effects of
dietary fat on gene expression has only been proposed
recently, based on studies run in rat liver or intestine
(1, 11). Fatty acids might directly mediate these shortterm regulatory effects by interacting with the a-isoform of PPAR, a nuclear receptor of the steroid-thyroid
hormone receptor superfamily. In fact, cotransfection
experiments indicate that, after activation by fatty
acids, PPARa mediates transcriptional stimulation of
MCAD, ACO, and other genes involved in fatty acid
catabolism (27). Fatty acids, clofibrate, and other agonists were recently shown to act as ligands of PPARa
(15, 23).
PPARa is highly expressed in the proximal tubule of
rat kidney (7, 22), in which its function(s) remains
unclear. The expression of PPARa gene during development had not yet been studied in the rat kidney. A faint
PPARa signal was found by in situ hybridization in the
proximal convoluted tubule of 8-day-old mouse kidney,
which increased during the postnatal period up to
adulthood (5). The present data establish that PPARa
gene is expressed in the cortex, and to a lower level in
the ISOM, of 3-wk-old rat kidney. Clofibrate supplementation for 24 h resulted in marked increases in cortex
mRNA levels of LCAD, MCAD, and ACO. Pups receiving clofibrate by gavage also exhibited, within 6 h,
significant increases in cortex MCAD gene expression.
PPARa mRNA steady-state levels in the kidney cortex
of clofibrate-treated rats were similar to those found in
control or high-fat fed animals. Thus PPARa can effectively control gene expression of b-oxidation enzymes
in kidney cortex of 3-wk-old rats and could mediate
short-term changes in gene expression of these enzymes, and this does not require upregulation of PPARa
gene expression. This contrasts with the data obtained
from the ISOM in which mRNA levels of mitochondrial
and peroxisomal b-oxidation enzymes were found unchanged in response to clofibrate, despite the presence
of detectable levels of PPARa mRNA. Studies of MCAD
gene 58-flanking regions indicate that the PPARa response element lies within a pleiotropic regulatory
DNA sequence that can bind various combinations of
nuclear receptors (8). The expression of specific transcription factors, competing with PPARa for binding on
DNA regulatory sequence, might possibly account for
the lack of changes in MCAD gene expression in
response to clofibrate in the ISOM.
Data obtained from rat liver suggest that glucagon
could represent an important factor in the regulation of
b-oxidation enzyme gene expression during the postnatal period, as well as in the adult (9, 17, 31). Since the
transition from a low- to a high-fat diet is accompanied
by an increase in glucagon plasma level, we studied
MCAD gene expression levels in the liver and kidney of
pups receiving glucagon. In agreement with data obtained in the adult liver, MCAD mRNA levels were
found increased in response to a single injection of
glucagon. However, expression of MCAD gene in the
cortex of the same animals was found unchanged. This
strongly suggests that glucagon is not involved in
mediating changes in b-oxidation enzyme gene expres-
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Fig. 6. Effects of coconut oil, clofibrate, or glucagon on renal and hepatic MCAD gene expression over a 6-h period.
Data were obtained from 21-day-old rats fed a low-fat diet from day 16 to 21. A: animals received by gavage 1 ml of
coconut oil or 1 ml sterile water (left columns), or 1 ml of 50 mg/ml clofibrate solution or 1 ml of clofibrate vehicle
(right columns). B: animals received a single injection of glucagon (150 µg/100 g body wt) or vehicle. Tissues were
always removed 6 h later. MCAD mRNA abundance was analyzed by Northern blots (top). Results are means 6 SE
of 4–6 animals. * P , 0.05, compared with vehicle-treated animals, taken as 100%.
REGULATION OF b-OXIDATION GENE BY DIETARY LIPIDS
We thank Drs. A. Strauss and D. P. Kelly for providing us with the
rat LCAD and MCAD cDNAs, respectively, and Dr. T. Gilbert for
careful reading of the manuscript.
This work was presented in abstract form at the Annual Meeting of
the American Society of Nephrology in San Antonio, TX, November
1997.
Address for reprint requests: J. Bastin, INSERM U 319, Université Paris 7, 2 Place Jussieu, F-75251 Paris Cedex 05, France.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Received 11 February 1998; accepted in final form 27 July 1998.
19.
REFERENCES
1. Asins, G., D. Serra, G. Arias, and F. G. Hegardt. Developmental changes in carnitine palmitoyltransferase I and II gene
expression in intestine and liver of suckling rats. Biochem. J.
306: 379–384, 1995.
2. Auestad, N., R. A. Korsak, J. D. Bergstrom, and J. Edmond.
Milk-substitutes comparable to rat’s milk: their preparation,
composition and impact on development and metabolism in the
artificially reared rat. Br. J. Nutr. 61: 495–518, 1989.
3. Bastin, J., E. Delaval, N. Freund, F. Djouadi, M. Razanoelina, J. Bismuth, and J. P. Geloso. Effects of birth on energy
metabolism in the rat kidney. Biochem. J. 252: 337–341, 1988.
4. Bastin, J., F. Djouadi, J. P. Geloso, and C. Merlet-Benichou.
Postnatal development of oxidative enzymes in various rat
nephron segments: effect of weaning on different diets. Am. J.
Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F895–F901,
1990.
5. Beck, F., S. Plummer, P. V. Senior, S. Byrne, S. Green, and
W. J. Brammar. The ontogeny of PPAR gene expression in the
mouse and rat. Proc. R. Soc. Lond. B. 247: 83–87, 1992.
6. Bhuiyan, J., P. H. Pritchard, S. V. Pande, and D. W.
Seccombe. Effects of high-fat and fasting on levels of acylcoenzyme A binding protein in liver, kidney and heart of rat.
Metabolism 44: 1185–1189, 1995.
7. Braissant, O., F. Foufelle, C. Scotto, M. Dauça, and W.
Wahli. Differential expression of peroxisome proliferator-
20.
21.
22.
23.
24.
25.
activated receptors (PPARs): tissue distribution of PPAR alpha,
beta and gamma in the adult rat. Endocrinology 137: 354–366,
1996.
Carter, M. E., T. G. Gulick, D. D. Moore, and D. P. Kelly.
A pleiotropic element in the medium chain acyl coenzyme A
dehydrogenase gene promoter mediates transcriptional regulation by multiple nuclear receptor transcription factors and
defines novel receptor-DNA binding motifs. Mol. Cell. Biol. 14:
4360–4372, 1994.
Chatelain, F., C. Kohl, V. Esser, J. D. Mc Garry, J. Girard,
and J. P. Pegorier. Cyclic AMP and fatty acids increase
carnitine palmitoyltransferase I gene transcription in cultured
rat hepatocytes. Eur. J. Biochem. 235: 789–798, 1996.
Clarke, S. D. Dietary polyunsaturated fatty acid regulation of
gene transcription. Annu. Rev. Nutr. 14: 83–98, 1994.
Clarke, S. D., M. K. Armstrong, and D. B. Jump. Dietary
polyunsaturated fats uniquely suppress rat liver fatty acid
synthase and S14 mRNA content. J. Nutr. 120: 225–231, 1990.
Djouadi, F., J. Bastin, D. P. Kelly, and C. Merlet-Benichou.
Transcriptional regulation of mitochondrial oxidative genes in
the developing rat kidney by glucocorticoids. Biochem. J. 315:
555–562, 1996.
Djouadi, F., B. Riveau, C. Merlet-Bénichou, and J. Bastin.
Tissue-specific regulation of medium chain acylCoA dehydrogenase gene by thyroid hormones in the developing rat. Biochem. J.
324: 289–294, 1997.
Djouadi, F., A. Wijkhuisen, and J. Bastin. Coordinate development of oxidative enzymes and Na-K-ATPase in thick ascending
limb: role of corticosteroids. Am. J. Physiol. 263 (Renal Fluid
Electrolyte Physiol. 32): F237–F242, 1992.
Forman, B. C., J. Chen, and R. M. Evans. Hypolipidemic
drugs, polyunsaturated fatty acids and eicosanoids are ligands
for PPAR a and d. Proc. Natl. Acad. Sci. USA 94: 4312–4317,
1997.
Froyland, L., L. Madsen, H. Vaagenes, G. K. Totland, J.
Auwerx, H. Kryvi, B. Staels, and R. K. Berge. Mitochondrion
is the principal target for nutritional and pharmacological control of triglyceride metabolism. J. Lipid Res. 38: 1851–1858,
1997.
Girard, J., P. Ferré, J. P. Pégorier, and P. H. Duée. Adaptation of glucose and fatty acid metabolism during perinatal period
and suckling-weaning transition. Physiol. Rev. 72: 507–562,
1992.
Girard, J., D. Perdereau, F. Foufelle, C. Prip-Buus, and
P. Ferre. Regulation of lipogenic enzyme gene expression by
nutrients and hormones. FASEB J. 8: 36–42, 1994.
Glass, R. L., H. A. Troolin, and R. Jenness. Comparative
biochemical studies of milks. IV. Constituent fatty acids of milk
fats. Comp. Biochem. Physiol. 22: 415–425, 1967.
Hainline, B. E., D. J. Kahlenbeck, J. Grant, and A. W.
Strauss. Tissue specific and developmental expression of rat
long- and medium-chain acyl-CoA dehydrogenases. Biochim.
Biophys. Acta 1216: 460–468, 1993.
Kelly, D. P., J. J. Kim, J. J. Billadello, B. E. Hainline, T. W.
Chu, and A. W. Strauss. Nucleotide sequence of medium-chain
acyl-coA dehydrogenase mRNA and its expression in enzymedeficient human tissue. Proc. Natl. Acad. Sci. USA 84: 4068–
4072, 1987.
Kliewer, S. A., B. M. Forman, B. Blumberg, E. S. Ong, U.
Borgmeyer, D. J. Mangelsdorf, K. Umesono, and R. M.
Evans. Differential expression and activation of a family of
murine peroxisome proliferator-activated receptors. Proc. Natl.
Acad. Sci. USA 91: 7355–7359, 1994.
Kliewer, S. A., S. S. Sundseth, S. A. Jones, P. J. Brown, G. B.
Wisely, C. S. Koble, P. Devchand, W. Wahli, T. M. Willson,
J. M. Lenhard, and J. M. Lehmann. Fatty acids and eicosanoids regulate gene expression through direct interactions with
PPAR a and g. Proc. Natl. Acad. Sci. USA 94: 4318–4323, 1997.
Kunau, W. H., V. Dommes, and H. Schulz. b-oxidation of fatty
acids in mitochondria, peroxisomes, and bacteria: a century of
continued progress. Prog. Lipid Res. 34: 267–342, 1995.
Laposata, M. Fatty acids: biochemistry to clinical significance.
Am. J. Clin. Pathol. 104: 172–179, 1995.
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sion in response to variations in lipid supply in the
immature kidney cortex.
Taken together, these data allow one to conclude that
gene expression of mitochondrial and peroxisomal b-oxidation enzyme in the immature kidney cortex can be
physiologically coregulated according to variations in
the lipid supply to the rat pups. This supports the
recent hypothesis suggesting that both peroxisomes
and mitochondria might represent a major target for a
nutritional control of lipid metabolism (16). The signaling pathway(s) involved in regulating b-oxidation enzyme gene expression as a function of dietary lipid
supply cannot be inferred from the present data. However, since activation of PPARa led to a marked stimulation of both peroxisomal and mitochondrial b-oxidation
genes in the kidney cortex of rat pups, it is tempting to
speculate that fat supply-dependent gene regulation
might operate via PPARa. It was recently demonstrated that PPARa can recognize a broad array of fatty
acids and lipid-derived metabolites and can regulate
gene expression in all fatty acid catabolism pathways
(27). Nevertheless, the physiological relevance of these
functions, unique among the nuclear receptors, remains far from being completely elucidated. Further
studies will help to delineate whether, as suggested by
the present data, PPARa might play a major role in
triggering the postnatal development of fatty acid
utilization in organs like kidney, which closely depend
upon these mechanisms to ensure their energy homeostasis.
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F784
REGULATION OF b-OXIDATION GENE BY DIETARY LIPIDS
32.
33.
34.
35.
36.
37.
a-subunit of electron transfer flavoprotein in rat. J. Biol. Chem.
268: 24114–24124, 1993.
Neat, C., M. S. Thomassen, and H. Osmundsen. Induction of
peroxisomal b-oxidation in rat liver by high-fat diets. Biochem. J.
186: 369–371, 1980.
Osmundsen, H., J. Bremer, and J. I. Pedersen. Metabolic
aspects of peroxisomal b-oxidation. Biochim. Biophys. Acta 1085:
141–158, 1991.
Prip-Buus, C., S. Thumelin, F. Chatelain, J. P. Pegorier,
and J. Girard. Hormonal and nutritional control of liver fatty
acid oxidation and ketogenesis during development. Biochem.
Soc. Trans. 23: 500–505, 1995.
Wijkhuisen, A., F. Djouadi, J. Vilar, C. Merlet-Benichou,
and J. Bastin. Thyroid hormones regulate development of
energy metabolism enzymes in rat proximal convoluted tubule.
Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F634–
F642, 1995.
Wirthensohn, G., and W. G. Guder. Renal lipid metabolism.
Miner. Electrolyte Metab. 9: 2O3–211, 1983.
Zaar, K. Structure and function of peroxisomes in the mammalian kidney. Eur. J. Cell Biol. 59: 233–254, 1992.
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.2 on June 17, 2017
26. Leibowitz, S. F., D. J. Lucas, K. L. Leibowitz, and Y. S.
Jhanwar. Developmental patterns of macronutrient intake in
female and male rats from weaning to maturity. Physiol. Behav.
50: 1167–1174, 1991.
27. Lemberger, T., B. Desvergne, and W. Wahli. Peroxisome
proliferator-activated receptors: a nuclear receptor signaling
pathway in lipid physiology. Ann. Rev. Cell Dev. Biol. 12:
335–363, 1996.
28. Lemberger, T., R. Saladin, M. Vasquez, F. Assimacopoulos,
B. Staels, B. Desvergne, W. Wahli, and J. Auwerx. Expression of the PPAR a gene is stimulated by stress and follows a
diurnal rhythm. J. Biol. Chem. 271: 1764–1769, 1996.
29. Messer, M., E. B. Thoman, A. G. Terrasa, and P. R. Dallman.
Artificial feeding of infant rats by continuous gastric infusion. J.
Nutr. 98: 404–410, 1969.
30. Miyazawa, S., H. Hayashi, M. Hijikata, N. Ishii, S. Furuta,
H. Kagamiyama, T. Osumi, and T. Hashimoto. Complete
nucleotide sequence of cDNA and predicted amino acid sequence
of rat acylCoA oxidase. J. Biol. Chem. 262: 8131–8137, 1987.
31. Nagao, M., B. Parimoo, and K. Tanaka. Developmental,
nutritional, and hormonal regulation of tissue-specific expression of the genes encoding various acyl-CoA dehydrogenases and