# Cardiac PPARα Protein Expression is Constant as Alternate Nuclear Receptors and PGC-1 Coordinately Increase During the Postnatal Metabolic Transition

Authors: Norman E. Buroker; Xue-Han Ning; Michael Portman
Journal: PPAR Research
 (2008)

---

## Abstract

Gene expression data obtained in mouse heart indicate that increased expression for the nuclear receptor, peroxisomal proliferator activated receptorα (PPARα), prompts the postnatal transition from predominantly carbohydrate to fatty acid oxidation preference. However, no phenotypic or proteomic data are available to confirm downstream signaling and metabolic transition in mice. We studied the hypothesis that shifts in nuclear receptor expression trigger the newborn metabolic switch in a newborn sheep. This species is well characterized with regards to developmental changes in substrate oxidative metabolism. Heart tissues from fetal (130 days
gestation), newborn ≤24 hours, and 30-day old lambs were evaluated for protein expression from multiple enzymes controlling oxidative metabolism as well as principal nuclear receptors and coactivators. Although muscle and liver type carnitine palmitoyl transferases I showed no significant changes to correspond to the metabolic transition, hexokinase II protein content
showed a profound transient drop, and pyruvate dehydrogenase kinase steadily increased. PPARα showed no increases preceding or during the transition, while peroxisomal proliferator activated receptor gamma coactivator-1 (PGC-1) increased approximately 20-fold transiently in newborn heart in conjunction with significant increases in thyroid hormone receptor α1 and retinoid-activated receptor α. These data challenge the paradigm that increases in PPARα prompt the postnatal metabolic switch, and suggest that other nuclear receptors play a major role. As thyroid hormone (TH) modulates PGC-1 expression in sheep during development, these data further suggest that well-characterized perinatal TH surge in sheep contributes to this metabolic switch.


---

## Body

## 1. Introduction

The fetal heart uses glucose and lactate   as the main oxidative substrate sources,
then switches to fatty acids  as the predominant fuel shortly after birth [1]. Rapid expansion of mitochondrial pool as well
as total oxidative capacity accompanies the perinatal metabolic transition. Initial newborn suckling likely stimulates
release of hormonal factors, which trigger modification of substrate
preference. Recent data indicate that multiple nuclear receptors and their coactivators
initiate transcriptional events controlling both newborn substrate switching
and perinatal mitochondrial biogenesis.The nuclear receptor family includes classical endocrine receptors
activated by ligands such a thyroid hormone or steroid hormones. Other more
recently identified nuclear receptors (NR) respond to dietary-derived lipid intermediates
involved in the metabolism of these activating ligands. In particular, the fatty acid-activated peroxisome
proliferator-activated receptors (PPAR) participate as key regulators of
cardiac energy metabolism. The hormones
or ligands for the nuclear receptors attach to their respective ligand binding
domains. These receptors will attach to
DNA response elements (REs) of their target genes as monomers, homodimers, or partner
as heterodimers [2, 3]. Many NRs involved in metabolic regulation
heterodimerize with retinoid X receptor, creating a potential mechanism for
regulatory integration. Ligand binding promotes a permissive receptor
conformation for coactivator interaction. Coactivators such as PPARγ coactivator-1
(PGC-1) bind the receptor in the process of establishing a transcriptional
complex with RNA polymerase-2 to initiate transcription of target genes [3–5]. PGC-1 enhances transactivation mediated by
numerous nuclear receptors involved in energy metabolism and mitochondrial
biogenesis, thereby providing a second mechanism for integration of these
processes.Although NR-mediated control of cardiac energy
metabolism has been examined with gain or loss of function using transgenic
models, developmental regulation of these processes has not been studied in
detail. Messenger RNA expression studies
have provided the prevailing evidence for the PPAR and PGC-1 triggering of the
cardiac metabolic switch after birth.
However, multiple NRs are subject to several post-transcriptional
processes including alternate heteronuclear RNA splicing and differing
translation site initiation, as well as end-product feedback inhibition. For the most part, regulatory patterns have
not been confirmed at the protein expression level. Accordingly, we studied developmental
integration and coordination of protein expression for three major nuclear
receptor families involved in regulation of both cardiac metabolism and
mitochondrial biogenesis: thyroid
hormone receptors (TRs), PPARs, and RXRs.
This study included analyses of PGC-1, as it provides a coactivator
function for both TRs and PPARs.
Furthermore, previous studies involving NR regulation of developmental
metabolic switching have been performed in mice, a poorly characterized species
with regard to newborn cardiac metabolism [6]. Since substantial data is available defining
developmental cardiac energy metabolism in sheep, we used this species to test
the
hypothesis
that nuclear receptor signaling prompts the newborn metabolic switch. The data obtained in this well-characterized
species challenge existing paradigms regarding triggering for the postnatal
metabolic transition.


## 2. Materials and Methods

### 2.1. Animal Samples

Domestic sheep (Ovis aries) were used for our
study. Heart samples were collected at
130 days into gestation (F), one day after birth (N), and 30 days after birth
(C) (n
=
6, from each group). Heart
tissue from the left ventricle was quickly blotted dry, frozen in liquid
nitrogen, and stored at −80°C. Our
investigation conforms to the Guide for the Care and Use of Laboratory
Animals published by the US National Institutes of Health (NIH Publication
No. 85-23, revised 1996). The Animal Care Committee of the University of Washington
approved all animal protocols.


### 2.2. Protein Isolation

The frozen heart tissue was diced and
homogenized at 4°C in a three-fold amount of protein lyses buffer
(50 mM Tris-Cl, pH 8.0, 100 mM NaCl, 20 mM HEPES, pH7.9, 1% Triton X-100, 10 mM
NaF, 1 mM Na
3
VO
4, 100 ug/ml C
7
H
7
FO
2S, 5 ug/ml Aprotinin, and 5 ug/ml
Leupeptin). The samples were then incubated on ice for 30 minutes and
centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was transferred to another
microfuge tube and centrifuged one more time.
The supernatant was stored at −80°C for immunoblotting
analysis.


### 2.3. Immunoblotting

Fifty micrograms of total protein
extracts from sheep heart tissue were electrophoresed along with one lane
containing thirty micrograms of human HeLa cells as a positive control and one
lane of molecular weight size markers (Chemichrome Western Control, Sigma-Aldrich Co., Mo, USA)
in a 4.5% stacking and 7.5, 10, or 12% running SDS-polyacrylamide gel depending
on the molecular weight of the protein of interest. The gels were then electroblotted onto
polyvinylidene fluoride (PVDF) plus membranes.
The western blots were blocked for one hour at room temperature with
either a 1% or 5% nonfat milk (depending on the antibodies requirements) in
Tris-buffered saline plus Tween-20 (TBST)[10 mM Tris-HCl, pH 7.5, 150 mM NaCl,
and 0.05% Tween-20], followed by overnight incubation at 4°C with
each primary antibody diluted in the appropriate blocking solution as recommended
by the supplier. The primary antibodies used in the study are β-actin (SC-1616), HK2 (SC-6521), MTCO3 (SC-23986), PGC-1 (SC-5814), PPARα
(SC-9000), and RXRα (SC-553) obtained from (Santa Cruz Biotechnology, Inc.,
Calif, USA). The primary antibody TRα (PA1-211A) was obtained from (Affinity BioReagents, Inc., CO, USA). The primary antibodies L&M-CPTI and PDK2
were obtained as personal gifts from Gebre Woldegiorgis and Robert Harris,
respectively. After two five-minute washes
with TBST and one five-minute
wash with Tris-buffered saline (TBS), membranes were incubated at room
temperature for one hour with the appropriated secondary antibody conjugated to
horseradish peroxidase (HRP). The
membranes were washed twice for ten minutes with TBST and visualized with
enhanced chemiluminescence after exposure to Kodak biomax light ML-2 film. The membranes were stripped by washing them
two times for 30 minutes with 200 mM Glycine, 0.1% SDS, and 1% Tween-20 (pH
adjusted to 2.2), followed by three ten-minute washes with TBS. The membranes were again blocked for one hour
as above, followed by overnight incubation at 4°C with β-Actin antibody diluted
1 : 200 in blocking solution. The next day,
the membranes were washed (as above), the appropriate secondary-HRP antibody
was applied, and the remaining procedures (as described above) were
followed. The β-actin was used to verify
protein lane loadings.


### 2.4. Statistical Analysis

The film expression was determined using the ImageJ 1.32j program produced by Wayne Rasband for (the National Institute of Health, Md, USA). The protein expression was standardized
againstβ-Actin and the means and standard errors
for the three stages of heart development are displayed in a histogram (Figure 1). Statistical significance was
determined with Student's t-test (two tailed) comparison between all
stages of development.Immunoblots and expression patterns for
enzymes controlling substrate oxidation.
Data is shown for left ventricle from three development periods (F, N, C) as defined in text. Abbreviations are
carnitine palmitoyltransferase I (muscle isoform M-CPTI), carnitine
palmitoyltransferase I (liver isoform L-CPTI), hexokinase 2 (HK2), and pyruvate
dehydrogenase kinase 2 (PDK2). M-CPTI
protein expression decreases occurred between N and C (*
P
<
.01). L-CPTI protein expression differences
decreased immediately after birth F (*
P
<
.01) and continued to drop in C (+
P
<
0.001, versus N). HK2 protein expression exhibited
a marked but transient decrease after birth N (*
P
<
.05, versus F & C). PDK2
protein expression increased with significance noted in C compared to F and N
(*
P
<
.05, F & N).
(a)
(b)
(c)
(d)


## 2.1. Animal Samples

Domestic sheep (Ovis aries) were used for our
study. Heart samples were collected at
130 days into gestation (F), one day after birth (N), and 30 days after birth
(C) (n
=
6, from each group). Heart
tissue from the left ventricle was quickly blotted dry, frozen in liquid
nitrogen, and stored at −80°C. Our
investigation conforms to the Guide for the Care and Use of Laboratory
Animals published by the US National Institutes of Health (NIH Publication
No. 85-23, revised 1996). The Animal Care Committee of the University of Washington
approved all animal protocols.


## 2.2. Protein Isolation

The frozen heart tissue was diced and
homogenized at 4°C in a three-fold amount of protein lyses buffer
(50 mM Tris-Cl, pH 8.0, 100 mM NaCl, 20 mM HEPES, pH7.9, 1% Triton X-100, 10 mM
NaF, 1 mM Na
3
VO
4, 100 ug/ml C
7
H
7
FO
2S, 5 ug/ml Aprotinin, and 5 ug/ml
Leupeptin). The samples were then incubated on ice for 30 minutes and
centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was transferred to another
microfuge tube and centrifuged one more time.
The supernatant was stored at −80°C for immunoblotting
analysis.


## 2.3. Immunoblotting

Fifty micrograms of total protein
extracts from sheep heart tissue were electrophoresed along with one lane
containing thirty micrograms of human HeLa cells as a positive control and one
lane of molecular weight size markers (Chemichrome Western Control, Sigma-Aldrich Co., Mo, USA)
in a 4.5% stacking and 7.5, 10, or 12% running SDS-polyacrylamide gel depending
on the molecular weight of the protein of interest. The gels were then electroblotted onto
polyvinylidene fluoride (PVDF) plus membranes.
The western blots were blocked for one hour at room temperature with
either a 1% or 5% nonfat milk (depending on the antibodies requirements) in
Tris-buffered saline plus Tween-20 (TBST)[10 mM Tris-HCl, pH 7.5, 150 mM NaCl,
and 0.05% Tween-20], followed by overnight incubation at 4°C with
each primary antibody diluted in the appropriate blocking solution as recommended
by the supplier. The primary antibodies used in the study are β-actin (SC-1616), HK2 (SC-6521), MTCO3 (SC-23986), PGC-1 (SC-5814), PPARα
(SC-9000), and RXRα (SC-553) obtained from (Santa Cruz Biotechnology, Inc.,
Calif, USA). The primary antibody TRα (PA1-211A) was obtained from (Affinity BioReagents, Inc., CO, USA). The primary antibodies L&M-CPTI and PDK2
were obtained as personal gifts from Gebre Woldegiorgis and Robert Harris,
respectively. After two five-minute washes
with TBST and one five-minute
wash with Tris-buffered saline (TBS), membranes were incubated at room
temperature for one hour with the appropriated secondary antibody conjugated to
horseradish peroxidase (HRP). The
membranes were washed twice for ten minutes with TBST and visualized with
enhanced chemiluminescence after exposure to Kodak biomax light ML-2 film. The membranes were stripped by washing them
two times for 30 minutes with 200 mM Glycine, 0.1% SDS, and 1% Tween-20 (pH
adjusted to 2.2), followed by three ten-minute washes with TBS. The membranes were again blocked for one hour
as above, followed by overnight incubation at 4°C with β-Actin antibody diluted
1 : 200 in blocking solution. The next day,
the membranes were washed (as above), the appropriate secondary-HRP antibody
was applied, and the remaining procedures (as described above) were
followed. The β-actin was used to verify
protein lane loadings.


## 2.4. Statistical Analysis

The film expression was determined using the ImageJ 1.32j program produced by Wayne Rasband for (the National Institute of Health, Md, USA). The protein expression was standardized
againstβ-Actin and the means and standard errors
for the three stages of heart development are displayed in a histogram (Figure 1). Statistical significance was
determined with Student's t-test (two tailed) comparison between all
stages of development.Immunoblots and expression patterns for
enzymes controlling substrate oxidation.
Data is shown for left ventricle from three development periods (F, N, C) as defined in text. Abbreviations are
carnitine palmitoyltransferase I (muscle isoform M-CPTI), carnitine
palmitoyltransferase I (liver isoform L-CPTI), hexokinase 2 (HK2), and pyruvate
dehydrogenase kinase 2 (PDK2). M-CPTI
protein expression decreases occurred between N and C (*
P
<
.01). L-CPTI protein expression differences
decreased immediately after birth F (*
P
<
.01) and continued to drop in C (+
P
<
0.001, versus N). HK2 protein expression exhibited
a marked but transient decrease after birth N (*
P
<
.05, versus F & C). PDK2
protein expression increased with significance noted in C compared to F and N
(*
P
<
.05, F & N).
(a)
(b)
(c)
(d)


## 3. Results

### 3.1. Carnitine Palmitoyltransferases

The enzyme  carnitine palmitoyltransferase I
(CPT I; palmitoyl-CoA:L-carnitine O-palmitoyltransferase; EC 2.31.21) is a
rate-limiting step in mitochondria transport during FA oxidation. It catalyzes the initial reaction of acyl-CoA
and carnitine to acylcarnitine during the mitochondria import of long-chain FAs
into the inner mitochondria membrane.
PPAR regulates CPTI and turns the gene on during the FA oxidation
cascade of events shortly after birth [7, 8]. In mammalian heart, there are two CPTI
isoenzymes: a liver, L-CPTI isoenzyme, also known as CPTIα: and a muscle, M-CPTI isoenzyme, also
known as CPTIβ [9, 10] In human tissue, the L-CPTI protein is
composed of 773 amino acids that corresponds to a molecular weight of 88.3 kDa
[P50416]. In sheep heart ,the L-CPTI
isoenzyme corresponds to a molecular weight of 82 kDa (Figure 3), which is
smaller than that reported in human tissue, but in agreement with what has been
reported in fetal and newborn lambs [9]. In human tissue, the M-CPT I protein is
composed of 772 amino acids which corresponds to a molecular weight of 87.8 kDa
[Q92523]. The adult sheep protein
sequence is highly conserved with relation to other mammals with an 89%
similarity to humans and 88% similarity to mouse and rat [11]. In sheep heart, the M-CPTI antibody
recognizes an 88-kDa isoenzyme consistently expressed in the three study groups
(Figure 1). The 88-kDa protein
corresponds to the 771 amino acids reported for M-CPTI in sheep [C81315], but
is in disagreement with the 80-kDa protein that has been previously reported in
sheep [9]. In this study, the M-CPTI (Figure 3(a)) and
L-CPTI (Figure 3(b)) protein expression was significantly greater in the day old
samples then in the 30-day old samples, while the L-CPTI protein expression was
significantly greater in the fetal then in either the day old or 30-day old
samples (Figure 1).Content for nuclear receptors peroxisome
proliferator-activated receptor (PPARα), (b)
retinoid X receptor alpha (RXRα), and
thyroid hormone receptor (TRα
1)
among three stages (F, N, C). Modest
transient but significant elevations for RXRα and
TRα
1 occurred in N compared to
both F and C (*
P
<
.01). PPARα expression did not change immediately after
birth, but later decreased compared to F and N (*
P
<
.01).
(a)
(b)
(c)


### 3.2. Hexokinase

Hexokinase (HK, E.C.2.7.1.1) in mammalian  tissues exist as four isoenzymes (HK1-4) with
distinct kinetic properties  and tissue distribution [12]. The hexokinases are rate limiting glycolytic
enzymes that catalyze the phosphorylation of glucose to glucose-6-phosphate [13]. HK type 2 and HK type 4 are found in heart
tissue [14] . In sheep heart, the HK2 antibody detects a
102-kDa protein among the three study groups (Figure 1). The 102-kDa protein
corresponds to the molecular weight of the human HK2 isoenzyme with 917 amino
acids [P52789]. A 102-kDa protein was
also detected by this antibody in a HeLa human cell line obtained from (Santa Cruz Biotechnology, Inc.,
Calif, USA) (data not included). In
this study, we monitored  the appearance of HK2 during the three stages of sheep
development and found a significant decrease in HK2 protein expression for the
day old samples compared to either the fetal or 30-day old samples. This
finding indicates that a reduction in glycolysis at birth due to the onset of
FAO.


### 3.3. Pyruvate Dehydrogenase Kinase

The isoenzyme pyruvate dehydrogenase kinase 2 (PDK2;
pyruvate dehydrogenase [lipoamide] kinase isoenzyme 4; EC 2.7.1.99) is one of
four PDK isoenzymes found in mammalian tissues.
PDK2 is expressed at high levels in heart tissue [15–17] and is regulated by PPARα [18]. The enzyme is responsible for phosphorylation
of pyruvate dehydrogenase (PDH), a mitochondrial multienzyme complex, rendering
it inactive. The PDH catalyzes the
oxidative decarboxylation of pyruvate, linking glycolysis to the tricarboxylic
acid cycle and FA synthesis [18]. Increased levels of PDK in early postnatal
life for PDH inactivation are thought to be the result of changes in lipid
supply and a switch from glucose to FAs as an energy supply [19]. In addition to hexokinase, PDK is also a rate-limiting
step in the glycolytic pathway [20, 21]. In human tissue, the PDK2 isoenzyme is
composed of 407 amino acids which corresponds to a molecular weight of 46 kDa
for the isoenzyme [Q15119]. PDK2 has its
strongest expression in the day old (N) sheep samples. In sheep heart,  the PDK2 antibody detects a
46-kDa protein among the three study groups (Figure 1). A 47-kDa protein was also detected by this
antibody in a HeLa
human cell line obtained from (Santa Cruz Biotechnology, Inc., Calif,
USA)
(data not included). In this study,
PDK2 protein expression levels were significantly greater in the 30-day old (C)
samples when compared to either fetal (F) or the one-day old (N) samples;
(Figure 1) indicating the switch from glucose to FAO.


### 3.4. Mitochondrial Proteins Involved with Cardiac Energy Metabolism

With the beginning of aerobic development at
birth there is a rapid deployment of new mitochondria in cardiac cells to
handle the FA energy metabolism and ATP output [22]. The mitochondrial genome must be running near
full capacity with genome replication for new mitochondria as well as the
transcription of mitochondria genes [23] in order to accommodate this
expansion. We used protein expression for
the cytochrome c oxidase 3 as a reporter for mitochondrial biogenesis. Cytochrome
c oxidase 3 (MTCO3; Cytochrome c oxidase polypeptide 3; E.C. 1.9.3.1) is one of
three subunits transcribed in the mitochondria from a total of 13 subunits that
make up cytochrome c oxidase. The
remaining ten subunits are transcribed from nuclear genes [23]. In sheep heart, the MTCO3 antibody detects a
30-kDa molecular weight protein, which represents the cytochrome c oxidase 3 in
the three study groups. MTCO3 protein
expression is greater in N then either the F or C (Figure 2). We
did not see comparable changes for the nuclear cytochrome c oxidase, subunit 4,
(NCO4) gene (data not included). This
difference between the expression of mitochondria and nuclear cytochrome c
oxidase subunits during mitochondria biogenesis has previously been reported [24].Cytochrome c oxidase 3 expression serves a
reporter for the mitochondrial genome (MTCO3; Cytochrome c oxidase polypeptide
3; E.C. 1.9.3.1). MTCO3 increased transiently in the newborn (**
P
<
.01, versus F
& C). Coordinate changes in protein
content occurred for PGC-1α, implicated
as a regulator of mitochondrial biogenesis.
(a)
(b)


### 3.5. Peroxisome Proliferator-Activated Receptor-γ Co-Activator 1 (PGC-1)

In sheep, the PGC-1 antibody recognizes the
PGC-1α protein or the 91-kDa isoform, while
the PRC and PGC-1β isoforms were not detected among the
three study groups (Figure 2). PGC-1α displays a significant increase in
protein expression in the day old samples (N) compared with either the fetal
(F) or 30-day old samples (C), while the 30-day old samples (C) were found to
have a significantly greater protein expression level than the fetal samples
(F). In a previous sheep study, we noted
that PGC-1 protein expression was near threefold greater in C than in F.


### 3.6. Peroxisome Proliferator-Activated Receptors (PPARα)

The PPARα antibody detects the 52-kDa nuclear
receptor at each stage of development (Figure 3). In human tissues, the PPARα protein is composed of 478 amino acids
that reflects a molecular weight of 52 kDa [Q07869], which coincides with the
molecular weight observed in sheep. The
52-kDa protein was also detected by this antibody in a HeLa human cell line obtained from (Santa Cruz Biotechnology, Inc., Calif, USA) (data not
included). The protein expression of the
PPARα nuclear receptor was found to be significantly lower in the 30-day old
samples (C) when compared to the Fetal (F) and the day old samples (N) (Figure 3).


### 3.7. Retinoid X Receptors (RXRα)

In sheep heart, the RXRα
antibody detects the 51-kDa nuclear receptor at each stage of development
(Figure 3) corresponding to the full length RXRα is
462 amino acids, which represents a molecular weight of 51 kDa [P19793]. We noted a small but significant surge in RXRα expression in the one-day old sheep
heart (Figure 2(b)).


### 3.8. Thyroid Hormone Receptor (TRα
1)

In
sheep heart, the TRα1 antibody detects the 47-kDa nuclear
receptor among the three study groups (Figure 3). In humans and rodents, the
full length of TRα is 410 amino acids, which corresponds
to a molecular weight of 47 kDa. In this
study, the THRα1 nuclear receptor has a significantly
greater level of protein expression in the day old samples (N) compared with
either the fetal (F) or the 30-day old samples (C) (Figure 3).


### 3.9. Other Nuclear Receptor Isoforms and Metabolic Proteins of Interest

Antibodies
against other protein expression were used.
These include PPARβ, PPARγ, RXRβ, TRα2, TRβ, MYLCD, and PDK4. Unfortunately, a cross reactivity exists
between the secondary antibodies (i.e., those with goat, mouse, and rabbit hosts
for the primary antibodies) and the one-day and 30-day old stages of
development in our sheep samples. Consequently, we could not get reliable data
for these proteins of interest.


## 3.1. Carnitine Palmitoyltransferases

The enzyme  carnitine palmitoyltransferase I
(CPT I; palmitoyl-CoA:L-carnitine O-palmitoyltransferase; EC 2.31.21) is a
rate-limiting step in mitochondria transport during FA oxidation. It catalyzes the initial reaction of acyl-CoA
and carnitine to acylcarnitine during the mitochondria import of long-chain FAs
into the inner mitochondria membrane.
PPAR regulates CPTI and turns the gene on during the FA oxidation
cascade of events shortly after birth [7, 8]. In mammalian heart, there are two CPTI
isoenzymes: a liver, L-CPTI isoenzyme, also known as CPTIα: and a muscle, M-CPTI isoenzyme, also
known as CPTIβ [9, 10] In human tissue, the L-CPTI protein is
composed of 773 amino acids that corresponds to a molecular weight of 88.3 kDa
[P50416]. In sheep heart ,the L-CPTI
isoenzyme corresponds to a molecular weight of 82 kDa (Figure 3), which is
smaller than that reported in human tissue, but in agreement with what has been
reported in fetal and newborn lambs [9]. In human tissue, the M-CPT I protein is
composed of 772 amino acids which corresponds to a molecular weight of 87.8 kDa
[Q92523]. The adult sheep protein
sequence is highly conserved with relation to other mammals with an 89%
similarity to humans and 88% similarity to mouse and rat [11]. In sheep heart, the M-CPTI antibody
recognizes an 88-kDa isoenzyme consistently expressed in the three study groups
(Figure 1). The 88-kDa protein
corresponds to the 771 amino acids reported for M-CPTI in sheep [C81315], but
is in disagreement with the 80-kDa protein that has been previously reported in
sheep [9]. In this study, the M-CPTI (Figure 3(a)) and
L-CPTI (Figure 3(b)) protein expression was significantly greater in the day old
samples then in the 30-day old samples, while the L-CPTI protein expression was
significantly greater in the fetal then in either the day old or 30-day old
samples (Figure 1).Content for nuclear receptors peroxisome
proliferator-activated receptor (PPARα), (b)
retinoid X receptor alpha (RXRα), and
thyroid hormone receptor (TRα
1)
among three stages (F, N, C). Modest
transient but significant elevations for RXRα and
TRα
1 occurred in N compared to
both F and C (*
P
<
.01). PPARα expression did not change immediately after
birth, but later decreased compared to F and N (*
P
<
.01).
(a)
(b)
(c)


## 3.2. Hexokinase

Hexokinase (HK, E.C.2.7.1.1) in mammalian  tissues exist as four isoenzymes (HK1-4) with
distinct kinetic properties  and tissue distribution [12]. The hexokinases are rate limiting glycolytic
enzymes that catalyze the phosphorylation of glucose to glucose-6-phosphate [13]. HK type 2 and HK type 4 are found in heart
tissue [14] . In sheep heart, the HK2 antibody detects a
102-kDa protein among the three study groups (Figure 1). The 102-kDa protein
corresponds to the molecular weight of the human HK2 isoenzyme with 917 amino
acids [P52789]. A 102-kDa protein was
also detected by this antibody in a HeLa human cell line obtained from (Santa Cruz Biotechnology, Inc.,
Calif, USA) (data not included). In
this study, we monitored  the appearance of HK2 during the three stages of sheep
development and found a significant decrease in HK2 protein expression for the
day old samples compared to either the fetal or 30-day old samples. This
finding indicates that a reduction in glycolysis at birth due to the onset of
FAO.


## 3.3. Pyruvate Dehydrogenase Kinase

The isoenzyme pyruvate dehydrogenase kinase 2 (PDK2;
pyruvate dehydrogenase [lipoamide] kinase isoenzyme 4; EC 2.7.1.99) is one of
four PDK isoenzymes found in mammalian tissues.
PDK2 is expressed at high levels in heart tissue [15–17] and is regulated by PPARα [18]. The enzyme is responsible for phosphorylation
of pyruvate dehydrogenase (PDH), a mitochondrial multienzyme complex, rendering
it inactive. The PDH catalyzes the
oxidative decarboxylation of pyruvate, linking glycolysis to the tricarboxylic
acid cycle and FA synthesis [18]. Increased levels of PDK in early postnatal
life for PDH inactivation are thought to be the result of changes in lipid
supply and a switch from glucose to FAs as an energy supply [19]. In addition to hexokinase, PDK is also a rate-limiting
step in the glycolytic pathway [20, 21]. In human tissue, the PDK2 isoenzyme is
composed of 407 amino acids which corresponds to a molecular weight of 46 kDa
for the isoenzyme [Q15119]. PDK2 has its
strongest expression in the day old (N) sheep samples. In sheep heart,  the PDK2 antibody detects a
46-kDa protein among the three study groups (Figure 1). A 47-kDa protein was also detected by this
antibody in a HeLa
human cell line obtained from (Santa Cruz Biotechnology, Inc., Calif,
USA)
(data not included). In this study,
PDK2 protein expression levels were significantly greater in the 30-day old (C)
samples when compared to either fetal (F) or the one-day old (N) samples;
(Figure 1) indicating the switch from glucose to FAO.


## 3.4. Mitochondrial Proteins Involved with Cardiac Energy Metabolism

With the beginning of aerobic development at
birth there is a rapid deployment of new mitochondria in cardiac cells to
handle the FA energy metabolism and ATP output [22]. The mitochondrial genome must be running near
full capacity with genome replication for new mitochondria as well as the
transcription of mitochondria genes [23] in order to accommodate this
expansion. We used protein expression for
the cytochrome c oxidase 3 as a reporter for mitochondrial biogenesis. Cytochrome
c oxidase 3 (MTCO3; Cytochrome c oxidase polypeptide 3; E.C. 1.9.3.1) is one of
three subunits transcribed in the mitochondria from a total of 13 subunits that
make up cytochrome c oxidase. The
remaining ten subunits are transcribed from nuclear genes [23]. In sheep heart, the MTCO3 antibody detects a
30-kDa molecular weight protein, which represents the cytochrome c oxidase 3 in
the three study groups. MTCO3 protein
expression is greater in N then either the F or C (Figure 2). We
did not see comparable changes for the nuclear cytochrome c oxidase, subunit 4,
(NCO4) gene (data not included). This
difference between the expression of mitochondria and nuclear cytochrome c
oxidase subunits during mitochondria biogenesis has previously been reported [24].Cytochrome c oxidase 3 expression serves a
reporter for the mitochondrial genome (MTCO3; Cytochrome c oxidase polypeptide
3; E.C. 1.9.3.1). MTCO3 increased transiently in the newborn (**
P
<
.01, versus F
& C). Coordinate changes in protein
content occurred for PGC-1α, implicated
as a regulator of mitochondrial biogenesis.
(a)
(b)


## 3.5. Peroxisome Proliferator-Activated Receptor-γ Co-Activator 1 (PGC-1)

In sheep, the PGC-1 antibody recognizes the
PGC-1α protein or the 91-kDa isoform, while
the PRC and PGC-1β isoforms were not detected among the
three study groups (Figure 2). PGC-1α displays a significant increase in
protein expression in the day old samples (N) compared with either the fetal
(F) or 30-day old samples (C), while the 30-day old samples (C) were found to
have a significantly greater protein expression level than the fetal samples
(F). In a previous sheep study, we noted
that PGC-1 protein expression was near threefold greater in C than in F.


## 3.6. Peroxisome Proliferator-Activated Receptors (PPARα)

The PPARα antibody detects the 52-kDa nuclear
receptor at each stage of development (Figure 3). In human tissues, the PPARα protein is composed of 478 amino acids
that reflects a molecular weight of 52 kDa [Q07869], which coincides with the
molecular weight observed in sheep. The
52-kDa protein was also detected by this antibody in a HeLa human cell line obtained from (Santa Cruz Biotechnology, Inc., Calif, USA) (data not
included). The protein expression of the
PPARα nuclear receptor was found to be significantly lower in the 30-day old
samples (C) when compared to the Fetal (F) and the day old samples (N) (Figure 3).


## 3.7. Retinoid X Receptors (RXRα)

In sheep heart, the RXRα
antibody detects the 51-kDa nuclear receptor at each stage of development
(Figure 3) corresponding to the full length RXRα is
462 amino acids, which represents a molecular weight of 51 kDa [P19793]. We noted a small but significant surge in RXRα expression in the one-day old sheep
heart (Figure 2(b)).


## 3.8. Thyroid Hormone Receptor (TRα
1)

In
sheep heart, the TRα1 antibody detects the 47-kDa nuclear
receptor among the three study groups (Figure 3). In humans and rodents, the
full length of TRα is 410 amino acids, which corresponds
to a molecular weight of 47 kDa. In this
study, the THRα1 nuclear receptor has a significantly
greater level of protein expression in the day old samples (N) compared with
either the fetal (F) or the 30-day old samples (C) (Figure 3).


## 3.9. Other Nuclear Receptor Isoforms and Metabolic Proteins of Interest

Antibodies
against other protein expression were used.
These include PPARβ, PPARγ, RXRβ, TRα2, TRβ, MYLCD, and PDK4. Unfortunately, a cross reactivity exists
between the secondary antibodies (i.e., those with goat, mouse, and rabbit hosts
for the primary antibodies) and the one-day and 30-day old stages of
development in our sheep samples. Consequently, we could not get reliable data
for these proteins of interest.


## 4. Discussion

Several investigations have established the time course for maturation of cardiac
energy metabolism in the sheep model in vivo. For instance, Bartelds et al. [1] showed that glucose and lactate were the prime
energy substrates during ovine fetal life, and the switch to fatty acids as
prime oxidative substrate occurred within 2–16 days after birth [1, 25]. Our laboratory has shown that regulation of
myocardial oxidative phosphorylation matures within the same age period in
parallel with accumulation of the adenine nucleotide translocator protein [26]. Considered in summation,
these studies indicate that postnatal transitions in oxidative phosphorylation
and substrate oxidation occur coordinately and their regulation is integrated
through a unifying signaling mechanism.
The nuclear receptors, operating in conjunction with their coactivators,
offer a potential mechanism for rapidly integrating these processes shortly
after birth.Fatty acid and
carbohydrate metabolism generally exhibit reciprocal type regulatory patterns,
where one decreases as the other increases [27]. Prior work in the newborn
sheep heart has focused on carnitine palmitoyl transferase I as the pivotal
enzyme determining the preferential shift for fatty acid oxidation over
carbohydrate utilization. Bartelds et al. [1] detected postnatal
increases in CPTI activity and protein content, which were much lower than the
increase in the rate of LC-FA oxidation in vivo in the same animals.
Furthermore, they found relatively high rates of CPTI activity in fetal lambs [9]. These data led to their contention that
substrate supply was the major determinant for the increase
preference for LC-FA oxidation around birth.
Similarly, our data show steady decline in L-CPTI immediately after
birth with maintained M-CPTI protein levels, and imply that CPTI activity or
content does not regulate the postnatal metabolic transition in sheep. Studies
in rabbit heart have also shown that the postnatal increases in fatty acid flux
do not relate to CPTI-content, isoform pattern or activity. The increase in fatty acid oxidation in the
rabbit heart relates directly to a reduction in levels of malonyl-CoA
decarboxylase (MYLCD), an inhibitor of CPTI.
PDH activity also increases in postnatal rabbit myocardium despite
decreases in glucose oxidation.In our sheep
studies, we show for the first time changes in ovine expression for myocardial hexokinase
2 during development. This enzyme
catalyzes glucose phosphorylation, a rate limiting step in glucose oxidation
and positioned upstream from pyruvate dehydrogenase. Although,
glucose transport across the sarcolemma is controlled in part by glucose
transporters (GLUT1 and GLUT4), which are expressed abundantly in the fetal sheep
heart [28], hexokinase-2 has emerged recently
as the rate limiting glucose oxidation step during periods of stress [21, 29]. We also found modest but late postnatal increases
for PDK2, which are consistent with inhibition of pyruvate dehydrogenase
flux. Thus, our data imply that inhibition
of the glucose oxidation pathway contributes to the postnatal metabolic switch
with reciprocal and indirect activation of fatty acid oxidation accompanying.The paradigm of PPARα activation serving as the primary signaling
mechanism for the postnatal myocardial metabolic switch has been propagated
solely by studies demonstrating increases in PPARα and PGC-1 mRNA levels in mice or rats during
development [4, 30]. Supportive developmental expression studies
for the corresponding proteins or their activity are lacking in the literature
for these species. Furthermore, no
published data, that we are aware of, is available regarding the occurrence and
timing of the postnatal metabolic switching in either of these species. Extensive literature search reveals no studies
regarding myocardial substrate oxidation patterns near or after birth in these
species. Rather, mRNA data from rodents has been
extrapolated to describe signaling for postnatal phenomenon characterized in
larger species such as sheep, rabbit [31], and pigs [32–34]. This interpretative approach is of dubious
value, as we have previously shown that mRNA levels for these nuclear receptors
and coactivators demonstrate no coordination with protein content in the
postnatal period [35]. Results from previous work
in sheep imply that steady-state mRNA levels from PPARα and PGC-1 are subject to auto feedback from respective
proteins. The dramatic changes in content for the proteins controlling cardiac substrate
and oxidative metabolism, such as hexokinase-2, and cytochrome c oxidase, are
not preceded by elevation in PPARα protein
abundance. Hence, our study indicates
that PPARα protein content
plays no role in initiating the immediate postnatal upregulation of these
metabolic proteins.Though the data challenge
the concept that postnatal increases in PPARα steady-state mRNA coordinates the metabolic
shift, they do not eliminate a role for enhanced PPARα-mediated transactivation of target genes. In the current model, PPARα activity might be increased through
simultaneous coactivation by PGC-1α,
heterodimer formation through increased RXR availability, and enhanced ligand
availability generated by newborn suckling and fatty acid intake. However, we have little downstream evidence
for increased PPARα
transcriptional activity. On the
contrary, known PPARα targets,
such as LCPTI, MCPTI, and PDK2 are not overtly elevated in terms of protein
content during the immediate period after birth. Finally, hexokinase-2, rapidly upregulated by
the PPARα antagonist
WY14643 in mice [36], shows a marked depression in
this model.PGC-1 remains an
attractive candidate as a primary regulator for the postnatal myocardial
transition, as this factor also appears to coordinate cross-talk between
mitochondrial and nuclear genomes during development [24]. We have previously shown that gene and
protein expression for nuclear-encoded adenine nucleotide translocator
increases in sheep heart by 28–30 days after birth [26]. In the current study, we demonstrate that
the mitochondrial encoded respiratory chain component, MTCO3 increases near
four-fold in conjunction with the robust change in PGC-1. As PGC-1 and MTCO3 change coordinately, the
data imply that PGC-1 coordinates substrate switching along with mitochondrial
membrane expansion by affecting the mitochondrial genome. PGC-1 also closely links to total functional
cytochrome c oxidase (Cyt aa
3) and cytochrome c (Cyt c) content and respiratory capacity during postnatal ovine heart
development [35]. However, we found no concomitant change in
the nuclear-encoded cytochrome c oxidase, subunit 4. This finding suggests that content for
nuclear-encoded components are adequate in the fetus, but that rapid postnatal
mitochondrial biogenesis depends on PGC-1 promotion of mitochondrial encoded
components.This study was designed to
sample three points during sheep development, which would determine the
temporal relationship between protein expression and the well-documented late
fetal and immediate postnatal surge in circulating thyroid hormone [37]. Since, multiple changes in
hormone concentrations and environment occur during this time period, we cannot
prove without a doubt that this change in thyroid hormone homeostasis
exclusively causes increases in PGC-1 and nuclear receptors. However, thyroidectomy immediately after birth
abrogates the postnatal thyroid hormone surge, reduces PGC-1α levels [35], and attenuates expansion of
mitochondrial membrane protein content and respiratory capacity [38]. This observation supports the contention that
thyroid hormone plays an important role in signaling the postnatal metabolic
transition in heart. Our data from prior
work and the current study suggest that T3 simultaneously elevates
PGC-1α, and binds as a ligand to the TRs,
which also exhibit increased expression, during this critical developmental
period. These coordinated events would
lead to increased transactivation of metabolic target genes by TRs and PPARs
when binding as heterodimers with RXRs.In
summary, our data challenge specific concepts regarding the importance of PPARα protein expression in control of the postnatal
metabolic switch. First, we showed that
alterations in hexokinase-2, a rate limiting step in glucose oxidation,
accompany the transition. These data suggest that inhibition of glucose
oxidation reciprocally stimulates fatty acid flux, as opposed to a direct
increase in enzyme expression and /or activity of CPTI. Secondly, we demonstrate that no increase in PPARα protein occurs prior to the postnatal
metabolic switch in sheep. Thus,
transcriptional mediation of this protein does not trigger the change in
substrate preference. However, a robust
postnatal increase in PGC-1 and the PPARα binding partner, RXRα, does provide conditions for enhanced PPARα activity.



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*Source: 279531-2008-01-17.xml*