From the Division of Cardiovascular and Respiratory
Medicine, Department of Internal Medicine, the § Division of
Molecular Medicine, International Center for Medical Research, Kobe
University Graduate School of Medicine, Kobe 6500017, Japan
Received for publication, May 9, 2002, and in revised form, November 21, 2002
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ABSTRACT |
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Energy metabolism is the most fundamental
capacity for mammals, impairment of which causes a variety of diseases
such as type 2 diabetes and insulin resistance. Here, we identified a
novel gene, termed diabetes-related ankyrin repeat protein (DARP) that is up-regulated in the heart of KKAy mouse, a type 2 diabetes and insulin resistance model animal. DARP contains putative
nuclear localization signals and four tandem ankyrin-like repeats. Its
expression is restricted in heart, skeletal muscle, and brown adipose.
Western blot analysis and immunocytochemistry of DARP-transfected
Chinese hamster ovary (CHO) and COS-7 cells reveal that DARP is a
nuclear protein. When DARP is expressed in CHO cells,
[1-14C]palmitate uptake is significantly decreased,
whereas the palmitate oxidation does not show significant change.
Furthermore, DARP expression is altered by the change of energy supply
induced by excess fatty acid treatment of skeletal myotube in
vitro and fasting treatment of C57 mouse in vivo. We
confirmed that DARP expression is also altered in Zucker fatty rat,
another insulin resistance model animal. Taken together, these data
suggest that DARP is a novel nuclear protein potentially involved in
the energy metabolism. Detailed analysis of DARP may provide new
insights in the energy metabolism.
Metabolic disorders cause wide variety of diseases including
hyperlipidemia, hyperuricaemia, diabetes, and insulin resistance. Among
these diseases, diabetes and insulin resistance are epidemic worldwide
and are expected to affect 300 million people by 2025 (1). Recently,
abnormalities of fatty-acid metabolism are recognized as key components
of the pathogenesis of type 2 diabetes and insulin resistance (2-5).
High fat diet and raised levels of circulating free fatty acids are
sufficient to induce insulin resistance that is related to the fat
content of skeletal muscle in rats (6). Accumulation of lipids inside
muscle cells and, specifically, an increase in muscle long chain fatty
acyl-CoA content are reported to cause insulin resistance. This
suggests that abnormal fatty acid metabolism and the accumulation of
lipid in skeletal muscle play crucial roles in the pathogenesis of
insulin resistance (7, 8). Moreover, the relation between insulin
resistance and muscle triglyceride content is independent of total
adiposity. Although the details of the mechanisms connecting lipid
accumulation and insulin resistance are still unclear, studies of
insulin receptor signaling reveal that the accumulation of lipid
products causes the phosphorylation of insulin receptor as well as
insulin receptor substrate (IRS)-1 through protein kinase C activation.
This results in the inhibition of insulin receptor signaling (4).
Recently identified molecules involved in the pathogenesis of insulin
resistance act at least partially through the alteration of fatty acid
metabolism. Adiponectin, whose secretion from white adipose tissue is
reduced in insulin-resistant animal models, induces tissue fatty acid
oxidation, leading to a reduction of tissue steatosis and reduced
plasma glucose, triglycerides, and free fatty acids concentrations (9).
The new class of insulin-sensitizing agents, thiazolidinediones,
affects a wide variety of metabolic genes in insulin-sensitive tissues
and has direct effects on mitochondrial fuel oxidation (10-12).
Here, we report a novel nuclear protein, termed diabetes-related
ankyrin repeat protein
(DARP),1 that is up-regulated
in the heart of KKAy mouse, a type 2 diabetes and insulin
resistance model animal. DARP-expressing CHO cells demonstrate
significantly decreased [1-14C]palmitate uptake.
Furthermore, DARP expression in skeletal muscle is altered by a change
of energy supply both in vitro and in vivo. Also,
DARP expression is altered in Zucker fatty rat, another insulin
resistance model animal. These results suggest that DARP is a novel
nuclear protein that is potentially involved in energy metabolism.
Cloning of DARP--
Total RNA was extracted from the heart of
10-week-old KKAy and C57BL/6J mice (Nihon Crea) using
ISOGEN (Wako Pure Chemical Industries). Suppression-subtractive
hybridization was performed using PCR-Select cDNA Subtraction Kit
(BD Biosciences Clontech) with 2 µg of
poly(A)+ RNA purified from total RNA using the FastTrack
2.0 Kit (Invitrogen) as recommended by the manufacturers. Subtracted
cDNAs were subcloned into pT7 vector (Novagen) and sequenced. The
5' end of DARP cDNA was cloned by 5'-rapid amplification of
cDNA ends (5'-RACE) (Invitrogen) against C57BL/6J mouse
heart. The primers for 5'-RACE were designed according to the sequences
obtained by the search of GenBankTM
(5'-TTCACCAGCTGTCTGTGGCCCTTCAGACA-3' for synthesis of first strand cDNA, 5'-CAGGTACTTGTCAATCAGGGCCTCCTGGT-3' for first PCR, and
5'-TTTAGTTTCTCCCGGGGCCACAGCCTCTT-3' for nested PCR). The full-length
cDNA was obtained by RT-PCR as described (13). Human DARP cDNA
was obtained by RT-PCR using human skeletal muscle total RNA (Sawady
Technology). The primers were designed according to the sequences
obtained by the search of GenBank (forward primer:
5'-GGACCATGGACTTCATCAGCATTCAGCA-3'; reverse:
5'-TCAGCACCGGGTGCGGGGATGCGCCACGT-3'). For nucleotide sequencing,
overlapping restriction fragments of the cDNA were subcloned into
pBluescript vector (Stratagene) and sequenced. Both strands of cDNA
were covered at least twice.
Northern Blot Analysis--
338-bp cDNA, corresponding to
the 3' end of mouse DARP coding region, labeled with
[ Cell Culture and Transfection--
Rat skeletal myoblasts were
isolated from rat soleus muscles as described (14). Confluent myoblasts
were differentiated into myotube by incubating in Dulbecco's modified
Eagle's medium containing 2% horse serum (differentiation
medium) for 4 days. Differentiated myotube were cultured for another
24 h with or without 250 µM oleic acid-albumin
(Sigma). CHO-K1 cells were cultured as previously described (15). Oligo
nucleotides that encode a FLAG tag (DYKDDDDK) were inserted at the end
of the DARP coding region by PCR-based mutagenesis (DARP-FLAG
cDNA). The primers used were
5'-GGACCATGGACTTCATCAGCATTGAGGAG-3' and
5'-TCATTTGTCGTCGTCGTCCTTGTAGTCGCACCGGGTACGGGGATGGCCCAC-3'. The
DARP-FLAG cDNA was subcloned into the pME18Sf expression vector (14). Stable transfection of CHO cells and isolation of the transfectant clones were performed as described (14). Briefly, DARP-FLAG expression construct was co-transfected with pSV2neo vector
into CHO cells by lipofection. 24 h after transfection, cells were
seeded sparsely and incubated in the medium containing 0.1% G418 for 1 week. Isolated colonies were picked up using a cloning cup, and we
confirmed the expression of DARP-FLAG mRNA and protein. Transient
transfection of DARP-FLAG into COS-7 cells was performed using
LipofectAMINE PLUS (Invitrogen) as recommended by the manufacturer.
Western Blot Analysis and Immunocytochemistry--
For Western
blot analysis, cells were lysed in the lysis buffer (1% zwittergent,
50 mM phosphate, and 150 mM NaCl), and the concentrations of which were measured by Bio-Rad Protein Assay (Bio-Rad) based on the method of Bradford using bovine serum albumin as
the standard. 30 µg of each protein were separated on 10%
SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride
membrane. After blocking with TBS containing 5% (v/v) skim milk for 30 min, the membrane was incubated for 1 h with 10 µg/ml anti-FLAG
M2 antibody (Sigma) followed by washing with TBS containing 0.1% Tween
20 three times for 5 min each. The membrane was then incubated with horseradish peroxidase-conjugated anti-mouse IgG antibody for 1 h.
After washing the membrane three times for 15 min each, signals were
detected using ECL system (Amersham Biosciences). Nuclear and
cytoplasmic fractions were extracted using NE-PER Nuclear and
Cytoplasmic Extraction Reagents (Pierce) as recommended by the
manufacturer. 30 µg of each fraction were subjected to Western blot
analysis. For immunocytochemistry, cells were fixed and permeabilized in methanol for 5 min at Assay of Metabolism of
[1-14C]Palmitate--
[1-14C]Palmitate-BSA
was prepared as described (16). Briefly, [1-14C]palmitic
acid dissolved in toluene (Amersham Biosciences) was incubated at
35 °C under nitrogen gas to evaporate toluene. After evaporation,
the palmitic acid was dissolved in water containing KOH by incubating
at 40 °C. Subsequently, palmitate solution was mixed well with
BSA-containing solution in the ratio of 700 mg of BSA and 7.7 mg of
palmitate. To examine the effect of DARP on
[1-14C]palmitate metabolism, approximately an equal
number of CHO cells with or without expression of DARP-FLAG were
incubated in serum-free Dulbecco's modified Eagle's medium/F-12
containing 40 µM [1-14C]palmitate for
6 h. For determination of the palmitate uptake, cells were washed
three times with PBS and harvested. After collection by brief
centrifugation, the cell pellet was dissolved in scintillation fluid
assayed for radioactivity. For determination of palmitate oxidation,
cells were cultured in the sealed flask containing a suspended filter
paper. The 14CO2 in the medium was liberated by
addition of 1 ml of 6 N hydrochloric acid. The
14CO2 collected overnight on the filter paper
alkalized with 2 N sodium hydroxide was quantified by
scintillation counting.
In Vivo Experiments--
Animal care and procedures were in
accordance with guidelines and regulations of the institutional animal
care committee. To examine DARP expression, 8-week-old Zucker fatty and
lean rats (Charles River, Japan) were used. For fasting, 8-week-old
C57BL/6J mice were divided into three groups. One group was maintained on chow, a second was fasted for 48 h, and a third was fasted for
48 h followed by unrestricted access to chow for 48 h.
Statistical Analysis--
All data are presented as mean ± S.E. as indicated. Statistical analyses of the characteristics of
Zucker rats and Northern blot analyses were performed with a
Mann-Whitney's U test. Differences between mean values
obtained for palmitate metabolism studies were determined by a
Student's t test. p < 0.05 was considered significant.
Identification of DARP--
In the heart, energy metabolism is
appreciably active and dynamic. Alteration of heart energy metabolism
is reported in diabetes and insulin resistance model animals (17, 18).
To isolate genes that are involved in energy metabolism, we have
performed suppression-subtractive hybridization using the heart of the
KKAy mouse, a model mouse of type 2 diabetes and insulin
resistance. Since the KKAy mouse shows obesity and insulin
resistance due to polygene impairment, there is no authentic normal
control mouse with same genetic background. Although the KK mouse is
used as a control for the KKAy mouse in several studies,
the KK mouse shows mild diabetic phenotype. Thus, KK mouse is not an
appropriate control mouse for our experiments, and we used the C57
mouse as a control.
We then successfully identified a novel gene, termed DARP, whose
expression is up-regulated in KKAy mouse heart as compared
with C57 mouse heart (Fig. 1). Nucleotide homology search of the GenBank and 5'-RACE using mouse heart total RNA
allowed us to isolate a full-length DARP cDNA. DARP encodes 306 amino acids containing putative nuclear localization signals and four
tandem ankyrin (ANK)-like repeats. The amino acid sequence of DARP
showed high similarity to cardiac ankyrin-repeat protein (CARP) and
ankyrin-repeat domain 2 (Ankrd2) with 45 and 36% identities, respectively (Fig. 2A). We
also isolated human DARP cDNA by RT-PCR using human skeletal muscle
total RNA (Fig. 2B). Searching the GenBank of human DARP
gene revealed that it is located at chromosome 2p11.1-2q11.1 where no
genetic diseases are reported.
Tissue Distribution of DARP mRNA--
Northern blot analysis
of adult mouse tissues revealed that DARP is expressed most abundantly
in skeletal muscle and also highly expressed in heart and brown adipose
tissue (Fig. 3). All of these tissues are
metabolically highly active. No signal was observed in other tissues,
including brain, lung, liver, kidney, intestine, and white adipose.
Expression of DARP in Eukaryotic Cells--
Since DARP contains
putative nuclear localization signals, we investigated whether DARP is
indeed a nuclear protein. We prepared CHO cells that stably express
DARP with FLAG tag attached at its C terminus (CHO/DARP-FLAG). Western
blot analysis of the cell lysate of CHO/DARP-FLAG demonstrated
appropriate DARP expression in the size of ~34.3 kDa, although
nonspecific signals were observed in both CHO/DARP-FLAG and
vector-transfected CHO cells (CHO/MOCK) (Fig.
4A). Immunocytochemistry of
CHO/MOCK demonstrated relatively strong immunoreactivities dispersed
diffusely, presumably due to nonspecific cross-reaction with anti-FLAG
antibody (data not shown). We then performed Western blot analysis of
nuclear and cytoplasmic fractions of CHO/DARP-FLAG as described.
Appreciable amount of DARP-FLAG expression was observed in the nuclear
fraction, although a significant amount of protein still remained in
the cytoplasmic fraction, presumably due to ongoing protein synthesis (Fig. 4B). To further confirm its nuclear localization, we
performed Western blot analysis of nuclear and cytoplasmic fractions of COS-7 cells in which DARP-FLAG was transiently transfected
(COS/DARP-FLAG). In COS/DARP-FLAG cells, we also detected the
appreciable amount of DARP-FLAG expression in the nuclear fraction
(Fig. 4B). Immunocytochemistry of COS/DARP-FLAG cells
demonstrated strong immunoreactivities in the nucleus, whereas no
significant immunoreactivities were observed in vector-transfected
COS-7 cells (COS/MOCK) (Fig. 4C). These findings indicate
that DARP is a nuclear protein.
Effects of DARP on Fatty Acid Metabolism--
Recently, evidence
that abnormalities of fatty acid metabolism in skeletal muscle play
crucial roles in the pathogenesis of insulin resistance is increasing.
Abundant expression of DARP in skeletal muscle and its altered
expression in the KKAy mouse led us to investigate its
function in fatty acid metabolism. We prepared three individual stable
transfectants of CHO cells that stably express DARP-FLAG (CHO/DARP-1, 2 and 3) and compared their [1-14C]palmitate metabolism to
that of parental CHO cells (CHO/control). The expression of DARP-FLAG
mRNA and protein were confirmed by Northern blot and Western blot
analysis (Fig. 5, A and
B). After 6 h of incubation in medium containing 40 µM [1-14C]palmitate, palmitate uptake,
measured as the amount of radioactivity in the cells,
slightly but significantly decreased in all three stable transfectants
that express DARP-FLAG compared with control (Fig. 5C). On
the other hand, palmitate oxidation in DARP-FLAG transfectants did not
show significant differences from that of control cells (Fig.
5D).
DARP Expression Is Regulated by Energy Supply--
We then
examined the effect of exogenous energy supply on DARP expression. In
skeletal myotube, addition of 250 µM oleate in
differentiation medium containing glucose significantly increased DARP
expression after 24 h of incubation, indicating that DARP expression is altered by exogenous energy supply in vitro
(Fig. 6A). There was no
significant morphological change after addition of oleate, and the
expression of acetylcholine receptor DARP Expression Is Altered in Insulin Resistance Model
Animals--
We cannot exclude the possibility that enhanced DARP
expression in KKAy mouse heart is due to the difference of
genetic background between KKAy and C57 mice. Therefore, we
examined DARP expression in another type 2 diabetes and insulin
resistance model animal, Zucker fatty rats. Zucker fatty rats showed
significantly higher body weight and plasma insulin level than those of
Zucker lean control rats, whereas the blood glucose of both groups was
not significantly different, indicating that they are appropriate model
animal for insulin resistance (Table I).
DARP expression in heart and skeletal muscle was significantly higher
in Zucker fatty rats than Zucker lean control rats (Fig.
7, A and B). In
contrast, DARP expression in brown adipose was significantly lower in
Zucker fatty rats as compared with that of control rats (Fig.
7C). These results indicate that DARP expression is indeed
altered in insulin resistance animals.
We have described the cloning and characterization of DARP, a
novel nuclear protein, whose mRNA expression is altered in type 2 diabetes and insulin resistance model animals. From the data presented
in this manuscript, we are unable to determine whether DARP has a clear
function in free fatty acid metabolism. However, its restricted
expression in heart, skeletal muscle, and brown adipose and relevance
to fatty acid metabolism suggest that analysis of DARP may reveal new
insights in the energy metabolism.
Amino acid sequencing of DARP revealed that it contains putative
nuclear localization signals and four tandem ANK-like repeats, sharing
high homology with CARP and Ankrd2. CARP was initially identified as a
cytokine-inducible nuclear protein from human endothelial cells (19).
Later, CARP was reported to be a downstream molecule in the Nkx2-5
homeobox gene pathway in cardiomyocyte (20) and to be a downstream
target of TGF- Immunocytochemistry of COS/DARP-FLAG cells and Western blot analysis of
nuclear and cytoplasmic fractions of COS/DARP-FLAG and CHO/DARP-FLAG
demonstrated that DARP is a nuclear protein. Its nuclear localization
suggests that DARP may play a role in the regulation of gene
expression. It was reported that subcellular localization of CARP is
altered by a change of circumstance of the cell, such as serum
depletion in vitro (20). Since the function of protein is
sometimes regulated by its subcellular localization (25, 26), detailed
analysis of DARP localization may provide important clues to clarify
the physiological function of DARP.
DARP expression in CHO cells caused a slight but significant decrease
of palmitate uptake, suggesting that DARP may be involved in fatty acid
metabolism. However, in CHO cells, it appeared that the effect of DARP
on fatty acid metabolism was not enhanced proportionately to its
expression level. Because DARP possesses ANK-like repeats that may
mediate protein-protein interaction, DARP may functionally require
partner molecule(s). Therefore, over-expression of DARP in CHO cells
may not be sufficient. However, our results strongly suggest that DARP
is potentially involved in fatty acid metabolism. Further experiments
are required to elucidate the detailed mechanism of DARP effect on
fatty acid metabolism.
Because skeletal muscle is the principal tissue for insulin-mediated
glucose disposal and a major site of peripheral insulin resistance, the
correlation between skeletal muscle fuel handling and insulin
resistance has been extensively investigated. Recent studies revealed
that skeletal muscle in insulin resistance shows increased glucose
oxidation and decreased fatty acid oxidation under basal conditions and
decreased glucose oxidation and increased fatty acid oxidation under
insulin-stimulated conditions. This is referred to as a state of
"metabolic inflexibility" (4). Decreased fatty acid oxidation under
basal conditions could lead to lipid accumulation within skeletal
muscle that is strongly associated with insulin resistance. Since
DARP-expressing CHO cells demonstrated a significant decrease of
palmitate uptake, DARP expression may be up-regulated in
insulin-resistant animals to partially compensate for abnormalities in
fatty acid accumulation in skeletal muscle. However, further analyses
are required to address this point.
DARP expression is altered by a change of energy supply and energy
metabolic condition, induced by excess fatty acid treatment in
vitro and fasting in vivo. Initially, we expected that
fasting would enhance the expression of DARP as well as excess fatty
acid treatment in vitro since fasting was shown to increase
plasma fatty acid level (27). Unexpectedly, fasting resulted in a
decrease in DARP expression. This could be due to a significantly
reduced glucose supply under fasted conditions. Although detailed
mechanisms are unknown, our observations suggest that DARP expression
is, at least partially, regulated by energy supply. Since energy supply appreciably affects energy metabolism (27, 28), these findings further
suggest that DARP is implicated in energy metabolism. To clarify the
physiological function of DARP, gene targeting and transgenic animal
studies will likely be required. Detailed analysis of DARP will provide
new insights of energy metabolism and crucial information as to the
molecular regulatory mechanisms of energy metabolism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP, was used for Northern analysis with
QuickHyb solution (Stratagene) as recommended by the manufacturer. The
washed blots were exposed to imaging plate analyzed by BAS
analyzer (Fuji film).
20 °C. After washing in PBS, PBS
containing 10% (v/v) normal goat serum (NGS/PBS) was added. Following
a 1-h incubation at 37 °C, the NGS/PBS was replaced with buffer
containing anti-FLAG M2 antibody (1:300). After incubation for 90 min
at 37 °C, the cells were washed with PBS three times for 10 min each and then incubated in NGS/PBS containing 1.7 µg/ml of RITC-goat anti-mouse IgG antibody (EY Laboratories) for 1 h at 37 °C.
Following incubation, cells were washed six times with PBS for 10 min
each. The coverslips were mounted on the slides with 90% (v/v)
glycerol, 50 mM Tris-HCl (pH 9.0), and 2.5% (v/v)
1,4-diazabicyclo-[2.2.2]octane and observed with a confocal
fluorescent microscopy.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
DARP expression in C57 and
KKAy mouse heart. 10 µg of total RNA
extracted from the hearts of C57 and KKAy mice were
separated by electrophoresis on 1% agarose/formaldehyde gel and
transferred onto a nylon membrane subjected to Northern blot analysis
of DARP. The same blot was stripped and re-probed for -actin
mRNA.
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Fig. 2.
Amino acid sequence of human and mouse DARP.
A, amino acid sequence of mouse DARP aligned with mouse CARP
and Ankrd2. Conserved residues are marked by closed boxes.
Underlined residues are the four tandem ankyrin-like
repeats. Putative nuclear localization signals are marked by
asterisks. B, amino acid sequence of human DARP
aligned with mouse DARP. Dots represent identical amino
acids to those in human DARP. The sequences are available under GenBank
accession number AF492400 and AF492401.
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Fig. 3.
Tissue distribution of mouse DARP. 15 µg of total RNA isolated from a wide variety of mouse tissues were
subjected to Northern blot analysis. 28 S rRNA is shown at the
bottom.
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Fig. 4.
Western blot analysis and
immunocytochemistry of CHO/DARP-FLAG and COS/DARP-FLAG
cells. A, CHO/DARP-FLAG cell lysate was subjected to Western
blot analysis using anti-FLAG M2 antibody. B, nuclear
(N) and cytoplasmic (C) fractions of
CHO/DARP-FLAG and COS/DARP-FLAG cells were prepared as described under
"Experimental Procedures," and 30 µg of each fraction were
subjected to Western blot analysis using anti-FLAG M2 antibody.
C, immunocytochemistry of COS/DARP-FLAG and COS/MOCK using
anti-FLAG M2 antibody.
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Fig. 5.
Effect of DARP expression on
[1-14C]palmitate metabolism. A,
total RNAs were extracted from each stable transfectant and subjected
to Northern blot analysis. All three transfectants express DARP-FLAG
mRNA, whereas no expression of DARP was detected in parental CHO
cell. B, cell lysate of each stable transfectant was
subjected to Western blot analysis using anti-FLAG M2 antibody. All
three transfectants express DARP-FLAG protein. C, palmitate
uptake measured as the amount of radioactivity in CHO/DARP and
CHO/control cells incubated with [1-14C]palmitate as
described under "Experimental Procedures." Counts per minute was
determined by scintillation counting and was normalized to the value of
CHO/control cells (= 100%) (n = 10 for control group,
and n = 5 for each CHO/DARP group). Mean ± S.E.
are shown; *, p < 0.0005 versus
CHO/control; and **, p < 0.005 versus
CHO/control. D, total 14CO2 produced
from [1-14C]palmitate in CHO/DARP and CHO/control cells
(n = 9 for control group and n = 5 for
each CHO/DARP group). Palmitate oxidation did not show any significant
difference between groups.
subunit (AchR
), a marker of
differentiated myotubes, did not change. These findings suggest that
addition of oleate in the medium does not affect the differentiation
state of skeletal myotube. To confirm the regulation of DARP expression
by energy supply, we further examined the effect of fasting on DARP
expression using the C57 mouse in vivo. After 48 h of
fasting, DARP expression in skeletal muscle was significantly reduced.
Interestingly, 48 h of re-feeding after 48 h of fasting
increased DARP expression in skeletal muscle to an even higher level
than that of control mice without fasting (Fig. 6B).
However, fasting showed no significant effects on DARP expression in
either the heart or brown adipose tissue (data not shown). These
observations indicate that DARP expression is, at least partially,
regulated by the energy supply in skeletal muscle both in
vitro and in vivo.
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Fig. 6.
DARP expression is regulated by energy
supply. A, 30 µg of total RNA isolated from skeletal
myotube incubated with or without 250 µM oleate for
24 h were subjected to Northern blot analysis of DARP and AchR .
The same blot was stripped and reprobed for
-actin mRNA.
B, 10 µg of total RNA extracted from skeletal muscle of
mice with unrestricted feeding, fasted, or fasted and then refed were
subjected to Northern blot analysis. Radioactivities of DARP mRNA
signals were normalized with actin signal (n = 5 for
control and fasted group, and n = 6 for fasted and then
refed group). Values (mean ± S.E.) are presented as a percent of
control (= 100%); *, p < 0.01 versus
control; **, p < 0.01 versus fasted group;
and ***, p < 0.05 versus control.
Characteristics of Zucker lean and fatty rats
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Fig. 7.
DARP expression is altered in insulin
resistance model animals. 20 µg of total RNA from the heart
(A) and brown adipose (C) and 5 µg of total RNA
from skeletal muscle (B) of Zucker fatty rats and Zucker
lean rats were subjected to Northern blot analysis of DARP. The same
blots were stripped and reprobed with -actin probe. Radioactivities
of DARP mRNA signals were normalized with actin signal. Values
(mean ± S.E.) are presented as a percent of lean control (=
100%) (n = 5 for each group); *, p < 0.05 versus lean rats; and **, p < 0.01 versus lean rats.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/Smad signaling in vascular smooth muscle cell (21).
However, its physiological function is still unclear. Ankrd2 was
identified from mouse skeletal muscle as a gene putatively responsible
for stretch-induced muscle hypertrophy (22, 23). Their
identical structural features are nuclear localization signals and the
ANK repeat motif. Although ANK repeats were initially reported to
mediate protein-protein interactions, their function is more diverse.
ANK repeat proteins carry out a wide variety of biological activities,
and this motif has been recognized in more than 400 proteins including
cyclin-dependent kinase inhibitors, transcriptional
regulators, cytoskeletal organizers, developmental regulators, and
toxins (24). Thus, the ANK repeat motif does not determine the specific
function of DARP, although it may play key roles in DARP function.
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ACKNOWLEDGEMENT |
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We thank Dr. Cheryl E. Gariepy for her critical reading of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF492400 and AF492401 (mouse DARP and human DARP, respectively).
¶ To whom correspondence should be addressed: Div. of Cardiovascular and Respiratory Medicine, Dept. of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki, Chuo, Kobe 6500017, Japan. Tel.: 81-78-382-5846; Fax: 81-78-382-5859; E-mail: emoto@med.kobe-u.ac.jp.
Published, JBC Papers in Press, November 26, 2002, DOI 10.1074/jbc.M204563200
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ABBREVIATIONS |
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The abbreviations used are: DARP, diabetes-related ankyrin repeat protein; CHO, Chinese hamster ovary; RACE, rapid amplification of cDNA ends; RT, reverse transcription; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; NGS, normal goat serum; BSA, bovine serum albumin; ANK, ankyrin; CARP, cardiac ankyrin-repeat protein.
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