1 The John B. Pierce Laboratory, 2 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06519
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ABSTRACT |
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Fasting elicits a progressive increase in lipid metabolism within skeletal muscle. To determine the effects of fasting on the transcriptional regulation of genes important for metabolic control in skeletal muscle composed of different fiber types, nuclei from control and fasted (24 and 72 h) rats were subjected to nuclear run-on analysis using an RT-PCR-based technique. Fasting increased (P < 0.05) transcription rate of the muscle-specific uncoupling protein-3 gene (UCP3) 14.3- to 21.1-fold in white gastrocnemius (WG; fast-twitch glycolytic) and 5.5- to 7.5-fold in red gastrocnemius (RG; fast-twitch oxidative) and plantaris (PL; mixed) muscles. No change occurred in soleus (slow-twitch oxidative) muscle. Fasting also increased transcription rate of the lipoprotein lipase (LPL), muscle carnitine palmitoyltransferase I (CPT I), and long-chain acyl-CoA dehydrogenase (LCAD) genes 1.7- to 3.7-fold in WG, RG, and PL muscles. Transcription rate responses were similar after 24 and 72 h of fasting. Surprisingly, increasing metabolic demand during the initial 8 h of starvation (two 2-h bouts of treadmill running) attenuated the 24-h fasting-induced transcriptional activation of UCP3, LPL, CPT I, and LCAD in RG and PL muscles, suggesting the presence of opposing regulatory mechanisms. These data demonstrate that fasting elicits a fiber type-specific coordinate increase in the transcription rate of several genes involved in and/or required for lipid metabolism and indicate that exercise may attenuate the fasting-induced transcriptional activation of specific metabolic genes.
starvation; metabolism; medium-chain acyl-coenzyme A dehydrogenase; hexokinase II; exercise
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INTRODUCTION |
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SKELETAL MUSCLE, BY VIRTUE of its mass and total energy requirement, is the primary tissue responsible for the clearance of dietary glucose and lipids from the circulation and thus plays a key role in maintaining overall metabolic homeostasis (33, 42). The increasing recognition that subtle changes in energy balance, when considered over prolonged periods of time, represent a significant risk factor for the development of such metabolic abnormalities as insulin resistance, hypertriglyceridemia, obesity, and non-insulin-dependent diabetes mellitus has heightened the search for specific cell signaling and regulatory proteins that may influence metabolic control in skeletal muscle (32).
Most of the advances in our understanding of how acute challenges to intermediary metabolism may be sensed and responded to at the molecular level have arisen from work in liver, kidney, and adipose tissue. For example, transition from the fed to the fasted state dramatically activates transcription of a number of genes encoding enzymes with rate-limiting roles in hepatic gluconeogenesis, fatty acid oxidation, and ketogenesis. Aided by molecular studies in transgenic mice and various cell culture systems, detailed characterization of regulatory elements within the promoter regions of these genes has led to the identification of key signaling proteins and transcription factors that respond to various nutritional and/or hormonal manipulations (13, 19, 30).
Skeletal muscle also possesses a remarkable capacity to adapt to changes in metabolic demand, particularly in response to the challenges imposed by changes in contractile activity (45). Progress in deciphering the molecular mechanisms mediating the adaptive changes in gene expression in skeletal muscle have been hampered, however, by the fact that skeletal muscle cells grown in culture lack the important influences of the motor nerve (31), express only the embryonic forms of some proteins (6), and rely almost exclusively on glycolytic metabolism (23), factors that limit their ability to faithfully model critical features of adult myofibers. In the present study, we report a simplified procedure for isolating nuclei from small amounts of skeletal muscle tissue and an RT-PCR-based technique for performing nuclear run-on analysis, the combination of which permits the determination of gene-specific and fiber type-specific changes in transcription rate in rodent skeletal muscle. The purpose of the present study was to examine the extent to which the metabolic challenges imposed by 24-72 h of fasting influence the transcriptional regulation of genes important for metabolic control in skeletal muscle composed of different fiber types. In addition, we tested the hypothesis that overall metabolic demand/energy expenditure in skeletal muscle may directly influence transcriptional regulation by examining the effects of fasting in combination with low-intensity exercise performed during the initial portion of a 24-h fast. We focused particularly on uncoupling protein-3 (UCP3), a recently identified member of the uncoupling protein family that is expressed exclusively in skeletal muscle (10, 25, 43) and that is acutely increased at the mRNA level by fasting (2, 35, 40). The fiber type-specific transcriptional responses of several genes with critical roles in fatty acid oxidation, namely lipoprotein lipase (LPL), muscle carnitine palmitoyltransferase I (CPT I), long-chain acyl-CoA dehydrogenase (LCAD), and medium-chain acyl-CoA dehydrogenase (MCAD), were also determined.
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MATERIALS AND METHODS |
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Materials. Male Sprague-Dawley rats were bred in-house or were purchased from Charles River Laboratory (Wilmington, MA). All rats were housed individually in a temperature (22°C)- and light-controlled room (dark 9:00 AM-9:00 PM) and were given free access to food (Purina Rodent Diet) and water. Radiolabeled compounds were purchased from Amersham Pharmacia Biotech. All other chemicals were of molecular biology grade and were purchased from Boehringer Mannheim, GIBCO-BRL, Promega, or Sigma Chemical.
Experimental design. Rats weighed 340-360 g at the time of each experiment. Food was removed from experimental rats at the beginning of the dark cycle (9:00 AM) and was withheld for 24 or 72 h while maintaining free access to water. Control rats continued to have free access to food and water. In a second set of experiments, metabolic demand was increased during fasting (24 h) by having rats complete two 2-h bouts of treadmill exercise (18 m/min, 5° incline) beginning 1 and 6 h after removal of food (i.e., 10:00 AM and 3:00 PM). Rats were killed at the 24-h mark (~16 h after the last exercise bout) and were compared with additional control and 24-h-fasted rats. At the completion of the experiments, rats were anesthetized (35 mg/kg ip pentobarbital sodium) and placed on a heating pad to maintain body temperature during surgery.
Nuclei isolation.
The procedure for isolating nuclei from small amounts of skeletal
muscle tissue was developed from our previous work (27) and from
techniques described by Hahn and Covault (11). To examine the effects
of fasting on skeletal muscles with different metabolic characteristics, nuclei were isolated from soleus (slow-twitch oxidative), plantaris (mixed fast-twitch oxidative/glycolytic), and
portions of red (fast-twitch oxidative) and white (fast-twitch glycolytic) gastrocnemius muscle. Skeletal muscles from one hindlimb were quickly removed and dissected free of connective tissue. Muscle
weights immediately after dissection ranged from ~160 mg (soleus) to
~400 mg (red and white gastrocnemius). Harvested muscle was
immediately placed in 35 ml of ice-cold 15 mM HEPES, pH 7.5, 60 mM KCl,
3 mg/ml BSA, 300 mM sucrose, 5 mM each of EDTA and EGTA, 1 mM
dithiothreitol (DTT), 0.5 mM spermidine, 0.15 mM spermine, 0.5 mM
phenylmethylsulfonyl fluoride (PMSF), and 2 µg/ml leupeptin (buffer A) and was allowed to cool for ~2 min. The tissue was then finely minced, returned to buffer A, rotated for 5 min at 4°C, and homogenized for 20 s using a Brinkman Polytron (setting 4.5). After incubation on ice for an additional 5 min, homogenates were
centrifuged at 700 g for 10 min at 4°C. The crude nuclear pellets were gently resuspended in 10 ml of ice-cold 15 mM HEPES, pH
7.5, 60 mM KCl, 3 mg/ml BSA, 300 mM sucrose, 0.1 mM each of EDTA and
EGTA, 0.5% Triton X-100, 1 mM DTT, 0.5 mM spermidine, 0.15 mM
spermine, 0.5 mM PMSF, and 2 µg/ml leupeptin and were passed through
prewetted cheesecloth to remove connective tissue. Nuclei were pelleted
at 700 g for 10 min at 4°C, gently resuspended in 10 ml of
ice-cold 15 mM HEPES, pH 7.5, 60 mM KCl, 3 mg/ml BSA, 300 mM sucrose,
0.1 mM each of EDTA and EGTA, 5 mM MgAc, 1 mM DTT,
0.5 mM spermidine, 0.15 mM spermine, 0.5 mM PMSF, and 2 µg/ml
leupeptin, and repelleted. Final nuclei pellets were gently resuspended
in 230 µl of storage buffer (40% glycerol, 75 mM HEPES, pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgAc
, 0.1 mM each of EDTA
and EGTA, 1 mM DTT, 0.5 mM spermidine, 0.15 mM spermine, and 2 µg/ml
each of aprotinin and leupeptin), quick-frozen in liquid nitrogen, and
stored at
70°C.
Determination of transcription rate by RT-PCR.
Nuclear run-on reactions were performed by incubating 160 µl of
nuclei (thawed on ice) with 2× reaction buffer (20% glycerol, 100 mM KCl, 10 mM MgCl2, 4.5 mM DTT, 1.2 mM ATP, 0.6 mM
each of CTP, GTP, and UTP, 0.5 mM spermidine, 0.15 mM spermine, and 80 U/ml RNase inhibitor) for 15 min at 22°C. Nuclei were then
subjected to DNase I (20 units, RNase free) digestion in the presence
of 1 mM CaCl2 for 15 min at 37°C. This was followed by
digestion of nuclear proteins by addition of 10× 100 mM Tris, pH
8.0, 10 mM EDTA, 5% SDS, and 100 µg of proteinase K and incubation
for 30 min at 37°C. To extract the nascent RNA transcripts, 1 ml of TRIzol (GIBCO-BRL), 100 µg of yeast tRNA (to aid in visualization of
RNA pellet), and 200 µl of chloroform were added to each sample. Samples were mixed vigorously, incubated on ice for 5 min, and centrifuged at 12,000 g for 15 min at 4°C. Transcribed RNA
was precipitated from the aqueous phase by addition of an equal volume of ice-cold isopropanol followed by a 10-min incubation at
20°C and a 10-min centrifugation at 12,000 g and
4°C. The resulting RNA pellets were rinsed with cold 70% ethanol
(EtOH), transferred to 1.5-ml tubes, dried briefly, and resuspended in
100 µl of 10 mM Tris buffer (pH 8.0). To ensure complete removal of
genomic DNA, samples were subjected to a second digestion (37°C, 30 min) with DNase I (10 units) in the presence of 1 mM CaCl2.
RNA transcripts were reextracted by addition of 500 µl of TRIzol and
100 µl of chloroform and were centrifuged for 15 min at 12,000 g and 4°C. RNA was precipitated from the aqueous phase by
addition of an equal volume of isopropanol and incubation at
20°C for 30 min. After centrifugation (12,000 g, 10 min, 4°C), RNA pellets were rinsed two times in cold 70% EtOH,
dried briefly, and resuspended overnight at 4°C in 22 µl of 10 mM
Tris (pH 8.0) and 0.1 mM EDTA.
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Isolation and quantification of genomic DNA.
To correct for initial differences in nuclei content among samples,
genomic DNA was isolated from a portion of each sample of nuclei on the
same day as the nuclear run-on reaction. A 20-µl aliquot of nuclei
was placed in 380 µl of digestion buffer (10 mM Tris, pH 8.0, 100 mM
NaCl, 25 mM EDTA, 0.5% SDS, and 100 µg of proteinase K) and
incubated at 50°C for ~6 h. After adding an additional 380 µl
of nuclease-free H2O, DNA was isolated by extraction with
an equal volume of phenol-chloroform-isoamyl OH (25:24:1), separated by
centrifugation (12,000 g, 4°C), and precipitated from the
resulting aqueous phase by addition of vol 3 M
NaOAc
, 100 µg of tRNA (to aid in visualization of
DNA pellet), and 2.5 vol of 100% EtOH. DNA was pelleted (12,000 g, 10 min, 4°C), rinsed with 70% EtOH, and resuspended in
50 µl of 10 mM Tris and 1 mM EDTA (TE, pH 8.0) overnight at 4°C.
Relative quantification of genomic DNA (initial nuclei content) was
determined by PCR amplification of the
-actin gene. These data were
used to adjust final dilutions of the corresponding RT reaction
products from the nuclear run-on reactions (before PCR, see above) to
account for small differences in initial nuclei content across samples.
Determination of total transcriptional activity.
During starvation, metabolic rate slows, particularly within skeletal
muscle, in an effort to conserve energy (24). In the present study, we
tested the possibility that fasting may also influence total
transcriptional activity in skeletal muscle by subjecting a portion of
each nuclei preparation to the nuclear run-on reaction in the presence
of [32P]UTP. Nuclei (40 µl) were incubated
with 40 µl of 2× reaction buffer (omitting cold UTP) containing
1.4 µM [32P]UTP (400 Ci/mmol) for 15 min at
room temperature, as described above. In preliminary tests,
supplementing the reaction with cold UTP did not increase total
32P incorporation, indicating that the concentration of
labeled UTP was not rate limiting under the existing reaction
conditions (data not shown). Upon completion of the reaction,
radiolabeled nascent RNA transcripts were extracted by addition of 200 µl TRIzol, 100 µg yeast tRNA, and 40 µl of chloroform, separated
by centrifugation (12,000 g, 4°C), and precipitated from
the resulting aqueous phase by addition of an equal volume of cold
isopropanol. After incubation (20°C for 30 min), the RNA was
pelleted by centrifugation (12,000 g, 4°C), rinsed two
times with cold 75% EtOH, and resuspended in 75 µl of RNase-free TE.
To separate radiolabled RNA transcripts from unincorporated
[32P]UTP, the entire 75 µl were loaded on a
freshly prepared spin column (PGC Scientifics) containing G-50 Sephadex
(swollen in 10 mM Tris, pH 8.0, 1 mM EDTA, and 100 mM NaCl) and were
centrifuged for 4 min at 1,100 g. Total radioactivity within
the resulting column effluent was measured in triplicate by liquid
scintillation spectrometry. Background activity due to flow through of
free [32P]UTP was determined by omission of
nuclei in the run-on reaction and was found to represent <1.5% of
sample effluent activity.
Statistical analysis.
Transcription rate data for all metabolic genes were expressed relative
to the transcription rate of the -actin gene. All data across
experimental treatments were expressed relative to data from control
rats with mean data set to 1.0. Statistical analyses were performed
using a one-way ANOVA with all pairwise multiple comparisons among
groups performed using the Student-Newman-Keuls method. The level of
significance was set at P < 0.05.
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RESULTS |
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Validation of RT-PCR-based nuclear run-on analysis.
Primers specific for the rat -actin gene were designed and selected
(Fig. 1A) to test whether RT-PCR
can be used to detect the formation of RNA transcripts produced by
nuclear run-on analysis of nuclei isolated from ~400 mg of rat
skeletal muscle. An additional intent was to determine whether
transcripts produced during the run-on reaction represent unprocessed
(intron containing) or processed (spliced) RNA. PCR using a forward
primer within exon 4 (FP exon 4) and a reverse primer within exon 5 (RP
exon 5) yielded the appropriate 364-bp product from rat genomic DNA
(containing intron 4; Fig. 1B, lane 2) and a 277-bp
product from total muscle RT-RNA (intron 4 spliced out; Fig.
1B, lane 3). When RNA was isolated from nuclei not
subjected to the nuclear run-on reaction (native RNA; Fig. 1B,
lane 4), only a very faint 277-bp product was detected (visible
with 0.3 s integration), indicating that only a small amount of RNA is
initially present in the nuclei preparations. In contrast, a major
277-bp product and a minor 374-bp product were detected from RNA
isolated from nuclei after the run-on reaction (Fig. 1B,
lanes 5 and 7, duplicate experiments), demonstrating that transcript formation is detected by RT-PCR and that the majority of RNA transcripts produced are immediately processed (major 277-bp product). As a negative control, a portion of the RNA isolated from
nuclei after the run-on reaction was used as a template for PCR without
being reverse transcribed; no PCR products were detected (Fig.
1B, lanes 6 and 8), demonstrating that the
minor 364-bp product present in lanes 5 and 7 was not
due to the presence of residual or contaminating genomic DNA. Varying
the run-on reaction time from 30 s to 30 min did not influence the
ratio of spliced to unspliced transcript (unpublished data). PCR using
the intron-specific reverse primer (RP intron 4, Fig. 1A)
generated the appropriate 209-bp product from genomic DNA (Fig.
1B, lane 9) and no product from total muscle RT-RNA
(lane 12). When RP intron 4 was used with nuclear run-on RT-RNA
as the template, only a faint 209-bp product (Fig. 1B,
lanes 10 and 11) was obtained, approximately the same
intensity as the 364-bp product obtained using RP exon 5 (Fig.
1B, lanes 5 and 7), providing further evidence
that the majority of RNA transcripts produced during the run-on
reaction are immediately processed. Our results are similar to a
previous report from Rolfe and Sewell (34), also using a PCR-based
nuclear run-on procedure, and collectively support recent biochemical and cytological evidence demonstrating that RNA synthesis is intimately linked and coordinated with processing through the presence of processing factors at the carboxy-terminal domain of RNA polymerase II,
which catalyzes the capping, splicing, and polyadenylation of nascent
transcripts during transcriptional elongation (28).
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Effect of fasting on total transcriptional activity.
To determine the potential influence of fasting on the overall
transcriptional activity in skeletal muscle, a portion of nuclei from
each sample was subjected to the nuclear run-on reaction in which cold
UTP was replaced with [32P]UTP. Total
radioactivity of the isolated nuclear run-on RNA, normalized to genomic
DNA, was taken as an index of total transcriptional activity. As shown
in Fig. 2, fasting significantly reduced
total transcriptional activity by 20-53% in plantaris and white
gastrocnemius muscle. The decrease was evident within the first 24 h
and was not further depressed after 72 h. Although responses were
somewhat variable, fasting also tended (P = 0.051) to decrease
total transcriptional activity in red gastrocnemius muscle. Total
transcriptional activity in soleus muscle was not affected by fasting.
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Effect of fasting on the transcription rate of specific metabolic
genes.
Fasting elicited clear and marked changes in the transcription rate of
several metabolic genes (relative to -actin) in the plantaris, red
gastrocnemius, and white gastrocnemius muscles while having little
effect on transcriptional regulation within the soleus muscle (Fig.
3). As summarized in Fig.
4, fasting elicited a 5.5- to 7.5-fold
increase in the transcription rate of the UCP3 gene in both the
plantaris and the red gastrocnemius muscles. In white portions of the
gastrocnemius muscle, UCP3 transcription rate was increased by
14.3-fold after 24 h and by 21.1-fold after 72 h of fasting. In
contrast, the UCP3 transcription rate in the soleus muscle was not
significantly elevated after 24 or 72 h of fasting.
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Effect of increased metabolic demand on transcriptional regulation
during fasting.
To determine the potential interaction between substrate supply and
metabolic demand on the transcriptional regulation of metabolic genes,
a second study was performed, in which metabolic demand was increased
during fasting (24 h) by having the rats complete two 2-h bouts of
moderate-intensity exercise (18 m/min, 5° incline) performed 1 and
6 h after removal of food. Surprisingly, increasing metabolic demand
during fasting attenuated the increases in transcription rate evident
with fasting alone (Figs. 5 and 6), a response that appeared to be most
apparent in muscles composed predominantly of oxidative fiber types.
For example, UCP3 transcription rate in 24-h-fasted-plus-exercised rats
was significantly lower than 24-h-fasted rats and was not significantly
different from control rats in the red gastrocnemius muscle. Similarly,
increasing metabolic demand also attenuated the fasting-induced
increase in LPL, CPT I, and LCAD transcription rate in the red
gastrocnemius muscle as well as CPT I and LCAD induction in the
plantaris muscle. With the exception of LPL, increasing metabolic
demand did not affect the fasting-induced transcriptional regulation of
any of the genes examined in white gastrocnemius muscle. Fasting plus exercise also did not significantly influence the transcription rate of
the -actin gene relative to fasting alone.
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DISCUSSION |
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The primary purpose of the present study was to test the hypothesis that a severe metabolic challenge to skeletal muscle, such as fasting, triggers a coordinate adaptive response in the transcriptional regulation of genes with critical roles in substrate metabolism. The results of the present study demonstrate that fasting induces a coordinate increase in the transcription rate of a number of metabolically related genes, specifically in fast-twitch skeletal muscle. Chief among the responses was a dramatic increase in transcription rate of the UCP3 gene in plantaris, red gastrocnemius, and white gastrocnemius muscles within 24 h after removal of food. Transcriptional activation of several genes required for lipid metabolism also occurred in response to fasting, suggesting a common regulatory mechanism. Surprisingly, however, the added metabolic demand imposed by exercise performed during the initial hours after food removal actually attenuated the transcriptional activation found with 24 h of fasting alone, suggesting the presence of opposing regulatory mechanisms.
The discovery of an uncoupling protein whose expression is restricted to skeletal muscle, a major site of energy expenditure, has led to speculation that UCP3 may play a role in regulating nonshivering thermogenesis and whole body energy expenditure (10, 25, 43). Fueling this hypothesis is the fact that the human UCP2 and UCP3 genes map to within 100 kb of one another on chromosome 11(q13), a region that is coincident with several independently mapped quantitative trait loci for obesity and resting metabolic rate (3, 8, 10, 41). Although UCP3 displays uncoupling activity and increases thermogenesis when overexpressed in yeast (8-10, 15), its physiological role in skeletal muscle has not been firmly established. For example, a number of investigators have found that both food restriction and fasting induce a marked increase in skeletal muscle UCP3 mRNA (2, 35, 40). Our data extend these findings, demonstrating that fasting increases the transcription rate of the UCP3 gene by 5- to 20-fold in both red and white fast-twitch skeletal muscle. Collectively, these findings appear to be counterintuitive, given that skeletal muscle is a recognized site of energy conservation during starvation (24). One possible explanation is that changes in UCP3 expression do not correlate with uncoupling activity because other factors, such as posttranslational modifications, may be required for activation. For example, UCP1 activity in brown adipose tissue is inhibited by purine nucleotide binding and is activated by fatty acids (17). Similar allosteric regulation has not as yet been ascribed to UCP3, although posttranslational control of UCP3 has recently been implicated in the control of resting metabolic rate in thyroid-treated mice (16). Another possibility is that UCP3 may have other functions apart from its putative role in regulating thermogenesis. Samec et al. (36) have postulated that UCP3 may be involved in regulating the use of lipids as fuel substrates in skeletal muscle. This hypothesis is based on the fact that changes in UCP3 mRNA expression in response to various thermoregulatory and/or nutritional transitions (i.e., cold exposure, fasting, food restriction, refeeding, weaning) do not coincide with corresponding changes in thermogenesis but are more closely linked with the demand for lipid metabolism in skeletal muscle (1, 5, 36, 37, 44). Thus, although UCP3 expression changes dramatically in response to different metabolic challenges, its role in skeletal muscle with respect to mitochondrial function and fatty acid metabolism has yet to be determined.
Fasting coordinately activated transcription of three genes with
critical roles in mediating fatty acid metabolism in skeletal muscle
(LPL, CPT I, and LCAD). LPL catalyzes the hydrolysis of triglycerides
in circulating chylomicrons and very low density lipoproteins,
representing the rate-limiting step in the utilization of
triglyceride-derived fatty acids. Although regulation of LPL activity
in adipose tissue has been shown to be primarily at the translational
and posttranslational level, regulation within skeletal muscle is
believed to involve both translational and pretranslational control
(7). Ladu et al. (21) found in rats that 1 day of fasting significantly
increased LPL mRNA levels in soleus and red and white portions of the
vastus lateralis muscle. LPL activity did not increase after 1 day of
fasting but was significantly increased after 6 days (21). Data from
the present study provide the first direct evidence that the increase
in skeletal muscle LPL expression in response to fasting is mediated,
at least in part, at the level of transcription of the LPL gene.
Fasting also elicited increases in transcription of the genes encoding
for CPT I and LCAD, two enzymes that are responsible for catalyzing the
first steps in the transport of fatty acids across the mitochondrial membrane and subsequent -oxidation within the mitochondrial matrix, respectively (20, 26). Although no other data are available from
skeletal muscle, fasting has been reported to increase CPT I mRNA
levels and enzyme activity in liver tissue of rats (26). Interestingly,
with the exception of white gastrocnemius muscle, fasting did not
significantly influence transcription of the MCAD gene, suggesting the
presence of control mechanisms distinct from that of LPL, CPT I, and LCAD.
The soleus muscle in rats is composed primarily of slow-twitch fibers that rely heavily on oxidative metabolism. LPL mRNA has been shown to increase by ~50% in soleus muscle after 1 day of fasting (21). In contrast, LPL transcription rate in the present study did not change in soleus muscle, suggesting that changes in LPL expression induced by fasting in muscle composed primarily of slow-twitch oxidative fibers may involve posttranscriptional control mechanisms.
In tissues such as liver and kidney, shifts in nutritional and/or hormonal status often evoke on the order of 5- to 20-fold changes in the expression of genes required for fatty acid oxidation and gluconeogenesis (13, 19, 30). In the present study, fasting elicited no more than a two- to fourfold increase in transcription of the LPL, CPT I, LCAD, and MCAD genes, prompting us to consider whether accelerating the demand for fatty acid oxidation in skeletal muscle by combining exercise with fasting would augment the transcriptional induction of these genes relative to fasting alone. To test for this possibility, rats performed two 2-h bouts of moderate-intensity treadmill exercise (separated by 3 h of rest) within the first 8 h of a 24-h fast. In contrast to our hypothesis, the added metabolic demand imposed by exercise did not elicit a persistent, enhanced, or additive effect but, rather, partially attenuated the fasting-induced (24-h) increase in transcription of CPT I and LCAD in plantaris and UCP3, LPL, CPT I, and LCAD in red gastrocnemius muscle (Fig. 6). Interestingly, with the exception of LPL, increasing metabolic demand did not significantly affect the fasting-induced transcriptional induction of UCP3, CPT I, or LCAD in white gastrocnemius muscle, possibly due to lack of recruitment of these muscle fibers during low-intensity exercise. It is important to emphasize that the effects of fasting plus exercise were evaluated 16 h after the last exercise bout to examine the more general effects of increased metabolic demand as opposed to the specific effects of exercise, i.e., the marked transient increases in transcription rates of metabolic genes that occurs during the initial 3- to 4-h recovery period after exercise (14, 27, 29). The surprising conclusion that exercise attenuates the transcriptional response to fasting, at least under the limited experimental conditions of the present study, raises the possibility that fasting and exercise may trigger opposing regulatory mechanisms that persist well beyond the cessation of exercise.
The signaling events mediating the transcriptional induction of
metabolic genes in skeletal muscle are unknown. One possibility is that
increased free fatty acid (FFA) levels associated with fasting or
exercise may activate signaling pathways targeted to genes encoding
enzymes involved in the lipid oxidation, possibly through activation of
the peroxisome proliferator-activated receptor (PPAR) family of
transcription factors (22). Brun et al. (4) have recently provided
evidence that induction of UCP3 expression in skeletal muscle of
newborn mice is linked to the initiation of lipid intake during
suckling and, specifically, to the activation of PPAR-. The suckling
period is also characterized by marked increases in the expression of
other lipid metabolism genes, including LPL, CPT I, LCAD, and MCAD (12,
18, 26, 39), all of which contain PPAR response elements within their
promoter regions (38). Whether similar regulatory mechanisms operate in
response to elevated FFA levels during fasting in adult animals, as
well as whether such effects may be overridden by exercise, has not
been determined. Moreover, the impact of factors such as substrate
supply, metabolic demand, and circulating hormonal milieu on the
transcriptional regulation of metabolic genes in skeletal muscle,
particularly during recovery from exercise, remains virtually undefined.
In summary, the results from the present study demonstrate that fasting induces a marked increase in transcription of the UCP3 gene and a coordinate increase in transcription of several genes required for lipid metabolism in fast-twitch red and white skeletal muscle, likely reflecting the increased reliance of muscle on fatty acid metabolism during starvation. Surprisingly, however, increasing the metabolic demand within skeletal muscle during the initial 8 h of a 24-h fast significantly attenuates the transcriptional activation of several metabolic genes associated with lipid metabolism in red skeletal muscle, raising the possibility that fasting and exercise may trigger opposing regulatory mechanisms.
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ACKNOWLEDGEMENTS |
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We thank Drs. Henritte Pilegaard and David Cameron-Smith for helpful discussions and review of the manuscript.
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FOOTNOTES |
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This work was supported by a grant from the Yale Diabetes Endocrinology Research Center and by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-45372.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. D. Neufer, The John B. Pierce Laboratory, Yale Univ. School of Medicine, 290 Congress Ave., New Haven, CT 06519 (E-mail: dneufer{at}jbpierce.org).
Received 13 September 1999; accepted in final form 14 January 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Boss, O,
Samec S,
Kuhne F,
Bijlenga P,
Assimacopoulos-Jeannet F,
Seydoux J,
Giacobino JP,
and
Muzzin P.
Uncoupling protein-3 expression in rodent skeletal muscle is modulated by food intake but not by changes in environmental temperature.
J Biol Chem
273:
5-8,
1998
2.
Boss, O,
Samec S,
Paoloni-Giacobino A,
Rossier C,
Dulloo A,
Seydoux J,
Muzzin P,
and
Giacobino JP.
Uncoupling protein-3: a new member of the mitochondrial carrier family with tissue-specific expression.
FEBS Lett
408:
39-42,
1997[ISI][Medline].
3.
Bouchard, C,
Perusse L,
Chagnon YC,
Warden C,
and
Ricquier D.
Linkage between markers in the vicinity of the uncoupling protein 2 gene and resting metabolic rate in humans.
Hum Mol Genet
6:
1887-1889,
1997
4.
Brun, S,
Carmona MC,
Mampel T,
Vinas O,
Giralt M,
Iglesias R,
and
Villarroya F.
Activators of peroxisome proliferator-activated receptor-alpha induce the expression of the uncoupling protein-3 gene in skeletal muscle: a potential mechanism for the lipid intake-dependent activation of uncoupling protein-3 gene expression at birth.
Diabetes
48:
1217-1222,
1999[Abstract].
5.
Brun, S,
Carmona MC,
Mampel T,
Vinas O,
Giralt M,
Iglesias R,
and
Villarroya F.
Uncoupling protein-3 gene expression in skeletal muscle during development is regulated by nutritional factors that alter circulating non-esterified fatty acids.
FEBS Lett
453:
205-209,
1999[ISI][Medline].
6.
Duclert, A,
and
Changeux JP.
Acetylcholine receptor gene expression at the developing neuromuscular junction.
Physiol Rev
75:
339-368,
1995
7.
Enerback, S,
and
Gimble JM.
Lipoprotein lipase gene expression: physiological regulators at the transcriptional and post-transcriptional level.
Biochim Biophys Acta
1169:
107-125,
1993[ISI][Medline].
8.
Fleury, C,
Neverova M,
Collins S,
Raimbault S,
Champigny O,
Levi-Meyrueis C,
Bouillaud F,
Seldin MF,
Surwit RS,
Ricquier D,
and
Warden CH.
Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia.
Nat Genet
15:
269-272,
1997[ISI][Medline].
9.
Gimeno, RE,
Dembski M,
Weng X,
Deng N,
Shyjan AW,
Gimeno CJ,
Iris F,
Ellis SJ,
Woolf EA,
and
Tartaglia LA.
Cloning and characterization of an uncoupling protein homolog: a potential molecular mediator of human thermogenesis.
Diabetes
46:
900-906,
1997[Abstract].
10.
Gong, DW,
He Y,
Karas M,
and
Reitman M.
Uncoupling protein-3 is a mediator of thermogenesis regulated by thyroid hormone, beta3-adrenergic agonists, and leptin.
J Biol Chem
272:
24129-24132,
1997
11.
Hahn, CG,
and
Covault J.
Isolation of transcriptionally active nuclei from striated muscle using Percoll density gradients.
Anal Biochem
190:
193-197,
1990[ISI][Medline].
12.
Hainline, BE,
Kahlenbeck DJ,
Grant J,
and
Strauss AW.
Tissue specific and developmental expression of rat long-and medium-chain acyl-CoA dehydrogenases.
Biochim Biophys Acta
1216:
460-468,
1993[ISI][Medline].
13.
Hanson, RW,
and
Reshef L.
Regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression.
Annu Rev Biochem
66:
581-611,
1997[ISI][Medline].
14.
Hildebrandt, AL,
and
Neufer PD.
Transcriptional regulation of metabolic genes during recovery from acute exercise (Abstract).
FASEB J
13:
A418,
1999[ISI].
15.
Hinz, W,
Faller B,
Gruninger S,
Gazzotti P,
and
Chiesi M.
Recombinant human uncoupling protein-3 increases thermogenesis in yeast cells.
FEBS Lett
448:
57-61,
1999[ISI][Medline].
16.
Jekabsons, MB,
Gregoire FM,
Schonfeld-Warden NA,
Warden CH,
and
Horwitz BA.
T3 stimulates resting metabolism and UCP-2 and UCP-3 mRNA but not nonphosphorylating mitochondrial respiration in mice.
Am J Physiol Endocrinol Metab
277:
E380-E389,
1999
17.
Jezek, P,
and
Garlid KD.
Mammalian mitochondrial uncoupling proteins.
Int J Biochem Cell Biol
30:
1163-1168,
1998[ISI][Medline].
18.
Kelly, DP,
Gordon JI,
Alpers R,
and
Strauss AW.
The tissue-specific expression and developmental regulation of two nuclear genes encoding rat mitochondrial proteins. Medium chain acyl- CoA dehydrogenase and mitochondrial malate dehydrogenase.
J Biol Chem
264:
18921-18925,
1989
19.
Kersten, S,
Seydoux J,
Peters JM,
Gonzalez FJ,
Desvergne B,
and
Wahli W.
Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting.
J Clin Invest
103:
1489-1498,
1999
20.
Kurtz, DM,
Rinaldo P,
Rhead WJ,
Tian L,
Millington DS,
Vockley J,
Hamm DA,
Brix AE,
Lindsey JR,
Pinkert CA,
O'Brien WE,
and
Wood PA.
Targeted disruption of mouse long-chain acyl-CoA dehydrogenase gene reveals crucial roles for fatty acid oxidation.
Proc Natl Acad Sci USA
95:
15592-15597,
1998
21.
Ladu, MJ,
Kapsas H,
and
Palmer WK.
Regulation of lipoprotein lipase in adipose and muscle tissues during fasting.
Am J Physiol Regulatory Integrative Comp Physiol
260:
R953-R959,
1991
22.
Latruffe, N,
and
Vamecq J.
Peroxisome proliferators and peroxisome proliferator activated receptors (PPARs) as regulators of lipid metabolism.
Biochimie
79:
81-94,
1997[ISI][Medline].
23.
Li, K,
Neufer PD,
and
Williams RS.
Nuclear responses to depletion of mitochondrial DNA in human cells.
Am J Physiol Cell Physiol
269:
C1265-C1270,
1995
24.
Ma, SW,
and
Foster DO.
Starvation-induced changes in metabolic rate, blood flow, and regional energy expenditure in rats.
Can J Physiol Pharmacol
64:
1252-1258,
1986[ISI][Medline].
25.
Matsuda, J,
Hosoda K,
Itoh H,
Son C,
Doi K,
Tanaka T,
Fukunaga Y,
Inoue G,
Nishimura H,
Yoshimasa Y,
Yamori Y,
and
Nakao K.
Cloning of rat uncoupling protein-3 and uncoupling protein-2 cDNAs: their gene expression in rats fed high-fat diet.
FEBS Lett
418:
200-204,
1997[ISI][Medline].
26.
McGarry, JD,
and
Brown NF.
The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis.
Eur J Biochem
244:
1-14,
1997[Abstract].
27.
Neufer, PD,
and
Dohm GL.
Exercise induces a transient increase in transcription of the GLUT-4 gene in skeletal muscle.
Am J Physiol Cell Physiol
265:
C1597-C1603,
1993
28.
Neugebauer, KM,
and
Roth MB.
Distribution of pre-mRNA splicing factors at sites of RNA polymerase II transcription.
Genes Dev
11:
1148-1159,
1997[Abstract].
29.
O'Doherty, RM,
Bracy DP,
Granner DK,
and
Wasserman DH.
Transcription of the rat skeletal muscle hexokinase II gene is increased by acute exercise.
J Appl Physiol
81:
789-793,
1996
30.
Ouali, F,
Djouadi F,
Merlet-Benichou C,
and
Bastin J.
Dietary lipids regulate beta-oxidation enzyme gene expression in the developing rat kidney.
Am J Physiol Renal Physiol
275:
F777-F784,
1998
31.
Pette, D,
and
Staron RS.
Cellular and molecular diversities of mammalian skeletal muscle fibers.
Rev Physiol Biochem Pharmacol
116:
1-76,
1990[Medline].
32.
Reaven, GM.
Pathophysiology of insulin resistance in human disease.
Physiol Rev
75:
473-486,
1995
33.
Richter, EA.
Glucose utilization.
In: The Handbook of Physiology. Exercise: Regulation and Inntegration of Multiple Systems. Bethesda, MD: Am. Physiol. Soc, 1996, sect. 12, chapt. 20, p. 912-951.
34.
Rolfe, FG,
and
Sewell WA.
Analysis of human interleukin-5 gene transcription by a novel nuclear run on method based on the polymerase chain reaction.
J Immunol Methods
202:
143-151,
1997[ISI][Medline].
35.
Samec, S,
Seydoux J,
and
Dulloo AG.
Interorgan signaling between adipose tissue metabolism and skeletal muscle uncoupling protein homologs: is there a role for circulating free fatty acids?
Diabetes
47:
1693-1698,
1998[Abstract].
36.
Samec, S,
Seydoux J,
and
Dulloo AG.
Role of UCP homologues in skeletal muscles and brown adipose tissue: mediators of thermogenesis or regulators of lipids as fuel substrate?
FASEB J
12:
715-724,
1998
37.
Samec, S,
Seydoux J,
and
Dulloo AG.
Post-starvation gene expression of skeletal muscle uncoupling protein 2 and uncoupling protein 3 in response to dietary fat levels and fatty acid composition: a link with insulin resistance.
Diabetes
48:
436-441,
1999
38.
Schoonjans, K,
Staels B,
and
Auwerx J.
Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression.
J Lipid Res
37:
907-925,
1996[Abstract].
39.
Semenkovich, CF,
Chen SH,
Wims M,
Luo CC,
Li WH,
and
Chan L.
Lipoprotein lipase and hepatic lipase mRNA tissue specific expression, developmental regulation, and evolution.
J Lipid Res
30:
423-431,
1989[Abstract].
40.
Sivitz, WI,
Fink BD,
and
Donohoue PA.
Fasting and leptin modulate adipose and muscle uncoupling protein: divergent effects between messenger ribonucleic acid and protein expression.
Endocrinology
140:
1511-1519,
1999
41.
Solanes, G,
Vidal-Puig A,
Grujic D,
Flier JS,
and
Lowell BB.
The human uncoupling protein-3 gene. Genomic structure, chromosomal localization, and genetic basis for short and long form transcripts.
J Biol Chem
272:
25433-25436,
1997
42.
Van Der Vusse, GJ
and Reneman RS. Lipid metabolism in muscle.
In: The Handbook of Physiology. Exercise: Regulation and Inntegration of Multiple Systems. Bethesda, MD: Am. Physiol. Soc, 1996, sect. 12, chapt. 21, p. 952-994.
43.
Vidal-Puig, A,
Solanes G,
Grujic D,
Flier JS,
and
Lowell BB.
UCP3: an uncoupling protein homologue expressed preferentially and abundantly in skeletal muscle and brown adipose tissue.
Biochem Biophys Res Commun
235:
79-82,
1997[ISI][Medline].
44.
Weigle, DS,
Selfridge LE,
Schwartz MW,
Seeley RJ,
Cummings DE,
Havel PJ,
Kuijper JL,
and
BeltrandelRio H.
Elevated free fatty acids induce uncoupling protein 3 expression in muscle: a potential explanation for the effect of fasting.
Diabetes
47:
298-302,
1998[Abstract].
45.
Williams, RS,
and
Neufer PD.
Regulation of gene expression in skeletal muscle by contractile activity.
In: The Handbook of Physiology. Exercise: Regulation and Inntegration of Multiple Systems. Bethesda, MD: Am. Physiol. Soc, 1996, sect. 12, chapt. 25, p. 1124-1150.