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,2
* Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, Caserta, Italy;
Dipartimento di Scienze Biologiche ed Ambientali, Università degli Studi del Sannio, Benevento, Italy; and
Dipartimento delle Scienze Biologiche, Università degli Studi di Napoli "Federico II," Napoli, Italy
2Correspondence: A.L.: Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, Via Vivaldi 43, 81100, Caserta, Italy; E-mail: antonia.lanni{at}unina2.it, or F.G.: Dipartimento di Scienze Biologiche ed Ambientali, Università degli Studi del Sannio, Via Port’Arsa 11, 82100, Benevento, Italy; E-mail: goglia{at}unisannio.it
ABSTRACT
Coping with reduced energy sources entails drastic morphological and functional changes in skeletal muscle, but the sequence of events required classification. We found that gastrocnemius muscle from food-deprived rats shows acute rises in peroxisome proliferator activated receptor (PPAR) 
coactivator (PGC) -1
/PPAR 
nuclear protein and myosin heavy chain (MHC) Ib protein, while type I fibers accumulate and the muscle tissue appears redder. AMP levels, phosphorylation of both AMP-activated protein kinase (AMPK) and its downstream target acetyl coenzyme A carboxylase (ACC) are induced within 6 h. Rapidly increased MyoD mRNA levels are followed by an increase in uncoupling protein (UCP) 3 (UCP3) transcription. Increased serum fatty acid levels coincide with increases in mitochondrial UCP3 protein levels and fatty acid oxidation. Accompanying this is a decrease in AMPK phophorylation, reversible upon nicotinic acid treatment, indicating that fatty acids may modulate this kinase’s activity after the metabolic challenges posed by food deprivation.—de Lange, P., Farina, P., Moreno, M., Ragni, M., Lombardi, A., Silvestri, E., Burrone, L., Lanni, A., Goglia, F. Sequential changes in the signal transduction responses of skeletal muscle following food deprivation.
Key Words: mitochondria • fatty acid oxidation • UCP3 • fasting
THE METABOLIC RESPONSE to food deprivation involves hormonal and metabolic adaptations accompanied by transcriptional reprogramming events (1
, 2)
. Food deprivation, which represents a metabolic challenge to skeletal muscle, necessitates an increased reliance on lipid and protein metabolism as part of the body’s overall effort to minimize total carbohydrate utilization. Skeletal muscle is a major site in the regulation of whole-body fatty acid and glucose (Glc) metabolism. Muscle fibers undergo adaptive changes in gene expression when faced with sudden or sustained increases in metabolic demand (1
2
3
4)
. Alterations in mRNA/protein levels during fasting go hand in hand with activation of multiple metabolic enzymes and transcription factors, leading to a suppression of anabolic pathways and stimulation of Glc uptake and fatty acid oxidation, which together act to restore intracellular ATP levels (5
6
7
8
9)
. In conditions involving ATP depletion/ADP accumulation (such as fasting or exercise), AMP levels are elevated due to the activity of members of the adenylate kinase (AK) family, which catalyze the reaction 2ADP 
ATP + AMP and maintain it close to equilibrium at all times (9
, 10)
. The resulting elevated AMP level triggers the activity of the AMPK, which may be the central factor in the metabolic shift after food deprivation (9
10
11)
.
Fasting may be a crucial area in which to observe structural and biochemical changes. These events may be time dependent, species/organ dependent, and transient (i.e., appear, then disappear). An important example comes from the abovementioned AMPK. Fasts lasting 24 h are reported to induce AMPK activation in rat liver (12
, 13)
and heart (14)
, but not in rat soleus muscle (15)
or mouse gastrocnemius muscle (16)
. A second example is provided by the PPAR , playing a key role in attenuating the metabolic syndrome through control of mitochondrial fatty acid oxidation (17
18
19)
. After a 24 h fast, PPAR expression has been reported to increase in mouse muscle (19)
while remaining unchanged in rat muscle (20)
, and we and others have even reported reduced levels of PPAR in gastrocnemius muscle (7)
and heart (21)
from 48 h fasted rats. These seemingly discrepant findings may point toward organ-specific responses to food deprivation, including possible differences in the time course of gene expression/protein activation. This would apply especially to skeletal muscles, which differ in their response to metabolic challenges due to their differing fiber structures.
It is clear that although a systematic and coordinated pattern of in vivo changes in key factors may be involved in reprogramming the enzymatic/transcriptional events that follow food deprivation in metabolically adaptive, mixed-fiber skeletal muscles, the actual sequence of events remains unknown. This is important, since making single time point measurements could cause one to miss evidence of some important stimuli required for adaptive changes in gene expression and/or enzyme activity. The aim of the present study of food-deprived rats was therefore to shed light on the temporal sequence of biochemical and molecular events (and their interrelationships) leading to adaptive responses at the morphological/functional level. By obtaining time course data from the gastrocnemius muscle (at 0, 6, 12, 24, and 48 h after food deprivation), we aimed to establish expression levels of the MHCIb gene, which directs the formation of type I muscle fibers, together with those of two factors known to be involved in the formation of type I fibers, namely PGC-1 (22)
and PPAR (23)
. Next we examined cellular ATP/AMP levels, AMPK, and ACC phosphorylation and the mRNA/protein levels of several genes that are hypothesized or known to be involved in the metabolic response to fasting, including UCP 3 (24
, 25)
. In addition, to verify whether fatty acids might in some way influence the biochemical events related to food deprivation, we measured their circulating levels and examined their mitochondrial oxidation, and also determined the effect on AMPK phosphorylation induced by lowering their circulating levels (through in vivo nicotinic acid treatment).
MATERIALS AND METHODS
Animals
Male Wistar rats (250–300 g) (Charles River, Lecco, Italy) were kept one per cage in a temperature-controlled room at 28°C under a 12 h light, 12 h dark cycle. A commercial mash and water were available ad libitum. Animals were food deprived for 0, 6, 12, 24, or 48 h. All rats were housed under an inverted light/dark cycle to make it easier to deprive them of food nocturnally. In a subgroup of 48 h fasted rats, the antilipolytic agent nicotinic acid (Fluka Biochimica, Buchs, Switzerland) was administered at 100 mg/kg body weight intraperitoneally (i.p.) every 2 h during the last 8 h of the fasting period. At the end of the treatments, rats were anesthetized by an i.p. injection of chloral hydrate (40 mg/100 g body weight), then killed by decapitation. Gastrocnemius and soleus muscles were excised, photographed, weighed, and either immediately processed for preparation of mitochondria or immediately frozen in liquid nitrogen and stored at –80°C for later processing. All experiments were performed in accordance with general guidelines regarding animal experiments and were approved by our institutional committee for animal care.
Identification of type I fibers
Gastrocnemius muscle was dissected to provide tissue to be cut transversally on a cryostat. Tissue pieces were fixed in TissueTek (Varini Sr. L, Naples, Italy) and frozen at –20°C. Cryostat sections were dried-frozen until staining. Metachromatic staining of muscle fibers was performed according to the protocol of Ogilvie and Feeback (26)
.
Preparation of total lysates
For Western blot analysis, gastrocnemius muscle was homogenized in lysis buffer containing 20 mM Tris-HCl pH7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM Na2H2P2O7, 1 mM b-CH3H7O6PNa2, 1 mM Na3VO4, 1 mM PMSF, 1 mg/ml leupeptin, and 1% Triton X-100 (all from Sigma-Aldrich Corp., St. Louis, MO, USA) using an ultraturrax and centrifuged at 12,000 rpm for 10 min at 4°C (Beckman Optima TLX, Beckman Coulter S.p.A., Milan, Italy). To determine muscle fiber content, supernatants were used without further processing. For determination of other intracellular proteins, supernatants were ultracentrifuged at 40,000 rpm for 10 min at 4°C (Beckman Optima TLX, Beckman Coulter S.p.A.). The protein concentrations of the supernatants and of the ultracentrifuged cleared lysates were determined using Bio-Rad’s DC method (Bio-Rad Laboratories, Hercules, CA, USA).
Preparation of nuclei and mitochondria.
Gastrocnemius muscle mitochondria were isolated as described previously (25)
. Briefly, tissue pieces were homogenized in medium A (consisting of 220 mM mannitol, 70 mM sucrose, 20 mM Tris-HCl, 1 mM EDTA, 5 mM EGTA, and 5 mM MgCl2, pH 7.4, all from Sigma-Aldrich Corp.). The homogenate was centrifuged at 700 gav for 10 min, the supernatant containing the mitochondria was then centrifuged at 8000 gav. The resulting mitochondrial pellet was washed twice, resuspended in a minimal volume of medium A, and kept on ice. The freshly prepared mitochondria were used immediately to measure respiratory rate (fatty acid oxidation). Nuclei and mitochondria prepared for Western blot analysis were isolated in medium A supplemented with the following protease inhibitors: 1 mM benzamidine, 4 µg/ml aprotinin, 1 µg/ml pepstatin, 2 µg/ml leupeptin, 5 µg/ml betastatin, 50 µg/ml N-tosyl-L-phenylalanine-chloromethyl ketone, and 0.1 mM phenylmethylsulfonylfluoride (all from Sigma-Aldrich Corp.). The homogenate was centrifuged at 700 gav for 10 min, with the resulting pellet containing the nuclei being lysed and ultracentrifuged in lysis buffer at 40,000 rpm for 10 min at 4°C (Beckman Optima TLX), the final supernatant containing nuclear protein. The homogenate supernatant containing the mitochondria was centrifuged at 8000 gav. The resulting mitochondrial pellet was washed twice, resuspended in a minimal volume of antiprotease-containing medium A, and kept on ice. Protein concentrations of the nuclear and mitochondrial fractions (with or without antiprotease) were determined using Bio-Rad’s DC method (Bio-Rad Laboratories).
RNA isolation
Total RNA was isolated using TRIZOL standard protocol (Invitrogen Life Technologies, Milan, Italy). Tissue/TRIZOL mixtures were homogenized using a polytron, keeping the viscosity of the solution to a minimum to ensure effective inactivation of endogenous RNase activity.
RT-polymerase chain reaction (RT-PCR) assays
One microgram of total RNA was reverse transcribed using 100 pmol random hexamers (Invitrogen Life Technologies, Milan, Italy), 2.0 U Superscript reverse transcriptase, 0.5 U RNase inhibitor, and 1 mM deoxynucleotide triphosphates (dNTPs) in reverse transcriptase buffer (all from HT Biotechnology, Cambridge, UK). The total volume was adjusted to 20 µl with distilled H2O, and the reaction was carried out for 1 h at 40°C. One-quarter of the RT reaction mixture was used directly for the polymerase chain reaction (PCR) reaction in a total volume of 25 µl containing 0.25 U of SuperTaq polymerase, 0.25 mM dNTPs, SuperTaq PCR buffer (all from HT Biotechnology), 5% (v/v) dimethyl sulfoxide (DMSO, Sigma-Aldrich Corp.), and 0.38 pmol of the relevant oligonucleotide primers (Sigma Genosys, Cambridge, UK). Gene expression signals were normalized with respect to the nonregulated 40S ribosomal protein S12 (RPS12) signal. The primers used had the following sequences: RPS12 sense 5'-GCTGCTGGAGGTGTAATGGA-3', RPS12 antisense 5'-CTACAACGCAACTGCAACCA-3'; CPT1b sense 5'-CTCAGCCTCTACGGCAAATC-3', CPT1b antisense 5'-CTTCTTGATCAGGCCTTTGC-3'; PPAR sense 5'-TCCACGAAGCCTACCTGAAG-3', PPAR antisense 5'-GAACTCTCGGGTGATGAAGC-3'; PPAR sense 5'-AACATCCCCAACTTCAGCAG-3', PPAR antisense 5'-GGAAGAGGTACTGGCTGTCG-3'; UCP3 sense 5'-ATGGATGCCTACAGAACCAT-3', UCP3 antisense 5'-CTGGGCCACCATCCTCAGCA-3'; PGC-1 sense 5'-AGAGGGCCCGGTACAGTGAG-3', PGC-1 antisense 5'-TGGTGCTGCAAGGAGAGACC-3', MyoD sense 5'-CTGCTCTGATGGCATGATGG-3', MyoD antisense 5'-GGACACTGAGGGGTGGAGTC-3'; MHCIb sense 5'-ACAGAGGAAGACAGGAAGAACCTAC-3', MHCIb antisense 5'-GGGCTTCACAGGCATCCTTAG-3', MTE-I sense 5'-CCTCGTCTTTCGCTGTCCTG-3'; MTE-I antisense 5'-GTGTCCGTCCAGCACCTCCA-3'. Parallel amplifications (20, 25, and 30 cycles) of the same cDNA were used to determine the optimum number of cycles. After 30 cycles, a readily detectable signal within the linear range was observed. For the actual analysis, samples were heated for 5 min at 94°C, then 30 cycles were carried out, each consisting of 1 min at 94°C, 1.5 min at 61°C, and 1.5 min at 72°C. This was followed by a final 10 min extension at 72°C. The quantities of the PCR products were determined in separate preparations from three rats. Separation of the PCR reaction products was performed on a 2% agarose gel containing EtBr, and the products were readily visualized. Reverse image signals of the RT-PCR bands were quantified by means of a Bio-Rad Molecular Imager FX using the supplied software (Bio-Rad Laboratories). Primary RT-PCR data are shown in the figures, with quantities displayed above each gel. At the 48 h time point, induction quantities for all tested genes were directly comparable to those obtained by microarray detection (7)
.
Measurement of intracellular ATP and AMP levels
In the same muscle tissues that have been used for protein measurements (see the next section), we measured intracellular ATP and AMP levels using the "CheckLite AN" kit, a kind gift from Kikkoman Corp. (Chiba, Japan). This kit allows the measurement of absolute AMP levels after enzymatic conversion into ATP by pyruvate orthophosphate dikinase (PPDK) based on the luciferase reaction in a standard luminometer. The measurable ATP concentration range is from 10–13 to 10–5 M. Each muscle sample was dissolved at 10% (w/v) in 0.01% benzalkonium chloride (Fluka Biochimica) to inhibit all ATPase activity, homogenized, centrifuged at 4000 gav for 10 min at 4°C, then diluted to 0.001% with distilled water. Luciferase activity was assessed in reaction medium with and without PPDK. The luminescence coming from the conversion of all AMP into ATP by PPDK was calibrated for the ATP concentration. Luminescence was measured using a manually programmed TD20/20n luminometer (Promega s.r.l., Milan, Italy) according to the instructions provided with the CheckLite AN kit.
Western immunoblot analysis
MHCIb protein was determined using muscle lysates centrifuged at 12,000 g as described earlier and separated on an 8% SDS-polyacrylamide gel using a monoclonal antibody (mAb) (Chemicon International, Temecula, CA, USA). Protein levels (PPAR ) from supernatants of the ultracentrifuged lysates (see above) and from the nuclear extracts (30 µg of protein) were determined using a 10% SDS-polyacrylamide gel by employing a polyclonal antibody (pAb) directed against the amino terminus (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Nuclear PGC-1 protein levels were measured in a similar fashion using a pAb against the carboxyl terminus of PGC-1 (Chemicon International). Protein levels (total AMPK and ACC) in the supernatants of the ultracentrifuged lysates (see above; 40 µg of protein) were determined using 13% and 5% SDS-polyacrylamide gels, respectively, by employing specific antibodies against AMPK (Cell Signaling Technology Inc., Beverly, MA, USA) and ACC (Upstate Biotechnology, Lake Placid, NY, USA). Phosphorylation of the -subunit of AMPK was examined using an antibody (Ab) against phosphopeptides based on the amino acid sequence surrounding Thr 172 of the -subunit of human AMPK (Cell Signaling Technology Inc.). Phosphorylation of ACC was examined using an Ab against phosphopeptides based on the amino acid sequence surrounding Ser-79 of human ACC (Cell Signaling Technology Inc.). UCP3 and MTEI protein levels in mitochondrial lysates (30 µg of protein) were determined using 13% SDS-polyacrylamide gels, with a pAb raised either against the C-terminal region of human UCP3 protein (Chemicon International) or against human MTE I (kindly provided by Dr. Stefan Alexson, Karolinska Institute, Huddinge, Sweden) being used as primary Ab.
The protein concentrations of the samples were determined using Bio-Rad’s DC method (Bio-Rad Laboratories, Milan, Italy). Protein levels were detected by a chemiluminescence protein detection method based on the protocol supplied with a commercially available kit (NEN, Boston, MA, USA) and using the indicated antibodies and secondary antibodies. Signals were quantified by means of a Bio-Rad Molecular Imager FX using the supplied software (Bio-Rad Laboratories).
Measurement of mitochondrial proton conductance
Throughout we used freshly isolated mitochondria from gastrocnemius muscles obtained from rats treated as described above. The value of D
was determined from the distribution of the lipophilic cation triphenylmethylphosphonium (Ph3MeP+), which was measured using a Ph3MeP+-sensitive electrode. A Ph3MeP+ binding correction of 0.4 was applied, and D was measured in the presence of nigericine so that the whole proton motive force could be expressed as D. Nonphosphorylating mitochondrial respiration was measured in the presence of oligomycin using a Clarke-type oxygen electrode as described previously (25)
. The analyses were performed in a final volume of 1 ml containing 80 mM KCl, 50 mM HEPES (pH 7.2), 1 mM EGTA, 5 mM K2HPO4, 5 mM MgCl2, 1 µg oligomycin, 80 ng nigericin, 5 µM rotenone, 1% BSA, 0.5 mM oleate (to exclude putative variations in endogenous fatty acid content), and 15 µg/ml carboxyatractylate (to inhibit adenine nucleotide translocase activity). Mitochondria were energized with 6 mM succinate and respiration was titrated with an increasing amount of malonate of up to 2 mM. The involvement of UCP3 was assessed by performing the measurements in presence and absence of 500 µM GDP, a specific inhibitor of UCP3.
Measurement of fatty acid oxidation rate
The rate of mitochondrial fatty acid oxidation was assessed polarographically using a Clark-type electrode (25)
at 30°C in a final volume of 0.5 ml of 80 mM KCl, 50 mM HEPES (pH 7.0), 1 mM EGTA, 5 mM K2HPO4, 1% BSA (w/v), and 2.5 mM malate in the presence of ADP (120 µg/ml). The reaction was started by the addition of palmitoyl-L-carnitine (40 µM). All the required chemicals were purchased from Sigma-Aldrich Corp.
Measurement of circulating free fatty acid levels
Serum fatty acid levels were measured using a WAKO NEFA C kit (WAKO Chemicals GmbH, Neuss, Germany).
Statistical analysis
Results are expressed as means ± SE. The statistical significance of differences between groups was determined using 1-way ANOVA, followed by a Student-Newman-Keul’s test.
RESULTS
Structural features of skeletal muscle after food deprivation
Within 6 h after food deprivation, gastrocnemius muscle displayed rapid increases in MHCIb mRNA and protein levels (Fig. 1
A, B). Quantitation of the bands by densitometry indicated that each of these increases was 
8-fold, and band intensities were similar to those obtained at the 48 h time point. The food-deprived muscles (at 48 h) appeared redder than those of the fed controls, and the white parts of the muscle were smaller in area (Fig. 1C
, cross sections of gastrocnemius muscle; the clearer regions represent the white parts, indicated by arrows). Histochemical staining of the fibers demonstrated a tendency toward an increase in fiber type I content at 48 h (representative sections are shown in Fig. 1D
). These results demonstrate that structural adaptive responses to food deprivation occur very rapidly, as manifested by a measurable change in type I fiber content within 48 h.
|
PPAR , PGC-1, and MyoD expression on food deprivation
In view of the known stimulatory effects of the muscle-specific transcription factors PPAR , PGC-1, and MyoD on the expression of genes involved in preferential lipid utilization and mitochondrial fatty acid oxidation, we evaluated the induction of their expression after food deprivation. Within 6 h, food deprivation led to up-regulations of the mRNAs for MyoD, PGC-1, and PPAR (Fig. 2
A). To verify whether PPAR mRNA levels reflected protein expression, total PPAR protein levels were measured. In accordance with the mRNA levels, PPAR protein levels were elevated 6-fold at 6 h, then declined to control level from 12 to 48 h (Fig. 2B
). The pattern was similar for the nuclear PPAR protein level: it was increased by 2-fold at 6 h and was nearly zero at 48 h. Similarly, PGC-1 nuclear protein increased 4-fold at 6 h and had declined at 48 h (Fig. 2C
). These rapid inductions of transcription factors indicate acute in vivo reprogramming of rat gastrocnemius gene expression on food deprivation.
|
Subsequent induction of expression of target genes involved in mitochondrial lipid handling after food deprivation and mitochondrial fatty acid oxidation levels
We next evaluated the response of several representative target genes for the above-mentioned transcription factors. Expression patterns of the PPAR/PGC-1 targets MTE I, CPT1, and UCP3 (the latter depending also on MyoD) displayed different kinetics, but all these mRNAs exhibited up-regulation by 6 or 12 h; mRNA levels for CPT1 were already maximal at 6 h whereas those for UCP3 and MTE I peaked at 12 h (Fig. 3
A).
|
The mitochondrial protein levels for both UCP3 and MTE I were elevated by 5-and 3-fold, respectively, at 12 h. The MTE I protein level was even further elevated at 24 h, whereas the UCP3 protein level showed a slight tendency to decline at this time point (Fig. 3B
).
The elevations in UCP3 and MTE I mitochondrial protein levels at 12 h coincided with an increase in mitochondrial fatty acid oxidation, which continued to rise throughout the 48 h period (Fig. 3C
).
These in vivo results show a tight temporal relationship between the mitochondrial protein levels of "lipid handling" genes and the actual mitochondrial fatty acid oxidation rate, and also demonstrate that this is achieved in vivo by acute activation of their respective transcription factor pathways.
Cellular ATP/AMP levels, transient phosphorylation of AMPK and ACC, and serum fatty acid levels after food deprivation
To determine whether AMPK activity participates in nutritional energy-sensing in vivo, both the upstream causes and the downstream consequences of its phosphorylation were explored by measuring the effects of food deprivation on the intracellular AMP/ATP ratio, phosphorylation of AMPK and ACC, and serum free fatty acid levels, respectively. In the fed controls, the intramuscular (i.m.) ATP level was 0.8 µM and the AMP level was 5.0 µM (6-fold higher than the ATP level, see Table 1
). ATP levels did not vary significantly at 6 and 12 h, then displayed a 20-fold drop from 24 to 48 h. Intramuscular AMP levels were doubled at 6 h, remained constant at 12 h, and declined to control levels at 24 h, decreasing further to 1 µM at 48 h. The AMP/ATP ratio significantly increased 1.5 fold at 6 h up to 12.5 fold at 24 h, then started to decrease at 48 h but was still 5-fold increased with respect to the fed controls. A transient rise in the phosphorylations of AMPK and ACC (P-AMPK and P-ACC at positions Thr 172 and Ser-79, respectively, see Fig. 4
A) was observed. Concomitant with the rise in i.m. AMP levels, the P-AMPK level at 6 h was elevated 4-fold. It then declined, the increase above control being 2-fold at 12 h and zero at 24 h. At 48 h the level was below control. A similar tendency was observed for P-ACC (4-fold the control level at 6 h to 4-fold from 12 to 24 h, and back to control levels at 48 h). Total AMPK and ACC protein levels remained constant throughout the measurement period (Fig. 4A
, AMPK and ACC). These data seem to imply that food deprivation acutely activates AMPK through an elevation of i.m. AMP levels, but once this kinase has effected the appropriate metabolic adjustment, its activity returns to baseline levels. The transient rise in ACC phosphorylation and the subsequent increase in fatty acid oxidation from 12 h onward is consistent with this idea. Serum fatty acid levels were elevated at 12 h, peaked at 24 h, and had declined to 2.5-fold the fed control level at 48 h (Fig. 4B
). These results indicate that an increase in serum fatty acid availability may be the cause of the observed inhibition of AMPK activity.
|
|
Mitochondrial membrane potential and respiration rate at 12, 24, and 48 h after food deprivation
To verify whether mitochondrial proton conductance varied over time on food deprivation, we measured proton leak kinetics in 12, 24, and 48 h food-deprived rats. No change in the kinetics was measured despite the clear increase in mitochondrial UCP3 protein levels (data not shown).
Effect of removal of exogenous fatty acids on AMPK phosphorylation and UCP3 expression in gastrocnemius muscle from food-deprived rats
To explore the idea that a rise in fatty acid levels could effectively switch off AMPK activity, we assessed whether the converse was true (i.e., if causing a decrease in serum fatty acid levels during fasting would result in increased AMPK phosphorylation). To this end, we induced a decrease in the concentration of circulating fatty acids in 48 h food-deprived rats by in vivo nicotinic acid treatment (see "Animals" in Materials and Methods) and measured the effect on AMPK phosphorylation. Figure 5
A shows that at 48 h treatment with nicotinic acid had reduced the serum fatty acid concentration to about half that seen at the same time point in control fasted rats. The same nicotinic acid treatment induced an increase of 5-fold in AMPK phosphorylation in gastrocnemius muscle (vs. 48 h food-deprived controls; Fig. 5B
), supporting the idea that AMPK phosphorylation (and thus AMPK activity) is inversely correlated with the serum fatty acid level. Treatment with nicotinic acid neither influenced the absolute ATP and AMP levels at 48 h (being 0.04±0.02 µM and 1.08±0.28 µM, respectively) nor the AMP/ATP ratio (27±4.3). Finally, since it is known that UCP3 expression depends on fatty acids, we measured UCP3 mRNA in gastrocnemius muscle from 48 h food-deprived rats pretreated with nicotinic acid. Indicative of a decrease in muscular fatty acid levels, UCP3 expression was halved in such gastrocnemius muscle (vs. control 48 h fasted rats, Fig. 5C
). These results show that a decrease in the serum fatty acid level correlates with a decrease in the muscular fatty acid level. Taken together, our results demonstrate an inverse correlation between the muscular fatty acid level and AMPK phosphorylation.
|
DISCUSSION
Recent evidence suggests that muscle structure and metabolic activity, established targets for adaptive changes in response to altered functional demands, are tightly linked at the molecular level. Here, we provide for the first time a sequential analysis of the morphological and functional adaptations to food deprivation. Our results demonstrate that in this condition there are rapid, transient elevations in the expression of the nuclear receptor PPAR , transcriptional coactivator PGC-1, and transcription factor MyoD in rat gastrocnemius muscle. The PPAR protein level (also at the nuclear level) shows a similar tendency, as do the nuclear levels of PGC-1. Nuclear accumulation of PGC-1 and PPAR and increased MyoD expression is followed by increased expression of some of their downstream target genes (such as CPT1, MTE1, and UCP3), as well as by an increase in the mitochondrial fatty acid oxidation rate. These data illustrate the capacity of rat gastrocnemius muscle to be rapidly reprogrammed at the metabolic level upon food deprivation and highlight the roles played by PGC-1 and PPAR as in vivo determinants of such reprogramming. Furthermore, we observed rapid increases in the levels of the mRNA and protein for the MHCIb gene indicative of the formation of oxidative fibers. This increase coincided with rapid inductions of PGC-1 and PPAR nuclear proteins. These factors have a clear role in inducing a muscle fiber transition from glycolytic (type II) to oxidative (type I) (22
, 23)
. Experiments on mice overexpressing an activated form of PPAR have revealed that type I fiber formation can be directly stimulated without induction of PGC-1 (23)
. However, in physiological situations an up-regulation of PGC-1 is likely to be the first signal in the cascade leading to muscle fiber shifts, since PGC-1 is a coactivator of PPAR. A physical interaction between PGC-1 and PPAR has been demonstrated by coimmunoprecipitation in the nuclei of gastrocnemius muscles obtained from PPAR transgenic mice (27)
, and here we have shown a nuclear accumulation of both factors within 6 h after food deprivation.
In the present study we have shown an early increase in MHCIb mRNA and protein level reflecting a structural change in the muscle fibers toward a slow, oxidative phenotype, including an increase in the number of type I fibers and a redder appearance of cross sections of whole muscle at 48 h. Collectively, the above data underline the existence of species based differences in the structural/metabolic adaptations to food deprivation by skeletal muscle. In vitro, one metabolism-regulating factor known to play an important role as a "fuel provider" during the adaptation of cells to nutrient deprivation is AMPK (9)
. However, its role in vivo is less clear, and in 24 h fasted mouse muscle no change in AMPK phosphorylation/activity was observed (16)
. In contrast to data so far obtained from mice, here we report that in the rat in vivo, a very rapid, transient phosphorylation of AMPK occurs, highlighting a role for AMPK in the rat gastrocnemius muscle response to food deprivation. Phosphorylation of AMPK occurs in concert with a doubling of i.m. AMP concentrations at 6 h. In parallel with this AMPK phosphorylation, there is an increased phosphorylation of ACC (a downstream target of AMPK), with a timing that underlines its involvement in the increase in the rate of fatty acid oxidation. These data showing a coordinated activation of AMPK and ACC in the metabolic pathway leading to increased mitochondrial fatty acid oxidation point to AMPK participating in nutritional energy-sensing in vivo, as it does in vitro (9)
, at least in rats. In addition, AMPK phosphorylation showed a negative correlation with the increase in the circulating fatty acid level (see Fig. 6
A). In fact, at the time that this level and mitochondrial fatty acid oxidation began to increase, AMPK phosphorylation started to decrease despite high AMP levels and an elevated AMP/ATP ratio. This process was reversed by nicotinic acid treatment, which did not influence the AMP/ATP ratio, suggesting that fatty acids may also be involved in the activation of pathways capable of decreasing AMPK phosphorylation (see Fig. 6B
).
|
Reduced serum and intracellular fatty acid levels seem to go hand in hand, since we show here that treatment with nicotinic acid during food deprivation not only reduced the circulating FFA level and increased AMPK phosphorylation, but also inhibited transcription of the UCP3 gene in gastrocnemius muscle. This gene is known to be up-regulated specifically by fatty acids and PPAR agonists both in L6 myotubes (18
, 28)
and in rats fed a high-fat diet (29)
as well as in PPAR transgenic mice (27)
. Increased FFA and decreased Glc serum levels during fasting lead to an increased mitochondrial uptake of FFA over Glc as fuel substrate. In turn, the increased intracellular FFA level that inhibits AMPK phosphorylation guarantees the sparing of Glc for use by the brain. Randle and colleagues first used the term "glucose-fatty acid cycle" to describe the reciprocal inhibition of Glc metabolism by high-level fatty acid utilization in the heart (30)
. Our study is the first to describe an inhibitory effect of fatty acids or their oxidation products on AMPK activity in vivo in fasted skeletal muscle. The data provided by our experimental approach, summarized in Fig. 6
, prompt us to propose an in vivo regulatory role for AMPK in the economy of fatty acid utilization in skeletal muscle after food deprivation.
Uncoupling proteins (UCPs) may play a role in conditions of altered metabolism such as fasting. Although extensive research has been undertaken to unravel the putative functions of the novel UCPs (UCP2 and UCP3), there is considerable uncertainty about their physiological roles, and proposals that they might play a role in mediation of thermogenesis, regulation of lipids as fuel substrate, the control of insulin secretion, and/or controlling the production of reactive oxygen species have been made (31)
. The effects of physiological interventions consistently support a role for UCP3 in promoting fatty acid oxidation (32
, 33)
or translocation of peroxidized fatty acids (34)
. We (25)
and others (35)
have demonstrated that although fasting increases rat UCP3 expression and protein levels, it does not lead to increased mitochondrial proton leak in muscle mitochondria. In this study we confirm these results by measuring the proton leak in 12, 24, and 48 h food-deprived rats.
In addition, numerous reports have proposed an interplay between UCP3 and AMPK, AMPK activity being a trigger for UCP3 expression (36
37
38)
or vice versa (39)
. Here, we present evidence that in the gastrocnemius muscle of food-deprived rats, increased AMPK activity is unlikely to be the consequence of an increased expression of UCP3. Indeed, our results imply that the increase in mRNA levels and mitochondrial protein levels of UCP3 are downstream events of AMPK phosphorylation (see Figs. 3
, 4
). AMPK phosphorylation also preceded the increased expression of MyoD, an essential transcription factor for UCP3 (40)
. These data lead us to conclude that UCP3 is not responsible for the increased phosphorylation of AMPK, but rather that fluctuating muscular levels of fatty acids are most likely involved in AMPK activation/deactivation.
By elucidating the physiological sequence of events in the in vivo coordinated pattern of biochemical/molecular changes involved in the reprogramming of gastrocnemius muscle after food deprivation, this study highlights the roles played in vivo by several factors (including PPAR , AMPK, and fatty acids) through the chronology of events and their possible relationship.
ACKNOWLEDGMENTS
This work was supported by Grant MIUR-COFIN 2003 Protocol No. 2003052503–002. The authors wish to thank the Kikkoman Corporation (Chiba, Japan) for kindly providing the CheckLite AN kit.
FOOTNOTES
1 These authors contributed equally to this work. 
Received for publication March 9, 2006. Accepted for publication July 6, 2006.
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  Pieter de Lange, M. Moreno, E. Silvestri, A. Lombardi, F. Goglia, and A. Lanni Fuel economy in food-deprived skeletal muscle: signaling pathways and regulatory mechanisms FASEB J, November 1, 2007; 21(13): 3431 - 3441. [Abstract] [Full Text] [PDF]
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