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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 4, 2003 as doi:10.1096/fj.03-0342fje. |
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* Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, 81100 Caserta, Italy;
Dipartimento di Scienze Biologiche ed Ambientali, Università degli Studi del Sannio, 82100 Benevento, Italy; and
Dipartimento di Fisiologia Generale ed Ambientale, Università degli Studi di Napoli "Federico II," 80134 Napoli, Italy
3 Correspondence: Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli (SUN), Via Vivaldi 43, 81100 Caserta, Italy. E-mail: antonia.lanni{at}unina2.it; pieter.delange{at}unina2.it
SPECIFIC AIM
Fasting results in drastic changes in the metabolic activity of skeletal muscle directed toward the use of lipids and amino acids as alternative to glucose. The alterations in gene transcription in muscles during fasting related to these metabolic adaptations in rat gastrocnemius muscle are largely unknown. In this study performed on rats subjected to a 48 h fast, we determined the key transcriptional events related to fiber differentiation, intermediary metabolism, hormonal transduction, and protein turnover (including transcriptional effects on the proteasome machinery) by using a combined RT-PCR/cDNA array approach. Since several lines of evidence point to the existence of species-specific mechanisms of adaptation to starvation stimuli such as fasting, the data were compared with those already reported in the gastrocnemius muscle of the fasted mouse.
PRINCIPAL FINDINGS
1. Fasting increases the mRNA levels of myosin heavy chain Ib (MHC Ib) as well as those of factors determining the shift toward "slow" myosin fibers in rat gastrocnemius muscle
In agreement with the well-known shift toward lipid metabolism during fasting, the mRNA level of myosin heavy chain Ib (MHC Ib, also termed "slow" myosin) that directs the formation of slow oxidative fibers was elevated by 
12 fold (see Fig. 1
A). This rise in MHCIb mRNA corresponded to a threefold increase in MHCIb protein in fasting rats (Fig. 1B
). mRNA levels of the fast oxidative glycolytic myosins [MHCIIa and MHCIId(x)] and those of the fast glycolytic myosin (MHCIIb) did not differ significantly between fasted rats and controls (not shown). mRNA levels of two factors involved in muscle differentiation toward type I fibers, termed p27kip1 and muscle lim protein (MLP), were elevated by 15- and 9-fold, respectively. To our knowledge, these results demonstrate for the first time a structural shift toward lipid metabolism in skeletal muscle, induced by fasting.
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2. Expression of genes involved in lipid uptake and utilization increases during fasting
The mRNA levels of all genes present on the array and involved in lipid metabolism increased during fasting, including those of the very long, long, and medium chain acyl CoA dehydrogenase (VLCAD, LCAD, and MCAD) by 5, 7, and 4-fold, respectively. The expression of lipoprotein lipase (LPL) and acyl CoA synthetase (ACS) increased by threefold (see Fig. 1
). mRNA of carnitine palmitoyl transferase 1 and 2 (CPT1ß and CPT2) was up-regulated by seven- and fivefold, respectively (Fig. 1)
. Thus, the fasting stimulus provokes an activation of transcription of these interrelated gene products, representing an important adaptive metabolic response.
3. Fasting elevates the expression of genes involved in facilitating transport of oxidative phosphorylation products but reduces the expression of genes involved in mitochondrial ubiquinone synthesis
Uncoupling protein 3 (UCP3) mRNA and protein levels were elevated by twofold in fasting rats (see Fig. 1A, B
). In addition to UCP3, the mRNA levels of carbonic anhydrase III (CAIII) and adenine nucleotide translocator (ANT), facilitating the transport of oxidative phosphorylation products (CO2 and nucleotides, respectively), were elevated by threefold. mRNA levels of mitochondrial membrane protein COQ7 and 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) were reduced by threefold (see Fig. 1
). Decreased activity and expression levels of COQ7 and HMGR would explain the low intramitochondrial coenzyme Q (CoQ) levels we earlier observed in the gastrocnemius muscle of the fasted rat.
4. Fasting down-regulates the expression of the peroxisome proliferator-activated receptors (PPARs) 
and 
but up-regulates expression of the receptors for retinoic acid (RXR), tumor necrosis factor (TNFR1), and insulin (INSR)
Surprisingly, fasting lowered the expression of PPAR and by 3 and twofold, respectively (Fig. 1)
. The mRNA levels of adipose-specific PPAR
1 and 2 were undetectable in gastrocnemius muscle from both fasted and control rats. Levels of RXR, TNFR1, and INSR were markedly up-regulated, by at least sevenfold.
5. Expression of all genes encoding free radical scavengers, kinases, heat shock proteins, and transcription factors present on the cDNA array is up-regulated by fasting
Fasting markedly increased mRNA levels of various transcription factors, among which were activator protein-1 and subunit p105 of nuclear factor-
B (both by 5-fold). Expression of adenylate kinase 3 (AK3), which is located within the mitochondrial matrix and produces AMP in response to lowered ATP levels, was elevated 3.6-fold during fasting. mRNA levels for several heat shock proteins (including mitochondrial hsp60) were also increased by fasting, as were scavengers of free radicals such as cytosolic superoxide dismutase and phospholipid hydroperoxide glutathione peroxidase (2.1 and 8.6-fold, respectively). These variations protect the cell against the damaging effects of the accumulation of free radicals, including peroxidated lipids, during fasting.
6. Fasting increases the expression of genes involved in protein breakdown, with the exception of ubiquitin and ubiquitin-conjugating enzyme (E214k)
As a result of fasting there were increases in all proteasome genes analyzed although our cDNA array results revealed unaltered mRNA levels for ubiquitin and ubiquitin-conjugating enzyme (E214k) (data not shown). In rat gastrocnemius muscle, both normal and fasted, polyubiquitin was one of the most abundant mRNAs analyzed on the array (arbitrary units: 8683 in the controls and 7882 in the fasted rats). We observed a marked elevation in mRNA levels of two cysteine proteases, cathepsin L and cathepsin C/J, by 2.1- and 2.9-fold, respectively. Cathepsin L was preferentially expressed in rat gastrocnemius muscle.
CONCLUSIONS AND SIGNIFICANCE
In the rat gastrocnemius muscle, fasting is accompanied by a structural and functional shift toward lipid metabolism, reflected by increased MHC Ib expression (implying type I fiber formation) and increased expression of all analyzed genes involved in lipid metabolism (for an overview, see Fig. 2
). As it has been shown that MLP, a factor involved in type I fiber formation, enhances the activity of MyoD (an essential transcription factor for the expression of UCP3) and that LPL enzyme activity positively affects UCP3 transcription, the above data may be directly associated with elevated UCP3 levels and point to a role for UCP3 in this metabolic shift. The changes in expression of the myosin genes as well as the aforementioned genes involved in lipid metabolism and oxidative phosphorylation do not occur in gastrocnemius muscle from fasted mice. These observations point out that major differences exist in the mechanisms involved in the adaptation to fasting between smaller and larger animals and underscore the need for caution in comparing data related to mechanisms of metabolic adaptations between different species.
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Fasting is accompanied by drastic changes in receptor expression, indicative of active metabolic adaptations (Fig. 2)
. Lowered PPAR expression during fasting is surprising, since PPARs are involved in ligand free fatty acid-dependent transcriptional activation of genes involved in lipid metabolism as well as UCP3 through binding to PPAR response elements in their promoters. PPAR has been shown to mediate at least part of the increased UCP3 expression induced by fatty acids in skeletal muscle in vivo. In contrast, the increased RXR and TNFR1 mRNA levels correlate well with increased UCP3 expression since 1) retinoids and TNF have been shown to stimulate UCP3 expression in muscle cells and 2) RXR may form heterodimers with thyroid hormone receptor (THR) and PPAR to bind to specific regulatory motifs in the UCP3 promoter. The increase in insulin receptor (INSR) expression may imply that upon refeeding, the fasted gastrocnemius muscle would be able to shift back rapidly to glucose metabolism.
In conditions involving ATP depletion/ADP accumulation such as fasting, AMP levels are elevated. An enzyme responsible for this elevation is AK3. The resulting elevated AMP level triggers the activity of AMP-activated kinase (AMPK), known to be involved in increased fatty acid oxidation and associated with increased UCP3 mRNA and protein levels. Taken together, these data indicate good correlations between fasting and increased expression of AK3, increased activity of AMPK, increased fatty acid oxidation, and increased UCP3 expression.
With regard to its proposed role as an uncoupler of energy production, increased expression of UCP3 seems puzzling, since during fasting energy dissipation needs to be suppressed. Since a role has recently been attributed to CoQ as an essential cofactor for the uncoupling activity of UCP3, the down-regulation of HMGR and COQ7, genes involved in the production of COQ (as well as increased phosphorylation of AMPK, shown to inactivate HMGR activity), is in line with the known lack of uncoupling activity of UCP3.
To our knowledge, this is the first study to report unaltered mRNA levels of ubiquitin in fasted rat gastrocnemius muscle. In line with the data obtained in fasted mice, mRNA levels of ubiquitin-conjugating enzyme (E214k) remained unaltered. The mRNA level of an early marker of muscle wasting through the lysosomal pathway, cathepsin L, was increased in fasted rat gastrocnemius muscle. The powerful increases in the levels of expression of proteasome components and the insulin-like growth factor binding proteins (which, by forming heterodimers with RXR, inhibit ubiquitin and E214k mRNA turnovers), as well as of TNF and the cathepsins, favor increased protein breakdown through increased action of both the proteasomal and lysosomal pathways (Fig. 2)
.
In conclusion, the transcriptional data obtained in this study show that rat gastrocnemius muscle adapts to fasting by undergoing a structural and metabolic shift to the use of metabolic fuels other than glucose. These results also reveal significant differences in the response to fasting of mice vs. rats.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0342fje 
2 These two authors contributed equally to the work.
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