The purpose of this study was to identify novel differences in genes related to metabolism, muscle development, and fiber-type determination and differentiation in mRNA and protein content in skeletal muscle between men and women at rest using microarray, Real Time-PCR analysis, and protein analysis. Microarrays are a useful tool for the identification of novel mRNA expression patterns and can help to understand potential pathways involved in regulating cellular activity in skeletal muscle 
. The results from these studies indicate that there are significant differences in mRNA content between men and women. Roth et al. (2002) and Welle et al. (2008) have previously shown that there are significant differences in skeletal muscle mRNA content between men and women, as well as showing sex differences are significantly greater than age and/or strength training effects on mRNA content 
. Approximately 30% of the genes we found to have changed due to sex correspond to the results of Welle and colleagues 
. Results likely vary between studies due to differences in age of the subject populations, fitness variations, low sample numbers, pooled samples on one gene chip versus individual gene chips per subject, and different gene array technology 
. The methodology used in this study greatly strengthens the data regarding sex based differences in skeletal muscle mRNA for we examined over 23,000 genes with updated annotation, with 12 subjects per group (N
24 total) hybridized to individual gene chips for analysis and used stringent statistical analysis with an NLOGP>4. The criteria for differential expression is stringent compared to other array studies that generally use an NLOGP>2, however, necessary for the most accurate unbiased account of gene content differences. For the microarray experiment we used samples from study one which included 7 women in the follicular phase and 5 women in the luteal phase, 6 on oral contraceptives and 6 not on oral contraceptives which gives a good representation of the female population. Given the high n-value of the analysis and stringent array analysis criteria we wanted to reduce any variability in gene expression due to these factors in order to only identify specific and significant gene differences that can be applied to a larger population. The goal of the microarray was not to identify genetic differences due menstrual cycle phase but identify sex related differences between an average population of men and women. In another manuscript “in preparation” from study 2 we used a targeted PCR approach to examine differences in metabolic related genes in men compare to women in both the follicular and luteal phases of the menstrual cycle and found that menstrual cycle had little effect on metabolic related mRNA species, compared to the robust difference that sex has (Figures S1
). Due to these findings, the little amount of precious human muscle sample, and the relatively consistent use of follicular phase women in other gender related studies 
we compared the mRNA and protein of women in the follicular phase only. It is also important to note that Devries et al (2006) report no physiological difference in exercise performance due to menstrual cycle phase including average RER, glycogen utilization, glucose rate of appearance, rate of disappearance, and metabolic clearance rate averaged over the exercise period 
The first focus was on the mRNA content of genes involved in intermediary substrate metabolism due to the known fact that women oxidize more fat during endurance exercise as compared with men 
. Array results identified six genes related to metabolism that were differentially regulated between men and women. We chose to confirm the mRNA content of five of these genes by RT-PCR. Results identified a novel sex-based difference in the mRNA content of ACAA2 and HADHB (TFP-β). Also, the mRNA content for catalase, lipoprotein lipase, and uncoupling protein-2 were higher in women compared with men. Lastly, ALDH1A1 mRNA content was lower in women compared with men. There was also sex based differences in the mRNA content of genes involved in protein biosynthesis, cell proliferation, signal transduction, transcription, and translation with a particular interest in those genes which are directly involved in muscle function and/or structure.
HADHB (TFPβ) is a multi-enzyme complex found in the mitochondria that is involved in the β-oxidation of fatty acids 
. Specifically, the TFP enzyme catalyzes the last three steps of long chain fatty acid β-oxidation for long-chain specific acyl-CoA moieties. We are the first to measure and report a sex difference in the mRNA content for this gene. An enhanced β-oxidation capacity distal to transport of FFA into the mitochondria may allow for maintenance of β-oxidation when the cell is under metabolic stress. For example, male transgenic peripheral peroxisome activating receptor knockout (PPARα−/−
) animal develop severe hypoglycemia when an inhibitor of CPT activity (etomoxir) was given, yet the majority of female mice survived 
. Furthermore, CPTII deficiency is an autosomal recessive condition and yet many more cases have been documented in men as compared with women, possible due to enhanced β-oxidation capacity in women. Others have reported that the short-chain specific isoform of HAD (SCHAD) has higher mRNA and protein abundance in women compared with men 
These results also indicate a significant sex based difference in the mRNA content of ACAA2 (acetyl-Coenzyme A acyltransferase 2). ACAA2 is one of two isoforms of ACAA, which is an intracellular enzyme that biosynthesizes cholesteryl esters. Specifically, ACAA is involved in storing cholesteryl esters as lipid droplets, in absorbing dietary cholesterol, and in providing cholesteryl esters as part of the core lipid for lipoprotein synthesis and assembly 
. Although ACAA appears to be present in many cell types such as hepatocytes, adrenal cells, skin cells, intestinal enterocytes, neurons, and macrophages it has not been well characterized in skeletal muscle 
. In the aforementioned cell types it has been shown that the ACAA1 isoform is the predominant enzyme compared to ACAA2 
. Our gene array results suggest that men and women have no significant difference in the expression of ACAA1 but women have an increased expression of ACAA2 compared to men. Therefore, the total ACAA expression should be significantly higher in women than men, which may explain why lipids are more readily available for substrate utilization during endurance exercise in women. Future studies are needed to determine the expression profile of ACAA1 and ACAA2 in human skeletal muscle as well as the importance of ACAA2 over-expression in women compared to men.
Catalase is one of the three primary antioxidant enzymes. RT-PCR confirmed that mRNA content for catalase is significantly higher in women compared to men. Interestingly, Fano et al (2001) have shown that the enzyme activity of catalase, in the vastus lateralis
, is significantly higher in women than men 
. Sex differences have also been observed in other antioxidant enzyme, including manganese-superoxide dismutase 
, suggesting that women are better protected against reactive oxygen species (ROS) as compared with men. We also demonstrated that mRNA content for UCP2 was higher in women compared to men. UCP's can affect energy metabolism efficiency by uncoupling ATP production from mitochondrial respiration. UCP2 is involved in the regulation of energy metabolism and might play a role in obesity 
. UCP2 has also been suggested to affect the production of reactive oxygen species (ROS) 
, and regulate the [ATP]/[ADP] ratio 
. Although we are the first to show a sex specific difference in mRNA content in the vastus lateralis
, this may help to explains why young women are protected against ROS (reviewed in 
). Furthermore, if UCP2 does play a role in obesity the higher expression in women might help to regulate lipid oxidation.
ALDH1A1 is an isoform of the aldehyde dehydrogenase superfamily primarily responsible for the oxidation of endogenous and exogenous aliphatic and aromatic aldehydes including acetaldehyde, benzaldehyde, 4-hydroxynonenal, malondialdehyde, and retinaldehyde 
. Recently ALDH1A1 was also shown to convert 3-deoxyglucosone into 2-keto-3-deoxygluconate 
. In this study we found that the mRNA content was lower in women compared with men, suggesting that men might be able to metabolize aldehydes (i.e. alcohol) more efficiently than women. ALDH1A1 has not been well characterized in humans, let alone skeletal muscle, but in the mouse liver and human colon there appears to be no sex differences in activity 
. Further studies into the implications of ALDH1A1 mRNA sex differences in skeletal muscle need to be conducted.
These results also showed sex specific differences in genes involved in cell transport, protein biosynthesis, cell proliferation, signal transduction pathways, transcription and translation. Of interest, women had a 2.2-fold reduction in the solute carrier family 1, member 4 that is involved in glutamate/neutral amino acid transport which could be important in metabolism. Women had a 1.7-fold increase in mRNA content of angiopoietin 1, a factor involved in increasing vascularization to a specific tissue, in this case, muscle; however, there does not seem to be a significant difference in capillarization of the tibialis anterior
or vastus lateralis
muscle of women compared to men 
. Women also had a 1.7- and 1.5-fold reduction in Dishevelled associated activator of morphogenesis 2, and spectrin- beta- non-erythrocytic 1 which are involved in actin cytoskeleton organization and biogenesis, and barbed-end actin filament capping, respectively. Both are important in muscle cell shape and function. There was an interesting trend in the expression of signal transduction genes and transcription factor genes. Women had a significant up-regulation of the majority of signal transduction related genes and a significant down-regulation of the majority of transcription factor genes. Despite these trends, there was no evidence that one signal transduction pathway was favored over another as the identified genes spanned multiple pathways including the MAPK pathway, frizzled signaling pathway, G-protein coupled receptor protein signaling pathway, and insulin receptor signaling pathway.
It is important to note that gene array analysis works on the assumption that basal mRNA equates into changes at the protein level and thus activity level. However, recent studies have been demonstrating that is not always the case. For example Kiens et al. (2004) demonstrated that women have a significantly higher LPL mRNA content; however there was no observed differences in LPL activity between men and women 
. Similarly, Roepstorff et al. demonstrated that although mRNA and protein expression of hormone sensitive lipase (HSL) was higher in women, phosphorylation activation was significantly higher in men 
. Another study in skeletal muscle biology also found discrepancies in the correlation between mRNA and protein content of a number of genes related to fatty acid oxidation 
. Part of the discrepancy between mRNA abundance and protein and enzyme assays may relate to higher variance in Western blots and activity assaystechnique, and/or that the transcriptome abundance regulates multiple interacting and synergistic pathways that combine to influence flux through metabolic pathways at the protein level that is below the detectable threshold for statistical changes in a single given protein to be manifested. In order to fully understand cellular differences between men and women it is important to understand pre-translational (mRNA abundance), translational (protein) and post-translational (phosphorylation) levels of control.
It has recently been hypothesized that some of the sex differences in exercise substrate selection may be due to fiber-type compositional differences 
. Subject fiber type characteristics were the same in this study as previously reported 
; specifically, the proportionate area (area%) of type I fibers was higher, while that of type II fibers was lower, in women compared with men. Previous findings of a sex difference in type I fiber proportion 
and a larger type I fiber area 
, in women as compared with men were not confirmed. Nevertheless, it is the proportion of the total muscle area represented by a given fiber type (area X proportion
area%) that should determine the overall abundance of a given transcript or protein in a homogenate of skeletal muscle. Examination of mRNA expression of myosin heavy chain genes, which are specifically expressed in their corresponding muscle fiber types 
, are good markers of the terminal differentiation of muscle fibers. In this study, we found a significantly higher mRNA content of MHCI and a similar mRNA content of MHCIIa and MHCIIx in the skeletal muscles of women compared with men. The difference in MHCI mRNA did not translate into differences in MHCI protein expression, consistent with previous findings 
Similarly, we found sex differences in the mRNA content, but not the protein content, of PPARδ; which plays a role in the conversion of muscle fiber type II into type I and maintenance of the number of type I fibers 
, and increases the capacity for oxidative metabolism of muscle fibers through hyperplasia of type I fibers 
in transgenic mice.
Strong evidence in transgenic mice 
, and controversial evidence in humans 
, suggests that PGC-1α is important in the determination of muscle fiber type and induces a fiber type transformation from type II into type I muscle fibers. We did not find an influence of sex on the mRNA or protein content of PGC-1α in skeletal muscle in spite of the fact that women had a higher% area of type I fibers. We also found that there was no sex difference in the mRNA content of myostatin, a negative regulator of skeletal muscle growth 
At rest, there are no significant differences in protein content of the select genes examined, which are involved in metabolism or fiber type. Consistently, there are no observed sex differences in substrate utilization at rest 
. However, mRNA content suggest that men and women are “primed” differently for specific cellular events, and future studies are need to determine if exercise induces changes at the translational and post-translational levels.
Overall, these results identified sex-based differences in mRNA content of metabolic related genes that might lead the way towards an understanding of the sex-based differences in metabolic fuel selection during endurance exercise. Furthermore, this study emphasizes the importance of the influence of sex based differences in gene expression. At the mRNA level there are no inconsistencies in our data or in the literature, which supports that women have higher mRNA abundance for genes involved in fat metabolism as compared with men. Furthermore, men and women demonstrate varied regulation of genes involved in mitochondrial function, transport, protein biosynthesis, cell proliferation, signal transduction pathways, transcription and translation, even at rest.