Skeletal Muscle FOXO1 (FKHR) Transgenic Mice Have Less Skeletal Muscle Mass, Down-regulated Type I (Slow Twitch/Red Muscle) Fiber Genes, and Impaired Glycemic Control * □S

FOXO1, a member of the FOXO forkhead type transcription factors, is markedly up-regulated in skeletal muscle in energy-deprived states such as fasting and severe diabetes, but its functions in skeletal muscle have remained poorly understood. In this study, we created transgenic mice specifically overexpressing FOXO1 in skeletal muscle. These mice weighed less than the wild-type control mice, had a reduced skeletal muscle mass, and the muscle was paler in color. Microarray analysis revealed that the expression of many genes related to the structural proteins of type I muscles (slow twitch, red muscle) was decreased. Histological analyses showed a marked decrease in size of both type I and type II fibers and a significant decrease in the number of type I fibers in the skeletal muscle of FOXO1 mice. Enhanced gene expression of a lysosomal proteinase, cathepsin L, which is known to be up-regulated during skeletal muscle atrophy, suggested increased protein degradation in the skeletal muscle of FOXO1 mice. Running wheel activity (spontaneous locomotive activity) was significantly reduced in FOXO1 mice compared with control mice. Moreover, the FOXO1 mice showed impaired glycemic control after oral glucose and intraperitoneal insulin administration. These results suggest that FOXO1 negatively regulates skeletal muscle mass and type I fiber gene expression and leads to impaired skeletal muscle function. Activation of FOXO1 may be involved in the pathogenesis of sarcopenia, the age-related decline in muscle mass in humans, which leads to obesity and diabetes.

FOXO1, a member of the FOXO forkhead type transcription factors, is markedly up-regulated in skeletal muscle in energy-deprived states such as fasting and severe diabetes, but its functions in skeletal muscle have remained poorly understood. In this study, we created transgenic mice specifically overexpressing FOXO1 in skeletal muscle. These mice weighed less than the wildtype control mice, had a reduced skeletal muscle mass, and the muscle was paler in color. Microarray analysis revealed that the expression of many genes related to the structural proteins of type I muscles (slow twitch, red muscle) was decreased. Histological analyses showed a marked decrease in size of both type I and type II fibers and a significant decrease in the number of type I fibers in the skeletal muscle of FOXO1 mice. Enhanced gene expression of a lysosomal proteinase, cathepsin L, which is known to be up-regulated during skeletal muscle atrophy, suggested increased protein degradation in the skeletal muscle of FOXO1 mice. Running wheel activity (spontaneous locomotive activity) was significantly reduced in FOXO1 mice compared with control mice. Moreover, the FOXO1 mice showed impaired glycemic control after oral glucose and intraperitoneal insulin administration. These results suggest that FOXO1 negatively regulates skeletal muscle mass and type I fiber gene expression and leads to impaired skeletal muscle function. Activation of FOXO1 may be involved in the pathogenesis of sarcopenia, the age-related decline in muscle mass in humans, which leads to obesity and diabetes.
Skeletal muscle is the largest organ in the human body, comprising about 40% of the body weight. The mass and composition of skeletal muscle are critical for its functions, such as exercise, energy expenditure, and glucose metabolism (1,2). Elderly humans are known to undergo a progressive loss of muscle fibers associated with diabetes, obesity, and decreased physical activity (sarcopenia) (3). In human skeletal muscle, there are two major classifications of fiber type: type I (slowtwitch oxidative, so-called red muscle) and type II (fast-twitch glycolytic, so-called white muscle) fibers (2). Mass, fiber size, and fiber composition in adult skeletal muscle are regulated in response to changes in physical activity, environment, or pathological conditions. For example, space flight experiments using rats showed a reduction in total skeletal muscle mass of up to 37% as well as a significant loss of contractile proteins in type I but not type II fibers by 1-2 weeks of microgravity (4). Furthermore, the ratio of type I to type II fibers is associated with obesity and diabetes; the number of type I fibers is reduced in obese subjects and diabetic subjects compared with that in controls (5)(6)(7).
Skeletal muscle mass is positively regulated by hormones such as insulin-like growth factors (IGFs) 1 and growth hormone (8). Induction of hypertrophy in adult skeletal muscle by increased load is accompanied by the increased expression of IGF-1 (9). Systemic administration of IGF-1 results in increased skeletal muscle protein and reduced protein degradation (10). In addition, overexpression of IGF-1 blocks the agerelated loss of skeletal muscle (11). Supplementation of IGF-1 to muscle cells in vitro promotes myotube hypertrophy, suggesting that hypertrophy can be mediated by autocrine-or paracrine-produced IGF-1 (12). Thus, delivery of the IGF-1 gene specifically into skeletal muscle has been proposed as a genetic therapy for skeletal muscle disorders. A better understanding of the role of IGF-1 in skeletal muscle is therefore of great importance.
Specialized/differentiated myofiber phenotypes, including type I and type II fibers, are plastic and are physiologically controlled by variations in motor neuron activity. The influence of motor neuron activity on different types of skeletal muscle fibers is considered to be transduced via calcium signaling and downstream molecules such as calcineurin and the calmodulindependent kinase (CaMK) pathway (13). Signals generated by calcium/calcineurin/CaMK augment the transactivating function of Mef2 and/or NFAT and enhance type I fiber-specific gene expression (13)(14)(15)(16)(17)(18). More recently, it has been shown that a nuclear receptor cofactor (19,20), peroxisome proliferator activated receptor-␥ coactivator-1␣ (PGC-1␣) (21), drives the formation of type I fibers. Specifically, in transgenic mice expressing PGC-1␣, type II fibers are red in color, and PGC-1␣ activates expression of type I fiber-specific genes (22). We also reproduced the PGC-1␣-induced red appearance of skeletal muscle; both type I and type II fibers appear redder in transgenic mice overexpressing PGC-1␣ in skeletal muscle (23). FOXO1 (FKHR), FOXO4 (AFX), and FOXO3a (FKHRL1) are a subfamily of the forkhead type transcription factors (24,25). FOXO1 was originally cloned from a rhabdomyosarcoma because of its aberrant fusion with another transcription factor, PAX3, resulting from a chromosomal translocation (26). Recent studies have shown that the FOXO protein can also act as a cofactor of nuclear receptor activity (27)(28)(29)(30). FOXO family members have been shown to regulate various cellular functions. FOXOs influence the transcription of genes involved in metabolism (31)(32)(33)(34), the cell cycle (35,36), and apoptosis (37,38). In addition, FOXO1 can modulate cell differentiation; the constitutive active form of FOXO1 prevents the differentiation of preadipocytes (39) and stimulates myotube fusion of primary mouse myoblasts (40). Moreover, a FOXO1 knockout mouse has been reported; Foxo1 haploinsufficiency restores insulin sensitivity and rescues the diabetic phenotype in insulin-resistant mice by reducing the hepatic expression of glucogenetic genes and by increasing the adipocytic expression of insulinsensitizing genes (41). We have shown that FOXO1 expression is increased in skeletal muscle in energy-deprived states, such as in fasting mice, in mice with streptozotocin (STZ)-induced diabetes, and in mice after treadmill running (42). However, the physiological role of FOXO1 in skeletal muscle is still unclear. Although many studies have been performed using cultured cells, studies using animals with genetic modifications focused to the skeletal muscle remain to be conducted in order to understand the function of the FOXO family proteins in vivo. Meanwhile, it has been reported that FOXO1 and PGC-1␣ can physically interact and regulate gene expression in the liver (43). Given that PGC-1␣ is important for the differentiation of type I fibers, FOXO1 might be involved in this process. (Hereafter, we use "differentiation of muscle fiber" to mean "a switch from one fiber type to another fiber type.") On the other hand, a genetic study of Caenorhabditis elegans showed that DAF16, the worm counterpart of FOXO, functions as a suppressor of insulin receptor-like signaling (44). Thus, the FOXO family may act negatively in mammals as a downstream player in insulin or IGF signaling. As IGF-1 plays an important role in controlling skeletal muscle mass, FOXO1 might also be involved in this process.
To gain insight into the potential role of FOXO1 in skeletal muscle, including the control of skeletal muscle mass and the control of differentiation of muscle fiber type, we established transgenic mice specifically overexpressing FOXO1 in their skeletal muscle. Most interestingly, these mice showed reduced skeletal muscle mass, and the muscle was paler in color. Histochemical, physiological, and microarray analyses of these FOXO1 transgenic mice showed that FOXO1 is involved in the regulation of skeletal muscle mass and type I fiber gene expression. In addition, our results suggest that FOXO1 activa-tion may play a role in the impairment of skeletal muscle function including glycemic control.
Generating Transgenic Mice-The human skeletal muscle ␣-actin promoter (45) was provided by Drs. E. D. Hardeman and K. Guven (Children's Medical Research Institute, Australia). The human FOXO1 cDNA was as described previously (42). The transgene (Fig. 1A) was excised from agarose gel and purified for injection (2 ng l Ϫ1 ). Fertilized eggs were recovered from C57BL/6 females crossed with C57BL/6 males and microinjected at Japan SLC Inc. (Hamamatsu, Japan). The mice were maintained at a constant temperature of 22°C with fixed artificial light (12-h light and 12-h dark cycle). Care of the mice was conducted in accordance with the institutional guidelines.
Histological Analyses-Skeletal muscle (soleus) samples were frozen in liquid nitrogen-cooled isopentane, and transverse serial sections were stained with ATPase at pH 4.3 to detect type I fibers and at pH 10.5 to detect type II fibers (48). The ratio of type I fibers to type II fibers and the size (area) of skeletal muscle cells were determined by counting cell numbers in six randomly selected cross-section areas (each 900 m 2 ) stained with ATPase at pH 4.3.
Blood Analysis-Blood samples were obtained from mice tail tips for hormone and metabolite determination under feeding conditions. Immunoreactive insulin was measured by an insulin assay kit (Morinaga, Kanagawa, Japan), free fatty acid by NEFA C-test Wako (Wako Biochemicals, Osaka, Japan), lactate by the lactate reagent (Sigma), and glucose by the TIDEX glucose analyzer (Sankyo, Tokyo, Japan).
Running Wheel Activity-Mice were housed individually in cages (9 ϫ 22 ϫ 9 cm) equipped with a running wheel (20-cm in diameter, Shinano Co., Tokyo, Japan). Each wheel revolution was registered by a magnetic switch, which was connected to a counter. The number of revolutions was recorded daily for 6 days.
Oral Glucose and Insulin Tolerance Test-For the oral glucose tolerance test, D-glucose (1 mg/g of body weight, 10% (w/v) glucose solution) was administered with a stomach tube after an overnight fast. Blood samples were obtained by cutting the tail tip before and 30, 60, and 120 min after glucose administration. For the insulin tolerance test, human insulin (Humulin R; Lilly) was injected intraperitoneally (0.75 milliunits/g of body weight) into fed animals. Blood glucose concentrations were measured using a TIDEX glucose analyzer (Sankyo, Tokyo, Japan).
Microarray Analyses-RNA was isolated from skeletal muscle (quadriceps) of sex-and age-matched FOXO1 mice (A1 and A2 lines) and control mice (males at 4 months of age, RNA from three mice of each group were combined). Each of the combined samples was hybridized to the Affymetrix MGU74A microarray, which contains 12,489 genes including ESTs, and analyzed with the Affymetrix Gene Chip 3.1 software as described previously (49). Of the 12,489 genes including ESTs analyzed, 2500 (nontransgenic control mice), 2490 (line A1, transgenic), and 2510 (line A2, transgenic) genes were expressed at a substantial level (absolute call is present and average difference is above 150). Genes were classified on the basis of the biological function of the encoded protein, using a previously established classification scheme (50). The classification scheme was composed of seven major functional categories and several minor functional categories within the major categories.
Statistical Analyses-Statistical comparisons of data from the experimental groups were performed by the one-way analysis of variance, and groups were compared using the Fisher's protected least significant difference test (Statview 5.0, Abacus Concepts, Inc., Berkeley, CA). The glucose and insulin tolerance curves were compared by repeated measure analysis (Statview 5.0, Abacus Concepts). When significant, groups were compared by the Fisher's protected least significant difference test. Statistical significance was defined as p Ͻ 0.05.

RESULTS
Creation of FOXO1 Mice-The human skeletal muscle ␣-actin promoter (45) was used to drive the expression of the human FOXO1 transgene in mice (Fig. 1A). During development, cardiac muscle ␣-actin is the predominant isoform of sarcomeric ␣-actin in mice, and the switch to skeletal muscle ␣-actin occurs postpartum (45). Thus, by using the skeletal muscle ␣-actin promoter, the possibility that embryonic expression of FOXO1 might interfere with development was minimized. We obtained two independent lines of transgenic mice (lines A1 and A2). Southern blot analysis of DNA obtained from mouse tails was performed as shown in Fig. 1B. The transgene copy number of each animal was estimated by densitometric scanning of the autoradiographs from the Southern blots.
Expression of the FOXO1 transgene was evaluated by Northern blot analysis with RNA isolated from the tissues of FOXO1 mice and age-matched control mice at 8 weeks of age (Fig. 1C). The use of this promoter resulted in predominantly high expression levels of the FOXO1 transgene in skeletal muscle (about 3.5 kb). The A2 line showed expression levels of the FOXO1 transgene in skeletal muscle that were similar to or slightly higher than that in the A1 line. Transgene expression was observed not only in the gastrocnemius and quadriceps but also in other areas of skeletal muscle including the tibialis anterior, extensor digitorum longus (EDL), and soleus (not shown). The blot was then re-hybridized with a cDNA probe of Gadd45␣, an authentic target gene of FOXO1 (51,52). As expected, induction of the expression of Gadd45␣ was observed in skeletal muscle but not in other tissues in both FOXO1 transgenic mouse lines (Fig. 1C), indicating that the transgene expressed a functional FOXO1 protein. By using an antibody that recognizes both human and mouse FOXO1, we confirmed FIG. 1. Creation of FOXO1 transgenic mice. A, map of the 5-kb construct used for transgenic microinjection. The transgene was under the control of the human skeletal muscle ␣-actin promoter and included exon 1 and the intron of the human skeletal muscle ␣-actin gene as well as the bovine growth hormone polyadenylation site (45). B, characterization of FOXO1 mice. Two transgenic lines, A1 and A2, were identified by Southern blot analyses of DNA obtained from the tail of each mouse. The copy number was 2 for A1 and 10 for A2, as estimated by densitometric scanning of the autoradiographs of the Southern blot. C, expression of the FOXO1 transgene in mice. Northern blot analysis of human FOXO1 mRNA expression in tissues from FOXO1 mice (line A1 and A2) and nontransgenic control mice. RNAs from brain, brown adipose tissue (BAT), heart, kidney, liver, lung, skeletal muscle (gastrocnemius (Gastro.) and quadriceps (Quadri.)), and white adipose tissue (WAT) were analyzed. The blots were re-hybridized with the Gadd45␣ probe. Each lane contained 20 g of total RNA. 28 S ribosomal RNA staining of a sample from control mice is shown. Similar staining was observed in samples from transgenic mice (not shown). D, expression of the FOXO1 protein in the skeletal muscle of FOXO1 mice. Protein extracts (30 g per lane) were subjected to SDS-PAGE. The FOXO1 protein was detected by immunoblotting. The densitometric ratio is shown below the autoradiogram (the control was set as 100). E, comparison of representative samples of dissected skeletal muscle (TA, tibialis anterior; Sol, soleus; Gastro, gastrocnemius; Quadri, quadriceps) between FOXO1 mice and littermate control mice. Legs were removed from 4-month-old (lines A1 and A2) transgenic mice and agematched control mice. Tibialis anterior, gastrocnemius, and quadriceps contain a mixture of type I and II fibers; EDL is enriched in type II fibers, and soleus is enriched in type I fibers (control). Average dry mass (n ϭ 3 in each group) is shown below the panel. Muscles were smaller in size and paler in color in FOXO1 mice than in control mice. the presence of the FOXO1 protein in the skeletal muscle of FOXO1 mice (Fig. 1D). An ϳ2.2-fold (line A1) and 3-fold (line A2) increase in FOXO1 protein levels was observed. These increases were at the physiological level, since 24-h fasting has been shown to increase FOXO1 protein content by 2.5-3-fold (Ref. 53 and data not shown).
FOXO1 Mice Are Small-The apparent phenotype observed in FOXO1 mice was small stature and thinner legs than the control mice. Both male and female transgenic mice weighed about 10% less than the control mice at 5 weeks of age (not shown). We used DEXA to measure the lean body mass (body weight excluding fat weight) and the content of fat in the whole body of the A1 line (at 5 months of age) and the A2 line (at 4 months of age) in age-and sex-matched control mice (Table I). Both body weight and lean body mass were significantly lower in both male and female FOXO1 mice (both lines) than in control mice. However, the fat content per total body weight of both FOXO1 mouse lines was comparable with that of nontransgenic mice (Table I). Thus, the decrease in body weight of the FOXO1 mice is not caused by a decrease in body fat but by a decrease in lean body mass. Consistent with the data on decreased lean body mass, the skeletal muscles in FOXO1 mice were smaller in size and dry mass, as well as paler in color than those of control mice (Fig. 1E). Consumption of food per body weight was not significantly different between FOXO1 mice and control mice (Table I). Blood metabolite (free fatty acid, lactate, and glucose) and insulin levels did not differ significantly between FOXO1 mice and the controls (Table I).
Microarray Analysis-To obtain information on changes in gene expression in FOXO1 mice, we performed microarray analysis using RNA samples from skeletal muscle (quadriceps) of transgenic and control mice. Most interestingly, the largest category of genes with suppressed expression in the transgenic mice was those involved in cell structure. Namely, about half of the down-regulated genes were classified as cytoskeletal proteins (Table II). The FOXO1-induced genes were distributed throughout various categories (not shown, see Supplemental Material 1).
In the skeletal muscle of FOXO1 mice, there was a decrease in the expression levels of genes related to structural proteins of the type I fiber (slow twitch oxidative, red muscle), such as slow muscle isoforms of myosins (Table II, line numbers 1, 4, and 6), slow isoforms of troponins (Table II, line numbers 2, 5,  and 7), ␣-tropomyosin slow type (Table II, line number 13), myoglobin (Table II, line number 12), and mtCK (Table II, line  number 15), which are abundant in type I fibers (54). This is consistent with the observation that the skeletal muscles of FOXO1 mice are pale (Fig. 1E). In the microarray, the expression of mitochondrial oxidative metabolism genes, such as the electron transport system, did not differ between FOXO1 mice and controls (not shown). In large mammals such as humans, type I fibers are higher in mitochondrial content and more dependent on oxidative metabolism than type II fibers. In small mammals (e.g. mouse and rat), a large amount of mitochondria is seen in type II fibers as well as type I fibers (2). The large amount of mitochondria in both type I and type II fibers in mice would explain the unchanged gene expression of the mitochondrial electron transport system, although expression of type I fiber genes was markedly suppressed. In addition, the gene expression of type II fiber isoforms did not differ (not shown). Namely, expression of genes preferentially abundant in type I fibers appears to be suppressed in the skeletal muscle of FOXO1 mice.
Northern Blot Analysis of Representative Genes-We recognize the limitation of single microarray assays, as they can contain certain noise in the data. Thus, to verify the changes of gene expression found in the microarray analysis, we performed Northern blot analysis by using probes for several genes. In addition to representative genes in the list (Table II), we also analyzed several additionally selected genes of type I fiber or type II fiber markers or genes that may be involved in fiber differentiation. FOXO1 overexpression did not significantly affect mRNA levels of the other FOXO members, FOXO4 and FOXO3a ( Fig. 2A). Consistent with the microarray data, a reduction in gene expression was confirmed for type I fiber proteins, such as troponin C (slow) (  (Fig. 2A). On the other hand, expression levels of genes for components of the mitochondrial electron transport system, such as cytochrome c oxidase II and IV (COX II and IV), and the F 0 ,F 1 -ATPase, were not markedly changed in the skeletal muscle of FOXO1 mice. Next, we examined type II fiber genes. The expression of genes for troponin I (fast), troponin T (fast), and MLC (fast) did not differ between FOXO1 mice and control mice. Thus, the results of the microarray analysis were confirmed by Northern blot analysis. In addition, given that Mef2, NFAT, CaMK, and PGC-1␣ have been implicated recently in regulating gene expression in type I fibers (14 -18, 22), we also examined the level of their expression in skeletal muscle of control and FOXO1 mice. PGC-1␣ mRNA levels were slightly increased in the skeletal muscle of FOXO1 mice (line A2). Most interestingly, expression levels of Mef2c and CaMK were reduced in FOXO1 mice. FOXO1-mediated down-regulation of type I fiber genes may, in part, be regulated by Mef2c and CaMK.
Moreover, we examined the expression levels of genes whose expression levels are known to be changed during skeletal

FOXO1 Reduces Skeletal Muscle Function
muscle atrophy such as caused by fasting, cachexia, and STZinduced diabetes (55). Specifically, gene expression of atrogin 1/MAFbx, MuRF1 (both are ubiquitin ligases), and cathepsin L (a lysosomal protease) is up-regulated and IGFBP5 is downregulated during skeletal muscle atrophy (55). In our Northern blot analysis, the level of atrogin 1 expression was increased in the A2 line of FOXO1 mice, which has less skeletal muscle, but not in the A1 line, which also has less skeletal muscle mass than nontransgenic controls. In both the A1 and A2 lines of FOXO1 mice, the expression of cathepsin L and IGFBP5 was increased and decreased, respectively. The MuRF1 mRNA level was not changed. Thus, atrophy-related gene expression changes including that of protein degradation likely occurred in the skeletal muscle of FOXO1 mice. Western Blot Analysis of the Skeletal Muscle of FOXO1 Mice and PGC-1␣ Mice-We examined the expression of various gene products of FOXO1 mice at the protein level by Western blot analysis (Fig. 2B). Protein extracts from the skeletal muscle of FOXO1 mice (A1 and A2 lines) and wild-type control mice were used. For comparison, we analyzed protein extracts from the skeletal muscle of PGC-1␣ transgenic mice, which we previously analyzed (23). Protein levels of troponin I (slow) and myoglobin, which are rich in type I fibers, were increased in PGC-1␣ mice but decreased in FOXO1 mice (Fig. 2B). On the other hand, the protein level of troponin I (fast), which is rich in type II fibers, was decreased in PGC-1␣ mice but not in FOXO1 mice (Fig. 2B). Thus, Western blot analysis of the protein expression of genes for type I and type II fibers was consistent with the results of mRNA expression analysis.
Histological Analysis of Skeletal Muscle of FOXO1 Mice-We examined the relationship between the change in type I fiber gene expression and actual muscle fiber morphology in the skeletal muscle (soleus) of transgenic mice using light microscopy and histochemical procedures (A1 line, 4 months after birth; A2 line, 3 months after birth). Distinction between type I and type II fibers can be made by myosin ATPase staining at different pH values. Specifically, at pH 10.5, type II fibers are well stained but not type I fibers, and at pH 4.3, type I fibers are well stained but not type II fibers (2). ATPase staining revealed that skeletal muscle cells (both type I and type II fibers) in the FOXO1 mice are smaller than those of the control mice (average cross-sectional area of muscle fibers; A1 line, 11.5 Ϯ 0.8 m 2 in FOXO1 mice and 20.0 Ϯ 2.7 m 2 in control mice; A2 line, 9.8 Ϯ 0.5 m 2 in FOXO1 mice and 14.1 Ϯ 1.9 m 2 in control mice) and had fewer type I fibers than those in the control mice (average; A1 line, 28.6 Ϯ 1.3% in FOXO1 mice and TABLE II Gene with decreased expression in the skeletal muscle of FOXO1 mice The expression levels of 22 genes were significantly decreased in both the A1 and A2 lines of FOXO1 mice. The genes are listed in the order of greatest fold change in expression in skeletal muscle from line A1 mice relative to control mice. Fold change calculations were carried out as an indication of the relative change of each transcript represented on the probe array. The average difference value is a marker of abundance of each gene. Categories and subcategories are based on a previously established classification scheme (50) and literature review. Change (2) indicates that expression is significantly decreased compared with control mice.  (Fig. 3A). Immunohistochemistry with antibodies to myoglobin (present at high concentrations in type I fibers) confirmed the reduction in the number of type I fibers in the skeletal muscle of FOXO1 mice (not shown). Skeletal muscle samples from FOXO1 mice had no structural abnormalities such as mitochondrial abnormalities, glycogen accumulation, vacuolar formation, and muscle fiber degeneration (not shown).
Running Wheel Activity of FOXO1 Mice-The mass and fiber composition of skeletal muscle are important for physical ex-ercise. Type I fibers are more resistant to fatigue than type II fibers (2). As the FOXO1 mice had decreased total skeletal muscle mass and fewer type I fibers, they may have a low capacity for endurance, such as that needed in a marathon. We then compared the running wheel activity (spontaneous locomotive activity) in FOXO1 mice and control mice. Mice were transferred to cages with a running wheel and monitored daily for the number of wheel revolutions made for 6 days. Both lines of FOXO1 mice showed significantly fewer wheel revolutions (Fig. 3B). The decrease in running wheel activity suggested that FOXO1 mice were less able to sustain continuous muscle contractions than control mice, which is consistent with the reduction in the mass of skeletal muscle and the number of type I fibers.
Oral Glucose Tolerance Test and Insulin Tolerance Test on FOXO1 Mice-Skeletal muscle is important for glucose metabolism. To examine whether the decreased skeletal muscle mass of FOXO1 mice is affecting their systemic glucose homeostasis, we examined oral glucose tolerance and insulin tolerance in FOXO1 mice. Glucose tolerance was impaired in both lines of FOXO1 mice, namely peak blood glucose values in FOXO1 mice were elevated significantly above those of the control mice (Fig.  3C). The insulin tolerance test clearly demonstrated that the glucose-lowering effects of insulin were impaired in both the A1 and A2 lines of FOXO1 mice, compared with those in age-and sex-matched control mice (Fig. 3D). FOXO1 mice showed a low capacity for glucose metabolism and decreased insulin sensitivity. Adipose tissue, another organ playing a role in glucose metabolism, appears not to be involved in this impaired glycemic control because 1) body fat did not differ between FOXO1 mice and control mice (Table I), and 2) gene expression of glucose transporter 4, which is a rate-limiting molecule of insulin-dependent glucose intake (56), was not decreased in adipose tissue of FOXO1 mice (see Supplemental Material 2). FOXO1 mice may therefore represent a certain type of diabetic state in humans.
Change in Endogenous FOXO1 Expression by Physical Inactivity-We performed Northern blot analysis with RNA from the skeletal muscle of mice maintained under a long period of physical inactivity. The right hindlimbs of wild-type mice were immobilized in plaster casts, and the left hindlimbs were left freely moving for the control sample. After 3 weeks in the plaster casts, skeletal muscle (gastrocnemius) weight of the right hindlimbs was significantly decreased compared with that in the controls (average, 88 Ϯ 12 mg for immobilized and 149 Ϯ 6 mg for freely moving controls, n ϭ 3, p Ͻ 0.05). As shown in Fig. 4, the gene expression of troponin C (slow), myoglobin, and mtCK but not MLC (fast) and troponin T (fast) was markedly decreased in the plaster-casted muscle. At the same time, endogenous FOXO1 mRNA was increased in the immobilized muscle (Fig. 4). Furthermore, Gadd45␣ was increased in the same sample. In addition, cathepsin L, but not atrogin 1 and MuRF1, were increased (Fig. 4). Thus, mRNAs of endogenous FOXO1, Gadd45␣, and cathepsin L were increased; skeletal muscle mass was decreased, and the expression of type I fiber genes but not type II fiber genes were decreased. The gene expression changes observed in the plaster-casted skeletal muscle were similar to the changes observed in the FOXO1 mice ( Fig. 2A). These results further support the involvement of FOXO1 in the negative regulation of skeletal muscle mass and the expression of type I fiber genes. DISCUSSION To gain insight into the role of FOXO1 in skeletal muscle in vivo, we established transgenic mice overexpressing human FOXO1. The FOXO1 transgene was predominantly expressed in the skeletal muscle, and the increase in FOXO1 protein expression was within physiological levels. Most interestingly, the skeletal muscle of FOXO1 mice weighed less and was paler FIG. 3. A, histological analysis of skeletal muscle. Light microscopy of ATPase (pH 4.3 for type I fibers and pH 10.5 for type II fibers)-stained transverse sections of skeletal muscle (soleus) specimens from FOXO1 mice (line A2) and control littermates at 3 months of age. Bars, 50 m. Skeletal muscle fibers of FOXO1 mice were thinner and contained fewer type I fibers than that of control mice. B, running wheel activity of FOXO1 mice. Mice were housed individually in cages equipped with a running wheel (20 cm in diameter). The number of revolutions made was recorded daily for 6 days, and the cumulative values are shown. Open column, control; closed column, FOXO1 mice. Running wheel activity was significantly (p Ͻ 0.05) reduced in FOXO1 mice (line A1, left; line A2, right) compared with control mice. Mice used were females at 10 weeks (line A1) and 9 weeks (line A2) of age. Numbers of animals used are as follows: line A1, control, n ϭ 6; FOXO1 mice, n ϭ 5; line A2, control, n ϭ 4; FOXO1 mice, n ϭ 3. Because male mice responded similarly, only the data from female mice are shown. C and D, oral glucose tolerance tests (C) and insulin tolerance tests (D) on FOXO1 mice. For the oral glucose tolerance test, mice were fasted overnight and given D-glucose (1 mg/g body weight) orally by a stomach tube. Blood glucose levels were determined at the times indicated. For the insulin tolerance test, mice were allowed free access to food and then given 0.75 milliunits of human insulin/g of body weight. Blood glucose levels were measured at the indicated time points. Mice used were males at 10 weeks (line A1) and 9 weeks (line A2) of age. The numbers of animals used were: line A1, control, n ϭ 6; FOXO1 mice, n ϭ 5; line A2, control, n ϭ 5; FOXO1 mice, n ϭ 4. in color. The results of gene expression analyses showed that type I (red muscle) fiber-related gene expression was decreased in the skeletal muscle of FOXO1 mice. In addition, histological examinations showed that the skeletal muscle of FOXO1 mice had fewer type I fibers and smaller type I and type II fibers. Consistently, under long time physical inactivity by immobilizing skeletal muscle in plaster casts, an increased expression of endogenous FOXO1 mRNA and a markedly decreased expression of genes related to type I fibers were observed. These results suggest that FOXO1 is a negative regulator of skeletal muscle mass and expression of type I fiber-related genes. Moreover, FOXO1 mice showed poor glycemic control and low capacity for physical exercise, which involves the skeletal muscles, especially type I fibers. These phenotypes are consistent with the decreased mass of skeletal muscle including type I fibers in FOXO1 mice.
How does FOXO1 affect the skeletal muscle, including the reduction of mass of both type I and type II fibers and the suppressed expression of type I fiber genes? In the following, we discuss the possibility of involvement of FOXO1 in 1) growth, 2) protein degradation, and 3) differentiation of skeletal muscle.
1) FOXO1 may suppress increase of skeletal muscle mass. A genetic study of C. elegans showed that DAF16, the worm counterpart of FOXO, functions as a suppressor of insulin receptor-like signaling (44). Thus, the FOXO family might act negatively in mammals as a downstream player in insulin or IGF signaling. IGF-1 stimulates the proliferation of skeletal muscle satellite cells (57). Mature skeletal muscle fibers are not able to proliferate. Skeletal muscle satellite cells, mononuclear cells located between the basement membrane and the plasma membrane of myofibers in mature cells, are important in postnatal skeletal muscle hypertrophy because of their ability to add new myonuclei into growing myofibers. Machida et al. (58) showed that FOXO1 inhibited IGF-1-mediated skeletal muscle cell proliferation. In primary skeletal muscle satellite cells, FOXO1 activates the promoter of p27 Kip1, an inhibitor of the cell cycle at the G 1 stage, which leads to inhibition of cell proliferation, and addition of IGF-1 reverses the FOXO1-mediated activation of the p27 Kip1 promoter (58). Unexpectedly, p27 Kip1 mRNA expression was unchanged in the skeletal muscle of FOXO1 mice compared with that of controls (not shown). As the ratio of satellite cells is very small in total skeletal muscle, the increased expression of p27 Kip1 in satellite cells may not have been detected in our assay. On the other hand, we showed enhanced expression of Gadd45␣, an inhibitor of the cell cycle at the G 2 stage (51,52), in the skeletal muscle of FOXO1 mice (Figs. 1 and 2A). As a 0.7-kb stretch of the rat skeletal muscle ␣-actin promoter is active in skeletal muscle satellite cells (59), the FOXO1 transgene, driven by a 2-kb stretch of the human skeletal muscle ␣-actin promoter (45), is likely to be expressed in the skeletal muscle satellite cells of the FOXO1 mice. Thus, the increased amount of Gadd45␣ and possibly p27 Kip1 in the skeletal muscle satellite cells of FOXO1 mice may have suppressed the proliferation of satellite cells and caused a decrease in skeletal muscle mass (size).

FOXO1 Reduces Skeletal Muscle Function
2) FOXO1 may increase the degradation rate of skeletal muscle proteins. Gene expression of atrogin 1, MuRF1 (both are ubiquitin ligases), and cathepsin L (a lysosomal protease) is up-regulated and IGFBP5 is down-regulated during skeletal muscle atrophy caused by fasting, cachexia, STZ-induced diabetes, and other diseases (55). After we submitted our manuscript, a member of the FOXO family, FOXO3a, was reported to activate the gene expression of atrogin 1, and addition of IGF-1 was found to reverse the FOXO3a-mediated activation of the atrogin 1 promoter (60). Overexpression of an active form of FOXO3a reduces the size of skeletal muscle fibers, both in vivo and in vitro (60). In addition, another group reported that overexpression of an active form of FOXO1 in C2C12 muscle cells did not change the base-line expression of atrogin 1 and MuRF1, but the active form of FOXO1 suppresses IGF-1-mediated repression of atrogin 1 and MuRF1 expression induced by glucocorticoids (61). This suggests that FOXO1 expression is not sufficient for inducing atrophy-related genes, but FOXO1 is negatively involved in IGF-1-mediated suppression of atrophy of skeletal muscle. In our Northern blot analysis, the level of atrogin 1 was increased in the A2 line but not in the A1 line of FOXO1 mice, although both had less skeletal muscle mass than the nontransgenic controls. In both the A1 and A2 lines of FOXO1 mice, the expression of cathepsin L and IGFBP5 was increased and decreased, respectively. MuRF1 mRNA levels were not altered in both lines. Thus, atrophy-related protein degradation probably occurs in the skeletal muscle of FOXO1 mice and could explain, in part, the decrease in skeletal muscle mass of the FOXO1 mice. However, the increase in atrogin 1 is unlikely to be enough to cause the decrease in skeletal muscle mass of FOXO1 mice, because the expression level did not change in the A1 line of FOXO1 mice. This is consistent with the description by Sandri et al. (60) that overexpression of atrogin 1 alone does not cause myotube or muscle atrophy. On the other hand, IGFBP5 is reported to modulate the activity of IGF-1 (62), and hence decreased expression of IGFBP5 may contribute to the decrease in skeletal muscle mass by affecting IGF-1 action. FOXO1 transgene expression was observed in both type I fiber-rich soleus and type II fiber-rich EDL. Thus, changes in the expression of atrophy-related genes may be an alternative molecular explanation for the decreased skeletal muscle mass, including the size of both type I and type II fibers of FOXO1 mice.
3) Does FOXO1 inhibit the differentiation of type I fibers? The FOXO1 transgene is expressed in muscles rich in both type I and type II fibers. How does it cause the selective reduction of gene expression in type I fibers but not in type II fibers? It is possible that FOXO1 suppresses the function of a factor(s) that is preferentially expressed in type I fibers and therefore activates gene expression only in type I fibers. One candidate for such a factor is PGC-1␣, which is known to be preferentially expressed in type I fibers and enhances type I fiber gene expression (22). As the FOXO1 protein can interact with the PGC-1␣ protein (43), FOXO1 may affect certain functions of PGC-1␣. FOXO1 may inhibit PGC-1␣ function via its binding to PGC-1␣. FOXO1 itself is a transcription factor. In addition, several reports (27)(28)(29)(30) have shown that FOXO1 acts as a corepressor of nuclear receptors, whereas PGC-1␣ can activate many nuclear receptors (21,63). Although to our knowledge nuclear receptors have not been shown to be involved in type I fiber-specific gene expression, a certain nuclear receptor(s) and transcription factor(s), which can interact with both FOXO1 and PGC-1␣, may be involved in a process positively and negatively regulated by PGC-1␣ and FOXO1, respectively. Further studies are required to examine this possibility. Besides, although PGC-1␣ stimulates the differentiation of type I fibers, in FOXO1 mice, gene expression was reduced in type I fibers but was not affected in type II fibers. Thus, fiber differentiation (switching) from type I to type II is not likely to occur in FOXO1 mice, and FOXO1 appears not to be involved in fiber differentiation.
FOXO1 mice showed a clear phenotype related to the function of skeletal muscle. Specifically, spontaneous locomotor activity was lower in FOXO1 mice than in control mice (Fig.  3B). In addition, FOXO1 mice had impaired oral glucose tolerance and impaired insulin-mediated glucose-lowering effects (Fig. 3, C and D). Elderly humans have been reported to show FIG. 4. Gene expression in skeletal muscle immobilized in plaster casts. The right hindlimbs of mice at 9 weeks of age were immobilized in plaster casts, and left hindlimbs of the mice were kept free for the control sample. After 3 weeks of immobilization in plaster casts, Northern analysis was performed on total RNA (20 g per lane) isolated from the skeletal muscle (gastrocnemius) of right hindlimbs and left hindlimbs. Plus and minus denote with or without immobilization, respectively. The names of the genes examined are on the right of the autoradiograms. A typical autoradiogram, representative of three independent mice with similar results, is shown. The densitometric ratio is shown below the autoradiograms (the control was set as 100). a progressive loss of muscle fibers associated with diabetes, obesity, and decreased physical activity (sarcopenia). Overexpression of IGF-1 in skeletal muscle prevents the age-related decline in muscle mass (11,57). As described above, the reduced skeletal muscle mass in FOXO1 mice may be caused by the suppression of IGF signaling during skeletal muscle formation, and FOXO1 may therefore be involved in age-related sarcopenia in humans. FOXO1 mice may be valuable as a model for human diseases related to loss of muscle fibers. Further analysis of the molecular mechanisms of FOXO1 action in skeletal muscle is important from a clinical as well as a sports science perspective.