Skeletal muscle is one of the primary tissues responsible for insulin resistance and type 2 diabetes (T2D). The fetal stage is crucial for skeletal muscle development. Obesity induces inflammatory responses, which might regulate myogenesis through Wnt/β-catenin signaling. This study evaluated the effects of maternal obesity (>30% increase in body mass index) during pregnancy on myogenesis and the Wnt/β-catenin and IKK/NF-κB pathways in fetal skeletal muscle using an obese pregnant sheep model. Nonpregnant ewes were assigned to a control group (C; fed 100% of National Research Council recommendations; n = 5) or obesogenic (OB; fed 150% of National Research Council recommendations; n = 5) diet from 60 days before to 75 days after conception (term ∼148 days) when fetal semitendenosus skeletal muscle was sampled for analyses. Myogenic markers including MyoD, myogenin, and desmin contents were reduced in OB compared with C fetal semitendenosus, indicating the downregulation of myogenesis. The diameter of primary muscle fibers was smaller in OB fetal muscle. Phosphorylation of GSK3β was reduced in OB compared with C fetal semitendenosus. Although the β-catenin level was lower in OB than C fetal muscle, more β-catenin was associated with FOXO3a in the OB fetuses. Moreover, we found phosphorylation levels of IKKβ and RelA/p65 were both increased in OB fetal muscle. In conclusion, our data showed that myogenesis and the Wnt/β-catenin signaling pathway were downregulated, which might be due to the upregulation of inflammatory IKK/NF-κB signaling pathways in fetal muscle of obese mothers.
- nuclear factor-κB
- skeletal muscle
about 29% of pregnant women are obese (26). Although accumulating evidence shows that maternal obesity (MO) predisposes offspring to obesity and type 2 diabetes (T2D; Ref. 4, 37, 60), the causative mechanisms remain poorly defined.
Skeletal muscle is one of the principal sites for glucose and fatty acid utilization and composes ∼40–50% of body mass of adults (32). It is the primary tissue responsible for insulin resistance in obese and T2D subjects (33, 40, 45). The fetal period is crucial for skeletal muscle development, because no net increase in the number of muscle fibers occurs after birth (59). Skeletal myogenesis involves the differentiation of mesodermal cells to myogenic precursor cells, myoblasts, and then myotubes, which is regulated by sequential expression of Pax7, a marker for myogenic precursor cells, and myogenic regulatory factors comprising Myf-5, MyoD, myogenin, and myogenic regulatory factor (MRF)-4 (44, 49). The initial stage of myogenesis occurs during the embryonic stage forming primary myofibers. The second wave of myogenesis occurs around midgestation in sheep and human fetuses, which forms the majority of muscle fibers (7) and is susceptible to the availability of maternal nutrients, with maternal undernutrition reducing secondary myofiber numbers (59). Rodents are born highly immature and secondary myogenesis occurs late in gestation and during the neonatal period, which is quite different from human fetal muscle development. Due to the similarity between sheep and human pregnancy, sheep have been extensively used for pregnancy and fetal developmental studies (10, 15, 29). Currently, the impact of MO on fetal muscle development is poorly understood. In our previous study, we observed that MO enhanced the expression of adipogenic markers in fetal muscle, which was associated with systemic inflammatory response (60). In this study, we further examined the impact of MO (>30% increase in body mass index) on fetal myogenesis and associated signaling pathways.
The canonical Wnt/β-catenin signaling pathway is essential for myogenesis (5, 35). Activation of Wnt/β-catenin signaling enhances the stability of β-catenin, which enters the nuclei, where β-catenin forms a complex with the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to increase expression of MRFs, including Myf5 and MyoD, enhancing myogenesis (14).
Obesity leads to low-grade inflammation (19, 48, 56). NF-κB is a primary regulator of the inflammatory response (3). An increasing number of studies (6) have implicated NF-κB in skeletal muscle differentiation, which may be mediated through forkhead transcription factors (FOXOs). FOXOs (FOXO1, FOXO3a, and FOXO4) are activated by inflammatory signaling (1, 54). β-Catenin interacts with FOXO to promote exit from the cell cycle and entry into quiescence (9). In cultured cells, oxidative stress causes diversion of the limited pool of β-catenin from TCF-mediated transcription to FOXO-mediated transcription (2). Since inflammation and oxidative stress are associated (20, 21), it is plausible to hypothesize that inflammation associated with MO in fetal muscle diverts β-catenin from TCF/LEF transcription factors to combine with FOXO, downregulating myogenesis.
MATERIALS AND METHODS
Care and use of animals.
All animal procedures were approved by the University of Wyoming Animal Care and Use Committee. The animal procedures have been previously described (60). Briefly, from 60 days before conception to day 75 of gestation (day of mating, day 0; term ∼148 days), multiparous Rambouillet/Columbia ewes were fed either a highly palatable diet at 100% [control (C)] of National Research Council recommendations or the same diet at 150% [obesogenic (OB)] of National Research Council recommendations on a metabolic body weight basis (BW0.75). Ewes on the OB diet developed severe obesity (60). At day 75 of gestation, the body condition score was 5.0 ± 0.3 vs. 8.3 ± 0.2 (body condition score: 1 = emaciated and 9 = extremely obese) and body weight was 70.1 ± 4.5 vs. 100.7 ± 4.9 kg (C vs. OB), respectively. The body fat content of C ewes was 17.7 ± 1.3%, while OB ewes had 28.6 ± 1.6% body fat (P < 0.01). After a 12-h overnight fast on day 75 of gestation, 10 pregnant sheep (5 C and 5 OB) with twin pregnancy were weighed and anaesthetized as previously described (60). Briefly, sedation was induced by intravenous ketamine (10 mg/kg) and anesthesia was induced and maintained by isoflurane inhalation. Under general anesthesia, fetuses were euthanized by exsanguination and then quickly removed to obtain weight and length, and the semitendenosus (St) muscles on both sides were collected and snap frozen in liquid nitrogen for biological analyses. Only one fetus from each ewe was used for further analyses. Although fetal sex did not affect fetal weight, fetal sex was balanced between treatments.
Antibodies against GSK3β, phospho-GSK3β at Ser9, β-catenin, phospho-IKKα/β at Ser176/180, phospho-IκBα at Ser32, phospho-NF-κB p65 at Ser536, FOXO3a, and horseradish peroxidase linked secondary antibody were purchased from Cell Signaling (Danvers, MA). MyoD antibody was purchased from GenScript (Piscataway, NJ). Antibodies against desmin, myogenin, and Pax7 were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences (Iowa City, IA). Anti-GAPDH antibody was purchased from Ambion (Austin, TX).
St muscle (0.1 g) was powdered in liquid nitrogen and homogenized in a polytron homogenizer (7-mm diameter generator) with 400 μl of ice-cold buffer containing 137 mM NaCl, 50 mM HEPES, 2% SDS, 1% NP-40, 10% glycerol, 2 mM PMSF, 10 mM sodium pyrophosphate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 mM Na3VO4, and 100 mM NaF, pH 7.4. The protein content of lysates was determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA; Ref. 58) and used for immunoblotting analyses as previously described (59).
St muscle (0.05 g) was powdered in liquid nitrogen and homogenized in a polytron homogenizer (7-mm diameter generator) with 500 μl of ice-cold lysis buffer containing 20 mM Tris (pH 7.5), 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 2.5 mM sodium pyrophosphate, 1 μg/ ml leupeptin, 1 mM Na3VO4, and 100 mM NaF. Samples were then microcentrifuged for 10 min at 14,000 g at 4°C, and 300 μl supernatant were precleared with 30 μl protein A-Sepharose bead slurry (50%; Rockland, Gilbertsville, PA) and incubated at 4°C for 1 h. The supernatant (200 μl) was taken and immunoprecipitated with anti-β-catenin antibody (1:200 dilution) and incubated with gentle rocking overnight at 4°C. Protein A-Sepharose bead slurry (50%, 20 μl) was added, and incubation was continued with gentle rocking for 2 h at 4°C. Immunoprecipitated samples were collected and washed with 500 μl of lysis buffer five times, and then they were resuspended with 20 μl of SDS sample buffer containing 4% β-mercaptoethanol. After being heated to 95°C for 5 min, the samples were analyzed by SDS-PAGE and Western blotting using anti-FOXO3a or anti-β-catenin antibodies.
Real-time quantitative RT-PCR.
Total RNA was extracted from the fetal sheep St muscle using TRI reagent (Invitrogen, Carlsbad, CA) and quantified by spectrophotometry. Total RNA (1 μg) was reverse transcribed into cDNA using a qScript cDNA synthesis kit (Quanta BioScience, Gaithersburg, MD). Primer sets used are shown in Table 1. RT-PCR was performed on an iQ5 RT-PCR detection system (Bio-Rad, Hercules, CA) using SYBR Green RT-PCR kit from Bio-Rad and the following cycle parameters: 94°C for 2 min and 40 cycles at 94°C for 15 s, 58°C for 30 s. Melting point dissociation curves and agarose gel electrophoresis were performed to confirm that only a single product was amplified. Results were expressed using the comparative cycle threshold (Ct) method (realquant 1.0; Roche Diagnostics). The ΔCt values were calculated for each gene of interest as the following: Ct of the detecting gene − Ct of the control gene tubulin. Tubulin was selected as the control gene since no difference in tubulin content was detected between treatments.
St muscle samples were fixed in 4% (w/v) paraformaldehyde in a phosphate buffer (0.12 M, pH 7.4), embedded in paraffin, and cut into sections (thickness = 5 μm). Sections were rehydrated using a series of incubations in xylene and ethanol solutions and then stained with Masson's trichrome stain for standard light microscopy. The relative diameter of primary myofibers and the space between muscle fibers were measured in 10 different microscopic fields of each section at ×400 magnifications using the Image J 1.41 software (National Institutes of Health, Bethesda, MD). Ten sections (every fifth section) were viewed for each sample. Only those fasciculi where primary and secondary myofibers were clearly differentiated were measured.
Statistical analyses were conducted according to our previous studies in sheep (58, 59). Briefly, each animal was considered as an experimental unit. Only one fetus from each ewe was used for analyses. Data were analyzed using the General Linear Model of Statistical Analysis System (SAS, 2000). Data are means ± SE. Statistical significance was considered as P < 0.05.
Fetal muscle and body weight increased due to MO.
As previously described, ewes fed the OB diet had much higher body weights and body condition scores than C ewes and developed severe obesity (60). The weight of fetal St muscle (C vs. OB = 0.49 ± 0.01 vs. 0.65 ± 0.05 g; P < 0.05) was dramatically increased due to MO, as well as the fetal body weight (C vs. OB = 268 ± 12 vs. 374 ± 10 g; P < 0.05; Ref. 60). No difference in the ratio of muscle to body weight was observed.
Expression of myogenic markers in fetal St muscle.
As shown in Fig. 1, MyoD, myogenin, and desmin protein expression was decreased in OB fetal muscle, indicating downregulation of myogenesis. No difference in Pax7 expression was observed. Since MyoD and myogenin are transcription factors regulating myogenesis, we further analyzed the mRNA expression of MyoD and myogenin. The mRNA for both transcription factors was downregulated, consistent with their protein expression (Fig. 2).
Diameter of primary muscle fiber was smaller in OB fetal St muscle.
The diameter of primary muscle fibers was smaller in OB compared with C fetal St muscle (Fig. 3). The diameter of secondary muscle fibers was too small to measure accurately. There were more spaces surrounding the muscle fibers and bundles of OB fetal St muscle (Fig. 3). Connective tissue, which was stained blue, was clearly observed in these spaces (Fig. 3).
Wnt/β-catenin expression was downregulated in OB fetal St muscle.
GSK3β is a negative regulator of β-catenin and in its unphosphorylated form promotes β-catenin degradation (38). The ratio of phosphorylation of GSK3β at Ser9 to total GSK3β protein levels was decreased (P < 0.05) in the fetal muscle of OB sheep (Fig. 4).
As shown in Fig. 5, the contents of β-catenin at both the mRNA and protein levels were reduced in OB fetal St muscle, with a 27.0 ± 3.8% decrease at the protein level and a 31.9 ± 7.8% decrease at the mRNA level (P < 0.05).
As shown in Fig. 6, the FOXO3a content increased in OB fetal St muscle but no change was detected in its mRNA expression. Immunoprecipitation results demonstrated that a higher amount of FOXO3a was associated with β-catenin in OB fetal St muscle (Fig. 6).
IKK/NF-κB signaling pathway was upregulated in OB fetal St muscle.
We further analyzed the IKK/NF-κB signaling pathway in fetal skeletal muscle. As shown in Fig. 7, the phosphorylation of all three key components (IKK, IκB, and NF-κB p65) of the IKK/NF-κB signaling pathway was increased in OB fetal St muscle (P < 0.05).
Consequences of MO on fetal skeletal muscle development.
Myocytes, adipocytes, and fibroblasts are all derived from mesenchymal stem cells. Fetal muscle development is largely separated into two stages. Primary myofibers are first formed in the embryonic stage, followed by the formation of secondary myofibers around midgestation in humans and sheep (59). Primary myofibers have peripherally located myofibrils surrounding an axial core of nuclei and cytoplasm (7, 50). The secondary myofibers are derived from myogenic progenitor cells, which are initially maintained in a proliferating, undifferentiated state (50). Those precursor cells differentiate into myoblasts and fuse to form secondary myofibers parallel to primary myofibers, forming the majority of skeletal muscle (7). The timing of formation of secondary myofibers coincides with adipogenesis, which is initiated at midgestation in the human and sheep fetuses, as well as fibrogenesis, which forms the perimysium and epimysium in fetal skeletal muscle (60). Up to now, most studies (11) regarding myogenesis have been focused on primary myogenesis in rodents. Studies mainly in rodents show that Wnt, Pax3, and Pax7, and four MRFs, including MyoD, Myf-5, myogenin, and MRF-4, are important regulators of myogenesis. The mechanisms regulating the formation of secondary myofibers, adipogenesis, and fibrogenesis are poorly studied.
The downregulation of myogenesis is expected to decrease the density of myocytes by diverting mesenchymal stem cells to differentiate in other directions such as adipogenesis and fibrogenesis. Previously, we (60) found no difference in the ratio of primary to secondary muscle fibers between C and OB fetal muscle. We report here that the diameter of primary myofibers was reduced in OB fetal muscle. In keeping with this view, key regulators of myogenesis, MyoD, and myogenin were reduced at both the protein and mRNA levels. In this study, we also observed that MO increased the spaces between muscle fibers and bundles. Although midgestation is too early to detect mature adipocytes, the markers for adipogenesis were enhanced in OB fetal muscle (60). The spaces appeared to be filled with collagens, indicating that fibrogenesis might also be enhanced. In summary, these data clearly show that MO altered fetal skeletal muscle development by changing both intramuscular matrix and muscle fibers themselves.
Satellite cells are a heterogeneous group of cells with myogenic capacity, which reside in the basal lamina of muscle fibers and are crucial for muscle growth and regeneration (16). The satellite cell niche is mainly composed of muscle fibers, basal lamina, intramuscular adipocytes, and fibroblasts, and it guides the activation, proliferation, and differentiation of satellite cells (31). Local inflammation and cytokines are crucial for satellite cell activation and differentiation (12, 51–53). Therefore, in this study, the downregulation of myogenesis, the detected inflammation response, and the enhancement of intracellular spaces change the satellite cell niche, which could potentially permanently change the behavior of progeny satellite cells during muscle regeneration and hypertrophy, thereby exerting long-term physiological effects. In addition, the downregulation of myogenesis is likely to reduce the functionality of skeletal muscle in offspring, such as reducing the force of contraction.
Wnt signaling and myogenesis.
The canonical Wnt/β-catenin signaling pathway plays a crucial role in the regulation of the embryonic, postnatal, and oncogenic growth of many tissues (5, 35). The Wnt family has more than 10 members that act by binding to Frizzled (Fzd) proteins on target cells (43), which in turn activate β-catenin-dependent and/or -independent signaling pathways (28). Binding of Wnt to Fzd activates Disheveled (Dvl), leading to GSK-3β phosphorylation and inactivation. As a result, β-catenin, the target of GSK-3β, is stabilized and enters the nucleus. By acting in a complex with members of the TCF/LEF family of transcription factors, β-catenin activates its target gene transcription (17, 25), including Myf5 and MyoD (13). β-catenin is a key mediator in this canonical Wnt/β-catenin pathway, which plays a critical role in embryonic myogenesis (24, 41, 57). In this study, we found that the ratio of phosphorylated GSK-3β to total GSK-3β was decreased and that it was correlated with decreased β-catenin levels in the fetal muscle of OB sheep. As GSK-3β phosphorylation leads to its inactivation and more β-Catenin accumulation and translocation to the nucleus-activating myogenic genes, both reduced GSK-3β phosphorylation and β-catenin levels are indicative of the downregulation of Wnt/β-catenin downstream signaling in OB fetal muscle.
Downregulation of Wnt/β-catenin signaling would be expected to decrease myogenesis. Downregulation of MyoD and myogenin expression as well as the myogenic marker desmin in St muscle from fetuses of OB ewes is associated with the observed downregulation of myogenesis in OB fetal muscle (44, 49). In our previous study (60), using the same animal model, the expression of adipogenic markers was enhanced in fetal muscle due to MO. When the two studies are combined, the data suggest that MO downregulates myogenesis but enhances adipogenesis in fetal muscle and Wnt/β-catenin signaling pathway might play an important role in this shift. Consistent with our findings, activation of the Wnt signaling pathway enhances myogenesis and inhibits adipogenesis in cultured mesenchymal stem cells (46).
The expression of MRFs is controlled by signals such as Wnts and Sonic hedgehog (Shh; Refs. 36, 42, 49) via Pax3 and Pax7 (30). Pax7 is a marker of cells committed to myogenetic lineage (39), and, thus, its expression was analyzed. However, no difference in Pax7 expression was observed between C and OB fetal muscle. The exact reason for this lack of difference is unclear but might be due to the fact that Pax7 is a marker of myogenic progenitor cells and it is rapidly downregulated in myogenic cells. The occurrence of the majority of myogenic commitment before midgestation may well explain lack of difference and why Pax7 expression at this stage is low.
β-Catenin, inflammation, and IKK/NF-κB signaling pathway.
In our previous study (60), we observed increased oxidative stress and higher TNFα levels in fetal muscle from OB ewes. In response to oxidative stress, FOXO is activated to promote mammalian cell survival by stimulating exit from the cell cycle and entry into quiescence (9). Inflammation activates FOXOs (1, 54). Chronic activation of NF-κB is detrimental to muscle development (6) by activating FOXO and promoting the association of β-catenin and FOXO (34). β-Catenin binds directly to FOXO and enhances FOXO transcriptional activity in mammalian cells, which is regulated by both insulin and oxidative stress signaling (18). In OB6 cells, oxidative stress has been demonstrated to cause diversion of the limited pool of β-catenin from TCF-mediated transcription to FOXO-mediated transcription (2). FOXO competes with TCF for interaction with β-catenin, thereby inhibiting TCF transcriptional activity and reducing the expression of its targeted genes, such as MyoD. Reduced binding between TCF and β-catenin is observed after FOXO overexpression and cellular oxidative stress (27). Oxidative stress decreased the amount of nuclear β-catenin- and TCF/LEF-dependent transcription (47), suggesting that oxidative stress in fetal muscle might be responsible for the downregulation of β-catenin and myogenesis.
Obesity leads to low-grade inflammation (19, 48, 56), which is associated with oxidative stress (20, 21). NF-κB is a primary regulator of inflammatory responses, and IKKβ is required for its activation (3). An increasing number of studies (6) have implicated NF-κB in skeletal muscle differentiation. In this study, the IKK/NF-κB signaling pathway was upregulated in OB fetal muscle. Although whether NF-κB promotes or inhibits skeletal muscle differentiation process remains to be well defined, more data seem to support the notion that it functions as an inhibitor of myogenesis (6). The underlying mechanisms may be that NF-κB p65 represses myogenesis in response to TNFα by suppressing MyoD synthesis (23) or through the induction of cyclin D1 (22). As demonstrated in the current study, such an effect is likely mediated by activating FOXOs and limiting the formation of the β-catenin/TCF transcription complex. It might also inhibit myogenesis by stimulating expression of the Polycomb group protein YY1 (6, 55).
In conclusion, this study showed that β-catenin content was downregulated in the fetal skeletal muscle of obese, overnourished sheep, and an associated downregulation of myogenesis was detected in OB fetal muscle. Inflammatory response in OB fetal muscle might be responsible for the upregulation of FOXO, which links to the downregulation of myogenesis by diverting β-catenin from forming an active transcription complex with TCF to induce the expression of MRFs.
This work was supported by Research Initiative Grants 2007-35203-18065 and 2006-55618-16914 from the U.S. Department of Agriculture-Cooperative State Research, Education, and Extension Service and National Institute of Child Health and Human Development Grant 1R03HD-057506 to M. Du and by the Division of Research Resources (IDeA Network of Biomedical Research Excellence) Grant P20 RR-016474-04 to S. P. Ford.
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