Mei J. Zhu

Fetal programming of skeletal muscle development in ruminant animals.

Enhancing skeletal muscle growth is crucial for animal agriculture because skeletal muscle provides meat for human consumption. An increasing body of evidence shows that the level of maternal nutrition alters fetal skeletal muscle development, with long-term effects on offspring growth and performance. Fetal skeletal muscle development mainly involves myogenesis (i.e., muscle cell development), but also involves adipogenesis (i.e., adipocyte development) and fibrogenesis (i.e., fibroblast development). These tissues in fetal muscle are mainly derived from mesenchymal stem cells (MSC). Shifting the commitment of MSC from myogenesis to adipogenesis increases intramuscular fat (i.e., marbling), improving the quality grade of meats. Strong experimental evidence indicates that Wingless and Int (Wnt)/beta-catenin signaling regulates MSC differentiation. Upregulation of Wnt/beta-catenin promotes myogenesis, and downregulation enhances adipogenesis. A lack of nutrients in early to midgestation reduces the formation of secondary muscle fibers in ruminant animals. Nutrient deficiency during mid- to late gestation decreases the number of intramuscular adipocytes and muscle fiber sizes. Knowledge of this regulatory mechanism will allow the development of strategies to enhance muscle growth and marbling in offspring, especially in the setting of nutrient deficiency.

Maternal obesity downregulates myogenesis and β-catenin signaling in fetal skeletal muscle

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/beta-catenin signaling. This study evaluated the effects of maternal obesity (>30% increase in body mass index) during pregnancy on myogenesis and the Wnt/beta-catenin and IKK/NF-kappaB 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 approximately 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 GSK3beta was reduced in OB compared with C fetal semitendenosus. Although the beta-catenin level was lower in OB than C fetal muscle, more beta-catenin was associated with FOXO3a in the OB fetuses. Moreover, we found phosphorylation levels of IKKbeta and RelA/p65 were both increased in OB fetal muscle. In conclusion, our data showed that myogenesis and the Wnt/beta-catenin signaling pathway were downregulated, which might be due to the upregulation of inflammatory IKK/NF-kappaB signaling pathways in fetal muscle of obese mothers.

Maternal Obesity, Inflammation, and Fetal Skeletal Muscle Development

Abstract Maternal obesity coupled with Western-style high-energy diets represents a special problem that can result in poor fetal development, leading to harmful, persistent effects on offspring, including predisposition to obesity and type 2 diabetes. Mechanisms linking maternal obesity to the increased incidence of obesity and other metabolic diseases in offspring remain poorly defined. Because skeletal muscle is the principal site for glucose and fatty acid utilization and composes 40%–50% of total body mass, changes in the properties of offspring skeletal muscle and its mass resulting from maternal obesity may be responsible for the increase in type 2 diabetes and obesity. Fetal stage is crucial for skeletal muscle development because there is no net increase in the muscle fiber number after birth. Fetal skeletal muscle development involves myogenesis, adipogenesis, and fibrogenesis, which are all derived from mesenchymal stem cells (MSCs). Shifting commitment of MSCs from myogenesis to adipogenesis and fibrogenesis will result in increased intramuscular fat and connective tissue, as well as reduced numbers of muscle fiber and/or diameter, all of which have lasting negative effects on offspring muscle function and properties. Maternal obesity leads to low-grade inflammation, which changes the commitment of MSCs in fetal muscle through several possible mechanisms: 1) inflammation downregulates wingless and int (WNT) signaling, which attenuates myogenesis; 2) inflammation inhibits AMP-activated protein kinase, which promotes adipogenesis; and 3) inflammation may induce epigenetic modification through polycomb group proteins. More studies are needed to further explore the underlying mechanisms associated with maternal obesity, inflammation, and the commitment of MSCs.

Up-Regulation of Toll-Like Receptor 4/Nuclear Factor-κB Signaling Is Associated with Enhanced Adipogenesis and Insulin Resistance in Fetal Skeletal Muscle of Obese Sheep at Late Gestation

Maternal obesity is increasing at an alarming rate. We previously showed that maternal obesity induces an inflammatory response and enhances adipogenesis in fetal skeletal muscle at midgestation. The objective of this study was to evaluate effects of maternal obesity on adipogenesis, inflammatory signaling, and insulin pathways at late gestation when ovine fetal skeletal muscle matures. Nonpregnant ewes were assigned to a control diet (Con, fed 100% of National Research Council nutrient recommendations, n = 6) or obesogenic diet (OB, fed 150% of National Research Council recommendations, n = 6) from 60 d before to 135 d after conception (term 148 d) when the fetal semitendenosus skeletal muscle was sampled. Expression of the adipogenic marker, peroxisome proliferator-activated receptor-gamma, was increased in OB compared with Con fetal semitendenosus muscle, indicating up-regulation of adipogenesis. More intramuscular adipocytes were observed in OB muscle. Phosphorylation of inhibitor-kappaB kinase-alpha/beta and nuclear factor-kappaB RelA/p65 were both increased in OB fetal muscle, indicating activation of nuclear factor-kappaB pathway. Phosphorylation of c-Jun N-terminal kinase and c-Jun (at Ser 63 and Ser 73) was also elevated. Toll-like receptor 4 expression was higher in OB than Con fetal muscle. Moreover, despite higher insulin concentrations in OB vs. Con fetal plasma (2.89 +/- 0.53 vs. 1.06 +/- 0.52 ng/ml; P < 0.05), phosphorylation of protein kinase B at Ser 473 was reduced, indicating insulin resistance. In conclusion, our data show maternal obesity-induced inflammatory signaling in late gestation fetal muscle, which correlates with increased im adipogenesis and insulin resistance, which may predispose offspring to later-life obesity and diabetes.

Maternal obesity markedly increases placental fatty acid transporter expression and fetal blood triglycerides at midgestation in the ewe

Obesity of women at conception is increasing, a condition associated with offspring obesity. We hypothesized that maternal obesity increases placental fatty acid transporter (FATP) expression, enhancing delivery of fatty acids to their fetuses. Sheep are a commonly utilized biomedical model for pregnancy studies. Nonpregnant ewes were randomly assigned to a control group [100% of National Research Council (NRC) recommendations] or obese group (OB, 150% of NRC) from 60 days before conception to 75 or 135 days of gestation (dG; term = 150 dG), when placental cotyledonary tissue was collected for analysis. Fetuses of OB ewes were markedly heavier (P < 0.05) on 75 dG than fetuses from control ewes, but this difference disappeared by 135 dG. Maternal obesity markedly increased (P < 0.05) cholesterol and triglyceride concentrations of both maternal and fetal blood. There is no difference in lipoprotein lipase mRNA expression between control and OB group at either gestational age. On 75 dG, the mRNA expression of FATP1 (P < 0.05), FATP4 (P = 0.08), and fatty acid translocase CD (cluster of differentiation) 36 (P < 0.05) proteins were more enhanced in cotyledonary tissue from OB than control ewes; consistently, protein expression of FATP1 and FATP4 was increased (P < 0.05). Similarly, on 135 dG, the mRNA levels of FATP1, FATP4, and CD36 were all higher (P < 0.05), but only FATP4 protein content was enhanced (P < 0.05) in OB cotyledonary tissue. Peroxisome proliferator-activated receptor (PPAR)-γ regulates the expression of FATPs. Both the mRNA expression and protein content of PPARγ were increased in OB cotyledonary in the midgestation. In conclusion, maternal obesity enhances the mRNA expression and protein content of FATPs in cotyledonary in the midgestation, which is associated with higher PPARγ content in cotyledonary.

AMP-Activated Protein Kinase Enhances the Expression of Muscle-Specific Ubiquitin Ligases Despite Its Activation of IGF-1/Akt Signaling in C2C12 Myotubes

Two muscle‐specific ubiquitin ligases (UL), muscle atrophy F box (MAFbx) and muscle RING finger 1 (MuRF1), are crucial for myofibrillar protein breakdown. The insulin like growth factor‐1 (IGF‐1) pathway inhibits muscle UL expression through Akt‐mediated inhibition of FoxO transcription factors, while AMP‐activated protein kinase (AMPK) promotes UL expression. The underlying cellular mechanism, however, remains obscure. In this study, the effect of AMPK and its interaction with IGF‐1 on ubiquitin ligases expression was investigated. C2C12 myotubes were treated with 0, 0.1, 0.3, and 1.0 mM 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR) in the presence or absence of 50 ng/ml IGF‐1. IGF‐1 activated Akt, which enhanced phosphorlytion of FoxO3a at Thr 318/321 and reduced the expression of UL. Intriguingly, though activation of AMPK by 0.3 and 1.0 mM AICAR synergized IGF‐1‐induced Akt activation, the expression of UL was not attenuated, but strengthened by AMPK activation. AICAR treatment decreased FoxO3a phosphorylation at 318/321 in the cytoplasm and induced FoxO3 nuclear relocation. mTOR inhibition increased basal MAFbx expression and reversed the inhibitory effect of IGF‐1 on UL expression. In conclusion, our data show that AMPK activation by AICAR stimulates UL expression despite the activation of Akt signaling, which may be due to the possible antagonistic effect of FoxO phosphorylation by AMPK on phosphorylation by Akt. In addition, AMPK inhibition of mTOR may provide an additional explanation for the enhancement of UL expression by AMPK. J. Cell. Biochem. 108: 458–468, 2009.

Pre-slaughter transport, AMP-activated protein kinase, glycolysis, and quality of pork loin.

Numerous studies have revealed that pre-slaughter stress, like transport, increases the occurrence of pale, soft, and exudative (PSE) pork meat. The molecular mechanism underlying this phenomenon, however, is poorly defined. In this study, we investigated the effects of pre-slaughter transport and subsequent rest on energy metabolism, AMP-activated protein kinase (AMPK) activation, and glycolysis in postmortem pork loin. Results indicated that pre-slaughter transport accelerated ATP depletion, which led to lower energy status in postmortem muscle immediately post-exsanguination when compared with control. The lower energy status led to AMPK activation within 1h postmortem, subsequently increasing glycolysis, leading to rapid glycolysis and high incidence of PSE meat. Allowing pigs to rest after transport restored energy status in muscle ante-mortem. Higher energy status then prevented premature and rapid AMPK activation in postmortem muscle and lessened the negative effects of pre-slaughter transport on meat quality. AMPK regulated glycolysis in postmortem muscle, at least partially, through phosphorylation and activation of phosphofructose kinase-2, since fructose-2,6-diphosphate content, an allosteric activator of phosphofructose kinase-1, was well correlated with AMPK activation and glycolytic rate. This suggests that AMPK is a potential molecular target for the control of PSE incidence in pork.

Maternal obesity and overnutrition alter fetal growth rate and cotyledonary vascularity and angiogenic factor expression in the ewe

In pregnant sheep, maternal:fetal exchange occurs across placentomes composed of placental cotyledonary and uterine caruncular tissues. Recently, we reported that fetal weights of obese (OB) ewes [fed a diet of 150% of National Research Council (NRC) recommendations] were approximately 30% greater than those of control (C) ewes (fed a diet 100% of NRC recommendations) at midgestation (MG), but fetal weights were similar in late gestation (LG). Transplacental nutrient exchange is dependent on placental blood flow, which itself is dependent on placental vascularity. The current study investigated whether the observed initial faster and subsequent slower fetal growth rate of OB compared with C was associated with changes in cotyledonary vascularity and expression of angiogenic factors (vascular endothelial growth factor, fibroblast growth factor-2, placental growth factor, angiopoietin-1 and -2). Cotyledonary arteriole diameters were markedly greater (P < 0.05) in OB than C ewes at MG, but while arteriole diameter of C ewes increased (P < 0.05) from MG to LG, they remained unchanged in OB ewes. Cotyledonary arterial angiogenic factors mRNA and protein expression were lower (P < 0.05) in OB than C ewes at MG and remained low from MG to LG. In contrast, mRNA levels of angiogenic factors in C ewes declined from high levels at MG to reach those of OB ewes by LG. The increase in cotyledonary arteriole diameter in early to MG may function to accelerate fetal growth rate in OB ewes, while the decreased cotyledonary arterial angiogenic factors from MG-LG may function to protect the fetus from excessive placental vascular development, increased maternal nutrient delivery, and excessive weight gain.

AMP-activated protein kinase (AMPK) cross-talks with canonical Wnt signaling via phosphorylation of β-catenin at Ser 552

AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism; its activity is regulated by a plethora of physiological conditions, exercises and many anti-diabetic drugs. Recent studies show that AMPK involves in cell differentiation but the underlying mechanism remains undefined. Wingless Int-1 (Wnt)/beta-catenin signaling pathway regulates the differentiation of mesenchymal stem cells through enhancing beta-catenin/T-cell transcription factor 1 (TCF) mediated transcription. The objective of this study was to determine whether AMPK cross-talks with Wnt/beta-catenin signaling through phosphorylation of beta-catenin. C3H10T1/2 mesenchymal cells were used. Chemical inhibition of AMPK and the expression of a dominant negative AMPK decreased phosphorylation of beta-catenin at Ser 552. The beta-catenin/TCF mediated transcription was correlated with AMPK activity. In vitro, pure AMPK phosphorylated beta-catenin at Ser 552 and the mutation of Ser 552 to Ala prevented such phosphorylation, which was further confirmed using [gamma-(32)P]ATP autoradiography. In conclusion, AMPK phosphorylates beta-catenin at Ser 552, which stabilizes beta-catenin, enhances beta-catenin/TCF mediated transcription, expanding AMPK from regulation of energy metabolism to cell differentiation and development via cross-talking with the Wnt/beta-catenin signaling pathway.

Down-Regulation of Growth Signaling Pathways Linked to a Reduced Cotyledonary Vascularity in Placentomes of Over-Nourished, Obese Pregnant Ewes

Both protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) are down-stream components of the insulin/insulin like growth factor-1 (IGF-1) signaling pathway. AMP-activated protein kinase (AMPK) is known to sensitize cells to insulin/IGF-1 signaling. The objective of this study was to assess the activity of AMPK and its role in the observed down-regulation of insulin/IGF-1 signaling in cotyledonary (COT) arteries supplying the placental component of the ewe placentome. Nonpregnant ewes were randomly assigned to a control (C, 100% of NRC recommendations) or obesogenic (OB, 150% of NRC) diet from 60 days before conception until necropsy on day 75 of gestation (n=5/group) or until lambing (n=5/group). At necropsy on day 75 of gestation, the smallest terminal arteries that entered the COT tissues (0.5-1.0 mm in diameter) were collected for analyses. Fetal weights were approximately 20% greater (P<0.05) on OB than C ewes, but birth weights of lambs were similar across dietary groups. Fetal plasma concentrations of glucose, insulin and IGF-1 were higher (P<0.05) in the blood of fetuses from OB than C ewes. Total AMPK and phosphorylated AMPK at Thr 172 (the active form) were reduced (P<0.05) by 19.7+/-8.4% and 25.9+/-7.7%, respectively in the COT arterial tissues of OB ewes. Total acetyl-CoA carboxylase (ACC), a down-stream target of AMPK, and its phosphorylated form were also reduced (P<0.05) by 32.9+/-9.2% and 45.4+/-14.6%, respectively. The phosphorylation of IRS-1 at Ser 789, a site phosphorylated by AMPK, was 24.5+/-9.0% lower (P<0.05) in COT arteries of OB than C ewes. No alteration in total insulin receptor, total IGF-1 receptor or their phosphorylated forms was observed, down-stream insulin signaling was down-regulated in COT arteries of OB ewes, which may have resulted in the observed decrease in COT vascular development in OB ewes.