Administration of cyclic glycine-proline during infancy improves adult spatial memory, astrocyte plasticity, vascularization and GluR-1 expression in rats.

Cyclic glycine-proline (cGP) is a natural nutrient of breast milk and plays a role in regulating the function of insulin-like growth factor-1 (IGF-1). IGF-1 function is essential for post-natal brain development and adult cognitive function. We evaluated the effects of cGP on spatial memory and histological changes in the hippocampus of the adult rats following infancy administration. Infant rats were treated with either cGP or saline between post-natal days 8 and 22 via oral administration to lactating dams. The spatial memory was evaluated between post-natal days 70 and 75 using Morris water maze tests. The changes of capillaries, astrocytes, synaptophysin and glutamate receptor-1 were examined in the CA1 stratum radiatum of the hippocampus. Compared to saline-treated group, cGP-treated group showed higher path efficiency of entry and lower average heading errors to the platform zone. cGP-treated group also showed longer, larger and more astrocytic processes, more capillaries and higher glutamate receptor-1 expression. The rats made less average heading error to the platform zone have more capillaries, larger and longer astrocytic branches. Thus cGP treatment/supplementation during infancy moderately improved adulthood spatial memory. This long-lasting effect of cGP on memory could be mediated via promoting astrocytic plasticity, vascularization and glutamate trafficking. Therefore, cGP may have a role in regulating IGF-1 function during brain development.

Effects of feeding high volumes of milk replacer on reproductive performance and on concentrations of metabolites and hormones in blood of Japanese black heifer calves.

We evaluated the effects of feeding high volumes of milk replacer on growth and reproductive performances in Japanese black heifers. Fifty-one heifers were fed milk replacer at 9 L/day for 60 days (9 L × 60 days; n = 18) or 41 days (9 L × 41 days; n = 15), or at 7 L/day for 40 days (7 L × 40 days; n = 18). Artificial insemination (AI) was performed on heifers with ≥270 kg body weight and ≥116 cm body height at 300 days of age. The age at the first AI was 0.35 month later for 7 L × 40 days than the other groups (p < .01). However, age at calving did not differ among treatments (22.1 months). The interval from the first AI to pregnancy tended to be ~2 months longer for the 9 L × 60 days than the other groups (p = .07). Our results showed that feeding high volumes of milk replacer may reduce the age at calving via an improved rate of growth. In addition, we propose that feeding a maximum of 7 L milk replacer for 40 days may be the most appropriate rearing regime because the success of pregnancy per AI may be reduced in calves fed a maximum of 9 L for 41 and 60 days.

IGF1 does not overcome sexual dimorphism of body and muscle size in Mstn-/- mice.

Insulin-like growth factor-1 (IGF1) is crucial for regulating post-natal growth and, along with myostatin (MSTN), regulates muscle size. Here, we sought to clarify the roles of these two genes in regulating sexually dimorphic growth of body and muscle mass. In the first study, we established that Igf1 mRNA was increased to a greater extent and Igf1 receptor mRNA increased earlier in male, than in female, gastrocnemius muscles during the rapid phase of growth (from 2 to 6 weeks) were unchanged, thereafter, to 32 weeks of age in WT mice (P < 0.001). In the second study, we sought to determine if supplemental IGF1 could overcome the sexual dimorphism of muscle and body mass, when myostatin is absent. We crossed myostatin null (Mstn-/-) mice with mice over-expressing Igf1 in skeletal muscle (Igf1+) to generate six genotypes; control (Mstn+/+), Mstn+/-, Mstn-/-, Mstn+/+:Igf1+, Mstn+/-:Igf1+ and Mstn-/-:Igf1+ (n = 8 per genotype and sex). In both sexes, body mass at 12 weeks was increased by at least 1.6-fold and muscle mass by at least 3-fold in Mstn-/-:Igf1+ compared with Mstn+/+ mice (P < 0.001). The abundance of AKT was increased in muscles of mice transgenic for Mstn, while phosphorylation of AKTS473 was increased in both male and female mice transgenic for Igf1+. The ratio of phosphorylated to total AKT was 1.9-fold greater in male mice (P < 0.001). Thus, despite increased growth of skeletal muscle and body size when myostatin was absent and IGF1 was in excess, sexual dimorphism persisted, an effect consistent with greater IGF1-induced activation of AKT in skeletal muscles of males.

IGF1 stimulates greater muscle hypertrophy in the absence of myostatin in male mice.

Insulin-like growth factors (IGFs) and myostatin have opposing roles in regulating the growth and size of skeletal muscle, with IGF1 stimulating, and myostatin inhibiting, growth. However, it remains unclear whether these proteins have mutually dependent, or independent, roles. To clarify this issue, we crossed myostatin null (Mstn-/-) mice with mice overexpressing Igf1 in skeletal muscle (Igf1+) to generate six genotypes of male mice; wild type (Mstn+/+ ), Mstn+/-, Mstn-/-, Mstn+/+:Igf1+, Mstn+/-:Igf1+ and Mstn-/-:Igf1+ Overexpression of Igf1 increased the mass of mixed fibre type muscles (e.g. Quadriceps femoris) by 19% over Mstn+/+ , 33% over Mstn+/- and 49% over Mstn-/- (P < 0.001). By contrast, the mass of the gonadal fat pad was correspondingly reduced with the removal of Mstn and addition of Igf1 Myostatin regulated the number, while IGF1 regulated the size of myofibres, and the deletion of Mstn and Igf1+ independently increased the proportion of fast type IIB myosin heavy chain isoforms in T. anterior (up to 10% each, P < 0.001). The abundance of AKT and rpS6 was increased in muscles of Mstn-/-mice, while phosphorylation of AKTS473 was increased in Igf1+mice (Mstn+/+:Igf1+, Mstn+/-:Igf1+ and Mstn-/-:Igf1+). Our results demonstrate that a greater than additive effect is observed on the growth of skeletal muscle and in the reduction of body fat when myostatin is absent and IGF1 is in excess. Finally, we show that myostatin and IGF1 regulate skeletal muscle size, myofibre type and gonadal fat through distinct mechanisms that involve increasing the total abundance and phosphorylation status of AKT and rpS6.

Cyclic-glycine-proline accelerates mammary involution by promoting apoptosis and inhibiting IGF-1 function.

In rodents, post-lactational involution of mammary glands is characterized by the loss of mammary epithelial cells via apoptosis, which is associated with a decline in the expression of insulin-like growth factor-1 (IGF-1). Overexpression of IGF-1 delays involution by inhibiting apoptosis of epithelial cells and preserving the remaining secretory alveoli. Cyclic-glycine-proline (cGP), a metabolite of IGF-1, normalizes IGF-1 function under pathological conditions by regulating the bioavailability of IGF-1. The present study investigated the effect of cGP on the physiological decline in IGF-1 function during post-lactational mammary involution. Rat dams were gavaged with either cGP (3 mg/kg) or saline once per day from post-natal d8-22. Before collecting tissue on post-natal d23, a pair of mammary glands were sealed on d20 (72 hr-engorgement, thus representative of late-involution) and d22 (24 hr-engorgement, thus representative of mid-involution), while the remaining glands were allowed to involute naturally (early-involution). During early-involution, cGP accelerated the loss of mammary cells through apoptosis, resulting in an earlier clearance of intact secretory alveoli compared with the control group. This coincided with an earlier up-regulation of the cell survival factors, Bcl-xl and IGF-1R, in the early-involution cGP glands compared with the control glands. During late-involution, cGP reduced the bioactivity of IGF-1, which was evident through decreased phosphorylation of IGF-1R in the regressed alveoli. Maternal administration of cGP did not alter milk production and composition during early-, peak-, or late-stage of lactation. These data show that cGP accelerates post-lactational involution by promoting apoptosis and the physiological decline in IGF-1 function.

Tight Junction Protein Abundance and Apoptosis During Involution of Rat Mammary Glands.

To examine tight junction protein abundance and apoptosis of epithelial cells at the onset of involution in rodent mammary glands, milk accumulation and mammary engorgement were induced by teat-sealing with an adhesive for 0, 6, 12, 18, 24, and 36 h (n = 6 per group) at peak lactation. In non-sealed control glands, histological analysis confirmed a lactating phenotype, indicating suckling by pups throughout the experiment. In contrast, alveoli of teat-sealed glands were distended within 6 h, with maximal luminal size observed by 12 h of non-suckling. By 18 h following teat-sealing, an involuting phenotype was observed, indicated by alveolar lumina engorged with milk vesicles and increased leukocytes. Relative to non-sealed glands, mammary apoptosis was increased in engorged glands 18 h following teat-sealing. The abundance of ZO-1 and occludin proteins was decreased in engorged glands by 12 and 18 h, respectively, following teat-sealing. In contrast, the claudin-1 22 kDa band was increased by 6 h and peaked at 12-18 h, whereas the 28 kDa band declined by 36 h, relative to controls. There were no temporal changes in ZO-1, occludin, and claudin-1 22 kDa proteins within control glands, although there were minor differences in claudin-1 28 kDa. These data indicate that intramammary milk accumulation due to cessation of milk removal is associated with mammary apoptosis. The apoptotic event is preceded by a rapid loss of abundance of ZO-1, occludin and an initial increase in claudin-1. The loss of cell-cell communication may initiate involution and apoptosis of mammary epithelial cells and is a localized intramammary event, occurring only in non-suckled glands. J. Cell. Physiol. 232: 2075-2082, 2017. © 2016 Wiley Periodicals, Inc.

Effects of nutritional status on hormone concentrations of the somatotropin axis and metabolites in plasma and colostrum of Japanese Black cows.

We aimed to determine the effects of nutritional status on concentrations of somatotropic axis hormones (growth hormone (GH) and insulin-like growth factor 1 (IGF-1)), insulin and metabolites (glucose, total protein and nonesterified fatty acids (NEFA)) in the plasma and colostrum in late antepartum cows. Eight pregnant Japanese Black cows were randomly assigned to two experimental groups (n = 4 per group). Control cows (CON) received 100% of their nutritional requirements until parturition, whereas restricted group cows (RES) received 60% of their nutritional requirements. Blood samples were taken during the antepartum period, and blood and colostrum samples were collected on days 0, 1, and 3 after calving. Compared to the CON group, the RES group had higher concentrations of GH and NEFA in plasma, but significantly lower concentrations of glucose and insulin in plasma. The concentrations of GH in plasma after calving were significantly higher, but total plasma protein was significantly lower in RES than in CON cows. Compared to the CON group, the RES group had significantly higher concentrations of GH in colostrum, but significantly lower total concentrations of protein in colostrum. Concentrations of IGF-1 were not different between the two groups. These findings suggest that maternal nutritional status during late gestation influences concentrations of GH and total protein in the blood and colostrum of Japanese Black cows.

Maternally Administered Cyclic Glycine-Proline Increases Insulin-Like Growth Factor-1 Bioavailability and Novelty Recognition in Developing Offspring.

Cyclic glycine-proline (cGP), a metabolite of IGF-1, is an endogenous neuropeptide that improves memory in adult rats. The presence and concentrations of endogenous cGP, and its association with IGF-1 and IGF binding protein-3 (IGFBP-3) in rat milk and plasma, were evaluated during postnatal development. Maternal-infantile transfer of cGP during lactation and its efficacy on the memory of developing offspring were also investigated. Dams were gavaged with either cGP (3 mg/kg) or saline daily from postnatal days 8-22. Concentrations of cGP were measured in dams' milk, and concentrations of cGP, IGF-1, and IGFBP-3 were measured in the plasma of dams, pups, and young adults. The recognition memory, locomotor function, and anxiety-like behavior of offspring were evaluated using behavioral tests. Endogenous cGP was detected in rat milk, and its concentration was higher during peak lactation compared with late lactation. Comparisons within control groups showed low endogenous IGF-1 and IGFBP-3 and high endogenous cGP concentrations in the plasma of male pups. The reduced IGFBP-3 and increased cGP may be a response to increase the bioavailability of IGF-1 during infancy. Exogenous cGP showed oral bioavailability and effective maternal-infantile transfer through milk. Maternally transferred cGP also led to improved recognition memory in the developing offspring, possibly through increased IGF-1 bioavailability, with no effect on locomotor activity and anxiety-like behavior. These results show that cGP is an essential endogenous peptide during early postnatal development as it improves the bioavailability of IGF-1 during infancy. Furthermore, maternal cGP supplementation offers an effective and natural route of administration for improving memory in the developing offspring.

Effects of a high milk intake during the pre-weaning period on nutrient metabolism and growth rate in Japanese Black cattle.

This study aimed to determine the effects of feeding an increased volume of high-fat milk during the early post-natal life on metabolite concentrations in the blood, the expression of key genes regulating intermediary metabolism in the skeletal muscles, and the rate of growth of Japanese Black cattle. All calves were fed a high-fat milk replacer (crude protein, 26%; crude fat, 25.5%; total dissolved nitrogen, 116%). Control calves (n = 4) were nursed with 500 g milk replacer until 3 months of age, whereas calves in the experimental group (n = 4) were nursed with 1800 g milk replacer until 3 months, and then the volume was gradually reduced until 5 months. Body weight was significantly higher in the experimental group than in the control group at 7 months. Plasma glucose concentrations were significantly lower in the experimental group. Expression of glucose-transporter-4 messenger RNA (mRNA) was lower, whereas that of glucose transporter 1, cluster of differentiation 36, and carnitine palmitoyltransferase-1b mRNA was significantly higher in the Longissimus thoracis of the experimental group. Nutritional status during early post-natal life appears to strongly influence the growth rate and glucose and lipid metabolism in Japanese Black cattle.

Translational signalling, atrogenic and myogenic gene expression during unloading and reloading of skeletal muscle in myostatin-deficient mice.

Skeletal muscles of myostatin null (Mstn(-/-)) mice are more susceptible to atrophy during hind limb suspension (HS) than are muscles of wild-type mice. Here we sought to elucidate the mechanism for this susceptibility and to determine if Mstn(-/-) mice can regain muscle mass after HS. Male Mstn(-/-) and wild-type mice were subjected to 0, 2 or 7 days of HS or 7 days of HS followed by 1, 3 or 7 days of reloading (n = 6 per group). Mstn(-/-) mice lost more mass from muscles expressing the fast type IIb myofibres during HS and muscle mass was recovered in both genotypes after reloading for 7 days. Concentrations of MAFbx and MuRF1 mRNA, crucial ligases regulating the ubiquitin-proteasome system, but not MUSA1, a BMP-regulated ubiquitin ligase, were increased more in muscles of Mstn(-/-) mice, compared with wild-type mice, during HS and concentrations decreased in both genotypes during reloading. Similarly, concentrations of LC3b, Gabarapl1 and Atg4b, key effectors of the autophagy-lysosomal system, were increased further in muscles of Mstn(-/-) mice, compared with wild-type mice, during HS and decreased in both genotypes during reloading. There was a greater abundance of 4E-BP1 and more bound to eIF4E in muscles of Mstn(-/-) compared with wild-type mice (P<0.001). The ratio of phosphorylated to total eIF2α increased during HS and decreased during reloading, while the opposite pattern was observed for rpS6. Concentrations of myogenic regulatory factors (MyoD, Myf5 and myogenin) mRNA were increased during HS in muscles of Mstn(-/-) mice compared with controls (P<0.001). We attribute the susceptibility of skeletal muscles of Mstn(-/-) mice to atrophy during HS to an up- and downregulation, respectively, of the mechanisms regulating atrophy of myofibres and translation of mRNA. These processes are reversed during reloading to aid a faster rate of recovery of muscle mass in Mstn(-/-) mice.

Identification and expression of a novel transcript of the growth and differentiation factor-11 gene.

The growth and differentiation factor-11 (GDF-11) gene is thought to code for a single protein that plays a crucial role in regulating the development of multiple tissues. In this study, we aimed to investigate if the GDF-11 gene has another transcript and, if so, to characterise this transcript and determine its tissue-specific and developmental expression. We have identified a novel transcript of GDF-11 in mouse muscle, which contains the 3' region of intron 1, exon 2, exon 3 and 3'UTR, and has two transcription initiation sites and a single termination site. We named the novel transcript GDF-11ΔEx1 because it does not contain exon 1 of canonical GDF-11. The GDF-11ΔEx1 transcript was expressed in the skeletal muscles, heart, brain and kidney, but was undetectable in the liver and gut. The concentration of the GDF-11ΔEx1 transcript was increased in gastrocnemius muscles from three to 6 weeks of age, a period of accelerated muscle growth, steadily declined thereafter and was higher in male than female mice (P < 0.001 for age and sex). GDF-11ΔEx1 cDNA was predicted to code for a putative N-terminal-truncated propeptide and the canonical ligand for GDF-11. However, propeptide-specific antibodies could not identify proteins of the expected size in skeletal muscle. Interestingly, in silico analysis of the GDF-11ΔEx1 RNA predicted a secondary structure with the potential to coordinate multiple protein interactions as a molecular scaffold. Therefore, we postulate that GDF-11ΔEx1 may act as a long non-coding RNA to regulate the transcription of canonical GDF-11 and/or other genes in skeletal muscle and other tissues.

Discovery of a mammalian splice variant of myostatin that stimulates myogenesis.

Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over-expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P<0.05), which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant directly antagonizes the biological activity of the canonical gene product.

Botulinum toxin-induced paralysis leads to slower myosin heavy chain isoform composition and reduced titin content in juvenile rat gastrocnemius muscle.

Intramuscular injections of botulinum toxin A (Btx-A) and exercise are used in the treatment of muscle spasticity in children with cerebral palsy. However, little is known about the biological changes within muscle subsequent to Btx-A-induced paralysis and how the combination of Btx-A and exercise might affect the growing muscle. The wet mass, myosin heavy chain (MHC) composition, and titin content of the juvenile rat gastrocnemius muscle were determined 3 weeks after Btx-A injections and subsequent voluntary wheel-running exercise. Btx-A increased the proportion of type IIa (+121%) and IIx (+65%) MHC while decreasing the proportion of type IIb MHC (-51%) and reducing the titin content (-18%). Exercise did not amplify or reduce the changes induced by Btx-A. Thus, we conclude that although the sarcomeric stability of paralyzed muscle might be impaired, moderate mechanical loading does not seem to affect paralyzed muscle protein composition.

The decrease in mature myostatin protein in male skeletal muscle is developmentally regulated by growth hormone.

Myostatin inhibits myogenesis and there is reduced abundance of the mature protein in skeletal muscles of adult male compared with female mice. This reduction probably occurs after translation, which suggests that it is a regulated mechanism to reduce the availability of myostatin in males. Reduced myostatin may, thereby, contribute to the development of sexually dimorphic growth of skeletal muscle. Our first objective was to determine if the decrease in mature myostatin protein occurs before the linear growth phase to aid growth, or afterwards to maintain the mass of adult muscle. Mice were killed from 2 to 32 weeks and the gastrocnemius muscle was excised. Myostatin mRNA increased from 2 to 32 weeks and was higher in males than females (P < 0.001). In contrast, mature protein decreased in males after 6 weeks (P < 0.001). Our second objective was to determine if growth hormone (GH) induces the decrease in mature myostatin protein. GH increased myostatin mRNA and decreased the abundance of mature protein in hypophysectomised mice (P < 0.05). Our final objective was to determine if the decrease in mature protein occurs in skeletal muscles of male Stat5b(-/-) mice (Stat5b mediates the actions of GH). As expected, mature myostatin protein was not reduced in Stat5b(-/-) males compared with females. However, myostatin mRNA remained higher in males than females irrespective of genotype. These data suggest that: (1) the decrease in mature myostatin protein is developmentally regulated, (2) GH acting via Stat5b regulates the abundance of mature myostatin and (3) GH acts via a non-Stat5b pathway to regulate myostatin mRNA.

Effect of botulinum toxin A-induced paralysis and exercise training on mechanosensing and signalling gene expression in juvenile rat gastrocnemius muscle.

Intramuscular injections of the paralytic botulinum neurotoxin A (Btx) and physical exercise are used in the treatment of chronic spasticity in children with cerebral palsy. We tested whether Btx-induced paralysis and/or exercise training would have differential effects on the expression of mechanosensing and signalling genes implicated in the adaptive remodelling of skeletal muscle. Juvenile (29-day-old) male rats were injected with Btx or saline (NoBtx) into the right gastrocnemius and housed in standard cages (NoEx) or with running wheels (Ex), for 3 weeks (n = 6 per group). The mRNA expression of nine sarcomere-associated genes in the medial gastrocnemius was then determined by quantitative reverse transcriptase-polymerase chain reaction. The Btx-injected muscles weighed 50% less than NoBtx muscles, but Ex had no effect on the wet mass of Btx or NoBtx muscles. Atrogenic MuRF1, sarcomeric Titin and myogenic MyoD were upregulated (2-fold) with the elimination of contractile activity in Btx muscle. Expression of CARP, Ankrd2 and MLP was increased with mechanical stimuli associated with Btx (5- to 10-fold) or Ex (2- to 4-fold). Expression of CARP and Ankrd2 increased synergistically in Btx-Ex muscle (> or = 20-fold), indicating that these genes may be sensitive to passive stretch of the sarcomeric I-band region of titin to which their proteins bind. Tcap, Myopalladin and Atrogin1 were not, or were no longer responsive to the altered mechanical stimuli after 3 weeks of Btx or Ex. The expression of Ankrd2, CARP and MLP may thus be enhanced by passive stretch within the Btx-paralysed and/or exercising gastrocnemius and contribute to adaptations, other than muscle mass, in juvenile rats.

Myostatin regulates glucose uptake in BeWo cells.

Myostatin is a member of the transforming growth factor (TGF)-beta superfamily, known for its ability to inhibit muscle growth. It can also regulate metabolism and glucose uptake in a number of tissues. To determine the mechanism of myostatin's effect on glucose uptake, we evaluated its actions using choriocarcinoma cell lines that are widely used as models for placental cells. Protein and mRNA were determined using immunoblotting and RT-PCR/PCR, respectively. Glucose uptake was assessed by uptake of radiolabeled deoxyglucose in vitro. All choriocarcinoma cell lines tested i.e., BeWo, JEG, and Jar, are used as models of placental cells, and all expressed myostatin protein and mRNA. Treatment of BeWo cells with myostatin resulted in inhibition of glucose uptake in a concentration-dependent manner (P < 0.01). At all concentrations tested, follistatin, a functional inhibitor of myostatin, completely blocked the inhibitory effect of myostatin (40 nM) on glucose uptake by BeWo cells (0.4 nM, P < 0.05). Follistatin treatment alone also increased glucose uptake (0.4 and 4 nM, P < 0.001; 40 nM, P < 0.05). Because BeWo cells proliferated and greater cell densities were achieved, glucose uptake declined irrespective of treatment. Myostatin treatment of BeWo cells did not alter the levels of myostatin receptor, ActRII A/B proteins. The levels of glucose transport proteins also remained unaltered in BeWo cells with myostatin treatment. This study has shown that myostatin specifically inhibits glucose uptake into BeWo cells, suggesting that locally produced myostatin may control glucose metabolism within the placenta.

Myostatin is a human placental product that regulates glucose uptake.

CONTEXT: Myostatin is a member of the TGF-beta superfamily and is primarily known for its ability to inhibit muscle growth. It also has actions on glucose metabolism. We hypothesized that it may act as a paracrine regulator of glucose uptake in the placenta, potentially contributing to fetal and placental growth. OBJECTIVES: The objective of this study was to determine whether myostatin is present in and formed by the human placenta and to evaluate its effects on glucose uptake. MATERIALS AND METHODS: Myostatin protein and mRNA were measured using Western immunoblotting and real-time PCR, respectively. Glucose uptake was assessed by uptake of radiolabeled deoxyglucose in vitro. Placental tissues were obtained at term (n = 8), preterm (n = 8; 24-34 wk), and early in pregnancy (n = 6; 9-13 wk). RESULTS: Human placentas were shown to express myostatin protein, with a significantly lower expression in term samples compared with samples collected in preterm samples. Human placentas express myostatin mRNA throughout gestation, which does not change. Myostatin treatment of human term placental explants resulted in an increase in deoxyglucose uptake compared with controls. CONCLUSIONS: Myostatin is synthesized, released, and acts within the human placenta. It contributes to placental glucose homeostasis and may be a therapeutic target in diseases ranging from placental insufficiency to diabetes in pregnancy.

Decreased expression of beta1-integrin and focal adhesion kinase in epithelial cells may initiate involution of mammary glands.

The mechanisms regulating involution of mammary glands after weaning are not clear, but engorgement with milk is a key trigger. Many cell types require to be anchored to an extracellular matrix (ECM) as a prerequisite for survival and this is achieved via intregrins binding to specific motifs and signalling their attachment, intracellularly, via focal adhesion kinase (FAK). We sought to determine firstly, if expression of beta1-integrin and FAK is reduced during the first stage of involution. Expression of beta1-integrin and FAK was significantly reduced at 6 h after sealing teats and this was accompanied with a decreased abundance of cytochrome C in mitochondria. Secondly, we sought to determine if expression of beta1-integrin and FAK was restored during the first, partially reversible stage of involution (at 24 h), but not during the second irreversible stage, which occurs after 72 h. Re-suckling restored full expression of the 80 kDa fragment of FAK, but not of the 125 kDa protein or beta1-integrin at 24 h after weaning. Re-suckling did not restore expression of either peptide after 72 h. Changes in expression of cytochrome C and pro-caspase-3 (apoptotic markers) were similar to that of the 80 kDa fragment of FAK. These data suggest that epithelial cells can restore partial contact with their basement membrane during the first, reversible stage, but not during the second irreversible stage of involution. We speculate that decreased contact between epithelial cells and their basement membrane initiates apoptosis in mammary glands at weaning. This process begins within 6 h of pup withdrawal.

Myostatin-deficient mice lose more skeletal muscle mass than wild-type controls during hindlimb suspension.

Myostatin inhibits myogenesis. Therefore, we sought to determine if mice lacking the myostatin gene [Mstn(-/-)] would lose less muscle mass than wild-type mice during 7 days of hindlimb suspension (HS). Male Mstn(-/-) and wild-type (C57) mice were subjected to HS or served as ground-based controls (n = 6/group). Wild-type mice lost 8% of body mass and approximately 13% of wet mass from biceps femoris, quadriceps femoris, and soleus, whereas the mass of extensor digitorum longus (EDL) was unchanged after HS. Unexpectedly, Mstn(-/-) mice lost more body (13%, P < 0.05) and quadriceps femoris (17%, P < 0.05) mass than wild-type mice and lost 33% of EDL mass (P < 0.01) after HS. Protein expression of myostatin in biceps femoris and quadriceps femoris was not altered, whereas expression of MyoD, Myf-5, and myogenin increased in wild-type mice and tended to decrease in muscles of Mstn(-/-) mice. These data suggest that HS induced myogenesis in wild-type mice to counter atrophy, whereas myogenesis was not induced in Mstn(-/-) mice, thereby resulting in a greater loss of muscle mass.

Sexual dimorphism is associated with decreased expression of processed myostatin in males.

Myostatin inhibits skeletal muscle development. Therefore, we sought to determine whether larger body and muscle mass in male mice was associated with lower mRNA and protein expression of myostatin compared with females. Ten male and ten female mice of the C57 strain were killed at 16-18 wk of age, and their biceps femoris, gastrocnemius, and quadriceps femoris muscles were collected. Body and muscle masses were 40% heavier (P < 0.001) in males than in females. Northern analysis showed no difference in mRNA between males and females. In contrast, Western analysis showed that processed myostatin (26 kDa) was 40-60% lower (P < 0.001) in males compared with females. These data show first that decreased processed myostatin is a posttranscriptional and posttranslational event and, second, that decreased abundance of processed myostatin is associated with increased body mass and skeletal muscle mass in male compared with female mice.