Analysis of the Molecular Signaling Signatures of Muscle Protein Wasting Between the Intercostal Muscles and the Gastrocnemius Muscles in db/db Mice.

Type 2 diabetes (T2D) patients suffer from dyspnea, which contributes to disease-related morbidity. Although T2D has been reported to induce a catabolic state in skeletal muscle, whether T2D induces muscle wasting in respiratory muscles has not yet been investigated. In this study, we examine the difference in the molecular signaling signature of muscle wasting between the intercostal and gastrocnemius muscles using db/db mice, a well-known diabetic mouse model. Akt phosphorylation was significantly decreased in both the intercostal and gastrocnemius muscles of db/db mice and was accompanied by a decrease in mTORC1 activity. In addition, FoxO phosphorylation was suppressed, and ubiquitin-proteasome degradation, characterized by the level of Atrogin-1 and MuRF1, was subsequently enhanced in both muscle types of db/db mice. An increase in LC3BII levels and a decrease in p62 levels marked the occurrence of substantial autophagy in the gastrocnemius muscle but not in the intercostal muscles of db/db mice. Therefore, we suggest that the signaling events of muscle wasting in the intercostal muscles of db/db mice are different from those in the gastrocnemius muscle of db/db mice.

C-Peptide Promotes Cell Migration by Controlling Matrix Metallopeptidase-9 Activity Through Direct Regulation of ß-Catenin in Human Endometrial Stromal Cells.

The motility of endometrial stromal cells (ESCs) contributes to the restoration of the endometrial functional layer and subsequently supports the trophoblast invasion during early pregnancy. Following ESCs differentiation through decidualization in response to progesterone during the menstrual cycle and embryo implantation, decidualized ESCs (D-ESCs) have greater motility and invasive activity. The human proinsulin-connecting peptide (C-peptide) is produced in equimolar amounts during the proteolysis of insulin in pancreatic ß-cells. However, the function of C-peptide in the cellular motility of the human endometrium remains unexamined. In the present study, C-peptide was identified as a determinant of undecidualized human endometrial stromal cells (UnD-ESCs) migration. C-peptide promoted the migration and invasion of UnD-ESCs and trophoblast-derived Jeg3 cells, but not that of ESCs post decidualization, a functional and biochemical differentiation of UnD-ESCs. Both Akt and protein phosphatase 1 regulated ß-catenin phosphorylation in UnD-ESCs, not D-ESCs, thereby promoting ß-catenin nuclear translocation in C-peptide-treated UnD-ESCs. C-peptide was also observed to increase matrix metallopeptidase-9 (MMP9) activity by increasing MMP9 expression and decreasing the expression of metallopeptidase inhibitor 1 (TIMP1) and TIMP3. Their expression was modulated by the direct binding of ß-catenin in the regulatory region of the promoter of MMP9, TIMP1, and TIMP3. Inhibition of either ß-catenin or MMP9 dampened C-peptide-enhanced migration in UnD-ESCs. Together, these findings suggest that C-peptide levels are critical for the regulation of UnD-ESC migration, providing evidence for the association between C-peptide levels and the failure rate of trophoblast invasion by inducing abnormal migration in UnD-ESCs in hyperinsulinemia or PCOS patients.

Self-transducible LRS-UNE-L peptide enhances muscle regeneration.

BACKGROUND: Muscle regeneration includes proliferation and differentiation of muscle satellite cells, which involves the mammalian target of rapamycin (mTOR). We identified the C-terminal unique attached sequence motif (UNE) domain of leucyl-tRNA synthetase (LRS-UNE-L) as an mTORC1 (mTOR complex1)-activating domain that acts through Vps34 and phospholipase D1 (PLD1) when introduced in the form of a muscle-enhancing peptide. METHODS: In vitro Vps34 lipid kinase assay, phosphatidylinositol 3-phosphate (PI(3)P) measurement, in vivo PLD1 assay, and western blot assay were performed in HEK293 cells to test the effect of the LRS-UNE-L on the Vps34-PLD1-mTOR pathway. Adeno-associated virus (AAV)-LRS-UNE-L was transduced in C2C12 cells in vitro, in BaCl2 -injured tibialis anterior (TA) muscles, and in 18-month-old TA muscles to analyse its effect on myogenesis, muscle regeneration, and aged muscle, respectively. The muscle-specific cell-permeable peptide M12 was fused with LRS-UNE-L and tested for cell integration in C2C12 and HEK293 cells using FACS analysis and immunocytochemistry. Finally, M12-LRS-UNE-L was introduced into BaCl2 -injured TA muscles of 15-week-old Pld1+/+ or Pld1-/- mice, and its effect was analysed by measurement of cross-sectional area of regenerating muscle fibres. RESULTS: The LRS-UNE-L expression restored amino acid-induced S6K1 phosphorylation in LRS knockdown cells in a RagD GTPases-independent manner (421%, P = 0.007 vs. LRS knockdown control cells). The LRS-UNE-L domain was directly bound to Vps34; this interaction was accompanied by increases in Vps34 activity (166%, P = 0.0352), PI(3)P levels (146%, P = 0.0039), and PLD1 activity (228%, P = 0.0294) compared with amino acid-treated control cells, but it did not affect autophagic flux. AAV-delivered LRS-UNE-L domain augmented S6K1 phosphorylation (174%, P = 0.0013), mRNA levels of myosin heavy chain (MHC) (122%, P = 0.0282) and insulin-like growth factor 2 (IGF2) (146%, P = 0.008), and myogenic fusion (133%, P = 0.0479) in C2C12 myotubes. AAV-LRS-UNE-L increased the size of regenerating muscle fibres in BaCl2 -injured TA muscles (124%, P = 0.0279) (n = 9-10), but it did not change the muscle fibre size of TA muscles in old mice. M12-LRS-UNE-L was preferentially delivered into C2C12 cells compared with HEK293 cells and augmented regeneration of BaCl2 -injured TA muscles in a PLD1-dependent manner (116%, P = 0.0022) (n = 6). CONCLUSIONS: Our results provide compelling evidence that M12-LRS-UNE-L could be a muscle-enhancing protein targeting mTOR.

MicroRNA-4516 in Urinary Exosomes as a Biomarker of Premature Ovarian Insufficiency.

Premature ovarian insufficiency (POI) is a typical disorder of amenorrhea that lasts for a minimum of four months in women < 40 years old and is typically characterized by reduced estrogen levels and elevated serum concentrations of follicle-stimulating hormone. We collected urine samples from two participant cohorts from Gil Hospital of Gachon University (Incheon, Korea): a sequencing cohort of 19 participants (seven patients with POI (POI patients without Turner syndrome), seven patients with Turner syndrome (POI patients with Turner syndrome), and five control individuals (age-matched controls with confirmed ovarian sufficiency)) and a validation cohort of 46 participants (15 patients with POI, 11 patients with Turner syndrome, and 20 control individuals). Among differentially expressed miRNAs, hsa-miR-4516 was significantly upregulated in patients with POI in both cohorts, independent of the presence of Turner syndrome. Moreover, the upregulation of miR-4516 was confirmed in the ovary-but not in the uterus-of a cyclophosphamide and busulfan-induced POI mouse model. This was accompanied by a decrease in STAT3 protein level, a predicted target of miR-4516, via miRTarBase2020. Our study provides compelling evidence that miR-4516 is highly expressed in patients with POI and POI mouse models, suggesting that miR-4516 is a diagnostic marker of POI.

Deterioration of mitochondrial function in the human intercostal muscles differs among individuals with sarcopenia, obesity, and sarcopenic obesity.

BACKGROUND & AIMS: Sarcopenic obesity (SO) increases the risk of mortality more than sarcopenia or obesity alone. Sarcopenia weakens the peripheral and respiratory muscles, leading to respiratory complications. It also induces mitochondrial dysfunction in the peripheral muscle; however, whether mitochondrial dysfunction in respiratory muscles differs among individuals with obesity, sarcopenia, and SO remains unknown. We evaluated the deterioration of respiratory muscle strength and mitochondrial function among normal, sarcopenia, obesity, and SO subjects. METHODS: Twenty-five patients who underwent lung resections were enrolled between April 2017 and January 2021, and their intercostal muscles were harvested. Based on their L3 muscle index and visceral fat area, the patients were divided into four groups (normal, obesity, sarcopenia, and SO). The clinical data, mRNA expression, and protein expressions associated with mitochondrial biogenesis/fusion/fission in the intercostal muscles were compared among the four groups. RESULTS: The respiratory muscle strength was evaluated using peak expiratory flow rate (PEFR). The PEFR values of the four groups were not significantly different. The levels of pAkt/Akt and mTOR (a marker of protein synthesis) were not significantly different among the four groups; however, those in the SO group were substantially lower than those in the sarcopenia or obesity groups. The levels of Atrogen-1 and MuRF1 (a marker of protein degradation) were not significantly different among the four groups; however, those in the SO group were substantially higher than those in the sarcopenia or obesity groups. Expression of PGC1-α (a marker of mitochondrial biogenesis) in the SO group was significantly lower than that in the normal group. MFN1 and MFN2 (marker of mitochondrial fusion) levels were significantly lower in the SO group than those in the normal group. DRP1 (a marker of mitochondrial fission) level in the SO group was substantially lower than that in the normal group. The expression of TNF-α (a pro-inflammatory cytokine) in the SO group was substantially lower than that in the normal group. CONCLUSION: Our results suggest that the deterioration of protein synthesis and degradation of mitochondrial function in the respiratory muscles was most prominent in the SO before the weakening of the respiratory muscles. The deterioration mechanism may differentially regulate obesity, sarcopenia, and SO.

C-Peptide Inhibits Decidualization in Human Endometrial Stromal Cells via GSK3ß-PP1.

Decidualization refers to the functional differentiation of endometrial stromal cells and plays a significant role in embryo implantation and pregnancy. C-peptide is excreted in equimolar concentrations as that of insulin during the metabolism of proinsulin in pancreatic beta-cells. High levels of C-peptide are correlated with hyperinsulinemia and polycystic ovarian syndrome, which show a defect in decidualization. However, the role of C-peptide in decidualization has not yet been studied. Here, we identified C-peptide as an endogenous antideciduogenic factor. This inhibitory function was confirmed by the reduced expression of decidual markers, including prolactin, insulin-like growth factor-binding protein-1, and Forkhead box protein O1 as well as by the fibroblastic morphological change in the presence of C-peptide. C-peptide also enhanced cellular senescence and decreased the proportion of apoptotic cells during decidualization. In addition, C-peptide potentiated the inhibitory effects of both insulin and palmitic acid in an AKT- and autophagy-independent manner, respectively. Furthermore, C-peptide augmented protein phosphatase 1 (PP1) activity, leading to a reduction in the inhibitory phosphorylation of glycogen synthase kinase (GSK)3ß, which resulted in enhanced cellular senescence and decreased apoptosis during decidualization. Taken together, our findings suggest that C-peptide is an antideciduogenic factor acting via the regulation between PP1 and GSK3ß in patients with hyperinsulinemia.

Simulated microgravity inhibits C2C12 myogenesis via phospholipase D2-induced Akt/FOXO1 regulation.

The skeletal muscle system has evolved to maintain body posture against a constant gravitational load. Mammalian target of rapamycin (mTOR) regulates the mechanically induced increase in the skeletal muscle mass. In the present study, we investigated mTOR pathway in C2C12 myoblasts in a model of mechanical unloading by creating a simulated microgravity (SM) using 3 D clinorotation. SM decreased the phosphorylation of Akt at Ser 473, which was mediated by mTOR complex 2 (mTORC2), in C2C12 myoblasts, leading to a decrease in the cell growth rate. Subsequently, SM inhibited C2C12 myogenesis in an Akt-dependent manner. In addition, SM increased the phospholipase D (PLD) activity by enhancing PLD2 expression, resulting in the dissociation of mSIN1 from the mTORC2, followed by decrease in the phosphorylation of Akt at Ser 473, and FOXO1 at Ser 256 in C2C12 myoblasts. Exposure to SM decreased the autophagic flux of C2C12 myoblasts by regulation of mRNA level of autophagic genes in a PLD2 and FOXO1-dependent manner, subsequently, resulting in a decrease in the C2C12 myogenesis. In conclusion, by analyzing the molecular signature of C2C12 myogenesis using SM, we suggest that the regulatory axis of the PLD2 induced Akt/FOXO1, is critical for C2C12 myogenesis.

Microgravity inhibits decidualization via decreasing Akt activity and FOXO3a expression in human endometrial stromal cells.

Decidualization is characterized by the differentiation of endometrial stromal cells (eSCs), which is critical for embryo implantation and maintenance of pregnancy. In the present study, we investigated the possible effect of simulated microgravity (SM) on the process of proliferation and in vitro decidualization using primary human eSCs. Exposure to SM for 36 h decreased the proliferation and migration of eSCs significantly, without inducing cell death and changes in cell cycle progression. The phosphorylation of Akt decreased under SM conditions in human eSCs, accompanied by a simultaneous decrease in the level of matrix metalloproteinase (MMP)-2 and FOXO3a. Treatment with Akti, an Akt inhibitor, decreased MMP-2 expression, but not FOXO3a expression. The decreased level of FOXO3a under SM conditions impeded autophagic flux by reducing the levels of autophagy-related genes. In addition, pre-exposure of eSCs to SM significantly inhibited 8-Br-cAMP induced decidualization, whereas restoration of the growth status under SM conditions by removing 8-Br-cAMP remained unchanged. Treatment of human eSCs with SC-79, an Akt activator, restored the reduced migration of eSCs and decidualization under SM conditions. In conclusion, exposure to SM inhibited decidualization in eSCs by decreasing proliferation and migration through Akt/MMP and FOXO3a/autophagic flux.

Differential regulation of mTORC1 and mTORC2 is critical for 8-Br-cAMP-induced decidualization.

Human endometrium decidualization, a differentiation process involving biochemical and morphological changes, is a prerequisite for embryo implantation and successful pregnancy. Here, we show that the mammalian target of rapamycin (mTOR) is a crucial regulator of 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP)-induced decidualization in human endometrial stromal cells. The level of mSin1 in mTOR complex 2 (mTORC2) and DEPTOR in mTOR complex 1 (mTORC1) decreases during 8-Br-cAMP-induced decidualization, resulting in decreased mTORC2 activity and increased mTORC1 activity. Notably, DEPTOR displacement increases the association between raptor and insulin receptor substrate-1 (IRS-1), facilitating IRS-1 phosphorylation at serine 636/639. Finally, both S473 and T308 phosphorylation of Akt are reduced during decidualization, followed by a decrease in forkhead box O1 (FOXO1) phosphorylation and an increase in the mRNA levels of the decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1). Taken together, our findings reveal a critical role for mTOR in decidualization, involving the differential regulation of mTORC1 and mTORC2.