Leucine and arginine regulate trophoblast motility through mTOR-dependent and independent pathways in the preimplantation mouse embryo

AbstractUterine implantation is a critical element of mammalian reproduction and is a tightly and highly coordinated event. An intricate and reciprocal uterine-embryo dialog exists to synchronize uterine receptivity with the concomitant activation of the blastocyst, maximizing implantation success. While a number of pathways involved in regulating uterine receptivity have been identified in the mouse, less is understood about blastocyst activation, the process by which the trophectoderm (TE) receives extrinsic cues that initiate new characteristics essential for implantation. Amino acids (AA) have been found to regulate blastocyst activation and TE motility in vitro. In particular, we find that arginine and leucine alone are necessary and sufficient to induce TE motility. Both arginine and leucine act individually and additively to propagate signals that are dependent on the activity of the mammalian target of rapamycin complex 1 (mTORC1). The activities of the well-established downstream targets of mTORC1, p70S6K and 4EBP, do not correlate with trophoblast motility, suggesting that an independent-rapamycin-sensitive pathway operates to induce trophoblast motility, or that other, parallel amino acid-dependent pathways are also involved. We find that endogenous uterine factors act to induce mTORC1 activation and trophoblast motility at a specific time during pregnancy, and that this uterine signal is later than the previously defined signal that induces the attachment reaction. In vivo matured blastocysts exhibit competence to respond to an 8-hour AA stimulus by activating mTOR and subsequently undergoing trophoblast outgrowth by the morning of day 4.5 of pregnancy, but not on day 3.5. By the late afternoon of day 4.5, the embryos no longer require any exposure to AA to undergo trophoblast outgrowth in vitro, demonstrating the existence and timing of an equivalent in vivo signal. These results suggest that there are two separate uterine signals regulating implantation, one that primes the embryo for the attachment reaction and another that activates mTOR and initiates invasive behavior

Inhibited Insulin Signaling in Mouse Hepatocytes Is Associated with Increased Phosphatidic Acid but Not Diacylglycerol

Although an elevated triacylglycerol content in non-adipose tissues is often associated with insulin resistance, the mechanistic relationship remains unclear. The data support roles for intermediates in the glycerol-3-phosphate pathway of triacylglycerol synthesis: diacylglycerol (DAG), which may cause insulin resistance in liver by activating PKCϵ, and phosphatidic acid (PA), which inhibits insulin action in hepatocytes by disrupting the assembly of mTOR and rictor. To determine whether increases in DAG and PA impair insulin signaling when produced by pathways other than that of de novo synthesis, we examined primary mouse hepatocytes after enzymatically manipulating the cellular content of DAG or PA. Overexpressing phospholipase D1 or phospholipase D2 inhibited insulin signaling and was accompanied by an elevated cellular content of total PA, without a change in total DAG. Overexpression of diacylglycerol kinase-θ inhibited insulin signaling and was accompanied by an elevated cellular content of total PA and a decreased cellular content of total DAG. Overexpressing glycerol-3-phosphate acyltransferase-1 or -4 inhibited insulin signaling and increased the cellular content of both PA and DAG. Insulin signaling impairment caused by overexpression of phospholipase D1/D2 or diacylglycerol kinase-θ was always accompanied by disassociation of mTOR/rictor and reduction of mTORC2 kinase activity. However, although the protein ratio of membrane to cytosolic PKCϵ increased, PKC activity itself was unaltered. These data suggest that PA, but not DAG, is associated with impaired insulin action in mouse hepatocytes

The Signal Transducer and Activator of Transcription 1 (STAT1) Inhibits Mitochondrial Biogenesis in Liver and Fatty Acid Oxidation in Adipocytes

The transcription factor STAT1 plays a central role in orchestrating responses to various pathogens by activating the transcription of nuclear-encoded genes that mediate the antiviral, the antigrowth, and immune surveillance effects of interferons and other cytokines. In addition to regulating gene expression, we report that STAT1-/- mice display increased energy expenditure and paradoxically decreased release of triglycerides from white adipose tissue (WAT). Liver mitochondria from STAT1-/- mice show both defects in coupling of the electron transport chain (ETC) and increased numbers of mitochondria. Consistent with elevated numbers of mitochondria, STAT1-/- mice expressed increased amounts of PGC1α, a master regulator of mitochondrial biogenesis. STAT1 binds to the PGC1α promoter in fed mice but not in fasted animals, suggesting that STAT1 inhibited transcription of PGC1α. Since STAT1-/-mice utilized more lipids we examined white adipose tissue (WAT) stores. Contrary to expectations, fasted STAT1-/- mice did not lose lipid from WAT. β-adrenergic stimulation of glycerol release from isolated STAT1-/- WAT was decreased, while activation of hormone sensitive lipase was not changed. These findings suggest that STAT1-/- adipose tissue does not release glycerol and that free fatty acids (FFA) re-esterify back to triglycerides, thus maintaining fat mass in fasted STAT1-/- mice

SEIPIN Regulates Lipid Droplet Expansion and Adipocyte Development by Modulating the Activity of Glycerol-3-phosphate Acyltransferase

Berardinelli-Seip congenital lipodystrophy 2 (BSCL2) is caused by loss-of-function mutations in SEIPIN, a protein implicated in both adipogenesis and lipid droplet expansion but whose molecular function remains obscure. Here, we identify physical and functional interactions between SEIPIN and microsomal isoforms of glycerol-3-phosphate acyltransferase (GPAT) in multiple organisms. Compared to controls, GPAT activity was elevated in SEIPIN-deficient cells and tissues and GPAT kinetic values were altered. Increased GPAT activity appears to underpin the block in adipogenesis and abnormal lipid droplet morphology associated with SEIPIN loss. Overexpression of Gpat3 blocked adipogenesis, and Gpat3 knockdown in SEIPIN-deficient preadipocytes partially restored differentiation. GPAT overexpression in yeast, preadipocytes, and fly salivary glands also formed supersized lipid droplets. Finally, pharmacological inhibition of GPAT in Seipin-/- mouse preadipocytes partially restored adipogenesis. These data identify SEIPIN as an evolutionarily conserved regulator of microsomal GPAT and suggest that GPAT inhibitors might be useful for the treatment of human BSCL2 patients