Function (Oxford, England)最新文献

Mechanistic target of rapamycin (mTOR) signaling is a positive regulator of human placental function including System A/L amino acid transport activity. Placental mTOR signaling is inhibited in fetal growth restriction (FGR) and activated in fetal overgrowth in women, however the causes of these changes in placental mTOR signaling are unknown. DEP-domain containing mTOR-interacting protein (DEPTOR) is an endogenous inhibitor of mTOR. We tested the hypothesis that trophoblast-specific Deptor knockdown activates placental mTOR signaling and amino acid transport and causes fetal overgrowth. Using lentiviral transduction of blastocyst trophectoderm with a small hairpin RNA, we achieved 47% knockdown of placental Deptor mRNA expression, without altering fetal Deptor mRNA expression. Trophoblast-specific Deptor knockdown activated placental mTORC1 and mTORC2 signaling and increased trophoblast plasma membrane (TPM) LAT1 and SNAT2 protein abundance, and TPM System L and System A transporter activity. In addition, Deptor knockdown increased in vivo transplacental System A and L amino acid transport and stimulated placental and fetal growth. In human FGR, placental DEPTOR protein expression was higher and negatively correlated with birth weight and microvillus plasma membrane System A activity. In conclusion, we provide mechanistic evidence that DEPTOR regulates placental mTOR signaling and amino acid transport and fetal growth in vivo. We speculate that modulation of placental Deptor is a promising target for intervention in pregnancies characterized by abnormal placental function and fetal growth.

Apolipoprotein E4 (APOE4) is the greatest genetic risk factor for Alzheimer's (AD) and is linked to whole-body metabolic dysfunction. However, it's unclear how APOE4 interacts with modifiable factors like diet to impact tissues central to regulating whole-body metabolism. We examined APOE4- and Western diet-driven effects in skeletal muscle using APOE3 (control) and APOE4 targeted replacement mice on a C57BL/6NTac background fed a high-fat diet (HFD, 45% kcal fat) or low-fat diet (LFD, 10% kcal fat) for four months (n=7-8 per genotype/diet/sex combination). We assessed body composition and whole-body outcomes linked to skeletal muscle function including respiratory exchange ratio (RER) and resting energy expenditure (REE). In skeletal muscle, we evaluated the proteome and mitochondrial respiration. In males only, APOE4 drove greater gains in fat mass and lower gains in lean mass on both diets. APOE4 did not affect daily RER but was associated with elevated REE in males and lower REE in HFD females after covarying for body composition. Skeletal muscle proteomics showed APOE4 exerts several diet- and sex-specific effects on mitochondrial pathways, including elevations in branched-chain amino catabolism in HFD males and reductions in oxidative phosphorylation in LFD females. This did not translate to differences in skeletal muscle mitochondrial respiration, suggesting that compensatory mechanisms may sustain mitochondrial function at this age. Our work indicates that genetic risk may mediate early life effects on skeletal muscle mitochondria and energy expenditure that are partially dependent on diet. This has important implications for mitigating AD risk in APOE4 carriers.

We simulated the dynamics of a group of 10 nephrons supplied from an arterial network and subjected to acute increases in blood pressure. Arterial lengths and topology were based on measurements of a vascular cast. The model builds on a previous version exercised at a single blood pressure with 2 additional features: pressure diuresis and the effect of blood pressure on efferent arteriolar vascular resistance. The new version simulates autoregulation, and reproduces tubule pressure oscillations. Individual nephron dynamics depended on mean arterial pressure and the axial pressure gradient required to cause blood flow through the arteries. Rhythmic blood withdrawal into afferent arterioles caused blood flow fluctuations in downstream vessels. Blood pressure dependent changes in nephron dynamics affected synchronization metrics. The combination of vascular pressure gradients and oscillations created a range of arterial pressures at the origins of the 10 afferent arterioles. Because arterial blood pressure in conscious animals has ${1}/{f}$ dynamics, we applied an arterial pressure pattern with such dynamics to the model. Amplitude of tubule pressure oscillations were affected by the ${1}/{f}$ blood pressure fluctuations, but the oscillation frequencies did not change. The pressure gradients required to deliver blood to all afferent arterioles impose a complexity that affects nephrons according to their locations in the network, but other interactions compensate to ensure the stability of the system. The sensitivity of nephron response to location on the network, and the constancy of the tubular oscillation frequency provide a spatial and time context.

Excess dietary salt and salt-sensitivity contribute to cardiovascular disease. Distinct T cell phenotypic responses to high salt and hypertension, as well as influences from environmental cues, are not well understood. The aryl hydrocarbon receptor (AhR) is activated by dietary ligands, promoting T cell and systemic homeostasis. We hypothesized that activating AhR supports CD4+ homeostatic functions, such as cytokine production and mobilization, in response to high salt intake while mitigating salt-sensitive hypertension. In the intestinal mucosa, we demonstrate that a high-salt diet (HSD) is a key driving factor, independent of hypertension, in diminishing interleukin 17A (IL-17A) production by CD4+ T (Th17) cells without disrupting circulating cytokines associated with Th17 function. Previous studies suggest that hypertensive patients and individuals on a HSD are deficient in AhR ligands or agonistic metabolites. We found that activating AhR augments Th17 cells during experimental salt-sensitive hypertension. Further, we demonstrate that activating AhR in vitro contributes to sustaining Th17 cells in the setting of excess salt. Using photoconvertible Kikume Green-Red mice, we also revealed that HSD drives CD4+ T cell mobilization. Next, we found that excess salt augments T cell mobilization markers, validating HSD-driven T cell migration. Also, we found that activating AhR mitigates HSD-induced T cell migration markers. Using telemetry in a model of experimental salt-sensitivity, we found that activating AhR prevents the development of salt-sensitive hypertension. Collectively, stimulating AhR through dietary ligands facilitates immunologic and systemic functions amid excess salt intake and restrains the development of salt-sensitive hypertension.

The spatiotemporal interplay between the second messenger cyclic AMP (cAMP) and its main effector, protein kinase A (PKA), is crucial for the pleiotropic nature of this cascade. To maintain a high degree of specificity, the cAMP/PKA axis is organised into functional units called microdomains, precisely distributed within the cell. While the subcellular allocation of PKA is guaranteed by a family of tethers called A-Kinase-anchoring Proteins (AKAPs), the mechanisms underlying the efficient confinement of a microdomain's functional effects are not fully understood. Here, we used FRET-based sensors to detect cAMP levels and PKA-dependent phosphorylation within specific subcellular compartments. We find that cellular cAMP levels may depend on different mechanisms and are responsible for the activation of local PKA enzymes. On the other hand, the dephosphorylating actions of phosphatases dictate the duration of the microdomain's effects. To test the range of action of PKA microdomains, we used rigid aminoacidic nanorulers to distance our FRET sensors from their original location for 10 or 30 nm. Interestingly, we established that cAMP levels do not affect the spatial range of the microdomain while on the contrary, phosphatase activity provides a functional boundary for phosphorylated PKA targets. Finally, using the same strategy to distance phosphatases from the mitochondria, we found that enzymes close to the outer mitochondrial membrane produced a fragmented phenotype that was not observed when phosphatases were moved to 30 nm from the organelle's surface. Our findings contribute to the design of a picture where 2 microdomain-forming events have distinct roles. Cyclic AMP elevations trigger the initial activation of subcellular PKA moieties, while the temporal and spatial extent of the PKA's actions are regulated by phosphatases.

Extracellular ATP is a signaling molecule that acts as a paracrine and autocrine modulator of cell function. Here we characterized the role of luminal ATP in the regulation of epithelial principal cells (PCs) in the epididymis, an understudied organ that plays crucial roles in male reproduction. We previously showed that ATP secretion by PCs is part of a complex communication system that ensures the establishment of an optimal luminal acidic environment in the epididymis. However, the molecular mechanisms regulating ATP release and the role of ATP-mediated signaling in PCs acidifying functions are not fully understood. In other cell types, Pannexin 1 (PANX-1) has been associated with ATP-induced ATP release through interaction with the purinergic P2 × 7 receptor. Here, we show that PANX-1 and P2 × 7 are located in the apical membrane of PCs in the mouse epididymis. Functional analysis using the immortalized epididymal PCs cell line (DC2) and the mouse epididymis perfused in vivo showed that: 1) PANX-1 and P2 × 7 participate in ATP release by DC2 cells, together with Cystic Fibrosis Transmembrane Conductance Regulator (CFTR); 2) Several ATP-activated P2Y and P2X purinergic receptors are expressed in DC2 cells; 3) The non-hydrolysable ATP analogue ATPγS induces a dose-dependent increase in intracellular Ca2+ concentration in DC2 cells, a process that is mainly mediated by P2 × 7; and 4) Perfusion of the epididymal lumen in vivo with ATPγS induces the internalization of apical sodium-hydrogen exchanger 3 (NHE3) in PCs. Altogether, this study shows that luminal ATP, regulated by CFTR, PANX-1 and P2 × 7, modulates sodium-proton exchange in PCs in an autocrine-manner through activation of purinergic receptor-mediated intracellular calcium signaling.

LncRNAs are engaged in signaling pathways in human physiological and pathological states. However, LncRNAs mediate the onset of human labor still remains unknown. RNA sequencing of lower segment myometrium (in labor vs. not in labor) was analyzed. N6-Methyladenosine (m6A) complexes were detected by RIP and meRIP in human myometrial cells. Plasmid and siRNA transfection was performed, and contraction ability was assessed. RNA pulldown, silver staining, protein mass spectrometry, and RIP were used to identify binding proteins. FISH and immunofluorescence costaining were applied to assess the coexpression. GAS5 was upregulated in human myometrium after labor onset. METTL3 and IGF2BP1 maintained GAS5 RNA stability based on actinomycin assay, thus strengthening the contraction of myometrial cells. RIP and meRIP revealed the binding sites of GAS5 with METTL3 and IGF2BP1, respectively. Furthermore, GAS5 binds TPM4 in cytoplasm of myometrium cells and transports TPM4 to the contraction filaments. m6A RNA modifications were also noted in the mouse myometrium after labor onset. These findings highlighted the critical role of m6A modification in GAS5, providing a new method to explore RNA epigenetic regulatory patterns in human parturition.