Estrogen Modulates Glycerol Permeability in Sertoli Cells through Downregulation of Aquaporin-9.
ABSTRACT: High 17?-Estradiol (E2) levels are known to cause alterations of spermatogenesis and environments throughout the male reproductive tract. Sertoli cells (SCs) ensure an adequate environment inside the seminiferous tubule. Glycerol stands as essential for the maintenance of blood?testis barrier created by SCs, however, the role of E2 in this process is not known. Herein, we hypothesized that the effect of E2 on glycerol permeability in mouse SCs (mSCs) could be mediated by aquaglyceroporins. The expression of aquaglyceroporins was assessed by RT-PCR and qRT-PCR. Glycerol permeability was evaluated by stopped-flow light scattering. We were able to identify the expression of AQP3 and AQP9 in mSCs where AQP9 is more abundant than AQP3. Our results show that high E2 levels decrease AQP9 mRNA abundance with no influence on AQP3 in mSCs. Interestingly, high E2 levels decreased mSCs' permeability to glycerol, while downregulating AQP9 expression, thus suggesting a novel mechanism by which E2 modulates fluid secretion in the testis. In conclusion, E2 is an important regulator of mSCs physiology and secretion through changes in AQP9 expression and function. Thus, alterations in glycerol permeability induced by E2 may be the cause for male infertility in cases associated with the presence of high E2 levels.
Project description:The aquaglyceroporins are a subfamily of aquaporins that conduct both water and glycerol. Aquaporin-3 (AQP3) has an important physiological function in renal water reabsorption, and AQP3-mediated hydrogen peroxide (H2O2) permeability can enhance cytokine signaling in several cell types. The related aquaglyceroporin AQP7 is required for dendritic cell chemokine responses and antigen uptake. Selective small-molecule inhibitors are desirable tools for investigating the biological and pathological roles of these and other AQP isoforms. Here, using a calcein fluorescence quenching assay, we screened a library of 7360 drug-like small molecules for inhibition of mouse AQP3 water permeability. Hit confirmation and expansion with commercially available substances identified the ortho-chloride-containing compound DFP00173, which inhibited mouse and human AQP3 with an IC50 of ?0.1-0.4 ?m but had low efficacy toward mouse AQP7 and AQP9. Surprisingly, inhibitor specificity testing revealed that the methylurea-linked compound Z433927330, a partial AQP3 inhibitor (IC50, ?0.7-0.9 ?m), is a potent and efficacious inhibitor of mouse AQP7 water permeability (IC50, ?0.2 ?m). Stopped-flow light scattering measurements confirmed that DFP00173 and Z433927330 inhibit AQP3 glycerol permeability in human erythrocytes. Moreover, DFP00173, Z433927330, and the previously identified AQP9 inhibitor RF03176 blocked aquaglyceroporin H2O2 permeability. Molecular docking to AQP3, AQP7, and AQP9 homology models suggested interactions between these inhibitors and aquaglyceroporins at similar binding sites. DFP00173 and Z433927330 constitute selective and potent AQP3 and AQP7 inhibitors, respectively, and contribute to a set of isoform-specific aquaglyceroporin inhibitors that will facilitate the evaluation of these AQP isoforms as drug targets.
Project description:Aquaglyceroporins (AQP3, AQP7, AQP9 and AQP10) encompass a subfamily of aquaporins that allow the movement of water and other small solutes, especially glycerol, through cell membranes. Adipose tissue constitutes a major source of glycerol via AQP7. We have recently reported that, in addition to the well-known expression of AQP7 in adipose tissue, AQP3 and AQP9 are also expressed in omental and subcutaneous fat depots. Moreover, insulin and leptin act as regulators of aquaglyceroporins through the PI3K/Akt/mTOR pathway. AQP3 and AQP7 appear to facilitate glycerol efflux from adipose tissue while reducing the glycerol influx into hepatocytes via AQP9 to prevent the excessive lipid accumulation and the subsequent aggravation of hyperglycemia in human obesity. This Extra View focuses on the control of glycerol release by aquaglyceroporins in the adipose tissue and briefly discusses the importance of glycerol as a substrate for hepatic gluconeogenesis, pancreatic insulin secretion and cardiac ATP production.
Project description:Glycerol is an important metabolite for the control of lipid accumulation in white adipose tissue (WAT) and liver. We aimed to investigate whether exogenous administration of leptin improves features of non-alcoholic fatty liver disease (NAFLD) in leptin-deficient ob/ob mice via the regulation of AQP3 and AQP7 (glycerol channels mediating glycerol efflux in adipocytes) and AQP9 (aquaglyceroporin facilitating glycerol influx in hepatocytes). Twelve-week-old male wild type and ob/ob mice were divided in three groups as follows: control, leptin-treated (1 mg/kg/d) and pair-fed. Leptin deficiency was associated with obesity and NAFLD exhibiting an AQP3 and AQP7 increase in WAT, without changes in hepatic AQP9. Adipose Aqp3 and hepatic Aqp9 transcripts positively correlated with markers of adiposity and hepatic steatosis. Chronic leptin administration (4-weeks) was associated with improved body weight, whole-body adiposity, and hepatosteatosis of ob/ob mice and to a down-regulation of AQP3, AQP7 in WAT and an up-regulation of hepatic AQP9. Acute leptin stimulation in vitro (4-h) induced the mobilization of aquaglyceroporins towards lipid droplets (AQP3) and the plasma membrane (AQP7) in murine adipocytes. Our results show that leptin restores the coordinated regulation of fat-specific AQP7 and liver-specific AQP9, a step which might prevent lipid overaccumulation in WAT and liver in obesity.
Project description:Aquaporin 3 (AQP3), a member of the aquaglyceroporin family, which transports water and glycerol, is robustly expressed in epidermis and plays an important role in stratum corneum hydration, permeability barrier function and wound healing. PPAR and LXR activation regulates the expression of many proteins in the epidermis and thereby can affect epidermal function. Here, we report that PPARgamma activators markedly stimulate AQP3 mRNA expression in both undifferentiated and differentiated cultured human keratinocytes (CHKs). The increase in AQP3 mRNA by PPARgamma activator occurs in a dose- and time-dependent fashion. Increased AQP3 mRNA levels are accompanied by an increase in AQP3 protein in undifferentiated keratinocytes and a significant increase in glycerol uptake. Activation of LXR, RAR and RXR also increases AQP3 mRNA levels in undifferentiated and differentiated CHKs, but to a lesser extent. PPARdelta activation stimulates AQP3 expression in undifferentiated CHKs but decreases expression in differentiated CHKs. In contrast, PPARalpha activators do not alter AQP3 expression. AQP9 and AQP10, other members of aquaglyceroporin family, are less abundantly expressed in CHKs, and their expression levels are not significantly altered by treatment with LXR, PPAR, RAR or RXR activators. Finally, when topically applied, the PPARgamma activator, ciglitazone, induces AQP3 but not AQP9 gene expression in mouse epidermis. Our data demonstrate that PPAR and LXR activators stimulate AQP3 expression, providing an additional mechanism by which PPAR and LXR activators regulate epidermal function.
Project description:Aquaglyceroporins, belonging to the family of aquaporins (AQPs), are integral plasma membrane proteins permeable to water and glycerol that have emerged as key players in obesity. The aim of this study was to investigate the expression profile of AQPs in undifferentiated and differentiated 3T3-L1 cells and to investigate the changes in expression of aquaglyceroporins in 3T3-L1 cells differentiated into adipocytes and subjected to lipopolysaccharide (LPS) mimicking inflammation occurring during obesity. Furthermore, the study aimed at identifying the signaling cascade involved in the regulation of aquaglyceroporins expression upon LPS stimulation. 3T3-L1 cells were grown as undifferentiated cells (UDC; preadipocytes) or cells differentiated into adipocytes (DC, adipocytes). DC were incubated in the presence or absence of LPS with or without inhibitors of various protein kinases. AQPs mRNA expression levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR). AQP1, AQP2, AQP3, AQP9 and AQP11 mRNA were expressed in both UDC and DC, whereas AQP4, AQP7 and AQP8 mRNA were expressed only in DC. In DC, LPS up-regulated AQP3 mRNA levels (p < 0.05) compared to control; these effects were inhibited by CLI095, SP600125 and BAY11-7082 (p < 0.05). LPS decreased both AQP7 and AQP11 mRNA levels (p < 0.01) in DC as compared to control; this decrease was inhibited by CLI095 and BAY11-7082 (p < 0.05) and additionally by SP00125 for AQP7 (p < 0.05). SB203580 had no effect on LPS-induced AQP3, AQP7 and AQP11 mRNA levels modulations. In conclusion, our results clearly show that many AQPs are expressed in murine 3T3-L1 adipocytes. Moreover, in DCs, LPS led to decreased AQP7 and AQP11 mRNA levels but to increased AQP3 mRNA levels, resulting from the Toll-like receptor 4 (TLR4)-induced activation of JNK and/or NF?B pathway.
Project description:To investigate if intracellular glycerol content plays a role in the regulation of insulin secretion in pancreatic beta cells, we studied the expression of the glycerol channels, or aquaglyceroporins, encoded by the aquaporin 3 (Aqp3), Aqp7, and Aqp9 genes in mouse islets. We found expression of Aqp7 only, not that of Aqp3 or Aqp9, in the endocrine pancreas at both the mRNA (by reverse transcription-PCR) and protein (by immunohistochemistry) levels. Immunohistochemistry revealed a complete overlap between insulin and Aqp7 immunostaining in the pancreatic islet. Inactivation of Aqp7 by gene targeting produced viable and healthy mice. Aqp7-/- mice harbored an increased intraislet glycerol concentration with a concomitant increase of the glycerol kinase transcript level and enzyme activity. The islet triglyceride content in the Aqp7-/- mice was also increased compared to that in the Aqp7+/+ mice. Interestingly, Aqp7-/- mice displayed reduced beta-cell mass and insulin content but increased insulin-1 and insulin-2 mRNAs. The reduction of beta-cell mass in Aqp7-/- mice can be explained at least in part by a reduction in cell proliferation through protein kinase C and the c-myc cascade, with a reduction in the transcript levels of these two genes. Concomitantly, there was a decreased rate of apoptosis, as reflected by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and caspase 3 and Bax expression in Aqp7-/- mice. Compared with Aqp7+/+ islets, islets isolated from Aqp7-/- mice secreted insulin at a higher rate under basal low-glucose conditions and on exposure to a high (450 mg/dl) glucose concentration. Aqp7-/- mice exhibited normal fasting blood glucose levels but elevated blood insulin levels. Their plasma glucose response to an intraperitoneal (i.p.) glucose tolerance test was normal, but their plasma insulin concentrations were higher than those of wild-type mice during the 2-h test. An i.p. insulin tolerance test showed similar plasma glucose lowering in Aqp7-/- and Aqp7+/+ mice, with no evidence of insulin resistance. In conclusion, we found that pancreatic beta cells express AQP7, which appears to be a key regulator of intraislet glycerol content as well as insulin production and secretion.
Project description:BACKGROUND: Increasing cell membrane permeability to water and cryoprotectants is critical for the successful cryopreservation of cells with large volumes. Artificial expression of water-selective aquaporins or aquaglyceroporins (GLPs), such as mammalian aquaporin-3 (AQP3), enhances cell permeability to water and cryoprotectants, but it is known that AQP3-mediated water and solute permeation is limited and pH dependent. To exploit further the possibilities of using aquaporins in cryobiology, we investigated the functional properties of zebrafish (Danio rerio) GLPs. RESULTS: Water, glycerol, propylene glycol and ethylene glycol permeability of zebrafish Aqp3a, -3b, -7, -9a, -9b, -10a and -10b, and human AQP3, was examined. Expression in Xenopus laevis oocytes indicated that the permeability of DrAqp3a and -3b to ethylene glycol was higher than for glycerol or propylene glycol under isotonic conditions, unlike other zebrafish GLPs and human AQP3, which were more permeable to glycerol. In addition, dose-response experiments and radiolabeled ethylene glycol uptake assays suggested that oocytes expressing DrAqp3b were permeated by this cryoprotectant more efficiently than those expressing AQP3. Water and ethylene glycol transport through DrAqp3a and -3b were, however, highest at pH 8.5 and completely abolished at pH 6.0. Point mutations in the DrAqp3b amino acid sequence rendered two constructs, DrAqp3b-T85A showing higher water and ethylene glycol permeability at neutral and alkaline pH, and DrAqp3b-H53A/G54H/T85A, no longer inhibited at acidic pH but less permeable than the wild type. Finally, calculation of permeability coefficients for ethylene glycol under concentration gradients confirmed that the two DrAqp3b mutants were more permeable than wild-type DrAqp3b and/or AQP3 at neutral pH, resulting in a 2.6- to 4-fold increase in the oocyte intracellular concentration of ethylene glycol. CONCLUSION: By single or triple point mutations in the DrAqp3b amino acid sequence, we constructed one mutant with enhanced ethylene glycol permeability and another with reduced pH sensitivity. The DrAqp3b and the two mutant constructs may be useful for application in cryobiology.
Project description:As ambient temperatures fall in the autumn, freeze-tolerant Cope's gray treefrogs, Dryophytes chrysoscelis (formerly Hyla chrysoscelis), accumulate glycerol as a cryoprotective agent. We hypothesized that these treefrogs express an ortholog of the mammalian aquaglyceroporin AQP9 and that AQP9 expression is upregulated in the cold to facilitate glycerol transport. We sequenced 1790 bp from cloned cDNA that codes for a 315 amino acid protein, HC-9, containing the predicted six transmembrane spanning domains, two Asn-Pro-Ala (NPA) motifs, and five amino acid residues characteristic of aquaglyceroporins. Functional characterization after heterologous expression of HC-9 cRNA in Xenopus laevis oocytes indicated that HC-9 facilitates glycerol and water permeability and is partially inhibited by 0.5 mmol/L phloretin or 0.3 mmol/L HgCl2 HC-9 mRNA (qPCR) and protein (immunoblot) were expressed in most treefrog tissues analyzed (muscle, liver, bladder, stomach, kidney, dorsal skin, and ventral skin) except the protein fraction of red blood cells. Contrary to our prediction, both mRNA and protein expression were either unchanged or downregulated in most tissues in response to cold, freezing, and thawing. A notable exception to that pattern occurred in liver, where protein expression was significantly elevated in frozen (~4-fold over warm) and thawed (~6-fold over warm) conditions. Immunofluorescence labeling of HC-9 protein revealed a signal that appeared to be localized to the plasma membrane of hepatocytes. Our results indicate that gray treefrogs express an AQP9-like protein that facilitates glycerol permeability. Both the transcriptional and translational levels of HC-9 change in response to thermal challenges, with a unique increase in liver during freezing and thawing.
Project description:The structures of several aquaglyceroporins have been resolved to atomic resolution showing two or more glycerols bound inside a channel and confirming a glycerol-facilitator's affinity for its substrate glycerol. However, the kinetics data of glycerol transport experiments all point to unsaturated transport that is characteristic of low substrate affinity in terms of the Michaelis-Menten kinetics. In this article, we present an in silico-in vitro research focused on AQP3, one of the human aquaglyceroporins that is natively expressed in the abundantly available erythrocytes. We conducted 2.1 ?s in silico simulations of AQP3 embedded in a model erythrocyte membrane with intracellular-extracellular asymmetries in leaflet lipid compositions and compartment salt ions. From the equilibrium molecular dynamics (MD) simulations, we elucidated the mechanism of glycerol transport at high substrate concentrations. From the steered MD simulations, we computed the Gibbs free-energy profile throughout the AQP3 channel. From the free-energy profile, we quantified the kinetics of glycerol transport that is unsaturated due to glycerol-glycerol interactions mediated by AQP3 resulting in the concerted movement of two glycerol molecules for the transport of one glycerol molecule across the cell membrane. We conducted in vitro experiments on glycerol uptake into human erythrocytes for a wide range of substrate concentrations and various temperatures. The experimental data quantitatively validated our theoretical-computational conclusions on the unsaturated glycerol transport through AQP3 that has high affinity for glycerol.
Project description:It has been hypothesized that aquaporin-9 (AQP9) is part of the unknown route of hepatocyte glycerol uptake. In a previous study, leptin receptor-deficient wild-type mice became diabetic and suffered from fasting hyperglycemia whereas isogenic AQP9(-/-) knock-out mice remained normoglycemic. The reason for this improvement in AQP9(-/-) mice was not established before. Here, we show increased glucose output (by 123% ± 36% S.E.) in primary hepatocyte culture when 0.5 mM extracellular glycerol was added. This increase depended on AQP9 because it was absent in AQP9(-/-) cells. Likewise, the increase was abolished by 25 μM HTS13286 (IC(50) ~ 2 μM), a novel AQP9 inhibitor, which we identified in a small molecule library screen. Similarly, AQP9 deletion or chemical inhibition eliminated glycerol-enhanced glucose output in perfused liver preparations. The following control experiments suggested inhibitor specificity to AQP9: (i) HTS13286 affected solute permeability in cell lines expressing AQP9, but not in cell lines expressing AQPs 3, 7, or 8. (ii) HTS13286 did not influence lactate- and pyruvate-dependent hepatocyte glucose output. (iii) HTS13286 did not affect glycerol kinase activity. Our experiments establish AQP9 as the primary route of hepatocyte glycerol uptake for gluconeogenesis and thereby explain the previously observed, alleviated diabetes in leptin receptor-deficient AQP9(-/-) mice.