Hepatocyte Aquaporins AQP8 and AQP9 Are Engaged in the Hepatic Lipid and Glucose Metabolism Modulating the Inflammatory and Redox State in Milk-Supplemented Rats.

Milk is an important source of nutrients and energy, but there are still many uncertainties regarding the health effects of milk and dairy products consumption. Milk from different species varies in physicochemical and nutritional properties. We previously showed that dietary supplements with different milks in rats trigger significant differences in metabolic and inflammatory states, modulating mitochondrial functions in metabolically active organs such as the liver and skeletal muscle. Here, we have deepened the effects of isoenergetic supplementation of milk (82 kJ) from cow (CM), donkey (DM) or human (HM) on hepatic metabolism to understand the interlink between mitochondrial metabolic flexibility, lipid storage and redox state and to highlight the possible role of two hepatocyte aquaporins (AQPs) of metabolic relevance, AQP8 and AQP9, in this crosstalk. Compared with rats with no milk supplementation, DM- and HM-fed rats had reduced hepatic lipid content with enhanced mitochondrial function and decreased oxidative stress. A marked reduction in AQP8, a hydrogen peroxide channel, was seen in the liver mitochondria of DM-fed rats compared with HM-fed, CM-fed and control animals. DM-fed or HM-fed rats also showed reduced hepatic inflammatory markers and less collagen and Kupffer cells. CM-fed rats showed higher hepatic fat content and increased AQP9 and glycerol permeability. A role of liver AQP8 and AQP9 is suggested in the different metabolic profiles resulting from milk supplementation.

Characterization of the Aquaporin-9 Inhibitor RG100204 In Vitro and in db/db Mice.

Aquaporin-9 (AQP9) is a facilitator of glycerol and other small neutral solute transmembrane diffusion. Identification of specific inhibitors for aquaporin family proteins has been difficult, due to high sequence similarity between the 13 human isoforms, and due to the limited channel surface areas that permit inhibitor binding. The few AQP9 inhibitor molecules described to date were not suitable for in vivo experiments. We now describe the characterization of a new small molecule AQP9 inhibitor, RG100204 in cell-based calcein-quenching assays, and by stopped-flow light-scattering recordings of AQP9 permeability in proteoliposomes. Moreover, we investigated the effects of RG100204 on glycerol metabolism in mice. In cell-based assays, RG100204 blocked AQP9 water permeability and glycerol permeability with similar, high potency (~5 × 10-8 M). AQP9 channel blocking by RG100204 was confirmed in proteoliposomes. After oral gavage of db/db mice with RG100204, a dose-dependent elevation of plasma glycerol was observed. A blood glucose-lowering effect was not statistically significant. These experiments establish RG100204 as a direct blocker of the AQP9 channel, and suggest its use as an experimental tool for in vivo experiments on AQP9 function.

RG100204, A Novel Aquaporin-9 Inhibitor, Reduces Septic Cardiomyopathy and Multiple Organ Failure in Murine Sepsis.

Sepsis is caused by systemic infection and is a major health concern as it is the primary cause of death from infection. It is the leading cause of mortality worldwide and there are no specific effective treatments for sepsis. Gene deletion of the neutral solute channel Aquaporin 9 (AQP9) normalizes oxidative stress and improves survival in a bacterial endotoxin induced mouse model of sepsis. In this study we described the initial characterization and effects of a novel small molecule AQP9 inhibitor, RG100204, in a cecal ligation and puncture (CLP) induced model of polymicrobial infection. In vitro, RG100204 blocked mouse AQP9 H2O2 permeability in an ectopic CHO cell expression system and abolished the LPS induced increase in superoxide anion and nitric oxide in FaO hepatoma cells. Pre-treatment of CLP-mice with RG100204 (25 mg/kg p.o. before CLP and then again at 8 h after CLP) attenuated the hypothermia, cardiac dysfunction (systolic and diastolic), renal dysfunction and hepatocellular injury caused by CLP-induced sepsis. Post-treatment of CLP-mice with RG100204 also attenuated the cardiac dysfunction (systolic and diastolic), the renal dysfunction caused by CLP-induced sepsis, but did not significantly reduce the liver injury or hypothermia. The most striking finding was that oral administration of RG100204 as late as 3 h after the onset of polymicrobial sepsis attenuated the cardiac and renal dysfunction caused by severe sepsis. Immunoblot quantification demonstrated that RG100204 reduced activation of the NLRP3 inflammasome pathway. Moreover, myeloperoxidase activity in RG100204 treated lung tissue was reduced. Together these results indicate that AQP9 may be a novel drug target in polymicrobial sepsis.

Ablation of Aquaporin-9 Ameliorates the Systemic Inflammatory Response of LPS-Induced Endotoxic Shock in Mouse.

Septic shock is the most severe complication of sepsis, being characterized by a systemic inflammatory response following bacterial infection, leading to multiple organ failure and dramatically high mortality. Aquaporin-9 (AQP9), a membrane channel protein mainly expressed in hepatocytes and leukocytes, has been recently associated with inflammatory and infectious responses, thus triggering strong interest as a potential target for reducing septic shock-dependent mortality. Here, we evaluated whether AQP9 contributes to murine systemic inflammation during endotoxic shock. Wild type (Aqp9+/+; WT) and Aqp9 gene knockout (Aqp9-/-; KO) male mice were submitted to endotoxic shock by i.p. injection of lipopolysaccharide (LPS; 40 mg/kg) and the related survival times were followed during 72 h. The electronic paramagnetic resonance and confocal microscopy were employed to analyze the nitric oxide (NO) and superoxide anion (O2-) production, and the expression of inducible NO-synthase (iNOS) and cyclooxigenase-2 (COX-2), respectively, in the liver, kidney, aorta, heart and lung of the mouse specimens. LPS-treated KO mice survived significantly longer than corresponding WT mice, and 25% of the KO mice fully recovered from the endotoxin treatment. The LPS-injected KO mice showed lower inflammatory NO and O2- productions and reduced iNOS and COX-2 levels through impaired NF-κB p65 activation in the liver, kidney, aorta, and heart as compared to the LPS-treated WT mice. Consistent with these results, the treatment of FaO cells, a rodent hepatoma cell line, with the AQP9 blocker HTS13268 prevented the LPS-induced increase of inflammatory NO and O2-. A role for AQP9 is suggested in the early acute phase of LPS-induced endotoxic shock involving NF-κB signaling. The modulation of AQP9 expression/function may reveal to be useful in developing novel endotoxemia therapeutics.

In Vitro and In Vivo Nutraceutical Characterization of Two Chickpea Accessions: Differential Effects on Hepatic Lipid Over-Accumulation.

Dietary habits are crucially important to prevent the development of lifestyle-associated diseases. Diets supplemented with chickpeas have numerous benefits and are known to improve body fat composition. The present study was undertaken to characterize two genetically and phenotypically distinct accessions, MG_13 and PI358934, selected from a global chickpea collection. Rat hepatoma FaO cells treated with a mixture of free fatty acids (FFAs) (O/P) were used as an in vitro model of hepatic steatosis. In parallel, a high-fat diet (HFD) animal model was also established. In vitro and in vivo studies revealed that both chickpea accessions showed a significant antioxidant ability. However, only MG_13 reduced the lipid over-accumulation in steatotic FaO cells and in the liver of HFD fed mice. Moreover, mice fed with HFD + MG_13 displayed a lower level of glycemia and aspartate aminotransferase (AST) than HFD mice. Interestingly, exposure to MG_13 prevented the phosphorylation of the inflammatory nuclear factor kappa beta (NF-kB) which is upregulated during HFD and known to be linked to obesity. To conclude, the comparison of the two distinct chickpea accessions revealed a beneficial effect only for the MG_13. These findings highlight the importance of studies addressing the functional characterization of chickpea biodiversity and nutraceutical properties.

Stopped-flow Light Scattering Analysis of Red Blood Cell Glycerol Permeability.

Stopped-Flow Light Scattering (SFLS) is a method devised to analyze the kinetics of fast chemical reactions that result in a significant change of the average molecular weight and/or in the shape of the reaction substrates. Several modifications of the original stopped-flow system have been made leading to a significant extension of its technical applications. One of these modifications allows the biophysical characterization of the water and solute permeability of biological and artificial membranes. Here, we describe a protocol of SFLS to measure the glycerol permeability of isolated human red blood cells (RBCs) and evaluate the pharmacokinetics properties (selectivity and potency) of isoform-specific inhibitors of AQP3, AQP7 and AQP9, three mammalian aquaglyceroporins allowing transport of glycerol across membranes. Suspensions of RBCs (1% hematocrit) are exposed to an inwardly directed gradient of 100 mM glycerol in a SFLS apparatus at 20 °C and the resulting changes in scattered light intensity are recorded at a monochromatic wavelength of 530 nm for 120 s. The SFLS apparatus is set up to have a dead time of 1.6-ms and 99% mixing efficiency in less than 1 ms. Data are fitted to a single exponential function and the related time constant (τ, seconds) of the cell-swelling phase of light scattering corresponding to the osmotic movement of water that accompanies the entry of glycerol into erythrocytes is measured. The coefficient of glycerol permeability ( Pgly , cm/s) of RBCs is calculated with the following equation: Pgly = 1/[(S/V)τ] where τ (s) is the fitted exponential time constant and S/V is the surface-to-volume ratio (cm-1) of the analyzed RBC specimen. Pharmacokinetics of the isoform-specific inhibitors of AQP3, AQP7 and AQP9 are assessed by evaluating the extent of RBC Pgly values resulting after the exposure to serial concentrations of the blockers.

Aquaporin-9 is involved in the lipid-lowering activity of the nutraceutical silybin on hepatocytes through modulation of autophagy and lipid droplets composition.

Hepatic steatosis is the hallmark of non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome and insulin resistance with potential evolution towards non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma. Key roles of autophagy and oxidative stress in hepatic lipid accumulation and NAFLD progression are recognized. Here, we employed a rat hepatoma cell model of NAFLD progression made of FaO cells exposed to oleate/palmitate followed or not by TNFα treatment to investigate the molecular mechanisms through which silybin, a lipid-lowering nutraceutical, may improve hepatic lipid dyshomeostasis. The beneficial effect of silybin was found to involve amelioration of the fatty acids profile of lipid droplets, stimulation of the mitochondrial oxidation and upregulation of a microRNA of pivotal relevance in hepatic fat metabolism, miR-122. Silybin was also found to restore the levels of Aquaporin-9 (AQP9) and glycerol permeability while reducing the activation of the oxidative stress-dependent transcription factor NF-κB, and autophagy turnover. In conclusion, silybin was shown to have molecular effects on signaling pathways that were previously unknown and potentially protect the hepatocyte. These actions intersect TG metabolism, fat-induced autophagy and AQP9-mediated glycerol transport in hepatocytes.

Ectopic expression of a rice plasma membrane intrinsic protein (OsPIP1;3) promotes plant growth and water uptake.

Plasma membrane intrinsic proteins (PIPs) are known to be major facilitators of the movement of a number of substrates across cell membranes. From a drought-resistant cultivar of Oryza sativa (rice), we isolated an OsPIP1;3 gene single-nucleotide polymorphism (SNP) that is mostly expressed in rice roots and is strongly responsive to drought stress. Immunocytochemistry showed that OsPIP1;3 majorly accumulated on the proximal end of the endodermis and the cell surface around the xylem. Expression of GFP-OsPIP1;3 alone in Xenopus oocytes or rice protoplasts showed OsPIP1;3 mislocalization in the endoplasmic reticulum (ER)-like neighborhood, whereas co-expression of OsPIP2;2 recruited OsPIP1;3 to the plasma membrane and led to a significant enhancement of water permeability in oocytes. Moreover, reconstitution of 10×His-OsPIP1;3 in liposomes demonstrated water channel activity, as revealed by stopped-flow light scattering. Intriguingly, by patch-clamp technique, we detected significant NO3- conductance of OsPIP1;3 in mammalian cells. To investigate the physiological functions of OsPIP1;3, we ectopically expressed the OsPIP1;3 gene in Nicotiana benthamiana (tobacco). The transgenic tobacco plants exhibited higher photosynthesis rates, root hydraulic conductivity (Lpr ) and water-use efficiency, resulting in a greater biomass and a higher resistance to water deficit than the wild-type did. Further experiments suggested that heterologous expression of OsPIP1;3 in cyanobacterium altered bacterial growth under different conditions of CO2 gas supply. Overall, besides shedding light on the multiple functions played by OsPIP1;3, this work provides insights into the translational value of plant AQPs.

Corrigendum: Aquaporin-9 Contributes to the Maturation Process and Inflammatory Cytokine Secretion of Murine Dendritic Cells.

[This corrects the article DOI: 10.3389/fimmu.2018.02355.].

Identification and characterization of potent and selective aquaporin-3 and aquaporin-7 inhibitors.

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 µm 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 µm), is a potent and efficacious inhibitor of mouse AQP7 water permeability (IC50, ∼0.2 µm). 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.

CSRP3 mediates polyphenols-induced cardioprotection in hypertension.

Berries contain bioactive polyphenols, whose capacity to prevent cardiovascular diseases has been established recently in animal models as well in human clinical trials. However, cellular processes and molecular targets of berries polyphenols remain to be identified. The capacity of a polyphenol-enriched diet (i.e., blueberries, blackberries, raspberries, strawberry tree fruits and Portuguese crowberries berries mixture) to promote animal survival and protect cardiovascular function from salt-induced hypertension was evaluated in a chronic salt-sensitive Dahl rat model. The daily consumption of berries improved survival of Dahl/salt-sensitive rats submitted to high-salt diet and normalized their body weight, renal function and blood pressure. In addition, a prophylactic effect was observed at the level of cardiac hypertrophy and dysfunction, tissue cohesion and cardiomyocyte hypertrophy. Berries also protected the aorta from fibrosis and modulated the expression of aquaporin-1, a channel involved in endothelial water and nitric oxide permeability. Left ventricle proteomics analysis led to the identification of berries and salt metabolites targets, including cystein and glycin-rich protein 3 (CSRP3), a protein involved in myocyte cytoarchitecture. In neonatal rat ventricular cardiomyocytes, CSRP3 was validated as a target of a berries-derived polyphenol metabolite, 4-methylcatechol sulfate, at micromolar concentrations, mimicking physiological conditions of human plasma circulation. Accordingly, siRNA silencing of CSRP3 and 4-methylcatechol sulfate pretreatment reversed cardiomyocyte hypertrophy and CSRP3 overexpression induced by phenylephrine. Our systemic study clearly supports the modulation of CSRP3 by a polyphenol-rich berries diet as an efficient cardioprotective strategy in hypertension-induced heart failure.

Modulation of Aquaporins by Dietary Patterns and Plant Bioactive Compounds.

Healthful dietary patterns and bioactive compounds supplementation can be adopted as simple and easy intervention to prevent, attenuate or cure clinical disorders, especially when it comes to degenerative and chronic diseases. In the recent years, a growing body of evidence indicates Aquaporins (AQPs), a family of membrane channel proteins widely expressed in the human body, among the targets underlying the beneficial action played by some food nutrients and phytochemical compounds. Here, we provide an overview of what is known regarding the AQP modulation exerted by healthful dietary patterns and plant polyphenols.

Aquaporin 9 Contributes to the Maturation Process and Inflammatory Cytokine Secretion of Murine Dendritic Cells.

Dendritic cells (DCs) are the most potent antigen-presenting cells able to trigger the adaptive immune response to specific antigens. When non-self-antigens are captured, DCs switch from an "immature" to a "mature" state to fulfill their function. Among the several surface proteins involved in DCs maturation, the role of aquaporins (AQPs) is still poorly understood. Here we investigated the expression profile of Aqps in murine bone marrow derived dendritic cells (BMDCs). Among the Aqps analyzed, Aqp9 was the most expressed by DCs. Its expression level was significantly upregulated 6 h following LPS exposure. Chemical inhibition of Aqp9 led to a decreased inflammatory cytokines secretion. BMDCs from AQP9-KO mice release lower amount of inflammatory cytokines and chemokines and increased release of IL-10. Despite the reduced release of inflammatory cytokines, Aqp9-KO mice were not protected from DSS induced colitis. All together, our data indicate that AQP9 blockade can be an efficient strategy to reduce DCs inflammatory response but it is not sufficient to protect from acute inflammatory insults such as DSS induced colitis.

A Stopped-Flow Light Scattering Methodology for Assessing the Osmotic Water Permeability of Whole Sertoli Cells.

Movement of water into and out of the cell is fundamental to life. In the male reproductive tract, at the level of the seminiferous epithelium, Sertoli cells are pivotal in mediating the movement of water into the luminal compartment, a process providing a means of transport of spermatozoa into the epididymal ducts. Here, we describe a stopped-flow light-scattering methodology to easily assess the Sertoli cell osmotic water permeability. The devised methodology is well suited for studying how the Sertoli cell's permeability changes following exogenous stimuli.

Lipopolysaccharide Modifies Glycerol Permeability and Metabolism in 3T3-L1 Adipocytes.

Aquaglyceroporins-aquaporin membrane channels (AQP) that conduct glycerol and other small neutral solutes in addition to water-play major roles in obesity. In adipocytes, aquaglyceroporins mediate glycerol uptake and release across the plasma membrane, which are two key steps for triacylglycerols (TAGs) synthesis (lipogenesis) and hydrolysis (lipolysis). The aim of this study was to assess both glycerol permeability and metabolism in undifferentiated 3T3-L1 cells (UDCs) as well as in untreated (CTL-DCs) versus lipopolysaccharide (LPS-DCs)-treated differentiated 3T3-L1 adipocytes. Glycerol release, TAGs content and whole membrane glycerol permeability were significantly increased in DCs as compared to UDCs. Moreover, in DCs, LPS treatment significantly increased TAGs content and decreased glycerol permeability. In addition, a significant reduction in whole membrane glycerol permeability was observed in LPS-DCs as compared to CTL-DCs. The relative contributions of AQP3, AQP7 and AQP9 (facilitated diffusion), as well as that of the phospholipid bilayer (simple diffusion), to the whole membrane glycerol permeability, were estimated biophysically in UDCs, CTL-DCs and LPS-DCs, using selective AQP inhibitors. Further studies will be required to determine if modifications in either subcellular localization and/or activity of aquaglyceroporins could account for the data herein. Nevertheless, our findings provide novel insights in understanding the LPS-induced adipocyte hypertrophy that accompanies obesity.

Dynamical modeling of liver Aquaporin-9 expression and glycerol permeability in hepatic glucose metabolism.

Liver is crucial in the homeostasis of glycerol, an important metabolic intermediate. Plasma glycerol is imported by hepatocytes mainly through Aquaporin-9 (AQP9), an aquaglyceroporin channel negatively regulated by insulin in rodents. AQP9 is of critical importance in glycerol metabolism since hepatic glycerol utilization is rate-limited at the hepatocyte membrane permeation step. Glycerol kinase catalyzes the initial step for the conversion of the imported glycerol into glycerol-3-phosphate, a major substrate for de novo synthesis of glucose (gluconeogenesis) and/or triacyglycerols (lipogenesis). A model addressing the glucose-insulin system to describe the hepatic glycerol import and metabolism and the correlation with the glucose homeostasis is lacking so far. Here we consider a system of first-order ordinary differential equations delineating the relevance of hepatocyte AQP9 in liver glycerol permeability. Assuming the hepatic glycerol permeability as depending on the protein levels of AQP9, a mathematical function is designed describing the time course of the involvement of AQP9 in mouse hepatic glycerol metabolism in different nutritional states. The resulting theoretical relationship is derived fitting experimental data obtained with murine models at the fed, fasted or re-fed condition. While providing useful insights into the dynamics of liver AQP9 involvement in male rodent glycerol homeostasis our model may be adapted to the human liver serving as an important module of a whole body-model of the glucose metabolism both in health and metabolic diseases.

Hepatocyte and Sertoli Cell Aquaporins, Recent Advances and Research Trends.

Aquaporins (AQPs) are proteinaceous channels widespread in nature where they allow facilitated permeation of water and uncharged through cellular membranes. AQPs play a number of important roles in both health and disease. This review focuses on the most recent advances and research trends regarding the expression and modulation, as well as physiological and pathophysiological functions of AQPs in hepatocytes and Sertoli cells (SCs). Besides their involvement in bile formation, hepatocyte AQPs are involved in maintaining energy balance acting in hepatic gluconeogenesis and lipid metabolism, and in critical processes such as ammonia detoxification and mitochondrial output of hydrogen peroxide. Roles are played in clinical disorders including fatty liver disease, diabetes, obesity, cholestasis, hepatic cirrhosis and hepatocarcinoma. In the seminiferous tubules, particularly in SCs, AQPs are also widely expressed and seem to be implicated in the various stages of spermatogenesis. Like in hepatocytes, AQPs may be involved in maintaining energy homeostasis in these cells and have a major role in the metabolic cooperation established in the testicular tissue. Altogether, this information represents the mainstay of current and future investigation in an expanding field.

Increased water flux induced by an aquaporin-1/carbonic anhydrase II interaction.

Aquaporin-1 (AQP1) enables greatly enhanced water flux across plasma membranes. The cytosolic carboxy terminus of AQP1 has two acidic motifs homologous to known carbonic anhydrase II (CAII) binding sequences. CAII colocalizes with AQP1 in the renal proximal tubule. Expression of AQP1 with CAII in Xenopus oocytes or mammalian cells increased water flux relative to AQP1 expression alone. This required the amino-terminal sequence of CAII, a region that binds other transport proteins. Expression of catalytically inactive CAII failed to increase water flux through AQP1. Proximity ligation assays revealed close association of CAII and AQP1, an effect requiring the second acidic cluster of AQP1. This motif was also necessary for CAII to increase AQP1-mediated water flux. Red blood cell ghosts resealed with CAII demonstrated increased osmotic water permeability compared with ghosts resealed with albumin. Water flux across renal cortical membrane vesicles, measured by stopped-flow light scattering, was reduced in CAII-deficient mice compared with wild-type mice. These data are consistent with CAII increasing water conductance through AQP1 by a physical interaction between the two proteins.

Functional reconstitution of a rice aquaporin water channel, PIP1;1, by a micro-batchwise methodology.

Assessing the selectivity, regulation and physiological relevance of aquaporin membrane channels (AQPs)requires structural and functional studies of wild type and modified proteins. In particular, when characterizing their transport properties, reconstitution in isolation from native cellular or membrane processes is of pivotal importance. Here, we describe rapid and efficient incorporation of OsPIP1;1, a rice AQP, in liposomes at analytical scale. PIP1;1 was produced as a histidine-tagged form, 10 His-OsPIP1;1, in an Escherichia coli-based expression system. The recombinant protein was purified by affinity chromatography and incorporated into liposomes by a micro-batchwise technology using egg-yolk phospholipids and the non-polar Amberlite resin. PIP1;1 proteoliposomes and control empty liposomes had good size homogeneity as seen by quasi-elastic light scattering and electron microscopy analyses. By stopped-flow light scattering, indicating correct protein folding of the incorporated protein, the osmotic water permeability exhibited by the PIP1;1 proteoliposomes was markedly higher than empty liposomes. Functional reconstitution of OsPIP1;1 was further confirmed by the low Arrhenius activation energy (3.37 kcal/mol) and sensitivity to HgCl2, a known AQP blocker, of the PIP1;1-mediated osmotic water conductance. These results provide a valuable contribution in fully elucidating the regulation and water-conducting property of PIP1;1, an AQP that needs to hetero-multimerize with AQPs of the PIP2 subgroupto reach the native plasma membrane and play its role. The micro-batchwise methodology is suitable for the functional reconstitution of whichever AQPs and other membrane transport proteins.

Liver glycerol permeability and aquaporin-9 are dysregulated in a murine model of Non-Alcoholic Fatty Liver Disease.

One form of liver steatosis, namely Non-Alcoholic Fatty Liver Disease (NAFLD), is a worrisome health problem worldwide characterized by intrahepatic triacylglycerol (TG) overaccumulation. NAFLD is a common feature of metabolic syndrome being often associated with obesity, dyslipidemia and diabetes and mostly closely linked to insulin resistance. The mechanism of NAFLD pathogenesis is object of intense investigation especially regarding complex systems ultimately resulting in excessive TG deposition in hepatocytes. However, scarce is the attention about the relevance of hepatic import of glycerol, the other primary source (as glycerol-3-phosphate) of increased TG in hepatocytes. Obese leptin-deficient (ob/ob) mice, an animal model of NAFLD, were used to evaluate the functional involvement of Aquaporin-9 (AQP9), the major pathway of liver glycerol entry, in hepatosteatosis. By RT-PCR and qPCR, the level of Aqp9 mRNA in the liver of starved obese mice was comparable with the corresponding control lean littermates. By immunoblotting, the AQP9 protein at the hepatocyte sinusoidal plasma membrane of obese mice was markedly lower (33%) than lean mice, a finding fully confirmed by immunohistochemistry. By stopped-flow light scattering, the liver glycerol permeability of ob/ob mice was significantly lower (53%) than lean mice, a finding consistent with both the observed down-regulation of AQP9 protein and increased level of plasma glycerol characterizing obese mice. In summary, our results suggest implication of AQP9 in liver steatosis. The reduction of hepatocyte AQP9 and, consequently, glycerol permeability might be a defensive mechanism to counteract further fat infiltration in liver parenchyma.