Glycerol Permeability Research Articles

Endothelial Aquaporins and Hypomethylation: Potential Implications for Atherosclerosis and Cardiovascular Disease.

Aquaporins (AQPs) are transmembrane channels that facilitate water and glycerol permeation through cell membranes. Recently, the water channel AQP1 was suggested to contribute to endothelial homeostasis and cardiovascular health. Less is known about endothelial aquaglyceroporins expression and its implication in cardiovascular disease (CVD). We have previously used cultured human endothelial cells under a hypomethylating environment to study endothelial dysfunction and activation, a phenotype implicated in the establishment of atherosclerosis and CVD. Here, we used the same cell model to investigate aquaporin’s expression and function in healthy or pro-atherogenic phenotype. We first confirmed key features of endothelium dysfunction and activation in our cell model, including an augmented endothelial transmigration under hypomethylation. Subsequently, we found AQP1 and AQP3 to be the most predominant AQPs accounting for water and glycerol fluxes, respectively, in the healthy endothelium. Moreover, endothelial hypomethylation led to decreased levels of AQP1 and impaired water permeability without affecting AQP3 and glycerol permeability. Furthermore, TNF-α treatment-induced AQP1 downregulation suggesting that the inflammatory NF-κB signaling pathway mediates AQP1 transcriptional repression in a pro-atherogenic endothelium, a possibility that warrants further investigation. In conclusion, our results add further support to AQP1 as a candidate player in the setting of endothelial dysfunction and CVD.

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.

Expression of the aquaglyceroporin HC-9 in a freeze-tolerant amphibian that accumulates glycerol seasonally.

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.

Temperature‐Induced Changes to Aquaporin Localization in the Freeze‐Tolerant Cope's Gray Treefrog

Cope's gray treefrogs alter their physiology in response to cold as part of their freeze tolerance mechanism. One such change involves the accumulation, both extracellularly and intracellularly, of cryoprotective glycerol, which likely crosses cellular membranes using glycerol‐permeable channels called aquaglyceroporins (GLPs). Three GLPs (HC‐3, HC‐7, and HC‐9) have been identified in the Cope's gray treefrog, representing homologs to the mammalian AQP3, AQP7, and AQP9, respectively.Regulation of transmembrane glycerol diffusion depends on GLP expression in the plasma membrane resulting from changes to transcriptional and translational output of the GLPs and/or by changes in protein trafficking to the plasma membrane. We have previously reported that GLP mRNA and protein expression are modulated during cold acclimation and freezing; however, localization of GLP proteins in the plasma membrane has not been studied thoroughly. We hypothesized that changes in acclimation temperature would alter the localization of the GLPs HC‐3 and HC‐9 in the plasma membrane and confirmed this with immunofluorescence, plasma membrane fractionation, and biotinylation.For HC‐9 in liver, quantitation of protein expression (by immunoblot) showed a 4‐ to 6‐fold increase in the higher molecular weight band of frozen (−2.5°C for 24 hours) and thawed (5°C for 24 hours after freezing) over warm (20°C) treefrogs. In immunohistochemistry images, HC‐9 signal appeared to be localized to the plasma membrane of hepatocytes of warm‐ and cold‐acclimated animals but dispersed intracellularly in frozen treefrog hepatocytes. In preliminary studies, fractionation of cellular protein (Biovision, Milpitas, CA) indicated that between freezing and thawing, the nominal (~34 kDa) molecular weight band of HC‐9 diminishes in the plasma membrane fraction while the (putatively) glycosylated high molecular weight band of HC‐9 increases.For HC‐3 in erythrocytes, protein expression is enhanced by cold acclimation and by incubation in medium containing glycerol. We incubated erythrocytes taken from warm‐acclimated animals at 4°C for 48 hours and biotinylated the surface proteins. In immunoblots of the biotinylated fraction probed with anti‐HC‐3 primary antibody, the high molecular weight (glycosylated) band was enriched and the nominal (~32 kDa) molecular weight band was diminished. These results suggest that water and glycerol permeability during cold acclimation and freezing can be modulated by protein trafficking, which may be influenced by the post‐translational modification of glycosylation.Support or Funding InformationSupported by NSF IOS‐1121457.

The mechanism of aquaporin inhibition by gold compounds elucidated by biophysical and computational methods

The inhibition of water and glycerol permeation via human aquaglyceroporin-3 (AQP3) by gold(iii) complexes has been studied by stopped-flow spectroscopy and, for the first time, its mechanism has been described using molecular dynamics (MD), combined with density functional theory (DFT) and electrochemical studies. The obtained MD results showed that the most effective gold-based inhibitor, anchored to Cys40 in AQP3, is able to induce shrinkage of pores preventing glycerol and water permeation. Moreover, the good correlation between the affinity of the Au(iii) complex to Cys binding and AQP3 inhibition effects was highlighted, while no influence of the different oxidative character of the complexes could be observed.

Higher membrane fluidity mediates the increased subcutaneous fatty acid content in pigs fed reduced protein diets

The production of pork with moderate amounts of intramuscular fat (IMF) without an increase in subcutaneous fat is highly desirable for the meat industry. Several studies indicate that dietary protein reduction during the growing–finishing period of pigs enhances IMF content, but its consequence on carcass fat deposition is still contradictory. In this study, we hypothesized that the effects of reduced protein diets (RPD), corrected or not with the limiting amino acid lysine, on subcutaneous fat deposition from pigs with distinct genotypes are mediated by adipose membranes biophysical properties. In total, 36 crossbred (Large White×Landrace×Pietrain – a lean genotype) and purebred (Alentejana breed – a fatty genotype) male pigs were randomly assigned to the control group, the RPD group or the reduced protein diet equilibrated for lysine (RPDL) group, allowing a 2×3 factorial arrangement (n=6). Backfat thickness and total fatty acid content were higher in Alentejana relative to crossbred pigs. Although dietary treatments did not change backfat thickness, RPD and RPDL increased total fatty acids content of subcutaneous fat. In order to understand this effect, adipose tissue membranes isolated from pig’s subcutaneous fat were assayed for glycerol permeability and fluidity, using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-(trimethylamino)-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) probes. The glycerol transport across adipose membranes was not mediated by aquaglyceroporins and remained unchanged across dietary groups. Regardless of lysine correction, RPD increased membrane fluidity at the hydrocarbon region (lower DPH fluorescence anisotropy) in both genotypes of pigs. This result was associated with a lower ratio between oleic acid and linoleic acid on membrane’s fatty acid composition. Adipose membrane’s cholesterol content was independent from genotype and diet. Taken together, the present study shows that dietary protein reduction is successful in maintaining backfat thickness, although a negative side effect was observed on total fatty acids in subcutaneous fat, which may be due to changes in the fluidity of adipose membranes.

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.

Cloning and in vitro characterization of a Schistosoma japonicum aquaglyceroporin that functions in osmoregulation.

As one of the three major human pathogens that cause schistosomiasis, Schistosoma japonicum is the only one that is endemic in China. Despite great progress on schistosomiasis control over the past 50 years in China, S. japonicum transmission still occurs in certain endemic regions, which causes significant public health problems and enormous economic losses. During different life stages, parasites are able to survive dramatic osmolality changes between its vector, fresh water, and mammal host. However, the molecular mechanism of parasite osmoregulation remains unknown. To address this challenging question, we report the first cloning of an S. japonicum aquaglyceroporin (SjAQP) from an isolate from Jiangsu province, China. Expressing SjAQP in Xenopus oocytes facilitated the permeation of water, glycerol, and urea. The water permeability of SjAQP was inhibited by 1 mM HgCl2, 3 mM tetraethylammonium, 1 mM ZnCl2, and 1 mM CuSO4. SjAQP was constitutively expressed throughout the S. japonicum life cycle, including in the egg, miracidia, cercaria, and adult stages. The highest expression was detected during the infective cercaria stage. Our results suggest that SjAQP plays a role in osmoregulation throughout the S. japonicum life cycle, especially during cercariae transformation, which enables parasites to survive osmotic challenges.

Aquaporin 9 in the Freeze Tolerance Mechanism of Cope's Gray Treefrogs

Cope's Gray Treefrogs Hyla chrysoscelis accumulate glycerol in response to cold as part of their freeze tolerance mechanism. This glycerol may derive from glucose stores in the liver that are broken down and released through glycerol transporters. We therefore hypothesized that these treefrogs would express aquaporin 9 (AQP9), a transmembrane protein from the aquaglyceroporin family that facilitates rapid glycerol transport, predominately in the liver. We also hypothesized that this protein would be up‐regulated during cold acclimation to promote glycerol permeability. To test this hypothesis, we sequenced the AQP9 homologue HC‐9, analyzed RNA and protein expression, and characterized water and glycerol permeability through a Xenopus oocyte expression system.HC‐9 has 315 amino acids which include 2 conserved NPA motifs unique to aquaporins, 5 conserved residues typical of glyceroporins, a high homology (69%) to human AQP9, and a nearly identical homology (97%) to the hyla japonica. The mRNA strand also possesses a long untranslated tail unique to amphibians that appears to be necessary for translation. Furthermore, characterization using the Xenopus expression system has shown that HC‐9 is permeable to glycerol but has a poor water permeability compared to human AQP1.In warm‐acclimated animals, HC‐9 mRNA (RT‐PCR) and protein (western blotting) were expressed in many tissues, with some of the highest levels in liver, stomach, ventral skin, and muscle. Across most tissues, realtime mRNA expression (qRT‐PCR) decreased in cold‐acclimated (5ºC) and frozen (−2.5 ºC) treefrogs, with recovery in treefrogs returned to 5ºC for 24 hours. Meanwhile, protein expression increased in the liver of cold and frozen treefrogs, but remained unchanged or decreased in all other tissues.HC‐9 expression is clearly regulated as a part of the Cope's Gray Treefrogs freeze tolerance mechanism. Contrary to our hypothesis, most tissues have decreased expression, which may reduce glycerol permeability to protect cells during the freeze process. Liver is a notable exception by increasing protein expression, which may facilitate the re‐uptake of glycerol during ice formation. Liver appears to play a critical role in the distribution of the cryoprotectant glycerol, and this study suggests HC‐9 facilitates in this role.Support or Funding InformationSupported by NSF IOS‐1121457.

Glyceroporin HC‐9 in the Freeze Tolerance Mechanism of Cope's Gray Treefrogs

Cope's gray treefrogs Hyla chrysoscelis accumulate glycerol in response to cold as part of their freeze tolerance mechanism. This glycerol may derive from glucose stores in the liver that are broken down and released through glycerol transporters. We therefore hypothesized that these treefrogs would express aquaporin 9 (AQP9), a transmembrane protein from the aquaglyceroporin family that facilitates glycerol transport, predominately in the liver. We also hypothesized that this protein would be up‐regulated during cold acclimation to promote glycerol permeability. To test this hypothesis, we sequenced the AQP9 homolog HC‐9, analyzed RNA and protein expression, and characterized water and glycerol permeability through a Xenopus oocyte expression system.HC‐9 has 315 amino acids which include 2 conserved NPA motifs characteristic of aquaporins, 5 conserved residues typical of glyceroporins, and high sequence homology to human AQP9 (69%) and to AQP9 from Hyla japonica (97%). The mRNA strand also possesses a long untranslated tail that appears to be unique to amphibians and necessary for translation. Characterization using the Xenopus oocyte expression system indicates that HC‐9 is permeable to glycerol but has a poor water permeability compared with human AQP1.In warm‐acclimated treefrogs, HC‐9 mRNA (RT‐PCR) and protein (Western blotting) were expressed in many tissues, with highest levels in liver, stomach, ventral skin, and muscle. Across most tissues, realtime mRNA expression (qRT‐PCR) decreased in cold‐acclimated (5°C) and frozen (−2.5°C) treefrogs, with return to levels near those in warm acclimated animals after return to 5°C for 24 hours. In contrast, protein expression increased in the liver and ventral skin of cold and frozen treefrogs, but remained unchanged or decreased in all other tissues.HC‐9 expression is clearly regulated as a part of the Cope's gray treefrog freeze tolerance mechanism. Contrary to our hypothesis, most tissues have decreased expression of HC‐9 in the cold, which may reduce glycerol permeability to protect cells during the freezing process. Liver is a notable exception by increasing protein expression from warm to cold to frozen conditions. Liver appears to play a critical role in the distribution of the cryoprotectant glycerol during the freeze‐thaw cycle, and this study suggests HC‐9 facilitates in this role.Support or Funding InformationSupported by NSF IOS‐1121457

Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2.

The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis.

Aquaporin 3 protects against lumbar intervertebral disc degeneration via the Wnt/β-catenin pathway.

Previous studies have demonstrated that the expression of aquaporin 3 (AQP3), a water channel which promotes glycerol permeability and water transport across cell membranes, is reduced in degenerative lumbar intervertebral disc (IVD) tissues. However, the role of AQP3 in the pathogenesis of IVD degeneration has not recieved much scholarly attention. The objective of the present study was to investigate the effect of AQP3 on cell proliferation and extracellular matrix (ECM) degradation in human nucleus pulposus cells (hNPCs) using gain-of-function and loss-of-function experiments, and to determine whether Wnt/β-catenin signaling is involved in the effect of AQP3 on IVD degeneration. hNPCs were transfected with the AQP3-pcDNA3.1 plasmid or AQP3 siRNA to overexpress or suppress AQP3. An MTT assay was performed to determine cell proliferation, and we found that AQP3 promoted hNPC proliferation. The expression of aggrecan, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4 and ADAMTS5 was detected using western blot analysis, to examine the effect of AQP3 on ECM degradation in hNPCs. The results revealed that AQP3 inhibited ECM degradation in hNPCs. In addition, we found that Wnt/β-catenin signaling was suppressed by AQP3. However, the effect of AQP3 on hNPC proliferation and ECM degradation was reversed by treatment with lithium chloride, a known activator of Wnt/β‑catenin signaling. In conclusion, using in vitro and in vivo tests, we have reported for the first time, to the best of our knowledge, that AQP3 exerts protective effects against IVD degeneration, and these are effected, at least partially, through the inhibition of Wnt/β-catenin signaling.

Exploring the gating mechanisms of aquaporin-3: new clues for the design of inhibitors?

The pH gating of human AQP3 and its effects on both water and glycerol permeabilities have been fully characterized for the first time using a human red blood cell model (hRBC). For comparison, the effects of pH on the gating of rat AQP3 have also been characterized in yeast. The obtained results highlight similarities as well as differences between the two isoforms. In addition, we investigated the molecular mechanism of hAQP3 pH gating in silico, which may disclose new pathways to AQP regulation by small molecule inhibitors, and therefore may be important for drug development.

The inhibition of glycerol permeation through aquaglyceroporin-3 induced by mercury(II): A molecular dynamics study

Mercurial compounds are known to inhibit water permeation through aquaporins (AQPs). Although in the last years some hypotheses were proposed, the exact mechanism of inhibition is still an open question and even less is known about the inhibition of the glycerol permeation through aquaglyceroporins. Molecular dynamics (MD) simulations of human aquaporin-3 (AQP3) have been performed up to 200ns in the presence of Hg2+ ions. For the first time, we have observed the unbiased passage of a glycerol molecule from the extracellular to cytosolic side. Moreover, the presence of Hg2+ ions covalently bound to Cys40 leads to a collapse of the aromatic/arginine selectivity filter (ar/R SF), blocking the passage of both glycerol and water. Interestingly, the local conformational changes of the protein follow mercury coordination by water and by aminoacidic donor atoms. Overall, the obtained results are important to improve the design of selective AQP inhibitors for future therapeutic and imaging applications.

Sexual Dimorphism of Adipose and Hepatic Aquaglyceroporins in Health and Metabolic Disorders.

Gender differences in the relative risk of developing metabolic complications, such as insulin resistance or non-alcoholic fatty liver disease (NAFLD), have been reported. The deregulation of glycerol metabolism partly contributes to the onset of these metabolic diseases, since glycerol constitutes a key substrate for the synthesis of triacylglycerols (TAGs) as well as for hepatic gluconeogenesis. The present mini-review covers the sex-­related differences in glycerol metabolism and aquaglyceroporins (AQPs) and its impact in the control of adipose and hepatic fat accumulation as well as in whole-body glucose homeostasis. Plasma glycerol concentrations are increased in women compared to men probably due to the higher lipolytic rate and larger AQP7 amounts in visceral fat as well as the well-known sexual dimorphism in fat mass with women showing higher adiposity. AQP9 represents the primary route for glycerol uptake in hepatocytes, where glycerol is converted by the glycerol-kinase enzyme into glycerol-3-phosphate, a key substrate for de novo synthesis of glucose and TAG. In spite of showing similar hepatic AQP9 protein, women exhibit lower hepatocyte glycerol permeability than men, which might contribute to their lower prevalence of insulin resistance and NAFLD.

Molecular Cloning of cDNA Encoding an Aquaglyceroporin, AQP-h9, in the Japanese Tree Frog, Hyla japonica: Possible Roles of AQP-h9 in Freeze Tolerance.

In order to study the freeze-tolerance mechanism in the Japanese tree frog, Hyla japonica, wecloned a eDNA encoding aquaporin (AQP) 9 from its liver. The predicted amino acid sequence ofH. japonica AQP9 (AQP-h9) contained six putative transmembrane domains and two conservedAsn-Pro-Aia motifs, which are characteristic of AQPs. A swelling assay using Xenopus laevisoocytes injected with AQP-h9 cRNA showed that AQP-h9 facilitated water and glycerol permeation,confirming its property as an aquaglyceroporin. Subsequently, glycerol concentrations in serumand tissue extracts were compared among tree frogs that were hibernating, frozen, or thawed afterfreezing. Serum glycerol concentration of thawed frogs was significantly higher than that of hibernatingfrogs. Glycerol content in the liver did not change in the freezing experiment, whereas thatin the skeletal muscle was elevated in thawed frogs as compared with hibernating or frozen frogs. Histological examination of the liver showed that erythrocytes aggregated in the sinusoids during hibernation and freezing, and immunoreactive AQP-h9 protein was detected over the erythrocytes. The AQP-h9 labeling was more intense in frozen frogs than in hibernating frogs, but nearly undetectable in thawed frogs. For the skeletal muscle, weak labels for AQP-h9 were observed in the cytoplasm of myocytes of hibernating frogs. AQP-h9 labeling was markedly enhanced by freezing and was decreased by thawing. These results indicate that glycerol may act as a c;:ryoprotectant in H. japonica and that during hibernation, particularly during freezing, AQP-h9 may be involved in glycerol uptake in erythrocytes in the liver and in intracellular glycerol transport in the skeletal muscle cells.

Sequencing and Gene Expression of HC‐9, an Aquaporin‐9 Homolog, in Freeze‐tolerant Cope's Gray Treefrog

Cope's gray treefrogs, Hyla chrysoscelis, accumulate glycerol in response to cold as part of their freeze tolerance mechanism. The glycerol is suspected to derive from glucose that is stored in the liver before it is exported to the bloodstream for distribution to other tissues. We therefore hypothesized that tissues from H. chrysoscelis, including hepatocytes, would express a homolog of aquaporin 9 (AQP9), a transmembrane protein from the aquaglyceroporin family that facilitates glycerol transport in mammalian liver. We also hypothesized that AQP9 would be up-regulated during cold acclimation to promote glycerol permeability. To test this hypothesis, we designed primers based on published AQP9 sequences, generated cDNA from mRNA extracted from liver, and identified a 945 base pair gene, HC-9, with high homology (69%) to human AQP9. Quantitative PCR analysis revealed HC-9 expression in liver, stomach, bladder, kidney, eye, lung, muscle, and skin (and at low levels in intestine, brain, heart, fat, and red blood cells). In tissues from frogs that had been cold acclimated, HC-9 was not up-regulated in any tissue and was down regulated in muscle and stomach, contrary to our hypothesis. In frozen and thawed animals, expression of HC-9 RNA mRNA was down-regulated in most tissues. Ongoing studies are characterizing the transport properties of HC-9 and the pattern of HC-9 protein expression in cold acclimation and freezing. Supported by NSF IOS-1121457.

Adipocyte membrane glycerol permeability is involved in the anti-adipogenic effect of conjugated linoleic acid

Conjugated linoleic acid (CLA), a group of minor fatty acids from ruminant origin, has long been recognized as a body fat lowering agent. Given the trans(t)10,cis(c)12-CLA well documented interference on lipolysis, we hypothesized for adipocytes altered permeation to glycerol when supplemented with this isomer. 3T3-L1 murine differentiated adipocytes were medium supplemented with linoleic acid (LA) and individual or combined c9,t11 and t10,c12-CLA isomers. Adipocytes treated with the t10,c12-CLA isomer and CLA mixture showed reduced triacylglycerols content (p < 0.001), re-enforcing the t10,c12-CLA as the anti-adipogenic CLA isomer. This finding was supported by decreased Δ9-desaturase index and adipocyte differentiation markers for the t10,c12-CLA group (p < 0.001), which suggest reduced lipogenesis and differentiation, respectively. The glycerol permeability was higher in all CLA treated cells compared to control and LA groups (p < 0.05). The increase in glycerol permeability agrees with both reduced triacylglycerols and non-osmotic cellular volume in the t10,c12-CLA and CLA mixture groups. Taken together, our data suggest that the increased adipocyte plasma membrane glycerol fluxes may be part of the anti-adipogenic response to CLA treatments.

Cross-evaluation of optimal glycerol concentration to enhance optical tissue clearing efficacy.

The efficacy of light therapeutic and diagnostic applications can be enhanced by employing optical tissue clearing (OTC) agents to minimize light scattering in tissue. This study aimed to investigate the optimal concentration of glycerol, so that it can be efficiently used as an OTC agent in dermatology. Glycerol was topically applied to avoid the possibility of edema that could be caused by dermal injection. The efficacy of glycerol was quantitatively evaluated for various concentrations using optical coherence tomography (OCT) to evaluate light scattering and ultrasound imaging modality to evaluate collagen dissociation. The intensity in the OCT images in the deeper regions increased over time after glycerol application owing to enhanced light penetration caused by glycerol permeation into the sample. A comparable decrease over time in the collagen distribution was observed in the ultrasound images after glycerol application. The optimal concentration of glycerol to maximize OTC was found to be 70%. The finding of this study may provide a guideline regarding the use of glycerol for efficient light diagnosis and therapy in dermatology.

C-2020: Determination of membrane permeability of porcine adipose derived-stem cell

Adipose-derived stem cells (ADSCs), as an abundant and accessible source of adult stem cells, self-renew and differentiate into various cell lines and have promising potential in tissue engineering and as an alternative to the use of regenerative medicine strategies. In order to meet clinically urgent demand of ADSCs, the cell cryopreservation was applied to the ADSCs long-term preservation. To optimize the cryoprotective agents (CPAs) addition, removal and the freezing as well as thawing processes, we measured the transport properties of porcine adipose-derived stem cells (pADSCs). In this study, the microperfusion system was used to measure transport properties of porcine adipose-derived stem cells (pADSCs) membrane. The membrane permeability coefficient to water (Lp), the membrane permeability coefficient to Me2SO, EG and glycerol (Ps), and the activation energies and reference membrane coefficient have been investigated at 28, 18, 8 and −2 °C, respectively. We determined the inactive cell (Vb), the hydraulic conductivity (Lp) and the CPAs permeability (Ps) at various temperature in 2-paramerter (2 − p) model. No significant difference in the Me2SO and EG permeability was obtained at experimental temperature and the effect of the temperature on the transport properties of the pADSC membrane was analyzed. However, the glycerol permeability of pADSCs was too small to be precisely measured. We obtained the parameter of pADSCs with 1.5 M glycerol at 32 °C and determined the accurate activation energies of glycerol permeability and the referent of glycerol permeability. Membrane permeability of porcine Adipose Derived-Stem cell quantitatively measured was used to minimize the excessive volume shrinkage and the toxicity with CPAs addition.