Glycerol Transport Research Articles

An Atherogenic Diet Disturbs Aquaporin 5 Expression in Liver and Adipocyte Tissues of Apolipoprotein E-Deficient Mice: New Insights into an Old Model of Experimental Atherosclerosis.

The dysfunction of vascular endothelial cells is profoundly implicated in the pathogenesis of atherosclerosis and cardiovascular disease, the global leading cause of death. Aquaporins (AQPs) are membrane channels that facilitate water and glycerol transport across cellular membranes recently implicated in the homeostasis of the cardiovascular system. Apolipoprotein-E deficient (apoE−/−) mice are a common model to study the progression of atherosclerosis. Nevertheless, the pattern of expression of AQPs in this atheroprone model is poorly characterized. In this study, apoE−/− mice were fed an atherogenic high-fat (HF) or a control diet. Plasma was collected at multiple time points to assess metabolic disturbances. At the endpoint, the aortic atherosclerotic burden was quantified using high field magnetic resonance imaging. Moreover, the transcriptional levels of several AQP isoforms were evaluated in the liver, white adipocyte tissue (WAT), and brown adipocyte tissue (BAT). The results revealed that HF-fed mice, when compared to controls, presented an exacerbated systemic inflammation and atherosclerotic phenotype, with no major differences in systemic methylation status, circulating amino acids, or plasma total glutathione. Moreover, an overexpression of the isoform AQP5 was detected in all studied tissues from HF-fed mice when compared to controls. These results suggest a novel role for AQP5 on diet-induced atherosclerosis that warrants further investigation.

Genome-wide association study for beef fatty acid profile using haplotypes in Nellore cattle

The objective of this study was to identify genomic regions associated with beef fatty acid (FA) profile in Nellore cattle finished in feedlot, using haplotype based analyzes. Also, the metabolic pathway enrichment analysis was performed to seek for genes with known functions, close to the haplotype blocks, associated with the evaluated traits, providing subsidies for a better understanding of how the genes affect this trait. A total of 963 Nellore bulls with phenotype for FA, were genotyped using the Illumina BovineHD BeadChip. Samples from the Longissimus thoracis muscle were taken for FAs profile measurement by gas chromatography using a SP-2560 capillary column. Haplotype blocks were defined based on the linkage disequilibrium (LD), where pairs of SNPs are in strong LD if the confidence interval is equal to or greater than 95% with D’ equal to 0.98 and the lower limit is above 0.70. The model used to estimate the haplotypes effects for the sum of beef fatty acid groups included the fixed effects of contemporary group, animal age at slaughter (as linear covariable), haplotypes (as linear regression over the number of haplotype copies: 0, 1 e 2), and the additive genetic effect as random (polygenic effect). Genome-wide association analyzes were performed for each haplotype block (single marker association) by a Restricted Maximum Likelihood model (REML) using ASREML v. 4.3 software. A total of 15, 15, 4, 3, 6, 5, and 10 haplotypes were significantly associated (FDR< 0.10) with monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), total amount of saturated fatty acids (SFA), PUFA:SFA ratio, omega 3 (n3), omega 6 (n6), and n6:n3 ratio, respectively. The statistically significant haplotypes harbored 38 genes, among which we highlighted those involved in lipid biosynthesis and metabolism, low-density lipoprotein oxidation, transport of fatty acids and cholesterol, adipocyte functions, biosynthesis of glycoprotein hormones, regulation of serum triglyceride and high-density lipoprotein-cholesterol levels, and adipogenesis. In addition, several novel regions were identified, including a putative quantitative trait locus for lipid metabolism and energy homeostasis. The enrichment analysis by DAVID tool revealed 14 significant (P< 0.05) terms related to fatty acids such as cellular response to retinoic acid, glycerol transport, regulation of Notch signaling pathway, and Ras signaling pathway. These results would contribute to the improvement of nutritional and health value of beef lipids in indicine cattle by including information from causal mutations in genetic evaluation through weighted their effects differently in genomic predictions.

Aquaporin-3 is involved in NLRP3-inflammasome activation contributing to the setting of inflammatory response.

Inflammasomes are large immune multiprotein complexes that tightly regulate the production of the pro-inflammatory cytokines, being dependent on cell regulatory volume mechanisms. Aquaporins (AQPs) are protein channels that facilitate the transport of water and glycerol (aquaglyceroporins) through membranes, essential for cell volume regulation. Although these membrane proteins are highly expressed in monocytes and macrophages, their role in the inflammatory process is still unclear. Here, we investigated the role of aquaglyceroporin AQP3 in NLRP3-inflammasome activation by complementary approaches based either on shRNA silencing or on AQP3 selective inhibition. The latter has been achieved using a reported potent gold-based inhibitor, Auphen. AQP3 inhibition or silencing partially blocked LPS-priming and decreased production of IL-6, proIL-1β, and TNF-α, suggesting the possible involvement of AQP3 in macrophage priming by Toll-like receptor 4 engagement. Moreover, AQP3-dependent cell reswelling increased IL-1β release through caspase-1 activation. NLRP3-inflammasome activation induced by reswelling, nigericin, and ATP was also blocked when AQP3 was inhibited or silenced. Altogether, these data point towards AQPs as potential players in the setting of the inflammatory response.

Inhibition of aquaporin-3 in macrophages by a monoclonal antibody as potential therapy for liver injury

Aquaporin 3 (AQP3) is a transporter of water, glycerol and hydrogen peroxide (H2O2) that is expressed in various epithelial cells and in macrophages. Here, we developed an anti-AQP3 monoclonal antibody (mAb) that inhibited AQP3-facilitated H2O2 and glycerol transport, and prevented liver injury in experimental animal models. Using AQP3 knockout mice in a model of liver injury and fibrosis produced by CCl4, we obtained evidence for involvement of AQP3 expression in nuclear factor-κB (NF-κB) cell signaling, hepatic oxidative stress and inflammation in macrophages during liver injury. The activated macrophages caused stellate cell activation, leading to liver injury, by a mechanism involving AQP3-mediated H2O2 transport. Administration of an anti-AQP3 mAb, which targeted an extracellular epitope on AQP3, prevented liver injury by inhibition of AQP3-mediated H2O2 transport and macrophage activation. These findings implicate the involvement of macrophage AQP3 in liver injury, and provide evidence for mAb inhibition of AQP3-mediated H2O2 transport as therapy for macrophage-dependent liver injury.

Seasonal effect in expression of AQP1, AQP3 and AQP5 in skin of Murrah buffaloes.

Aquaporins are transmembrane protein channels which are known to help the passage of water and solutes across the cell membranes. AQP1, AQP3 and AQP5 are isoforms of aquaporin known to aid in transepithelial water movement. AQP3 is also known to aid in glycerol transport. The present study was conducted to investigate the role of AQP1, AQP3 and AQP5 in thermoregulation of buffaloes by probing the expression of the genes in skin of buffaloes during different season viz. winter, spring and summer. The skin tissue samples were collected from the neck region of Murrah buffaloes (n=12) and analyzed for gene expression by RT-PCR and immunolocalization. The physiological responses including respiration rate, rectal temperature and neck skin temperature observed during summer were significantly higher than winter and spring seasons. The study revealed the expression of AQP1, AQP3 and AQP5 genes in skin samples. The relative mRNA expressions of AQP1, AQP3 and AQP5 in skin relative to spring season were 1.41±0.47, 1.95±0.22 and 6.77±1.02 folds during summer which were significantly higher than other seasons. The up-regulation of the expression of the studied AQPs were concomitant with the increase in physiological responses including skin temperature and sweating rate during summer. During summer season, AQP1 were mostly immunolocalized in the walls of skin blood capillaries, while AQP3 were observed mostly in the epidermal layer of the skin. The immunolocalization of AQP5 were mostly observed in the secretory glands of skin. The up-regulation of AQP1, AQP3 and AQP5 in skin during summer season indicates their role in thermoregulation of buffaloes.

The application of electrodialysis in synthesis of oligoglycerols from glycerol and epichlorohydrin via Williamson reaction

The effectiveness of electrodialytic (ED) desalination of an exemplary oligoglycerols synthesis post-reaction mixture prepared by a typical Williamson ether synthesis route was examined. The mixture, prepared using a previously reported method, contained water, unreacted glycerol, di-, tri- and tetraglycerols as well as approx. 5% of KCl. The effect of ED voltage on desalination efficiency, water and glycerol transport rates are discussed, and expected ED energy consumption was calculated. It was shown that ED was successfully utilized in the desalination of the post-reaction mixtures in question; its application allows for production of individual oligoglycerols with low chloride content.

Maternal physical activity significantly alters the placental transcriptome

IntroductionMaternal lifestyle, in particular physical activity (PA), influences many of the physiological adaptations during pregnancy associated with feto-placental development and growth. There is limited to no information on the link between PA during pregnancy and the molecular mechanisms governing placental function. The aim of this study was to investigate the molecular mechanisms through which maternal PA may influence placental function. MethodsThe level of PA was measured by accelerometry and gene expression was measured in term placenta with custom polymerase chain reaction (PCR) arrays and microarray analysis followed by a pathway analyses on significantly differentially expressed genes (DEGs). ResultsMicroarray analysis showed 43 significantly DEGs between active and non-active participants. RT-qPCR validation of a sub-sample of DEGs revealed significant changes in the level of expression between active and non-active moms (student's t-test, p < 0.05, n = 11). Genes involved in transport of water (p = 0.00236) and uptake of glycerol (p = 0.00219) were enriched in active moms. PA was also associated with the alteration of alternative splicing patters. The most consistent splicing changes were observed for AQP9 where active moms lacked exon 2. DiscussionVariations in maternal PA influences placental gene. We show significant expression changes of genes that are involved in transport and localization between active and non-active women. Most notably, the expression of the aquaporin family of genes (e.g. AQP1 and AQP9) were found to be significantly higher in the placentas of active women suggesting an adaptive response for the transport of water and glycerol in this population.

Glycerol uptake and synthesis systems contribute to the osmotic tolerance of Kluyveromyces marxianus

The accumulation of glycerol is essential for yeast viability upon hyperosmotic stress. In this study, the STL1 homolog KmSTL1, encoding a putative glycerol transporter contributing to cell osmo-tolerance, was identified in Kluyveromyces marxianus NBRC1777. We constructed the KmSTL1, KmGPD1, and KmFPS1 single-deletion mutants and the KmSTL1/KmGPD1 and KmSTL1/KmFPS1 double-deletion mutants of K. marxianus. Deletion of KmSTL1 or KmGPD1 resulted in K. marxianus cell sensitization to hyperosmotic stress, whereas deletion of KmFPS1 improved stress tolerance. The expression of KmSTL1 was osmotically induced, whereas that of KmFPS1 was osmotically inhibited. The expression of KmGPD1 was constitutive and continuous in the ΔKmSTL1 mutant strain but inhibited in the ΔKmFPS1 mutant strain due to feedback suppression by glycerol. In summary, our findings indicated that K. marxianus would increase glycerol synthesis by increasing GPD1 expression, increase glycerol import from the extracellular environment by increasing STL1 expression, and reduce glycerol efflux by reducing FPS1 expression under hyperosmotic stress.

Hepatic Glycerol Metabolism-Related Genes in Carnivorous Rainbow Trout (Oncorhynchus mykiss): Insights Into Molecular Characteristics, Ontogenesis, and Nutritional Regulation

Glycerol metabolism in rainbow trout is poorly studied even though it is at the interface between lipid and glucose metabolism. Moreover, glycerol can be an important ingredient in new aquafeed formulation to decrease the catabolism of dietary amino acids. Thus, the present study aimed to characterize for the first time the different genes coding for key enzymes and proteins involved in hepatic glycerol metabolism. From the trout genomes, all the paralogous genes coding for glycerol transport (aqp9b), glycerol kinase (gk2a and gk5), glycerol-3-phosphate phosphatase (pgp), and glycerol-3-phosphate dehydrogenase (gpd1a, gpd1b, and gpd1c) were identified. The ontogenesis determined that the capacity to metabolize glycerol begins with the apparition of the liver during the development (stage 22) and are more expressed at the endogenous–exogenous feeding period (stage 35). The postprandial regulation of the expression of these genes in juvenile trout showed that the postprandial peak of expression is between 4 and 24 h after the last meal for many of the genes, demonstrating that glycerol metabolism could be nutritionally regulated at a molecular level. However, surprisingly, no regulation of the mRNA abundance for the glycerol metabolism-related genes by different levels of dietary glycerol (0, 2.5, and 5%) have been detected, showing that hepatic glycerol metabolism is poorly regulated at a molecular level by dietary glycerol in rainbow trout juveniles.

Glycerol transporter 1 (Gt1) and zinc-regulated transporter 1 (Zrt1) function in different modes for zinc homeostasis in Komagataella phaffii (Pichia pastoris).

To identify the zinc transport function of the membrane proteins Gt1 and Zrt1 in Komagataella phaffii (Pichia pastoris) and study their regulatory mode. Two membrane proteins that might have zinc transport function were found in K. phaffii. GT1 was known to encode a glycerol transporter belonging to the Major Facilitator Superfamily. ZRT1 was predicted to resemble the zinc transporter gene in Saccharomyces cerevisiae. Consistent with the prediction, protein plasma-membrane localizations were confirmed by ultracentrifugation and confocal microscopy. Their zinc binding abilities were identified by ITC in vitro, and the impaired zinc uptake activity caused by their deficiencies was confirmed by zinc fluorescence quantification in vivo. Furthermore, zinc excess could turn the two channels off, while zinc deficiency induced their expressions. Gt1 could only function to maintain zinc homeostasis in glycerol, while the block of Gt1 function might lead to Zrt1 upregulation in glucose. The zinc transport capabilities of Gt1 and Zrt1 were identified in vivo and in vitro. Their regulatory mode to maintain zinc homeostasis in K. phaffii is a new inspiration.

Unravelling the Complex Duplication History of Deuterostome Glycerol Transporters.

Transmembrane glycerol transport is an ancient biophysical property that evolved in selected subfamilies of water channel (aquaporin) proteins. Here, we conducted broad level genome (>550) and transcriptome (>300) analyses to unravel the duplication history of the glycerol-transporting channels (glps) in Deuterostomia. We found that tandem duplication (TD) was the major mechanism of gene expansion in echinoderms and hemichordates, which, together with whole genome duplications (WGD) in the chordate lineage, continued to shape the genomic repertoires in craniates. Molecular phylogenies indicated that aqp3-like and aqp13-like channels were the probable stem subfamilies in craniates, with WGD generating aqp9 and aqp10 in gnathostomes but aqp7 arising through TD in Osteichthyes. We uncovered separate examples of gene translocations, gene conversion, and concerted evolution in humans, teleosts, and starfishes, with DNA transposons the likely drivers of gene rearrangements in paleotetraploid salmonids. Currently, gene copy numbers and BLAST are poor predictors of orthologous relationships due to asymmetric glp gene evolution in the different lineages. Such asymmetries can impact estimations of divergence times by millions of years. Experimental investigations of the salmonid channels demonstrated that approximately half of the 20 ancestral paralogs are functional, with neofunctionalization occurring at the transcriptional level rather than the protein transport properties. The combined findings resolve the origins and diversification of glps over >800 million years old and thus form the novel basis for proposing a pandeuterostome glp gene nomenclature.

Leptin and Nutrition in Gestational Diabetes.

Leptin is highly expressed in the placenta, mainly by trophoblastic cells, where it has an important autocrine trophic effect. Moreover, increased leptin levels are found in the most frequent pathology of pregnancy: gestational diabetes, where leptin may mediate the increased size of the placenta and the fetus, which becomes macrosomic. In fact, leptin mediates the increased protein synthesis, as observed in trophoblasts from gestational diabetic subjects. In addition, leptin seems to facilitate nutrients transport to the fetus in gestational diabetes by increasing the expression of the glycerol transporter aquaporin-9. The high plasma leptin levels found in gestational diabetes may be potentiated by leptin resistance at a central level, and obesity-associated inflammation plays a role in this leptin resistance. Therefore, the importance of anti-inflammatory nutrients to modify the pathology of pregnancy is clear. In fact, nutritional intervention is the first-line approach for the treatment of gestational diabetes mellitus. However, more nutritional intervention studies with nutraceuticals, such as polyphenols or polyunsaturated fatty acids, or nutritional supplementation with micronutrients or probiotics in pregnant women, are needed in order to achieve a high level of evidence. In this context, the Mediterranean diet has been recently found to reduce the risk of gestational diabetes in a multicenter randomized trial. This review will focus on the impact of maternal obesity on placental inflammation and nutrients transport, considering the mechanisms by which leptin may influence maternal and fetal health in this setting, as well as its role in pregnancy pathologies.

Glycerol Utilization By Phytoplankton1.

Dissolved organic carbon (DOC) is an important source of carbon and energy for microbes, but whether it can be used by phytoplankton has not been systematically studied, and the underlying molecular metabolism remains poorly understood. Here, we investigated the ability of phytoplankton to utilize glycerol as a representative of DOC. The widespread presence and expression of glycerol transporter genes were found in transcriptomes and genomes, suggesting the glycerol utilization potential in the diverse marine phytoplankton. We surveyed 29 representative phytoplankton species (31 strains) from six phyla for their ability to use glycerol. Three types of responses were found: positive utilization (Type I), no response (Type II), and negative response (Type III). In all, 11 Type I species were further investigated in axenic cultures with different glycerol concentrations, and five species showed intrinsic glycerol utilizing ability without the aid of bacteria. The ability of species to use glycerol to support non-photosynthetic (DCMU treated) growth was consistent with their possession and expression of glycerol transporter genes. However, some species from the Type II and Type III also possess and express the genes, raising a question whether glycerol transporter in algae might have diversified its function to glycerol export or even non-glycerol transport. Our results show that glycerol could serve as organic carbon source, harmless xenobiotics, or growth inhibitors for phytoplankton depending on species, indicating that glycerol, including natural sources or human discharges, may shape the marine phytoplankton community structure, especially under low photosynthetic efficiency conditions.

P0011THE ROLE OF AQUAPORIN-11 (AQP11) IN THE THYMUS DEVELOPMENT

Abstract Background and Aims Some aquaporins transport not only water but also small non-ionic molecules such as glycerol. In fact, aquaporin-9 (AQP9) has been shown to transport glycerol which is critical to support memory T cell survival through metabolic fitness (Cui G et al. Cell. 161:750, 2015). As AQP11 is widely expressed and transport glycerol as well as water, AQP11 may also play a role in immunological system, which will expand the repertoire of the aquaporin research field. Method The AQP11 expression in the thymus was examined by RT-PCR, Western blot and immunohistochemistry in mice. Phenotypic analysis and microarray analysis of the thymus were conducted in wild and AQP11 null mice at 21 day old before the development of uremia due to polycystic kidneys. Results Both mRNA and protein expressions of AQP11 in the thymus were higher than those of the kidney by RT-PCR and Western blot. Interestingly, the size of the thymus from AQP11 null mice was smaller than that of the wild mice: 28mg vs. 56mg, even after the correction by the body weight: 0.59 % vs. 0.82 %, as the body size of AQP11 null mice was smaller. The vacuolated medullary epithelial cells were observed in the thymus of AQP11 null mice with narrowed cortex. The immunohistochemical analysis revealed the AQP11 expression at the stromal-epithelial cells in the thymus. The microarray analysis of the gene expression in the thymus was compared between the wild and AQP11 null thymus by the annotation analysis based on the David Bioinformatics Resources 6.8 (beta). The up-regulated 66 genes more than 1.5 folds in AQP11 null thymus are mainly related to the PI3K/Akt signaling pathway to promote metabolism, proliferation, cell survival, growth and angiogenesis. On the other hand, the down-regulated 55 genes less than half in AQP11 null thymus are related to energy production and the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Further RT-qPCR analysis confirmed the enhanced expression of Egfr (Epidermal Growth Factor Receptor), Itgb4 (Integrin beta-4) and Il2ra (interleukin 2 receptor alpha) and the diminished expression of aquaglyceroporin AQP7, Pck1 (Phosphoenolpyruvate carboxy-kinase 1) and Ucp1 (Mitochondrial uncoupling protein 1). The up-regulated genes for growth signaling was unexpected with the smaller thymus but may support the survival of the hypoplastic thymus as a compensation. On the other hand, the down-regulated genes may compromise fat-glucose-energy metabolism in the thymus leading to the smaller thymus, which may be aggravated by the decreased expression of AQP7, another glycerol channel. Conclusion AQP11 may play an important role in the metabolic control of the developing thymus possibly through its glycerol transport. The smaller thymus with narrower cortex in AQP11 null mice may lead to immunological dysfunction, which is currently under study.

P0980IMPLICATIONS OF ADIPOSE EARLY DOWNREGULATION OF INTEGRIN-LINKED KINASE (ILK) IN THE DEVELOPMENT OF METABOLIC SYNDROME IN MICE

Abstract Background and Aims Insulin resistance (IR)-related metabolic disorders, such as diabetes type 2 and obesity, are linked to renal and cardiovascular risk. Unfortunately, they lack a better prognosis before their full establishment. An important mechanism associated to the IR development is the dysregulation of lipolysis in white adipose tissue (WAT), which is driven by the excessive lipid accumulation, together with inflammation and the excessive deposition of ECM components such as collagens and fibronectin, known as fibrosis. The intracellular lipid adjustment is affected by changes in the expression and activity of the hormone-sensitive lipase (HSL) and the intracellular and circulating balance of the lipolysis products glycerol and free fatty acids. We published that transgenic depletion of the scaffold intracellular protein Integrin-linked kinase (ILK), one of the adhesome translators of the ECM signals, modulates IR in WAT [Hatem-Vaquero et al. J Endocrinol. 2017]. We also published that ILK downregulation in WAT is an early event in a short-term high fat diet (HFD) mice model that correlates with a faster insulin sensitivity loss, accompanied with an early increase of body weight and the erratic expression of WAT adipokines and metabolites transporters. [Hatem-Vaquero et al. Cell Physiol Biochem 2020]. Here we used in vivo and in vitro approaches to decode the role of ILK downregulation during WAT expansion and altered lipolysis. Method Adult mice with global transgenic downregulation of ILK expression (cKD-ILK) and littermates without that depletion (CT) were fed with either standard (STD) or high fat (HFD) diets during 2 and 6 weeks. We determined the weight changes on body and WAT depots, epidydimal (eWAT) and subcutaneous (scWAT), and the circulating lypolisis product glycerol. The expression of ECM component fibronectin (FN), fibrosis marker TGF-beta and the glycerol transporter AQP3 were determined in eWAT by RT-qPCR or Western blot. Comparative studies were performed in cultured C3H10t1/2-based adipocytes where ILK expression was deliberately downregulated by the transfection of siRNAs against ILK or in control adipocytes (scramble siRNAs). We determined the expression of ILK, total and active HSL isoforms (HSL, p-HSL s660) by RT-qPCR or Western blot and the intracellular fat droplet content (by adipo-red dye) Results HFD increased progressively CT eWAT, scWAT and whole body weight gains. HFD-fed cKD-ILK have an earlier increase of body and eWAT weights. However, the scWAT weight ratio against eWAT was inverted in the same cKD-ILK animals. This early depot-specific response to HFD coincide with increase in IR [Wajchenberg BL. Endocr Rev 2000]. HFD-fed cKD-ILK WAT early expressed altered levels of fibrotic markers (TGF-beta and Fibronectin). HFD-fed cKD-ILK shown a temporary increase of circulating glycerol and its WAT transporter AQP3. In cultured adipocytes with downregulated expression of ILK, the intracellular fat droplet was increased and the HSL contain and its lipolysis activity (measured as levels of p-HSL isoform) were reduced. Conclusion We suggest ILK as a modulator during the obesity establishment. Considering the observed relation between ILK expression with the altered lipolysis and fibrosis patterns observed in visceral WAT, ILK downregulation may be a predictive value and probably its pharmacological upregulation may be a novel therapeutic strategy against obesity and IR.

AQP3 Facilitates Proliferation and Adipogenic Differentiation of Porcine Intramuscular Adipocytes.

The meat quality of animal products is closely related to the intramuscular fat content. Aquaglyceroporin (AQP) defines a class of water/glycerol channels that primarily facilitate the passive transport of glycerol and water across biological membranes. In this study, the AQP3 protein of the AQP family was mainly studied in the adipogenic function of intramuscular adipocytes in pigs. Here, we found that AQP3 was increased at both mRNA and protein levels upon adipogenic stimuli in porcine intramuscular adipocytes in vitro. Western blot results showed knockdown of AQP3 by siRNA significantly suppressed the expression of adipogenic genes (PPARγ, aP2, etc.), repressed Akt phosphorylation, as well as reducing lipid accumulation. Furthermore, deletion of AQP3 by siRNA significantly downregulated expression of cell cycle genes (cyclin D, E), and decreased the number of EdU-positive cells as well as cell viability. Collectively, our data indicate that AQP3 is of great importance in both adipogenic differentiation and proliferation in intramuscular adipocytes, providing a potential target for modulating fat infiltration in skeletal muscles.

Cooperative Response of Pichia kudriavzevii and Saccharomyces cerevisiae to Lactic Acid Stress in Baijiu Fermentation.

Lactic acid is a universal metabolite, as well as a growth inhibitor of ethanol producers in Baijiu fermentation. Revealing the mechanism of lactic acid tolerance is essential for the yield of fermented foods. Here, we employed reverse transcription-quantitative polymerase chain reaction to explore the degradation mechanism of lactic acid, based on the coculture of Pichia kudriavzevii and Saccharomyces cerevisiae. Under high lactic acid stress, P. kudriavzevii decreased lactic acid from 40.00 to 35.46 g L-1 within 24 h. Then, S. cerevisiae restored its capacity to degrade lactic acid. Finally, lactic acid decreased to 26.29 g L-1. Coculture significantly enhanced lactic acid consumption compared to the monoculture of P. kudriavzevii (90% higher) or S. cerevisiae (209% higher). We found that lactate catabolism, H+ extrusion, and glycerol transport were the lactic acid tolerance pathways in yeasts. This study reveals the novel acid tolerance mechanisms of microbiota and would provide new strategies for ethanol production under acid stress.

Integrin Linked Kinase (ILK) Downregulation as an Early Event During the Development of Metabolic Alterations in a Short-Term High Fat Diet Mice Model.

Diabetes type 2, metabolic syndrome or non-alcoholic fatty liver disease are insulin resistance-related metabolic disorders, which lack a better prognosis before their full establishment. We studied the importance of the intracellular scaffold protein integrin linked kinaes (ILK) as a key modulator in the initial pathogenesis and the early progression of those insulin resistance- related disorders. Adult mice with a global transgenic downregulation of ILK expression (cKD-ILK) and littermates without that depletion (CT) were fed with either standard (STD) or high fat (HFD) diets during 2 and 6 weeks. Weights, blood glucose and other systemic biochemical parameters were determined in animals under fasting conditions and after glucose or pyruvate intraperitoneal injections to test their tolerance. In RNA or proteins extracted from insulin-sensitive tissues, we determined by reverse transcription-quantitative PCR and western blot the expression of ILK, metabolites transporters and other metabolism and inflammatory markers. Glucose uptake capacity was studied in freshly isolated tissues. HFD feeding was able to early and progressively increase glycaemia, insulinemia, circulating glycerol, body weight gain, liver-mediated gluconeogenesis along this time lapse, but cKD-ILK have all these systemic misbalances exacerbated compared to CT in the same HFD time lapse. Interestingly, the tisular expression of ILK in HFD-fed CT was dramatically downregulated in white adipose tissue (WAT), skeletal muscle and liver at the same extent of the original ILK downregulation of cKD-ILK. We previously published that basal STD-fed cKD-ILK compared to basal STD-CT have different expression of glucose transporters GLUT4 in WAT and skeletal muscle. In the same STD-fed cKD-ILK, we observed here the increased expressions of hepatic GLUT2 and WAT pro-inflammatory cytokines TNF-α and MCP-1. The administration of HFD exacerbated the expression changes in cKD-ILK of these and other markers related to the imbalanced metabolism observed, such as WAT lipolysis (HSL), hepatic gluconeogenesis (PCK-1) and glycerol transport (AQP9). ILK expression may be taken as a predictive determinant of metabolic disorders establishment, because its downregulation seems to correlate with the early imbalance of glucose and glycerol transport and the subsequent loss of systemic homeostasis of these metabolites.

Molecular and Functional Characterization of Grapevine NIPs through Heterologous Expression in aqy-Null Saccharomyces cerevisiae.

Plant Nodulin 26-like Intrinsic Proteins (NIPs) are multifunctional membrane channels of the Major Intrinsic Protein (MIP) family. Unlike other homologs, they have low intrinsic water permeability. NIPs possess diverse substrate selectivity, ranging from water to glycerol and to other small solutes, depending on the group-specific amino acid composition at aromatic/Arg (ar/R) constriction. We cloned three NIPs (NIP1;1, NIP5;1, and NIP6;1) from grapevine (cv. Touriga Nacional). Their expression in the membrane of aqy-null Saccharomyces cerevisiae enabled their functional characterization for water and glycerol transport through stopped-flow spectroscopy. VvTnNIP1;1 demonstrated high water as well as glycerol permeability, whereas VvTnNIP6;1 was impermeable to water but presented high glycerol permeability. Their transport activities were declined by cytosolic acidification, implying that internal-pH can regulate NIPs gating. Furthermore, an extension of C-terminal in VvTnNIP6;1M homolog, led to improved channel activity, suggesting that NIPs gating is putatively regulated by C-terminal. Yeast growth assays in the presence of diverse substrates suggest that the transmembrane flux of metalloids (As, B, and Se) and the heavy metal (Cd) are facilitated through grapevine NIPs. This is the first molecular and functional characterization of grapevine NIPs, providing crucial insights into understanding their role for uptake and translocation of small solutes, and extrusion of toxic compounds in grapevine.

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.