Aquaporin adipose, a putative glycerol channel in adipocytes

Adipose tissue is a major site of glycerol production in response to energy balance. However, molecular basis of glycerol release from adipocytes has not yet been elucidated. We recently cloned a novel member of the aquaporin family, aquaporin adipose (AQPap), which has glycerol permeability. The current study was designed to examine the hypothesis that AQPap serves as a glycerol channel in adipocytes. Adipose tissue expressed AQPap mRNA in high abundance, but not the mRNAs for the other aquaglyceroporins, AQP3 and AQP9, indicating that AQPap is the only known aquaglyceroporin expressed in adipose tissue. Glycerol release from 3T3-L1 cells was increased during differentiation in parallel with AQPap mRNA levels and suppressed by mercury ion, which inhibits the function of AQPs, supporting AQPap functions as a glycerol channel in adipocytes. Fasting increased and refeeding suppressed adipose AQPap mRNA levels in accordance with plasma glycerol levels and oppositely to plasma insulin levels in mice. Insulin dose-dependently suppressed AQPap mRNA expression in 3T3-L1 cells. AQPap mRNA levels and adipose glycerol concentrations measured by the microdialysis technique were increased in obese mice with insulin resistance. Accordingly, negative regulation of AQPap expression by insulin was impaired in the insulin-resistant state. Exposure of epinephrine translocated AQPap protein from perinuclear cytoplasm to the plasma membrane in 3T3-L1 adipocytes. These results strongly suggest that AQPap plays an important role in glycerol release from adipocytes.     

Role of aquaporin-7 in the pathophysiological control of fat accumulation in mice

Aquaporins are channels that allow the movement of water across the cell membrane. Some members of the aquaporin family, the aquaglyceroporins, also allow the transport of glycerol, which is involved in the biosynthesis of triglycerides and the maintenance of fasting glucose levels. Aquaporin-7 (AQP7) is a glycerol channel mainly expressed in adipocytes. The deletion of AQP7 gene in mice leads to obesity and type 2 diabetes. AQP7 modulates adipocyte glycerol permeability thereby controlling triglyceride accumulation and fat cell size. Furthermore, the coordinated regulation of fat-specific AQP7 and liver-specific AQP9 may be key to determine glucose metabolism in insulin resistance.     

Knockdown of aquaporin-8 induces mitochondrial dysfunction in 3T3-L1 cells

BackgroundAquaporin-8 (AQP8), a member of the aquaporin water channel family, is expressed in various tissue and cells, including liver, testis, and pancreas. AQP8 appears to have functions on the plasma membrane and/or on the mitochondrial inner membrane. Mitochondrial AQP8 with permeability for water, H₂O₂ and NH₃ has been expected to have important role in various cells, but its information is limited to a few tissues and cells including liver and kidney. In the present study, we found that AQP8 was expressed in the mitochondria in mouse adipose tissues and 3T3-L1 preadipocytes, and investigated its role by suppressing its gene expression.MethodsAQP8-knocked down (shAQP8) cells were established using a vector expressing short hairpin RNA. Cellular localization of AQP8 was examined by western blotting and immunocytochemistry. Mitochondrial function was assessed by measuring mitochondrial membrane potential, oxygen consumption and ATP level measurements.ResultsIn 3T3-L1 cells, AQP8 was expressed in the mitochondria. In shAQP8 cells, mRNA and protein levels of AQP8 were decreased by about 75%. The shAQP8 showed reduced activities of complex IV and ATP synthase; it is probable that the impaired mitochondrial water handling in shAQP8 caused suppression of the electron transport and ADP phosphorylation through inhibition of the two steps which yield water. The reduced activities of the last two steps of oxidative phosphorylation in shAQP8 cause low routine and maximum capacity of respiration and mitochondrial hyperpolarization.ConclusionMitochondrial AQP8 contributes to mitochondrial respiratory function probably through maintenance of water homeostasis.General significanceThe AQP8-knocked down cells we established provides a model system for the studies on the relationships between water homeostasis and mitochondrial function.     

Over-expression of AQP7 contributes to improve insulin resistance in adipocytes

OBJECTIVE: Aquaglyceroporin 7 (AQP7) is required for efflux of glycerol from adipocytes. In this study, we aimed to analyze expression profiles of AQP7 in the different differentiation phases of adipocytes and to investigate the role of AQP7 in the insulin resistance of adipocytes. Methods: 3T3-L1 pre-adipocyte cells were induced to be fully differentiated adipocytes and then insulin resistance was induced by Dexamethasone (DXM) or TNF-α. Adenovirus vector with over-expression AQP7 (Ad-AQP7) was constructed and transfected into adipocytes. The expression level of AQP7 and phosphorylated PKB (p-PKB) were measured. The glycerol released from adipocytes and glucose consuming rate were tested too. Results: AQP7 expression was gradually up-regulated along with the differentiation processing of 3T3-L1 preadipocytes, which was consistent with the expression level of p-PKB. Dexamethasone down-regulated the expression of AQP7, p-PKB and the glycerol content in adipocytes. Over-expression of AQP7 by transfecting Ad-AQP7 to insulin resistant adipocytes restored the phosphorylation of PKB and attenuated the glycerol secretion and glucose consuming rate of adipocytes. Conclusions: AQP7 is down-regulated in adipocytes with insulin resistance. The over-expression of AQP7 contributes to improve insulin resistance in adipocytes, which is potentially correlated with the increased phosphorylation of PKB.     

Glycerol metabolism alteration in adipocytes from n3-PUFA-depleted rats, an animal model for metabolic syndrome

Aquaglyceroporin 7 (AQP7) is a glycerol transporter expressed in adipocytes. Its expression has been shown to be modulated in obesity. Metabolic syndrome is characterized by abdominal obesity, insulin resistance, dyslipidemia, and hypertension. An animal model displaying several features of metabolic syndrome was used to study the AQP7 expression at both mRNA and protein level and glycerol flux in adipocytes. Second generation n3-PUFA depleted female rats is a good animal model for metabolic syndrome as it displays characteristic features such as liver steatosis, visceral obesity, and insulin resistance. Our data show a reduced expression of AQP7 at the protein level in adipose tissue from n3-PUFA-depleted rats, without any changes at the mRNA levels. [U-(14)C]-Glycerol uptake was not modified in adipocytes from n3-PUFA-depleted animals.     

Aquaglyceroporins serve as metabolic gateways in adiposity and insulin resistance control

Aquaglyceroporins (AQP3, AQP7, AQP9 and AQP10) encompass a subfamily of aquaporins that allow the movement of water and other small solutes, especially glycerol, through cell membranes. Adipose tissue constitutes a major source of glycerol via AQP7. We have recently reported that, in addition to the well-known expression of AQP7 in adipose tissue, AQP3 and AQP9 are also expressed in omental and subcutaneous fat depots. Moreover, insulin and leptin act as regulators of aquaglyceroporins through the PI3K/Akt/mTOR pathway. AQP3 and AQP7 appear to facilitate glycerol efflux from adipose tissue while reducing the glycerol influx into hepatocytes via AQP9 to prevent the excessive lipid accumulation and the subsequent aggravation of hyperglycemia in human obesity. This Extra View focuses on the control of glycerol release by aquaglyceroporins in the adipose tissue and briefly discusses the importance of glycerol as a substrate for hepatic gluconeogenesis, pancreatic insulin secretion and cardiac ATP production.     

Activin B inhibits lipolysis in 3T3-L1 adipocytes

Activin B, consisting of two inhibin βB (INHBB) subunits, is a hormone known to affect gonadal function, reproduction and fetal development. We have reported that INHBB and activin B receptors are highly expressed in adipocytes suggesting that activin B may have local effects in adipose tissue. In this study, we investigate the effect of activin B on lipolysis, measured as release of non-esterified fatty acids and free glycerol. Recombinant activin B decreased lipolysis in a concentration-dependent manner and increased intracellular triglyceride content in 3T3-L1 adipocytes. siRNA-mediated knock-down of INHBB expression increased lipolysis, and this effect was abolished by addition of recombinant activin B. In line with its inhibitory effect on lipolysis, activin B caused a down regulation of the expression of adipose triglyceride lipase and hormone sensitive lipase, key genes involved in lipolysis. In summary, we suggest that activin B is a novel adipokine that inhibits lipolysis in a paracrine or autocrine manner.     

Aquaglyceroporins serve as metabolic gateways in adiposity and insulin resistance control

Aquaglyceroporins (AQP3, AQP7, AQP9 and AQP10) encompass a subfamily of aquaporins that allow the movement of water and other small solutes, especially glycerol, through cell membranes. Adipose tissue constitutes a major source of glycerol via AQP7. We have recently reported that, in addition to the well-known expression of AQP7 in adipose tissue, AQP3 and AQP9 are also expressed in omental and subcutaneous fat depots. Moreover, insulin and leptin act as regulators of aquaglyceroporins through the PI3K/Akt/mTOR pathway. AQP3 and AQP7 appear to facilitate glycerol efflux from adipose tissue while reducing the glycerol influx into hepatocytes via AQP9 to prevent the excessive lipid accumulation and the subsequent aggravation of hyperglycemia in human obesity. This Extra View focuses on the control of glycerol release by aquaglyceroporins in the adipose tissue and briefly discusses the importance of glycerol as a substrate for hepatic gluconeogenesis, pancreatic insulin secretion and cardiac ATP production.Key words: glycerol, aquaporin, fat accumulation, glucose homeostasis, insulin secretion, ATP production     

Molecular characterization, chromosomal and expression patterns of three aquaglyceroporins (AQP3, 7, 9) from pig

As one subgroup of aquaporin, aquaglyceroporin including AQP3, 7, 9, 10 facilitates glycerol transport as well as water transport. In this study, we cloned the full length coding sequences of porcine (Sus scrofa) AQP3, 7 and 9 and the genomic sequence of AQP3 including 6 exons and 5 introns. Additionally, as a first step toward understanding the regulatory mechanisms of AQP9 in pig, we cloned and analyzed the upstream genomic sequence of the ATG translation initiation codon and found two negative insulin response elements (TGTTTTC and TATTTTG.), glucocorticoid-responsive elements, several CCAAT enhancer binding protein (C/EBP) sites, hepatocyte nuclear factor (HNF) sites, and NF-kappaB sites in this region. Subsequently, semi-quantitative analysis showed that AQP3 selectively expressed in spleen, stomach, kidney and lung. AQP7 and AQP9 were ubiquitously detected in all tissues examined and highly expressed in adipose tissue and liver, respectively. Finally, both AQP3 and AQP7 were assigned to chromosome 10q while AQP9 was mapped to chromosome 1q. This is the first report of molecular characterization of aquaglyceroporin in pig, which provides basic observations useful for future assessing and characterizing the role of aquaglyceroporin.     

Implications of aquaglyceroporins 7 and 9 in glycerol metabolism and metabolic syndrome

The discovery of water channel protein (aquaporin [AQP]) has made a great impact on life sciences. So far, 13 AQPs have been identified in human. AQP3, 7, 9, and 10 are subcategorized as aquaglyceroporins which permeabilize glycerol as well as water. Many investigators have demonstrated that AQPs play a crucial role in the maintenance of water homeostasis, but the physiological significance of some AQPs as glycerol channels remains elusive. Adipocyte is a major source of glycerol, which is one of the substrates for hepatic gluconeogenesis. This review focuses on recent studies on glycerol metabolism through AQP7 and AQP9, and briefly discusses the importance of glycerol channel in adipocytes, liver, and heart.     

Effects and Molecular Mechanism of GST-Irisin on Lipolysis and Autocrine Function in 3T3-L1 Adipocytes

Irisin, which was recently identified as a myokine and an adipokine, transforms white adipose tissue to brown adipose tissue and has increasingly caught the attention of the medical and scientific community. However, the signaling pathway of irisin and the molecular mechanisms responsible for the lipolysis effect remain unclear. In this study, we established an efficient system for the expression and purification of GST-irisin in Escherichia coli. The biological activity of GST-irisin was verified using the cell counting kit-8 assay and by detecting the mRNA expression of uncoupling protein 1. Our data showed that GST-irisin regulates mRNA levels of lipolysis-related genes such as adipose triglyceride lipase and hormone-sensitive lipase and proteins such as the fatty acid-binding protein 4, leading to increased secretion of glycerol and decreased lipid accumulation in 3T3-L1 adipocytes. In addition, exogenous GST-irisin can increase its autocrine function in vitro by regulating the expression of fibronectin type III domain-containing protein 5. GST-irisin could regulate glucose uptake in 3T3-L1 adipocytes. Hence, we believe that recombinant GST-irisin could promote lipolysis and its secretion in vitro and can potentially prevent obesity and related metabolic diseases.     

Aquaporins and glycerol metabolism

The discovery of aquaporin (AQP) has made a great impact on life sciences. AQPs are a family of homologous water channels widely distributed in plants, unicellular organisms, invertebrates, and vertebrates. So far, 13 AQPs have been identified in human. AQP3, 7, 9, and 10 are subcategorized as aquaglyceroporins which permeabilize glycerol as well as water. Many investigators have demonstrated that AQPs play a crucial role in maintaining water homeostasis, but the physiological significance of some AQPs as a glycerol channel is not fully understood. Adipose tissue is a major source of glycerol and glycerol is one of substrates for gluconeogenesis. This review focuses on recent studies of glycerol metabolism through aquaglyceroporins, and briefly discusses the importance of glycerol channel in adipose tissues and liver.     

Aquaporins and glycerol metabolism

The discovery of aquaporin (AQP) has made a great impact on life sciences. AQPs are a family of homologous water channels widely distributed in plants, unicellular organisms, invertebrates, and vertebrates. So far, 13 AQPs have been identified in human. AQP3, 7, 9, and 10 are subcategorized as aquaglyceroporins which permeabilize glycerol as well as water. Many investigators have demonstrated that AQPs play a crucial role in maintaining water homeostasis, but the physiological significance of some AQPs as a glycerol channel is not fully understood. Adipose tissue is a major source of glycerol and glycerol is one of substrates for gluconeogenesis. This review focuses on recent studies of glycerol metabolism through aquaglyceroporins, and briefly discusses the importance of glycerol channel in adipose tissues and liver.     

Progressive adipocyte hypertrophy in aquaporin-7-deficient mice: adipocyte glycerol permeability as a novel regulator of fat accumulation

Aquaporin-7 (AQP7) is a water/glycerol transporting protein expressed in adipocyte plasma membranes. We report here remarkable age-dependent hypertrophy in adipocytes in AQP7-deficient mice. Wild type and AQP7 null mice had similar growth at 0-16 weeks as assessed by body weight; however, by 16 weeks AQP7 null mice had 3.7-fold increased body fat mass. Adipocytes from AQP7 null mice of age 16 weeks were greatly enlarged (diameter 118 mum) compared with wild type mice (39 mum). Adipocytes from AQP7 null mice also accumulated excess glycerol (251 versus 86 nmol/mg of protein) and triglycerides (3.4 versus 1.7 mumol/mg of protein). In contrast, at age 4 weeks, adipocyte volume and body fat mass were comparable in wild type and AQP7 null mice. To investigate the mechanism(s) responsible for the progressive adipocyte hypertrophy, glycerol permeability and fat metabolism were studied in adipocytes isolated from the younger mice. Plasma membrane glycerol permeability measured by [(14)C]glycerol uptake was 3-fold reduced in AQP7-deficient adipocytes. However, adipocyte lipolysis, measured by free fatty acid release and hormone-sensitive lipase activity, and lipogenesis, measured by [(14)C]glucose incorporation into triglycerides, were not affected by AQP7 deletion. These data suggest that adipocyte hypertrophy in AQP7 deficiency results from defective glycerol exit and consequent accumulation of glycerol and triglycerides. Increasing AQP7 expression/function in adipocytes may reduce adipocyte volume and fat mass in obesity.     

The mitogenic and antiapoptotic actions of ghrelin in 3T3-L1 adipocytes

Ghrelin, a stomach-derived hormone, induces adiposity when administered to rodents. Because ghrelin receptor is abundantly expressed in adipose tissue, we investigated the role of ghrelin in adipocyte biology. We observed ghrelin receptor expression in 3T3-L1 preadipocytes and adipocytes. Treatment of preadipocytes with ghrelin induced cellular proliferation and differentiation to mature adipocytes, as well as basal and insulin-stimulated glucose transport, but it inhibited adipocyte apoptosis induced by serum deprivation. Exposure of 3T3-L1 cells to ghrelin caused a rapid activation of MAPKs, especially ERK1/2. Chemical inhibition of MAPK blocked the mitogenic and antiapoptotic effects of ghrelin. Ghrelin also stimulated the insulin receptor substrate-associated phosphatidylinositol 3-kinase/Akt pathway in 3T3-L1 preadipocytes and adipocytes, whereas inhibition of this pathway blocked the effects of ghrelin on cell proliferation, antiapoptosis and glucose uptake. These findings suggest that the direct effects of ghrelin on proliferation, differentiation, and apoptosis in adipocytes may play a role in regulating fat cell number. These effects may be mediated via activation of the MAPK and phosphatidylinositol 3-kinase/Akt pathways.     

Expression and function of aquaporins in human skin: Is aquaporin-3 just a glycerol transporter?

The aquaporins (AQPs) are a family of transmembrane proteins forming water channels. In mammals, water transport through AQPs is important in kidney and other tissues involved in water transport. Some AQPs (aquaglyceroporins) also exhibit glycerol and urea permeability. Skin is the limiting tissue of the body and within skin, the stratum corneum (SC) of the epidermis is the limiting barrier to water loss by evaporation. The aquaglyceroporin AQP3 is abundantly expressed in keratinocytes of mammalian skin epidermis. Mice lacking AQP3 have dry skin and reduced SC hydration. Interestingly, however, results suggested that impaired glycerol, rather than water transport was responsible for this phenotype. In the present work, we examined the overall expression of AQPs in cells from human skin and we reviewed data on the functional role of AQPs in skin, particularly in the epidermis. By RT-PCR on primary cell cultures, we found that up to 6 different AQPs (AQP1, 3, 5, 7, 9 and 10) may be selectively expressed in various cells from human skin. AQP1, 5 are strictly water channels. But in keratinocytes, the major cell type of the epidermis, only the aquaglyceroporins AQP3, 10 were found. To understand the role of aquaglyceroporins in skin, we examined the relevance to human skin of the conclusion, from studies on mice, that skin AQP3 is only important for glycerol transport. In particular, we find a correlation between the absence of AQP3 and intercellular edema in the epidermis in two different experimental models: eczema and hyperplastic epidermis. In conclusion, we suggest that in addition to glycerol, AQP3 may be important for water transport and hydration in human skin epidermis.     

Expression and function of aquaporins in human skin: Is aquaporin-3 just a glycerol transporter?

The aquaporins (AQPs) are a family of transmembrane proteins forming water channels. In mammals, water transport through AQPs is important in kidney and other tissues involved in water transport. Some AQPs (aquaglyceroporins) also exhibit glycerol and urea permeability. Skin is the limiting tissue of the body and within skin, the stratum corneum (SC) of the epidermis is the limiting barrier to water loss by evaporation. The aquaglyceroporin AQP3 is abundantly expressed in keratinocytes of mammalian skin epidermis. Mice lacking AQP3 have dry skin and reduced SC hydration. Interestingly, however, results suggested that impaired glycerol, rather than water transport was responsible for this phenotype. In the present work, we examined the overall expression of AQPs in cells from human skin and we reviewed data on the functional role of AQPs in skin, particularly in the epidermis. By RT-PCR on primary cell cultures, we found that up to 6 different AQPs (AQP1, 3, 5, 7, 9 and 10) may be selectively expressed in various cells from human skin. AQP1, 5 are strictly water channels. But in keratinocytes, the major cell type of the epidermis, only the aquaglyceroporins AQP3, 10 were found. To understand the role of aquaglyceroporins in skin, we examined the relevance to human skin of the conclusion, from studies on mice, that skin AQP3 is only important for glycerol transport. In particular, we find a correlation between the absence of AQP3 and intercellular edema in the epidermis in two different experimental models: eczema and hyperplastic epidermis. In conclusion, we suggest that in addition to glycerol, AQP3 may be important for water transport and hydration in human skin epidermis.     

Chemerin enhances insulin signaling and potentiates insulin-stimulated glucose uptake in 3T3-L1 adipocytes

To explore a novel adipokine, we screened adipocyte differentiation-related gene and found that TIG2/chemerin was strongly induced during the adipocyte differentiation. Chemerin was secreted by the mature 3T3-L1 adipocytes and expressed abundantly in adipose tissue in vivo as recently described. Intriguingly, the expression of chemerin was differently regulated in the liver and adipose tissue in db/db mice. In addition, serum chemerin concentration was decreased in db/db mice. Chemerin and its receptor/ChemR23 were expressed in mature adipocytes, suggesting its function in autocrine/paracrine fashion. Finally, chemerin potentiated insulin-stimulated glucose uptake concomitant with enhanced insulin signaling in the 3T3-L1 adipocytes. These data establish that chemerin is a novel adipokine that regulates adipocyte function.     

11β-hydroxysteroid Dehydrogenase 1 in Adipocytes: Expression is Differentiation-dependent and Hormonally Regulated

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) catalyses the reversible metabolism of physiological glucocorticoids (cortisol, corticosterone) to inactive metabolites (cortisone, 11-dehydrocorticosterone), thus regulating glucocorticoid access to receptors. 11β-HSD-1 expression is regulated during development and by hormones in a tissue specific manner. The enzyme is highly expressed in liver, where it may influence glucocorticoid action on fuel metabolism, processes also important in adipose tissue. Here we show that 11β-HSD-1 is expressed in white adipose tissue, in both the adipocyte and stromal/vascular compartments, and in the adipocyte cell lines 3T3-F442A and 3T3-L1. In these cells, 11β-HSD-1 expression is induced upon differentiation into adipocytes and is characteristic of a ‘late differentiation’ gene, with maximal expression 6-8 days after confluence is reached. In intact 3T3-F442A adipocytes the enzyme direction is predominantly 11β-reduction, activating inert glucocorticoids. The expression of 11β-HSD-1 mRNA is altered in fully differentiated 3T3-F442A adipocytes treated with insulin, dexamethasone or a combination of the hormones, in an identical manner to glycerol-3-phosphate dehydrogenase (GPDH) mRNA (encoding a key enzyme in triglyceride synthesis and a well-characterised marker of adipocyte differentiation). The demonstration of 11β-HSD-1 expression in adipocytes and its predominant reductase activity in intact 3T3-F442A adipocytes suggests that 11β-HSD-1 may play an important role in potentiating glucocorticoid action in these cells. 3T3-F442A and 3T3-L1 represent useful model systems in which to examine the factors which regulate 11β-HSD-1 gene expression and the role of 11β-HSD-1 in modulating glucocorticoid action in adipose tissue.