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.     

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.     

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.     

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.     

Biophysical Assessment of Human Aquaporin-7 as a Water and Glycerol Channel in 3T3-L1 Adipocytes

The plasma membrane aquaporin-7 (AQP7) has been shown to be expressed in adipose tissue and its role in glycerol release/uptake in adipocytes has been postulated and correlated with obesity onset. However, some studies have contradicted this view. Based on this situation, we have re-assessed the precise localization of AQP7 in adipose tissue and analyzed its function as a water and/or glycerol channel in adipose cells. Fractionation of mice adipose tissue revealed that AQP7 is located in both adipose and stromal vascular fractions. Moreover, AQP7 was the only aquaglyceroporin expressed in adipose tissue and in 3T3-L1 adipocytes. By overexpressing the human AQP7 in 3T3-L1 adipocytes it was possible to ascertain its role as a water and glycerol channel in a gain-of-function scenario. AQP7 expression had no effect in equilibrium cell volume but AQP7 loss of function correlated with higher triglyceride content. Furthermore it is also reported for the first time a negative correlation between water permeability and the cell non-osmotic volume supporting the observation that AQP7 depleted cells are more prone to lipid accumulation. Additionally, the strong positive correlation between the rates of water and glycerol transport highlights the role of AQP7 as both a water and a glycerol channel and reflects its expression levels in cells. In all, our results clearly document a direct involvement of AQP7 in water and glycerol transport, as well as in triglyceride content in adipocytes.     

Differential expression of aquaporin 7 in adipose tissue of lean and obese high fat consumers

Aquaporin 7 (AQP7) is an aquaglyceroprotein responsible for the secretion and uptake of glycerol from the adipocyte. The modulation of the expression of this membrane transport protein might play an important role in the susceptibility to the development of obesity. The aim of the present study was to compare the AQP7 gene expression in subcutaneous abdominal fat in lean vs. obese high fat intakers with a similar daily physical activity pattern. Twelve young men, 6 lean (BMI=23.2+/-0.4kg/m(2)) and 6 obese (35.0+/-1.1kg/m(2)) with a similar habitual dietary intake of fat (45.5+/-2.5 vs. 43.5+/-1.7% daily energy from fat for lean and obese, respectively) and physical activity (16.0+/-5.7 vs. 17.2+/-5.1 METsh/week for lean and obese, respectively), were recruited. Subcutaneous abdominal fat biopsies were obtained and total RNA was extracted and purified. Pools of RNA from lean and obese individuals were probed into Affymetrix GeneChip Human U133A. The microarray analysis revealed that AQP7 gene was down-regulated in obese compared to lean subjects. The results of the microarray analysis were confirmed by real-time PCR studies. In summary, our data show that the AQP7 gene is differentially expressed in adipose tissue of lean and obese individuals. The down-regulation of the AQP7 gene could be implicated in the susceptibility to obesity by reducing glycerol release and promoting the accumulation of lipids in the adipose tissue.     

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.     

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.     

Adipocytes as a Link Between Gut Microbiota-Derived Flagellin and Hepatocyte Fat Accumulation

While the role of both elevated levels of circulating bacterial cell wall components and adipose tissue in hepatic fat accumulation has been recognized, it has not been considered that the bacterial components-recognizing adipose tissue receptors contribute to the hepatic fat content. In this study we found that the expression of adipose tissue bacterial flagellin (FLG)-recognizing Toll-like receptor (TLR) 5 associated with liver fat content (r = 0.699, p = 0.003) and insulin sensitivity (r = -0.529, p = 0.016) in humans (n = 23). No such associations were found for lipopolysaccharides (LPS)-recognizing TLR4. To study the underlying molecular mechanisms of these associations, human HepG2 hepatoma cells were exposed in vitro to the conditioned culture media derived from FLG or LPS-challenged human adipocytes. The adipocyte-mediated effects were also compared to the effects of direct HepG2 exposure to FLG and LPS. We found that the media derived from FLG-treated adipocytes stimulated fat accumulation in HepG2 cells, whereas either media derived from LPS-treated adipocytes or direct FLG or LPS exposure did not. This is likely due to that FLG-treatment of adipocytes increased lipolysis and secretion of glycerol, which is known to serve a substrate for triglyceride synthesis in hepatocytes. Similarly, only FLG-media significantly decreased insulin signaling-related Akt phosphorylation, IRS1 expression and mitochondrial respiratory chain ATP5A. In conclusion, our results suggest that the FLG-induced TLR5 activation in adipocytes increases glycerol secretion from adipocytes and decreases insulin signaling and mitochondrial functions, and increases fat accumulation in hepatocytes. These mechanisms could, at least partly, explain the adipose tissue TLR5 expression associated with liver fat content in humans.     

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.     

Cloning and identification of a new member of water channel (AQP10) as an aquaglyceroporin

Recently, a new member of aquaporins was reported as AQP10 [Biochem. Biophys. Res. Commun. 287 (2001) 814], which is incompletely spliced to lose the sixth transmembrane domain and has poor water and no glycerol/urea permeabilities. Independently, we identified a similar clone in human. Our AQP10 consists of 301 amino acids with a highly conserved sixth transmembrane domain. AQP10 has higher identity with aquaglyceroporins (50% with AQP9, 48% with AQP3, 42% with AQP7) and lower identity with other aquaporins (32% with AQP1 and AQP8). AQP10 is expressed only in the small intestine with (approximately 2 kb). RNase protection assay revealed the absence of the unspliced form, supporting the authenticity of our clone. When expressed in Xenopus oocytes, AQP10 stimulated osmotic water permeability sixfold in a mercury-sensitive manner. Glycerol and urea uptakes were also stimulated, while adenine uptake was not. The genome structure of AQP10 is similar to those of other aquaglyceroporins (AQP3, AQP7, AQP9) with six exons. We conclude that AQP10 represents a new member of aquaglyceroporins functionally as well as structurally.     

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.     

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.     

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.     

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.     

Physiological roles of AQP7 in the kidney: Lessons from AQP7 knockout mice

The aquaporin7 (AQP7) water channel is known to be a member of the aquaglyceroporins, which allow the rapid transport of glycerol and water. AQP7 is abundantly present at the apical membrane of the proximal straight tubules in the kidney. In this paper, we review the physiological functions of AQP7 in the kidney. To investigate this, we generated AQP7 knockout mice. The water permeability of the proximal straight tubule brush border membrane measured by the stopped flow method was reduced in AQP7 knockout mice compared to wild-type mice (AQP7, 18.0+/-0.4 x 10(-3 )cm/s vs. wild-type, 20.0+/-0.3 x 10(-3) cm/s). Although AQP7 solo knockout mice did not show a urinary concentrating defect, AQP1/AQP7 double knockout mice showed reduced urinary concentrating ability compared to AQP1 solo knockout mice, indicating that the contribution of AQP7 to water reabsorption in the proximal straight tubules is physiologically substantial. On the other hand, AQP7 knockout mice showed marked glycerol in their urine (AQP7, 1.7+/-0.34 mg/ml vs. wild-type, 0.005+/-0.002 mg/ml). This finding identified a novel pathway of glycerol reabsorption that occurs in the proximal straight tubules. In two mouse models of proximal straight tubule injury, the cisplatin-induced acute renal failure (ARF) model and the ischemic-reperfusion ARF model, an increase of urine glycerol was observed (pre-treatment, 0.007+/-0.005 mg/ml; cisplatin, 0.063+/-0.043 mg/ml; ischemia, 0.076+/-0.02 mg/ml), suggesting that urine glycerol could be used as a new biomarker for detecting proximal straight tubule injury.     

Enhanced utilization of glycerol for glyceride synthesis in isolated adipocytes from early pregnant rats

Adipose tissue normally has low glycerol kinase activity, but its expression is enhanced under conditions of augmented insulin sensitivity and/or obesity. Since these conditions occur during early pregnancy, the comparative utilization of glucose or glycerol by isolated adipocytes from rats at 0, 7, 14, or 20 days of pregnancy was studied. Incubations were carried out in the presence of [U¹⁴C]-glucose or -glycerol in medium supplemented or not with 5 mM glucose and 100 nM insulin. The conversion of glucose into esterified fatty acids and glyceride glycerol was greatest in adipocytes from 7-day pregnant rats, the effect being further enhanced by insulin. Both the amount of aquoporin 7 and the in vitro conversion of glycerol into glyceride glycerol were greatest in adipocytes of 7-day pregnant rats, the later being unaltered by insulin. In the presence of glucose, the overall glycerol utilization was lower than in its absence and glycerol conversion into glyceride glycerol was further decreased by insulin, the effect only being significant in adipocytes from 7-day pregnant rats. It is proposed that the enhanced utilization of glycerol for glyceride glycerol synthesis in adipose tissue contributes to the net accumulation of fat depots that normally takes place in early pregnancy.