Mariko Hara‐Chikuma

Aquaporin-3 (AQP3) is a water/glycerol-transporting protein expressed strongly at the plasma membranes of basal epidermal cells in skin. We found that human skin squamous cell carcinoma strongly overexpresses AQP3. A novel role for AQP3 in skin tumorigenesis was discovered using mice with targeted AQP3 gene disruption. We found that AQP3-null mice were remarkably resistant to the development of skin tumors following exposure to a tumor initiator and phorbol ester promoter. Though tumor initiator challenge produced comparable apoptotic responses in wild-type and AQP3-null mice, promoter-induced cell proliferation was greatly impaired in the AQP3-null epidermis. Reductions of epidermal cell glycerol, its metabolite glycerol-3-phosphate, and ATP were found in AQP3 deficiency without impairment of mitochondrial function. Glycerol supplementation corrected the reduced proliferation and ATP content in AQP3 deficiency, with cellular glycerol, ATP, and proliferative ability being closely correlated. Our data suggest involvement of AQP3-facilitated glycerol transport in epidermal cell proliferation and tumorigenesis by a novel mechanism implicating cellular glycerol as a key determinant of cellular ATP energy. AQP3 may thus be an important determinant in skin tumorigenesis and hence a novel target for tumor prevention and therapy.

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 μm) compared with wild type mice (39 μm). Adipocytes from AQP7 null mice also accumulated excess glycerol (251 versus 86 nmol/mg of protein) and triglycerides (3.4 versus 1.7 μmol/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 [14C]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 [14C]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. 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 μm) compared with wild type mice (39 μm). Adipocytes from AQP7 null mice also accumulated excess glycerol (251 versus 86 nmol/mg of protein) and triglycerides (3.4 versus 1.7 μmol/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 [14C]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 [14C]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 aquaporins are a family of small integral membrane proteins that transport water and in some cases water and small molecules such as glycerol (“aquaglyceroporins”) and other small solutes. The aquaglyceroporin AQP3 1The abbreviations used are: AQP, aquaporin; PPARγ, peroxisome proliferator-activated receptor-γ; C/EBPα, CCAAT/enhancer-binding protein α; WAT, white adipose tissue; KRH, Krebs-Ringer Hepes; BSA, bovine serum albumin; FFA, free fatty acid; TG, triglyceride. was cloned initially from kidney and found to transport water and glycerol when expressed heterologously (1Ma T. Frigeri A. Hasegawa H. Verkman A.S. J. Biol. Chem. 1994; 269: 21845-21849Abstract Full Text PDF PubMed Google Scholar, 2Ishibashi K. Sasaki S. Fushimi K. Uchida S. Kuwahara M. Saito H. Furukawa T. Nakajima K. Yamaguchi Y. Gojobori T. Marumo F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6269-6273Crossref PubMed Scopus (544) Google Scholar, 3Echevarria M. Windhager E.E. Tate S.S. Frindt G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10997-11001Crossref PubMed Scopus (268) Google Scholar). AQP3 gene disruption in mice produced a urinary concentrating defect caused by reduced collecting duct water permeability (4Ma T. Song Y. Yang B. Gillespie A. Carlson E.J. Epstein C.J. Verkman A.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4386-4391Crossref PubMed Scopus (347) Google Scholar), as well as dry skin (5Ma T. Hara M. Sougrat R. Verbavatz J.M. Verkman A.S. J. Biol. Chem. 2002; 277: 17147-17153Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), which was related to impaired epidermal cell glycerol permeability (6Hara M. Ma T. Verkman A.S. J. Biol. Chem. 2002; 277: 46616-46621Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Impaired glycerol transport in AQP3-deficient epidermal cells produced a low glycerol concentration in epidermis and stratum corneum (with normal serum glycerol), resulting in reduced stratum corneum hydration, elasticity, and biosynthesis, each of which could be corrected by glycerol replacement therapy (7Hara M. Verkman A.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7360-7365Crossref PubMed Scopus (229) Google Scholar). These studies provided evidence for a physiological role for aquaporin-facilitated glycerol transport. The aquaglyceroporin AQP7 was cloned from human adipose tissue (originally named AQPap; Ref. 8Kuriyama H. Kawamoto S. Ishida N. Ohno I. Mita S. Matsuzawa Y. Okubo K. Biochem. Biophys. Res. Commun. 1997; 241: 53-58Crossref PubMed Scopus (166) Google Scholar) and rat testis (9Ishibashi K. Kuwahara M. Gu Y. Kageyama Y. Tohsaka A. Suzuki F. Marumo F. Sasaki S. J. Biol. Chem. 1997; 272: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar) and shown in heterologous expression systems to function as a water/glycerol transporter. Adipocyte AQP7 expression was found to be sensitive to fasting/refeeding (10Kishida K. Kuriyama H. Funahashi T. Shimomura I. Kihara S. Ouchi N. Nishida M. Nishizawa H. Matsuda M. Takahashi M. Hotta K. Nakamura T. Yamashita S. Tochino Y. Matsuzawa Y. J. Biol. Chem. 2000; 275: 20896-20902Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), insulin deficiency (11Kishida K. Shimomura I. Kondo H. Kuriyama H. Makino Y. Nishizawa H. Maeda N. Matsuda M. Ouchi N. Kihara S. Kurachi Y. Funahashi T. Matsuzawa Y. J. Biol. Chem. 2001; 276: 36251-36260Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 12Kuriyama H. Shimomura I. Kishida K. Kondo H. Furuyama N. Nishizawa H. Maeda N. Matsuda M. Nagaretani H. Kihara S. Nakamura T. Tochino Y. Funahashi T. Matsuzawa Y. Diabetes. 2002; 51: 2915-2921Crossref PubMed Scopus (209) Google Scholar), and steroids and adrenergic agonists (13Fasshauer M. Klein J. Lossner U. Klier M. Kralisch S. Paschke R. Horm. Metab. Res. 2003; 35: 222-227Crossref PubMed Scopus (119) Google Scholar). These findings provided indirect evidence for a role of AQP7 in adipocyte function. AQP7 is also expressed in testis and sperm (14Saito K. Kageyama Y. Okada Y. Kawakami S. Kihara K. Ishibashi K. Sasaki S. J. Urol. 2004; 172: 2073-2076Crossref PubMed Scopus (69) Google Scholar, 15Calamita G. Mazzone A. Bizzoca A. Svelto M. Biochem. Biophys. Res. Commun. 2001; 288: 619-625Crossref PubMed Scopus (81) Google Scholar, 16Kageyama Y. Ishibashi K. Hayashi T. Xia G. Sasaki S. Kihara K. Andrologia. 2001; 33: 16516-16519Crossref Scopus (43) Google Scholar) and kidney (17Nejsum L.N. Elkjaer M. Hager H. Frokiaer J. Kwon T.H. Nielsen S. Biochem. Biophys. Res. Commun. 2000; 277: 164-170Crossref PubMed Scopus (73) Google Scholar), where its function is at present unknown. Here, we investigated a possible role for AQP7-mediated glycerol transport in fat cell physiology by phenotype analysis of transgenic mice with targeted AQP7 gene disruption. During the preparation of our manuscript Maeda et al. (18Maeda N. Funahashi T. Hibuse T. Nagasawa A. Kishida K. Kuriyama H. Nakamura T. Kihara S. Shimomura I. Matsuzawa Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17801-17806Crossref PubMed Scopus (152) Google Scholar) reported a mild phenotype of reduced serum glycerol concentration in AQP7-deficient young male mice after adrenergic stimuli and prolonged fasting. Here, we report in older AQP7 null mice marked adipocyte hypertrophy and increased body fat. Mechanistic analysis of adipocyte hypertrophy in AQP7 deficiency suggests pharmacological modulation of adipocyte AQP7 as a possible therapy in obesity. Mice—AQP7 null mice were generated by targeted deletion of exon 2 (manuscript describing gene knock-out procedures in preparation) and backcrossed into a CD1 genetic background. The mice were maintained in air-filtered cages and fed normal mouse chow in the University of California, San Francisco, Animal Care Facility. All procedures were approved by the University of California, San Francisco, Committee on Animal Research. RNA and Protein Analysis—Total RNA was isolated from epididymal fat and kidney using TRIzol (Invitrogen). Reverse transcription PCR and quantitative real-time PCR were done using sequence-specific primers (GenBank™ accession numbers: NM_007473 (AQP7), NM_011146 (peroxisome proliferator-activated receptor-γ (PPARγ)), NM_007678 (CCAAT/enhancer-binding protein α (C/EBPα)). Immunoblotting was done using polyclonal anti-PPARγ and C/EBPα (Santa Cruz Biotechnology, Inc.). Fat Mass and Morphology—White adipose tissue (WAT) from epididymal or gonadal, mesenteric, and inguinal tissues of wild type and AQP7 null mice at various ages (4–16 weeks) was excised and weighed. WAT was fixed in formalin and embedded in paraffin, and sections were stained with hematoxylin/eosin. For measurement of diameter, adipocytes were isolated from epididymal fat pad by collagenase digestion (19Rodbell M. J. Biol. Chem. 1964; 239: 375-380Abstract Full Text PDF PubMed Google Scholar) and photographing at high magnification for image analysis. Glycerol Permeability and Release—Glycerol permeability was measured in isolated adipocytes by modification of the method of Ma et al. (5Ma T. Hara M. Sougrat R. Verbavatz J.M. Verkman A.S. J. Biol. Chem. 2002; 277: 17147-17153Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). The adipocyte suspension was incubated for specified times with 100 mm glycerol in Krebs-Ringer Hepes (KRH) containing tracer [3H]glycerol (Amersham Biosciences) at room temperature. After separating on glass fiber filters and washing five times with ice-cold KRH in a suction filtration apparatus, cells were disrupted with 10% sodium dodecyl sulfate. Cell-associated radioactivity was determined by scintillation counting. Protein concentration was measured using a Bio-Rad DC protein assay kit. Glycerol release from WAT was determined as described previously (20Tordjman J. Chauvet G. Quette J. Beale E.G. Forest C. Antoine B. J. Biol. Chem. 2003; 278: 18785-18790Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Epididymal fat tissue was diced and incubated in glucose-free Dulbecco's modified Eagle's medium containing 3% fatty acid-free bovine serum albumin (BSA) for 1–3 h. Released glycerol was assayed using a glycerol assay Kit (Sigma). Glycerol, Free Fatty Acids, and Triglycerides in WAT and Serum— WAT was excised from the gonadal fat pad (female mice, age 16 weeks) and homogenized in phosphate-buffered saline. Lipid and aqueous phases were extracted by method of Bligh and Dyer (21Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (44361) Google Scholar). Aliquots of the lipid phase were resolved by TLC for measurement of free fatty acid (FFA) and triglyceride (TG). Glycerol was assayed enzymatically (Roche Applied Science). For serum analysis, enzymatic assay kits were used for the determination of serum nonesterified fatty acids (Wako) and glycerol, glucose, and total TG (Sigma). Lipolysis, Hormone-sensitive Lipase Activity, and Lipogenesis— Lipolysis was assayed using isolated adipocytes as described previously (22Morimoto C. Tsujita T. Okuda H. J. Lipid Res. 1997; 38: 132-138Abstract Full Text PDF PubMed Google Scholar). Isolated adipocytes were incubated for 1 h at 37°C in KRH buffer supplemented with 2.5% BSA, and release of FFA was measured. Hormone-sensitive lipase activity was measured using a lipase kit (Research Diagnostics, Inc.) as described previously (23Zhou Z. Yon Toh S. Chen Z. Guo K. Ng C.P. Ponniah S. Lin S.C. Hong W. Li P. Nat. Genet. 2003; 35: 49-56Crossref PubMed Scopus (391) Google Scholar). Lipogenesis was assayed as described previously (24Kimura Y. Ohminami H. Okuda H. J. Ethnopharmacol. 1998; 59: 117-123Crossref PubMed Scopus (10) Google Scholar). Adipocytes were incubated with [U-14C]glucose (Amersham Biosciences) in Krebs-Ringer bicarbonate buffer containing 10 nm insulin and 4% BSA for 1 h at 37°C. The lipid fraction was extracted and resolved by TLC, and 14C radioactivity in the TG fraction was measured. RT-PCR analysis in Fig. 1A confirmed AQP7 transcript expression in adipocytes and kidney from wild type but not homozygous AQP7 null mice. Analysis of mouse growth through age 16 weeks showed similar mouse body weight, although mouse length was reduced in the AQP7 null mice at 16 weeks (Fig. 1B). AQP7 null mice had remarkably greater gonadal fat mass compared with wild type mice as seen grossly (Fig. 1C). Fig. 1D (left) shows that fat mass from indicated sites was significantly elevated in both male and female AQP7 null mice at 16 weeks. Epididymal fat mass was comparable in wild type and AQP7 null mice until age 4 weeks but became greatly different as the mice aged (Fig. 1D, right). Fig. 2A shows histology of gonadal fat at 16 weeks, which was representative of the appearance of fat at multiple sites. Adipocytes were remarkably larger in AQP7 null mice than in wild type mice. Averaged adipocyte area was increased in AQP7 null mice (5322 ± 157 versus 2014 ± 67 μm2), suggesting that the greater fat mass in the AQP7 null mice is a consequence of adipocyte hypertrophy. Fig. 2B (top) shows light micrographs of adipocytes isolated from wild type and AQP7 null mice at age 4 and 16 weeks, which were used for subsequent cell-based studies. While adipocyte size was comparable in mice at age 4 weeks, adipocytes were remarkably enlarged in AQP7 null mice at age 16 weeks. Adipocyte diameter in AQP7 null mice was increased ∼3-fold (Fig. 2B, bottom). Fig. 2C summarizes the concentrations of glycerol, FFA, and TG in serum and adipocytes from mice at age 16 weeks. Serum parameters were unaffected by AQP7 deletion; similar results were obtained from age 4 week mice (data not shown). However, adipocyte glycerol, FFA, and TG content, expressed per milligram of total protein, were significantly elevated in the AQP7 null mice. To investigate the mechanism for the progressive adipocyte hypertrophy in AQP7 deficiency, measurements were made of adipocyte plasma membrane glycerol permeability, glycerol release, lipolysis, and lipogenesis. Plasma membrane glycerol permeability was measured from the initial uptake of [14C]-glycerol into isolated adipocytes from the younger wild type and AQP7 null mice (Fig. 3A), where adipocyte size is comparable. Uptake of [14C]glycerol was measured at 5 min based on initial studies showing linear uptake at this time (Fig. 3A, inset). [14C]Glycerol uptake was significantly reduced by 3.0-fold in AQP7 null mice compared with wild type mice. Permeability coefficients, Pglycerol, computed from uptake rates and adipocyte surface-to-volume ratio, were 2.5 × 10–6 cm/s and 0.7 × 10–6 cm/s for wild type and AQP7-deficient adipocytes, respectively. Glycerol release from minced fat tissue was determined by assay of glycerol in the physiological bathing solution at 1 and 3 h after incubation at 37 °C. Fig. 3B shows that glycerol release, expressed per WAT weight, was significantly reduced in the AQP7 null mice. However, these release data do not provide direct information about adipocyte lipolysis because glycerol release depends on several factors, including the rate of lipolysis, cytoplasmic glycerol concentration, and plasma membrane glycerol permeability. To assess lipolysis, FFA release from isolated adipocytes was measured, as well as the activity of hormone-sensitive lipase, a key regulator of lipolysis. Fig. 3C shows that lipolysis was not affected by AQP7 deletion. Lipogenesis was assayed in isolated adipocytes from the incorporation of [14C]glucose into TG. Fig. 3D shows similar lipogenesis in wild type and AQP7 null mice. We also determined whether PPARγ and C/EBPα were induced, which are transcription factors involved in adipogenesis. Immunoblot analysis in Fig. 3E shows similar C/EBPα protein expression in wild type and AQP7 null mice (1.0 ± 0.03-fold protein; 1.1 ± 0.3-fold mRNA, n = 5), although PPARγ was slightly reduced in AQP7 null mice at 16 weeks (0.79 ± 0.1-fold protein; 0.42 ± 0.3-fold reduced mRNA, n = 5). We found marked adipocyte hypertrophy and increased fat mass in AQP7-deficient mice, which developed progressively after age 6 weeks. Mechanistic studies in isolated adipocytes and minced fat tissue suggested that the ∼3-fold reduced glycerol permeability of the adipocyte plasma membrane was responsible for the marked adipocyte hypertrophy in AQP7 deficiency, rather than primary defects in adipocyte lipolysis, lipogenesis, or PPARγ induction (25Evans R.M. Barish G.D. Wang Y.X. Nat. Med. 2004; 10: 355-361Crossref PubMed Scopus (1327) Google Scholar). We propose that reduced adipocyte glycerol permeability results in intracellular glycerol and TG accumulation, as was verified experimentally, resulting in progressive adipocyte expansion. The recent phenotype study of Maeda et al. (18Maeda N. Funahashi T. Hibuse T. Nagasawa A. Kishida K. Kuriyama H. Nakamura T. Kihara S. Shimomura I. Matsuzawa Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17801-17806Crossref PubMed Scopus (152) Google Scholar) did not report data on adipocyte morphology or fat mass but looked only at relatively young mice focusing on basal and stimulated release of glycerol into the blood. They concluded from adipocyte glycerol release measurements that impaired glycerol “gateway” function was responsible for the phenotype of reduced plasma glycerol. However, although we agree that adipocyte glycerol permeability is impaired in AQP7-deficient adipocytes (from glycerol uptake measurements in Fig. 3A), glycerol gateway function cannot be deduced from the glycerol release measurements of Maeda et al. (18Maeda N. Funahashi T. Hibuse T. Nagasawa A. Kishida K. Kuriyama H. Nakamura T. Kihara S. Shimomura I. Matsuzawa Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17801-17806Crossref PubMed Scopus (152) Google Scholar), which depend on glycerol permeability as well lipolysis rate and steady-state adipocyte glycerol content. Increased adipocyte size and fat mass in AQP7-deficient mice were not noted by Maeda et al. (18Maeda N. Funahashi T. Hibuse T. Nagasawa A. Kishida K. Kuriyama H. Nakamura T. Kihara S. Shimomura I. Matsuzawa Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17801-17806Crossref PubMed Scopus (152) Google Scholar), possibly because the phenotype of older mice was not studied. Fig. 3F shows a proposed mechanism for progressive TG accumulation in AQP7-deficient adipocytes. Reduced plasma membrane glycerol permeability in AQP7 deficiency results in an increase in steady-state glycerol concentration in adipocyte cytoplasm. The 3.0-fold reduced plasma membrane glycerol permeability is consistent with the 2.9-fold increase in steady-state adipocyte glycerol concentration. Increased adipocyte glycerol concentration would then increase glycerol 3-phosphate and hence TG biosynthesis. Our results thus focus attention on adipocyte glycerol permeability as a novel regulator of adipocyte size and whole body fat mass. Modulation of adipocyte AQP7 expression and/or function may thus alter fat mass, providing a rational basis for investigation of AQP7 up-regulation as therapy in some forms of obesity. We thank Liman Qian for mouse breeding and genotype analysis.