Defective glycerol metabolism in aquaporin 9(AQP9) knockout miceAleksandra M. Rojek*, Mariusz T. Skowronski*, Ernst-Martin Fuchtbauer†, Annette C. Fuchtbauer†, Robert A. Fenton*,Peter Agre‡§¶, Jørgen Frøkiær*, and Søren Nielsen*¶

*The Water and Salt Research Center, University of Aarhus, DK-8000 Aarhus C, Denmark; †Department of Molecular Biology, University of Aarhus,DK-8000 Aarhus C, Denmark; ‡Duke University School of Medicine, DUMC 3701, Durham, NC 27710; and §Departments of Biological Chemistry andMedicine, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185

Contributed by Peter Agre, December 13, 2006 (sent for review October 24, 2006)

Aquaporin-9 (AQP9) is an aquaglyceroporin membrane channelshown biophysically to conduct water, glycerol, and other smallsolutes. Because the physiological role/s of AQP9 remain undefinedand the expression sites of AQP9 remain incomplete and conflict-ing, we generated AQP9 knockout mice. In the absence of physi-ological stress, knockout mice did not display any visible behav-ioral or severe physical abnormalities. Immunohistochemicalanalyses using multiple antibodies revealed AQP9 specific labelingin hepatocytes, epididymis, vas deferens, and in epidermis of wildtype mice, but a complete absence of labeling in AQP9�/� mice. Inbrain, no detectable labeling was observed. Compared with controlmice, plasma levels of glycerol and triglycerides were markedlyincreased in AQP9�/� mice, whereas glucose, urea, free fatty acids,alkaline phosphatase, and cholesterol were not significantly dif-ferent. Oral administration of glycerol to fasted mice resulted in anacute rise in blood glucose levels in both AQP9�/� and AQP9�/�

mice, revealing no defect in utilization of exogenous glycerol as agluconeogenic substrate and indicating a high gluconeogenic ca-pacity in nonhepatic organs. Obese Leprdb/Leprdb AQP9�/� andobese Leprdb/Leprdb AQP9�/� mice showed similar body weight,whereas the glycerol levels in obese Leprdb/Leprdb AQP9�/� micewere dramatically increased. Consistent with a role of AQP9 inhepatic uptake of glycerol, blood glucose levels were significantlyreduced in Leprdb/Leprdb AQP9�/� mice compared with Leprdb/Leprdb AQP9�/� in response to 3 h of fasting. Thus, AQP9 isimportant for hepatic glycerol metabolism and may play a role inglycerol and glucose metabolism in diabetes mellitus.

aquaglyceroporin � diabetes mellitus � leptin receptor

Aquaporin 9 (AQP9) is a member of the aquaglyceroporinsubfamily of aquaporins and shares the highest amino acid

sequence homology with AQP3, AQP7 (1), and AQP10 (2). Inaddition to water, the aquaglyceroporins transport small un-charged molecules like glycerol, urea, purines, and pyrimidines(3, 4), but their physiological function(s) remains unknown.AQP9 expression has been reported in many tissues, usingdifferent immunohistochemical and molecular approaches. Inthe liver, AQP9 is expressed in hepatocytes within the sinusoidalsurfaces of hepatocyte plates, where, during starvation, it isspeculated to function in glycerol uptake from the bloodstreamfor gluconeogenesis (5, 6). In addition, AQP9 has been proposedto be involved in urea elimination from hepatocytes (3). AQP9gene expression in the liver is down-regulated by insulin, poten-tially via an ‘‘insulin responsive element’’ in the AQP9 promoter.

AQP9 expression has been reported in other tissues includingthe male reproductive tract, where it localizes to the efferentductule epithelium, epididymis, and vas deferens and may beinvolved in sperm maturation, concentration, and storage, re-spectively (7). The expression of AQP9 has also been shown tobe regulated by testosterone and estrogen (8, 9). AQP9 expres-sion has been reported in the plasma membranes of Leydig cellsin rat testis (10), in rat spleen white pulp (11), in rat brain(11–13), in rat spinal cord (14), in the apical membrane of the

trophectoderm of the mouse blastocyst (15), in syncytiotropho-blast of human term placenta (16), and in the peripheralleukocytes (3). The function of AQP9 in these locations remainsunknown.

It is important to emphasize that, despite an abundance ofdata from different studies, there are still major discrepanciesbetween the reported expression sites, especially within thebrain. In speculation, these differences may be due to eitherheterogeneity of expression in different species, existence ofdifferent splice variants, or may represent artifacts related tospecificity of the anti-AQP9 antibodies used in immunohisto-chemical and immunoblotting methods.

The purposes of the present studies were: (i) to generateAQP9 gene knockout mice; (ii) to identify the major expressionsites of AQP9 in mouse by exploiting the AQP9 gene knockoutmice; (iii) to identify the physiological role(s) of AQP9 byexamining the phenotype of AQP9 gene knockout mice; (iv) togenerate and phenotype obese Leprdb lacking AQP9 (Leprdb is aleptin receptor mutation with absence of leptin function leadingto severe obesity and type II diabetes). One specific purpose ofthe studies was to investigate whether there was evidence for arole of AQP9 in hepatic glycerol metabolism because glycerol isa gluconeogenic substrate potentially contributing to the ele-vated hepatic glucose production seen in obese type II diabeticpatients. These approaches have provided further insights intothe expression and function of AQP9 and evidence for a possiblerole of AQP9 in diabetes mellitus.

ResultsGeneration of AQP9 Knockout Mice. In the AQP9 targeted allele, 55nucleotides of exon 2 were substituted for sequence encoding aneomycin phosphotransferase expression cassette (Fig. 1A). Thisinsertion was predicted to result in correct translation of theinitial 47 aa, followed by 100 random amino acids and a stopcodon encoded by vector sequence and the inverted pA signal ofthe neomycin phosphotransferase. The presence of the mutatedAQP9 transcript was confirmed by RT-PCR of AQP9�/� mouseliver RNA using primers prim1/prim2 (Fig. 1B), followed bysequencing of the amplification product. No PCR product wasobserved in an identical reaction using liver RNA from AQP9�/�

mice. RT-PCR from liver RNA using the primer pair prim1/prim3 gave the expected 303-bp product in the AQP9�/� andAQP9�/�; however, a smaller 177-bp product was amplified fromcDNA from the AQP9�/� mice. DNA sequencing showed thatthe smaller product is an alternatively spliced transcript, where

Author contributions: A.M.R., E.-M.F., P.A., and S.N. designed research; A.M.R., M.T.S.,E.-M.F., and A.C.F. performed research; E.-M.F. and S.N. contributed new reagents/analytictools; A.M.R., P.A., and S.N. analyzed data; and A.M.R., R.A.F., J.F., and S.N. wrote the paper.

The authors declare no conflict of interest.

Abbreviations: AQP9, aquaporin 9; ALP, alkaline phosphatase.

¶To whom correspondence may be addressed. E-mail: sn@ana.au.dk or pagre@cellbio.duke.edu.

© 2007 by The National Academy of Sciences of the USA

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exon 1 is spliced directly to exon 3. Because this splice variant wasnot amplified from AQP9�/� cDNA, we believe that this is aninfrequent alternative splicing event, and probably does not takeplace for the wild-type allele, but only for the allele containingthe neomycin cassette in exon 2. The electronic translation of thistranscript predicts a correctly translated initial 37 amino acids(exon 1) and a frameshift in exon 3 sequence, thus is unlikely toresult in any form of AQP9 protein.

Immunoblotting of liver and epididymis protein extracts fromAQP9�/� and AQP9�/� mice revealed an absence of AQP9protein in knockout mice (Fig. 1C). Interestingly, the proteinisoform of AQP9 expressed in wild-type mouse epididymisappears to have a smaller molecular weight than the form ofAQP9 expressed in the liver (Fig. 1C).

Crossing of AQP9�/� heterozygous animals with C57BL/6mice produced an approximately equal ratio of AQP9�/�/AQP9�/� offspring (55:48 ratio). Initial heterozygote crossesproduced 35 wild-type mice, 97 heterozygote offspring, and 44AQP9�/� mice, consistent with a 1:2:1 Mendelian pattern andindicating no increased embryonic mortality of the AQP9�/� and

AQP9�/� mice. There were no detectable differences in physicalappearance, body weight, or behavior between age-matchedAQP9�/� mice and littermate controls. AQP9�/� females arefertile and produced litter sizes similar to female AQP9�/�

siblings (AQP9�/�, 7.1 � 0.5; AQP9�/�, 6.1 � 0.5 pups in litter,difference not statistically significant). Homozygous AQP9�/�

males are fertile, and microscopic examination of the spermshowed normal morphology and motility.

Expression of AQP9 Protein in Mouse Organs. The tissue localizationof AQP9 was examined using immunohistochemistry. Two dif-ferent peptide-targeted antibodies against AQP9 were used: (i)AQP9A1 (Alpha Diagnostics, San Antonio, TX) and (ii)RA2674–685 (11). The following organs were examined: liver,epididymis, testis, skin, spleen, muscle, brain, spinal cord, ova-ries, and intestine. In wild-type control mice, using RA2674–685and AQP9A1 antibodies, only liver, epididymis, and skin (notpreviously identified expression site) showed strong immuno-staining using both antibodies, whereas there was a completeabsence of staining in AQP9�/� mice. At high concentration, theAQP9A1 antibody also stained different structures in spleen,brain, and spinal cord; however, the same staining pattern wasseen in AQP9�/� mice, and we therefore believe it is caused bynonspecific binding. Consistent with these findings, RA2674–685 did not stain these tissues (not shown).

In liver from control mice, staining was restricted to the sinu-soidal surfaces of hepatocyte plates (Fig. 2B). Staining was strongestaround the central vein (perivenous zone), whereas the sinusoidsaround the portal vein (periportal zone) were stained weakly (Fig.2B), consistent with previous observations (5). This pattern ofstaining was observed both in males and females (not shown). InAQP9�/� mice, there was no staining of liver (Fig. 2C), andhistological examination showed no apparent abnormalities.

In epididymis and vas deferens of control mice, the RA2674–685 and AQP9A1 antibodies stained microvilli of the principal

Fig. 1. Generation of AQP9 knockout mice. (A) Schematic diagram of thetargeting strategy. (B) AQP9 transcript analysis by RT-PCR of liver RNA andsequencing of the products. Primer pair ‘‘prim1’’ and ‘‘prim2’’ produced412-bp-long PCR product, confirming the presence of the mutated transcriptin the AQP9�/� and AQP9�/� mice. Primer pair ‘‘prim1’’ and ‘‘prim3’’ producedthe correct wild-type 303-bp-long product in the AQP9�/� and AQP9�/� mice,which is absent in the AQP9�/� mice. However, in AQP9�/� mice, these primersamplified 177-bp-long PCR product, which is evidence of incorrect splicing ofAQP9 gene where whole exon 2 containing the neo expression cassette is lost.(C) Immunoblotting of liver and epididymis protein samples with antibodyagainst AQP9 (Alpha Diagnostics). AQP9 in the epididymis appears to haveslightly lower molecular weight than AQP9 expressed in the liver.

Fig. 2. Immunocytochemistry of liver from AQP9�/� (A and B) and AQP9�/� (Cand D) mice stained with polyclonal antibody RA2674–685 at dilution 1:100.Staining of the sinusoidal plates surrounding the central vein (CV) visible in theAQP9�/� mouse (A and B) is absent in the AQP9�/� mouse (C and D).

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cells (Fig. 3 A, B, and E). This staining was absent in AQP9�/�

mice (Figs. 3 C, D, and F). Histological analysis of epididymisfrom AQP9�/� mice showed no signs of epididymal tubuledilatation or other histological abnormalities and the weight ofthe whole epididymis (caput, corpus, and cauda segments) didnot differ statistically from control mice (2.37 � 0.14 mg per gof body weight in AQP9�/� and 2.50 � 0.13 mg per g of bodyweight AQP9�/�, n � 8 per group).

In control mice (AQP9�/�), AQP9 was detected in the singlelayer of epidermis cells in the stratum granulosum of dorsal skin(Fig. 4A). This staining was absent in the AQP9�/� mice (Fig.4B). Incision through the epidermis and dermis followed by a10-day period of healing resulted in proliferation of regeneratingtissue and multilayered stratum granulosum with prominentAQP9 expression (Fig. 4 C and D). No defects in wound healingwere observed in AQP9�/� mice.

Phenotyping of AQP9�/� Mice Regarding Glycerol Metabolism. Toexamine whether AQP9�/� mice exhibit defects in the metabo-lism of various solutes, including glycerol, the concentration ofglucose, glycerol, FFA, triglycerides, urea, total cholesterol, andalkaline phosphatase (ALP) in plasma samples from AQP9�/�

and AQP9�/� mice were determined (shown in Table 1).AQP9�/� mice exhibited a marked increase in plasma glyceroland triglyceride levels compared with AQP9�/� mice, revealinga deficient glycerol metabolism. There were no significantdifferences in the other measured parameters.

After 24 h of fasting of AQP9�/� and AQP9�/� control mice,both groups showed similar body weight loss of �12.5%, and asimilar decrease in blood glucose levels (Table 2). However,plasma glycerol and triglycerides levels were significantly in-

creased in the fasted AQP9�/� mice compared with fastedcontrol mice, whereas FFA, urea, total cholesterol and ALPlevels were unchanged (Table 3).

To investigate whether AQP9-deficient mice are able to use anacute minor load of exogenous glycerol, AQP9�/� and AQP9�/�

mice were fasted overnight for 16 h and subsequently adminis-tered 87% glycerol orally. Blood glucose levels were measured10 min before glycerol administration and at 0, 15, 30, 45, 60 and120 min after glycerol administration. Blood glucose levelsincreased comparatively in both the AQP9�/� and AQP9�/�

mice after glycerol administration and there was no significantdifference between the genotypes (Fig. 5A). These data indicatethat there is a sufficient capacity to generate glucose fromacutely administered exogenous glycerol in AQP9-deficientmice.

AQP9 Function in Obese Leprdb Mice. Because glycerol is a glu-coneogenic substrate potentially contributing to the elevatedhepatic glucose production seen in obese type II diabetespatients, we generated mice lacking AQP9 and leptin receptorfunction. Mice carrying a mutation in the leptin receptor gene,Leprdb (Taconic, Ry, Denmark), are severely obese and develop

Fig. 3. Immunocytochemistry of epididymis from AQP9�/� (A and B) andAQP9�/� (C and D) mice, and vas deferens from AQP9�/� (E) and AQP9�/� (F)mice stained with polyclonal antibody RA2674–685 at dilution 1:100. In theAQP9�/� mouse, AQP9 is present in the microvilli of the principal cells of theepididymis and vas deferens (A, B, and E), and the staining is completely absentin the AQP9�/� mouse (C, D, and F).

Fig. 4. AQP9 localization in the skin. (A and B) Immunocytochemistry ofnormal dorsal skin from AQP9�/� (A) and AQP9�/� (B) mice stained withpolyclonal antibody RA2674–685 at dilution 1:100. In the AQP9�/� mouse,AQP9 is present in the stratum granulosum of the epidermis (A), and thestaining is absent in the AQP9�/� mouse (B). (C and D) After wounding and a10-day period of healing, the regenerating skin shows hypertrophy of theepidermis. The multilayered stratum granulosum shows clear AQP9 staining inthe apical pole (C). The wounded skin from the AQP9�/� mouse shows nostaining (D).

Table 1. Plasma values: Basal conditions (mice with free accessto food)

AQP9��� AQP9���

n 8 6Glycerol, �mol�liter 339 � 24 530 � 28*Triglycerids, mmol�liter 0.53 � 0.05 0.83 � 0.02*Urea, mmol�liter 7.3 � 0.6 7.4 � 0.4Total cholesterol, mmol�liter 1.80 � 0.11 2.10 � 0.15Free fatty acids, mmol�liter 0.47 � 0.07 0.66 � 0.10ALP, units�liter 144 � 10 142 � 7

Values are means � SE; n, number of mice; *, P �0.05, AQP��� comparedwith AQP���.

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type II diabetes and increased lipolysis and glycerol production(reviewed in ref. 18). Leprdb and AQP9 knockout mice werecrossed to generate obese Leprdb/Leprdb AQP9�/� mice andobese Leprdb/Leprdb AQP9�/� controls. Both Leprdb/Leprdb

AQP9�/� and Leprdb/Leprdb AQP9�/� mice developed severeobesity, with no significantly different gain of body weight. Theincreased bodyweight was accompanied by development of typeII diabetes within 15 weeks of age, evidenced by increased bloodglucose levels and increased levels of plasma glycerol and freefatty acids (Fig. 5B and Table 4). Importantly, the plasmaglycerol levels in obese Leprdb/Leprdb AQP9�/� mice were mark-edly increased compared with the obese Leprdb/Leprdb AQP9�/�

controls. These results further support a major role of AQP9 inglycerol metabolism.

To test whether AQP9 may act as an entrance port for hepaticgluconeogenesis from nonhepatic glycerol, the ability of theLeprdb/Leprdb AQP9 �/� mice to generate glucose in response tofasting was examined. The blood glucose levels measured in theabsence of fasting did not differ significantly in obese Leprdb/Leprdb AQP9�/� mice and control obese Leprdb/Leprdb mice(Table 4). However, the blood glucose levels measured after 3 hof fasting were moderately lower in the Leprdb/Leprdb AQP9�/�

mice (Fig. 5C). Taken together, these results support the viewthat AQP9 plays a role in hepatic glycerol metabolism.

DiscussionAQP9 knockout mice were generated to identify the mainexpression sites and the physiological role of AQP9 that thus farhave remained undefined. Our data determined that AQP9 ishighly expressed in liver, which represents its main expressionsite. Plasma levels of glycerol and triglycerides were markedlyincreased in AQP9�/� mice, revealing a role of AQP9 in glycerolmetabolism. Obese Leprdb/Leprdb AQP9�/� double knockoutmice had a dramatic increase in plasma glycerol levels comparedwith obese Leprdb/Leprdb AQP9�/� control mice, which hadhigher plasma glycerol levels than non-obese control mice. Thisresult further supports a role of AQP9 in glycerol metabolism.Moreover the blood glucose levels measured after 3 h of fastingwere moderately lower in the Leprdb/Leprdb AQP9�/� micecompared with Leprdb/Leprdb AQP9�/� mice, supporting theview that Leprdb/Leprdb AQP9�/� have a deficiency in generatingglucose in response to fasting due to lack of AQP9 as an entranceport for glycerol for hepatic gluconeogenesis. Taken together,these results strongly support the view that AQP9 is importantfor hepatic glycerol metabolism and provide further evidence fora role of AQP9 in diabetes mellitus.

Defective Glycerol Metabolism in AQP9 Gene Knockout Mice.AQP9�/� mice have markedly increased plasma glycerol andtriglyceride levels, revealing a defect in glycerol metabolism. Theabundant expression in the liver of normal mice strongly suggeststhat the increased plasma glycerol levels in AQP9�/� mice arecaused by an absence of hepatic AQP9 and an impaired uptakeof glycerol through the hepatocyte plasma membrane. Although

the increased plasma glycerol level in AQP9 null mice is likely tobe mediated by absence of AQP9, it cannot be excluded that theuptake and release of glycerol by other organs expressingaquaglyceroporins, e.g., kidney cortex (AQP3 and AQP7), fattissue (AQP7), or intestine (AQP3 and AQP10), may alsoinfluence the final plasma glycerol level. Importantly, it cannotbe excluded that the AQP9�/� mice exhibit compensatoryregulation of glycerol metabolism, which may also be the reasonwhy they tolerate 24-h starvation.

Hypoglycemic (fasted) AQP9�/� mice were capable of effi-ciently using orally administered glycerol for acute production ofglucose. It is likely that administered glycerol is converted toglucose in secondary gluconeogenic organs, e.g., the kidney orthe intestine (19), compensating for the assumed defect inglycerol transport into the hepatocytes. Thus, our data revealthat the capacity to metabolize exogenous glycerol via anAQP9-independent pathway is substantial, although the relativecontribution is difficult to estimate due to potential compensa-tory mechanisms in AQP9�/� mice. It is also possible that, in thepresent setting, the rate of glycerol uptake was not a rate-limitingstep in hepatic gluconeogenesis.

The increased mass of adipose tissue in the Leprdb/Leprdb miceis associated with increased lipolysis resulting in the observedincreased plasma glycerol levels. This increased glycerol avail-ability could potentially lead to increased conversion to glucosein liver or other gluconeogenic organs and play a role in thedevelopment of type II diabetes. The obese Leprdb/Leprdb

AQP9�/� mice show a dramatically increased plasma glycerollevel compared with obese Leprdb/Leprdb AQP9�/� controls(consistent with the hypothesis of impaired glycerol utilization bythe liver). To further support the hypothesis that AQP9 isinvolved in glycerol uptake and gluconeogenesis, Leprdb/Leprdb

AQP9�/� and Leprdb/Leprdb AQP9�/� control mice were fastedfor 3 h. Without fasting, the mice showed similar blood glucoselevels, possibly due to the glucose ingestion (Table 4). After a 3-hfasting period, the Leprdb/Leprdb AQP9�/� mice exhibited lowerpostprandial plasma glucose levels compared with those ofLeprdb/Leprdb AQP9�/� control mice (Fig. 5C), suggesting thatthe absence of AQP9 in the hepatocyte membrane reduced thecapacity for glycerol entrance for gluconeogenesis, leading toreduced plasma glucose levels, which may indicate an improve-ment of the diabetic state. This result is consistent with a role ofAQP9 as a glycerol-importing pathway. Further studies arerequired on these findings, but the development of specificblockers of aquaglyceroporins could potentially be very useful inelucidating whether pharmacological blockade of AQP9 may beof potential benefit in type II diabetes. Moreover, furtherexperiments will be necessary to clarify whether insulin produc-tion and insulin action are improved in the obese Leprdb/Leprdb

AQP9�/� mice compared with Leprdb/Leprdb AQP9�/� controlmice.

Interestingly, according to the model of metabolic zonation ofthe liver, gluconeogenesis predominantly takes place in the

Table 2. Response to 24 h of starvation

AQP9��� AQP9���

n 10 12Start body weight, g 20.4 � 1.0 21.9 � 0.8Final body weight, g 17.9 � 0.9 19.2 � 0.7% Change in body weight 12.6 � 1.0 12.4 � 0.5Start blood glucose, mmol�liter 6.3 � 0.2 6.6 � 0.2Final blood glucose, mmol�liter 3.5 � 0.4 3.7 � 0.3

Values are means � SE; n, number of mice; *, P � 0.05, AQP9��� comparedwith AQP9���.

Table 3. Plasma values: Mice starved for 24 h

AQP9��� AQP9���

n 12 12Glycerol, �mol�liter 353 � 16 493 � 21*Triglycerids, mmol�liter 0.58 � 0.02 0.74 � 0.03*Urea, mmol�liter 6.9 � 0.3 7.0 � 0.4Total cholesterol, mmol�liter 2.29 � 0.09 2.46 � 0.14Free fatty acids, mmol�liter 1.34 � 0.10 1.23 � 0.07ALP (units�liter) 142 � 5 143 � 6

Values are mean � SE; n, number of mice; *, P � 0.05, AQP9��� comparedwith AQP9���.

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hepatocytes surrounding the portal vein (periportal) that expresslittle AQP9 (reviewed in ref. 20), whereas relatively less glu-coneogenesis takes place in the hepatocytes surrounding thecentral vein (perivenous), where highest AQP9 levels are de-tected. Thus, it cannot be excluded that AQP9 may haveadditional functions in the mouse liver other than glyceroltransport.

Expression of AQP9 in Skin and Male Reproductive Tract. Our studiesreport the presence of AQP9 in skin, specifically in the epidermisoutermost layer stratum granulosum. In addition to AQP9,another aquaglyceroporin, AQP3, is present in the epidermis ata different site: the basal cell layer (21). Previous studies havereported that mice deficient in AQP3 have a defective skinhydration, decreased skin elasticity, and impaired wound healing(22). It is therefore plausible that AQP9, together with AQP3,is involved in maintaining skin hydration. However, no apparentdefects in wound healing were observed in AQP9 knockout mice.

Male AQP9�/� mice are fertile and spermatozoa show normalmotility. Further studies exploiting AQP9 knockout mice will behelpful in determining the role of AQP9 in fluid transport.

Materials and MethodsGeneration of AQP9 Knockout Mice. The genomic sequence of themouse AQP9 gene was obtained from www.ensembl.org (tran-script ID ENSMUST00000074465). A 3.2-kb DNA fragment(targeting vector arm 1) was amplified using genomic DNA from129S1/Sv strain of mice and primer pair 5�-tttccgcggCTCAG-GTCTCATGCAATGTCAGC-3� and 5�-ttaccgcggACG-GCAGTTGTGATGGCTCTTTA (lowercase indicates the re-striction enzyme sequence; uppercase indicates the anchor to

genomic DNA sequence). The 2.8 kb fragment (targeting vectorarm 2) was amplified using primer pair 5�-cttctcgagcccgggGCT-TGAGCAATAGAGCCACATCC-3� and 5�-cctctcgagCAG-TAGTCAGTGCCACTCTGCAAC-3�. A targeting constructwas created by inserting the 2.8-kb vector arm 2 into the XhoIsite of pKO Scrambler 1903, and the 3.2-kb vector arm 1 into theSacII site. The targeting construct was linearized by using NotIand electroporated into CJ7 embryonic stem cells derived from129S1/Sv mice (23). G418-resistant colonies were selected andexpanded. Clones with homologous recombination were de-tected by Southern blot analysis using a probe flanking the 3.2-kbarm and confirmed by PCR. Six clones with the correct recom-bination event were obtained and one clone was used forinjection into 20 B6D2F2 (24) mouse blastocysts. The chimericmales were bred with C57BL/6 females and agouti offspring(indicating germ-line transmission of the manipulated 129S1/SvES cells) were tested for the presence of the disrupted AQP9allele by PCR using genomic tail DNA and 3 primers: GTGC-TACTTCCATTTGTCACGTCCT, GCCACTAGCCATGT-GTTGGTATTTC, and AACTGGGGATAGTGGGAT-TCAAAGA. The expected PCR product for the mutated alleleis 503 bp, and the expected PCR product for the wild-type alleleis 735 bp. Heterozygous mice were further bred to obtainhomozygous AQP9 KO mice on a mixed genetic C57BL6/129S1/Sv background.

RT-PCR. Liver RNA was isolated by using the RNeasy kit (Qiagen,Valencia, CA). RT-PCR was performed by using SuperScript IIand primer pair prim1: 5�-AAAGGGGAACTTGAAC-CACTCCA-3� and prim2: 5�-GTGCTACTTCCATTTGT-CACGTCCT-3� (the expected product for the disrupted AQP9

Fig. 5. Physiological responses of AQP9 knockout mice. (A) Five milligrams of 87% glycerol per g of body weight was administered orally to AQP9�/� and controlmice (AQP9�/�) starved for 16 h, and blood glucose level measured at different time intervals. There was no difference in the glucose levels after glyceroladministration in the AQP9�/� (filled circles) and the control (open circles) mice. (B) Obese (Leprdb/Leprdb) AQP9�/� mice show similar body weight gain as obeseAQP9�/� mice. n � 18 AQP9�/�, 19 AQP9�/�. (C) The postabsorptive (3-h fasting) blood glucose is lower in the AQP9�/� mice compared with AQP9�/� mice. n �18 AQP9�/�, 19 AQP9�/�

Table 4. Plasma values in lean and obese AQP9��� and AQP��� mice: Basal conditions (micewith free access to food)

AQP9��� AQP9���

ObeseLeprdb�Leprdb Lean

ObeseLeprdb�Leprdb Lean

n 17 18 12 20Blood glucose, mmol�liter 17.1 � 1.4 6.6 � 0.2* 17.4 � 2.4 6.6 � 0.2†

Glycerol, �mol�liter 553 � 35 351 � 40* 939 � 43‡ 505 � 37§

Free fatty acids, mmol�liter 1.17 � 0.07 0.92 � 0.06* 1.34 � 0.11 1.04 � 0.07†

Values are means � SE; n, number of mice; *, P � 0.05 for obese Leprdb�Leprdb AQP9��� vs. lean AQP9���; †,P � 0.05 for obese Leprdb�Leprdb AQP9��� vs. lean AQP9���; ‡, P � 0.05 for obese Leprdb�Leprdb AQP9��� vs. obeseLeprdb�Leprdb AQP9���; §, P � 0.05 for lean AQP9��� vs. lean AQP9���.

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allele 412 bp) or the primer pair prim 1 and prim 3: 5�-GAGAAGGACCGAGCCAAGAAGAA-3� (the expected prod-uct for the wild-type allele is 303 bp).

Blood/Plasma Measurements. Method A: blood glucose measure-ments were performed using an Accu-Chek Sensor (Roche, Mann-heim, Germany) and a drop of saphenous vein blood. Method B:for other tests, mice were anesthetized, their chest cavity opened,and blood samples collected from the right ventricle of the heartinto heparinized tubes. The blood cells were removed by centrifu-gation for 10 min at 4,000 � g, and plasma glycerol, triglycerides,urea, free fatty acids, and total cholesterol analysis using standardlaboratory tests. Total ALP was measured using a Vitros 950analyzer (Ortho Chemical Diagnostics, Copenhagen, Denmark).

Immunostaining. Six-week-old AQP9�/�, AQP9�/�, andAQP9�/� siblings were perfusion fixed through the heart using3% PFA/0.1 M cacodylate buffer. Tissues were extracted andpostfixed for 1 h in the same fixative, and washed three times for10 min in 0.1 M cacodylate buffer. Tissues were embedded inparaffin, 2-�m sections were cut on a rotary microtome (LeicaMicrosystems, Herlev, Denmark) and subjected to immunola-beling, as described (17). Primary antibodies used were (i)AQP9A-1 (Alpha Diagnostics) and (ii) RA2674–685 as de-scribed (11).

Protein Sample Preparation and Immunoblotting. Organs were ho-mogenized in dissection buffer (0.3 M sucrose/25 mM imida-zole/1 mM EDTA, pH 7.2, containing 8.5 �M leupeptin, and 1mM phenylmethylsulfonyl f luoride) using an ultra-turrax T8homogenizer (IKA Labortechnik, Staufen, Germany). For semi-quantitative immunoblotting using antibody AQP9A-1 (AlphaDiagnostics) the homogenate was centrifuged at 4,000 � g for 15min at 4°C to remove whole cells, nuclei, and mitochondria. Gelsamples were prepared from the supernatant by addition of

Laemmli sample buffer to a 0.5% SDS final concentration. Theimmunoblotting procedure was performed as described (11).

Skin Wounding and Regeneration. An �4-cm2 area on the mouseback was shaved to synchronize the hair follicle cycle. Two weekslater, the skin was shaved again and, under general anesthesia, a1-cm-long wound was generated through the epidermis and dermisusing scissors. The wound was closed using metal stitches andallowed to regenerate for 10 days. The shaven area of skin wassubsequently excised and immersion fixed in 3% PFA, 0.1 Mcacodylate buffer for 24 h and processed for immunostaining asdescribed above.

Oral Glycerol Administration to Fasted Mice. Mice were fasted for16 h (from 1600 until 0800 the following day), and 5 mg of 87%glycerol per g body weight was administered orally by sponta-neous feeding. The blood glucose level was measured at statedtime intervals before and after glycerol administration using anAccu-Chek Sensor (Roche) and a drop of saphenous vein blood.

Presentation of Data and Statistical Analyses. Quantitative data arepresented as means � SE. Statistical comparisons were ac-complished by unpaired t test (equal variances). P values �0.05 were considered statistically significant.

We thank Gitte Kall, Inger-Merete Paulsen, Mette Vistisen, and LineV. Nielsen for expert technical assistance and Dr. Janne Lebeck forassistance with immunoblotting. The Water and Salt Research Centreat the University of Aarhus is established and supported by the DanishNational Research Foundation (Danmarks Grundforskningsfond).Support for this study was provided by The WIRED program (NordicCouncil and the Nordic Centre of Excellence Program in MolecularMedicine), The Danish Medical Research Council, The Karen EliseJensen Foundation, The Human Frontier Science Program, TheCommission of the European Union (EU Aquaplugs and EU actionprograms), and the University of Aarhus.

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