Plasmid Construction. Standard methods were used (10). pXβG-ev1-AQP9 was constructed with AQP9 cDNA prepared from rat liver total RNA and inserted into the BglII site of pXβG-ev1 (11). pYES2-AQP9 was made from pXβG-ev1-AQP9 with rat AQP9 amplified with primers 5′EcoRA9 (5′-AGAAACGAATTCATGCCTTCTGAGAAGGAC-3′) and 3′XbaRA9 (5′-CTGGCCTCTAGACTACATGATGACACTGAG-3′). The product contained an EcoRI site at the 5′ end and an XbaI site at the 3′ end. pYES2 vector (Invitrogen) encoding MGHHHHHHHHHHSSGHIEGRHEF between the HindIII and EcoRI sites was digested with EcoRI and XbaI for ligation with AQP9.
Expression and Purification of AQP9. Rat AQP9 was purified from protease-deficient (pep4Δ) Saccharomyces cerevisiae expressing pYES2-AQP9 (12, 13). Membranes were solubilized in 200 ml of buffer A [100 mM K2HPO4/10% glycerol/200 mM NaCl/5 mM 2-mercaptoethanol/3% octyl glucoside (N-octyl-β-D-glucopyranoside, Calbiochem) and protease inhibitors (EDTA-free complete protease inhibitor mixture tablets, Roche Biochemicals)] and loaded on an Ni-NTA agarose column (nickel-charged nitrilotriacetic acid, Qiagen, Valencia, CA), washed, and then eluted with buffer A containing 100 mM and 600 mM imidazole.
AQP9 Proteoliposome Transport Studies. Purified AQP9 protein was reconstituted with Escherichia coli polar lipid extract (Avanti, Alabaster, AL) into proteoliposomes by dialysis at a lipid to protein ratio of ≈100:1 overnight at 4°C vs. 100 volumes of reconstitution buffer [RB; 50 mM Mops/150 mM N-methyl-D-glucamine (Calbiochem)/1 mM NaN3 (Sigma) and protease inhibitors (EDTA-free), pH 7.5] (12, 13).
AQP9 proteoliposomes and protein-free control liposomes were analyzed by light scattering using a stopped-flow apparatus (SF-2001, Kin Tek Instruments, University Park, PA) with a dead time of ≤1 ms. Osmotic water permeability of proteoliposomes was measured at 4°C as described (13). Glycerol, urea, and β-hydroxybutyrate permeability were measured with proteoliposomes preequilibrated with solutes >15 min on ice, and permeability was measured at 24°C as described (13). Some proteoliposomes were reconstituted by dialysis in the presence of DL-β-hydroxybutyrate (total osmolality 855 mosM). Second-order rate constants were calculated; the first constant represents AQP9 transport and the second represents background permeability.
Oocyte Transport Studies. Capped cRNAs were synthesized in vitro from linearized pXβG-ev1 plasmids (11). Defolliculated X. laevis oocytes were injected with 5 ng of cRNA or 50 nl of diethyl pyrocarbonate-treated water. Injected oocytes were incubated for 3 days at 18°C in 200 mosM modified Barth's solution. Osmotic water permeability (11) and 14C-labeled solute permeability methods were used (1). Nonisotopic solute permeabilities (Ps) were measured by placing oocytes in 200 mosM modified Barth's solution containing 100 mosM of solute, causing water influx and oocyte swelling. Ps was calculated from the oocyte surface area (S = 0.045 cm2), initial oocyte volume (Vo = 9 × 10−4 cm3), the initial slope of the relative volume increase d(V/Vo)/dt, the total osmolality of the system (osmtotal = 200 mosM), and the osmotic solute gradient (solout − solin) as follows: Ps = [osmtotal × Vo × d(V/Vo)/dt]/[S × (solout − solin)].
Animal Studies. Experimental protocols were approved by the Institutional Animal Care and Use Committee and conform to National Institutes of Health guidelines. Male 225- to 250-g Sprague–Dawley rats (Taconic) in individual cages with free access to water received standard rodent chow, 14% protein, and 6% fat (5P07, Purina); ketogenic diet, 91% fat and 9% protein (96355, Harlan Teklad) (14); or 60% protein diet (5787, Purina). Some rats were injected s.c. with human insulin (Sigma) at 5 units/kg. Streptozotocin (30 mg/ml, Sigma) in 50 mM NaCitrate (pH 4.5) was injected i.p. at 100 mg/kg into other rats; some were injected s.c. with 13 units/kg NPH insulin (Humulin N, Eli Lilly) at 0900 and 1800 hours. Rats were killed at 1300 hours. Glucose, urea nitrogen, and β-hydroxybutyrate concentrations were measured in serum (Sigma kits). Blood glucose was measured with a OneTouch Ultra Blood glucose monitoring system (Lifescan, Mountain View, CA).
Tissue Processing, Immunoblotting, and Microscopy. Tissues were immediately placed on ice and homogenized, and membranes were collected by centrifugation (15); 25 μg of membrane protein was analyzed by SDS/PAGE (16); immunoblots were reacted with 3.5 μg/ml rabbit anti-rat AQP9 (Chemicon). Sample loadings were confirmed with Ponceau S (Sigma), and band intensities were scanned with a MacBAS bioimaging analyzer system (Fuji).
Control rats, rats fasted 4 days, or rats injected with streptozotocin 9 days earlier were perfusion-fixed with 3% paraformaldehyde, in 0.1 M cacodylate buffer, via left ventricle and prepared for paraffin embedding and immunohistochemistry (17). Confocal laser microscopy (Leica DMRS) was performed by using rabbit anti-rat AQP9 (Alpha Diagnostics) and goat anti-rabbit secondary antibodies (Alexa 488, Molecular Probes), and the images were merged with differential interference contrast images.