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Aquaglyceroporins form the subset of the aquaporin water channel family that is …


Biology Articles » Biochemistry » Protein Biochemistry » Aquaglyceroporin AQP9: Solute permeation and metabolic control of expression in liver » Figures

Figures
- Aquaglyceroporin AQP9: Solute permeation and metabolic control of expression in liver

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Figure 1

Solute permeability measurements of rat AQP9 or rat AQP4 in oocytes. X. laevis oocytes were injected with 5 ng of rat AQP9 cRNA, 5 ng of rat AQP4 (M23) cRNA, 2.5 ng of AQP4 plus 2.5 ng of AQP9 cRNA, or 50 nl of water and cultured for 3 days. (A) Osmotic water permeability (Pf) was assayed by transferring oocytes from 200 to 70 mosM Barth's solution, and swelling was monitored by videomicroscopy. Solute permeability was assayed by placing oocytes in isotonic solution containing 100 mM indicated solute. The concentration gradient caused solute influx resulting in water influx and oocyte swelling. Data are mean ± SD from six oocytes. (B) Uptake of 14C-labeled solute was measured over 90 s at room temperature. Values represent mean ± SD from 6–10 oocytes.

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Figure 2

Purification of rat AQP9 protein and measurement of solute transport in AQP9 proteoliposomes. (A) Coomassie-stained SDS/polyacrylamide gel (Left) and anti-AQP9 immunoblot (Right) of purified AQP9 protein. (B) Permeability of AQP9 proteoliposomes (reconstituted from pure E. coli lipids and purified AQP9 protein) or control liposomes (reconstituted with no protein) were evaluated for permeability for glycerol, urea, DL-β-hydroxybutyrate, or water. For solute transport measurement, proteoliposomes or liposomes containing the indicated solute were rapidly mixed with an equal volume of isotonic solution containing sucrose as a nonpermeant osmolyte. The concentration gradient caused solute efflux resulting in osmotically driven water efflux and vesicle shrinkage measured by light scattering. To measure inhibition of solute transport, proteoliposomes were incubated with 1 mM HgCl2 at least 15 min before measuring light scattering. For water transport measurement, AQP9 proteoliposomes, AQP4 proteoliposomes, and control liposomes were subjected to an abrupt 50% increase in extravesicular osmolality resulting in water efflux, and shrinking was measured by light scattering. The kinetics of 10 measurements were normalized and fitted to a characteristic first- or second-order exponential equation dependent on the time course of water efflux.

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Figure 3

Effect of fasting and refeeding on AQP9 levels in liver. (A Upper) Anti-AQP9 immunoblot of liver membranes from three rats after the indicated time of fasting. (A Lower) Scanning densities of anti-AQP9 immunoblot. (B) Average serum levels of glucose, urea, and β-hydroxybutyrate from the same rats. (C) After 96 h of fasting, rats were allowed access to food. (Upper) Anti-AQP9 immunoblot of liver membranes from three rats after indicated time of refeeding. (Lower) Scanning densities of anti-AQP9 immunoblot. Values represent mean ± SD from three rats.

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Figure 4

Effect of insulin on AQP9 levels in liver. (A) Insulin injection. (Top) Anti-AQP9 immunoblot of liver membranes from three rats killed 24 h after injection of buffer or 5 units/kg regular insulin, as indicated. (Middle) Scanning densities of anti-AQP9 immunoblot. (Bottom) Average blood levels of glucose at death. (B) Streptozotocin pretreatment followed by insulin injection. (Top) Anti-AQP9 immunoblot of liver membranes from three rats killed 9 days after injection of buffer (control), 100 mg/kg streptozotocin (STZ), or streptozotocin followed by 4 days of bidaily injections of 13 units/kg NPH insulin (STZ + ins). (Middle) Scanning densities of anti-AQP9 immunoblot. (Bottom) Average blood levels of glucose at death. Values represent mean ± SD from three rats.

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Figure 5

Confocal laser immunofluorescence microscopy of AQP9 in rat liver. (A) Distinct AQP9 immunolabeling is seen associated with hepatocytes in liver from control rat. (B) Increased AQP9 immunolabeling associated with hepatocytes close to central vein in liver from rat after 4 days of fasting or (C) 9 days after streptozotocin injection. CV, central vein. Arrowheads show sinusoidal surface. Bars indicate 40 μm.

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