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

<div><p>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 <i>AQP7</i> 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.</p></div

<i>AQP7</i> expression has no effect in equilibrium cell volume but depletion of <i>AQP7</i> correlates with higher non-osmotic volume and triglyceride content.

<p>A- Equilibrium cell volumes of 3T3-L1 adipocytes infected with scramble shRNA (Scramble control), <i>AQP7</i> knockdown adipocytes (AQP7-shRNA) and human <i>AQP7</i> overexpressing adipocytes (hAQP7). Bars show mean ±SD from 40–50 cells analyzed on 4 coverslips in 2 cell platings. B- Non-osmotic volumes β of 3T3-L1 adipocytes infected with scramble shRNA (Scramble control), <i>AQP7</i> knockdown adipocytes (<i>AQP7</i>-shRNA) and human <i>AQP7</i> overexpressing adipocytes (h<i>AQP7</i>). Bars show mean ±SD from 40–50 cells analyzed on 4 coverslips in 2 cell platings. C- Intracellular triglyceride (TG) content in 3T3-L1 adipocytes infected with scramble shRNA (Control) and in <i>AQP7</i> knockdown adipocytes (<i>AQP7</i>-shRNA). Bars show the mean ±SD of 6 separate measurements. Significance levels: ns, not significant, <i>P</i>>0.05; * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001, given either by one-way ANOVA followed by Tukey's <i>post-hoc</i> test or by Student's <i>t</i> test.</p

Aquaglyceroporin expression levels (arbitrary units) in 3T3-L1 cell lines.

<p>Semi-quantitative real-time PCR analysis <i>AQP3</i>, <i>AQP7</i> and <i>AQP9</i> expression in 3T3-L1 cells, 3T3-L1 cells transduced with scramble shRNA (Scramble control), transduced with a lentivirus expressing short-hairpin RNA that targets <i>AQP7</i> (<i>AQP7-shRNA</i>), or transduced with a lentivirus encoding GFP (GFP) or human <i>AQP7</i> plus GFP (h<i>AQP7</i>).</p><p>Each value represents the mean ±SD of the ratio between each transcript and <i>aRP</i> (n = 5). ^a<i>P</i><0.01 vs Fibroblasts; ^b<i>P</i><0.001 vs. Scramble Control; nd, not detected.</p

Functional assessment of <i>AQP7</i> water transport.

<p>Water permeability was assayed by epifluorescence microscopy in adipocytes infected with scramble shRNA (Scramble control), <i>AQP7</i> knockdown adipocytes (<i>AQP7</i>-shRNA) and human <i>AQP7</i> overexpressing adipocytes (h<i>AQP7</i>). <b>A</b> – Representative illustration of calcein loaded cells with initial equilibrium volume V_o (left panel) and final equilibrium volume V_∞ (right panel) after an osmotic challenge of tonicity two with mannitol. <b>B</b> – Relationship between changes in cell volume (V/V₀) and fluorescence intensity (F/F₀) (n = 5 cells). Individual calibration was performed for each fluorescence mannitol experiment. Inset shows a typical fluorescence (F) trace together with measured cell volumes (V/Vo) along the experiment. <b>C</b> - Representative time course of the relative cell volume change V/V_o for 3T3-L1 adipocytes expressing different levels of <i>AQP7</i> after an osmotic shock of tonicity two with mannitol. <b>D</b> - Osmotic water permeability coefficient (<i>P</i>_f). <b>E</b> – β dependence on <i>P</i>_f. The linear fit and the 95% confidence band are shown. Bars show mean ±SD from 40–50 cells analyzed on 4 coverslips in 2 cell platings. Significance levels: ns, not significant, <i>P</i>>0.05; * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001, given by one-way ANOVA followed by Tukey's post-hoc test.</p

Functional assessment of <i>AQP7</i> glycerol transport.

<p>Glycerol permeability was assayed by epifluorescence microscopy in 3T3-L1 adipocytes infected with scramble shRNA (Control), <i>AQP7</i> knockdown adipocytes (<i>AQP7</i>-shRNA) and in human <i>AQP7</i> overexpressing adipocytes (h<i>AQP7</i>). <b>A</b> - Representative time course of the relative cell volume change V/V_o for 3T3-L1 adipocytes expressing different levels of <i>AQP7</i> but presenting equivalent non-osmotic volumes (β) after an osmotic shock of tonicity two with glycerol. <b>B</b> - Representative time course of the relative cell volume change V/V_o. Cells equilibrated in an isotonic solution containing mannitol were switched to the same solution where mannitol was replaced by glycerol. All data points were from cells presenting equivalent non-osmotic volumes (β). The inset extends the time scale, showing the first 20 seconds of relative cell volume measurements. <b>C</b> - Glycerol permeability coefficient (<i>P</i>_gly). <b>D</b> - <i>P_gly</i> dependence on <i>P</i>_f. The linear fit and the 95% confidence band are shown. Bars show mean ± SD from 40–50 cells analyzed on 4 coverslips in 2 cell platings. Significance levels: ns, not significant, <i>P</i>>0.05; * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001, given by one-way ANOVA followed by Tukey's post-hoc test.</p

<i>AQP7</i> expression is higher in isolated mice adipocytes than in the stromal vascular fraction (SVF) of adipose tissue.

<p>A- <i>AQP3</i>, <i>AQP7</i> and <i>AQP9</i> mRNA expression in isolated mice adipocytes (black bars) and SVF (white bars). B–F - mRNA levels of specific markers of mature adipocytes (<i>aP2</i>, <i>GLUT4</i>, <i>HSL</i> and <i>Perilipin</i>) and of the capillary endothelia of adipose tissue (<i>SOX9</i>). <i>eEF2</i> (eukaryotic translation elongation factor 2) mRNA levels were used as reference genes. Data represent mean ± SD derived from five independent experiments. Statistically significant differences detected by Student's t are indicated by ** <i>P</i><0.01; *** <i>P</i><0.001.</p

Cholesterol depletion by simvistatin and cholesterol-binding antibiotics causes loss of cavin-2 in fat cells (A) and fibroblasts (B).

<p>Adipocytes or NIH-3T3 fibroblasts were treated with or without Simvastatin (10 µM, 18 hrs) methyl-β-cyclodextrin (20 mM, 90 minutes), nystatin (76 µM, 4 hours) or filipin (7.6 µM, 4 hours). Whole cell extracts were then prepared in RIPA buffer and analyzed by SDS-PAGE and Western blotting as in prior figures.</p

Cav1 null adipocytes lack caveolae but cavin-2 is membrane associated and redistributes to the cytosol upon cholesterol depletion.

<p>Cav1 null adipocytes were treated with or without MβCD as in previous figures and lysates (<b>A</b>) were prepared and analyzed by Western blot or (<b>B</b>) labeled with anti-cavin-2 and secondary antibody prior to analysis by confocal microscopy.</p

Cavin-2 is degraded by the proteosome and Cavin-1 redistributes to the cytosol following cholesterol depletion.

<p>3T3-L1 adipocytes were treated with or without MβCD (20 mM, 90 minutes) and subjected to either (<b>A</b>) immunostaining with the indicated antibodies (red) and for nuclei with DAPI (4′,6-diamidino-2-phenylindole, blue) and analyzed by confocal microscopy as described in Methods or (<b>B</b>), subjected to subcellular separation into membrane and cytosolic fractions. Following centrifugation, an equal proportion of each fraction (ca. 6X more cytosol protein than membrane) were analyzed by SDS-PAGE and Western blotting. Glyceraldehyde phosphate dehydrogenase (GAPDH) is a loading control for the cytosolic fraction. (<b>C</b>) Cultured at cells were treated with or without methylβ-cyclodextrin in combination with the proteasome inhibitor MG-132 (10 µM), the lysosomal inhibitor chloroquine (40 µM), or the inhibitors alone for 90 minutes and cell extracts were prepared in lysis buffer and analyzed by SDS-PAGE and Western blotting for the proteins indicated. Shown are representative experiments.</p

Cholesterol repletion restores cavin-2 levels, which allows return of cavin-1 to the plasma membrane.

<p>3T3-L1 adipocytes were treated without or without methyl-β-cyclodextrin (20 mM, 90 minutes), and the medium was changed to that containing either 10% FBS, 10% FBS depleted of lipoproteins (LPDS), or, 10% FBS lipoprotein depleted serum containing cholesterol loaded cyclodextrin (25 µM cholesterol). Following incubation for the times indicated (time 0 being after MβCD removal), cell lysates were analyzed by SDS-PAGE and Western blotting (<b>A</b>) or were processed for immunofluorescence with antibodies for cavin-1 and -2 (<b>B</b>) and for nuclei (DAPI) as in prior figures. The percent cavin-2 rim staining was determined by scoring 104+/−3 cells that were also positive for DAPI staining.</p