Periprostatic adipose tissue (PPAT) supernatant from obese mice releases anticontractile substances and increases human prostate epithelial cell proliferation: the role of nitric oxide and adenosine

Figures

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  FIGURE 1. The schematic representation of the bioassay protocols. Periprostatic adipose tissue (PPAT) from obese mice was weighed (∼40 mg), incubated in Krebs-Henseleit solution (30 min, 37°C) with carboxygen, and the supernatant was then collected. Prostate segments from lean or obese mice were incubated (30 min, 37°C without and with PPAT supernatant, in the absence (A) or presence (B) of NOS inhibitors (L-NAME, a non-selective inhibitor; 1400W, an inhibitor of inducible NOS; ODQ, an inhibitor of soluble guanylate cyclase, and ZM241385, adenosine A2A receptor antagonist).

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  FIGURE 2. General characteristics. (A) Total body weight, (B) prostate weight, and (C) periprostatic adipose tissue (PPAT) weight. The prostate and PPAT weights were normalized relative to the tibial length of the mice. The columns are the mean ± SD of six mice/group. *p < 0.05, compared to lean mice. (D) Glucose tolerance test (GTT), with glucose concentrations expressed in mg/dL. Blood samples were obtained from lean and obese mice at 0, 15, 30, 60, 90, and 120 min. (E) The area under the glucose curve (A.U.C.) derived from the GTT test for lean and obese mice. (F) Insulin tolerance test (ITT), with glucose concentrations expressed in mg/dL. Blood samples were obtained from lean and obese mice at 0, 5, 10, 15, 20, 25, and 30 min. (G) Glucose decay constant (κITT; %/min). In panels (E–G), the points and columns represent the mean ± SD (n = 5 mice/group). *p < 0.05 compared to lean mice in all cases (Student’s unpaired t-test).

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  FIGURE 3. Hematoxylin-eosin-stained sections of prostate and periprostatic adipose tissue (PPAT) at different magnifications. 10x (A,D), 20x (B,E), and 40x (C,F,G, and H). Scale bars in micrometers. (I) Adipocyte area. The area of 10 adipocytes of PPAT per slide was calculated and the results were expressed as the mean ± SD (n = 5). *p < 0.0007 compared to adipocytes from lean mice. (J) The representative photomicrographs of prostate tissue stained for the cell proliferation marker Ki-67. (K) Quantitative analysis of proliferating prostatic cells in lean and obese mice. The columns represent the mean ± SD (n = 5). *p < 0.05 compared to lean mice (Student’s unpaired t-test).

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  FIGURE 4. Total nitrite and nitrate (NOx-) and adenosine quantification. NOx− were quantified in PPAT supernatant from obese mice or in the vehicle (KHS) for 30 min (A), without prostate) and 60 min (B), with prostate) at 37°C and continuously bubbled with carboxygen. (C) Adenosine was quantified in PPAT supernatant from obese mice (without prostate, 30 min) or in the vehicle (KHS) at 37°C and continuously bubbled with carboxygen. *p < 0.05 for–obese PPAT vs. + obese PPAT. The columns represent the mean ± SD (n = 5–6) (Student’s unpaired t-test).

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  FIGURE 5. Functional reactivity assays. (A) Concentration-response curves to phenylephrine (Phe, n = 7) or (B) Electrical field stimulation of the prostatic tissue from lean (n = 6) and obese (n = 10) mice in the absence (KHS alone) and the presence of supernatant from periprostatic adipose tissue (+PPAT) of obese mice. PPAT from obese mice was incubated with KHS for 30 min at 37°C with carboxygen, after which the supernatant was collected and tested for biological activity. The points and columns represent the mean ± SD of the indicated number of mice. *p < 0.05 for control vs. obese and # p < 0.05 for obese vs. obese + PPAT (Student’s unpaired t-test, or one-way ANOVA followed by the Bonferroni multiple comparisons test.) The experiment with PPAT from lean animals was not tested for ethical and cost reasons involved.

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  FIGURE 6. Functional reactivity assays in the presence of inhibitors/antagonists. Concentration-response curves to phenylephrine (Phe) in prostatic tissue from obese mice incubated in the absence or presence of periprostatic adipose tissue (PPAT) alone (+PPAT), and with (A) L-NAME, a non-selective NOS inhibitor (n = 6), (B) 1400W, an inhibitor of inducible NOS (n = 5), (C) ODQ, a soluble guanylate cyclase inhibitor (n = 6), and (D) ZM241385, an adenosine A2A receptor antagonist (n = 5). The points represent the mean ± SD of the indicated number of mice. *p < 0.05 for–PPAT vs. + PPAT and # p < 0.05 for + PPAT vs. + PPAT with inhibitors (one-way ANOVA followed by the Bonferroni multiple comparisons test).

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  FIGURE 7. Cell-based assays with normal epithelial cells. Normal human epithelial cells (PNT1-A) viability (% of the vehicle) in the presence of lean periprostatic adipose tissue (+ Lean PPAT) and obese periprostatic adipose tissue (+Obese). (A) Cell proliferation in PNT1-A in the absence (vehicle) and presence of supernatant from periprostatic adipose tissue (PPAT) of obese mice. The cells were incubated with and without PPAT for 24 h, 48 h, and 72 h, after which cell proliferation was assessed using CyQUANT® assay kits. (B) The columns represent the mean ± SD (Student´s unpaired t-test or one-way ANOVA followed by the Bonferroni multiple comparisons test) of the three separate occasions. Each time, the PPAT was obtained from two animals. Therefore, in panels (A) and (B), a total of N = 6 animals were included in this assay. The effect of PPAT supernatant on the level of cleaved caspase-3 protein in PNT1-A cells. The level of cleaved caspase-3 was normalized relative to caspase-3 protein expression levels, and the values are expressed in arbitrary units (a.u.). (C) Representative images of the WB analysis showing + obese PPAT and Vehicle. (D) The columns represent the mean ± SD, N = 6…

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  FIGURE 8. Cell-based assays with hyperplastic epithelial cells. Cell proliferation in a human benign prostatic hyperplasia cell line (BPH-1) in the absence (vehicle KHS) and presence of supernatant from periprostatic adipose tissue (PPAT) of obese mice (+PPAT) incubated alone or with (A) L-NAME, a non-selective NOS inhibitor, (B) 1400W, an inhibitor of inducible NOS, (C) ODQ, a soluble guanylate cyclase inhibitor, (D) SQ 22,536, an inhibitor of adenylate cyclase, (E) ZM241385, an adenosine A2A receptor antagonist, and (F) MRS 1754, an adenosine A2B receptor antagonist. (G) The proliferation of BPH-1 cells in the presence of inhibitors or antagonists alone. None of the compounds had any effect on cell proliferation at the concentrations tested. In all cases, cell proliferation was assessed using CyQUANT® assay kits. The columns represent the mean ± SD of three to four occasions (each done in triplicate). Each time, the PPAT was obtained from two animals. Therefore, a total of N = six to eight animals were included in this assay. *p < 0.05 for the vehicle vs. + PPAT and # p < 0.05 for + PPAT vs. + PPAT plus inhibitors (two-way ANOVA followed by the Bonferroni multiple comparisons test).