Image_1_Hypogonadism and sexual function in men affected by adrenocortical carcinoma under mitotane therapy.tif

PurposeAdrenocortical carcinoma (ACC) is a rare and aggressive tumor. ACC male patients under adjuvant mitotane therapy (AMT) frequently develop hypogonadism, however sexual function has never been assessed in this setting. The aim of this retrospective study was to evaluate in AMT treated ACC patients the changes in Luteinizing hormone (LH), Sex Hormone Binding Globulin (SHBG), total testosterone (TT) and calculated free testosterone (cFT), the prevalence and type of hypogonadism and sexual function, the latter before and after androgen replacement therapy (ART).MethodsLH, SHBG, TT and cFT were assessed in ten ACC patients at baseline (T0) and six (T1), twelve (T2), and eighteen (T3) months after AMT. At T3, ART was initiated in eight hypogonadal patients, and LH, SHBG, TT and cFT levels were evaluated after six months (T4). In six patients, sexual function was evaluated before (T3) and after (T4) ART using the International Index of Erectile Function-15 (IIEF-15) questionnaire.ResultsUnder AMT we observed higher SHBG and LH and lower cFT levels at T1-T3 compared to T0 (all pConclusionAMT was associated with hypergonatropic hypogonadism and ED, while ART led to a significant improvement of cFT levels and sexual function in the hypogonadal ACC patients. Therefore, we suggest to evaluate LH, SHBG, TT and cFT and sexual function during AMT, and start ART in the hypogonadal ACC patients with sexual dysfunction.</p

Table_1_Hypogonadism and sexual function in men affected by adrenocortical carcinoma under mitotane therapy.docx

PurposeAdrenocortical carcinoma (ACC) is a rare and aggressive tumor. ACC male patients under adjuvant mitotane therapy (AMT) frequently develop hypogonadism, however sexual function has never been assessed in this setting. The aim of this retrospective study was to evaluate in AMT treated ACC patients the changes in Luteinizing hormone (LH), Sex Hormone Binding Globulin (SHBG), total testosterone (TT) and calculated free testosterone (cFT), the prevalence and type of hypogonadism and sexual function, the latter before and after androgen replacement therapy (ART).MethodsLH, SHBG, TT and cFT were assessed in ten ACC patients at baseline (T0) and six (T1), twelve (T2), and eighteen (T3) months after AMT. At T3, ART was initiated in eight hypogonadal patients, and LH, SHBG, TT and cFT levels were evaluated after six months (T4). In six patients, sexual function was evaluated before (T3) and after (T4) ART using the International Index of Erectile Function-15 (IIEF-15) questionnaire.ResultsUnder AMT we observed higher SHBG and LH and lower cFT levels at T1-T3 compared to T0 (all pConclusionAMT was associated with hypergonatropic hypogonadism and ED, while ART led to a significant improvement of cFT levels and sexual function in the hypogonadal ACC patients. Therefore, we suggest to evaluate LH, SHBG, TT and cFT and sexual function during AMT, and start ART in the hypogonadal ACC patients with sexual dysfunction.</p

Electrophysiological properties of ASC and of the derived <i>in vitro</i>-differentiated adipocytes.

<p>Electrophysiological analysis was performed on ASC and their corresponding <i>in vitro</i>-differentiated adipocytes in patch clamp condition. RMP: resting membrane potential. C_m: membrane capacitance. G_m and G_m/C_m: total and specific membrane conductance, respectively. V_th: voltage threshold of activation of the three types of K⁺ currents investigated (I_BK, I_Ks and I_Kir). K⁺ current activation time constant (τ) for I_BK and I_Ks and current density I_BK/C_m and I_Ks/C_m values have been obtained at 50 mV and I_Kir/C_m at −110 mV. Mean ±SE values are reported. Student's <i>t</i> test was applied to compare paired samples: **,***P<0.01 and 0.001 V- versus S-ASC and V- versus S-ADIPO; §, §§, §§§ P<0.05, 0.01, 0.001 S- or V-ADIPO versus the corresponding ASC. Total number of analyzed cells (n) in populations obtained from 5 different subjects were: S-ASC (n = 84), V-ASC (n = 82), S-ADIPO (n = 54), V-ADIPO (n = 52); for I_Kir: S-ASC (n = 17), V-ASC (n = 18), S-ADIPO (n = 28), V-ADIPO (n = 24). mV: milli Volt; pF: pico Faraday; nS: nano Siemens; ms: milli seconds; pA: pico Ampere.</p

ASC and <i>in vitro</i>-differentiated derived adipocytes express two types of delayed rectifier K⁺ currents.

<p>Light microscopy of a S-ASC (A), V-ASC (B), S-ADIPO (C) and V-ADIPO (D) plated on glass coverslip and impaled by the patch pipette for electrophysiological records (magnification 40X). (E–F) Typical I_K,DR trace currents elicited by a voltage step to 50 mV from a holding potential of −60 mV in Control solution with 4-AP (2 mM), Nifedipine (10 µM) and Ba²⁺(0.1 mM) in S-ASC (Ea), S-ADIPO (Eb), V-ASC (Fa) and V-ADIPO (Fb). In each cell type, I_Ks traces are obtained in the presence of Ibtx (100 nM) and I_BK traces by subtracting I_Ks from I_K,DR. By adding Chr (50 µM) only a very small residual current was recorded (Ibtx+Chr current traces). (G–J) Family of I_BK (G and I) and I_Ks (H and J) family of current traces evaluated as in panels E and F (same cells) from pharmacological dissection with voltage steps of 10 mV increments (from −80 to 50 mV; HP of −60 mV. Note the different ordinate scale in all panels.</p

Different proliferation rate in S-ASC and V-ASC.

<p>Growth curves were obtained for each population by haemocytometer cell counting (A), evaluation of BrdU incorporation (B), and MTS assay (C) at each time point. Results are expressed as mean ± SE fold increase of cell counts (A), BrdU (B) and MTS absorbance (C) at each time point over day 1 in at least 4 ASC populations derived from at least 4 independent subjects. *P<0.001 versus respective day 1; §§P<0.01, §§§P<0.001 S- versus V-ASC at the corresponding time point. (D) Ki-67 proliferation index is calculated as mean ± SE percentage of positive cells counted in at least 20 fields for each slide. 4 ASC populations were obtained from 4 different subjects, *P<0.001 versus the corresponding V-ASC population. (E) Population doubling (PD) curves were obtained by cell counting at different passages of S-ASC and V-ASC cultures expanded for about 2 months. Results are expressed as mean ± SE PD numbers obtained by counting cells in triplicates from 3 independent subjects; *P<0.001, versus respective passage 0; §P<0.05, §§§P<0.001 versus the corresponding V-ASC. (F) Immunophenotype of V-ASC obtained at 2 different passages (P3 and 12). Data correspond to mean ± SE FACScan percentage of positive cells for the indicated surface markers previously demonstrated to characterize ASC populations (7); cells were obtained from n = 2 independent subjects. Similar data were obtained for the corresponding S-ASC at the same passage. No statistically significant differences have been observed in the expression of the indicated markers between the two passages. (G) Representative karyotype analysis of V-ASC at passage 12, corresponding to about 3 months of culture. Similar data has been obtained for the corresponding S-ASC at the same passage, in 2 different subjects. Karyotype analysis was performed on ASC obtained from 2 different subjects.</p

Staining of intracellular triglyceride depots in adipocytes obtained from <i>in vitro</i> differentiation of S- and V-ASC.

<p>Dark (A,I; 5× magnification) and bright field (E,M, G,O,H,P; 40× magnification) microscopy, ORO staining of neutral lipids (B,J; 5× magnification; F,N; 40× magnification) and fluorescence microscopy (40× magnification) of <i>in vitro</i>-differentiated S- (C) and V- (K) adipocytes and of the corresponding S- (D) and V- (L) ASC stained with AdipoRed evidentiate a qualitative higher number of lipid droplets and a higher number of differentiated adipocytes in the S- compared to V- populations. No staining was present in ASC, confirming the high specificity of AdipoRed binding to triglycerides. Representative of cell populations obtained at least from n = 4 different subjects.</p

Stemness marker expression in S-ASC and V-ASC.

<p>Quantitative Real Time RT-PCR was performed on mRNA extracted from both S- and V-ASC and from the corresponding <i>in vitro</i>-differentiated adipocytes, to evaluate BMI-1, (A, n = 10 subjects) NANOG (B, n = 5 subjects) and OCT-4 (C, n = 5 subjects) expression. Data are expressed as the mean±SE gene expression versus the housekeeping GAPDH gene. °P<0.001 S- versus V-ASC and *P<0.05 adipocytes versus respective ASC.</p

Differences in adipogenic potential and functional capabilities in ASC and in their <i>in vitro</i>-derived adipocytes.

<p>Expression of the adipogenic genes PPARgamma (A), FABP4 (B) and adiponectin (C) evaluated by quantitative real time RT-PCR is higher in S- compared to derived V-ADIPO. Data are expressed by box charts of gene expression ratio versus GAPDH (C) or fold increase between adipocytes and ASC (A, B). Boxes indicate the 25^th (lower) and 75^th (upper) percentiles. Horizontal lines and dots in the boxes indicate the 50^th percentile value (median) and mean value, respectively. Vertical lines give the 10^th and 90^th percentile limits of the data. Statistical analysis for non-parametric distribution was performed with Wilcoxon text: *P<0.05, ***P<0.001 S- versus V-ADIPO, n = 30 experiments with cells obtained from 14 independent subjects. D: Western Blot analysis of Adiponectin (upper panel) and FABP4 (lower panel) protein expression in ASC compared to the derived adipocytes. Molecular weight (MW) in kDa has been indicated for both standards and proteins of interest. Equal protein loading was verified by probing for the housekeeping protein actin. SAT and VAT samples from the same subject were run as positive controls. Representative of 5 independent experiments performed on cells obtained from 5 subjects. E: Immunofluorescence analysis of FABP4 (left panel) in <i>in vitro</i>-differentiated S-ADIPO (upper panel) and V-ADIPO (lower panel) revealed positivity for the enzyme around the intracellular lipid droplets. Right panels: corresponding bright field microscopy. F: Adipogenic potential of S- and V-ASC has been evaluated as AdipoRed staining of intracellular lipid droplets in the derived adipocytes. Results are expressed as mean ± SE fold increase of AdipoRed absorbance (left axis) in adipocytes versus the corresponding ASC. Lipolytic activity (right axis) of the same adipocytes evaluated as AdipoRed absorbance fold decrease following 12 h treatment with 1 µM isoproterenol. *P<0.001 S- versus V-ADIPO. G: Adiponectin secretion evaluated during <i>in vitro</i>-induced adipogenesis in S- and V-ASC at two different time points (T1 and T2, 14 and 21 days of differentiation respectively) by ELISA adiponectin kit. °P<0.001 T2 versus T1; *P<0.001 S- versus V-ADIPO.</p

Voltage dependence of the two kind of I_K,DR (I_BK and I_Ks) and of I_Kir.

<p>(A) Plots represent the maximum mean value of I_K,DR (a), I_BK and I_Ks (b,c) as a function of the applied voltage step recorded in the presence of Ba²⁺ from all experiments as in Fig. 6 G–J. Note the different ordinate scale for ASC and ADIPO. Panels put in evidence that I_K,DR, I_BK and I_Ks are reduced in size in the ADIPO versus the corresponding ASC. (B) K⁺ currents in representative cells elicited by voltage ramp stimulation in Control external solution without Ba²⁺ recording the total K⁺ currents (I_K,tot) (a, – Ba²⁺) and after adding 0.1 mM Ba²⁺ to block I_Kir and record I_K,DR (b, +Ba²⁺ 0.1 mM). I_Kir have been obtained by detracting current traces recorded in the presence of Ba²⁺ from those in the absence (c). I_K/C_m and V_th mean ± SE values and number of experiments are indicated in Tab 2.</p