Testosterone treatment is associated with reduced adipose tissue dysfunction and nonalcoholic fatty liver disease in obese hypogonadal men

Abstract

Purpose: In both preclinical and clinical settings, testosterone treatment (TTh) of hypogonadism has shown beneficial effects on insulin sensitivity and visceral and liver fat accumulation. This prospective, observational study was aimed at assessing the change in markers of fat and liver functioning in obese men scheduled for bariatric surgery.

Methods: Hypogonadal patients with consistent symptoms (n = 15) undergoing 27.63 ± 3.64 weeks of TTh were compared to untreated eugonadal (n = 17) or asymptomatic hypogonadal (n = 46) men. A cross-sectional analysis among the different groups was also performed, especially for data derived from liver and fat biopsies. Preadipocytes isolated from adipose tissue biopsies were used to evaluate insulin sensitivity, adipogenic potential and mitochondrial function. NAFLD was evaluated by triglyceride assay and by calculating NAFLD activity score in liver biopsies.

Results: In TTh-hypogonadal men, histopathological NAFLD activity and steatosis scores, as well as liver triglyceride content were lower than in untreated-hypogonadal men and comparable to eugonadal ones. TTh was also associated with a favorable hepatic expression of lipid handling-related genes. In visceral adipose tissue and preadipocytes, TTh was associated with an increased expression of lipid catabolism and mitochondrial bio-functionality markers. Preadipocytes from TTh men also exhibited a healthier morpho-functional phenotype of mitochondria and higher insulin-sensitivity compared to untreated-hypogonadal ones.

Conclusions: The present data suggest that TTh in severely obese, hypogonadal individuals induces metabolically healthier preadipocytes, improving insulin sensitivity, mitochondrial functioning and lipid handling. A potentially protective role for testosterone on the progression of NAFLD, improving hepatic steatosis and reducing intrahepatic triglyceride content, was also envisaged.

Clinical trial registration: ClinicalTrials.gov Identifier: NCT02248467, September 25th 2014.

Keywords: Adipose tissue; Hypogonadism; Liver; NAFLD; Obesity; Testosterone.

Fig. 1

Flow-chart for the observational study. T testosterone, TTh testosterone therapy, FU follow-up

Fig. 2

Variation of biochemical and clinical parameters relative to hypoandrogenism in hypogonadal (HYPO) vs. hypogonadal subjects treated with testosterone (HYPO + TTh), V1 (surgery) vs. baseline. Data were adjusted for the baseline value of the outcome variable and for age and baseline BMI. AMS Aging Males’ Symptoms scale, BMI body mass index, cFT calculated free testosterone, T testosterone, TTh testosterone therapy

Fig. 3

Nonalcoholic Fatty Liver Disease (NAFLD) Activity Score (NAS) (a) and Steatosis Score (b) derived from liver biopsies according to the three experimental groups. ^Op < 0.05 vs. Eugonadal; *p < 0.05 vs. Hypogonadal

Fig. 4

a Bar graph shows liver triglyceride levels obtained from liver biopsies in all three groups of patients. b, c Display the correlation of liver triglyceride levels with steatosis and NAS scores, respectively. d–f Show the H&E staining of liver sections from eugonadal (EUG), hypogonadal (HYPO) and hypogonadal subjects treated with testosterone (HYPO + TTh), respectively. ^Op < 0.05 vs. Eugonadal; *p < 0.05 vs. Hypogonadal; Scale bar 100 μm

Fig. 5

a Displays the liver tissue relative mRNA expression of smooth muscle/fibrosis markers and genes related to glucose transport, insulin signaling, glycogenesis and lipid handling and metabolism. Data are calculated per the 2^−ΔΔCt comparative method, using the 18S ribosomal RNA subunit as the reference gene for normalization. Results are expressed as fold-change vs. the eugonadal group and are reported in a box plot as interquartiles ± SEM. Statistical analysis was performed using Kruskal–Wallis and Mann–Whitney tests. b–f show the correlations between cFT and ACLY, FAS, HMGCR, HMGCS and GLUT4 liver mRNA expression, respectively. (Eugonadal, n = 15; Hypogonadal, n = 26; Hypogonadal + TTh, n = 15). ^Op < 0.05, ^OOp < 0.01 vs. Eugonadal; *p < 0.05, **p < 0.01, ***p < 0.001 vs. Hypogonadal

Fig. 6

Visceral adipose tissue relative mRNA expression of genes related to brown, beige and white adipogenesis, lipid catabolism, insulin signaling and mitochondrial life cycle. Data are calculated per the 2^−ΔΔCt comparative method, using the 18S ribosomal RNA subunit as the reference gene for normalization. Results are expressed as fold-change vs. the eugonadal group and are reported in a box plot as interquartiles ± SEM. Statistical analysis was performed using Kruskal–Wallis and Mann–Whitney tests. (Eugonadal, n = 15; Hypogonadal, n = 37; Hypogonadal + TTh, n = 15). ^Op < 0.05, ^OOp < 0.01 vs. Eugonadal; *p < 0.05, **p < 0.01, ***p < 0.001 vs. Hypogonadal

Fig. 7

Human preadipocytes (hPADs) relative mRNA expression of genes related to brown, beige and white adipogenesis, lipid catabolism and handling, insulin signaling and mitochondrial life cycle. Data are calculated per the 2^−ΔΔCt comparative method, using the 18S ribosomal RNA subunit as the reference gene for normalization. Results are expressed as fold-change vs. the eugonadal group and are reported in a box plot as interquartiles ± SEM. Experiments were performed in triplicate using four different hPADs preparations. Statistical analysis was performed using Kruskal–Wallis and Mann–Whitney tests. ^Op < 0.05, ^OOp < 0.01, ^OOOp < 0.001 vs. Eugonadal; *p < 0.05, **p < 0.01, ***p < 0.001 vs. Hypogonadal

Fig. 8

Panel A shows the insulin dose-dependent ³H-2-deoxy-d-glucose uptake in hPADs from all groups after exposure (30 min) to increasing concentrations of insulin. Results are expressed in percentage over baseline (no insulin) and are reported as mean ± SEM of four different experiments, each performed in duplicate and using a different cell preparation per group. b Shows the glucose uptake AUC correlation with total testosterone levels at pre-surgery V1 [AUC: incremental area under the curve of glucose blood level during oral glucose tolerance test (OGTT)]. ^Op < 0.05 vs. Eugonadal; *p < 0.05 vs. Hypogonadal

Fig. 9

a–c Show representative time-lapse images of the mitochondrial function in hPADs isolated from all groups of patients, visualized by incubation of the mitochondria-targeted fluorescent probe (MitoTracker staining) and imaged for 3 min. Computer-assisted measurement of mitochondria length is reported in d, and respective p values are reported within the panel. For morphometric analysis of mitochondrial length, well-resolved mitochondria in the cell periphery were analyzed by ImageJ software. At least 50 individual mitochondrial structures in at least 10 cells/group were measured to determine mitochondrial length (μm) distribution. Data were obtained from three independent experiments

Fig. 10

a Displays representative time-lapse images of hPADs isolated from EUG, HYPO and HYPO + TTh patients, stained with 10 μM dihydroethidium (DHE) and imaged for 3 min. b Bar graph shows the changes in integrated fluorescence intensity measured in the nuclei of hPADs during time-lapse imaging. c Shows a higher magnification of DHE-derived fluorescence in each group. Panel D shows the oxygen consumption in hPADs isolated from EUG, HYPO and HYPO + TTh patients after 10 days of spontaneous differentiation. It was measured by the Oxygraph system instrument. The bar graph shows the ratio of oxygen consumption normalized per mL of cell volume. Data are reported as the mean ± SEM. of at least three independent experiments. ^Op < 0.05, ^OOp < 0.01, ^OOOp < 0.001 vs. Eugonadal; **p < 0.01, ***p < 0.001 vs. Hypogonadal