Experimental hypogonadism: insulin resistance, biochemical changes and effect of testosterone substitution

Animals – study design

Male Wistar rats, 11–12 weeks old and 220–250 g in weight, were used. The animals were housed in the Laboratory for Experimental Surgery and Surgical Research, Athens University Medical School, in a controlled environment at 20 ± 2 °C, in cages compliant with the European standards (Tecniplast, West Chester, PA, USA), with 55% relative humidity, central ventilation (15 air changes/hour), and an artificial 12-h light–12-h dark cycle. Normal chow diet and water were provided ad libitum. The experimental protocol was approved by the competent Veterinary Directorate of Attica Region and was in compliance with the EU legislation (Directive 2010/63/EU) regarding the use of animals in biomedical science.

Twenty-nine rats were randomized into three groups: control (n = 10), in which animals underwent sham surgery; orchiectomy (ORX, n = 9), in which animals underwent orchiectomy; and orchiectomy and testosterone (ORX+T, n = 10), in which animals underwent orchiectomy and received an intramuscular injection of testosterone enanthate/Norma (Norma Hellas S.A., Athens, Greece) at a dose of 100 μg/100 g/b.w. 10 and 25 days, postoperatively.

Animals were euthanized at the 40th postoperative day. All animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” [4].

Surgical procedure

Rats were anesthetized with xylazine (10 mg/kg) and ketamine (70 mg/kg) through intramuscular injection. A 1-cm long midline incision was performed in the scrotum. Testicles were sequentially exposed and removed after the transection of spermatic cords. In sham controls, the testicles were visualized but not removed.

Blood collection – biochemical measurements

Blood samples were collected (at 9:00 AM, after a 12-h fast) at baseline (day of surgery, D1), 10 days (D10), 25 days (D25) and 40 days (D40) postoperatively using capillary tubes introduced into the medial retro-orbital venous plexus under light ether anesthesia. The serum was separated by centrifugation at 3000 rpm for 10 min.

Glucose (GLU), total cholesterol (CHOL), high-density lipoprotein cholesterol (HDL) and triglycerides (TG) were determined in serum, and low-density lipoprotein (LDL) was calculated by the Friedewald formula.

Hormonal measurements

Insulin, irisin and resistin levels were determined by enzyme-linked immunosorbent assay (ELISA) via commercially available kits from Elabscience Biotechnology, Co., Ltd. (Wuhan, China). All kits used the sandwich ELISA principle, and the optical density (OD) was measured spectrophotometrically at a wavelength of 450 ± 2 nm with the use of the Bio-Rad 680 microplate reader.

Homeostatic model assessment of β-cell function and insulin resistance (HOMA-IR) was calculated as follows: glucose (mg/dL) × insulin (mIU/L) / 405.

Statistical analysis

Continuous variables following normal distribution are expressed as mean ± standard deviation, and those not following normal distribution are expressed as median ± interquartile range.

Comparisons between more than two groups were performed with analysis of variance followed by Bonferroni correction. The Kruskal-Wallis test was used as a non-parametric test for multiple group comparisons, and the Mann-Whitney U test was used for post hoc multiple testing. Comparisons between multiple time points for non-normal distributions were performed using Friedman test with Wilcoxon signed rank test for post hoc comparisons.

All tests were two-sided. Differences were considered as statistically significant if the null hypothesis could be rejected with >95% confidence interval (p < 0.05).

All statistical analyses were conducted in SPSS (version 23 for Mac OS; SPSS, Inc., Chicago, IL, USA).

Lipidemic and glycemic profile alterations

At baseline (day 1), there were no difference in CHOL, HDL, LDL, TG, GLUC and body weight among groups. Orchiectomy led to an increase in serum levels of CHOL (117.50 ± 17.50 vs. 97.50 ± 8.75 mg/dL for ORX and control groups, respectively) and LDL (42.50 ± 14.25 vs. 29.00 ± 13.25 mg/dL for ORX and control groups, respectively) at the end of the experiment (p = 0.006 and p = 0.018 for CHOL and LDL, respectively). Hormonal replacement was associated with amelioration of both parameters (CHOL 92.50 ± 30.00 mg/dL and LDL 27.50 ± 12.75 mg/dL for the ORX+T group, p = 0.001 as compared with the ORX group, in both cases). No significant differences were observed for any of the other biochemical indices or body weight among groups at any time point.

Insulin resistance and longitudinal hormonal changes

Although insulin levels and HOMA-IR did not differ between the ORX and the ORX+T groups, they exhibited changes throughout the duration of the study in the ORX+T group (Figure 1). Namely, orchiectomy in this group induced an increase in insulin 10 days postoperatively (4.10 ± 2.47 vs. 1.44 ± 2.94 ng/mL, p = 0.028, for D10 and D1, respectively), which was reversed with the administration of testosterone at the end of the experiment (1.78 ± 0.68 ng/mL for D40, p = 0.022 as compared with D10). A similar trend was also noted in the case of HOMA-IR (1.36 ± 2.75 vs. 3.68 ± 1.87 vs. 1.74 ± 0.69 ng/mL, for D1, D10 and D40, respectively, p = 0.024 and p = 0.026, respectively). Resistin levels were lower in the ORX group compared with the ORX+T group at baseline (0.28 ± 0.26 vs. 1.95 ± 0.62 ng/mL, p = 0.030 for ORX and ORX+T groups, respectively); however, significant changes were not observed at any other time point. As far as irisin is concerned, significant differences were also shown in the ORX+T group through time. More specifically, irisin levels peaked at the 10th postoperative day and were decreased at the end of the experiment (0.27 ± 0.11 vs. 0.85 ± 0.54 vs. 0.02 ± 0.07 ng/mL for D1, D10 and D40, respectively, p = 0.028 in both cases).

Figure 1:

Box plots indicating fluctuations of HOMA-IR, insulin, resistin and irisin of the ORX+T group throughout the experiment. The p values illustrate the levels of significance between D1–D10 and D10–D40. HOMA-IR, homeostatic model assessment of β-cell function and insulin resistance.