Abstract
Low testosterone level is associated with increased risks of cardiovascular diseases. As obese-insulin-resistant condition could impair cardiac function and that the incidence of obesity is increased in aging men, a condition of testosterone deprivation could aggravate the cardiac dysfunction in obese-insulin-resistant subjects. However, the mechanism underlying this adverse effect is unclear. This study investigated the effects of obesity on metabolic parameters, heart rate variability (HRV), left ventricular (LV) function, and cardiac mitochondrial function in testosterone-deprived rats. Orchiectomized or sham-operated male Wistar rats (n=36per group) were randomly divided into groups and were given either a normal diet (ND, 19.77% of energy fat) or a high-fat diet (HFD, 57.60% of energy fat) for 12weeks. Metabolic parameters, HRV, LV function, and cardiac mitochondrial function were determined at 4, 8, and 12weeks after starting each feeding program. We found that insulin resistance was observed after 8weeks of the consumption of a HFD in both sham (HFS) and orchiectomized (HFO) rats. Neither the ND sham (NDS) group nor ND orchiectomized (NDO) rats developed insulin resistance. The development of depressed HRV, LV contractile dysfunction, and increased cardiac mitochondrial reactive oxygen species production was observed earlier in orchiectomized (NDO and HFO) rats at week 4, whereas HFS rats exhibited these impairments later at week 8. These findings suggest that testosterone deprivation accelerates the impairment of cardiac autonomic regulation and LV function via increased oxidative stress and impaired cardiac mitochondrial function in obese-orchiectomized male rats.
Introduction
Testosterone deprivation is a condition in which the oncentration of testosterone is lower than the normal physiological level (Handelsman & Zajac 2004). This state can be found in aging male individuals. The prevalence of testosterone deficiency increases substantially with age (Araujo et al. 2007) and is associated with the development of several disorders including obesity, dyslipidemia, insulin resistance, metabolic syndrome, and cardiovascular disease (Grossmann et al. 2008, Oskui et al. 2013). Moreover, testosterone deprivation alone significantly induces left ventricular (LV) contractile dysfunction and cardiac sympathovagal imbalance in orchiectomized rats (Pongkan et al. 2015).
Obesity is one of the most important risk factors contributing to the overall burden of disease worldwide, and is known as the initial main sign of metabolic syndrome, which can manifest itself as many diseases, such as cardiovascular disease, type 2diabetes mellitus, and several types of cancer (McMillen et al. 2009, WHO 2011). In men, there has been a significant increase in clinical obesity, rising from 31.8 to 37.1% in the past decade (Flegal et al. 2010). In animal studies, it has been shown that rats fed on a HFD for at least 8weeks developed obese-insulin resistance (Pratchayasakul et al. 2011), leading to cardiac sympathovagal imbalance, LV contractile dysfunction, increased oxidative stress, and cardiac mitochondrial dysfunction (Apaijai et al. 2014). Although either testosterone deprivation or obese-insulin resistance affected cardiac function, the temporal effects of combined obese-insulin resistance and the loss of testosterone on metabolic parameters, cardiac function, heart rate variability (HRV), and cardiac mitochondrial function have never been investigated. We tested the hypothesis that testosterone deprivation in rats, together with obese-insulin resistance, accelerates and aggravates the impairment of metabolic parameters, cardiac function, HRV, and cardiac mitochondrial function.
Materials and methods
Animal preparation
All experiments were approved by the Institutional Animal Care and Use Committee of the Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand. Male Wistar rats weighing ~200g (aged 5–6weeks) were obtained from the National Laboratory Animal Center, Mahidol University, Bangkok, Thailand. All animals were housed in a climate-controlled room (22–25°C, with a ratio of 12h light:12h darkness cycle) and fed with standard laboratory chow (normal diet (ND)) or a high-fat diet (HFD) and water. Both food and water were given ad libitum.
Orchiectomy procedure
Rats were anesthetized and maintained using isoflurane 2–3% and the scrotal area was shaved and scrubbed using a sterile technique. The orchiectomy was performed using the scrotal approach technique (Foley 2005). The blood vessels and vas deferens were ligated with an absorbable suture. Then, both testes and epididymal fat pads were removed. The skin incision was closed using a nonabsorbable suture. Rats were monitored carefully to prevent them from chewing of sutures and also any other complications. Analgesic drugs and antibiotics were injected subcutaneously for 3days after the operation.
ND and HFD preparation
Standard laboratory chow (Mouse Feed Food No. 082, C.P. Company, Bangkok, Thailand) was used as a ND (19.77% of energy fat) and fed to control rats. In addition, the HFD (57.60% of energy fat) was mixed with the following ingredients: standard rat diet, casein, lard, cholesterol, vitamins, dl-methionine, yeast powder, and sodium chloride (Pratchayasakul et al. 2011). All ingredients were mixed and molded into a spherical shape. Rats were fed with these diets for 12weeks.
Experimental protocol
Rats were randomly divided into two groups: a sham-operated group and an orchiectomized (ORX) group. One week after surgery, rats in each group were divided into two subgroups and were given either a ND or a HFD for 4, 8, and 12weeks (n=6per group for each duration in each dietary group). Animals in the ND group were fed on standard laboratory chow (Mouse Feed Food No. 082, C.P. Company, Bangkok, Thailand), which had an energy content of 4.02kcal/g and 19.77% of total energy (%E from fat). Animals in the HFD group were fed a HFD, which had an energy content of 5.35kcal/g and 59.60% E from fat. Body weight and food intake were recorded daily, and blood samples were collected at baseline and then at 4th, 8th, and 12th week after fasting for 5–6h. Plasma of blood samples were kept at −80°C and used for the determination of metabolic parameters. At the end of the study, the heart was removed and used to study cardiac mitochondrial function.
HRV determination
HRV is a noninvasive assessment of cardiac autonomic function, which is used to determine cardiac sympathovagal balance (Chattipakorn et al. 2007). HRV was analyzed before the operation and then at 4th, 8th, and 12th week after being fed a ND or HFD. An ECG was conducted on each rat using PowerLab (PowerLab 4/25T, AD Instruments, Bella Vista, NSW, Australia) with chart 5.0 continuously for 20min, while the animals were still conscious. Data from the ECG recording was analyzed using MATLAB program (Glos et al. 2007). A high frequency (HF, 0.6–0.3Hz) was taken as parasympathetic activity, while a low frequency (LF, 0.2–0.6Hz) was taken as a combination of sympathetic and parasympathetic activities (Chattipakorn et al. 2007). The LF/HF ratio was used as an index of cardiac sympathovagal balance (Latchman et al. 2011). An increased LF/HF ratio is indicative of cardiac sympathovagal imbalance (Apaijai et al. 2014).
Cardiac function determination
LV function was determined using an ECG (SONOS4500, Philips) and analyzed before the operation and then at 4-, 8-, and 12-week feeding program. Rats were stabilized in a supine position, and the probe was gently placed on the chest and moved to collect data along the short and long axes of the heart. Signals from an M-mode ECG at the level of the papillary muscles were recorded. Parameters obtained included right ventricular dimension during diastole, interventricular septum during systole and diastole, LV internal dimension during systole (LVIDs) and diastole (LVIDd), and LV posterior wall thickness during systole and diastole. Fractional shortening (FS) was calculated using the formula: %FS=(LVIDd−LVIDs)×100/LVIDd.
At the end of the experiment (week 12), LV pressure was evaluated using a pressure catheter cannulated via the right carotid artery into the LV chamber. The LV pressure was recorded using LabScribe2 program (iWorx Systems Inc., Dover, NH, USA) (Chinda et al. 2014). Systolic and diastolic blood pressure, heart rate (HR), LV end-systolic pressure, and LV end-diastolic pressure (LVEDP) were assessed.
Cardiac mitochondrial function study
At the end of the experiment, cardiac mitochondria were isolated from the whole heart in each rat. Rats were deeply anesthetized and the hearts were removed and homogenized in ice-cold buffer. The tissues were homogenized by the homogenizer and then the homogenate was centrifuged at 800g for 5min. The supernatant was collected and centrifuged at 8800g for 5min. Mitochondrial pellets were resuspended in an ice-cold buffer and centrifuged one more time at 8800g for 5min. Protein concentration was determined by bicinchoninic acid assay as described previously (Thummasorn et al. 2011). Cardiac mitochondrial function was determined by measuring mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential change (ΔΨm), and mitochondrial swelling. Transmission electron microscopy (TEM) was also used to determine the morphology of the isolated cardiac mitochondria (Chinda et al. 2014).
Measurement of cardiac mitochondrial ROS production
Mitochondrial ROS production was determined by using dichlorodihydrofluorescein diacetate dye (DCFDA) (Thummasorn et al. 2011). Cardiac mitochondrial proteins (0.4mg/mL) were incubated at 25°C with 2μM DCFDA for 20min. The DCFDA diffuses through the mitochondrial membrane and is deacetylated by intracellular esterases to a nonfluorescent compound, which is later oxidized by ROS into dichlorofluorescein (DCF). DCF is a highly fluorescent compound, which can be detected by fluorescence spectroscopy with maximum excitation. An increase in DCF intensity indicates an increase in ROS production (Chinda et al. 2014). Fluorescent intensity of the DCF was measured at excitation wavelength (λex) 485nm (bandwidth 5nm) and emission wavelength (λem) 530nm (bandwidth 10nm), using a fluorescent microplate reader, and represented as arbitrary units of fluorescent intensity.
Measurement of cardiac mitochondrial membrane potential change (ΔΨm)
Mitochondrial membrane potential change was determined using the dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1). The isolated cardiac mitochondria (0.4mg/mL) were stained with JC-1 at 37°C for 30min. The fluorescence intensity was determined by using a fluorescent microplate reader (Thummasorn et al. 2011).
In the mitochondrial membrane interaction, JC-1 could be changed to either a monomer form or an aggregate form, which have different fluorescent properties. The monomer form of JC-1 was a cation and caused a display of green fluorescence, whereas the aggregate form of JC-1 was a reaction between JC-1 (cation) and O2− (anion) within a mitochondrial matrix resulting in a display of red fluorescence. Inside unhealthy mitochondria, the levels of anions are lower, which leads to the reduction in the red (due to JC-1 (cation)/green (due to O2− (anion)) fluorescence ratio in the mitochondrial matrix; this represents mitochondrial depolarization. JC-1 monomer and aggregate form were excited at wavelengths 485 and 585nm, whereas the emission of JC-1 monomer and aggregate form was detected at wavelengths 530 and 590nm, respectively. The change in the mitochondrial membrane potential was calculated from the ratio of the intensity of red to green fluorescence. A decrease in the red/green fluorescence intensity ratio indicates mitochondrial membrane depolarization (Chinda et al. 2014).
Measurement of cardiac mitochondrial swelling
The isolated mitochondrial suspension was prepared for the determination of the level of mitochondrial swelling. Mitochondria (0.4mg/mL) were incubated in respiration buffer as described previously (Thummasorn et al. 2011). Mitochondrial swelling was determined by measuring the change in the absorbance of the suspension at 540nm (A540) using a microplate reader (Synergy HT, BioTek). Cardiac mitochondrial swelling was indicated by a decrease in the absorbance by the mitochondrial suspension (Chinda et al. 2014). TEM was also used to determine the morphology of the isolated cardiac mitochondria (Chinda et al. 2014).
Metabolic parameters, insulin resistance, and testosterone level determination
Fasting plasma glucose, total cholesterol, and triglyceride concentrations were determined by an enzymatic colorimetric assay from a commercially available kit (Biotech, Bangkok, Thailand). Fasting plasma HDL was determined using a sensitive, colorimetric and fluorometric assay from a commercially available kit (BioVision), whereas plasma LDL was determined using Friedewald’s equation (Friedewald et al. 1972). Fasting plasma insulin level was determined by Sandwich ELISA (Millipore).
Insulin resistance was assessed using the homeostasis model assessment (HOMA) (Appleton et al. 2005), which was calculated from fasting plasma insulin and fasting plasma glucose concentrations. A higher HOMA index indicates a higher degree of insulin resistance (Pratchayasakul et al. 2011). Total plasma testosterone (ng/dL) was analyzed by ELISA technique at the Central Laboratory Service of Maharaj Nakorn Chiang Mai Hospital, Faculty of Medicine, Chiang Mai University.
Determination of malondialdehyde in plasma and cardiac tissues
Plasma and tissue malondialdehyde (MDA) levels were measured using a HPLC system (Thermo Scientific) (Apaijai et al. 2012). Plasma or tissue MDA was mixed with 0.44M H3PO4 and 0.6% thiobabituric acid solution, resulting in the generation of pink-colored products called thiobarbituric acid reactive substances (TBARS). Plasma or tissue TBARS concentrations were determined directly from a standard curve, and reported as an MDA equivalent concentration using an HPLC-based assay (Mateos et al. 2005).
Western blot analysis
Heart tissue was frozen in liquid nitrogen and stored at −80°C until use (Chinda et al. 2014). Myocardial protein extract was prepared by homogenization of the nitrogen-frozen heart tissues in a lysis buffer (containing 1% Nonidet P-40 (Merck Millipore), 0.5% sodium deoxycholate, 0.1% SDS in 1×PBS), and subsequently homogenized at 4°C. The homogenate was then centrifuged at 16,707g for 15min. The total protein from tissue pellets was mixed with a loading buffer (10% mercaptoethanol, 0.05% bromophenol blue, 75mM Tris–HCl, pH 6.8, 2% SDS, and 10% glycerol) and heated at 95°C for 10min. The protein samples were then subjected to 10 or 15% SDS–PAGE, and transferred to polyvinylidene difluoride membranes using a semi-dry transfer system (Trans-Blot SD; Bio-Rad). The membranes were blocked with 5% nonfat skimmed milk in TBST (20mM Tris–HCl, pH 7.6, 137mM NaCl, and 0.1% Tween 20) for 1h. The membranes were subsequently exposed to anti-BAX (Santa Cruz Biotechnology), anti-BCL2 (Cell Signaling Technology), or β-actin (Santa Cruz Biotechnology) overnight at 4°C. On the following day, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology) for 1h at room temperature. The signals were developed by incubating with an enhanced chemiluminescense reagent and subjected to autoradiography. The immunoblot films were scanned, and the band density was analyzed using ImageJ software (NIH, Bethesda, MD, USA).
Statistical analysis
Data were expressed as mean±s.e.m. Statistical comparisons at the different time points in the feeding program for HRV, cardiac function, and cardiac mitochondrial function were analyzed using a one-way ANOVA, followed by post hoc least significant difference analysis. Statistical comparisons of the biochemical parameters were calculated using the Mann–Whitney U test. All statistical analyses were performed using SPSS (version 16; SPSS). The statistical significance was accepted at a P value <0.05.
Results
Obesity, but not testosterone deficiency, led to insulin resistance
At week 4, testosterone deprivation was confirmed by decreased plasma testosterone levels and the ratio of seminal vesicle per body weight (seminal vesicle ratio). Orchiectomized rats in both dietary groups (NDO and HFO) had significantly decreased plasma testosterone levels and seminal vesicle ratio, when compared with sham rats at the same time points. Animals fed on a HFD for 4weeks had significantly increased body weight compared with ND rats. In addition, orchiectomized rats in both dietary groups (NDO and HFO) had decreased body weight, food intake, and visceral fat when compared with sham rats in the same dietary groups. However, there was no significant difference in the metabolic parameters (the level of plasma insulin, plasma glucose, HOMA index, plasma total cholesterol, plasma triglyceride, plasma HDL and LDL in the blood) among the dietary groups at 4weeks (Table 1).
Effect of obese-insulin resistance in testosterone-deprived rats on metabolic parameters investigated at 4 weeks (n=6 per group).
| Normal diet | High-fat diet | |||
|---|---|---|---|---|
| Parameters | NDS | NDO | HFS | HFO |
| Body weight (g) | 448±12 | 420±9* | 493±15*,† | 460±10†,‡ |
| Food intake (g/day) | 20.7±0.2 | 18.8±0.4*,‡ | 21.1±0.1 | 20.4±0.3†,‡ |
| Visceral fat (g) | 22.8±2.07 | 15.8±2.16*,‡ | 35.2±2.22* | 29±0.6*,†,‡ |
| Plasma testosterone (ng/dL) | 1.5±0.4 | 0.1±0.01*,‡ | 1.0±0.01 | 0.1±0.01*,‡ |
| Seminal vesicle ratio (×103) | 3.3±0.26 | 0.37±0.1*,‡ | 3.4±0.41 | 0.3±0.12*,‡ |
| Plasma insulin (ng/mL) | 2.1±0.2 | 2.0±0.2 | 2.2±0.3 | 2.2±0.3 |
| Plasma glucose (mg/dL) | 116±11 | 127±7 | 125±8 | 125±7 |
| HOMA index | 11±2 | 11.2±1 | 12.2±1 | 12.5±2 |
| Plasma total cholesterol (mg/dL) | 90±3 | 91±5 | 95±4 | 102±7 |
| Plasma triglyceride (mg/dL) | 134±12 | 139±8 | 138±16 | 142±7 |
| Plasma HDL (mg/dL) | 32±1 | 31±3 | 29±2 | 28±2 |
| Plasma LDL (mg/dL) | 62±3 | 68±3 | 67±5 | 69±8 |
| Plasma MDA (mmol/mL) | 4.7±0.08 | 5.6±0.19*,‡ | 5.0±0.06 | 5.5±0.17*,‡ |
| Cardiac MDA (μmol/mL) | 1.53±0.26 | 3.7±0.76 | 1.57±0.15 | 5.2±0.79* |
P<0.05 vs NDS; †P<0.05 vs NDO;
P<0.05 vs HFS.
At week 8, the body weight, food intake, and visceral fat mass were similar to the results at week 4. Peripheral insulin resistance developed only in the HFD groups (HFS and HFO) at 8weeks, as shown by both an increased insulin level and HOMA index. The metabolic parameters did not differ between HFS and HFO, but they were significantly higher than those in ND rats (NDS and NDO) (Table 2).
Effect of obese insulin resistance in testosterone-deprived rats on metabolic parameters investigated at 8 weeks (n=6 per group).
| Normal diet | High-fat diet | |||
|---|---|---|---|---|
| Parameters | NDS | NDO | HFS | HFO |
| Body weight (g) | 460±10 | 443±8*,‡ | 535±15* | 497±10*,†,‡ |
| Food intake (g/day) | 19.2±0.7 | 17.7±0.6*,‡ | 21.9±0.1 | 19.8±0.3†,‡ |
| Visceral fat (g) | 30.3±0.8 | 18.3±1.8*,‡ | 48.5±3.1* | 37.2±2.38*,†,‡ |
| Plasma testosterone (ng/dL) | 2.1±0.7 | 0.03±0.01*,‡ | 1.2±0.31* | 0.08±0.01*,‡ |
| Seminal vesicle ratio (×103) | 3.8±0.22 | 0.21±0.01*,‡ | 3.1±0.1* | 0.34±0.04*,‡ |
| Plasma insulin (ng/mL) | 1.95±0.1 | 2.0±0.1 | 2.9±0.3*,† | 2.8±0.3*,† |
| Plasma glucose (mg/dL) | 123±7 | 133±7 | 128±4 | 130±9 |
| HOMA index | 11.0±1 | 11.2±1 | 16.6±1*,† | 16.7±3*,† |
| Plasma total cholesterol (mg/dL) | 91±4 | 93±4 | 108±6*,† | 111±5*,† |
| Plasma triglyceride (mg/dL) | 129±10 | 130±14 | 124±17 | 123±24 |
| Plasma HDL (mg/dL) | 31±2 | 33±2 | 23±2*,† | 21±3*,† |
| Plasma LDL (mg/dL) | 61±4 | 68±5 | 83±6* | 89±5*,† |
| Plasma MDA (mmol/mL) | 5.1±0.06 | 5.7±0.13* | 5.9±0.1* | 5.6±0.12* |
| Cardiac MDA (μmol/mL) | 1.98±0.26 | 4.7±1.02* | 4.39±0.79* | 4.53±0.71* |
P<0.05 vs NDS
P<0.05 vs NDO
P<0.05 vs HFS.
At week 12, HF rats (HFS and HFO) still demonstrated consistent impairments in metabolic parameters, as shown by increased levels of all metabolic parameters, except triglyceride level, compared with NDS and NDO rats (Table 3). Moreover, HFS rats still showed a significant increase in body weight compared with NDS rats, whereas orchiectomized rats from both dietary groups (NDO and HFO) had decreased body weight, food intake, and visceral fat mass compared with the sham rats in the same dietary groups (Table 3).
Effect of obese-insulin resistance in testosterone-deprived rats on metabolic parameters investigated at 12 weeks (n=6 per group).
| Normal diet | High-fat diet | |||
|---|---|---|---|---|
| Parameters | NDS | NDO | HFS | HFO |
| Body weight (g) | 485±15 | 447±16*,‡ | 575±21* | 508±17†,‡ |
| Food intake (g/day) | 20.7±0.2 | 16.4±1*,‡ | 20.3±0.4 | 18.4±0.3†,‡ |
| Visceral fat (g) | 30.2±2.6 | 21.8±0.6*, ‡ | 51±4.6* | 28.5±3.4†,‡ |
| Plasma testosterone (ng/dL) | 1.2±0.4 | 0.07±0.01*,‡ | 0.5±0.06* | 0.1±0.06*,‡ |
| Seminal vesicle ratio (×103) | 3.82±0.28 | 0.51±0.33*,‡ | 3.1±0.07* | 0.18±0.02*,‡ |
| Plasma insulin (ng/mL) | 1.9±0.2 | 2.2±0.3 | 3.3±0.2*,† | 3.0±0.3*,† |
| Plasma glucose (mg/dL) | 140±5 | 136±4 | 132±4 | 136±6 |
| HOMA index | 13.5±1 | 12.5±1 | 20.1±1*,† | 19.8±2*,† |
| Plasma total cholesterol (mg/dL) | 89±5 | 100±2 | 109±6*,† | 130±7*,†,‡ |
| Plasma triglyceride (mg/dL) | 133±32 | 135±10 | 146±9 | 133±4 |
| Plasma HDL (mg/dL) | 33±2 | 35±2 | 25±2*,† | 25±3*,† |
| Plasma LDL (mg/dL) | 63±5 | 73±4 | 82±8* | 95±8*,† |
| Plasma MDA (mmol/mL) | 4.04±0.4 | 5.6±0.27* | 5.8±0.5* | 5.4±0.15* |
| Cardiac MDA (μmol/mL) | 2.48±0.63 | 6.08±0.75* | 6.37±1.30* | 7.07±0.67* |
P<0.05 vs NDS
P<0.05 vs NDO
P<0.05 vs HFS.
Testosterone deprivation led to oxidative stress earlier than high-fat-induced obesity
Orchiectomized rats (NDO and HFO) had increased plasma MDA from week 4 onward, when compared with sham rats in the same dietary groups, whereas cardiac tissue MDA was increased only in the HFO group (Table 1). Plasma and cardiac tissue levels of MDA in orchiectomized rats (NDO and HFO) continued to show an increase at week 8 and 12. Plasma and cardiac tissue levels of MDA in the HFS group in week 8 (Table 2) and 12 (Table 3) were also elevated, showing the same levels of plasma MDA observed in the NDO and HFO groups, and was significantly different when compared with the NDS group.
Testosterone deprivation led to cardiac dysfunction and HRV impairment earlier than obesity
In ORX rats fed on either a normal diet (NDO) or a high-fat diet (HFO), both FS and ejection fraction (EF) showed a significant decrease at week 4, and continued to decrease by week 8 and 12, when compared with sham rats in the same dietary groups (NDS and HFS). Unlike ORX rats, HFS rats exhibited a significant reduction in both %FS and %EF later at week 8 and 12 of the study, when compared with rats in the NDS group (Fig. 1A and B).
The effect of obese-insulin resistance in testosterone-deprived rats on FS, EF, and HRV (represented as LF/HF ratio) at different time courses (4, 8, and 12 weeks). (A) %FS and (B) %EF were decreased in NDO and HFO groups at any time following week 4, whereas %FS in the HFS group was decreased at week 8 and onward, compared with the NDS group. (C) LF/HF ratio was significantly increased in NDO and HFO groups from week 4, whereas the LF/HF ratio in the HFS group was increased at week 8, compared with the NDS group. *P<0.05 vs NDS, †P<0.05 vs HFS, n=5–6 per group. NDS, normal diet+sham surgery; NDO, normal diet+orchiectomy; HFS, high-fat diet+sham surgery; HFO, high-fat diet+orchiectomy.
Citation: Journal of Endocrinology 229, 3; 10.1530/JOE-16-0002
Consistent with the %FS and %EF data, ORX rats (NDO and HFO) were the first groups to present as having impaired sympathovagal balance. The LF/HF ratio in ORX rats showed a significant increase from week 4 onward, and still showed this impairment at week 8 and 12, when compared with the NDS group. The HFS group exhibited sympathovagal imbalance later, by week 8 and 12 (Fig. 1C). No significant difference in the LF/HF ratio was found in the NDO, HFS, and HFO groups at weeks 8 and 12.
The changes in the LV pressure at week 12 in all groups are given in Table 4. Rats in the NDO, HFS, and HFO groups had increased systolic and diastolic blood pressure when compared with rats in the NDS group. Moreover, these groups exhibited diastolic dysfunction at week 12, as shown by increased LVEDP, when compared with the NDS group.
Effect of obese insulin resistance in testosterone-deprived rats on cardiac function at 12 weeks (n=6 per group).
| Cardiac function parameters | Normal diet | High-fat diet | ||
|---|---|---|---|---|
| NDS | NDO | HFS | HFO | |
| Heart rate (bpm) | 285±5 | 284±15 | 276±11 | 284±15 |
| Systolic BP (mmHg) | 109±2 | 128±7* | 127±6* | 129±6* |
| Diastolic BP (mmHg) | 86±1 | 106±7* | 99±7* | 103±5* |
| LVESP (mmHg) | 103±6 | 96±5 | 109±2 | 101±4 |
| LVEDP (mmHg) | 5±1 | 9±1* | 10±1* | 10±1* |
| Pmax (mmHg) | 106±6 | 104±6 | 115±3 | 106±4 |
| Pmin (mmHg) | 3±1 | 6±3 | 7±1 | 8±1 |
| dP/dtmax (mmHg/s) | 7056±522 | 6757±410 | 7057±502 | 6264±286 |
| dP/dtmin (mmHg/s) | −5458±461 | −4433±449 | −6463±662† | −5950±1001 |
BP, blood pressure; LVESP, left ventricular end-systolic pressure.
P<0.05 vs NDS
P<0.05 vs NDO.
Testosterone deprivation led to cardiac mitochondrial dysfunction earlier than obesity
In ORX rats (NDO and HFO), cardiac mitochondrial dysfunction had developed by week 4 and dysfunction continued to week 8 and 12, as indicated by increased mitochondrial ROS production (Fig. 2A), mitochondrial depolarization as indicated by a decrease in the red/green fluorescent intensity ratio (Fig. 2B) and mitochondrial swelling as indicated by a decrease in the absorbance at 540nm (Fig. 2C) when compared with sham rats at the same point in time. In HFS rats, it was not until week 8 that cardiac mitochondrial dysfunction developed (Fig. 2). No significant difference in these cardiac mitochondrial parameters was found between NDO, HFS, and HFO groups at week 8 and 12. Representative TEM pictures of cardiac mitochondria are shown in Fig. 2D. Cardiac mitochondrial swelling could be observed in ORX rats (NDO and HFO) at week 4, 8, and 12, whereas in the HFS group was observed later, at week 8 and 12.
The effect of obese-insulin resistance in testosterone-deprived rats on cardiac mitochondrial function at different points in the feeding program (4, 8, and 12 weeks). (A) Mitochondrial ROS production; (B) mitochondrial membrane depolarization; and (C) mitochondrial swelling were all increased in NDO and HFO groups from week 4 onward, whereas the HFS group showed an increase in these parameters beginning at week 8. (D) Representative electron micrographs showed that obese-insulin resistance and testosterone deprivation caused cardiac mitochondrial swelling. *P<0.05 vs NDS, †P<0.05 vs HFS, n=5–6 per group. a.u., arbitrary units; NDS, normal diet+sham surgery; NDO, normal diet+orchiectomy; HFS, high-fat diet+sham surgery; HFO, high-fat diet+orchiectomy.
Citation: Journal of Endocrinology 229, 3; 10.1530/JOE-16-0002
Increased cardiac apoptotic protein expression in testosterone-deprived rats with and without obese-insulin resistance
In the heart, the expression of BAX and BCL2 showed no significant difference between all groups at week 4. At week 8, when compared with the NDS group, the pro-apoptotic (BAX) level was increased in NDO, HFS, and HFO groups (Fig. 3A,D), whereas the anti-apoptotic (BCL2) levels showed no significant difference between the groups (Fig. 3B,D). At week 12, the BAX level in NDO, HFS, and HFO groups was continuously increasing (Fig. 3A,D), whereas the BCL2 level in NDO, HFS, and HFO groups was lower at this time point, compared with the NDS group (Fig. 3B,D). The BAX/BCL2 ratio increased earlier in the HFO group (week 4), whereas this ratio increased later (week 8) in the NDO and HFS groups (Fig. 3C).
The effects of obese-insulin resistance in testosterone-deprived rats on myocardial apoptosis at different time courses (4, 8, and 12 weeks). Anti-apoptotic (BCL2) expression (A), pro-apoptotic (BAX) expression (B), BAX/BCL2 ratio (C), and representative bands from western blot analysis (D). BCL2 expression was reduced in NDO, HFS, and HFO groups at week 12, whereas BAX expression was increased in NDO, HFS, and HFO groups at week 8 and 12. BAX/BCL2 ratio showed an increase earlier in the HFO group (week 4), whereas this ratio increased later (week 8) in NDO, HFS, and HFO groups, compared with the NDS group at the same point in the feeding program. *P<0.05 vs NDS at the same point in the feeding program, n=5–6 per group. NDS, normal diet+sham surgery; NDO, normal diet+orchiectomy; HFS, high-fat diet+sham surgery; HFO, high-fat diet+orchiectomy.
Citation: Journal of Endocrinology 229, 3; 10.1530/JOE-16-0002
Discussion
This study has demonstrated the adverse effects of testosterone deprivation and obese-insulin resistance on the heart. The major findings of this study are as follows. First, testosterone deprivation alone does not cause insulin resistance, but leads to earlier development of LV dysfunction, cardiac sympathovagal imbalance, cardiac mitochondrial dysfunction, and increased circulating oxidative stress. Secondly, long-term HFD consumption alone enhances the development of obese-insulin resistance, LV dysfunction, cardiac sympathovagal imbalance, cardiac mitochondrial dysfunction, and increased circulating and cardiac tissue oxidative stress. These adverse effects, however, developed later than those observed in the testosterone-deprived rats. Thirdly, although obese-insulin resistance does not aggravate these adverse effects of testosterone deprivation, it accelerates the apoptotic process in the heart of testosterone-deprived rats. A summary of these findings is given in Table 5.
A summary of the cardiometabolic impairment of the experimental groups at different points in the feeding program (4, 8, and 12 weeks).
| 4 week | 8 week | 12 week | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Impairment | NDS | NDO | HFS | HFO | NDS | NDO | HFS | HFO | NDS | NDO | HFS | HFO |
| Metabolic disturbance | ✓ | ✓ | ✓ | ✓ | ||||||||
| LV dysfunction | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ||||
| Cardiac autonomic imbalance | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ||||
| Oxidative stress | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ||||
| Mitochondrial impairment | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ||||
In this study, testosterone deprivation did not have adverse effects on metabolic parameters, except for weight loss, at all measured time points. The weight loss found in NDO rats could be due to decreased food intake. This finding is consistent with previous studies, which reported that an orchiectomy could cause permanent hypophagia (Gentry & Wade 1976), an increase in oxidative stress (Pongkan et al. 2015), and an increase in leptin (Baumgartner et al. 1999). The possible mechanism for this could be due to the induction of proinflammatory cytokines (via nuclear factor-κ-B signaling molecules) causing oxidative stress (Vlantis & Pasparakis 2010). This may result in an increased concentration of circulating leptin via activation of the leptin gene through the p38-MAPK pathway (Finck & Johnson 2002) and lead to decreased food intake and a resulting drop in body weight (Aoyama et al. 2015). Although testosterone deprivation did not appear to have any measurable effects on metabolic parameters, it caused LV dysfunction and cardiac autonomic imbalance as observed from week 4 onward. Cardiac autonomic control has been shown to be mainly influenced by oxidative stress (Brook et al. 2010). It has been shown that increased oxidative stress is associated with cardiac autonomic imbalance (Sivasinprasasn et al. 2015). Moreover, cardiac mitochondrial function has been shown to play an important role on cardiac contractile function (Marchini et al. 2013). Therefore, the adverse effects observed in testosterone-deprived rats could be due to the increased oxidative stress and impaired cardiac mitochondrial function, thus resulting in cardiac autonomic imbalance and LV dysfunction, respectively.
Long-term consumption of a HFD has been shown to cause obese-insulin resistance (Pratchayasakul et al. 2011), which is characterized by increased plasma insulin, HOMA index, and plasma total cholesterol levels (Pratchayasakul et al. 2011). Although body weight, food intake, and visceral fat were decreased in obese-testosterone deficiency (HFO) rats, the insulin resistance index (HOMA index) was not improved in this study. Since a previous report showed that the reduction of food intake and body weight could improve insulin sensitivity (Schenk et al. 2009), our finding emphasized the importance of testosterone deficiency condition, which might aggravate insulin resistance as observed in this study. Supporting this hypothesis is due to the fact that testosterone deficiency has been shown to cause insulin resistance (Dubois et al. 2015, Pintana et al. 2015a).
In this study, insulin resistance was not observed until after 8weeks of consumption of a HFD, despite the fact that weight gain had been observed in these rats since week 4. However, the levels of metabolic impairment, LV dysfunction, and cardiac autonomic imbalance in HFS and HFO rats at weeks 8 and 12 were no different, suggesting that the obese-insulin-resistant condition did not aggravate these adverse effects caused by testosterone deprivation as observed in NDO rats. This is also supported by the findings that both HFO and HFS rats had similar levels of oxidative stress and cardiac mitochondrial dysfunction. However, these findings indicate that testosterone deprivation accelerated the development of cardiac autonomic disturbance and LV dysfunction as HFO rats had already developed these impairments by week 4, whereas HFS rats showed no signs of this at this period. Therefore, the dysfunctions in week 4 could be due to the influence of testosterone deprivation alone. Moreover, HFO rats at week 8 have testosterone deprivation combined with obese-insulin resistance and did not show any additive adverse effect, compared with the same group at 4 weeks. In addition, these abnormalities were similar between 8 and 12weeks. These findings suggest an unclear relationship between the effects produced by testosterone deprivation on cardiac function and those caused by obesity and insulin resistance since no additive adverse effect of these two abnormal conditions was observed. This is consistent with the previous study demonstrating the effects of testosterone deprivation on the cognitive function (Pintana et al. 2016). However, whether additive adverse effects from these two conditions can be observed at a longer duration of obese-insulin-resistant condition are not known and need further investigation in the future. Nevertheless, at this point our study demonstrated that testosterone deprivation did not aggravate these impairments, but accelerated the development of these impairments in obese-insulin-resistant rats.
Regarding apoptotic markers, the HFO rats demonstrated an increased ratio of BAX/BCL2 from week 4, which was earlier than that observed in the HFS rats. The possible mechanism could be due to the increasing level of plasma and cardiac MDA, and increased cardiac mitochondrial ROS production in HFO rats. The increase in the level of oxidative stress in the heart may have aggravated an apoptotic pathway (Zorov et al. 2006) and led to a decrease in anti-apoptotic (BCL2) levels, and an increase in pro-apoptotic protein (BAX) levels (Pongkan et al. 2015), therefore, leading to cardiomyocyte apoptosis and finally LV dysfunction (Aon et al. 2009).
Our findings are consistent with previous clinical studies. The cohort clinical study demonstrated that the prevalence of cardiovascular morbidity and mortality increased in patients suffering from prostate cancer (PCa) who have undergone to androgen deprivation therapy (ADT), suggesting that testosterone deficiency was the risk factor of cardiovascular diseases (Gandaglia et al. 2015). Moreover, diastolic dysfunction was found in patients with type 2 diabetes with low testosterone level compared with similar patients with normal testosterone levels (Tinetti et al. 2015). Furthermore, patients suffering from congestive heart failure (Jankowska et al. 2006) and coronary artery disease (Li et al. 2012) have been shown to have lower levels of testosterone compared with healthy men, and the severity of these conditions had a strong correlation with the degree of testosterone deficiency (Li et al. 2012, Oskui et al. 2013). Recent clinical studies demonstrated that testosterone replacement could improve insulin sensitivity, increase lean mass, and decrease subcutaneous fat (Dhindsa et al. 2016) and also improve cardiometabolic parameters in type 2 diabetes men with testosterone deprivation (Hackett et al. 2014) and in hypogonadal men with metabolic syndrome (Saad et al. 2016).
Although low testosterone level may be a risk factor for diabetes or insulin resistance (Muraleedharan & Jones 2010), the effects of testosterone replacement on insulin resistance and metabolic parameters may depend on its dose. Physiological level of testosterone exhibited the beneficial effect on insulin resistance and metabolic parameters (Pintana et al. 2015b). However, supraphysiological level of testosterone had been shown to provide adverse effect on insulin resistance (Yasui et al. 2012, Garrido et al. 2014). In healthy and menopausal women, supraphysiological level of testosterone could induce the development of breast cancer, heart disease, and insulin resistance (Polderman et al. 1994, Yasui et al. 2012). Moreover, high-dose testosterone significantly decreased plasma HDL, increased LDL cholesterol in healthy men and postmenopausal women (Taggart et al. 1982, Hurley et al. 1984, Thompson et al. 1989), and induced insulin resistance in a rat model (Holmang & Bjorntorp 1992). High-dose testosterone also increased oxidative stress levels by decreasing the expression of endothelial nitric oxide synthase and consequently the formation of NO, resulting in endothelial dysfunction (Skogastierna et al. 2014). Moreover, in an ischemia and reperfusion (I/R) injury rabbit model, supraphysiological level of testosterone increased fibrosis and oxidative stress and exacerbated such detrimental effects of I/R (Chuang et al. 2013), whereas testosterone replacement at the physiological level exerted antioxidative effects by reducing mitochondrial oxidative stress (Pongkan et al. 2015, Pintana et al. 2015b) and serum MDA levels (Pintana et al. 2015b).
In conclusion, testosterone deprivation did not cause metabolic disturbance, but caused cardiac autonomic imbalance and LV dysfunction due to increased oxidative stress and impaired cardiac mitochondrial function. Obese-insulin resistance caused by long-term consumption of a HFD could lead to metabolic disorders, cardiac autonomic imbalance, and LV dysfunction due to increased oxidative stress and impaired cardiac mitochondrial function. Despite these similar adverse effects on the heart, the findings of this study demonstrated that testosterone deprivation did not aggravate these impairments, but accelerated the development of these impairments in obese-insulin-resistant rats, the obesity and resulting insulin resistance being caused by consumption of a HFD. These findings may suggest the necessity for early testosterone replacement to ensure cardioprotection in obese-insulin-resistant conditions linked with testosterone deprivation. Further studies are needed to investigate the roles of testosterone replacement therapy under these conditions.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the National Science and Technology Development Agency (NSTDA) Research Chair Grant, Thailand (N C) and grants from the Thailand Research Fund BRG5780016 (S C), the Royal Golden Jubilee Program (N C and W P), the National Research Council Thailand (N C), and the Chiang Mai University Excellent Center Award (N C).
Acknowledgments
The authors thank Aimee Cole, Cicely Proctor, Drs Sirinart Kumfu, and Nattayaporn Apaijai for their editorial and technical assistance.
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