Sage Journals: Discover world-class research

Abstract

Background:

Testosterone deficiency (TD), brain mitochondrial uncoupling protein 2 (UCP2), and the low-density lipoprotein receptor (LDLR) are independently linked to the triggering of several behavioral disorders. Here, we investigated how TD impacts mitochondrial activity and apoptosis in different brain domains in the presence and absence of LDLR.

Materials and Methods:

Sham-operated or surgically castrated C57BL/6 or LDLR-deficient (Ldlr^−/−) mice were fed a western-type diet for 12 weeks. Then, mitochondrial UCP2 and cytochrome c (Cytc) relative protein levels were measured as markers of uncoupling and oxidative phosphorylation, respectively, in tissue specimens from different brain domains. The ratio of the relative cytoplasmic Cytc levels over the relative mitochondrial Cytc levels (CM ratio) was used as a biochemical marker of Cytc-dependent apoptosis.

Results:

In cerebral hemispheres and the cerebellum area of both C57BL/6 and Ldlr^−/− mice, castration resulted in increased UCP2, suggesting protection from hypogonadism-induced oxidative stress. In the broader midbrain, castration reduced UCP2 expression in C57BL/6 mice but not in Ldlr^−/− mice, suggesting that LDLR deficiency protects from oxidative stress in this region. CM ratio was ≤1 after castration in these three brain domains, suggesting increased protection from Cytc-dependent apoptosis. In diencephalon, castration had no effect on UCP2 and CM ratio in C57BL/6 mice but led to increased UCP2 expression and increased CM ratio in Ldlr^−/− mice, suggesting an increase in apoptosis in the diencephalon of castrated Ldlr^−/− mice.

Conclusions:

TD in Ldlr^−/− mice fed a western-type diet is associated with protection from oxidative stress but could contribute to the emergence of behavioral or systemic impairments linked to apoptosis in the diencephalon.

Introduction

Testosterone is a steroid hormone with an important role in male sex differentiation.¹ It is primarily produced in the testes (95%) and secreted by Leydig cells, as a result of multiple enzymatic reactions starting in the mitochondria that use cholesterol as the precursor.² Other organs can also produce and utilize testosterone in an autocrine fashion, including the adrenal gland, white adipose tissue, skin, and brain.³

Testosterone deficiency (TD) has been identified as an independent risk factor for the development of obesity, metabolic syndrome, and related diseases, among other disorders,^4–6 and vice versa obesity is correlated with a decrease in testosterone levels.⁷ Similarly, TD in combination with diet-induced obesity is proven to increase inflammation in the nervous system, and contribute to observed impairments in neural function.⁸ Age-related low testosterone metabolite levels, are also associated with cognitive and behavioral decline.⁹ Meanwhile, testosterone replacement therapy has been proposed for the treatment of neurological and neuropsychiatric disorders.¹⁰

Previous work¹¹ showed that the effects of testosterone on adipose tissue metabolism and diet-induced obesity are dependent on the expression of the low-density lipoprotein receptor (LDLR), the principal member of the LDLR superfamily that mediates the uptake of apolipoprotein E-containing lipoproteins from circulation.^12,13 Lack of a functional LDLR has been identified as the genetic cause of familial hypercholesterolemia and atherosclerosis¹⁴ and as a risk factor for the development of several cognitive disorders.¹⁵ Lack of LDLR has also been associated with the emergence of Alzheimer's disease, memory impairments, and the development of anxiety and depression.^16–20

Energy-consuming brain activities such as neurotransmission and synaptogenesis are supported by mitochondria.²¹ Neuronal plasticity is also accomplished by the specialized brain-expressed uncoupling protein 2 (UCP2).²² UCP2 is placed in the inner mitochondrial membrane and leaks protons in the mitochondrial matrix, thus reducing the production of reactive oxygen species (ROS).²³

In the brain, increased UCP2-mediated uncoupling leads to an increased number of mitochondria, therefore resulting in overall increased adenosine triphosphate (ATP) production.^22,24 Lack of UCP2 results in increased ROS production, leading to neurotoxicity, which contributes to the emergence of neurodegenerative diseases.²⁵ Similarly, immunological responses caused by oxidative stress have been associated with depression.²⁶ Therefore, an increased expression of UCP2 could constitute an adaptive response to oxidative stress caused by increased ROS in mitochondria.²⁷

Given the important cooperative roles of testosterone and diet-induced obesity in the regulation of neuroinflammation,⁸ the role of brain mitochondria in neurotransmission and synaptogenesis,²¹ and the role of LDLR in neurodegenerative diseases,¹⁵ it is conceivable to hypothesize a functional cross-talk among hypogonadism, mitochondrial function, and LDLR expression in the brain. Therefore, in the present work, we investigated how hypogonadism impacts mitochondrial activity and apoptosis in different brain domains in the presence and absence of functional LDLR.

Materials and Methods

Methods are broadly similar to those in Constantinou et al.¹¹

Animals

The Ldlr^−/− and C57BL/6 mice used in our studies were purchased from Jackson Labs (Bar Harbor, ME) and bred in our animal facility. Male mice 10–12 weeks old were used in these studies. Mice in each group were caged individually (one mouse per cage) and were allowed unrestricted access to food and water under a 12 h light/dark cycle. During breeding and colony maintenance, mice were fed the typical chow diet (1324 TPF; Altromin Spezialfutter GmbH & Co). In all studies involving a high-fat diet, the standard western-type diet was used (Mucedola SRL, Milano, Italy), which is composed of 17.3% protein, 48.5% carbohydrate, 21.2% fat, and 0.2% cholesterol (0.15% added, 0.05% from fat source) and contains 4.5 Kcal/g.

The content of the main ingredients expressed as g per kg of diet is: Casein 195, DL-Methionine 3, Sucrose 341.46, Corn Starch 150, Anhydrous Milkfat 210, Cholesterol 1.5, Cellulose 50, Mineral Mix 35, Calcium Carbonate 4, Vitamin Mix 10, Ethoxyquin, and antioxidant 0.04. For our studies, two groups of mice of the same strain were formed to have similar average starting age and body weights. One group of each strain (n = 6) was surgically castrated, and the other group (n = 6) was sham operated.

After a recovery period of 4 weeks on chow diet, to allow for T clearance from the circulation of castrated mice, all mouse groups were switched to a western-type diet (considered as week 0) for an additional period of 12 weeks. At the end of each experiment, mice were sacrificed and plasma and tissue samples were collected. Carcasses were stored at −80°C. All animal studies were governed by the European Union guidelines of the Protocol for the Protection and Welfare of Animals. The work was authorized and received Institutional Review Board (IRB) approval by the appropriate committee of the Laboratory Animal Center of the University of Patras Medical School and the Veterinary Authority of the Prefecture of Western Greece. Animals were monitored by a certified veterinarian throughout the course of the study.

Surgical castration and tissue isolation

Surgical castration of mice was performed as previously described.¹¹ Briefly, sham-operated mice underwent only a skin incision. Castrated animals were bilaterally castrated through 1 cm scrotal incisions. Both groups were operated under isoflurane (CP Pharma, Burgdorf, Germany) anesthesia. The animals could recover from the operation for a period of 4 weeks, and plasma T levels were determined to confirm the success of the operation.

Then, both groups were switched to a western-type diet for the indicated period of time. At the end of the 12-week period of feeding a western-type diet, mice were euthanized and blood samples, the cerebellum, diencephalon, midbrain, and cerebral hemispheres of the brain were collected. All samples were stored at −80°C.

Plasma testosterone determination

Blood was drawn from the tail vein at the indicated times, and plasma samples were separated by centrifugation (4000 rpm, 10 min) and stored at −20°C until they were used for T measurements. Plasma T levels were determined by using the Mouse/Rat Total T Elisa Kit (Cat. No. TE187S-100; Calbiotech, Spring Valley, CA) according to the manufacturer's instructions.

Determination of body weight and daily food consumption

During the experiment, animal body weight was determined by a Mettler-Toledo^® precision microscale (Mettler-Toledo, Columbus, OH) as previously described.¹¹ Food intake was assessed by determining the difference in food weight during a 7-day period to ensure reliable measurements.

Plasma lipid determination

Blood sample collection was performed after a 16 h fasting period. At the beginning of the experiment, blood samples were collected from the tail vein. At the end of the study, plasma samples were collected from the retro-orbital plexus during sacrifice of the animals. Plasma cholesterol and triglyceride (TG) levels were measured by using the Multi-purpose Kit: Cholesterol FS 10’ and Triglyceride FS 10’ (DiaSys Diagnostic System GmbH, Germany), respectively, according to the manufacturer's instructions.

Tissue homogenization and isolation of mitochondria

Mitochondria from the four brain regions were isolated as previously described.¹¹ All samples were stored at −80°C until an analysis was conducted by Western blotting. The protein concertation of each cytoplasmic and mitochondrial sample was determined by using the DC™ Protein Assay Kit II (Bio-Rad Laboratories Inc.).

Western blot analysis

Mitochondrial and cytoplasmic samples from cerebral hemispheres (60 μg/lane), cerebellum (25 μg/lane), midbrain (25 μg/lane), and diencephalon (18 μg/lane) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (12% acrylamide and 0.5% N, N′-diallyltartardiamide), and they were transferred to polyvinylidene difluoride membranes. The membranes were then probed with primary rabbit anti-mouse antibodies for UCP2, cytochrome c (Cytc), cytochrome c oxidase subunit 4 (COX4), and beta-tubulin (Cat. No. 1108-1-AP, Proteintech; Cat. No. 4272, Cell Signaling; Cat. No. 4844, Cell Signaling; and Cat. No. 2146, Cell Signaling; respectively).

Semiquantitative determination of the relative protein amounts was performed by using ImageJ free software, and band intensity was reported in arbitrary units (au). Relative UCP2 and Cytc expression in mitochondria fractions was normalized by using COX4, and relative Cytc expression in cytoplasm fractions was normalized by using β-tubulin.

Statistical analysis

Data are presented as mean ± standard error of the mean (*p < 0.05; **p < 0.001; ***p < 0.0001; n indicates the number of animals tested in each experiment). Data from two groups of mice were compared by using the Student's t-test. All statistical tests were performed by using SPSS software (released 2009, PASW statistics for Windows, Version 18.0; SPSS Inc., Chicago, IL).

Results

Body weight, food consumption, and plasma lipid levels

During the course of the study, body weight and average food intake were measured (Fig. 1). Wild-type mice, both castrated and sham-operated, and sham-operated Ldlr^−/− mice showed increased body weight after the 12-week period of feeding a Western-type diet, whereas castrated Ldlr^−/− mice maintained their weight unchanged throughout the course of the experiment (Fig. 1A).

FIG. 1.

(A) Body weight taken in weeks 0 and 12 of the experiment. (B) Food intake measured in weeks 0 and 12 of the experiment. (C) Plasma cholesterol levels. (D) Plasma triglycerides levels. Results are presented as mean ± SEM (n = 6; *p < 0.05; **p < 0.001; ***p < 0.0001; ^NSp > 0.05). SEM, standard error of the mean.

Specifically, sham-operated C57BL/6 mice increased their weight by 58.5% from 23.8 ± 0.6 g at week 0 to 37.7 ± 1.6 g at week 12 (p < 0.0001). Similarly, castrated mice increased their weight by 34.6% from 27.4 ± 1.2 g at week 0 to 36.9 ± 1.6 g at week 12 (p < 0.001). Similarly, sham-operated Ldlr^−/− mice showed increased body weight by 33%, from 25.2 ± 0.8 g at week 0 to 33.5 ± 0.2 g at week 12 (p < 0.0001). In contrast, castrated Ldlr^−/− mice exhibited no significant increase in body weight, changing from 26.0 ± 0.3 g at week 0 to 27.0 ± 0.3 g at week 12 (p > 0.05), in agreement with previous data.¹¹

The average daily food intake was similar among the four animal groups throughout the experiment (Fig. 1B). At weeks 0 and 12 of the experiment, food consumption was 4.00 ± 0.09 and 4.00 ± 0.04 g/day respectively (p > 0.05) for sham-operated C57BL/6 mice, 4.03 ± 0.11 and 3.95 ± 0.09 g/day respectively (p > 0.05) for castrated C57BL/6 mice, 3.95 ± 0.06 and 4.00 ± 0.11 g/day respectively (p > 0.05) for sham-operated Ldlr^−/−mice, and 4.0 ± 0.13 and 3.88 ± 0.06 g/day respectively (p > 0.05) for castrated Ldlr^−/− mice.

Plasma cholesterol levels of all experimental groups were measured at the beginning and at the end of the experiment, and as expected, mice showed increased total cholesterol levels in response to feeding a high-fat diet (Fig. 1C). Sham-operated C57BL/6 mice demonstrated a 237.4% increase in their cholesterol levels, from 71.0 ± 0.9 mg/dL at week 0 to 239.6 ± 13.2 mg/dL at week 12 (p < 0.0001). Castrated C57BL/6 mice also increased their cholesterol levels by 167.7% at week 12, changing from 73.5 ± 2.0 to 196.8 ± 33.6 mg/dL (p < 0.05).

In both Ldlr^−/− groups, cholesterol levels were elevated at the beginning of the experiment and were further increased at the end of the study. Sham-operated Ldlr^−/− mice had a starting cholesterol level of 257.5 ± 11.4 mg/dL at week 0, which increased by 846.6% at week 12 to 2437.2 ± 271.5 mg/dL (p < 0.0001). Similarly, castrated Ldlr^−/− mice entered the study with cholesterol levels of 297.4 ± 2.9 mg/dL at week 0 and reached 2405.5 ± 244.8 mg/dL at week 12, showing a 708.9% increase (p < 0.0001).

Plasma TG levels of sham-operated C57BL/6 animals were 271.9 ± 6.0 mg/dL at week 0 and 68.5 ± 11.0 mg/dL at week 12, showing a remarkable decrease of 74.8% (p < 0.0001). Likewise, in castrated C57BL/6 mice, plasma TG levels decreased after a high-fat diet by 75.2% (200.9 ± 9 mg/dL at week 0 and 49.8 ± 5.8 mg/dL at week 12, p < 0.0001).

Similarly, at week 0, sham-operated Ldlr^−/− mice also demonstrated higher TG levels of 464.2 ± 14.7 mg/dL compared with 249.7 ± 62.3 mg/dL at week 12, corresponding to a 46.2% decrease (p < 0.05). On the other hand, castrated Ldlr^−/− mice had TG levels of 433.4 ± 6.9 mg/dL at week 0 and 255.7 ± 20.6 mg/dL at week 12, showing a 41% decrease (p < 0.0001; Fig. 1D).

Brain mitochondrial activity

At the end of the study, the relative protein expression of UCP2 and Cytc was determined in mitochondrial extracts of different brain regions as described in the Materials and Methods section (Fig. 2). In cerebral hemispheres (Fig. 2A, B), the relative UCP2 expression was 0.61 ± 0.01 au in sham-operated C57BL/6 mice and 0.77 ± 0.02 au in their castrated counterparts, corresponding to a 26.2% increase (p < 0.05). Likewise, castrated Ldlr^−/− mice exhibited increased UCP2 expression by 12.2% compared with sham-operated Ldlr^−/− mice (0.92 ± 0.02 au and 0.82 ± 0.02 au respectively, p < 0.05).

FIG. 2.

Western blot analysis and quantification of relative mitochondrial UCP2 and Cytc expression over COX4 in different brain regions (A, B) Cerebral hemispheres, (C, D) Cerebellum, (E, F) Midbrain, (G, H) Diencephalon. Results are presented as mean ± SEM (n = 2 for sham-operated C57BL/6 and Ldlr^−/− mice; n = 3 for castrated C57BL/6 and Ldlr^−/− mice; *p < 0.05; **p < 0.001; ^NSp > 0.05). Cytc, cytochrome c; UCP2, uncoupling protein 2.

In the same brain region, mitochondrial Cytc expression of castrated C57BL/6 mice appears slightly increased compared with sham-operated C57BL/6 mice where the difference between C57BL/6 and Ldlr^−/− mice did not reach statistical significance (0.91 ± 0.02 au vs. 0.084 ± 0.03 au, p > 0.05). TD increased mitochondrial Cytc levels in Ldlr^−/− mice by 15.7%, changing from 0.7 ± 0.02 au in sham-operated Ldlr^−/− mice to 0.81 ± 0.01 au in castrated Ldlr^−/− animals (p < 0.05).

In the cerebellum (Fig. 2C, D), no significant difference in relative UCP2 expression was noted in sham-operated and castrated C57BL/6 mice (1.12 ± 0.00 au vs. 1.13 ± 0.02 au, p > 0.05), whereas castrated Ldlr^−/− mice showed a 14.7% increase in UCP2 expression compared with their sham-operated counterparts (1.13 ± 0.01 au and 0.99 ± 0.03 au respectively, p < 0.05). Mitochondrial Cytc was 0.8 ± 0.01 au in C57BL/6 sham-operated mice and 0.93 ± 0.03 au in castrated C57BL/6 mice, showing an increase of 16.2% (p < 0.05).

In sham-operated Ldlr^−/− animals, the relative expression of Cytc was 0.74 ± 0.01 au compared with 0.93 ± 0.02 au in castrated Ldlr^−/− mice, thus it increased by 26% (p = 0.001).

When midbrain mitochondria samples were analyzed (Fig. 2E, F), a notable 18.8% decrease in UCP2 expression of castrated C57BL/6 mice compared with their sham-operated counterparts was noted (0.76 ± 0.03 au and 0.93 ± 0.01 au, respectively, p < 0.05). However, UCP2 was comparable in sham-operated and castrated Ldlr^−/− mice and the difference was not statistically significant (0.85 ± 0.02 au vs. 0.92 ± 0.02 au, p > 0.05).

Cytc of midbrain mitochondria in wild-type animals also had no difference among the sham-operated and castrated group (0.53 ± 0.01 au vs. 0.53 ± 0.03 au, p > 0.05). However, a 20.4% increase in mitochondrial Cytc of castrated Ldlr^−/− mice compared with sham-operated Ldlr^−/− mice was found (0.65 ± 0.02 au and 0.54 ± 0.01 au, respectively, p < 0.05).

The relative expression of mitochondrial UCP2 and Cytc in the diencephalon of wild-type mice was not affected by TD (1.17 ± 0.03 au vs. 1.14 ± 0.05 au for UCP2, and 0.84 ± 0.00 au vs. 0.85 ± 0.02 au for Cytc, p > 0.05; Fig. 2G, H). In the Ldlr^−/− groups, castrated mice exhibited higher UCP2 expression by 15% compared with their sham-operated counterparts (1.3 ± 0.04 au and 1.13 ± 0.00 au, respectively, p < 0.05), but no statistically significant differences in mitochondrial Cytc expression was noted (0.97 ± 0.01 au in sham-operated vs. 0.89 ± 0.08 au in castrated mice, p > 0.05).

Cytc-dependent apoptosis

After the assessment of mitochondrial activity in the brain, we examined the effects of TD on apoptosis in cerebral hemispheres, cerebellum, midbrain, and diencephalon (Fig. 3). We measured the ratio of the relative cytoplasmic Cytc levels over the relative mitochondrial Cytc levels (CM ratio) as a biochemical marker of apoptosis.²⁸ The higher the CM ratio the higher the extent of apoptosis, as the release of Cytc from mitochondria to cytoplasm signals the activation of the caspase cascade, ultimately leading cells to apoptosis.²⁸

FIG. 3.

Western blot analysis and quantification of Cytc-dependent apoptosis as expressed by the cytoplasmic to mitochondrial Cytc relative expression ratio over β-tubulin for cytoplasm and COX4 for mitochondria (A, B) Cerebral hemispheres, (C, D) Cerebellum, (E, F) Midbrain, (G, H) Diencephalon. Results are presented as mean ± SEM (n = 2 for sham-operated C57BL/6 and Ldlr^−/− mice; n = 3 for castrated C57BL/6 and Ldlr^−/− mice p < 0.05; **p < 0.001; ^NSp > 0.05).

In cerebral hemispheres (Fig. 3A, B), CM ratio was decreased by 22.8% in castrated C57BL/6 mice compared with sham-operated C57BL/6 mice (1.05 ± 0.01 au and 1.36 ± 0.1 au respectively, p < 0.05). Likewise, in the Ldlr^−/− groups, surgical castration resulted in a decrease of CM ratio by 9.5% compared with the control Ldlr^−/− group (1.05 ± 0.02 au and 1.16 ± 0.01 au, respectively, p < 0.05).

When we analyzed samples from the cerebellum (Fig. 3C, D), we found no significant difference in CM ratio between sham-operated and castrated wild-type mice (1.16 ± 0.01 au vs. 1.12 ± 0.04 au, p > 0.05). On the other hand, castrated Ldlr^−/− animals showed a decrease of 12.8% in CM ratio compared with their sham-operated counterparts (0.98 ± 0.03 au and 1.12 ± 0.01 au, respectively, p < 0.05).

In the midbrain (Fig. 3E, H), there were no differences in the wild-type groups (1.92 ± 0.1 au for sham-operated vs. 1.79 ± 0.21 au for castrated mice, p > 0.05); whereas in Ldlr^−/− groups, castrated mice had decreased CM ratio by 12.6% compared with sham-operated mice (1.53 ± 0.06 au and 1.75 ± 0.0 au, respectively, p < 0.05).

Of all brain regions tested, the diencephalon (Fig. 3G, H) was the only one where we found increased CM ratio by 21.5% in castrated Ldlr^−/− mice, compared with their sham-operated counterparts (0.96 ± 0.05 au and 0.79 ± 0.01 au, respectively, p < 0.05). In the diencephalon of C57BL/6 animals, there was no statistically significant difference in CM ratio between sham-operated and castrated mice (0.71 ± 0.01 au vs. 0.77 ± 0.03 au, p > 0.05).

Discussion

In a previous study, we showed that LDLR modulates the effects of hypogonadism on processes associated with weight gain and related metabolic perturbations in mice.¹¹ Data from other investigators indicate that when mice lacking a functional LDLR are exposed to a high-fat diet, they develop cognitive disorders and increased locomotor activity, which are behaviors accompanied by changes in cholinergic transmission in the anterior cerebral cortex.²⁹

In a similar direction, obesity and low testosterone levels have been linked with neuroinflammation and subsequently the emergence of neural impairments.⁸ Multiple studies in the literature correlate brain mitochondrial activity, specifically brain-expressed UCP2, with the emergence and progress of several brain disorders of a metabolic background, such as ischemic strokes, anxiety disorders, and memory–learning impairments. It is believed that mitochondrial UCP2-induced uncoupling leads to reduced mitochondrial membrane potential, and thus reduced production of ROS from the organelle.³⁰

This effect of UCP2 is considered to be of particular importance in the development of the aging process, neurodegenerative diseases, and behavioral disorders, all conditions tightly connected to increased ROS production.²³ At the same time, recent studies have shown that consumption of a high-fat diet leads to increased uncoupling and ATP production in microglia by inducing changes in mitochondrial morphology and increasing mitochondrial number.²²

These data led us to investigate the effects of hypogonadism on mitochondrial activity and apoptosis in different brain regions, in the presence and absence of LDLR in mice fed a western-type diet.

In this direction, four groups of experimental animals consisting of sham-operated or castrated C57BL/6 mice and sham-operated or castrated Ldlr^−/− mice were formed. After surgery, a 4-week recovery period allowed the elimination of testosterone from the circulation of castrated animals. Then, mice were exposed to a western-type diet for 12 weeks.

In agreement with our previous studies¹¹ as well as unpublished data, we observed that both sham-operated and castrated C57BL/6 and sham-operated Ldlr^−/− mice showed a significant increase in body weight after feeding a high-fat diet for 12 weeks, whereas castrated Ldlr^−/− mice developed resistance to diet-induced obesity. Moreover, we found no significant differences in food consumption during the study, suggesting that the observed changes in body weight were not due to food intake.

Our results also show that at week 12, total cholesterol levels were increased in all groups, but C57BL/6 mice maintained plasma cholesterol within normal limits, unlike Ldlr^−/− mice, whose total cholesterol levels were far above normal. On the other hand, plasma TG levels appear lowered in both C57BL/6 and Ldlr^−/− castrated mice, indicating that TD has a positive impact on plasma TG levels in mice. The observed benefit on plasma TG levels could be explained by the lack of estrogens after surgical castration, as they are also mainly synthesized in the testis.¹¹

To assess the effects of testosterone and/or LDLR deficiency on brain mitochondrial activity, we measured UCP2 and Cytc protein levels as markers of uncoupling and oxidative phosphorylation, respectively, in all four mouse groups. Castration did not appear to affect UCP2 expression in the cerebellum and diencephalon of C57BL/6 mice (Fig. 2D, H; Table 1), suggesting no effect on antioxidant defense mechanisms.

Table 1.

A Graphical Summary of the Changes in Uncoupling Protein 2 and Cytochrome c Expression After Castration in the Different Brain Domains of the Test Groups of the Study

UCP2 and Cytc expression	C57BL/6, sham vs. castrated		Ldlr^−/−, sham vs. castrated
UCP2 and Cytc expression	UCP2	Cytc	UCP2	Cytc
CH	↑	—	↑	↑
Cb	—	↑	↑	↑
MB	↓	—	—	↑
Dien	—	—	↑	—

Up arrows show an increase when p < 0.05; down arrows show a decrease when p < 0.05; dash is used when p > 0.05 between the sham-operated and castrated groups of each mouse strain.

Cytc, cytochrome c; Cb, cerebellum; CH, cerebral hemispheres; Dien, diencephalon; MB, midbrain; UCP2, uncoupling protein 2.

However, castration resulted in increased UCP2 expression in cerebral hemispheres and a notable decrease in the midbrain in this strain (Fig. 2B, F; Table 1). Castration increased Cytc levels only in mitochondria isolated from the cerebellum, whereas no change was found in all other brain domains. Taken together, these findings suggest that in wild-type mice the absence of testosterone had a positive impact in cerebral hemispheres and a negative impact in the midbrain, on the mechanisms aiming at reducing the production of ROS.

When mitochondria from various brain regions of sham and castrated Ldlr^−/− mice were analyzed, our data showed that castration led to an increase in UCP2 expression in cerebral hemispheres, which was accompanied with increased mitochondrial Cytc levels (Table 1), suggesting increased uncoupling and oxidative phosphorylation.³¹

This suggested that in the absence of testosterone, cells in this brain region activated mechanisms to reduce the production of ROS, while they simultaneously increased the rate of oxidative phosphorylation as an effort to counterbalance the decrease in ATP synthesis that would be caused by uncoupling of oxidative phosphorylation from the electron transport chain. A similar profile was observed in the cerebellum and the broader midbrain area of castrated Ldlr^−/− mice, where relative UCP2 and mitochondrial Cytc increased in tandem (Table 1).

As expected, these brain regions (cerebral hemispheres, cerebellum, and midbrain) of castrated Ldlr^−/− mice, having reduced oxidative stress, were protected from Cytc-dependent apoptosis, as shown by the decreased CM ratio (Table 2). On the contrary, in the diencephalon of castrated Ldlr^−/− mice, UCP2 expression appeared increased, but a parallel increase of mitochondrial Cytc was not evident.

Table 2.

A Graphical Summary of the Changes in the CM Ratio After Castration in the Different Brain Domains of the Test Groups of the Study

CM ratio	C57BL/6, sham vs. castrated	Ldlr^−/−, sham vs. castrated
CH	↓	↓
Cb	—	↓
MB	—	↓
Dien	—	↑

Up arrows show an increase when p < 0.05; down arrows show a decrease when p < 0.05; dash is used when p > 0.05 between the sham-operated and castrated groups of each mouse strain.

As a result, an increased ratio of cytoplasmic to mitochondrial Cytc was noted, suggesting an increase in apoptosis in the diencephalon of castrated Ldlr^−/− mice, possibly due to reduced ATP production because of increased uncoupling. Since this brain region is composed of the thalamus and hypothalamus, it is conceivable to hypothesize that in the absence of LDLR, TD may have a significant impact in mood disorders such as depression, since the thalamus is an important component of circuits involved in depression and anxiety.^32,33 Likewise, apoptosis in the hypothalamus could also affect various functions such as circadian rhythm, glucose homeostasis, metabolism, and sex hormones.^34–36

Lack of functional LDLR has been linked to learning and memory impairments, whereas high plasma cholesterol levels contribute to the aggravation of oxidative stress.³⁷ Given the fact that testosterone production also takes place in the brain with the conversion of cholesterol into pregnenolone in mitochondria being the very first step in testosterone biosynthesis,² the effects of TD on brain function could be modulated by LDLR, which may determine intracellular cholesterol availability for this biosynthetic process.

Conclusions

Our data suggest that TD differentially impacts the mitochondrial activity in different brain domains of C57BL/6 mice in a fashion highly modulated by LDLR. This finding agrees with our previous work showing that the effects of castration on white and brown adipose tissue mitochondrial activity are also affected by LDLR.¹¹ The precise mechanism of this modulation remains unknown and may be rooted to the role of this receptor in the local management of cholesterol and production of steroid hormones.

To our surprise, despite the fact that LDLR deficiency and TD independently promote neural dysfunction,^8,19,20 their concurrent existence in our experimental mice leads to positive effects in mitochondrial activity of cerebral hemispheres, cerebellum, and the broader midbrain region. Possibly, LDLR deficiency and TD when present simultaneously could have a protective effect on these regions against behavioral disorders linked to oxidative stress.²⁵

The well-established role of the brain region, such as the dorsal and prefrontal cortex, the amygdala and hippocampus, the caudate nucleus, the raphe nuclei, and the substantia nigra in depression and anxiety,^32,38 allows us to hypothesize that the observed increase in mitochondrial activity in cerebral hemispheres, cerebellum, and the midbrain could improve these particular behaviors in castrated Ldlr^−/− mice.

By translating our findings into the clinical setup, we could hypothesize that given the importance of these brain areas in depression and anxiety, the concurrent existence of LDLR deficiency and TD may have a protective impact on the clinical manifestation of these behavioral traits. On the other hand, the negative effects on mitochondrial activity of diencephalon are expected to favor the emergence of these behavioral disorders.

Since behavior is controlled by neuronal circuits rather than isolated brain domains,³² the observed apoptosis in the diencephalon of castrated Ldlr^−/− mice is expected to attenuate or even reverse the beneficial effects of concurrent LDLR deficiency and TD on the other brain domains. The relative contribution of these different brain domains to the development of depression and anxiety in an individual will eventually dictate the overall effect. Future behavioral studies could enlighten the exact consequences of concurrent TD and LDLR deficiency on depression and anxiety.

Footnotes

Authors' Contributions

K.A. and A.N. participated in the execution of experiments, evaluation of data, drafting of the article, and approval of its final submitted version. K.E.K. participated in the design of the studies, execution of experiments, evaluation of data, drafting of the article, and approval of its final submitted version.

Acknowledgments

The authors would like to thank Dr. Caterina Constantinou for technical assistance and advice during brain dissection and mitochondria isolation.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

Abbreviations Used

References

Morales

. The long and tortuous history of the discovery of testosterone and its clinical application. J Sex Med. 2013; 10(4):1178–1183.

Zirkin

, Papadopoulos

. Leydig cells: Formation, function, and regulation. Biol Reprod. 2018; 99(1):101–111.

Schiffer

, Arlt

, Storbeck

. Intracrine androgen biosynthesis, metabolism and action revisited. Mol Cell Endocrinol. 2018; 465:4–26.

Traish

, Guay

, Feeley

, Saad

. The dark side of testosterone deficiency: I. metabolic syndrome and erectile dysfunction. J Androl. 2009; 30(1):10–22.

Traish

, Saad

, Guay

. The dark side of testosterone deficiency: II. Type 2 diabetes and insulin resistance. J Androl. 2009; 30(1):23–32.

Svartberg

, Von Mühlen

, Mathiesen

, Joakimsen

, Bønaa

, Stensland-Bugge

. Low testosterone levels are associated with carotid atherosclerosis in men. J Intern Med. 2006; 259(6):576–582.

Derby

, Zilber

, Brambilla

, Morales

, McKinlay

. Body mass index, waist circumference and waist to hip ratio and change in sex steroid hormones: The Massachusetts Male Ageing Study. Clin Endocrinol (Oxf). 2006; 65(1):125–131.

Jayaraman

, Lent-Schochet

, Pike

. Diet-induced obesity and low testosterone increase neuroinflammation and impair neural function. J Neuroinflammation. 2014; 11(1):1–14.

Frye

, Edinger

, Lephart

, Walf

. 3alpha-androstanediol, but not testosterone, attenuates age-related decrements in cognitive, anxiety, and depressive behavior of male rats. Front Aging Neurosci. 2010; 2:15.

10.

Gold

, Voskuhl

. Testosterone replacement therapy for the treatment of neurological and neuropsychiatric disorders. Curr Opin Investig Drugs. 2006; 7(7):625–630.

11.

Constantinou

, Mpatsoulis

, Natsos

, et al. The low density lipoprotein receptor modulates the effects of hypogonadism on diet-induced obesity and related metabolic perturbations. J Lipid Res. 2014; 55(7):1434–1447.

12.

Zannis

, Kypreos

, Chroni

, Kardassis

, Zanni

. Lipoproteins and atherogenesis. In: Molecular Mechanisms of Atherosclerosis (Loscalzo J, ed). London: Taylor & Francis, 2004; pp 111–174.

13.

, Mani

. Low-density lipoprotein receptor (LDLR) family orchestrates cholesterol homeostasis. Yale J Biol Med. 2012; 85(1):19–28.

14.

Defesche

, Gidding

, Harada-Shiba

, Hegele

, Santos

, Wierzbicki

. Familial hypercholesterolaemia. Nat Rev Dis Prim. 2017; 3:17093.

15.

Breteler

MMB

, Claus

, Grobbee

, Hofman

. Cardiovascular disease and distribution of cognitive function in elderly people: The Rotterdam study. BMJ. 1994; 308(6944):1604.

16.

de Bruijn

RFAG

, Ikram

. Cardiovascular risk factors and future risk of Alzheimer's disease. BMC Med. 2014; 12(1):1–9.

17.

Kim

, Basak

, Holtzman

. The role of apolipoprotein E in Alzheimer's disease. Neuron. 2009; 63(3):287–303.

18.

Basak

, Verghese

, Yoon

, Kim

, Holtzman

. Low-density lipoprotein receptor represents an apolipoprotein E-independent pathway of Aβ uptake and degradation by astrocytes. J Biol Chem. 2012; 287(17):13959–13971.

19.

Mulder

, Koopmans

, Wassink

, et al. LDL receptor deficiency results in decreased cell proliferation and presynaptic bouton density in the murine hippocampus. Neurosci Res. 2007; 59(3):251–256.

20.

Strekalova

, Evans

, Costa-Nunes

, et al. Tlr4 upregulation in the brain accompanies depression- and anxiety-like behaviors induced by a high-cholesterol diet. Brain Behav Immun. 2015; 48:42–47.

21.

Hermes

, Nagy

, Waterson

, et al. Role of mitochondrial uncoupling protein-2 (UCP2) in higher brain functions, neuronal plasticity and network oscillation. Mol Metab. 2016; 5(6):415–421.

22.

Kim

, Yoon

, Jin

, Diano

. Microglial UCP2 mediates inflammation and obesity induced by high-fat feeding. Cell Metab. 2019; 30(5):952.e5–962.e5.

23.

Erlanson-Albertsson

. The role of uncoupling proteins in the regulation of metabolism. Acta Physiol Scand. 2003; 178(4):405–412.

24.

Gimsa

, Kanitz

, Otten

, et al. Alterations in anxiety-like behavior following knockout of the uncoupling protein 2 (ucp2) gene in mice. Life Sci. 2011; 89(19–20):677–684.

25.

Uttara

, Singh

, Zamboni

, Mahajan

. Oxidative stress and neurodegenerative diseases: A review of upstream and downstream antioxidant therapeutic options. Curr Neuropharmacol. 2009; 7(1):65–74.

26.

Gong

, Chai

, Ding

, Sun

, Hu

. Chronic mild stress damages mitochondrial ultrastructure and function in mouse brain. Neurosci Lett. 2011; 488(1):76–80.

27.

Sun

, Liu

, Dai

, Ding

, Hu

. Uncoupling protein 2 knockout exacerbates depression-like behaviors in mice via enhancing inflammatory response. Neuroscience. 2011; 192:507–514.

28.

Cai

, Yang

, Jones

. Mitochondrial control of apoptosis: The role of cytochrome c. Biochim Biophys Acta Bioenerg. 1998; 1366(1–2):139–149.

29.

Moreira

ELG

, De Oliveira

, Nunes

, et al. Age-related cognitive decline in hypercholesterolemic LDL receptor knockout mice (LDLr^-/-): Evidence of antioxidant imbalance and increased acetylcholinesterase activity in the prefrontal cortex. J Alzheimers Dis. 2012; 32(2):495–511.

30.

Normoyle

, Kim

, Farahvar

, Llano

, Jackson

, Wang

. The emerging neuroprotective role of mitochondrial uncoupling protein-2 in traumatic brain injury. Transl Neurosci. 2015; 6(1):179–186.

31.

Hüttemann

, Lee

, Grossman

, Doan

, Sanderson

. Phosphorylation of mammalian cytochrome c and cytochrome c oxidase in the regulation of cell destiny: Respiration, apoptosis, and human disease. Adv Exp Med Biol. 2012; 748(Complex V):237–264.

32.

Drevets

, Price

, Furey

. Brain structural and functional abnormalities in mood disorders: Implications for neurocircuitry models of depression. Brain Struct Funct. 2008; 213(1–2):93–118.

33.

Vertes

, Linley

, Hoover

. Limbic circuitry of the midline thalamus. Neurosci Biobehav Rev. 2015; 54:89–107.

34.

Clarke

. Hypothalamus as an endocrine organ. Compr Physiol. 2015; 5:217–253.

35.

Saper

, Scammell

, Lu

. Hypothalamic regulation of sleep and circadian rhythms. Nature. 2005; 437(7063):1257–1263.

36.

Coll

, Yeo

GSH

. The hypothalamus and metabolism: Integrating signals to control energy and glucose homeostasis. Curr Opin Pharmacol. 2013; 13(6):970–976.

37.

Vitali

, Wellington

, Calabresi

. HDL and cholesterol handling in the brain. Cardiovasc Res. 2014; 103(3):405–413.

38.

Pandya

, Altinay

, Malone

, Anand

. Where in the brain is depression?. Curr Psychiatry Rep. 2012; 14(6):634–642.