Effects of high-fat diet-induced obesity on metabolism and sexual development of juvenile male rats

doi:10.21203/rs.3.rs-5302221/v1

Objective

To explore the effects of high-fat diet-induced obesity on the metabolism and sexual development of juvenile male rats.

Methods

Three-week-old male rats were divided into the control group and the model group. The two groups were fed normal and high-fat diets, respectively, for four weeks. Modeling was successful if Lee’s index exceeded the upper limit of Lee’s index of the control group. Levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), alanine transaminase (ALT), aspartate transaminase (AST), testosterone (T), estrogen (E2), and insulin-like growth factor 1 (IGF-1) of the rats were monitored. Morphological structures of hepatic and testicular tissues were examined by hematoxylin and eosin (H&E) staining; hepatic steatosis was investigated by Oil Red O staining; and aromatases were detected using the immunohistochemical method.

Results

Weight, waist-to-height ratio, Lee’s index, weight and thickness of visceral fat, levels of ALT, AST, TC, TG, LDL-C, E2, and IGF-1 of rats were significantly higher in the model group than in the control group (P < 0.05). H&E staining revealed that rats in the model group had hepatic steatosis, a disorder of seminiferous tubules, and a reduced quantity of spermatogenic cells. Oil Red O staining revealed a significantly increased accumulation of lipid droplets in hepatic cells. Immunohistochemical analysis revealed that aromatases in hepatic and testicular cells were up-regulated.

Conclusion

A high-fat diet induces dyslipidemia, thereby causing hepatic steatosis and liver dysfunction, and it significantly influences testicular development, as an increased level of aromatase leads to a reduced T level.

high-fat diet

lipid metabolism

liver

testis

sex hormones

Childhood obesity has become a global epidemic ^[1]. Its occurrence is influenced by several factors, among which changes in dietary structure, such as high-fat diets, greatly increase the risk of obesity. A high-fat diet increases the accumulation of lipids, inducing hyperlipidemia. Hyperlipidemia plays an essential role in metabolic diseases, leading to hepatic steatosis, Type 2 diabetes, and atherosclerosis ^[2]. It also affects the functions of the hypothalamo-pituitary-gonadal axis, such as reducing testosterone (T) levels and increasing the risk of functional hypogonadism ^{[3, 4]}. The influence of obesity on the reproductive system of male adults has been widely studied. However, some childhood obesity may persist into adulthood. Therefore, adult obesity is closely related to pubertal obesity, further highlighting the importance of studying the impact of pubertal obesity on the body.

The correlation between boy obesity and the onset of adolescence in boys remains controversial. A multicenter epidemiological survey in China analyzed 9812 6-18-year-old boys and found that obesity was significantly positively correlated with sexual maturity ^[5], consistent with other previous studies ^[6]. Crocker et al. reported a negative correlation between the degree of obesity in boys and testicular volume and pubic hair development. This might be related to the increased expression of aromatase in adipose tissue, promoting the conversion of T to estrogen (E2) and the occurrence of insulin resistance ^[7]. Oehme et al. conducted cross-sectional studies and found that boys with low body mass index (BMI) exhibited delayed sexual development, while boys with high BMI had similar sexual development time as boys with normal weight ^[8]. The inconsistency in these studies may be related to differences in the selection of indicators or the study population.

Herein, a pubertal obesity rat model was constructed by administering a high-fat diet before sexual maturation to explore the effects of obesity on metabolism and sexual development.

2.1 Materials

twenty-two specific-pathogen-free Sprague Dawley (SD) male rats (3 weeks old, weighing 50–55 g) were maintained at a room temperature of 23–25℃ and on a 12/12-h light/dark cycle, with ad libitum access to drinking water and food.

2.2 Grouping and feeding

The rats were randomly divided into the normal diet group and the high-fat diet group. The normal diet group received a regular diet as the control group (11 rats). The high-fat diet group received a high-fat and high-calorie diet as the model group (11 rats). All rats were raised until 7 weeks of age.

2.3 Indicators

Food and water were withdrawn 6 h before measurement. Body length (distance from nose tip to anus), BMI, waist circumference, penis length, and testicular size were measured. Lee’s index was calculated as follows: Lee’s index = (\(\:\sqrt[3]{\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:}\times\:1000\)/body length (cm)) ^[9].

Criteria for successful modeling: Lee’s index of the model rat exceeded the upper limit of Lee’s index of the control group (mean ± standard deviation).

2.4 Reagents

(1) Polyformaldehyde (Shanghai Sangon, China); (2) phosphate-buffered saline (PBS) powder (Zhongshan, China); (3) absolute alcohol (Sinopharm, China); (4) hematoxylin stain (Nanjing Jiangcheng, China); (5) eosin solution (Beyotime, China); (6) neutral resin (Sinopharm, China); (7) primary cytochrome P450 antibody (rabbit-derived) (Zen-bioscience, China); and (8) horseradish peroxidase-labeled secondary antibody (goat anti-rabbit) (Abcam, USA).

2.5 Methods

After anesthetizing rats with 7% chloral hydrate (0.5 mL/100 g), approximately 10 mL of blood was collected from the abdominal aorta and transferred to a pro-coagulant tube. The blood was left to settle for 2 h and then the serum was stored at -20℃. The levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), alanine transaminase (ALT), aspartate transaminase (AST), T, E2, and insulin-like growth factor 1 (IGF-1) were detected.

After blood collection, hepatic and testicular tissues were collected separately, and residual blood was washed with PBS. Samples were fixed with 4% paraformaldehyde for hematoxylin and eosin (H&E) staining and immunohistochemical detection. Some hepatic tissues were made into frozen sections and used for Oil Red O staining to observe hepatic steatosis.

Tissues were dehydrated, cleared, embedded in paraffin, and sliced (thickness = 5 µm). Sections were deparaffinized for further H&E staining. Morphological changes of the liver and testicles were observed under an optical microscope. Sections were dewaxed, antigen retrieved, blocked with goat serum, and incubated with a primary aromatase antibody (dilution ratio = 1:100) at 4℃ overnight. Sections were incubated with a goat anti-rabbit secondary antibody (dilution ratio = 1:50) at 37℃ for 30 min. After color development with diaminobenzidine, sections were counterstained with hematoxylin for 5 min, and expression changes of aromatase were observed under an optical microscope. Pale yellow indicates positive expression.The degree of hepatic steatosis was observed after staining, counterstaining and sealing of frozen sections, and the lipid droplets were orange.

2.6 Statistical analysis

Statistical analysis was performed using SPSS 23.0 software, and data were expressed as mean ± SD. Two independent sample t-tests were used to compare normally distributed data, and a non-parametric test was used for comparison of non-normally distributed data. P < 0.05 was considered statistically significant and P < 0.01 was considered highly statistically significant.

3.1 Analysis of body surface indicators

Rats were raised to 7 weeks of age for comparative analysis. The results showed that the weight, waist-to-height ratio and Lee’s index were significantly higher and penis length was significantly lower in the model group than in the control group (P < 0.05). No significant differences in testicular volume of rats were found between the two groups (P > 0.05).

3.2 Measurement of the fat pad and testicle

Three rats were randomly selected from each group for fat pad and testicular measurements. The results showed that visceral fat weight, fat thickness of epididymis, perirenal area, and the greater omentum were significantly increased in the model group had compared with the control group, with statistically significant differences (P < 0.05). However, no statistically significant differences in testicular weight of rats were found between the two groups (P > 0.05).

3.3 Serum biochemical indicators

ALT, AST, TC, TG, LDL-C, E2, and IGF-1 levels increased significantly and HDL-C and T levels decreased drastically in the model group compared with the control group, with statistically significant differences (P < 0.05).

3.4 H&E staining of liver cells and testicles

Liver cells had normal morphology and regular arrangement, and the nucleus was located in the center of the cell in control group rats. However, liver cells showed fat vacuoles that squeezed the nucleus in model group rats, resulting in varying degrees of steatosis. Additionally, the control group exhibited a regular arrangement of spermatogenic cells at all levels in the seminiferous tubules, with a complete and clear structure. However, intercellular connections in the upper layer of the seminiferous tubules were loose and the number of spermatogenic cells decreased in the model group.

3.5 Oil Red O staining of liver cells

The cytoplasm of liver cells appeared light blue, and the nucleus appeared dark blue in the control group, with a small amount of lipid droplets (red particulates). The model group showed a significant increase in lipid droplets in liver cells, with a red cytoplasm.

3.6 Immunohistochemical detection of aromatases in hepatic and testicular tissues

Accumulative and average optical densities of aromatases increased significantly in the hepatic cells of rats in the model group compared with those in the control group, with statistically significant differences (P < 0.05). Moreover, accumulative and average optical densities of aromatases increased markedly in the testicular cells of rats in the model group compared with those in the control group, with statistically significant differences (P < 0.05).

After excessive intake of high-calorie foods, adipocytes store excess glucose and fat as TGs ^[10], with limitation. After reaching a critical level, the body will contribute to fat production by increasing the number of adipocytes ^[11]. Hyperplasia of abdominal adipose tissues leads to abdominal obesity due to high-fat production activity in the mesenteric adipose tissues ^[12]. At the end of feeding, Lee’s index increased significantly in the model group compared with the control group, and the total fat content in the epididymis, perirenal area, and greater omentum was significantly higher in the model group than in the control group, confirming the above viewpoint. The waist-to-height ratio, an important index to evaluate abdominal obesity ^[13], provided guiding significance in predicting risk factors for cardiovascular diseases ^[14]. Clinically, hypertriglyceridemia, low HDL-C, and high LDL-C, termed “atherogenic lipid triad” ^[15], have been considered the main risk factors for cardiovascular diseases. Our data demonstrated that the waist-to-height ratio increased significantly in the model group compared with the control group. Moreover, TC, TG, and LDL-C levels were significantly higher and the HDL-C level was lower in the model group than in the control group, consistent with the research results of obesity-induced dyslipidemia in humans ^[16].

Under a high-fat diet, adipocytes continuously break down to produce a large amount of free fatty acids and enhance the activity of lipoprotein lipase, leading to dyslipidemia and non-alcoholic fatty liver diseases ^[17]. Meanwhile, H&E staining revealed that the arrangement of liver cells in the model group was disordered during this period, with some rats developing steatosis. Oil Red O staining showed a significant increase in lipid droplets in liver cells. Lipid metabolism disorder and the increased synthesis and decreased oxidation of free fatty acids led to the accumulation of lipids and lipoproteins in cells, causing liver cell dysfunction ^[18]. Additionally, ALT and AST levels were significantly higher in the model group than in the control group.

Currently, there is no uniform conclusion on the relationship between obesity and the onset of sexual development in boys. Lee et al. found that obese boys had delayed adolescence compared with normal and overweight boys based on a large-scale American population (3872 boys) ^[19]. Holmgren et al. defined adolescent development as the increase in height during adolescence and found that the adolescence of overweight and obese boys started 2.5–3.5 months earlier than normal-weight children ^[20]. A different study reported that obese children had increased insulin sensitivity and levels of gonadotropins (follicle-stimulating hormone and luteinizing hormone (LH)) and sex hormone-binding globulin (SHBG) after weight loss, suggesting that obesity had negative influences on the sexual development of boys ^[21]. The present study found that prepubertal obesity first affected the penis length of rats, which was significantly shorter than that of the control group. Although status changes in testicular tissues were observed under light microscopy, testicular volume and mass were not significantly affected.

Excess accumulation of adipose tissues in the body enhanced aromatase activity, promoting the conversion of T into E2 ^[22]. Increased E2 levels further inhibited the function of the hypothalamus (gonadotropin-releasing hormone)-pituitary (LH)-testicular (Leydig cells) axis. Moreover, E2 can further reduce T synthesis by inhibiting 17a-hydroxylase activity ^[23]. Additionally, obesity can lead to insulin resistance, and insulin can inhibit the production of androgen and increase its clearance rate in the liver ^[24], resulting in decreased serum levels of T. The current study found that the level of aromatase in the liver and testicular tissues increased in the model group compared with the control group. In addition, the level of E2 increased significantly and that of T decreased substantially in the model group compared with the control group, ultimately affecting the development of the penis. IGF-1, a polypeptide hormone composed of 70 amino acids, is mainly produced in the liver. It is also stimulated by growth hormones and insulin endocrine in the liver ^[25].

IGF-1 is closely related to various metabolic diseases, including obesity, Type 2 diabetes, and cardiovascular diseases ^{[26, 27]}. Our study showed that the level of IGF-1 was significantly higher in the model than in the control group, which is related to the regulation of the stability and biological activity of IGF-1 by insulin-like growth factor binding protein-1 (IGFBP-1) produced by the liver and insulin regulation. Adipose tissue, an endocrine organ, is closely related to insulin sensitivity. The accumulation of visceral fat leads to increased production of pro-inflammatory cytokines, including interleukin-6 (IL-6), tumor necrosis factor-alpha, and IL-8, which reduces insulin sensitivity and results in hyperinsulinemia and even insulin resistance ^[28]. However, we did not detect the insulin level but we speculate that insulin levels in the model group are elevated. Hyperinsulinemia not only reduces IGFBP-1 levels ^[29] but also directly stimulates the production of IGF-1. Low levels of IGFBP-1 can promote IGF-1 activity ^[30], ultimately leading to increased IGF-1 secretion. Elevated IGF-1 levels also indicate the occurrence of metabolic disorders in rats.

In summary, the sexual maturation of SD rats is approximately 6–10 weeks after birth ^[31]. We constructed a pubertal rat model of high-fat diet-induced obesity (7 weeks) to investigate the effects of prepubertal obesity on metabolism and sexual development. The findings demonstrated that a high-fat diet led to obesity, and the dyslipidemia induced by obesity not only affected the metabolic function of the liver but also damaged the reproductive system of male rats. Therefore, for children with prepubertal obesity, early dietary and weight management is essential to avoid the consequences of obesity.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Liaocheng People’s Hospital.

Consent for publication

All authors approved the final manuscript and the submission to this journal.

Competing interests

The authors declare no competing interests.

Funding

No funding was supported in this study.

Author Contribution

Conceptualization: Guimei Li.Data curation: Shujuan Guo.Formal analysis:Juan Zheng.Investigation: Shujuan Guo.Methodology:Juan Zheng.Project administration: Juan Zheng.Resources: Guimei Li.Software: Shujuan Guo.Supervision: Guimei Li.Validation: Juan Zheng.Visualization: Shujuan Guo.Writing – original draft: Shujuan Guo.Writing – review & editing: Juan Zheng.All authors reviewed the manuscript.

Acknowledgments

We would like to thank the study participants for their time.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Table 1

Comparison of physical parameters of model and control rats
	Model rats (n = 11)	Control rats (n = 11)	P-value
Body weight (g)	193.96 ± 18.07	177.78 ± 11.91	0.022^*
W/H	0.77 ± 0.03	0.62 ± 0.03	< 0.001^*
Lee’s index	312.73 ± 5.20	280.87 ± 5.40	< 0.001^*
Penis length (mm)	9.09 ± 0.39	11.06 ± 1.10	< 0.001^*
Testicular volume (cm³)	1.86 ± 0.24	2.10 ± 0.34	0.075
*: P < 0.05
W/H: ratio of waist circumference to body length

Table 2

Comparison of visceral fat and testis of model and control rats
	Model rats (n = 3)	Control rats (n = 3)	P-value
Visceral fat (g)	7.85 ± 0.89	4.95 ± 1.05	0.022^*
Testicular weight (g)	1.22 ± 0.08	1.11 ± 0.52	0.105
Epididymal fat (mm)	4.38 ± 0.44	3.36 ± 0.35	0.035^*
Perirenal fat (mm)	4.63 ± 0.25	3.27 ± 0.29	0.004^*
Greater omentum (mm)	3.11 ± 0.22	1.98 ± 0.30	0.006^*
^*: P < 0.05

Table 3

Comparison of serological indices of model and control rats
	Model rats (n = 3)	Control rats (n = 3)	P-value
ALT (IU/L)	117.89 ± 2.35	33.36 ± 0.62	< 0.001^*
AST (IU/L)	84.06 ± 0.86	28.61 ± 1.08	< 0.001^*
TC (mmol/L)	20.51 ± 0.08	6.37 ± 0.10	< 0.001^*
TG (mmol/L)	8.25 ± 0.05	2.54 ± 0.04	< 0.001^*
HDL-C (mmol/L)	1.27 ± 0.01	2.89 ± 0.18	< 0.001^*
LDL-C (mmol/L)	6.96 ± 0.08	2.93 ± 0.09	< 0.001^*
E2 (ng/L)	13.36 ± 0.22	5.19 ± 0.17	< 0.001^*
T (ng/mL)	0.53 ± 0.02	1.24 ± 0.01	< 0.001^*
IGF-1 (µg/L)	9.58 ± 0.23	5.14 ± 0.11	< 0.001^*
^*: P < 0.05
ALT, alanine transaminase; AST, aspartate aminotransferase; TC, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; E2, estrogen; T, testosterone; IGF-1, insulin-like growth factor 1.

No competing interests reported.

Effects of high-fat diet-induced obesity on metabolism and sexual development of juvenile male rats

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