| Background:Adipose tissue is identified as an important endocrine organ and participates in the regulation of diet intake, energy balance, glucose and lipid metabolism. Adipose tissue synthesize and secret a mass of bioactive peptides and proteins which are defined as adipokines. The adipokines can be divided into two groups, one originates from fat tissue and other tissues, such as THF a , and the other mainly comes from adipose tissue such as leptin. These fat-detrived adipokines can exert effects on the regulation of appetite, energy balance, insulin sensitivity and lipid metabolism and they also participate in immunological reaction. The adipokines, which can not only affect adipocyte’s function through autocrine and paracrine but also exert metabolic effects on other organs and systems through circulation, play an important role in explaining the link between visceral adipose accumulation and T2DM, cardiovascular diseases and nonalcoholic fatty liver disease.As the major triglyceride storing organ, disorders in the synthesis and secretion of adipokines induced by obesity has been widely studied. Primary studies demonstrated that excess cholesterol accumulation in adipocytes can lead to hypertrophied adipocytes and increased fluidity of plasma membrane, resulting to dysfunctional adipocytes under obesity state. However, there were few researches focused on effects of high cholesterol intake on the synthesis and secretion adipokine function of adipose tissue. Most studies observed the toxic effects under obesity state. Nevertheless, little attention has been paid to the obesity-independent effect of high cholesterol on adipose function. Our experiment adopted calories-equally added high cholesterol diet to contruct hypercholesterolemia animal model to observe the dynamic effects of high cholesterol diet on the level of fat-detrived adipokins in serum and visceral adipose tissue.Objectives:1. To investigate the effects of high cholesterol diet on rats insulin index.2. To investigate the effects of high cholesterol intake on adipokines (leptin, adiponectin, resistin, visfatin and glypican4) levels in serum and visceral adipose tissue (VAT).3. To observe the time-dependent effects of high cholesterol intake.Methods:1. Animal model:sixty SD rats were randomly divided into normal diet (NC) and high cholesterol diet (HC) groups according to their body weight after adaptive feeding for one week. And then the rats in two groups were assigned to standard feed and high cholesterol supplemented feed respectively.2. Specimen collection:during the treatment period, blood samples were obtained by subclavian venous puncture at the Oth,4th,12th, and 18th weeks. After killing rats at the Oth,4th, and 18th weeks, perirenal fat tissues were quickly dissected and stored in liquid nitrogen.3. Serum lipid analysis:serum levels of total cholesterol, triglycerides and low density lipoprotein cholesterol (LDL-C) were measured using Olympus AU5400 system.4. Measurement of Fasting blood glucose (FBG) levels and fasting serum insulin (FINS) levels:fasting blood glucose (FBG) levels were measured by the glucose oxidase method, fasting serum insulin (FINS) levels were measured by radioimmunoassay. The insulin resistance (HOMA-IR) value was calculated.5. Adipokines in serum and VAT were measured by enzyme-linked immunosorbent assays.Results:1. Serum cholesterol levels and serum LDL-C levels were increased in high cholesterol diet fed rats.2. Obvious increase of HOMA-IR value were only found in rats fed high cholesterol diet at the 18th week, suggesting the occurrence of insulin resistance.3. Long term high cholesterol diet (18 weeks) increased leptin levels in VAT.4. Resistin levels swung in serum and VAT during treatment of high cholesterol diet.5. High cholesterol diet induced the increase of visfatin levels in serum and VAT at the early stage and decreased at the late stage.6. With the lasting of treatment time, adiponectin levels in serum and VAT elevated gradually in rats fed with high cholesterol diet.7. Long term high cholesterol diet (18 weeks) increased glypican4 levels in VAT.Conclusions:1. Insulin resistance and adipose tissue dysfunction occurred in rats fed with long term high cholesterol diet. Long term high cholesterol diet (18 weeks) increased leptin and glypican4 levels in VAT, and it induced the increase of visfatin levels in serum and VAT at the early stage and decreased at the late stage. Resistin levels swung in serum and VAT during treatment of high cholesterol diet, and the adiponectin levels in serum and VAT elevated gradually in rats fed with high cholesterol diet.2. Under the excess cholesterol intake treatment, adipose tissue can secret insulin rasistance induced adipokines, most obviously characterized by resistin. Adipose tissue can also secret protective adipokines such as adiponectin at the same time. Such results may be resulted from the activated compensatory respons and fully inllustrated the important regulatory faction of adipose tissue under high cholesterol intake.Background:Male hypogonadism has high prevalence rate worldwide and seriously reduces quality of life. Male hypogonadism refers to a clinical syndrome caused by the failure of the testis to produce physiological concentrations of testosterone or normal amounts of sperm, mainly manifested as low testosterone level with some clinical syndromes such as low libido, erectile dysfunction and decreased virility. The testis, which is the most important male reproductive endocrine gland, has two seminal functions:production of testosterone and spermatogenesis. Testosterone, which is produced by Leydig cells, palys a vital role in the maintenance of the male phenotype and participates in the occurrence and development of osteoporosis, metabolic syndrome and cardiovascular diseases. Spermatogenesis occurs in sperm cells within the seminiferous tubules, depending on Sertoli cells to provide nutrition support. Both testosterone and sperm perform the physiological function of virilization, physical strength, behavior, and reproduction.Cholesterol is an essential component of the human diet and performs an important role in synthesis of steroid hormones and vitamin D. However, excess dietary cholesterol intake is a crucial risk factor for mortality and morbidity, not only for cardiovascular diseases, but also for nonalcoholic fatty liver disease, cancer and Alzheimer’s disease. Apart from the above, the effect of high cholesterol diet on reproductive function has also received clinical attention recently. Our previous epidemiological studies suggested that serum total testosterone levels were significantly and inversely correlated with serum total cholesterol levels in 4114 middle-aged and older Chinese subjects, consistent with Tobias’s conclusion. Limited animal studies have also demonstrated low serum testosterone levels and decreased sperm quality and quality in hypercholesterolemic rats and mice. Based on these studies, the underlying mechanism has not been elucidated in depth. The harmful effects of high cholesterol diet may associated with the testicular failure to synthesis physiological level of testosterone and sperm, but the further mechanism is unclear.Free cholesterol (FC) can induce cytotoxicity through the following mechanisms: 1. Loss of cellular membrane fluidity:a physiological FC/phospholipid ratio in cellular membranes is necessary to maintain proper membrane fluidity. However, when the FC/phospholipid ratio rises above a physiological level, the liquid-ordered rafts may become too rigid, and the liquid-crystalline domains may begin to lose their fluidity.2. Altered membrane proteins:high FC levels might kill cells in part by inhibiting one or more integral membrane proteins. Similar observations have been made with proteins residing in internal membranes, such as the Na+-Ca2+transporter in the endoplasmic reticulum.3. Cholesterol crystallization and oxysterol formation: other mechanisms of cellular toxicity associated with FC accumulation include intracellular cholesterol crystallization and oxysterol formation. Needle-shaped cholesterol crystals form when the FC/phospholipid ratio reaches a very high level. Excess intracellular FC accumulation can also promote the oxidation of cholesterol to oxysterols, some of which may be cytotoxic. Free cholesterol accumulation can change the the membrane fluidity and protein function of the endoplasmic reticulum (ER), causing an accumulation of misfolded and unfolded ER proteins and triggering ER stress. A high cholesterol diet reportedly contributes to the activation of ER stress in the mouse hippocampus and aortic valve, leading to cognitive deficits and aortic valve calcification. After cholesterol loading, unfolded protein response (UPR) is activated in hepatic L02 cell. The emerging evidence indicate that excess cholesterol may serve as an inducer of ER stress. However, it remains unanswered whether ER stress contributes to the occurrence and development of testicular dysfunction under high cholesterol.Objectives:1. To investigate the effects of high cholesterol diet on rats serum testosterone level.2. To investigate the effects of high cholesterol intake on Leydig cell, Sertoli cell and germ cells in rat testis.3. To observe underlying mechanism of high cholesterol diet induced testis dysfunction.Methods:1. Animal model:sixty SD rats were randomly divided into normal diet (NC) and high cholesterol diet (HC) groups according to their body weight after adaptive feeding for one week. And then the rats in two groups were assigned to standard feed and high cholesterol supplemented feed respectively.2. Specimen collection:during the treatment period, blood samples were obtained by subclavian venous puncture at the 4th,8th,12th, and 16th weeks. After killing rats at the 6th weeks, testis tissues were quickly dissected and fixed in 4% paraformaldehyde or stored in liquid nitrogen.3. Serum lipid analysis:serum levels of total cholesterol, triglycerides and low density lipoprotein cholesterol (LDL-C) were measured using Olympus AU5400 system.4. Measurement of serum testosterone levels:serum testosterone levels were measured by radioimmunoassay.5. Testis free cholesterol was extracted and assayed. Oil Red O Staining and Filipin Staining were used to assess testicular lipid accumulation.6. Hematoxylin and eosin (H&E) staining was used to evaluate the histological changes. Western blotting, immunohistochemistry and immunofluorescence were adopted to determine the expression of Leydig cell, Sertoli cell and germ cells makers-3β HSD, WT-1 and DAZL.7. RT-PCR was adopted to determine relative mRNA levels of testosterone synthesis related molecules such as StAR, P450scc,3β-HSD, P450c17 and 17β-HSD. Protein extraction and immunoblotting analysis were adopted to determine the protein expression of StAR, P450scc,3p-HSD, P450cl7.8. Protein extraction and western blotting analysis was adopted to determine the protein expression and phosphorylation of endoplasmic reticulum stress related proteins such as Bip、eIF2 α、IRE1 α、ATF6.Results:1. In high cholesterol diet fed rats, serum cholesterol levels and serum LDL-C levels were increased and serum testosterone levels were decreased.2. Free cholesterol deposition was discovered in the testes of rats fed a high cholesterol diet.3. High cholesterol diet decreased the expression of Leydig cell, Sertoli cell and germ cells makers.4. Compared with the NC group, expression of steroidogenic enzymes such as StAR, P450scc,3β-HSD, P450c17 were reduced in the HC group rats.5. Enhanced endoplasmic reticulum stress reflected by high expression of Bip、 ATF6 and strong eIF2a phosphorylation was discovered in the testes from Rats Fed a High Cholesterol Diet.Conclusions:1. Excess cholesterol intake can increase serum total cholesterol and LDL-C levels along with testicular lipid deposition.2. Excess cholesterol intake induce testicular disorder, manifested as low testosterone level and reduced expression of Leydig cell, Sertoli cell and germ cells makers-3 β HSD, WT-1 and DAZL.3. Excess cholesterol can trigger testicular endoplasmic reticulum stress, and it may serve as one of the underlying mechanisms in high cholesterol induced testicular dysfyction. |
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