The Effects Of Lipotoxicity On Pituitary Function

Background:The "Western Diet" is rich in lipids, including cholesterol and saturated fats, and is low in fiber, fruits and vegetables. This diet is well known for causing cardiovascular diseases, obesity, and diabetes. Studies are currently focused on the role of the "Western Diet", but there is a critical lack of investigation of the effect of cholesterol, which is one of the main lipid components. Previous studies have reported that lipid deposition in many non-adipose tissues, including liver, heart, pancreas, kidneys, even hypothalamus is detrimental to organ function. Araujo RL, et al and Sanchez-Garrido MA, et al reported that there are correlations between some pituitary hormones and high-fat lard diet. To date, however, few reports in the literature have investigated whether excessive intake of cholesterol uniquely contributes to the pathogenesis of pituitary dysfunction.The pituitary gland, a crucial neuroendocrine organ, includes adenohypophysis and neurohypophysis, according to the embryology and histology characteristics. It also is divided by anterior lobe, intermedia lobe and posterior lobe. The gland’s main function is to synthesize and secrete different types of hormones. Thyroid-stimulating hormone (TSH, also known as thyrotropin), follicular stimulating hormone (FSH), luteinizing hormone (LH) and adrenocorticotropin (ACTH) are derived from the basophilic cells of the anterior pituitary. FSH and LH also known as gonadotropic hormone (GnH). TSH, FSH and LH consist of a common a-subunit and a unique β-subunit; the latter is responsible for hormone specificity and confers biological activity on each hormone. All of these hormones play important roles in metabolic homeostasis in the body. For example, TSH controls energy metabolism, FSH and LH are indispensable for reproduction, and ACTH participates in the stress response. Inappropriate hormonal output can lead to metabolic disorders as well as disease, so maintenance of normal anterior pituitary structure and function is necessary for human health.Cholesterol is a component of the cell membrane, and excess cholesterol accumulation affects the stability and fluidity of the membrane, leading to cellular dysfunction. However, it is unclear whether a high-cholesterol diet leads to an accumulation of cholesterol in the pituitary gland and whether induction of pituitary dysfunction is secondary to this accumulation.In the present study, we assessed the relationship between cholesterol and pituitary hormone. The evidence suggests that a high-cholesterol diet could affect the anterior pituitary gland, inducing not only morphological but also potentially functional changes.Objectives:1. The purpose of this study was to investigate the effects of high-cholesterol diet on rat’s pituitary, including serologic and morphological changes.2. To observe the relationship between hypercholesterolemia and serum pituitary hormone levels.Methods:Part 1:Animal Study1. Animal model:Forty male Sprague-Dawley rats (180±10 g,6 weeks) were acclimatized feeding for 1-week, then randomly assigned to two groups:(1) normal control diet (n=20):100% standard rodent chow (NC group,3.37 kcal/g); (2) high-cholesterol diet (n=20):1% cholesterol+98.7% standard rodent chow+0.3% cholate (HC group,3.24 kcal/g). All rats feed for 28-week. We collected fasting blood samples by subclavian venous puncture at the 0-,4- and 28-week, respectively. All rats were sacrificed at the 28th week, and collecting the fasting blood samples and pituitaries.2. Serum lipid profile and fasting blood glucose analysis:Using automated spectrophotometry measured serum triglycerides (TG), total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C) and fasting plasma glucose (FPG) levels.3. Serum TSH, FSH, LH and ACTH analysis:Using ELISA kits to measure the serum TSH, FSH, LH and ACTH levels at the 0-,4- and 28-week, respectively.4. Pituitary lipid content assay:Using enzymatic methods to measure the pituitary total cholesterol, free cholesterol, triglycerides and free fatty acids contents.5. Pituitary morphological evaluation:The volume observation of rat pituitary was conducted. Observe the number of basophilic cells in rat pituitary by H&E.6. Pituitary tissue pituitary hormones assay:Using ELISA to measure the content of TSH, FSH, LH and ACTH in each anterior pituitary.7. The mRNA level of pituitary hormones assay:The mRNA expression of FSHβ、LHβ and ACTH in rat pituitary were measured by real time-PCR.8. The protein level of pituitary hormones assay:The protein expression of FSHβ、LHβ and ACTH in pituitary were measured by immunohistochemistry.Part 2:Epidemiological Study1. Subjects:This cross-sectional study was performed in Ningyang of Taian (Shandong Province, China), the people aged≥40 years old, lived here≥5 years were invited to receive an examination. According to the inclusion and exclusion criteria, then stratified according to the age, SBP, DBP, FPG and HbAlc, respectively. Finally,137 men aged 45-59 from the general population were evaluated.2. Detected index:Past medical history was evaluated with a questionnaire and blood pressure, height, weight, waist circumference and hip circumference were measured. We collected blood samples from all the patients during 8:00 to 10:00 after a minimum 8 h overnight fast. We used an Auto Biochemical Analyzer (OLYMPUS AU5400, Olympus, Japan) to measure the serum TC, TG, LDL-C, HDL-C and FPG levels. Using a Hemoglobin Testing System (VARIANT II, Bio-Rad Laboratories, USA) to measure HbAlc. Using electrochemiluminescent procedures (Cobas E601; Roche,Basel, Switzerland) to measure the serum TSH, FSH, LH and ACTH levels.3. Statistical analysis:Values were presented as the mean± standard deviation. Means were compared using unpaired Student’s t-tests or Mann-Whitney U test for comparisons between two groups. The relationships between serum total cholesterol and pituitary hormone levels were evaluated with Pearson’s correlation analysis. To adjust for several confounding factors, a partial correlation analysis was performed while evaluating their relationships.P<0.05 had a statistical significance.Results:1. A high-cholesterol diet increased the serum lipid profiles in ratsCompared with the normal control (NC) group, in the high-cholesterol (HC) group, serum TC was not significantly changed at the end of 4 weeks (P> 0.05) and was increased by approximately 70% at the end of 28 weeks (P< 0.01). Moreover, serum LDL-C levels were increased 1.8-fold at the end of 4 weeks (P< 0.05) and 3.6-fold at the end of 28 weeks (P<0.01). Serum TG and FPG were no significant differences between the two groups.2. A high-cholesterol diet increased rat serum levels of TSH, FSH and LHThe baseline levels of hormones such as TSH, FSH, LH and ACTH were similar between high-cholesterol and control diet at 0 weeks. When cholesterol levels were elevated, the increase in the TSH level was approximately 2.2-fold at 4 weeks (P<0.05) and was up to 3.2-fold at the end of 28 weeks (P<0.01) compared to the NC group. These changes showed a time-dependent effect. Compared to the NC group, no obvious change was seen until 28 weeks, when the cholesterol level was higher. These changes included an increase of 104% for FSH (P<0.05) and 4.6% for LH (which was slight but significant, P<0.05) with the high-cholesterol diet at the end of 28 weeks. For ACTH, no significant differences between the two groups were observed at either 4 or 28 weeks.3. A high-cholesterol diet induced pituitary morphological changes in ratsAt 28 weeks, the pituitary volumes were visually enlarged, and the relative pituitary weight, the ratio of pituitary weight to body weight, significantly increased (P<0.05) in rat fed high-cholesterol diet.H&E staining revealed that the anterior pituitary tissue from the NC group showed normal features, mainly comprising acidophilic cells, basophilic cells and chromophobic cells covered with rich capillaries. The proportion of acidophilic cells in the anterior pituitary was approximately 55-80%, and the size of the acidophilic cells was obviously smaller than that of the basophilic cells. However, in the HC group, the pituitary was characterized by an increase in the number of basophilic cells and a decrease in the number of acidophilic cells.4. A high-cholesterol diet increased rat pituitary total and free cholesterol contentsThe pituitary total and free cholesterol content increased approximately 1.4-fold (P< 0.01) and 1.3-fold (P< 0.05), respectively, while the pituitary triglyceride and free fatty acids content had no change, compared with the content in the NC group, suggesting that a high-cholesterol diet could increase the cholesterol content in the pituitary gland, not just in the serum.5. A positive correlation between pituitary TC content and TSH, FSH, LH in ratsA Pearson’s correlation analysis indicates that the pituitary TC content was positively associated with the serum TSH (r=0.648, P=0.023), FSH (r=0.650, P=0.022) and LH (r =0.648, P=0.023) concentrations, respectively. There was no association between pituitary TC content and ACTH (r=0.326, P=0.300).6. A high-cholesterol diet increased the expression of TSHβ, FSHβ and LHβReal time-PCR revealed the up-regulated mRNA expression of TSHβ、FSHβ、LHβ, and no change about ACTH mRNA. Immunohistochemistry confirmed the same variation trend of the protein expression of TSHβ、FSHβ、LHβ and ACTH in rat pituitary.7. A high-cholesterol diet increased the content of TSH, FSH and LHELISA revealed, compared with the NC group, in the HC group, the total pituitary content of TSH increased approximately 3.1-fold (P<0.01), that of FSH increased about 1.8-fold (P<0.01), and that of LH increased about 1.4-fold (P<0.05). ACTH content had no significant difference between the two groups (P>0.05).8. Hypercholesterolemia increased the serum levels of TSH, FSH and LH, and decreased ACTH levels in humansIn the human subjects with hypercholesterolemia, serum TSH (P=0.001), FSH (P< 0.001)and LH (P=0.027) levels increased relative to levels in the control subjects, whereas ACTH decreased (P=0.030).Significant positive correlations existed between the TC level and TSH, FSH and LH. Interestingly, after we adjusted for confounding factors that could affect these hormone levels, such as age, body mass index, FPG, and systolic blood pressure, these correlations still persisted in being significant. However, no correlation was observed between the levels of TC and ACTH, which were similar with the results of animal study.Conclusions:1. High-cholesterol feeding could increase the cholesterol content both in the serum and pituitary gland of rats.2. High-cholesterol feeding could increase serum TSH, FSH and LH levels, change the structure of the pituitary gland, and increase the TSHP, FSHβ and LHβ protein expression in rats.3. There are correlations between hypercholesterolemia and the levels of hormones derived from anterior pituitary basophilic cells.Background:Hypertriglyceridemia, characterized by elevated triglycerides (TGs) in the blood, has become one of the important risk factors threatening human health. Previous studies reported that high circulating TG levels promote the accumulation of neutral lipids as TGs and free fatty acids in non-adipose tissues, contributing to chronic cellular dysfunction and injury, referred to as lipotoxicity. In recent years, the serious and extensive impact of lipotoxicity on human health on the pathogenesis of metabolic disease (such as type 2 diabetes mellitus and non-alcoholic fatty liver disease) has become well known. In 2014, our previous study found that lipotoxicity induced abnormal function of the rat thyroid. Serum thyroid hormone levels are controlled by pituitary gland, but to date, no research has explored whether the pituitary is another organ affected by lipotoxicity.The pituitary gland, which is a crucial neuroendocrine organ, includes anterior lobe, intermedia lobe and posterior lobe. The gland’s main function is to synthesize and secrete different types of pituitary hormones, and the pituitary gland also participates in controlling multiple endocrine organs in the body. There are three classic, mature pituitary axes, including the pituitary-thyroid, pituitary-gonadal and pituitary-adrenal axes. Among the axes, the pituitary-thyroid axis produces several hormones, including thyroid-stimulating hormone (TSH, also known as thyrotropin) and thyroid hormone, control the function of the thyroid and are essential for metabolic homeostasis. Pituitary-gonadal axis hormones, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T) and estradiol (E2), control the function of the gonads and are essential for reproduction. Finally, pituitary-adrenal axis hormones, including adrenocorticotropin (ACTH) and cortisol (COR), control the function of the adrenal gland and are essential for stress responses. For example, the serum levels of thyroid hormones are directly regulated by the hypothalamic-pituitary-thyroid axis. The pituitary gland serves as a sophisticated biosensor of thyroid hormone levels and regulates TSH levels according to the feedback of thyroid hormones. Abnormal hormone levels can lead to metabolic disorder and even diseases. Thus, maintaining normal pituitary axis hormone levels is necessary for human health.In the present study, we first performed a cross-sectional epidemiological analysis to assess the correlation between serum TG levels and pituitary axis hormones. Second, to verify this clinical phenomenon and to investigate if long-term TG overload can affect the pituitary-thyroid axis function of male rats, we set up a hypertriglyceridemic rat models and assessed it not only for serologic changes but also for mRNA and protein level changes in this study. Our findings suggested that there was a correlation between lipotoxicity and pituitary-thyroid axis hormone levels. These results remind us to recognize the widespread deleterious effects of hypertriglyceridemia in the body.Objectives:1. To observe investigate the relationship between hypertriglyceridemia and serum pituitary hormone levels.2. To investigate the effects of lipotoxicity on pituitary, especially pituitary-thyroid axis.Methods:Part 1:Epidemiological Study1. Subjects:This cross-sectional study was performed in Ningyang of Taian (Shandong Province, China), the people aged≥40 years old, lived here≥ 5 years were invited to receive an examination. According to the inclusion and exclusion criteria, then stratified according to the age, SBP, DBP, FPG and HbAlc, respectively. Finally,90 men aged 45-59 from the general population were evaluated.2. Detected index:Past medical history was evaluated with a questionnaire and blood pressure, height, weight, waist circumference and hip circumference were measured. We collected blood samples from all the patients during 8:00 to 10:00 after a minimum 8 h overnight fast. We Used an Auto Biochemical Analyzer (OLYMPUS AU5400, Olympus, Japan) to measure the serum TC, TG, LDL-C, HDL-C and FPG levels. Using a Hemoglobin Testing System (VARIANT II, Bio-Rad Laboratories, USA) to measure HbAlc. Using electrochemiluminescent procedures (Cobas E601; Roche,Basel, Switzerland) to measure the serum TSH, FT3(free triiodothyronine), FT4(free thyroxine), FSH, LH, T, ACTH and COR levels.Part 2:Animal Study1. Animal model:Forty male SD rats (180±10 g,6 weeks) were acclimatized feeding for 1 week, then randomly assigned to two groups:(1) normal control diet (NC diet):100% standard rodent chow (NC group,3.37 kcal/g); (2) high-fat lard diet (HF diet):85% standard rodent chow supplemented with 15% lard (HF group,4.14 kcal/g). All rats feed for 28-week. We collected fasting blood samples by subclavian venous puncture at the 0-,4- and 28-week, respectively. All rats were sacrificed at the 28th week, and collecting the fasting blood samples and pituitaries.2. Serum lipid profile and fasting blood glucose parameters analysis:Using automated spectrophotometry measured serum triglycerides (TG), total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C) and fasting plasma glucose (FPG) levels.3. Serum TSH, FSH, LH and ACTH analysis:Using ELISA kits to measure the serum TSH, T3, T4, FSH, LH T, ACTH and COR levels at the 0-,4- and 28-week, respectively.4. Pituitary lipid content assay:Using enzymatic methods to measure the pituitary TC, free cholesterol (FC), TG and free fatty acids contents.5. Pituitary tissue pituitary hormones assay:Using ELISA to measure the content of TSH, FSH, LH and ACTH in each anterior pituitary.6. The mRNA level of pituitary hormones assay:The mRNA expression of TSHβ、 FSHβ、LHβ and ACTH in rat pituitary were measured by real time-PCR.7. The protein level of pituitary hormones assay:The protein expression of TSHβ、 FSHβ、LHβ and ACTH in pituitary were measured by immunohistochemistry.8. Statistical analysis:Values were presented as the mean±standard deviation. Means were compared using unpaired Student’s t-tests or Mann-Whitney U test for comparisons between two groups. The relationships between serum TG and pituitary hormone levels were evaluated with Pearson’s correlation analysis. To adjust for several confounding factors, a partial correlation analysis was performed while evaluating their relationships. To further explore the relationship between TG and TSH, FSH, LH and ACTH, we used a general linear model (GLM). P<0.05 had a statistical significance.Results:1. Hypertriglyceridemia was positively associated with the levels of serum TSH, FSH and LH and negatively associated with ACTH in the epidemiological study populationRegarding the pituitary-thyroid axis, serum TSH was significantly (approximately 51.7%) higher, coupled with decreased serum FT4 in the human subjects with hypertriglyceridemia than in the control subjects. Regarding the pituitary-gonadal axis, compared with the control group, in the human subjects with hypertriglyceridemia, serum FSH and LH levels were increased by 25.9% and 24.4%, respectively, accompanied by decreased serum T. Regarding the pituitary-adrenal axis, ACTH was decreased by 32.7%, accompanied by increased COR, in the human subjects with hypertriglyceridemia. These results showed an association of hypertriglyceridemia with pituitary axis hormone levels.Among all patients, significant positive correlations were identified between the serum TG level and the levels of TSH, FSH and LH. In contrast, significant negative correlations existed between the TG level and ACTH. Interestingly, the correlations remained significant after adjusting for age, BMI, FPG and SBP and even after an additional adjustment for the corresponding target gland hormone levels. Multiple regression analysis yielded similar results.We then analyzed the association between TG and serum pituitary hormone levels using the GLM. The significant linear trend between TG and TSH (P=0.001), FSH(P=0.013), LH (P=0.004) and ACTH (P< 0.001). Subjects with lower serum TG had slightly lower adjusted TSH, FSH and LH and higher ACTH compared with subjects with higher serum TG. These results clearly indicate a significant positive correlation of TG with TSH, FSH and LH and a significant negative correlation of TG with ACTH.2. An HF diet increased the body weight and serum lipid profiles in ratThe two groups of rats had similar body weights at the baseline. However, the rats in the high-fat lard diet (HF) group gained significantly more weight than those in the normal control diet (NC) group from the 18th week to the end of the study (P<0.05). Compared with the NC group, serum TG was increased by 61.8% at 4 weeks (P<0.05) and 77.9% at the end of 28 weeks (P<0.01) in the HF group. Serum TC, LDL-C and FPG were no significant differences between the two groups.3. An HF diet increased rat pituitary TG and free fatty acid contentPituitary TG content and free fatty acid content in the rats in the HF group were approximately 1.7-fold and 1.3-fold (both P< 0.05) higher than those in the NC group at the end of 28 weeks. Pituitary TC and FC contents had no differences between the two groups. These results suggested that an HF diet could increase the TG content in the pituitary gland, and not just in the serum.4. An HF diet increased the expression of TSHβ mRNA and proteinWe first analyzed pituitary extracts using ELISA to quantify the TSH content in each pituitary. Compared to the NC group, the pituitary content of TSH was increased by 207.3% (P<0.01) in the HF group.Compared with the NC group, the mRNA level of TSHβ was increased by 3.1-fold(P< 0.01) in the anterior pituitary gland tissue of the HF group. Immunohistochemistry confirmed the same variation trend of the protein expression of TSHβ in rat pituitary.5. An HF diet altered pituitary-thyroid axis hormone levelsThe baseline levels of TSH, T3 and T4 were similar between the two groups at 0 weeks. Compared with the NC group, in the HF group, in the early period (4 weeks), the increase in the TSH level was approximately 1.9-fold (P< 0.05), together with an increased serum T3 concentration (P< 0.05) and a normal T4 concentration. In the late period (28 weeks), the increase in the TSH level was approximately 3-fold (P< 0.01), together with a decreased T4 concentration (P< 0.01) and normal T3 levels.6. An HF diet did not alter pituitary-gonadal or pituitary-adrenal axis hormone levelsFor the pituitary-gonadal axis and pituitary-adrenal axis, FSHβ, LHβ and ACTH mRNA and protein expression did not significantly differ between the two groups and serum FSH, LH, T, ACTH and COR levels had no significant changes at either 4 weeks or 28 weeks after HF diet treatment.Conclusions:1. High-fat lard diet can increase triglyceride content both in serum and pituitary. It can change the pituitary-thyroid axis hormone levels, accompanied by the expression of TSHP mRNA and protein increase.2. Hypertriglyceridemia was associated with pituitary axis, especially pituitary-thyroid axis, hormone levels.