Lipotoxicity Suppresses The Synthesis Of Growth Hormone In Pituitary Somatotrophs Via Endoplasmic Reticulum Stress

BackgroundHigh-fat diet(HFD)-induced hyperlipidemia is constant threats to human health worldwide and can result in the development of multiple diseases,such as type 2 diabetes,coronary heart disease,musculoskeletal diseases and several cancers.Persistent hyperlipidemia,especially high triglyceride(TG)levels in the circulation,causes deleterious lipid accumulation in non-adipose tissues in a process,known as lipotoxicity.In recent years,lipotoxicity has been shown to cause dysfunction in many organs and tissues.According to our previous study,there are significant correlations between lipotoxicity and the levels of pituitary-thyroid axis hormones.These findings indicate that the pituitary may be a potential target organ of lipotoxicity.However,to date,it is unclear whether lipotoxicity can directly disrupt the function of the pituitary in synthesizing growth hormone(GH).GH is a kind of pleiotropic polypeptide that is synthesized and secreted by anterior pituitary somatotrophs,and its physiological function is to promote anabolic metabolism and growth.Circulating GH level is mainly regulated by the co-ordinated control of two hypothalamic neuropeptides,GH-releasing hormone(GHRH),which stimulates GH release,and somatostatin(SS),which has an inhibitory action.Many physiological and pathological factors,including sex,age,body weight and nutritional status,can significantly alter the secretion pattern of GH,which can then in turn influence its biological effects.Pit-1,also called GHF-1 or POU1F1,is a pituitary-specific transcription factor of GH that plays a major role in both GH activation and pituitary development.In the human pituitary,Pit-1 gene mutation can lead to congenital GH deficiency(GHD),Studies from epidemiology and animal have shown that circulating GH levels are reduced in the context of HFD-induced weight gain,however,whether this process is caused by suppression of Pit-induced GH synthesis needs to be explored.The endoplasmic reticulum(ER)is a key intracellular organelle that plays an important role in regulating the synthesis of newly secreted proteins.ER homeostasis is mainly regulated by the unfolded protein response(UPR),which is a complex signaling system that regulates transcription and translation in order to increase the protein folding capacity of the ER.In some organs,such as the heart,overnutrition induced by HFD consumption can result in ER dysfunction,lead to ER stress,and activate the UPR.Three major pathways involved in the UPR are the inositol-requiring enzyme 1?(IRE1?)signaling pathway,the protein kinase RNA-like endoplasmic reticulum kinase(PERK)signaling pathway and the activating transcription factor 6(ATF6)signaling pathway.Interestingly,some recent studies have suggested that ER stress also exists in the central nervous system and contributes to neurodegenerative diseases.In this study,we explored the effect of lipotoxicity on the function of pituitary somatotrophs.First,we analyzed the association between serum TG and GH levels in a cross-sectional population.Then,we fed rats either HFD or control diet(CD),and observed the levels of serum GH and the expression of proteins critical to GH synthesis and ER stress.We also used palmitic acid(PA)-treated GH3 cells to study the direct effects of lipotoxicity on GH synthesis.Finally,rat and GH3 cell models of ER stress alleviation were generated to study the role of ER stress in the suppression of pituitary GH synthesis caused by lipotoxicity.Our new findings provide a unique approach to manage GH deficiency in hypertriglyceridemia.Objectives1.To clarify the association between serum TG and GH levels in the population.2.In vivo,rats were fed either with CD or HFD diet.The levels of serum GH and the expression of proteins critical to GH synthesis and ER stress were observed.3.In vitro,we used PA-treated GH3 cells to study the direct effects of lipotoxicity on GH synthesis.4.Rat and GH3 cell models of ER stress alleviation were generated to study the role of ER stress in the suppression of pituitary GH synthesis caused by lipotoxicity.MethodsPart 1.Epidemiological Study1.SubjectsWe performed a cross-sectional epidemiological investigation in Ningyang in Shandong Province.All of the participants were more than 40 years old and had lived there for more than 5 years.According to the inclusion and exclusion criteria,and stratified the subjects according to age,fasting plasma glucose(FPG)levels,HbAlc levels,systolic blood pressure(SBP)and diastolic blood pressure(DBP).Ultimately,90 men were included in our study.2.Detected indexMeasure height,weight and blood pressure,and obtain past medical history information through questionnaires.All subjects fasted after 12 o'clock in the night,and fasting blood was drawn the next morning.Serum TG,total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C)and FPG were measured using Automatic Biochemical Analyzer(OLYMPUS AU5400,Olympus,Japan).Serum HbA1c levels were measured using Hemoglobin Testing System(VARIANT ?,Bio Rad Laboratories,USA).Serum GH levels were measured using electrochemiluminescent procedures(Cobas E601;Roche,Basel,Switzerland).Part 2.Animal and cell Study1.AnimalsAfter one week of adaptive rearing,the male SD rats were divided into two groups at random and fed either a CD(n=20)(100%standard chow,3.4 kcal/g)or an HFD(n=20)(85%standard chow supplemented with 15%lard,4.1 kcal/g).At the 0th,4th and 28th weeks of feeding,fasting blood samples were taken from the subclavian vein between 8:00 and 10:00 AM.Another 48 rats were randomly divided into four groups and fed a CD or HFD.From the 9th week,4-PBA was given daily at a dose of 100 mg/kg/d intraperitoneal injection(i.p.)to the rats in the CD+4-PBA group and the HFD+4-PBA group until the end of the 18th week.Correspondingly,equal volumes of PBS were given to the rats in the CD+PBS group and the HFD+PBS group at the same time,respectively.2.Serum lipid profile and liver function analysis:using Automatic Biochemical Analyzer measured serum TG,ALT and AST levels of HFD rats3.GH/IGF-1 axis hormone levels in serum and culture supernatant analysis:using ELISA kits to determine the concentration of GH and IGF-1 in rat serum or GH3 cell culture supernatant.4.Pituitary GH reserve function was evaluated by GH stimulation test:at the end of feeding a CD or an HFD for 28 weeks,the rats were injected with 5?g/kg GHRH via tail vein.The serum GH levels were tested by ELISA at 0,8,15,20 and 30 min after GHRH administration.5.Lipid content in pituitary tissue assay:using enzymatic method to measure the content of TG and free fatty acid(FFA)in rat pituitary tissue.6.Pituitary gland morphological observation:the general pathological morphology and ultrastructure changes of rat pituitary gland were observed by H?E staining and transmission electron microscope.7.Evaluation of lipid content in GH3 cells:lipid content in GH3 cells after PA treatment was observed by oil red staining.8.Assessing the mRNA levels of the key molecules for GH synthesis:detection of GH and Pit-1 mRNA levels by real time-PCR.9.Assessing the protein levels of the key molecules for GH synthesis:protein levels of GH and Pit-1 in pituitary and GH3 cells were observed by Western Blotting and immunofluorescence.10.Assessing the activation level of the ER stress signaling pathway:the activation levels of BiP,IRE1?,eIF2? and ATF6? in rat pituitary tissue and GH3 cells were observed by Western Blotting.11.Evaluation of the therapeutic effect of alleviating ER stress:by using the ER stress inhibitor 4-Phenylbutyric acid(4-PBA)or the IRE1? pathway inhibitor 4?8c,GH levels and the key proteins for GH synthesis were studied.Part 3.Statistical analysisThe values are expressed as the mean ±standard deviation(SD).All data were analyzed using SPSS 18.0(Chicago,IL,USA).The relationship between serum TG and GH levels was assessed through Spearman correlation analysis and multivariate linear regression.Means were compared using Student's t-test for comparisons between two groups and one-way ANOVA for comparisons among multiple groups.P<0.05 was considered to indicate significance.Results1.Serum GH levels were correlated negatively with TG levels in the population.Through a cross-sectional study,we found that serum basal GH levels and TG levels were negatively correlated(r=-0.494,P<0.001)in the population.The results of multiple linear regression showed that the negative correlations remained significant(P=-0.279,P=0.009)after adjustment for the known confounding factors such as age,BMI,FPG level and SBP.2.Long term high-fat diet increased the body weight and serum TG levels in rats.Since the 19th week of high-fat diet feeding,the rats in the HFD group showed higher body weights than those in the CD group(P<0.05).At the 28th week,the rats in the HFD group showed significantly higher serum TG levels than those in the CD group(P<0.01).These results indicate that we have successfully established hypertriglyceridemia model induced by an high-fat diet.3.Long term high-fat diet reduced serum GH/IGF-1 axis hormone levels in rats.At the 28th week,serum GH levels were obviously lower in the HFD group than in the CD group(P<0.01).The results of GH stimulation test showed that HFD consumption reduced the reserve function of somatotroph cells.Under the premise that the rat liver function remains unchanged,serum IGF-1 level was significantly reduced in the HFD group at the 28th week.4.Long term high-fat diet increased the content of TG and FFA in the pituitary with disturbance of anterior pituitary morphology.Both TG and FFA levels were significantly higher in the pituitary of HFD group(both P<0.05)than in the CD group,indicating excess lipid accumulation in pituitary tissues,which might be a prerequisite for lipotoxicity.In terms of morphology and structure,the H?E staining results showed that acidophilic cells in the HFD group were significantly reduced;the electron microscope results showed that the numbers of both secretory cells and granules were decreased in the HFD group.5.Liptotoxicity decreased the expression of GH in the pituitary.The Real time-PCR and Western blotting results revealed that both the mRNA and protein levels of GH were reduced in pituitary tissue after 28 weeks of HFD feeding.These indicated that the synthesis of GH had decreased.6.PA treatment increased the lipid content in GH3 cells and was accompanied by down-regulation of GH expression.Oil Red O staining revealed significant increases in lipid levels in GH3 cells after they were treated with PA,suggesting that GH3 cells are also susceptible to lipotoxicity.Lipotoxicity down-regulated the expression of GH mRNA and protein in GH3 cells,and reduced the concentration of GH in the supernatant of the culture medium.7.Liptotoxicity reduced the expression of Pit-1 in GH3 cells and the pituitary.Pit-1 is a major transcription factor that regulates GH synthesis.The Real time-PCR,Western blotting and immunofluorescence results revealed that the expression of Pit-1 mRNA and protein in PA-treated GH3 cells and pituitary tissue of the HFD group was significantly reduced.This confirmed that lipotoxicity could inhibit GH synthesis.8.Lipotoxicity triggered ER stress and IRE1? signaling pathway in pituitary somatotrophs.The expression of BiP and the phosphorylation of IRE1? in pituitary tissue were significantly greater in PA-treated GH3 cells and pituitary tissue of the HFD group,while the expression of eIF2?-and ATF6-related pathway members was not significantly increased.These results indicated that ER stress and the IRE1? signaling pathway were induced by lipotoxicity in the pituitary somatotrophs.9.The ER stress inhibitor 4-PBA could improve the GH synthesis function of high-fat diet rats.After treated with 4-PBA,the expression of Pit-1 and GH in the pituitary tissue and the concentration of GH in the serum increased in HFD+4-PBA group.This indicated that 4-PBA could improve the decrease in GH synthesis caused by lipotoxicity.10.Blocking the ER stress or IRE1? signaling pathway could improve the decrease in GH synthesis caused by lipotoxicity in GH3 cells.After treated with ER stress inhibitor 4-PBA or IRE1? pathway inhibitor4?8c,GH levels in GH3 cells and culture supernatants were higher,suggesting that ER stress and IRE1? signaling pathways played important roles in the regulation of GH synthesis.Conclusions1.Epidemiological analysis showed that serum GH levels were correlated negatively with TG levels in the population.2.Lipotoxicity suppressed the synthesis of GH in pituitary somatotrophs.3.Lipotoxicity triggered ER stress and IRE1? signaling pathway in pituitary somatotrophs,and blocking ER stress or IRE1? pathway attenuated the decrease in GH synthesis caused by lipotoxicity.