Protective Effect And Mechanism Of FGF21on Type1Diabetes-induced Testicular Apoptotic Cell

Diabetes mellitus is a chronic common metabolic disease，that is hypofunction ofislet induced by pathogenic factors included genetic factors, immune dysfunction,microbial infection, mental factors, obesity and hypertension. Diabetes is a metabolicdisorder syndrome that caused by glucose, protein, fat, water and electrolyte. In the21st century, diabetes is a major health crisis and diabetes complications are having aserious effect on the quality of life, even threatening the life of the patients. Theepidemic and development of diabetes has become an important global public healthand social problems, and how to prevent and delay the occurrence of complicationshas become a big problem in medical and biological research. Unfortunately there isno an effective drug or method that can completely cure diabetes and we can onlycontrolblood glucose so as to reduce and delay the occurrence of complications. Sothis study will have important theoretical and realistic significance.In recent years, reproductive system damage induced by diabetes is arousingmore and more attention as the increase of incidence of diabetes and lower age. Testisis important reproductive organs and testicular spermatogenic cells can produce sperm,but excessive testicular apoptotic cell will lead to cause infertility by thedevelopmental abnormalities of sperm. Diabetes can lead to testicular apoptotic celland attenuation of spermatogenic cells. Hyperglycemia can produce a large number ofROS, the products of oxidative stress, is regarded as one of the important factors thatlead to damage of mitochondria and the cell apoptosis. It is eventually led to theoccurrence and development of diabetes induced male reproductive complications.The fibroblast growth factor （FGF） family，as a multifunctional signalmolecules, plays multiple roles in regulating functions including promoting mitosisand the formation of vessels, the development and differentiation of embryonicorganization, wound healing and tissue regeneration and nerve nutritionin in someendocrine-relevant tissues or organs. Of the23known members of the family, FGF21is a novel member and expresses predominantly in pancreas, liver and adipose tissues, and relatively less in other organs. Accumulating evidence indicates the role of FGF21as a critical regulator of long-term energy balance of carbohydrate and fat metabolism.The expression of FGF21in the testicular tissue was found but its biological functionis still not clear. The role of FGF21in testis has not been well addressed but it hasbeen clear that other members of FGFs has obviously protective effect on theapoptosis of testicular cells. Whether FGF21as an important metabolic mediator isalso involved in the maintenance of the spermatogenesis and whether FGF21protectsthe germ cells from diabetes-induced apoptotic cell death have never beeninvestigated. There is no clear answer about it so we conducted a series of research.Firstly, the type1WT and FGF21-KO diabetes mouse model was induced withstreptozotocin （STZ） at single big dose. We have examined the mRNA expressionof FGF21in the testis of fasting and non-fasting mice or mice with type1diabetesbyreal-time RT-PCR assay.Then the mice in the KO-DM-FGF21group wereintraperitoneally injected with FGF21for10days. When these mice were sacrificedafter the last injection of FGF21on the10th day after the onset of diabetes bilateraltestes were harvested. The testicular tissue was adopted for histopathological andbiochemical studies by the ways of immunohistochemical staining,immunofluorescence and western blot and the corresponding mechanism has beendiscussed. The result is presented：1. We successfully established the mouse model of type1diabetes andexogenous FGF21can reduce blood glucose level of FGF21-KO-DM mice. Thetesticular expression of FGF21mRNA was not significantly changed under24hfasting condition. Testicular mRNA expression was significantly increased in diabeticmice compared to the WT mice. The testicular expression of FGF21mRNA was notaffected by supplementation of exogenous FGF21in FGF21-KO mice.2. FGF21-KO mice showed a significant elevation of spontaneous testicularapoptotic cell death, examined by TUNEL staining.FGF21-KO diabetic mice showeda significantly higher incidence of testicular apoptotic cell death than WT diabeticmice, which could be almost completely attenuated by supplementation of exogenousFGF21. It was no effect of Fgf21gene deletion or exogenous FGF21supplementationon the testicular cell proliferation in non-diabetic and diabetic conditions.3. Western blotting revealed a significant increase in the Bax to Bcl-2expressionratio, AIF and p53but no change of caspase-3and caspase-8cleavage level in WT-DM mice. These changes were significantly increased in FGF-KO diabetic mice,which was significantly prevented by supplementation of exogenous FGF21. Thediabetes-induced apoptotic cell death is caspase-3independentsignaling pathway thatis AIF dependent signaling pathways on the mitochondrial.4. Diabetes induced testicular ER stress, shown by the increased expression ofCHOP，caspase-12，BIP（GRP78）and ATF4.It means that FGF21can protect thediabetes-induced apoptotic cell death by ER stress apoptosis signaling pathways5. The diabetes-induced accumulation of3-NT and4-HNE was significantlyenhanced by Fgf21gene deletion in FGF21-KO diabetic mice and significantlyprevented by supplementation of exogenous FGF21, respectively. The expression ofAKT, GSK-3β and GS show that these indicators affects diabetes-induced apoptosisof mice testicular cell in Fgf21knockout mice diabetes.Deletion of Fgf21gene doesnot affect the expression of3-NT,4-HNE, GSK-3β and GS. There are other signalingpathways associated with AKT pathway for the increasing of apoptotic in FGF21-KOmice.6. We also checked the expression of ERK1/2, LKB1, AMPK and SirT-1andFGF21has the effect on diabetes-induced apoptotic cell death through the pathway offatty acid metabolismand the increase of testicular spontaneous apoptosis inFGF21-KO mice is closely related to this pathway.So we can draw the conclusion: FGF21plays a protective role in the process ofdiabetes-induced apoptosis of testicular cell. The effects are mediated by themitochondrial pathway, ER stress, glucose and fatty acid metabolism pathways. Therole of FGF21in the prevention and treatment of diabetes-induced reproductivecomplicationsprovidesa new target for clinical treatment and improves the life qualityof diabetic patients.