The Function Of ALK7 In High Glucose Induced Adipocytes Inflammation And Construction Of Adipose Tissue ALK7 Knockout Mice Model

BackgroundAdipose tissue,as a very dynamic endocrine organ in human body,is composed by lipid-filled adipocytes,pericytes,fibroblasts,preadipocytes,mast cells,immune cells and extracellular miatix which is contributed by each of these cells.In normal circumstances,pro-inflammation factors and anti-inflammation factors which are secreted by adipose tissue are kept in equilibrium.However,when patients suffer from obesity and diabetes,the adipose tissue will be hypertrophy,adipocytes will undergo pathological expansion.In addition,adipocytes are accompanied by increased lipid deposition,which will result in relative reduction of capillary density,Owing to insufficient supply of oxygen,adipose tissue secretes more pro-inflammation factors and less anti-inflammation factors.In response to alterations in the energy status,the adipose tissue is rapidly and dynamically remodeled through changes in the number and size of adipocyte,various stromal vascular cells in adipose tissue undergo numerical and functional changes,this series of events is called "adipose tissue remodeling".Adipose tissue pathological remodeling is one of the risk factors for variety of metabolic diseases and cardiovascular diseases,which causes great harm to human health.ALK7,as an orphan receptor,was firstly isolated from rat brain.ALK7 is one of type I of TGF-? super family,and is widely expressed in various tissues and organs.ALK7 is a transmembrane protein which is located on the surface of cell membrane,mainly including n-terminal(ligand binding zone),transmembrane zone,c-terminal(serine/threonine kinase zone).When the ligand binds to ALK7 and then they form ligand-receptor complex,the downstream signal molecules Smad2/3 are activated,and triggering various biological effects.Studies have shown that ALK7 was highly expressed in adipose tissue in human and rodent,and had closely relationship with adipose tissue metabolism.It has reported that ALK7 knockout mice could resist against diet-mediated obesity and reduce lipid accumulation.Thus,ALK7 may be involved in the energy metabolism of adipose tissue,but it is not clear whether ALK7 has a regulatory effect on the inflammation of adipose tissue.According to above studies,our study explored whether ALK7 could regulate the high glucose induced adipose tissue inflammation through activation of Smad2/3.Objective1.To explore the effect of high glucose on the expression of ALK7 on mature adipocytes2.To explore the effect of high glucose on mature adipocytes inflammation3.To explore whether ALK7 is invoved in high glucose induced adiopcytes'inflammationMaterial and methodsThe healthy people's and diabetics' visceral adipose tissues were collected,3T3-L1 cells were cultured,and RT-PCR,Western blot,immunofluorescence,immunohistochemical staining methods,transfection of ALK7shRNA was performed in this study.1.Immunofluorescence was performed to show the distribution and expression of ALK7 in healthy people's and diabetics'visceral adipose tissues2.Western blotting was used to test the expression of ALK7 in healthy people's and diabetics'visceral adipose tissues3.Immunohistochemical staining methods was performed to exam the expression of TNF-??IL-6 in healthy people's and diabetics' visceral adipose tissues4.Differentiated 3T3-L1 preadipocytes were cultured with DMEM containing 25mM glucose for 0h?6h?12h?24h?48h.The protein level of ALK7 was measured by Western blot5.3T3-L1 preadiopcytes were cultured with DMEM containing 5.5mM glucose,5.5mM glucose plus 19.5mM mannose,and 25mM glucose.The protein levels of ALK7?Smad2/3?P-Smad2?P-Smad3 were measured by Western blot,and mRNA levels of TNF-??IL-6 were measured by RT-PCR6.ALk7shRNA were transfected into 3T3-L1 preadipocytes to explore whether ALK7 could influence glucose-induced adipocyte inflammation.The protein levels of ALK7?Smad2/3 and phosphorylated of Smad2/3 were measured by Western blotting.The mRNA levels of TNF-??IL-6 were measured by RT-PCRResult1.The expression of ALK7 in visceral adipose tissue of diabetics was increased compared with it in healthy people.2.The expressions of TNF-??IL-6 in visceral adipose tissue of diabetics were increased compared with them in healthy people.3.High ambient glucose at a concentration of 25mM elevated the expression of ALK7 and phosphorylation of Smad2/3 in differentiated 3T3-L1 preadipocytes.4.High ambient glucose at a concentration of 25mM could increase the expression of TNF-? and IL-6 in adipocytes.5.Inhibitor the expression of ALK7,the phosphorylation of Smad2/3 and the expression of inflammation factors were decreased.Conclusion1.High glucose can elevate the expression of ALK7 in mature adipocytes2.High glucose can promote the expression of inflammatory cytokines(TNF-??IL-6)in mature adipocytes3.ALK7 regulates high glucose-induced mature adipocytes inflammation through activation of Smad2/3BackgroundALK7,known as activin receptor-like kinase 7,is a cell membrane receptor with serine/threonine kinase domain.As one of type ? transforming growth factor-? receptor,it broadly participates in the process of body metabolic reactions.Adipose tissue is a central energy regulation which can undergo a series of changes with variety of energy status in body,such as the changes of construction and function,and we call the changes "adipose tissue remodeling".Adipose tissue is dysfunctional when it undergoes pathological remodeling.Some studies shown that,ALK7 expresses in many tissues and organs.ALK7 not only involves in regulation of blood glucose,it also involves in the energy metabolism of adipose tissue.It indicates that ALK7 may play an important role in adipose tissue remodeling.With the rapid development of biological technology,Cre-loxp system has been widely used in gene editing in vivo and in vitro,the Cre recombinase can specifically identify the floxp sequence,and cut off the target gene labeled by floxp,and achieve the purpose of conditional knockout.In order to figure out how ALK7 works in adipose tissue,we constructed mice models of specific ALK7 knockout in adipose tissue.Objectives1.To construct conditional adipose tissue ALK7 knockout mice by using Cre-loxp system.Methods1.Constructed ALK7flox/flox mice: crossbred ALK7flox/- mice with ALK7flox/- mice;2.Constructed ALK7flox/--Cre(+)mice:crossbred ALK7flox/flox mice with Cre mice that expressed specifically in adipose tissue;3.Constructed ALK7flox/flox:Cre(+)mice.crossbred ALK7flox/-:Cre(+) mice with ALK7flox/flox mice;4.Genotype identification:The mice DNA were extracted from the mice tails,and the mice genotypes were identified by PCR amplification and agarose gel electrophoresis;5.Weston blot and immunofluorescenence:Western blot and immunofluorescenece were performed to determine the expression of ALK7;6.HE staining:HE staining was used to observe the morphological changes of adipose tissue of adipose tissue ALK7 knockout mice;Result1.The conditional adipose tissue ALK7 knockout mice were constructed;2.Compared with control group mice,the expression of ALK7 in adipose tissue of conditional adipose tissue ALK7 knockout mice were remarkably decreased;3.HE staining showed that there was no difference between the adipose tissue of ALK7flox/flox:Cre(+) mice and control group mice;ConclusionsWe successfully construct adipose tissue conditional ALK7 knockout mice by using floxp-Cre system,compared with control group mice,the expression of ALK7 in conditional knockout mice adipose tissue is significantly decreased.There is no difference in adipose tissue morphology between control mice and conditional knockout mice.