Mechanisms Of Macrophage-specific Deficiency Of Noc4l Leading To Insulin Resistance In Mice

With the improvement of life,the incidence of obesity worldwide has increased drastically and has caused an international health concern.Overweight and obesity are defined as abnormal or excessive fat accumulation that may impair health.Obesity is associated with a number of health problems such as insulin resistance,cardiovascular disease,diabetes,high cholesterol,high blood pressure.Over the past few years it has become increasingly evident that obesity-associated inflammation plays a crucial role in the development of obesity-associated insulin resistance,T2DM and related diseases.In fact,obesity is now considered as a state of chronic low-grade inflammation.Adipose tissue macrophage is believed to be the primary source of many inflammatory cytokines,such as tumour necrosis factor a(TNFa)and interleukin(IL)-6.However,the molecular mediators promoting the proinflammatory property of adipose tissue macrophages remain elusive.Therefore,finding a new protein which can regulates the activation of adipose tissue macrophage may become a new target for drugs to treat obesity-associated insulin resistance.NOC4L(Nucleolar complex associated 4 homolog)is a newly discovered protein in mammals.However,with few functional studies evaluated,the physiological contributions of NOC4L have yet to be defined.In this study,we found that in mice Noc4l was ubiquitously expressed in all tissues,particularly in highly proliferative tissues such as testes and lymphoid organs.By immunofluorence assay,Noc4l was mainly expressed on the macrophages,and its expression is directly regulated by obesity-associated factors,LPS and palmitate.In addition,Noc4l expression was downregulated in the adipose tissue of obese mice and obese patients.These results suggest NOC4L may be involved in the regulation of obesity-induced adipose tissue inflammation and insulin resistance.Because the general deficiency of Noc4l leads to embryonic lethality,we generated mice in which Noc4l was genetically and selectively inactivated in the myeloid cell compartment,named as Noc4lLKO.In this study,we found that compared to control mice,Noc4lLKO mice became high fat diet-induced obesity,elevated blood glucose and insulin resistance;the transcription level of proinflammatory cytokines in white adipose tissue ang liver and systemic inflammation were higher;the number of macrophages infiltrated in adipose tissue was increased.Noc4l inhibited M1 macrophage polarization and activation ex vivo.These results suggested that Noc4l-deficiency in macrophage exacerbated obesity-induced inflammation leading to insulin resistance.By further investigation,we found that Noc4l is not involved in regulating MyD88-dependent signaling pathway of TLR4.Noc4l knockout in macrophages had no effect on IKBa,Erk and JNK signal pathway after LPS treatment;and NOC4L can't inhibit MyD88 as well as downstream signal molecules TRAF6 and P65-induced NF-?B activation by dual luciferase assay.By contrasr,Noc4l is mainly involved in regulating TRIF-dependent signaling pathway,and Noc4l knockout in macrophages promotes the IRF3 phosphorylation;and NOC4L can inhibit TRIF-induced NF-?B activation by dual luciferase assay.The subcellular localization of NOC4L was analyzed and found that NOC4L mainly localized in the early endosome because NOC4L colocalized with the early endosomal markers RAB5.This result further confirmed that NOC4L can regulate TRIF signaling pathway,because TRIF signaling pathways mainly occur in the endosome.We found NOC4L regulates TRIF-dependent signaling pathway mainly through controling TLR4 endocytosis,because TLR4 expression of Noc4l-deficiency peritoneal macrophage(PM)cell surface was reduced,indicating TLR4 internalization increased.By immunoprecipitation assay,NOC4L can interact directly with TLR4,and after LPS treatment the interaction ability between NOC4L and TLR4 was reduced.Taken together,NOC4L-deficiency can aggravate the development of diet-induced obesity,obeisity-induced inflammation and insulin resistance;NOC4L inhibits TLR4 internalization by interacting with TLR4 to inhibit TRIF-dependent signaling pathway.Therefore NOC4L may become a new marker for the clinical detection of diabetes and therapeutic drug target of insulin resistance.