FOCXO1and CGI-58Involvement In Obesity-related Chronic Inflammation And Insulin Resistance

Obesity is a worldwide public health problem and is a high risk factor ofmetabolic disorders, such as cardiovascular desease and type2diabetes. Insulinresistance is the physophathological base of the development of metablic disorders.It is well established that obesity-related chronic inflammation initiates the insulinresistance in fat, liver and muscle.Forkhead box containing protein O subfamily-1(FOXO1), a transcription factordownstream of insulin signal, is negatively regulated by insulin. By controlling itstarget genes in liver, FOXO1regulates the gluconeogenesis, production of very lowdensity lipoprotein, oxidative stress and apoptosis. It is reported recently thatFOXO1can increase proinflammatory cytokine expression, such as IL-1β inmacrophages, MCP-1and IL-6in adipocytes, which indicates FOXO1might linkinsulin signal to proinflammatory cytokine production. And recent workdemonstrates that increased FOXO1expression is companied with the progression ofnon-alcoholic fatty liver desease (NAFLD) to non-alcoholic steatoheptitis (NASH).However, the relationship between FOXO1and proinflammatory cytokine productionin insulin resistant hepatocytes is still not well known.Obesity-induced ectopic deposition of lipid results in chronic inflammation andinsulin resistance in liver and muscle. Dysregulation of lipid metabolism induceslipotoxicity in macrophages and promotes the polarization of macrophage from anti-inflammatory (M2) to proinflammatory (M1) phenotype. Elevated macrophageinfiltration and M1/M2ratio in visceral fat play a causal role in obesity-relatedchronic inflammation. Comparative gene identification-58(CGI-58), an activator oftriglyceride hydrolysis, is expressed ubiquitously. Mutations in human CGI-58genecause Chanarin-Dorfman syndrome (CDS), a neutral lipid storage diseasecharacterized by excessive TG accumulation in the cytosolic lipid droplets (LDs) in most tissues. While the role of macrophage CGI-58in lipid metabolism ofmacrophage, systemic chronic inflammation and insulin resistance is largelyunknown.[Objectives]1. To investigate the correlation between FOXO1and chronic inflammation ininsulin resistant liver in both genetically obese (db/db) mice and diet-induced obese(DIO) mice, and to further explore the mechanism of FOXO1on the production ofproinflammatory cytokines in cultured insulin resistant hepatocytes.2. To investigate the role of ectopic lipid accumulation in macrophage inducedby specifically knocking-out of CGI-58gene in macrophage (MakO) on systemiclipid metabolism, chronic inflammation and insulin resitance in MakO mice.[Methods]1. FOXO1expression and activity and proinflammatory cytokine production inthe liver of obese db/db mice or diet-induced obese mice were detected by RT-PCR,real-time PCR, western blotting (WB) and ELISA. By enforced expression orknockdown of FOXO1, we further observed the regulatory role of FOXO1onproinflammatory cytokine production in cultured insulin resistance HepG2cell. Wefinally explored the transcriptional mechanism of FOXO1on CCL20gene promoteractivity by reporter gene assay and ChIP assay.2. To investigate the role of ectopic lipid accumulation in macrophage onsystemic lipid metabolism and inflammation, macrophage CGI-58specific knockout(MaKO) mouse model was generated, and the full spectrum of lipids in peritonealmacrophage, liver, fat and plasma were then measured. Glucose tolerance test (GTT),insulin tolerance test (ITT), acute insulin injection test and hyperinsulin-euglycemicclamp test were performed to detect the insulin sensitivity on MaKO and controlmice. Expression of proinflammatory cytokines in the visceral fat, liver and plasmawere determined with quantitative real-time PCR and ELISA. By administrating LPSto MaKO mice or its freshly prepared peritoneal macrophages, the tissue specific andsystemic inflammatory responses were obeserved. Finally, the therapeutic effect ofantioxidant N-Acetyl-L-cysteine (NAC) on macrophage CGI-58aggravated chronicinflammation and insulin resistance were tested. [Results]1. The role of FOXO1on hepatic insulin resistance related chronic inflammation1.1First, the correlation among FOXO1expression, proinflammatory cytokineproduction and inflammatory cell infiltration in insulin resistant liver of obese micewere observed. The results showed that30-week old db/db mice had significantlyincreased body weight, fasting serum glucose and insulin levels compared with thedb/+and WT mice. The db/db mice had increased lipids deposition, macrophage andT lymphocyte infiltration, FOXO1level and proinflammatory cytokines such asMCP-1, IL-1β, IL-6, iNOS and CCL20in the livers. Similar results were obtained onDIO C57/B6mice.1.2Then, the role of FOXO1on proinflammatory cytokine production in insulinresistant (IR) hepatocytes were determined. The results showed that insulin-stimulated phosphorylation of AKT, IRS-1and FOXO1in HepG2cells was inhibitedby treatment with TNF-α, indicating the successful set-up of the cultured IRhepatocyte model. In this model, TNF-α promoted nuclear translocation of p65andmRNA levels of proinflammatory cytokines, such as MCP-1, IL-1β and IL-6. Wefurther found FOXO1knockdown by small interfering RNA significantly inhibitedmRNA of proinflammatory cytokines, such as MCP-1, IL-1β and IL-6in TNF-α-treated HepG2cells.1.3Next, the mechanism of FOXO1on proinflammatory cytokine expressionwas intensively studied on CCL20, a potent T lymphocyte chemokine, by trancientgene transfection and reporter gene assay. The results showed: FOXO1increasedTNF-α-induced CCL20promoter activity through a NF-κB binding site (-81~-70).ChIP assay further verified that FOXO1could promote binding of p65/p50heterodimer to this concensus NF-κB binding site.1.4Finally, to explore the function of FOXO1-CCL20pathway on lymphocyteschemotaxis, Boyden cell migration test was performed. Results showed that the cellculture supernatants from FOXO1-transfected HepG2could enhance, while silenceof FOXO1could reduce TNF-α-induced PBMC migration. Moreover, chemotaxiseffect could be largely blocked by anti-CCL20antibody.2. Role of ectopic lipid accumulation induced by loss function of macrophage CGI-58on systemic lipid metabolism, chronic inflammation and insulin resistance2.1First, the macrophage CGI-58specific knockout (MaKO) mice weregenerated and the lipid levels in the peritoneal macrophages (PMs), liver, fat andplasma were observed. The results showed: increased lipid depositon were found inperitoneal macrophage of MaKO mice. The lipid composition analysis showed thatthe level of triglyceride (TG) increased, and the levels of some species of ceramide,phosphatidic acid (PA) and phosphatidylinositol (PI) decreased in the PMs fromMaKO mice compared with the fl/fl control mice.2.2Then, the insulin sensitivity of MaKO mice at different age were tested. Theresults showed: the MaKO on high fat diet (HFD) had significantly increased levelsof fasting and fed plasma insulin and HOMA-IR index. Glucuse tolerance test (GTT)and insulin tolerance test (ITT) showed the impaired glucuse tolerance and insulintolerance on MaKO-HFD mice. The acute insulin injection test revealed that MaKO-HFD mice had impaired insulin-stimulated AKT phosphorylation in the visceral fat,muscle and liver. Hyperinsulin-euglycemic clamp test showed that MaKO mice on a35-week HFD had significantly decreased glucose infusion rate. Together, theseresults indicate that macrophage CGI-58deficiency aggravates HFD-inducedsystemic insulin resistance.2.3Next, the proinflammatory cytokine production in the plasma, liver and fatin the MaKO-HFD mice were tested. Results showed that elevated mRNA levels ofproinflammatory cytokines (TNF-α, IL-β) and infiltration of inflammatory cellswere present in the epididymal fat (eFat) and liver, and increased levels of TNF-α,IL-6and IL-18in plasma. The fluorescence-activated cell sorting (FACS) revealedthat MaKO-HFD mice had increased stromal-vascular fraction (SVF) cells andincreased amount of M1macrophages, B cells, and T cells in the eFat. Takentogether, macrophage CGI-58deficiency aggravates HFD-induced systemic chronicinflammation.2.4Furthermore, the inflammatory response to LPS on MaKO mice and theprimarily isolated PMs were observed. The results showed: only IL-1β wereincreased in PMs and only IL-18increased in plasma of the MaKO-CD or MaKO-HFD mice, while no changes of TNF-α or IL-6levels were observed compared with their corresponding control mice.2.5To further explore the CGI-58deficiency-caused specific elevation of IL-1and IL-18levels in response to LPS stimulation in PMs and MaKO mice, theupstream inflammasome pathway was investigated. The results showed that increasedNlrp3mRNA were observed in the PMs isolated from MaKO compared with controlmice. Also observed were the increased Nlrp3mRNA levels in the eFat, liver tissuesas well as the eFat-derived macrophages. Taken together, macrophage CGI-58deficiency activates Nlrp3-related inflammasome pathway.2.6To get more insight into CGI-58deficiency-induced inflammasomeactivation, we measured the production of reactive oxygen species (ROS), a majorinducer of inflammasome pathway. The results showed: higher level of ROS weredetected in PMs from MaKO mice than the fl/fl control mice, and could be reducedby administration of antioxidant N-acetyl-L-cysteine (NAC). CGI-58deficiencyenhanced Caspase1activity and IL-1secretion. Again these effects could bealleviated by NAC treatment. Moreover, enhanced JNK and NF-κB signal anddecreased insulin-stimulated AKT phosphorylation were detected in eFat tissuewhich was co-cultured with PMs from MaKO mice. Similarly, these effects could berestored by NAC treatment. Taken together, these in vitro and ex vivo tests indicatethat macrophage CGI-58deficiency aggravates inflammation and insulin resistancein eFat via activation of ROS-Caspase1-IL-1β pathway.2.7Finally, to get further evidence in vivo, MaKO-HFD mice were given NACtreatment for4weeks and systemic inflammation and insulin sensitivity weremeasured. The results showed: impaired GTT and ITT in MaKO-HFD mice could berestored by NAC treatment. Also observed were the normalization of TNF-α, IL-1βand IL-18, and decreased IL-6level by this treatment. Taken together, these resultsindicate antioxidant NAC treatment could fully restore macrophage CGI-58deficiency-aggravated HFD-induced systemic chronic inflammation and insulinresistance.[Conclusion]1. FOXO1increases proinflammatory cytokine production in insulin resistantheptocytes; FOXO1enhances p65/p50heterodimer binding to CCL20promoter to increase CCL20production, which may induce infiltration of inflammatory cells andaggravation of inflammation in insulin resistant liver.2. Macrophage CGI-58deficiency promotes IL-1β and IL-18secretion by ROS-mediated inflammasome pathway, and further aggravates HFD-induced systemicchronic inflammation and insulin resistance.[Significance]FOXO1increases proinflammatory cytokines and chemokines production ininsulin resistant (IR) liver helps to understand the progression of IR-related chronicinflammation and may provide a potential theraperutic target to combat IR-relateddisorders.CGI-58deficiency in macrophage causes dysregulation of lipid metabolism bothlocally and systemically and results in systemic inflammation and insulin resistancevia ROS-dependent pathways. These results shed light to design new theraperuticstrateges to combat obesity-related diseases.