Chronic Inflammation Promotes Gluconeogenesis And Insulin Resistance In C57BL/6J Mice

PART ONE：EFFECTS OF CASEIN INJECTION ON THELEVELS OF PROINFLAMMATORY CYTOKINESObjectives: Metabolic disorders, including obesity, type2diabetes andatherosclerosis, have traditionally been viewed as lipid storage disorderscaused by overnutrition. It is now widely recognized that chronic low-gradeinflammation and oxidative stress play a key role in the initiation,propagation, and development of metabolic disorders. Population studieshave showed a strong correlation between proinflammatory biomarkers(such as C-reactive protein, IL-6, and TNF-α) and perturbations in glucosehomeostasis, obesity and atherosclerosis. We used casein injection inC57BL/6J mice to induce a chronic systemic inflammatory stress in vivo.The injections were done every other day for14weeks. Blood samples andthe tissues were taken for cytokines assay.Methods: Male C57BL/6J mice aged from8weeks were randomlyassigned to receive a normal chow diet (NCD, n=12) or NCD plussubcutaneous injection of0.5mL10%casein (NCD+Casein, n=12) or ahigh fat diet (84%fat,1.5%cholesterol)(HFD, n=6) or HFD plussubcutaneous injection of0.5mL10%casein (HFD+Casein, n=6).Subcutaneous injection of0.5mL normal saline was used as controls inthe NCD and HFD groups. The injections were done every other day and the mice were culled14weeks after first injection. Blood samples and thetissues were taken for further assessments. Serum tumor necrosis factor-α(TNF-α) and serum amyloid A protein (SAA) concentrations were measuredby ELISA kits. The mRNA and protein expression of TNF-α and MCP-1wasexamined by real-time reverse transcription polymerase chain reaction(real-time PCR) and Western blot.Results:(1) Serum levels of SAA and TNF-α were significantlyincreased in the casein-injected mice fed with normal chow diet or high-fatdiet in comparison with their respective controls, suggesting that chronicinflammatory stress was successfully induced in C57BL/6J mice.(2)Wedemonstrated that chronic inflammation and high-fat diet increasedTNF-α、MCP-1mRNA expression in the liver of C57BL/6J mice. Thelevels of TNF-α and MCP-1mRNA expression in HFD+Casein group micewere raised compared with HFD group.(3) Western blot showed thatchronic inflammation and high-fat diet increased TNF-α、MCP-1proteinexpression in the liver of C57BL/6J mice. The levels of TNF-α and MCP-1protein expression in HFD+Casein group mice were raised compared withHFD group.Conclusion: The levels of cytokines in serum and liver weresignificantly increased, suggesting that we successfully induced a chronicsystemic inflammation by subcutaneous injection of10%casein inC57BL/6J mice. PART TWO：EFFECTS OF CHRONIC INFLAMMATIONON GLYCOMETABOLISM IN C57BL/6J MICEObjectives: This study was designed to investigate the effect of caseininjection on the glycometabolism in C57BL/6J mice.Methods: Serum insulin concentrations were measured by ELISA kits.Glucose tolerance test and insulin tolerance test were used to evaluate bodyinsulin sensitivities.Results: The fasting glucose in casein-injected mice fed with NCD orHFD were increased compared with the respective controls. After glucoseloading, blood glucose concentrations in casein-injected mice fed withNCD or HFD were persistently higher than the respective controls,suggesting that chronic inflammation induced glucose intolerance inC57BL/6J mice. After the insulin injection, the levels of plasma glucoseremained high at all time points in the casein-injected group. In NCD mice,blood glucose levels dropped to40%of basal values at30min. Bloodglucose levels initially were decreased, but increased faster and higher incasein-injected mice fed with NCD or HFD than in the respective controls,suggesting that chronic systemic inflammation decreased insulin sensitivityin C57BL/6J mice.Conclusion: We demonstrated that casein injection induced impairedfasting glucose (IFG), glucose intolerance and decreased insulin sensitivityof C57BL/6J mice. PART THREE: EFFECTS OF CHRONIC INFLAMMATIONON HEPATIC GLUCONEOGENESIS AND INSULINSIGNALING IN C57BL/6J MICEObjectives: Hepatocytes play an important role in maintaining plasmaglucose homeostasis by adjusting the balance between hepatic glucoseproduction and utilization via the gluconeogenic and glycolytic pathways,respectively. It was proposed previously that increased hepatic glucoseproduction is a major feature of type2diabetes. Hepatic glucose productionand utilization are regulated at the transcriptional level of thephosphoenolpyruvate carboxykinase (PEPCK) and glucose6-phosphatase(G-6-Pase), which catalyze the last and the first rate-limitingsteps in gluconeogenesis. Chronic inflammation is associated with insulinresistance and impaired glucose tolerance. Numerous studies haveestablished that chronic inflammation participate in the pathogenesis ofinsulin resistance. In the state of insulin resistance, insulin loses its abilityto reduce gluconeogenesis, upragulates expressions of PEPCK andG-6-Pase, resulting in high fasting blood-glucose and impaired glucosetolerance. The mammalian target of rapamycin (mTOR), a nutritious sensor,is closely related to insulin resistance. We designed this study todemonstrate whether chronic inflammatory could increase hepaticgluconeogenesis and whether chronic inflammatory stress could increaseser/thr phosphorylation of IRS-1and induce insulin resistance viaactivating the mTOR pathway in the liver of C57BL/6J mice.Methods: We evaluated the effect of inflammation on the genes andprotein expression of PEPCK and G-6-Pase in livers of C57BL/6J mice byreal time PCR and Western blot, respectively. Glycogen granule wasevaluated by periodic acid-Schiff (PAS) staining. Western blot was used to examine protein expression involved in insulin signaling.Results: Casein injection increased the mRNA and protein expressionof PEPCK and G-6-Pase in liver, especially in the mice fed with HFD. PASstaining revealed an increase of glycogen granule in casein-injected mice,whether fed with normal chow diet or high fat diet. The protein expressionof mTOR, S6K and pIRS1-ser270were increased in inflamed mice, whilethe total IRS-1, pIRS1-tyr632, Akt, pAkt-473were decreased.Conclusion: Chronic systemic inflammation increased mRNA andprotein expression of PEPCK and G-6-Pase in liver of C57BL/6J mice.Also, chronic inflammatory enhances ser/thr phosphorylation of IRS-1andreduces tyrosine phosphorylation via activating mTOR/S6K1signalingpathway in liver of C57BL/6J mice. The ability of insulin to reducegluconeogenesis is reduced, resulting in an increase of glucose production,high fasting blood-glucose and impaired glucose tolerance.