The Effect And Mechanism Of Rheb1Deletion In Macrophages On Regulation Of Metabolism In Mice

Part I Effect of mTOR suppressed in Raw264.7macrophages on macrophage polarization and secretion of inflammatory mediatorsObjective To investigate effects of mTOR signaling pathway in macrophages timulated by lipopolysaccharide (LPS), palmitic acid (PA) or oleic acid (OA), on the M1/M2polarization and the relevant secretion of inflammatory mediators.Methods Raw264.7stable cell line were grown in12-well plates and grouped as:①non-intervention group (control group)②simulant group③rapamycin group④rapamycin+stimulant group. Stimulants were listed as LPS, PA and OA with selection of the best concentrations and durations. Cells were collected to extract mRNA after experiments. Expressions of macrophage recruitment, pro-inflammatory and anti-inflammatory mediators were measured via qRT-PCR. Double variant analysis of covariance with interaction was adopted to analysis the independent effect of the three stimulants (LPS, PA or OA) or rapamycin, and the interactive effect between stimulant and rapamycin.Results①LPS elicited classical proinflammatory responses as it raised level of IL-1β. Also rapamycin has a similar effect on the level of IL-1β. Combination of LPS and rapamycin proved additive effect on this same cytokine.②PA raised level of IL-1β, yet co-stimulation with rapamycin significantly decreased level of MCP-1, as well as increased level of IL-1β significantly.③OA raised level of IL-1β and Mrc1slightly, and co-stimulation with rapamycin also significantly reduced level of MCP-1, enhanced level of IL-1β significantly. Level of Mrc1was also higher in the costimulation group than rapamycin group.Conclusion LPS, PA or OA promotes M1polarization and secretion of pro-inflammatory mediators in macrophages. PartⅡ Effect of Rhebl specific deletion in Macrophages on diet-induced obesity and hepatosteatosisObjective To establish mice model with Rhebl specific deletion in macrophages, and to explore the diet-induced-obesity (DIO) and metabolism-related phenotype of this mice model.Methods Rheb1flox/flox F4/80CreTg/0(KO mice) and Rheb1flox/flox (wild-type mice, WT) were established and confirmed by identification of Rhebl expression in DNA, RNA and protein level. Littermates of KO and WT mice at5to6weeks were divided into high-fat diet (HFD) group and normal diet (ND) group. Body weight was monitored weekly. Food intake was monitored. The glucose tolerance and insulin sensitivity were determined by GTT and ITT. Gross and sectional morphology (including HE staining and oil red O staining) were displayed on organs regulating glucose metabolism, including liver, adipose tissue and muscle.Results①Genotype succeeded through tail DNA electrophoresis, assessment of Rhebl in mRNA level and protein level extracted from BMDM.②Since10-week age, mice fed ND had a lower body weight than mice fed HFD. KO mice gains weight more slowly in long-term HFD.③GTT results, including the area under curve (AUC) and separated detection spots tail glucose value showed no statistical differences at10-week HFD and peer ND, but significant differences between WT and KO mice after16-week HFD and peer ND. KO mice had severer glucose tolerance than WT mice in long-term HFD or ND.④ITT results, including AUC and separated detection spots tail glucose value showed no statistical differences at10-week HFD, but significant differences after16-week HFD. Insulin sensitivity was improved in KO mice after long-term HFD.⑤Both in fed and fasting status, liver from WT mice was larger, heavier and greyer in color than KO mice fed HFD. Results of red oil O staining and HE staining were in accordance as less fatty degeneration and cellular injury in KO mice. Moreover, HE staining showed more obvious inflammatory cells infiltration in crown-like region in KO mice. Additionally, gross and sectional observance showed no difference in fatty degeneration and muscle morphology.Conclusion Rhebl Macrophage-specific suppression of the mTORC1signaling pathway impairs glucose tolerance but improves insulin sensitivity possibly via ameliorating hepatosteatosis. PartⅢ Effect of Rhebl specific deletion in Macrophages on DIO-related steatosisObjective To investigate the effects of Rhebl specific deletion in macrophages on DIO-related steatosis, local macrophage infiltration and polarization, proinflammatory/antiinflammatory mediator spectrum, and function of glucose and lipid metabolism regulation in liver.Methods Function of liver glucogenesis was determined by PTT. Liver from fasting overnight DIO mice was obtained to measure mRNA levels of macrophage infiltration and polarization, pro-inflammatory/anti-inflammatory mediators, and enzymes related to glucose and lipid metabolism.Results①PTT results showed no statistical differences in AUC between the two groups fed at least4-month HFD, but not for fasting glucose and120’glucose. When compared to GTT in previous part, the extent of differences in the AUC between the two groups was much diminished.②qRT-PCR results showed lower levels of macrophage infiltration marker (F4/80), antiinflammatory mediators (IL-4, IL-10, Arg1, Mrc1, Mrc2, Mgl2) and most pro inflammatory mediators (IL-1β, IL-6, TNF-a) in liver from KO mice than that of WT mice, yet with no statistical differences. G6P related to liver glycogenolysis, ACC and SREBP1related to lipid synthesis were dramatically down-regulated in KO mice when compared to WT·mice.Conclusion Specific suppression of mTORCl in macrophages can ameliorate hepatosteatosis via reducing inflammatory responses, and improving glucogenesis and lipid synthesis. PartIV Effect of Rhebl specific deletion in Macrophages on adipose tissuesObjective To investigate the effects of Rhebl specific deletion in macrophages on DIO-related local macrophage infiltration and polarization, proinflammatory/antiinflammatory mediator spectrum, and function of thermogenesis and browning in various adipose tissues, including subcutaneous white adipose tissue (sWAT), gonadal white adipose tissue (gWAT), mesenteric white adipose tissue (mWAT) and brown adipose tissue (BAT).Methods sWAT, gWAT, mWAT and BAT from DIO mice were obtained to measure mRNA levels of macrophage infiltration and polarization, pro-inflammatory/anti-inflammatory mediators, molecules related to thermogenesis and browning. Ratios of different types of macrophages expressing specific surface markers from sWAT and gWAT were measured by flow cytometry.Results①qRT-PCR results showed KO group had a lower level of pro-inflammatory mediators and part of antiinflammatory mediators (IL-6and IL-4,) in sWAT; Most antiinflammatory mediators (IL-4, IL-10and Mgl2) were up-regulated in gWAT from KO group, but the trend of pro-inflammatory mediators was highly variable; Expressions of F4/80and most anti-inflammatory mediators were significantly raised in BAT from KO group.②Frequency of CD11c+CD206-macrophages ratio was reduced but CD11c-CD206+macrophages were slightly increased in sWAT and gWAT from KO mice. Meanwhile, the ratio of CD11c+CD206-/CD11c-CD206+was also in descent trend.③Conversely, KO group had a higher level of PAPRy, Prdm16, UCP1than WT group; Most thermogenesis and browning gene expression in gWAT had a resembled trend to sWAT, but without statistical differences; Expression of all thermogenesis and browning gene expression was highly enhanced in BAT from KO group compared to WT group, especially for PGC1α, Cidea, PAPRy, Prdm16and UCP1; Only PAPRy was up-regulated in mWAT from KO group, but this gene also was another anti-inflammatory mediator.Conclusion Effect of Rhebl specific deletion in macrophages on various adipose tissues as:①Increased infiltration of macropahges in BAT is positively related to enhanced function of thermogenesis and browning②M2polarization in macrophages in sWAT is positively related to enhanced function of thermogenesis and browning.