小鼠试验 z-bo 2020-11-19 427

　　Introduction: Macrophages accumulate in adipose tissue, causing chronic low-grade inflammation and promoting the development of systemic insulin resistance. Based on transcription profiles and expression markers from in vitro experiments, macrophages are generally classified as classic/inflammatory (M1) or alternate/anti-inflammatory (M2) activation. M1 macrophages are characterized by a high glycolysis rate, while M2 macrophages mainly rely on oxidative phosphorylation. The central role that drives the polarization of macrophages has been designated as hypoxia-inducible factor-1α (HIF-1α), which is the main regulator of glycolysis and is involved in the development of the M1 phenotype. The analysis of the intracellular metabolism of adipose tissue macrophages (ATMS) and the key regulatory factors involved are expected to further understand their metabolic functions, and ultimately may provide targets for regulating their inflammatory properties. Using different methods, we determined the unique metabolic activation of ATMS in obesity, which is not similar to M1 or M2 macrophages. The metabolic activation of macrophages is characterized by increased OXPHOS and glycolysis, which is induced in a dose-dependent manner when co-cultured with adipose tissue, and converted into increased secretion of cytokines. Although various metabolic pathways contribute to the release of cytokines from ATMS, glycolysis mainly comes from the higher cytokines produced by ATMs in obese mice. The inflammatory activation of ATMS in the early stages of obesity does not seem to be related to HIF-1α. A further understanding of the functional consequences of adipose tissue macrophage metabolism planning and the metabolic activation in the ATMS of obese adipose tissue is expected to lead to new therapeutic targets to reduce inflammation and ultimately reduce the metabolic complications caused by obesity.

　　Method: Mice: Male C57BL/6 mice are fed a high-fat diet (HFD) and a low-fat diet. To study the role of HIF-1α in ATMS of obese animals, chimeric HIF-1α male C57/BL6 mice aged 9-12 weeks were placed in the Cre recombinase driven by lysozyme M or the control group C57/BL6 was not placed The Cre recombinase driven by lysozyme M was exposed to HFD for 8 weeks. Seven weeks later, insulin was injected through the abdominal cavity, and an insulin tolerance test was performed in mice fasted for 5 hours. Blood was taken from the tail at a specific time point, and blood glucose was measured with an ACCU check blood glucose meter. All mice are housed individually and eat food and water ad libitum. All diets are obtained from research diets.

　　Cell culture: ATM and peritoneal macrophages were isolated from male, wild-type C57BL/6 mice. To cultivate.

　　ATMS: Freshly isolated ATMS was cultured in RPMI 1640, after 2 hours of culture, supplemented with 10% FCS and 1% penicillin/streptomycin for 24 hours. By providing 5.5 mmol/L 2-deoxyglucose (2-DG), 50 μmol/L Emoxel, 10 μmol/L UK5099 or 10 μmol/L BPTES until the end of the culture for 24 hours, the effects of various metabolic pathways on the release of cytokines were studied. contribution.

　　bone marrow-derived macrophages: culture bone marrow cells in DMEM supplemented with 10% FCS, 1% PS and 5% L929 conditioned medium. After 3 to 4 days, the adherent bone marrow-derived macrophages (BMDMS) were re-adherent and exposed for 3 days, and inserts containing 25 mg or 100 mg of epididymal adipose tissue collected from LFD-fed or HFD-fed mice Things. The control BMDMS is stored in DMEM/FCS/PS containing 5% (volume/volume) L929, and its empty insert has the same length of time. Incubate with 10 ng/ml lipopolysaccharide (LPS) (M1) or 25 ng/ml IL-4 (M2) for 24 hours to generate M1 and M2 macrophages. In order to measure the cytokine and lactate production of BMDMs after adipose tissue activation, the insert was removed and the BMDMs were stored in fresh DMEM/FCS/PS for 24 hours.

　　Adipose tissue: The epididymal adipose tissue is cultured and exposed to DMEM/PS containing 17.5 nmol/ml insulin for 20 minutes to measure insulin sensitivity. The tissues were stored in DMEM/FCS/PS, treated with LPS (10 ng/ml) or without LPS for 24 hours, and measured the release of IL-6. The tissues were cultured in DMEM/FCS/PS for 3 days for the determination of leptin and lactate.

　　Extracellular flux analysis: Use XF-96 extracellular flux analyzer to analyze real-time oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of ATMS and BMDMs.

　　Cytokine and lactate determination: Use mouse DuSET-ELISA kit to determine IL-6, chemokine ligand-1 (KC), TNF-α, IL-1β, IL-10 and leptin in cell culture supernatant s level. The lactic acid level was measured with a lactic acid determination kit.

　　Immunohistochemistry: Paraffin-embedded sections of epididymal fat tissue were stained with F4/80 antibody and counterstained with hematoxylin. 3,3-Diaminobenzene visualizes macrophages.

　　Microarray analysis and interpretation: In four separate experiments, four ATMS pools were isolated from the epididymal fat tissue of male C57BL/6 mice with LFD or HFD, and the expression profile was analyzed by microarray. In addition, raw transcriptome data from macrophages in different tissues, including ATMS, were obtained from LPS-stimulated BMDMs.

　　Results: The unique metabolic and inflammatory activation of ATMS in obese patients: In order to check whether the transcription of macrophages in adipose tissue is different from that of other tissues, we used macrophages isolated from the abdominal cavity, liver, spleen, lung, and internal organs. Principal component analysis (PCA) is performed on the gene expression profile published by phages. ATMS exhibits unique transcripts. The presence of obesity significantly affects the complete transcriptome of ATMS. The cluster analysis of ATMS sorted by obese mice and lean mice showed that PAN macrophage membrane markers EMR1-F4/80. Traditionally, macrophages in obese adipose tissue have features that enhance the inflammatory state. Using inflammatory genes as the input of PCA confirmed that macrophages present in obese adipose tissue have a unique inflammatory activation effect. Interestingly, the expression data of genes involved in glycolysis, OXPHOS and amino acid metabolism are also sufficient to distinguish the ATMS of obese mice from those of lean mice, suggesting robust changes in ATM energy metabolism in obese patients. In obese and lean mouse ATMS, many genes involved in glycolysis and OXPHOS are up-regulated. It is worth noting that the metabolic rearrangement of obesity is unique to ATMS, because these changes were not observed in peritoneal macrophages. In fact, the expression level of metabolic genes in ATMS is higher in peritoneal macrophages than in peritoneal macrophages, and is upregulated in obesity. ATMS derived from obese and lean animals produces more lactic acid, reflecting a higher rate of glycolysis. In contrast, no differences in the robustness of lactic acid secretion were found in the peritoneal macrophages of obese mice and lean mice.

　　The metabolic and inflammatory activation of macrophages in obese adipose tissue is different from the classic activation of LPS, and is related to the existence of type 2 diabetes in obese patients: To further understand the functional characteristics of metabolism and inflammatory rewiring in ATMS, use the metabolic and inflammatory gene set As input, perform gene set enrichment analysis (GSEA). Compared with obese and lean mice, the gene set in ATMS is significantly more depleted than ATM, and the gene set that is not depleted reflects the metabolic pathway. Metabolic gene sets including glycolysis and oxyphosphorus are enriched in obese ATMS. The transcriptional regulation of ATM in obesity is very different from the classic macrophage activation of LPS. Although some pathways are similarly regulated, most metabolic pathways include OXPHOS, glycolysis, and pentose phosphate pathways. Compared with lean mice, LPS-activated macrophages have less or negatively regulated LPS-activated macrophages Compared with obese ATMS, the cells are less or negatively regulated. According to the different metabolic regulation of ATMS and classic activated macrophages, we found that various pro-inflammatory and anti-inflammatory genes, including CD11c, MyD88, ARG1 and IL-10, are regulated differently in ATMs of obese and lean mice, not similar to M1 Macrophages.

　　The functional consequences of ATMS metabolic activation in obesity: To evaluate whether the transcriptional changes in ATMS translate into differences in energy metabolism. The OXPHOS and glycolysis rate of freshly separated ATMS were tested by measuring OCR and ECAR. With the increase of metabolic gene expression, ATMS of obese mice showed higher OXPHOS. The rate of glycolysis in ATMS of obese mice was higher. It confirmed that after 24 hours of culture, compared with lean mice, the lactic acid content in the ATMS supernatant of obese mice was significantly increased. The ATMs of obese mice produced more IL-6 and KC, and the ATMs of obese mice produced less TNF-α. IL-1β and IL-10 were not detected in the ATMS supernatant.

　　Conclusion: It is found that ATM has a unique metabolic activation in obesity, which is characterized by increasing OXPHOS and glycolysis. Blocking the ATMS metabolic pathway leads to glycolysis as a major factor in pro-inflammatory features, especially obesity. In the adipose tissue of obese patients, the metabolic characteristics observed in the adipose tissue of type 2 diabetic patients are similar to those found in ATMS during obesity, including induction of OXPHOS and lysosome genes. A better understanding of ATMS metabolism will most likely lead to new therapeutic targets that regulate macrophage metabolism and inhibit inflammation, which drives insulin resistance and type 2 diabetes in obese patients.