M1 Adipose Tissue Macrophages Research Articles

Subchronic exposure to 1,2-naphthoquinone induces adipose tissue inflammation and changes the energy homeostasis of mice, partially due to TNFR1 and TLR4

Air pollution affects energy homeostasis detrimentally. Yet, knowledge of how each isolated pollutant can impact energy metabolism remains incomplete. The present study was designed to investigate the distinct effects of 1,2-naphthoquinone (1,2-NQ) on energy metabolism since this pollutant increases at the same rate as diesel combustion. In particular, we aimed to determine in vivo effects of subchronic exposure to 1,2-NQ on metabolic and inflammatory parameters of wild-type mice (WT) and to explore the involvement of tumor necrosis factor receptor 1 (TNFR1) and toll-like receptor 4 (TLR4) in this process. Males WT, TNFR1KO, and TLR4KO mice at eight weeks of age received 1,2-NQ or vehicle via nebulization five days a week for 17 weeks. In WT mice, 1,2-NQ slightly decreased the body mass compared to vehicle-WT. This effect was likely due to a mild food intake reduction and increased energy expenditure (EE) observed after six weeks of exposure. After nine weeks of exposure, we observed higher fasting blood glucose and impaired glucose tolerance, whereas insulin sensitivity was slightly improved compared to vehicle-WT. After 17 weeks of 1,2-NQ exposure, WT mice displayed an increased percentage of M1 and a decreased (p = 0.057) percentage of M2 macrophages in adipose tissue. The deletion of TNFR1 and TLR4 abolished most of the metabolic impacts caused by 1,2-NQ exposure, except for the EE and insulin sensitivity, which remained high in these mice under 1,2-NQ exposure. Our study demonstrates for the first time that subchronic exposure to 1,2-NQ affects energy metabolism in vivo. Although 1,2-NQ increased EE and slightly reduced feeding and body mass, the WT mice displayed higher inflammation in adipose tissue and impaired fasting blood glucose and glucose tolerance. Thus, in vivo subchronic exposure to 1,2-NQ is harmful, and TNFR1 and TLR4 are partially involved in these outcomes.

Cellular and Molecular Mechanisms and Effects of Berberine on Obesity-Induced Inflammation.

Obesity represents chronic low-grade inflammation that precipitates type 2 diabetes, cardiovascular disease, and cancer. Berberine (BBR) has been reported to exert anti-obesity and anti-inflammatory benefits. We aimed to demonstrate the underlying immune-modulating mechanisms of anti-obesity effects of BBR. First, we performed in silico study to identify therapeutic targets, describe potential pathways, and simulate BBR docking at M1 and M2 adipose tissue macrophages (ATMs), tumor necrosis factor-α (TNF-α), C-C motif chemokine ligand 2 (CCL2), CCL4, CCL5, and C-X-C motif chemokine receptor 4 (CXCR4). Next, in vivo, we divided 20 C58BL/6 mice into four groups: normal chow, control (high fat diet (HFD)), HFD + BBR 100 mg/kg, and HFD + metformin (MET) 200 mg/kg. We evaluated body weight, organ weight, fat area in tissues, oral glucose and fat tolerance tests, HOMA-IR, serum lipids levels, population changes in ATMs, M1 and M2 subsets, and gene expression of TNF-α, CCL2, CCL3, CCL5, and CXCR4. BBR significantly reduced body weight, adipocyte size, fat deposition in the liver, HOMA-IR, triglycerides, free fatty acids, ATM infiltration, all assessed gene expression, and enhanced the CD206+ M2 ATMs population. In conclusion, BBR treats obesity and its associated metabolic dysfunctions, by modulating ATM recruitment and polarization via chemotaxis inhibition.

High-Fat Diet-Induced Dysregulation of Immune Cells Correlates with Macrophage Phenotypes and Chronic Inflammation in Adipose Tissue.

Obesity is a complex disease associated with various metabolic abnormalities, cardiovascular diseases, and low-grade chronic inflammation. Inflammation associated with T helper 1 (Th1) immune cells is dominant in adipose tissue (AT) and exerts metabolically deleterious impacts. The precise mechanism of alteration in AT immune system and its effect on metabolic homeostasis remains unclear. In this study, we investigated how a high-fat diet (HFD) alters the AT immune response and influences inflammation during obesity. HFD consumption amends the metabolic parameters, including body weight, glucose, and insulin levels. We observed increased infiltration of Th17 cells, a subset of dendritic cells (CD103+), and M1 macrophages in AT of mice fed HFD compared to those fed a normal diet (ND). In mice that were fed HFD, we also observed a reduction in regulatory T cells (Tregs) relative to the numbers of these cells in mice fed ND. Corresponding with this, mice in the HFD group exhibited higher levels of proinflammatory cytokines and chemokines than those in the ND group. We also observed alterations in signaling pathways, including increased protein expression of IRF3, TGFβ1, and mRNA expression of IL-6, KLF4, and STAT3 in the AT of the mice fed HFD as compared to those fed ND. Further, HFD-fed mice exhibited decreased protein expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) compared to mice fed ND, suggesting that PPAR-γ functions as a negative regulator of Th17 cell differentiation. These results suggest that HFD induces increased levels of inflammatory cytokines and key immune cells, including Th17, M1 macrophages, and CD103+ dendritic cells, and reduces levels of PPAR-γ and Tregs to sustain AT inflammation. This study supports the notion that dysregulation of Th17/Tregs, which polarizes macrophages towards M1 phenotypes in part through TGFβ1-IRF3-STAT3 and negatively regulates PPAR-γ mediated pathways, results in AT inflammation during obesity.

Puerarin Attenuates Obesity-Induced Inflammation and Dyslipidemia by Regulating Macrophages and TNF-Alpha in Obese Mice.

Obesity causes low-grade inflammation that results in dyslipidemia and insulin resistance. We evaluated the effect of puerarin on obesity and metabolic complications both in silico and in vivo and investigated the underlying immunological mechanisms. Twenty C57BL/6 mice were divided into four groups: normal chow, control (HFD), HFD + puerarin (PUE) 200 mg/kg, and HFD + atorvastatin (ATO) 10 mg/kg groups. We examined bodyweight, oral glucose tolerance test, serum insulin, oral fat tolerance test, serum lipids, and adipocyte size. We also analyzed the percentage of total, M1, and M2 adipose tissue macrophages (ATMs) and the expression of F4/80, tumor necrosis factor-α (TNF-α), C-C motif chemokine ligand 2 (CCL2), CCL4, CCL5, and C-X-C motif chemokine receptor 4. In silico, we identified the treatment-targeted genes of puerarin and simulated molecular docking with puerarin and TNF, M1, and M2 macrophages based on functionally enriched pathways. Puerarin did not significantly change bodyweight but significantly improved fat pad weight, adipocyte size, fat area in the liver, free fatty acids, triglycerides, total cholesterol, and HDL-cholesterol in vivo. In addition, puerarin significantly decreased the ATM population and TNF-α expression. Therefore, puerarin is a potential anti-obesity treatment based on its anti-inflammatory effects in adipose tissue.

Effects of interleukin-17 antibody on polarization of macrophages in adipose tissue of high-fat diet fed mice exposed to bisphenol A

To investigate the effects of interleukin-17(IL-17) antibody on polarization of adipose tissue macrophages(ATM) in mice fed with high-fat diet(HFD) exposed to bisphenol A(BPA). Four week-old male C57 BL/6 mice were divided into control group, IgG group, IL-17 antibody group, 1000 nmol/L BPA group, 1000 nmol/L BPA+IgG group, and 1000 nmol/L BPA+IL-17 antibody group according to random number table method. Eight mice per group were fed with HFD and BPA was exposed by drinking water. The IgG group and the 1000 nmol/L BPA+IgG group were intraperitoneally injected with 100 μg IgG antibody once a week, and the IL-17 group and the 1000 nmol/L BPA+IL-17 antibody group were intraperitoneally injected with 100 μg IL-17 antibody once a week. After 16 weeks, the mice were sacrificed, and serum samples were collected for serum separation. Leptin level was measured by enzyme-linked immunosorbent assay(ELISA). The expression of IL-1β, IL-6 and monocyte chemoattractant protein-1(MCP-1)inflammatory cytokines were observed by immunohistochemistry(IHC) of adipose tissue of epididymis. The activity of inducible nitric oxide synthase(iNOS)and arginase-1(Arg-1)was measured by ELISA, and the proportion of M1 and M2 ATMs was measured by flow cytometry(FCM). The expression of CD11 c and CD206 mRNA was measured by qRT-PCR. Compared with the control group, serum leptin, the expressions of inflammatory cytokines IL-6, IL-1β and MCP-1 in adipose tissue were increased in 1000 nmol/L BPA group, the proportion of M1 type ATM was increased(22.000%±0.500% vs. 31.467%±0.379%), iNOS activity was increased, CD11 c mRNA expression was increased, Arg-1 activity was decreased, CD206 mRNA expression was decreased, the differences were statistically significant(P<0.05), but the proportion of M2 type ATM was decreased insignificantly(P>0.05). There was no significant change in IgG group. Compared with 1000 nmol/L BPA group, IgG+1000 nmol/L BPA group had no significant change. In 1000 nmol/L BPA+IL-17 antibody group, serum leptin was decreased, the expressions of inflammatory factors IL-6, IL-1β and MCP-1 in adipose tissue were down-regulated, and the proportion of M1 type ATM was decreased(31.467%±0.379% vs. 22.933%±0.153%), iNOS activity was decreased, CD11 c mRNA expression was decreased, and the proportion of M2 type ATM was increased(4.847%±0.655% vs. 7.840%±0.555%), Arg-1 activity was enhanced, and CD206 mRNA expression was up-regulated, the differences were statistically significant(P<0.05). IL-17 antibody may reduce the secretion of ATM inflammatory factors by inhibiting the polarization of ATM to M1 type, thus improving the inflammation of adipose tissue in BPA-infected HFD-fed mice.

Mechanisms and Effect of Coptidis Rhizoma on Obesity-Induced Inflammation: In Silico and In Vivo Approaches.

Obesity is characterized as a chronic, low-grade inflammation state accompanied by the infiltration of immune cells into adipose tissue and higher levels of inflammatory cytokines and chemokines. This study aimed to investigate the mechanisms and effects of Coptidis Rhizoma (CR) on obesity and its associated inflammation. First, we applied a network pharmacology strategy to search the target genes and pathways regulated by CR in obesity. Next, we performed in vivo experiments to confirm the antiobesity and anti-inflammatory effects of CR. Mice were assigned to five groups: normal chow (NC), control (high-fat diet (HFD)), HFD + CR 200 mg/kg, HFD + CR 400 mg/kg, and HFD + metformin 200 mg/kg. After 16 weeks of the experimental period, CR administration significantly reduced the weight of the body, epididymal fat, and liver; it also decreased insulin resistance, as well as the area under the curve of glucose in the oral glucose tolerance test and triglyceride in the oral fat tolerance test. We observed a decrease in adipose tissue macrophages (ATMs) and inflammatory M1 ATMs, as well as an increase in anti-inflammatory M2 ATMs. Gene expression levels of inflammatory cytokines and chemokines, including tumor necrosis factor-α, F4/80, and C-C motif chemokine (CCL)-2, CCL4, and CCL5, were suppressed in adipose tissue in the CR groups than levels in the control group. Additionally, histological analyses suggested decreased fat accumulation in the epididymal fat pad and liver in the CR groups than that in the control group. Taken together, these results suggest that CR has a therapeutic effect on obesity-induced inflammation, and it functions through the inhibition of macrophage-mediated inflammation in adipose tissue.

Emodin inhibits lipid accumulation and inflammation in adipose tissue of high‐fat diet‐fed mice by inducing M2 polarization of adipose tissue macrophages

Adiposetissuemacrophages(ATMs)representthemostabundantleukocytes in adipose tissue (AT). An increase in number and a phenotypical switch of ATMs during the development of obesity contributetochronicinflammationand metabolic disorders, which have been regarded as potential therapeutic targets to restore AT homeostasis. Emodin has been shown to exert strong anti-inflammatory property via acting on macrophages in a range of disease models. However, whether emodin exerts a beneficial effect on obesity via modulating ATMs has not been reported. In high-fat diet (HFD)-induced obese mice, emodin significantly inhibited the increase of body weight and lipid accumulation in ATs. Emodin apparently reduced glucose and insulin levels and ameliorated serum lipid profiles in HFD-fed mice. Moreover, the local and systemic inflammation was dramatically alleviated by emodin. We next discovered that M2 macrophage percentage was greatly increased by emodin although total ATMs was not altered, which resulted in a net increase of M2 macrophages in AT. In vitro studies confirmed that emodin promoted the polarization of macrophages towards M2. Gene ontology (GO) analysis showed that myeloid leukocyte differentiation and activation were among the most significant biological processes in emodin-treated ATMs. We further identified that TREM2 was the most dramatically upregulated molecule by emodin and emodin-induced M2 macrophage polarization was dependent on TREM2. Furthermore, silencing TREM2 apparently abrogated the effect of emodin on AT inflammation and adipogenesis. We, for the first time, disclosed that emodin inhibited obesity by promoting M2 macrophage polarization via TREM2, suggesting that emodin may be explored as a clinical and translational candidate in preventing obesity and its related metabolic diseases.

Modulation of TRIB3 and Macrophage Phenotype to Attenuate Insulin Resistance After Downhill Running in Mice.

Eccentric exercise training accompanied by a low-fat diet can prevent insulin resistance (IR) and is currently an effective method for the treatment of IR induced by high-fat diet (HFD)-associated obesity. However, the molecular mechanisms underlying this improvement of IR in adipose tissue are still not completely clear. In this study, 5–6-week-old male mice were randomly divided into a standard control diet (SCD) group (SC, n = 12) and a HFD group (HF, n = 72). After 12 weeks, 12 mice in each group were randomly sacrificed. The remaining mice in the HF group were randomly submitted to one of the following experimental protocols for 8 weeks: obesity-HFD-sedentary (OHF-Sed, n = 14), obesity-HFD-exercise (OHF-Ex, n = 16), obesity-SCD-sedentary (OSC-Sed, n = 14), and obesity-SCD-exercise (OSC-Ex, n = 16). All obese mice in the exercise group were subjected to downhill running. Half of the mice in each group received an insulin injection (0.75 U/kg) before sample collection. Epididymal fat was removed and weighed. Adipocyte size and inflammatory cell infiltration were observed by H&E staining. Both basal and insulin-stimulated GLUT4 fluorescence and protein contents were detected by immunofluorescence and Western blot. Levels of IL-1β and IL-10 were detected by ELISA. Protein contents of iNOS, Arg-1, TRIB3, p-AKT, and AKT were determined by Western blot. CD86 and CD206 fluorescence were determined by immunofluorescence. The results showed that a HFD for 12 weeks induced IR accompanied by adipose tissue macrophages M1 polarization (increased iNOS protein content and CD86 fluorescence) and TRIB3-AKT activation. Downhill running accompanied by a low-fat diet attenuated IR (p < 0.01), reduced inflammation levels (increased IL-10 protein content and decreased IL-1β protein content), inhibited adipose tissue macrophages M1 polarization (decreased iNOS protein content and CD86 fluorescence) and promoted M2 polarization (increased Arg-1 protein content and CD206 fluorescence), and suppressed TRIB3-AKT signaling. We concluded that downhill running accompanied by dietary fat regulation attenuates HFD-related IR in mice, which may be associated with reduced TRIB3-AKT signaling and activated M2 macrophages in adipose tissue.

The Immunomodulating Effect of Baicalin on Inflammation and Insulin Resistance in High-Fat-Diet-Induced Obese Mice.

Background Obesity is a chronic low-grade systemic inflammation state, which causes insulin resistance, diabetes, and other metabolic diseases. Baicalin is known to have anti-inflammatory and antiobesity effects. In this study, we investigated the cellular and molecular immunological effects of baicalin on obesity-induced inflammation. Methods Male C57BL/6 mice were assigned to four groups: the normal chow, high-fat diet (HFD), BC2 (HFD + baicalin 200 mg/kg), and BC4 (HFD + baicalin 400 mg/kg) group; the three groups except normal chow were fed with a high-fat diet for 8 weeks to induce obesity followed by baicalin treatment with two doses for 8 weeks. The body weight, epididymal fat weight, liver weight, food intake, oral glucose tolerance test (OGTT), oral fat tolerance test (OFTT), and serum lipids were measured. We evaluated insulin resistance by measuring the serum insulin level and homeostatic model assessment of insulin resistance (HOMA-IR). Also, the major obesity-associated immune cells including monocytes, macrophages, T lymphocytes, and dendritic cells in the blood, fat, and liver and the inflammatory and insulin signaling-related gene expressions in the fat and liver were evaluated. Results Baicalin significantly reduced the body weight and liver weight and improved serum fasting glucose, insulin, HOMA-IR, free fatty acid, HDL cholesterol, and the levels of glucose and triglyceride at each time point in the OGTT and OFTT. In the analysis of immune cells, baicalin significantly decreased inflammatory Ly6Chi monocytes, M1 adipose tissue macrophages (ATMs), and M1 Kupffer cells. On the contrary, baicalin increased anti-inflammatory M2 ATMs and liver CD4+ T cells and CD4/CD8 ratio. In the analysis of inflammatory and insulin signaling molecules, baicalin significantly downregulated the gene expression of tumor necrosis factor-α, F4/80, and C-C motif chemokine 2 while upregulated the insulin receptor mRNA expression. Conclusion From these results, baicalin can be a promising treatment option for obesity and its related metabolic diseases based on its anti-inflammatory property.

The role of adipose tissue M1/M2 macrophages in type 2 diabetes mellitus.

Obesity and insulin resistance are closely associated with a state of low-grade inflammation in the body, and adipose tissue macrophages (ATMs) play central roles in this inflammation. ATMs are known to exhibit marked functional heterogeneity. M1 ATMs produce inflammatory cytokines and induce insulin resistance. On the other hand, the majority of ATMs in lean individuals are M2 ATMs, which have anti-inflammatory potential. We found that M1 and M2 ATMs can be clearly distinguished using CD11c and CD206 as markers, and that both the number of the M1 and M2 ATMs and the M1/M2 ratio are correlated with the degree of insulin resistance. M1/M2 polarity in the adipose tissue is influenced not only by the level of secretion of various polarizing adipokines and chemokines, but also by factors in the local microenvironment, such as hypoxia. M1 ATMs acquire their polarity via activation of hypoxia-inducible factor-1α (HIF-1α) by local hypoxia, and absence of HIF-1α in the myeloid cells appears to enhance insulin sensitivity by promoting angiogenesis in adipose tissue. On the other hand, the resident M2 ATMs interact with adipose tissue progenitors to control adiposity. Thus, beyond their role as immunoregulatory cells, the M1/M2 ATMs also regulate the microenvironment in the adipose tissue and control insulin sensitivity. Recently, we have shown that interventions in the gut microbiota may be effective in controlling obesity-induced chronic inflammation. Control of M1/M2 ATM polarity is a potential therapeutic target for the treatment of insulin resistance associated with obesity.

Molecular mechanism of obesity-induced adipose tissue inflammation; the role of Mincle in adipose tissue fibrosis and ectopic lipid accumulation.

Metabolic syndrome is a common metabolic disorder that involves multiple organs and is predominantly influenced by obesity, especially the accumulation of visceral fat. It is also known that macrophages that infiltrate obese adipose tissue play an important role in inflammation of the adipose tissue. Macrophage-inducible C-type lectin (Mincle), a new inflammatory regulator found in obese adipose tissue, is expressed in pro-inflammatory M1 macrophages in adipose tissue. In addition, Mincle is expressed in macrophages that form a crown-like structure, where dead or dying adipocytes are surrounded by pro-inflammatory M1 macrophages; within this crown-like structure, adipocyte-macrophage crosstalk may occur in close proximity. Although there is no significant difference in body weight between Mincle-deficient and wild-type mice under high-fat diet, the epididymal fat weight is significantly higher and the liver weight is significantly lower in Mincle-deficient mice than those in wild-type mice. It has been shown that adipose tissue inflammation and fibrosis are attenuated in Mincle-deficient mice when compared with wild-type mice. In addition, Mincle-deficient mice have reduced hepatic lipid accumulation and better glucose metabolism. These results suggest that Mincle signaling in adipose tissue macrophages activates adipose tissue fibroblasts, which leads to adipose tissue fibrosis.

Subcutaneous administration of α-GalCer activates iNKT10 cells to promote M2 macrophage polarization and ameliorates chronic inflammation of obese adipose tissue

ObjectiveThe role of iNKT cells was investigated in chronic adipose tissue inflammation in obese mice after administration of α-GalCer in different pathways. MethodsC57BL/6J mice were fed high-fat diet (HFD) for 12 weeks to establish the obese mouse model. The pathology of adipose tissue was observed by H&E staining. The rates of iNKT cells, macrophages and cell subsets in adipose tissue were detected by FCM. Cytokine levels in serum and adipose tissue lymphocyte-stimulated supernatants were assessed with the CBA kit. The expression levels of related transcription factor in adipose tissue were detected by Western blot. ResultsThe proportions of iNKT cells, iNKT10 cells and M2 macrophages were decreased, while those of iNKT1 and M1 macrophages were increased in adipose tissue of HFD-fed mice. The expression levels of the related transcriptional proteins E4BP4 and Arg-1 were decreased while iNOS expression was increased in adipose tissue. Administration of α-GalCer by subcutaneous injection resulted in increased rates of iNKT10 cells and M2 macrophages, and decreased amounts of M1 macrophages in adipose tissue of HFD-fed mice. The expression of E4BP4 and Arg-1 were up-regulated, but iNOS was down-regulated. Meanwhile, infiltration of inflammatory cells into adipose tissue was further reduced. ConclusionThe imbalance between the proportions of iNKT1 and iNKT10 cells may be involved in the development of chronic inflammation in obese adipose tissue. Administration of α-GalCer by subcutaneous injection in HFD-fed mice activates adipose tissue iNKT10 cells, which promote M2 macrophage polarization and improve chronic inflammation in obese adipose tissue.

Decitabine assists umbilical cord-derived mesenchymal stem cells in improving glucose homeostasis by modulating macrophage polarization in type 2 diabetic mice

BackgroundMesenchymal stem cells (MSCs) have emerged as a promising therapy for type 2 diabetes (T2D). Mechanistic researches demonstrate that the anti-diabetic effect of MSCs is partially mediated by eliciting macrophages into an anti-inflammatory phenotype thus alleviating insulin resistance. However, single MSC infusion is insufficient to ameliorate sustained hyperglycemia or normalize blood glucose levels. In this study, we used decitabine (DAC), which is involved in the regulation of macrophage polarization, to test whether MSCs combined with decitabine can prolong and enhance the anti-diabetic effect in T2D mice.MethodsHigh-fat diet (HFD) and streptozocin (STZ) were given to induce T2D mouse model. Successfully induced T2D mice were randomly divided into four groups: T2D group, MSC group, DAC group, and MSC + DAC group. Blood glucose was monitored, and glucose tolerance and insulin sensitivity were evaluated during the entire analysis period. Epididymal fat was extracted for analysis of macrophage phenotype and inflammation in adipose tissue. In vitro, we examined the effect of MSC + DAC on macrophage polarization in bone marrow-derived macrophages (BMDMs) and explore the possible mechanism.ResultsMSC infusion effectively improved insulin sensitivity and glucose homeostasis in T2D mice within 1 week, whereas combination therapy of MSCs + DAC extended the anti-diabetic effects of MSCs from 1 to 4 weeks (the end of the observation). Correspondingly, more M2 macrophages in adipose tissue were observed in the combination therapy group over the entire study period. In vitro, compared with the MSC group, MSCs combined with decitabine more effectively polarized M1 macrophages to M2 macrophages. Further analysis showed that the effect of MSC + DAC on macrophage polarization was largely abrogated by the peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662.ConclusionsOur data suggest that MSCs combined with decitabine can more effectively alleviate insulin resistance and prolong and enhance the anti-diabetic effect of MSCs in T2D mice in part by prompting M2 polarization in a PPARγ-dependent manner. Thus, decitabine may be an applicable addition to MSCs for diabetes therapy.Graphic UC-MSCs combined with decitabine activate the IL4R/STAT6/STAT3/PPARγ axis to further promote M2 macrophage polarization in adipose tissue, reduce inflammation, improve insulin sensitivity, and lead to better glucose metabolism and long-term hypoglycemic effects

Alcohol-induced adipose tissue macrophage phenotypic switching is independent of myeloid Toll-like receptor 4 expression.

Alcoholic liver disease results from a combination of immune and metabolic pathogenic events. In addition to liver injury, chronic alcohol consumption also causes adipose tissue inflammation. The specific immune mechanisms that drive this process are unknown. Here, we sought to determine the role of the innate immune receptor Toll-like receptor 4 (TLR4) in alcohol-induced adipose tissue inflammation. Using a model of chronic, multiple-binge alcohol exposure, we showed that alcohol-mediated accumulation of proinflammatory adipose tissue macrophages was absent in global TLR4 knockout mice. Proinflammatory macrophage accumulation did not depend on macrophage TLR4 expression; LysMCre-driven deletion of Tlr4 from myeloid cells did not affect circulating endotoxin or the accumulation of M1 macrophages in adipose tissue following alcohol exposure. Proinflammatory cytokine/chemokine production in the adipose stromal vascular fraction also occurred independently of TLR4. Finally, the levels of other adipose immune cells, such as dendritic cells, neutrophils, B cells, and T cells, were modulated by chronic, multiple-binge alcohol and the presence of TLR4. Together, these data indicate that TLR4 expression on cells, other than myeloid cells, is important for the alcohol-induced increase in proinflammatory adipose tissue macrophages.

1765-P: Adipose Sirtuin 6 Drives Macrophage Polarization Toward M2 through IL-4 Production and Maintains Systemic Insulin Sensitivity in Mice and Human

Adipose tissue inflammation is a reproducible feature of obesity and obesity-linked insulin resistance. Here, using adipocyte-specific Sirt6 knockout mice, we explored whether adipocyte Sirt6 inhibits adipose tissue inflammation and its underlying mechanism. aS6KO mice fed normal chow gained more body weight and fat mass than wild type mice and exhibited glucose intolerance and systemic insulin resistance. Measurement of plasma and tissue cytokines and flow cytometric analysis of adipose stromal vascular cells indicated a decrease in alternatively activated M2 macrophages in adipose tissue of aS6KO mice. Mechanistically, Sirt6 regulated the expression of a canonical type 2 cytokine IL-4 by adipocytes in a cell autonomous manner, which in turn affects M2 macrophage polarization. Consistent with animal experiment data, the degree of obesity and insulin resistance demonstrated by body mass index, fasting blood glucose and HbA1c correlated negatively with the expression of Sirt6 in human visceral fat tissues. Collectively, these results suggest that adipocyte Sirt6 regulates body weight gain and insulin sensitivity independent of diet, and the increased IL-4 production by Sirt6 and resultant M2 polarization of adipose tissue macrophages may attenuate proinflammatory responses in adipose tissue. Disclosure M. Song: None.

Cannabinoid Receptor 1 Blockade Attenuates Obesity and Adipose Tissue Type 1 Inflammation Through miR-30e-5p Regulation of Delta-Like-4 in Macrophages and Consequently Downregulation of Th1 Cells.

Obesity is characterized by chronic low-grade inflammation that contributes to development of cardiometabolic disorders. Cannabinoid receptor 1 (CB1) antagonists attenuate diet-induced obesity (DIO) and related inflammation, although the precise anti-inflammatory mechanisms involved have not been fully explored. In the current study we used a mouse model of DIO intervention to determine the microRNA (miRNA, miR)-mediated anti-obesity and anti-inflammatory effects of the CB1 antagonist, AM251. DIO mice that were fed high-fat diet (HFD) for 12 weeks were treated with AM251 (10 mg/kg) for an additional 4 weeks. HFD + AM251 mice experienced rapid and prolonged weight loss and reduced inflammatory M1 adipose tissue macrophage (ATM) infiltration. To investigate miRNA-mediated regulation of ATMs, F4/80+ cells from stromal vascular fractions (SVF) of epididymal fat were subjected to miR microarray analysis. Several miRs were differentially expressed in AM251-treated mice that were independent of calorie restriction. Prominently, miR-30e-5p was upregulated in ATMs from HFD + AM251 mice while the miR-30e-5p target, DLL4, was downregulated. Consistent with a decrease in DLL4-Notch signaling, fat storage and pro-inflammatory cytokine/chemokine expression was reduced following AM251 treatment. Furthermore, we found that AM251-treated macrophages can suppress DLL4-mediated Th1 polarization in CD4+ T cells. Together these data demonstrate that blocking CB1 receptors leads to upregulation of miR-30e-5p and down regulation of DLL4 in ATMs, which in turn suppress DLL4-Notch signaling-induced polarization of inflammatory Th1 cells and adipocyte energy storage. This combined effect of ATMs and T cells leads to an anti-inflammatory state and attenuation of DIO. These data support therapeutic potential of miR-30 in the treatment of cardiometabolic disorders.

Cannabinoid receptor 1 blockade attenuates obesity and adipose tissue type 1 inflammation through miR-30e-5p regulation of Delta-like-4

Abstract Obesity is characterized by chronic low-grade inflammation that contributes to development of cardiometabolic disorders. Cannabinoid receptor 1 (CB1) antagonists attenuate diet-induced obesity (DIO) and related inflammation, although the precise anti-inflammatory mechanisms involved have not been fully explored. In the current study we used a mouse model of DIO intervention to determine the microRNA (miR)-mediated anti-obesity and anti-inflammatory effects of the CB1 antagonist AM251. DIO mice that were fed high-fat diet (HFD) for 12 weeks were treated with AM251 (10mg/kg) for an additional four weeks. HFD+AM251 mice experienced rapid and prolonged weight loss and reduced inflammatory M1 adipose tissue macrophage (ATM) infiltration. To investigate miRNA-mediated regulation of ATMs, F4/80+ cells from stromal vascular fractions of epididymal fat were subjected to miR microarray analysis. Several miRs were differentially expressed in AM251-treated mice that were independent of calorie restriction. Prominently, miR-30e-5p was upregulated in ATMs from HFD+AM251 mice while the miR-30e-5p targeting DLL4 was downregulated. Consistent with a decrease in DLL4-Notch signaling, fat storage and pro-inflammatory cytokine/chemokine expression was reduced following AM251 treatment. Furthermore, we demonstrate that AM251-treated macrophages can suppress DLL4-mediated Th1 polarization in CD4+ T cells. Together these data indicate that weight-loss due to AM251 treatment mediates anti-inflammatory effects and amelioration of obesity through miR-30e-5p regulation of DLL4-Notch signaling-induced type 1 inflammation. These data support therapeutic potential of miR-30 in the treatment of cardiometabolic disorders.

Scutellaria baicalensis Alleviates Insulin Resistance in Diet-Induced Obese Mice by Modulating Inflammation.

Insulin resistance is strongly associated with the metabolic syndrome, and chronic inflammation is known to be a major mechanism of insulin resistance and is a therapeutic target. This study was designed to evaluate the effect of Scutellaria baicalensis (SB) in high-fat diet (HFD)-induced insulin-resistant mice and to investigate its mechanism based on inflammatory responses. Mice were fed a HFD to induce insulin resistance and then administered SB for nine weeks. Body weight, glucose, lipid, insulin, epididymal fat pad and liver weights, and histologic characteristics were evaluated to determine the effect on insulin resistance. In order to evaluate the effects on the inflammatory process, we analyzed the proportions of macrophages in liver and epididymal fat and measured inflammatory gene expression. Fasting and postprandial glucose, fasting insulin, HOMA-IR, triglycerides, and low density lipoprotein cholesterol levels were significantly decreased by SB administration. The epididymal fat and liver showed significant weight decreases and histological improvements. Total adipose tissue macrophages (ATMs) decreased (27.71 ± 3.47% vs. 45.26 ± 7.26%, p < 0.05), M2 ATMs increased (47.02 ± 6.63% vs. 24.28 ± 8.00%, p < 0.05), and CD11b+ Kupffer cells decreased. The expression levels of tumor necrosis factor alpha and F4/80 in the liver were significantly decreased (12.03 ± 1.47% vs. 25.88 ± 4.57%, p < 0.05) compared to HFD group. These results suggest that SB improved insulin resistance through inhibition of macrophage-mediated inflammation.

Berberine, a Traditional Chinese Medicine, Reduces Inflammation in Adipose Tissue, Polarizes M2 Macrophages, and Increases Energy Expenditure in Mice Fed a High-Fat Diet.

BackgroundThe uncoupling protein 1 (UCP1) gene has a role in mitochondrial energy expenditure in brown adipose tissue. This study aimed to investigate the effects of berberine, a benzylisoquinoline alkaloid used in traditional Chinese medicine, on energy expenditure, expression of the UCP1 gene, the cell stress protein inositol-requiring enzyme 1α (IRE1α), apoptosis genes, and macrophage phenotype (M1 and M2) in white and brown adipose tissue in an obese mouse model fed a high-fat diet.Material/MethodsFour-week-old C57BL/6J male mice (n=20) were divided into a high-fat diet group, a normal diet group, a group treated with berberine at 100 mg/kg/d in 0.9% normal saline, and a non-treated group. Whole-body fat mass, blood glucose, insulin resistance, and oxygen expenditure during physical activity were measured. After 16 weeks, the mice were euthanized for examination of liver and adipose tissue. The expression of pro-inflammatory cytokines, apoptosis genes, thermogenic genes (including UCP1), and IRE1α, were investigated using immunohistochemistry, Western blot, and quantitative reverse transcription polymerase chain reaction (qRT-PCR), in white and brown adipose tissue. Magnetic cell sorting harvested M1 and M2 macrophages in adipose tissue. Clodronate liposomes were used to inhibit macrophage recruitment.ResultsBerberine treatment in mice fed a high-fat diet increased energy metabolism, glucose tolerance, and expression of UCP1, and reduced expression of pro-inflammatory cytokines, macrophage recruitment, and resulted in M2 macrophage polarization in white adipose tissue. Polarized M2 macrophages showed reduced expression of apoptotic genes and IRE1α.ConclusionsBerberine improved metabolic function in a mouse model fed a high-fat diet.

Loss of Fractalkine-CX3CR1 Signaling Exacerbates Obesity-Induced Adipose Tissue Inflammation and Insulin Resistance through M1 Dominant Shift in Macrophages

Adipose tissue macrophages (ATMs) accumulation through CCR2 and its ligand MCP-1 is considered pivotal in the development of insulin resistance. Recently, we found that CX3C chemokine, fractalkine (CX3cl1) mRNA expression was persistently downregulated in epididymal white adipose tissue (eWAT) of HF diet (HFD)-induced obese (DIO) mice, as compared to lean controls. Furthermore, we demonstrated that loss of fractalkine receptor, CX3CR1, exacerbated HFD-induced insulin resistance and glucose intolerance. Here, we show that fractalkine-CX3CR1 signaling plays a crucial role in the inflammatory response to HF feeding by regulating macrophage M1/M2 status. To determine the effect of fractalkine-CX3CR1 signaling on ATM subsets, we performed flow cytometry analysis to quantify M1/M2 ATMs in eWAT of CX3CR1-/- (KO) and WT mice. Immunofluorescence study of eWAT in DIO mice revealed that CX3CR1 was predominantly expressed by F4/80+ macrophages in crown-like structures (CLS). On an HFD, KO mice had increased macrophage infiltration and formation of CLS in eWAT compared with those of WT mice, despite that weight and adipocyte size were similar. KO mice had 40% or 39% more CD11c+CD206- (M1) ATMs on normal chow (NC) or HFD, and 17% or 49% fewer CD11c-CD206+ (M2) ATMs on NC or HFD, respectively, than WT mice, resulting in predominance of the M1 over M2 ATM population. Furthermore, the predominance of the Ly6Chi over the Ly6C- monocyte population was observed in the peripheral blood and bone marrow (BM) of KO mice on NC or HFD. Importantly, HFD-induced adipose inflammation and hyperinsulinemia were aggravated in chimeric mice lacking CX3CR1 only in myeloid cells (BM transplant of KO to WT) as compared to control mice (BM transplant of WT to WT). Thus, loss of fractalkine-CX3CR1 signaling causes M1 dominant phenotypic shift in ATMs, which contributes to exacerbation of obesity-induced adipose tissue inflammation and insulin resistance. Disclosure M. Nagashimada: None. Y. Ni: None. T. Ota: None.