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Objective Macrophage accumulation in adipose tissue (AT) during obesity contributes to inflammation and insulin resistance. Recruitment of monocytes to obese AT has been the most studied mechanism explaining this accumulation. However, recent evidence suggests that recruitment-independent mechanisms may also regulate pro-inflammatory AT macrophage (ATM) numbers. The role of increased ATM survival during obesity has yet to be explored. Results We demonstrate that activation of apoptotic pathways is significantly reduced in ATMs from diet-induced and genetically obese mice. Concurrently, pro-survival Bcl-2 family member protein levels and localization to the mitochondria is elevated in ATMs from obese mice. This increased pro-survival signaling was associated with elevated activation of the transcription factor, NF-κB, and increased expression of its pro-survival target genes. Finally, an obesogenic milieu increased ATM viability only when NF-κB signaling pathways were functional. Conclusions Our data demonstrate that obesity promotes survival of inflammatory ATMs, possibly through an NF-κB-regulated mechanism., Graphical abstract, Highlights • Macrophage apoptosis is decreased in obese adipose tissue. • ATMs from obese mice display increased mitochondrial localization of Bcl-2. • The pro-survival targets of NF-κB are increased in ATMs from obese mice. • NF-κB activation in ATMs during metabolic stimulation increases their survival. • Decreased ATM apoptosis contributes to macrophage accumulation in obesity.

Adipose tissue expansion is accompanied by the infiltration and accumulation of adipose tissue macrophages (ATMs), as well as a shift in ATM polarization. Several studies have implicated recruited M1 ATMs in the metabolic consequences of obesity; however, little is known regarding the role of alternatively activated resident M2 ATMs in adipose tissue homeostasis, or how their function is altered in obesity. Herein, we report the discovery of a population of alternatively activated ATMs with elevated cellular iron content and an iron recycling gene expression profile. These iron-rich ATMs are referred to as MFe(hi), and the remaining ATMs are referred to as MFe(lo). In lean mice, approximately 25% of the ATMs are MFe(hi); this percentage decreases in obesity due to the recruitment of MFe(lo) macrophages. Similar to MFe(lo) cells, MFe(hi) ATMs undergo an inflammatory shift in obesity. In vivo, obesity reduces the iron content of MFe(hi) ATMs and the gene expression of iron importers as well as the iron exporter, ferroportin, suggesting an impaired ability to handle iron. In vitro, exposure of primary peritoneal macrophages to saturated fatty acids also alters iron metabolism gene expression. Finally, the impaired MFe(hi) iron handling coincides with adipocyte iron overload in obese mice. In conclusion, in obesity, iron distribution is altered both at the cellular and tissue levels, with adipose tissue playing a predominant role in this change. An increased availability of fatty acids during obesity may contribute to the observed changes in MFe(hi) ATMs phenotype and their reduced capacity to handle iron.

Macrophage and T cell infiltration into metabolic tissues contributes to obesity-associated inflammation and insulin resistance (IR). C-C chemokine receptor 5 (CCR5), expressed on macrophages and T cells, plays a critical role in the recruitment and activation of proinflammatory M1 and TH1 immune cells to tissues and is elevated in adipose tissue (AT) and liver of obese humans and mice. Thus, we hypothesized that deficiency of CCR5 would protect against diet-induced inflammation and IR. CCR5-deficient (CCR5−/−) mice and C57BL/6 (WT) controls were fed 10% low-fat (LF) or 60% high-fat (HF) diets for 16 wk. HF feeding increased adiposity, blood glucose, and plasma insulin levels equally in both genotypes. Opposing our hypothesis, HF-fed CCR5−/− mice were significantly more glucose intolerant than WT mice. In AT, there was a significant reduction in the M1-associated gene CD11c, whereas M2 associated genes were not different between genotypes. In addition, HF feeding caused a twofold increase in CD4+ T cells in the AT of CCR5−/− compared with WT mice. In liver and muscle, no differences in immune cell infiltration or inflammatory cytokine expression were detected. However, in AT and muscle, there was a mild reduction in insulin-induced phosphorylation of AKT and IRβ in CCR5−/− compared with WT mice. These findings suggest that whereas CCR5 plays a minor role in regulating immune cell infiltration and inflammation in metabolic tissues, deficiency of CCR5 impairs systemic glucose tolerance as well as AT and muscle insulin signaling.

Fish oil (FO) is a potent anti-inflammatory and lipid-lowering agent. Because inflammation can modulate lipid metabolism and vice versa, we hypothesized that combining FO with cyclooxygenase inhibitors (COXIBs), well-known anti-infl ammatory drugs, can enhance the antiinfl ammatory and lipid-lowering effect of FO. LDLR⁻/⁻ mice were fed a high-fat diet supplemented with 6% olive oil or FO for 12 wk in the presence or absence of indomethacin (Indo, 6 mg/l drinking water). FO reduced plasma total cholesterol by 30% but, in combination with Indo, exerted a greater decrease (44%). The reduction of liver cholesterol ester (CE) and triglycerides (TG) by FO (63% and 41%, respectively) was enhanced by Indo (80% in CE and 64% in TG). FO + Indo greatly increased the expression of genes modulating lipid metabolism and reduced the expression of inflammatory genes compared with control. The mRNA and/or protein expression of pregnane X receptor (PXR) and cytochrome P450 isoforms that alter inflammation and/or lipid metabolism are increased to a greater extent in mice that received FO + Indo. Moreover, the nuclear level of PXR is significantly increased in FO + Indo group. Combining FO with COXIBs may exert their beneficial effects on inflammation and lipid metabolism via PXR and cytochrome P450. This project was partly supported by the American Heart Association Scientist Development grant (0930335N) and a Pilot and Feasibility grant from the Digestive Disease Research Center at Vanderbilt University (DK 058404) (V.S.) and by National Institutes of Health grants GM15431 and ES13125 (G.L.M.) and DK-078765 (M.W.H.). Its contents are solely the responsibility of the authors and do not necessarily represent the offi cial views of the National Institutes of Health or other granting agencies.

OBJECTIVE Mice with CCR2 deficiency are protected from insulin resistance but only after long periods of high-fat diet (HFD) feeding, despite the virtual absence of circulating inflammatory monocytes. We performed a time course study in mice with hematopoietic and global CCR2 deficiency to determine adipose tissue–specific mechanisms for the delayed impact of CCR2 deficiency on insulin resistance. RESEARCH DESIGN AND METHODS Mice with global or hematopoietic CCR2 deficiency (CCR2−/− and BM-CCR2−/−, respectively) and wild-type controls (CCR2+/+ and BM-CCR2+/+, respectively) were placed on an HFD for 6, 12, and 20 weeks. Adipose tissue myeloid populations, degree of inflammation, glucose tolerance, and insulin sensitivity were assessed. RESULTS Flow cytometry analysis showed that two different populations of F4/80+ myeloid cells (CD11bloF4/80lo and CD11bhiF4/80hi) accumulated in the adipose tissue of CCR2−/− and BM-CCR2−/− mice after 6 and 12 weeks of HFD feeding, whereas only the CD11bhiF4/80hi population was detected in the CCR2+/+ and BM-CCR2+/+ controls. After 20 weeks of HFD feeding, the CD11bloF4/80lo cells were no longer present in the adipose tissue of CCR2−/− mice, and only then were improvements in adipose tissue inflammation detected. Gene expression and histological analysis of the CD11bloF4/80lo cells indicated that they are a unique undifferentiated monocytic inflammatory population. The CD11bloF4/80lo cells are transiently found in wild-type mice, but CCR2 deficiency leads to the aberrant accumulation of these cells in adipose tissue. CONCLUSIONS The discovery of this novel adipose tissue monocytic cell population provides advances toward understanding the pleiotropic role of CCR2 in monocyte/macrophage accumulation and regulation of adipose tissue inflammation.

Macrophages and T-lymphocytes are known to accumulate in the white adipose tissue (WAT) of obese mice and humans, but the factors that cause this infiltration are not yet determined. Chemokines, which attract leukocytes to inflammatory sites, are candidates for this process. Macrophage inflammatory protein-1alpha (MIP-1alpha) expression is significantly elevated in WAT of obese mice and humans and positively correlates with fasting plasma insulin, but its potential role in leukocyte recruitment to WAT is unknown. MIP-1alpha-deficient, heterozygous, and wild-type mice were fed a Western diet (WD) for 16 wk. Plasma lipids, adipose tissue mass, energy expenditure, food intake, liver triglyceride content, and inflammatory cytokine expression were not different among genotypes. Gene expression of macrophage markers F4/80 and CD68, as well as T-lymphocyte marker CD3epsilon was increased in perigonadal WAT of obese WD-fed mice but was not influenced by MIP-1alpha expression level. Immunohistochemical analysis of WAT also showed no effect of MIP-1alpha on macrophage content. Two related chemokines, MIP-1beta and RANTES, had reduced expression in obese male MIP-1alpha-deficient mice compared with wild-type controls (Por = 0.05). In mice fed the WD for 6 wk, WAT macrophage content was unchanged; however, CD8+ T-lymphocytes accumulated to a lesser extent in the MIP-1alpha-null mice. Therefore, expression of MIP-1alpha, as well as that of MIP-1beta and RANTES, increases as a consequence of weight gain, but these chemokines may not be required for the recruitment of monocytes to WAT during diet-induced obesity in mice and may impact T-lymphocyte recruitment only at early time points after WD feeding.

Quantitative fat-water MRI (FWMRI) methods provide valuable information about the distribution, volume, and composition of adipose tissue (AT). Ultra high field FWMRI of animal models may have the potential to provide insights into the progression of obesity and its comorbidities. Here, we present quantitative FWMRI with all known confounder corrections on a 15.2T preclinical scanner for noninvasive in vivo monitoring of an established diet-induced obesity mouse model. Male C57BL/6J mice were placed on a low-fat (LFD) or a high-fat diet (HFD). Three-dimensional (3-D) multiple gradient echo MRI at 15.2T was performed at baseline, 4, 8, 12, and 16 weeks after diet onset. A 3-D fat-water separation algorithm and additional processing were used to generate proton-density fat fraction (PDFF), local magnetic field offset, and [Formula: see text] maps. We examined these parameters in perirenal AT ROIs from LFD and HFD mice. The data suggest that PDFF, local field offset, and [Formula: see text] have different time course behaviors between LFD and HFD mice over 16 weeks. This work suggests FWMRI at 15.2T may be a useful tool for longitudinal studies of adiposity due to the advantages of ultra high field although further investigation is needed to understand the observed time course behavior.

Macrophage inflammatory protein-1α (CCL3) plays a well-known role in infectious and viral diseases; however, its contribution to atherosclerotic lesion formation and lipid metabolism has not been determined. Low density lipoprotein receptor deficient (LDLR(-/-)) mice were transplanted with bone marrow from CCL3(-/-) or C57BL/6 wild type donors. After 6 and 12 weeks on western diet (WD), recipients of CCL3(-/-) marrow demonstrated lower plasma cholesterol and triglyceride concentrations compared to recipients of C57BL/6 marrow. Atherosclerotic lesion area was significantly lower in female CCL3(-/-) recipients after 6 weeks and in male CCL3(-/-) recipients after 12 weeks of WD feeding (P