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While obesity and associated metabolic complications are linked to inflammation of white adipose tissue (WAT), the causal factors remain unclear. We hypothesized that the local metabolic environment could be an important determinant. To this end, we compared metabolites released from WAT of 81 obese and non-obese women. This identified glutamine to be downregulated in obesity and inversely associated with a pernicious WAT phenotype. Glutamine administration in vitro and in vivo attenuated both pro-inflammatory gene and protein levels in adipocytes and WAT and macrophage infiltration in WAT. Metabolomic and bioenergetic analyses in human adipocytes suggested that glutamine attenuated glycolysis and reduced uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) levels. UDP-GlcNAc is the substrate for the post-translational modification O-linked β-N-acetylglucosamine (O-GlcNAc) mediated by the enzyme O-GlcNAc transferase. Functional studies in human adipocytes established a mechanistic link between reduced glutamine, O-GlcNAcylation of nuclear proteins, and a pro-inflammatory transcriptional response. Altogether, glutamine metabolism is linked to WAT inflammation in obesity., Competing Interests: Declaration of Interests Following the initial submission of this work, M.M. has since June 2019 taken up a position as Senior Director and Staff Scientist at Genentech, Inc. and is a holder of Roche stock. However, neither he nor any other author of this work has any conflict of interest to report., (Copyright © 2019 Elsevier Inc. All rights reserved.)

White adipose tissue (WAT) inflammation contributes to the development of insulin resistance in obesity. While the role of adipose tissue macrophage (ATM) pro-inflammatory signalling in the development of insulin resistance has been established, it is less clear how WAT inflammation is initiated. Here, we show that ATMs isolated from obese mice and humans exhibit markers of increased rate of de novo phosphatidylcholine (PC) biosynthesis. Macrophage-specific knockout of phosphocholine cytidylyltransferase A (CCTα), the rate-limiting enzyme of de novo PC biosynthesis pathway, alleviated obesity-induced WAT inflammation and insulin resistance. Mechanistically, CCTα-deficient macrophages showed reduced ER stress and inflammation in response to palmitate. Surprisingly, this was not due to lower exogenous palmitate incorporation into cellular PCs. Instead, CCTα-null macrophages had lower membrane PC turnover, leading to elevated membrane polyunsaturated fatty acid levels that negated the pro-inflammatory effects of palmitate. Our results reveal a causal link between obesity-associated increase in de novo PC synthesis, accelerated PC turnover and pro-inflammatory activation of ATMs., Competing Interests: KP, SV, GB, BJ, CÇ, CM, MA, JD, MS, AK, AV No competing interests declared, (© 2019, Petkevicius et al.)

Signalling pathways that control endothelial cell (EC) permeability, leukocyte adhesion and inflammation are pivotal for atherosclerosis initiation and progression. Here we demonstrate that the Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), which has been implicated in inflammation, is abundantly expressed in ECs and in atherosclerotic plaques from mice and humans. On the basis of endothelial-specific MAP4K4 gene silencing and gene ablation experiments in Apoe(-/-) mice, we show that MAP4K4 in ECs markedly promotes Western diet-induced aortic macrophage accumulation and atherosclerotic plaque development. Treatment of Apoe(-/-) and Ldlr(-/-) mice with a selective small-molecule MAP4K4 inhibitor also markedly reduces atherosclerotic lesion area. MAP4K4 silencing in cultured ECs attenuates cell surface adhesion molecule expression while reducing nuclear localization and activity of NFκB, which is critical for promoting EC activation and atherosclerosis. Taken together, these results reveal that MAP4K4 is a key signalling node that promotes immune cell recruitment in atherosclerosis.

Adipose tissue (AT) of obese mice and humans accumulates immune cells, which secrete cytokines that can promote insulin resistance. AT macrophages (ATMs) are thought to originate from bone-marrow-derived monocytes, which infiltrate the tissue from the circulation. Here, we show that a major fraction of macrophages unexpectedly undergo cell division locally within AT, as detected by Ki67 expression and 5-ethynyl-2'-deoxyuridine incorporation. Macrophages within the visceral AT (VAT), but not those in other tissues (including liver and spleen), displayed increased proliferation in obesity. Importantly, depletion of blood monocytes had no impact on ATM content, whereas their proliferation in situ continued. Treatment with monocyte chemotactic protein 1 (MCP-1) induced macrophage cell division in AT explants, whereas mcp-1 deficiency in vivo decreased ATM proliferation. These results reveal that, in addition to blood monocyte recruitment, in situ proliferation driven by MCP-1 is an important process by which macrophages accumulate in the VAT in obesity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The pathophysiology of obesity and type 2 diabetes in rodents and humans is characterized by low-grade inflammation in adipose tissue and liver. The CD40 receptor and its ligand CD40L initiate immune cell signaling promoting inflammation, but conflicting data on CD40L-null mice confound its role in obesity-associated insulin resistance. Here, we demonstrate that CD40 receptor-deficient mice on a high-fat diet display the expected decrease in hepatic cytokine levels but paradoxically exhibit liver steatosis, insulin resistance, and glucose intolerance compared with their age-matched wild-type controls. Hyperinsulinemic-euglycemic clamp studies also demonstrated insulin resistance in glucose utilization by the CD40-null mice compared with wild-type mice. In contrast to liver, adipose tissue in CD40-deficient animals harbors elevated cytokine levels and infiltration of inflammatory cells, particularly macrophages and CD8(+) effector T cells. In addition, ex vivo explants of epididymal adipose tissue from CD40(-/-) mice display elevated basal and isoproterenol-stimulated lipolysis, suggesting a potential increase of lipid efflux from visceral fat to the liver. These findings reveal that 1) CD40-null mice represent an unusual model of hepatic steatosis with reduced hepatic inflammation, and 2) CD40 unexpectedly functions in adipose tissue to attenuate its inflammation in obesity, thereby protecting against hepatic steatosis.

Thoracic perivascular adipose tissue (PVAT) is a unique adipose depot that likely influences vascular function and susceptibility to pathogenesis in obesity and the metabolic syndrome. Surprisingly, PVAT has been reported to share characteristics of both brown and white adipose, but a detailed direct comparison to interscapular brown adipose tissue (BAT) has not been performed. Here we show by full genome DNA microarray analysis that global gene expression profiles of PVAT are virtually identical to BAT, with equally high expression of Ucp-1, Cidea, and other genes known to be uniquely or very highly expressed in BAT. PVAT and BAT also displayed nearly identical phenotypes upon immunohistochemical analysis, and electron microscopy confirmed that PVAT contained multilocular lipid droplets and abundant mitochondria. Compared with white adipose tissue (WAT), PVAT and BAT from C57BL6/J mice fed a high-fat diet for 13 wk had markedly lower expression of immune cell-enriched mRNAs, suggesting resistance to obesity-induced inflammation. Indeed, staining of BAT and PVAT for macrophage markers (F4/80 and CD68) in obese mice showed virtually no macrophage infiltration, and FACS analysis of BAT confirmed the presence of very few CD11b(+)/CD11c(+) macrophages in BAT (1.0%) compared with WAT (31%). In summary, murine PVAT from the thoracic aorta is virtually identical to interscapular BAT, is resistant to diet-induced macrophage infiltration, and thus may play an important role in protecting the vascular bed from inflammatory stress.

Toll-like receptors (TLRs) function as initiators of inflammation through their ability to sense pathogen-associated molecular patterns and products of tissue damage. Transcriptional activation of many TLR-responsive genes requires an initial de-repression step in which nuclear receptor co-repressor (NCoR) complexes are actively removed from the promoters of target genes to relieve basal repression. Ligand-dependent SUMOylation of liver X receptors (LXRs) has been found to suppress TLR4-induced transcription potently by preventing the NCoR clearance step, but the underlying mechanisms remain enigmatic. Here we provide evidence that coronin 2A (CORO2A), a component of the NCoR complex of previously unknown function, mediates TLR-induced NCoR turnover by a mechanism involving interaction with oligomeric nuclear actin. SUMOylated LXRs block NCoR turnover by binding to a conserved SUMO2/SUMO3-interaction motif in CORO2A and preventing actin recruitment. Intriguingly, the LXR transrepression pathway can itself be inactivated by inflammatory signals that induce calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ)-dependent phosphorylation of LXRs, leading to their deSUMOylation by the SUMO protease SENP3 and release from CORO2A. These findings uncover a CORO2A-actin-dependent mechanism for the de-repression of inflammatory response genes that can be differentially regulated by phosphorylation and by nuclear receptor signalling pathways that control immunity and homeostasis.

Gene silencing by double-stranded RNA, denoted RNA interference, represents a new paradigm for rational drug design. However, the transformative therapeutic potential of short interfering RNA (siRNA) has been stymied by a key obstacle-safe delivery to specified target cells in vivo. Macrophages are particularly attractive targets for RNA interference therapy because they promote pathogenic inflammatory responses in diseases such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease and diabetes. Here we report the engineering of beta1,3-D-glucan-encapsulated siRNA particles (GeRPs) as efficient oral delivery vehicles that potently silence genes in mouse macrophages in vitro and in vivo. Oral gavage of mice with GeRPs containing as little as 20 microg kg(-1) siRNA directed against tumour necrosis factor alpha (Tnf-alpha) depleted its messenger RNA in macrophages recovered from the peritoneum, spleen, liver and lung, and lowered serum Tnf-alpha levels. Screening with GeRPs for inflammation genes revealed that the mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) is a previously unknown mediator of cytokine expression. Importantly, silencing Map4k4 in macrophages in vivo protected mice from lipopolysaccharide-induced lethality by inhibiting Tnf-alpha and interleukin-1beta production. This technology defines a new strategy for oral delivery of siRNA to attenuate inflammatory responses in human disease.

Objective Obesity-induced accumulation of ectopic fat in the liver is thought to contribute to the development of insulin resistance, and increased activity of hepatic CB 1 R has been shown to promote both processes. However, lipid accumulation in liver can be experimentally dissociated from insulin resistance under certain conditions, suggesting the involvement of additional mechanisms. Obesity is also associated with pro-inflammatory changes which, in turn, can promote insulin resistance. Kupffer cells (KCs), the liver's resident macrophages, are the major source of pro-inflammatory cytokines in the liver, such as TNF-α, which has been shown to inhibit insulin signaling in multiple cell types, including hepatocytes. Here, we sought to identify the role of CB 1 R in KCs in obesity-induced hepatic insulin resistance. Methods We used intravenously administered β-D-glucan-encapsulated siRNA to knock-down CB 1 R gene expression selectively in KCs. Results We demonstrate that a robust knock-down of the expression of Cnr1 , the gene encoding CB 1 R, results in improved glucose tolerance and insulin sensitivity in diet-induced obese mice, without affecting hepatic lipid content or body weight. Moreover, Cnr1 knock-down in KCs was associated with a shift from pro-inflammatory M1 to anti-inflammatory M2 cytokine profile and improved insulin signaling as reflected by increased insulin-induced Akt phosphorylation. Conclusion These findings suggest that CB 1 R expressed in KCs plays a critical role in obesity-related hepatic insulin resistance via a pro-inflammatory mechanism. [ABSTRACT FROM AUTHOR]

Macrophages have diverse phenotypes and functions due to differences in their origin, location and pathophysiological context. Although their main role in the liver has been described as immunoregulatory and detoxifying, changes in macrophage phenotypes, diversity, dynamics and function have been reported during obesity-related complications such as non-alcoholic fatty liver disease (NAFLD). NAFLD encompasses multiple disease states from hepatic steatosis to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocarcinoma. Obesity and insulin resistance are prominent risk factors for NASH, a disease with a high worldwide prevalence and no approved treatment. In this Review, we discuss the turnover and function of liver-resident macrophages (Kupffer cells) and monocyte-derived hepatic macrophages. We examine these populations in both steady state and during NAFLD, with an emphasis on NASH. The explosion in high-throughput gene expression analysis using single-cell RNA sequencing (scRNA-seq) within the last 5 years has revolutionized the study of macrophage heterogeneity, substantially increasing our understanding of the composition and diversity of tissue macrophages, including in the liver. Here, we highlight scRNA-seq findings from the last 5 years on the diversity of liver macrophages in homeostasis and metabolic disease, and reveal hepatic macrophage function beyond their classically described inflammatory role in the progression of NAFLD and NASH pathogenesis. [ABSTRACT FROM AUTHOR]

While the role of transcription factors and coactivators in controlling enhancer activity and chromatin structure linked to gene expression is well established, the involvement of corepressors is not. Using inflammatory macrophage activation as a model, we investigate here a corepressor complex containing GPS2 and SMRT both genome-wide and at the Ccl2 locus, encoding the chemokine CCL2 (MCP-1). We report that corepressors co-occupy candidate enhancers along with the coactivators CBP (H3K27 acetylase) and MED1 (mediator) but act antagonistically by repressing eRNA transcription-coupled H3K27 acetylation. Genome editing, transcriptional interference, and cistrome analysis reveals that apparently related enhancer and silencer elements control Ccl2 transcription in opposite ways. 4C-seq indicates that corepressor depletion or inflammatory signaling functions mechanistically similarly to trigger enhancer activation. In ob/ob mice, adipose tissue macrophage-selective depletion of the Ccl2 enhancer-transcribed eRNA reduces metaflammation. Thus, the identified corepressor-eRNA-chemokine pathway operates in vivo and suggests therapeutic opportunities by targeting eRNAs in immuno-metabolic diseases. • Corepressor recruitment is a genome-wide feature of inflammatory enhancers • Corepressors control enhancer-silencer-promoter looping within the Ccl2 TAD • Corepressors antagonize eRNA transcription and CBP-mediated H3K27 acetylation • Ccl2 eRNA depletion in ob/ob mice supports eRNA function and targeting options Huang et al. dissect epigenetic mechanisms underlying the anti-inflammatory action of macrophage corepressors. By studying chromatin structure and histone modifications, transcription factor and coregulator dynamics, and eRNA transcription at the mouse Ccl2 locus, they uncover a role of corepressors in controlling enhancer and silencer remodeling linked to inflammatory gene expression. [ABSTRACT FROM AUTHOR]