linc-ADAIN, a human adipose lincRNA, regulates adipogenesis by modulating KLF5 and IL-8 mRNA stability.

Adipose tissue remodeling and dysfunction, characterized by elevated inflammation and insulin resistance, play a central role in obesity-related development of type 2 diabetes (T2D) and cardiovascular diseases. Long intergenic non-coding RNAs (lincRNAs) are important regulators of cellular functions. Here, we describe the functions of linc-ADAIN (adipose anti-inflammatory), an adipose lincRNA that is downregulated in white adipose tissue of obese humans. We demonstrate that linc-ADAIN knockdown (KD) increases KLF5 and interleukin-8 (IL-8) mRNA stability and translation by interacting with IGF2BP2. Upregulation of KLF5 and IL-8, via linc-ADAIN KD, leads to an enhanced adipogenic program and adipose tissue inflammation, mirroring the obese state, in vitro and in vivo. KD of linc-ADAIN in human adipose stromal cell (ASC) hTERT adipocytes implanted into mice increases adipocyte size and macrophage infiltration compared to implanted control adipocytes, mimicking hallmark features of obesity-induced adipose tissue remodeling. linc-ADAIN is an anti-inflammatory lincRNA that limits adipose tissue expansion and lipid storage.

Human Macrophage Long Intergenic Noncoding RNA, SIMALR, Suppresses Inflammatory Macrophage Apoptosis via NTN1 (Netrin-1).

BACKGROUND: Long noncoding RNAs (lncRNAs) have emerged as novel regulators of macrophage biology and inflammatory cardiovascular diseases. However, studies focused on lncRNAs in human macrophage subtypes, particularly human lncRNAs that are not conserved in rodents, are limited. METHODS: Through RNA-sequencing of human monocyte-derived macrophages, we identified suppressor of inflammatory macrophage apoptosis lncRNA (SIMALR). Lipopolysaccharide/IFNγ (interferon γ) stimulated human macrophages were treated with SIMALR antisense oligonucleotides and subjected to RNA-sequencing to investigate the function of SIMALR. Western blots, luciferase assay, and RNA immunoprecipitation were performed to validate function and potential mechanism of SIMALR. RNAscope was performed to identify SIMALR expression in human carotid atherosclerotic plaques. RESULTS: RNA-sequencing of human monocyte-derived macrophages identified SIMALR, a human macrophage-specific long intergenic noncoding RNA that is highly induced in lipopolysaccharide/IFNγ-stimulated macrophages. SIMALR knockdown in lipopolysaccharide/IFNγ stimulated THP1 human macrophages induced apoptosis of inflammatory macrophages, as shown by increased protein expression of cleaved PARP (poly[ADP-ribose] polymerase), caspase 9, caspase 3, and Annexin V+. RNA-sequencing of control versus SIMALR knockdown in lipopolysaccharide/IFNγ-stimulated macrophages showed Netrin-1 (NTN1) to be significantly decreased upon SIMALR knockdown. We confirmed that NTN1 knockdown in lipopolysaccharide/IFNγ-stimulated macrophages induced apoptosis. The SIMALR knockdown-induced apoptotic phenotype was rescued by adding recombinant NTN1. NTN1 promoter-luciferase reporter activity was increased in HEK293T (human embryonic kidney 293) cells treated with lentiviral overexpression of SIMALR. NTN1 promoter activity is known to require HIF1α (hypoxia-inducible factor 1 subunit alpha), and our studies suggest that SIMALR may interact with HIF1α to regulate NTN1 transcription, thereby regulating macrophages apoptosis. SIMALR was found to be expressed in macrophages in human carotid atherosclerotic plaques of symptomatic patients. CONCLUSIONS: SIMALR is a nonconserved, human macrophage lncRNA expressed in atherosclerosis that suppresses macrophage apoptosis. SIMALR partners with HIF1α (hypoxia-inducible factor 1 subunit alpha) to regulate NTN1, which is a known macrophage survival factor. This work illustrates the importance of interrogating the functions of human lncRNAs and exploring their translational and therapeutic potential in human atherosclerosis.

Cis-regulated expression of non-conserved lincRNAs associates with cardiometabolic related traits.

Many complex disease risk loci map to intergenic regions containing long intergenic noncoding RNAs (lincRNAs). The majority of these is not conserved outside humans, raising the question whether genetically regulated expression of non-conserved and conserved lincRNAs has similar rates of association with complex traits. Here we leveraged data from the Genotype-Tissue Expression (GTEx) project and multiple public genome-wide association study (GWAS) resources. Using an established transcriptome-wide association study (TWAS) tool, FUSION, we interrogated the associations between cis-regulated expression of lincRNAs and multiple cardiometabolic traits. We found that cis-regulated expression of non-conserved lincRNAs had a strikingly similar trend of association with complex cardiometabolic traits as conserved lincRNAs. This finding challenges the conventional notion of conservation that has led to prioritization of conserved loci for functional studies and calls attention to the need to develop comprehensive strategies to study the large number of non-conserved human lincRNAs that may contribute to human disease.

Palmitic Acid and Oleic Acid Differently Modulate TLR2-Mediated Inflammatory Responses in Microglia and Macrophages.

The relationship between systemic immunity and neuroinflammation is widely recognised. Infiltration of peripheral immune cells to the CNS during certain chronic inflammatory states contributes significantly to neuropathology. Obesity and its co-morbidities are primary risk factors for neuroinflammatory and neurodegenerative conditions, including Alzheimer's disease (AD). Dietary fats are among the most proinflammatory components of the obesogenic diet and play a prominent role in the low-grade systemic inflammation associated with the obese state. Saturated fatty acid (SFA) is largely implicated in the negative consequences of obesity, while the health benefits of monounsaturated fatty acid (MUFA) are widely acknowledged. The current study sought to explore whether SFA and MUFA differently modulate inflammatory responses in the brain, compared with peripheral immune cells. Moreover, we assessed the neuroinflammatory impact of high-fat-induced obesity and hypothesised that a MUFA-rich diet might mitigate inflammation despite obesogenic conditions. Toll-like receptor (TLR)2 mediates the inflammation associated with both obesity and AD. Using the TLR2 agonist lipoteichoic acid (LTA), we report that pre-exposure to either palmitic acid (PA) or oleic acid (OA) attenuated cytokine secretion from microglia, but heightened sensitivity to nitric oxide (NO) production. The reduction in cytokine secretion was mirrored in LTA-stimulated macrophages following exposure to PA only, while effects on NO were restricted to OA, highlighting important cell-specific differences. An obesogenic diet over 12 weeks did not induce prominent inflammatory changes in either cortex or hippocampus, irrespective of fat composition. However, we reveal a clear disparity in the effects of MUFA under obesogenic and non-obesogenic conditions.

Sodium salicylate rewires hepatic metabolic pathways in obesity and attenuates IL-1ß secretion from adipose tissue: The implications for obesity-impaired reverse cholesterol transport.

INTRODUCTION: High-fat diet (HFD)-induced obesity impairs clearance of cholesterol through the Reverse Cholesterol Transport (RCT) pathway, with downregulation in hepatic expression of cholesterol and bile acid transporters, namely ABCG5/8 and ABCB11, and reduced high-density lipoprotein (HDL) cholesterol efflux capacity (CEC). In the current study, we hypothesized that the development of hepatosteatosis, secondary to adipose-tissue dysfunction, contributes to obesity-impaired RCT and that such effects could be mitigated using the anti-inflammatory drug sodium salicylate (NaS). MATERIALS AND METHODS: C57BL/6J mice, fed HFD ± NaS or low-fat diet (LFD) for 24 weeks, underwent glucose and insulin tolerance testing. The 3H-cholesterol movement from macrophage-to-feces was assessed in vivo. HDL-CEC was determined ex vivo. Cytokine secretion from adipose-derived stromal vascular fraction (SVF) cells was measured ex vivo. Liver and HDL proteins were determined by mass spectrometry and analyzed using Ingenuity Pathway Analysis. RESULTS: NaS delayed HFD-induced weight gain, abrogated priming of pro-IL-1ß in SVFs, attenuated insulin resistance, and prevented steatohepatitis (ectopic fat accumulation in the liver). Prevention of hepatosteatosis coincided with increased expression of PPAR-alpha/beta-oxidation proteins with NaS and reduced expression of LXR/RXR-induced proteins including apolipoproteins. The latter effects were mirrored within the HDL proteome in circulation. Despite remarkable protection shown against steatosis, HFD-induced hypercholesterolemia and repression of the liver-to-bile cholesterol transporter, ABCG5/8, could not be rescued with NaS. DISCUSSIONS AND CONCLUSIONS: The cardiometabolic health benefits of NaS may be attributed to the reprogramming of hepatic metabolic pathways to increase fatty acid utilization in the settings of nutritional overabundance. Reduced hepatic cholesterol levels, coupled with reduced LXR/RXR-induced proteins, may underlie the lack of rescue of ABCG5/8 expression with NaS. This remarkable protection against HFD-induced hepatosteatosis did not translate to improvements in cholesterol homeostasis.

Nonconserved Long Intergenic Noncoding RNAs Associate With Complex Cardiometabolic Disease Traits.

OBJECTIVE: Transcriptome profiling of human tissues has revealed thousands of long intergenic noncoding RNAs (lincRNAs) at loci identified through large-scale genome-wide studies for complex cardiometabolic traits. This raises the question of whether genetic variation at nonconserved lincRNAs has any systematic association with complex disease, and if so, how different this pattern is from conserved lincRNAs. We evaluated whether the associations between nonconserved lincRNAs and 8 complex cardiometabolic traits resemble or differ from the pattern of association for conserved lincRNAs. Approach and Results: Our investigation of over 7000 lincRNA annotations from GENCODE Release 33-GRCh38.p13 for complex trait genetic associations leveraged several large, established meta-analyses genome-wide association study summary data resources, including GIANT (Genetic Investigation of Anthropometric Traits), UK Biobank, GLGC (Global Lipids Genetics Consortium), Cardiogram (Coronary Artery Disease Genome Wide Replication and Meta-Analysis), and DIAGRAM (Diabetes Genetics Replication and Meta-Analysis)/DIAMANTE (Diabetes Meta-Analysis of Trans-Ethnic Association Studies). These analyses revealed that (1) nonconserved lincRNAs associate with a range of cardiometabolic traits at a rate that is generally consistent with conserved lincRNAs; (2) these findings persist across different definitions of conservation; and (3) overall across all cardiometabolic traits, approximately one-third of genome-wide association study-associated lincRNAs are nonconserved, and this increases to about two-thirds using a more stringent definition of conservation. CONCLUSIONS: These findings suggest that the traditional notion of conservation driving prioritization for functional and translational follow-up of complex cardiometabolic genomic discoveries may need to be revised in the context of the abundance of nonconserved long noncoding RNAs in the human genome and their apparent predilection to associate with complex cardiometabolic traits.

Conjugated Linoleic Acid and Alpha Linolenic Acid Improve Cholesterol Homeostasis in Obesity by Modulating Distinct Hepatic Protein Pathways.

SCOPE: High-fat diet (HFD)-induced obesity impairs macrophage-to-feces reverse cholesterol transport (RCT). It is hypothesized that dietary supplementation with the polyunsaturated fatty acids conjugated linoleic acid (CLA) or alpha linolenic acid (ALA) would prevent HFD-impaired RCT by modulating hepatic protein pathways. METHODS AND RESULTS: ApoE3L.CETP mice are fed a HFD supplemented ± CLA or ALA for 12 weeks and in vivo macrophage-to-feces RCT is determined. Hepatic cholesterol transporters and the hepatic proteome are assessed by immunoblotting and mass spectrometry, respectively. Mice fed HFD alone, but not ALA-HFD or CLA-HFD, exhibit increased systemic cholesterol levels, increased 3 H-cholesterol levels in plasma and liver but not feces during RCT, and reduced hepatic ABCG5/8 expression relative to LFD. ALA-HFD significantly reduces liver weight, hepatic cholesterol levels, and expression of the cholesterol synthesis enzyme farnesyl pyrophosphate synthase relative to HFD. ALA further increases the expression of acetyl-coA oxidase-associated proteins and suppress PPARα-induced proteins relative to HFD. CLA does not significantly attenuate hepatic lipid levels but is associated with reduced hepatic expression of fatty acid binding protein (FABP)-1/FABP4 levels relative to HFD, and reduced inflammatory pathway activation relative to ALA-HFD. CONCLUSION: ALA and CLA exert distinct mechanistic advantages on cholesterol homeostasis and RCT in obesity.

Different monocyte phenotypes result in proresolving macrophages in conjugated linoleic acid-induced attenuated progression and regression of atherosclerosis.

Monocytes/macrophages drive progression and regression of atherosclerosis. Conjugated linoleic acid (CLA), an anti-inflammatory lipid, mediates atheroprotective effects. We investigated how CLA alters monocyte/macrophage phenotype during attenuated progression and regression of atherosclerosis. Apolipoprotein E knockout (ApoE-/-) mice were fed a high-fat (60%) high-cholesterol (1%) diet (HFHCD) for 2 wk, followed by 6-wk 1% CLA 80:20 supplementation to investigate disease progression. Simultaneously, ApoE-/- mice were fed a 12-wk HFHCD with/without CLA for the final 4 wk to investigate regression. Aortic lesions were quantified by en face staining. Proteomic analysis, real-time quantitative PCR and flow cytometry were used to interrogate monocyte/macrophage phenotypes. CLA supplementation inhibited atherosclerosis progression coincident with decreased proinflammatory and increased anti-inflammatory macrophages. However, CLA-induced regression was associated with increased proinflammatory monocytes resulting in increased proresolving M2 bone marrow-derived macrophages, splenic macrophages, and dendritic cells in lesion-draining lymph nodes. Proteomic analysis confirmed regulation of a proinflammatory bone marrow response, which was abolished upon macrophage differentiation. Thus, in attenuation and regression of atherosclerosis, regardless of the monocyte signature, during monocyte to macrophage differentiation, proresolving macrophages prevail, mediating vascular repair. This study provides novel mechanistic insight into the monocyte/macrophage phenotypes in halted atherosclerosis progression and regression of atherosclerosis.-Bruen, R., Curley, S., Kajani, S., Lynch, G., O'Reilly, M. E., Dillon, E. T., Fitzsimons, S., Mthunzi, L., McGillicuddy, F. C., Belton, O. Different monocyte phenotypes result in proresolving macrophages in conjugated linoleic acid-induced attenuated progression and regression of atherosclerosis.

Liraglutide Attenuates Preestablished Atherosclerosis in Apolipoprotein E-Deficient Mice via Regulation of Immune Cell Phenotypes and Proinflammatory Mediators.

We have shown that the glucagon-like peptide-1 receptor agonist (GLP-1RA) liraglutide (Lir) inhibits development of early atherosclerosis in vivo by modulating immune cell function. We hypothesized that Lir could attenuate pre-established disease by modulating monocyte or macrophage phenotype to induce atheroprotective responses. Human atherosclerotic plaques obtained postendarterectomy and human peripheral blood macrophages were treated ex vivo with Lir. In parallel, apolipoprotein E-deficient (ApoE-/-) mice received a high-fat, high-cholesterol diet to induce atherosclerosis for 8 weeks, after which ApoE-/- mice received 300 µg/kg of Lir daily or vehicle control for a further 4 weeks to investigate the attenuation of atherosclerosis. Lir inhibited proinflammatory monocyte chemoattractant protein-1 secretion from human endarterectomy samples and monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin (IL)-1ß secretion from human macrophages after ex vivo treatment. An increase in CD206 mRNA and IL-10 secretion was also detected, which implies resolution of inflammation. Importantly, Lir significantly attenuated pre-established atherosclerosis in ApoE-/- mice in the whole aorta and aortic root. Proteomic analysis of ApoE-/- bone marrow cells showed that Lir upregulated the proinflammatory cathepsin protein family, which was abolished in differentiated macrophages. In addition, flow cytometry analysis of bone marrow cells induced a shift toward reduced proinflammatory and increased anti-inflammatory macrophages. We concluded that Lir attenuates pre-established atherosclerosis in vivo by altering proinflammatory mediators. This is the first study to describe a mechanism through which Lir attenuates atherosclerosis by increasing bone marrow proinflammatory protein expression, which is lost in differentiated bone marrow-derived macrophages. This study contributes to our understanding of the anti-inflammatory and cardioprotective role of GLP-1RAs. SIGNIFICANCE STATEMENT: It is critical to understand the mechanisms through which liraglutide (Lir) mediates a cardioprotective effect as many type 2 diabetic medications increase the risk of myocardial infarction and stroke. We have identified that Lir reduces proinflammatory immune cell populations and mediators from plaque-burdened murine aortas in vivo and augments proresolving bone marrow-derived macrophages in attenuation of atherosclerotic disease, which provides further insight into the atheroprotective effect of Lir.

Nutritionally Derived Metabolic Cues Typical of the Obese Microenvironment Increase Cholesterol Efflux Capacity of Adipose Tissue Macrophages.

BACKGROUND: Cholesterol retention within plasma membranes of macrophages is associated with increased inflammatory signaling. Cholesterol efflux via the transporters ABCA1, ABCG1, and SR-BI to high-density lipoprotein (HDL) particles is a critical mechanism to maintain cellular cholesterol homeostasis. Little is known about the impact of the obese microenvironment on cholesterol efflux capacity (CEC) of macrophages. In this study, the CEC of obese-derived primary adipose-tissue macrophages (ATM) is evaluated and the in vivo microenvironment is modeled in vitro to determine mechanisms underlying modulated CEC. MATERIALS AND METHODS: F4/80+ ATM are labeled with 3 H-cholesterol ex vivo, and CEC and ABCA1/ABCG1 protein levels are determined. Total, ABCA1-dependent, and ABCA1-independent CECs are determined in J774 macrophages polarized to M1 (LPS&IFNγ), M2 (IL-4&IL-13), or metabolic phenotypes (glucose, insulin, and palmitic acid). RESULTS: Obese ATM exhibit enhanced CEC and ABCA1 and ABCG1 expression compared to lean ATM. In contrast, ABCA1-CEC is suppressed from M1 polarized macrophages compared to untreated in vitro, by activation of the JAK/STAT pathway. Incubation of macrophages in vitro in high glucose augments cAMP-induced ABCA1 protein expression and ABCA1-CEC. CONCLUSIONS: These novel findings demonstrate remarkable plasticity of macrophages to respond to their environment with specific modulation of ABCA1 depending on whether classical pro-inflammatory or metabolic cues predominate.

Liraglutide dictates macrophage phenotype in apolipoprotein E null mice during early atherosclerosis.

BACKGROUND: Macrophages play a pivotal role in atherosclerotic plaque development. Recent evidence has suggested the glucagon-like peptide-1 receptor (GLP-1R) agonist, liraglutide, can attenuate pro-inflammatory responses in macrophages. We hypothesized that liraglutide could limit atherosclerosis progression in vivo via modulation of the inflammatory response. METHODS: Human THP-1 macrophages and bone marrow-derived macrophages, from both wild-type C57BL/6 (WT) and apolipoprotein E null mice (ApoE-/-) were used to investigate the effect of liraglutide on the inflammatory response in vitro. In parallel, ApoE-/- mice were fed a high-fat (60% calories from fat) high-cholesterol (1%) diet for 8 weeks to induce atherosclerotic disease progression with/without daily 300 µg/kg liraglutide administration for the final 6 weeks. Macrophages were analysed for MΦ1 and MΦ2 macrophage markers by Western blotting, RT-qPCR, ELISA and flow cytometry. Atherosclerotic lesions in aortae from ApoE-/- mice were analysed by en face staining and monocyte and macrophage populations from bone marrow derived cells analysed by flow cytometry. RESULTS: Liraglutide decreased atherosclerotic lesion formation in ApoE-/- mice coincident with a reduction in pro-inflammatory and increased anti-inflammatory monocyte/macrophage populations in vivo. Liraglutide decreased IL-1beta in MΦ0 THP-1 macrophages and bone marrow-derived macrophages from WT mice and induced a significant increase in the MΦ2 surface marker mannose receptor in both MΦ0 and MΦ2 macrophages. Significant reduction in total lesion development was found with once daily 300 µg/kg liraglutide treatment in ApoE-/- mice. Interestingly, liraglutide inhibited disease progression at the iliac bifurcation suggesting that it retards the initiation and development of disease. These results corresponded to attenuated MΦ1 markers (CCR7, IL-6 and TNF-alpha), augmented MΦ2 cell markers (Arg-1, IL-10 and CD163) and finally decreased MΦ1-like monocytes and macrophages from bone marrow-derived cells. CONCLUSIONS: This data supports a therapeutic role for liraglutide as an atheroprotective agent via modulating macrophage cell fate towards MΦ2 pro-resolving macrophages.

Nutritional modulation of metabolic inflammation.

Metabolic inflammation is a very topical area of research, wherein aberrations in metabolic and inflammatory pathways probably contribute to atherosclerosis, insulin resistance (IR) and type 2 diabetes. Metabolic insults arising from obesity promote inflammation, which in turn impedes insulin signalling and reverse cholesterol transport (RCT). Key cells in the process are metabolically activated macrophages, which up-regulate both pro- and anti-inflammatory pathways in response to lipid spillover from adipocytes. Peroxisome proliferator-activated receptors and AMP-activated protein kinase (AMPK) are regulators of cellular homeostasis that influence both inflammatory and metabolic pathways. Dietary fats, such as saturated fatty acids (SFAs), can differentially modulate metabolic inflammation. Palmitic acid, in particular, is a well-characterized nutrient that promotes metabolic inflammation via the NLRP3 (the nod-like receptor containing a pyrin domain) inflammasome, which is partly attributable to AMPK inhibition. Conversely, some unsaturated fatty acids are less potent agonists of metabolic inflammation. For example, monounsaturated fatty acid does not reduce AMPK as potently as SFA and n-3 polyunsaturated fatty acids actively resolve inflammation via resolvins and protectins. Nevertheless, the full extent to which nutritional state modulates metabolic inflammation requires greater clarification.

Monounsaturated fatty acid-enriched high-fat diets impede adipose NLRP3 inflammasome-mediated IL-1ß secretion and insulin resistance despite obesity.

Saturated fatty acid (SFA) high-fat diets (HFDs) enhance interleukin (IL)-1ß-mediated adipose inflammation and insulin resistance. However, the mechanisms by which different fatty acids regulate IL-1ß and the subsequent effects on adipose tissue biology and insulin sensitivity in vivo remain elusive. We hypothesized that the replacement of SFA for monounsaturated fatty acid (MUFA) in HFDs would reduce pro-IL-1ß priming in adipose tissue and attenuate insulin resistance via MUFA-driven AMPK activation. MUFA-HFD-fed mice displayed improved insulin sensitivity coincident with reduced pro-IL-1ß priming, attenuated adipose IL-1ß secretion, and sustained adipose AMPK activation compared with SFA-HFD-fed mice. Furthermore, MUFA-HFD-fed mice displayed hyperplastic adipose tissue, with enhanced adipogenic potential of the stromal vascular fraction and improved insulin sensitivity. In vitro, we demonstrated that the MUFA oleic acid can impede ATP-induced IL-1ß secretion from lipopolysaccharide- and SFA-primed cells in an AMPK-dependent manner. Conversely, in a regression study, switching from SFA- to MUFA-HFD failed to reverse insulin resistance but improved fasting plasma insulin levels. In humans, high-SFA consumers, but not high-MUFA consumers, displayed reduced insulin sensitivity with elevated pycard-1 and caspase-1 expression in adipose tissue. These novel findings suggest that dietary MUFA can attenuate IL-1ß-mediated insulin resistance and adipose dysfunction despite obesity via the preservation of AMPK activity.