Adipose Tissue Inflammation In Obesity Research Articles

The Dietary Isoflavone Daidzein Reduces Expression of Pro-Inflammatory Genes through PPARα/γ and JNK Pathways in Adipocyte and Macrophage Co-Cultures.

Obesity-induced inflammation caused by adipocyte-macrophage interactions plays a critical role in developing insulin resistance, and peroxisome proliferator-activated receptors (PPARs) regulate inflammatory gene expression in these cells. Recently, the soy isoflavone daidzein was reported to act as a PPAR activator. We examined whether daidzein affected adipocyte-macrophage crosstalk via the regulation of PPARs. Co-cultures of 3T3-L1 adipocytes and RAW264 macrophages, or palmitate-stimulated RAW264 macrophages were treated with daidzein in the presence or absence of specific inhibitors for PPARs: GW6471 (a PPARα antagonist), and GW9662 (a PPARγ antagonist). Inflammatory gene expression was then determined. Daidzein significantly decreased chemokine (C-C motif) ligand 2 (Ccl2, known in humans as monocyte chemo-attractant protein 1 (MCP1)) and interleukin 6 (Il6) mRNA levels induced by co-culture. In 3T3-L1 adipocytes, daidzein inversed the attenuation of adiponectin gene expression by co-culture, and these effects were inhibited by the PPAR-γ specific inhibitor. Daidzein also decreased Ccl2 and Il6 mRNA levels in RAW264 macrophages stimulated with palmitate or conditioned medium (CM) from hypertrophied 3T3-L1 adipocytes. This inhibitory effect on Il6 expression was abrogated by a PPAR-α inhibitor. Additionally, we examined the activation of nuclear factor-kappa B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways and found that daidzein significantly inhibited palmitate-induced phosphorylation of JNK. Our data suggest that daidzein regulates pro-inflammatory gene expression by activating PPAR-α and -γ and inhibiting the JNK pathway in adipocyte and macrophage co-cultures. These effects might be favorable in improving adipose inflammation, thus, treatment of daidzein may be a therapeutic strategy for chronic inflammation in obese adipose tissue.

Fish Oil-Derived Long-Chain n-3 Polyunsaturated Fatty Acids Reduce Expression of M1-Associated Macrophage Markers in an ex vivo Adipose Tissue Culture Model, in Part through Adiponectin.

Adipose tissue (AT) macrophages (ATM) play a key role in obesity-associated pathologies, and their phenotype can be influenced by the local tissue microenvironment. Interestingly, long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) and the LC n-3 PUFA-upregulated adipokine, adiponectin (Ad), may mitigate excessive ATM inflammatory M1-polarization responses. However, to what extent LC n-3 PUFA and Ad work in concert to affect macrophage phenotype has not been examined. Thus, we used an established ex vivo AT organ culture model using visceral AT from mice fed a control (CON; 10% w/w safflower oil) n-6 PUFA-rich diet or an isocaloric fish oil (FO; 3% w/w menhaden oil + 7% w/w safflower oil)-derived LC n-3 PUFA-rich diet to generate AT conditioned media (ACM). We then evaluated if CON or FO ACM affected macrophage polarization markers in a model designed to mimic acute [18 h ACM plus lipopolysaccharide (LPS) for the last 6 h] or chronic (macrophages treated with LPS-challenged CON or FO ACM for 24 h) inflammation ± Ad-neutralizing antibody and the LPS-neutralizing agent, polymyxin B. In the acute inflammation model, macrophages treated with FO ACM had decreased lipid uptake and mRNA expression of M1 markers (Nos2, Nfκb, Il6, Il18, Ccl2, and Ccl5) compared with CON ACM (p ≤ 0.05); however, these effects were largely attenuated when Ad was neutralized (p > 0.05). Furthermore, in the chronic inflammation model, macrophages treated with FO ACM had decreased mRNA expression of M1 markers (Nos2, Tnfα, Ccl2, and Il1β) and IL-6 and CCL2 secretion (p ≤ 0.05); however, some of these effects were lost when Ad was neutralized, and were further exacerbated when both Ad and LPS were neutralized. Taken together, this work shows that LC n-3 PUFA and Ad work in concert to suppress certain M1 macrophage responses. Thus, future strategies to modulate the ATM phenotype should consider the role of both LC n-3 PUFA and Ad in mitigating obese AT inflammation.

16. Hyperinsulinemia Promotes Obesity-associated Adipose Tissue Inflammation (1847-P)

In obesity, adipose tissue (AT) dysfunction is associated with inflammation, systemic insulin resistance and hyperinsulinemia. However, the mechanisms that drive AT inflammation in obesity and disrupt adipocyte metabolic functions are incompletely understood. Here we show that reducing circulating insulin levels about 50% in obese mice by streptozotocin or diazoxide treatments surprisingly suppressed the elevated adipose tissue inflammation, as measured by a reduction of AT crown-like structures and by a decrease in macrophage markers and pro-inflammatory cytokine expression. This decreased cytokine expression in the AT of streptozotocin treated obese mice was associated with partial restoration of expression of enzymes in the de novo lipogenesis (DNL) pathway and of 14C-glucose conversion into triglyceride-fatty acids. Lowering insulin levels in obese mice also enhanced insulin stimulated Akt protein kinase phosphorylation and restored insulin responsiveness of AT DNL. Moreover, responsiveness of blood glucose to injected insulin was improved by streptozotocin and diazoxide treatments without changes in body weight and blood glucose levels. Additionally, chronic insulin treatment in mature 3T3-L1 adipocytes led to a 10-fold increase in CCL2 mRNA level within 6 h. Interest ingly, pre-treatment of 3T3-L1 adipocytes with the ERK inhibitor PD98059 ablated the insulin-induced increase in CCL2 expression, indicating that chronically elevated insulin promotes CCL2 expression in mature adipocytes in an ERK-dependent manner. Taken together, these results indicate that obesity-associated hyperinsulinemia unexpectedly drives AT inflammation in obese mice, which may in turn contribute to factors that suppress DNL and systemic insulin sensitivity.

Hematopoietic cyclooxygenase‐2 deficiency increases adipose tissue inflammation and adiposity in obesity

Adipose tissue (AT) macrophages mediate AT inflammation in obesity, and cyclooxygenase-2 (COX-2) is a major inflammatory gene. It was hypothesized that deletion of hematopoietic COX-2 will inhibit AT inflammation in obesity. Lethally irradiated wild-type (WT) mice were injected with bone marrow (BM) cells collected from WT or COX-2 knock-out (COX-2-/-) donor mice and fed a high-fat diet for 16 weeks. The mice that received BM cells from COX-2-/- mice (BM-COX-2-/-) gained increased body weight, fat mass, and visceral AT (VAT) mass. These mice exhibited reduced inflammatory markers in the VAT stromal vascular cells (SVC). However, the inflammatory markers were increased in adipocyte fraction and/or whole VAT. The activation of ERK1/2 MAPK, a pro-inflammatory signaling pathway, was increased in BM-COX-2-/- mice. The molecular markers of adipogenesis were increased in the VAT or adipocyte fraction. Wnt signaling markers which inhibit adipogenesis, including Wnt3A and DVL3, were reduced, and Wnt5a/b which promotes inflammation was increased in the VAT and/or adipocytes. Finally, an increase in hepatic triglyceride levels in BM-COX-2-/- mice was noted. The data suggest that COX-2 deletion in hematopoietic cells reduces SVC inflammation but increases VAT inflammation and promotes adiposity likely via altered Wnt signaling.

Abstract 414: Key Role of STAT4 Deficiency in the Hematopoietic Compartment on Insulin Resistance and Adipose Tissue Inflammation

Adipose tissue inflammation is a hallmark of obesity and contributes to insulin resistance (IR). Recent data from our lab showed that STAT4 global deficiency reduces inflammation and improves insulin sensitivity in obese mice. STAT4 is expressed in adipocytes and cells of hematopoietic lineage. The objective of this study was to determine the contribution of selective STAT4 deficiency in adipocyte vs hematopoietic cells to IR and adipose tissue inflammation. For this purpose we sub-lethally irradiated Stat-4 -/- C57Bl6 mice and reconstituted them with bone marrow cells (BMC) from Stat-4 +/+ C57Bl6 congenic donors to restore STAT4 expression in BM- derived cells (STAT4-/- recipients). These mice were compared to irradiated C57Bl6 wild-type mice reconstituted with Stat-4 -/- C57Bl6 BMCs to produce STAT4 deletion selectively in BM-derived cells (STAT4+/+ recipients). Mice received a HFD (60%kcal fat) for 12 weeks (n=7 mice/group). Body weights, plasma cholesterol, triglycerides and free fatty acids were not different between the STAT4+/+ and STAT4-/- recipients. However the STAT4-/- recipients had significantly increased fasted plasma glucose. Congruent with this data the IP-ITT and GTT showed significantly higher area under the curve for the STAT4-/- recipients indicating increased glucose intolerance and insulin resistance compared to STAT4+/+ recipients. Adipose tissue immune cell composition was determined by flow cytometry. The total number of CD45+ lymphocytes was significantly higher in the STAT4-/- recipients compared to STAT4+/+ recipients. Also, the numbers of CD3+CD4+ and CD3+CD8+ cells were significantly higher in the STAT4-/- recipient group. Adipocyte size was significantly higher and the number of crown-like structures was significantly increased in the STAT4 -/- recipients indicating adipocyte hypertrophy and increased macrophage infiltration. Following in vivo insulin stimulation, activation of IR and IRS-1 was increased in liver and muscle in the STAT4+/+ recipient group suggesting improved insulin signaling in the mice that received STAT4-/- BMCs. These studies provide clear evidence for the importance of STAT4 in hematopoietic cells in regulating IR and adipose tissue inflammation in obesity.

Macrophage very low density lipoprotein receptor regulates adipose tissue inflammation in obesity

Adipose tissue macrophages (ATMs) are key players to balance pro- and anti-inflammatory responses in fat tissues. In obesity, increased ATMs have been implicated in pro-inflammatory responses and lipid dysregulation. However, underlying mechanisms of lipid accumulation in ATMs of obese animals and their roles are largely unknown. In this study, we demonstrated that elevated expression of very low density lipoprotein receptor (VLDLR) could potentiate adipose tissue inflammation and insulin resistance in obesity. The level of adipose tissue VLDLR in obese animals were elevated, and its expression level was positively correlated with those of pro-inflammatory marker genes. Compared to wild-type mice, VLDLR homozygous-null mice exhibited attenuated insulin resistance and adipose tissue inflammation, concommitantly with less proinflammatory ATMs upon high-fat diet (HFD). In macrophages, the lack of VLDLR prevented lipid uptake and suppressed VLDL-dependent proinflammatory gene expression. Taken together, these data suggest that an elevated macrophage VLDLR expression in obesity would contribute to adipose tissue inflammation via lipid accumulation in ATMs. This work was supported by National Creative Research Initiative Program (2011-0018312) funded by the ministry of Education, Science, and Technology (MEST). K. C. Shin and S. S. Choe were supported by the BK21 program, Korea.

Strong association of serum- and glucocorticoid-regulated kinase 1 with peripheral and adipose tissue inflammation in obesity.

The serum- and glucocorticoid-regulated kinase 1 (SGK1) is an early transcriptional target of glucocorticoids and is activated via insulin. Here we investigate the regulation of SGK1 expression in human obesity, diet-induced murine obesity and human monocytic cell line THP-1 monocytes. SGK1 expression was studied in subcutaneous and omental adipose tissue (AT) of 20 morbidly obese and 20 age- and gender-matched non-obese controls in murine diet-induced obesity and the THP-1 cell line. The regulation of SGK1 by inflammatory signals was tested in THP-1 cells. Murine diet-induced obesity is associated with a significant upregulation of Sgk1 in gonadal AT. Sgk1 expression is highest in the macrophage-rich stromal vascular fraction and lower in adipocytes. In humans, AT SGK1 is predominantly expressed in CD14(+) macrophages and significantly upregulated in omental and subcutaneous AT of obese subjects. SGK1 mRNA expression in both omental and subcutaneous AT correlates with body mass index, circulating leptin and C-reactive protein, and the local expression of inflammatory markers including monocyte chemotactic protein-1 and macrophage inflammatory protein-1α. The expression of SGK1 in THP-1 cells is upregulated by inflammatory signals, such as lipopolysaccharide and tumour necrosis factor-α, as well as during the induction of monocyte-to-macrophage maturation. Our data present the first link between SGK1 and obesity-associated inflammation. SGK1 expression is stimulated in response to inflammatory signals and increased in AT macrophages. The characterisation of SGK1 functions in obesity and immunity may help identify potential therapeutic targets in the treatment of obesity.

Uncoupling lipid metabolism from inflammation through fatty acid binding protein-dependent expression of UCP2.

Chronic inflammation in obese adipose tissue is linked to endoplasmic reticulum (ER) stress and systemic insulin resistance. Targeted deletion of the murine fatty acid binding protein (FABP4/aP2) uncouples obesity from inflammation although the mechanism underlying this finding has remained enigmatic. Here, we show that inhibition or deletion of FABP4/aP2 in macrophages results in increased intracellular free fatty acids (FFAs) and elevated expression of uncoupling protein 2 (UCP2) without concomitant increases in UCP1 or UCP3. Silencing of UCP2 mRNA in FABP4/aP2-deficient macrophages negated the protective effect of FABP loss and increased ER stress in response to palmitate or lipopolysaccharide (LPS). Pharmacologic inhibition of FABP4/aP2 with the FABP inhibitor HTS01037 also upregulated UCP2 and reduced expression of BiP, CHOP, and XBP-1s. Expression of native FABP4/aP2 (but not the non-fatty acid binding mutant R126Q) into FABP4/aP2 null cells reduced UCP2 expression, suggesting that the FABP-FFA equilibrium controls UCP2 expression. FABP4/aP2-deficient macrophages are resistant to LPS-induced mitochondrial dysfunction and exhibit decreased mitochondrial protein carbonylation and UCP2-dependent reduction in intracellular reactive oxygen species. These data demonstrate that FABP4/aP2 directly regulates intracellular FFA levels and indirectly controls macrophage inflammation and ER stress by regulating the expression of UCP2.

Leucine-rich glioma inactivated 3 and tumor necrosis factor-α regulate mutually through NF-κB

Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein member of LGI family. We previously reported that LGI3 increased in obese adipose tissues and suppressed adipogenesis through its receptor, ADAM23. We proposed that LGI3 may be a pro-inflammatory adipokine secreted predominantly by preadipocytes and macrophages. In this study, we showed that LGI3 and tumor necrosis factor-α (TNF-α) upregulated each other in 3T3-L1 cells. Treatment of 3T3-L1 preadipocytes with LGI3 protein increased TNF-α mRNA and protein. LGI3 treatment led to NF-κB activation and binding to an NF-κB binding site (−523 to −514) in TNF-α promoter. TNF-α treatment increased mRNA and protein expression of LGI3 and ADAM23. TNF-α increased NF-κB binding to a predicted binding site (−40 to −31) in LGI3 promoter. High fat diet-fed mice showed that LGI3 and TNF-α were increased and colocalized in adipose tissue inflammation. Taken together, these results suggested that mutual upregulation of LGI3 and TNF-α may play a role in adipose tissue inflammation in obesity.

Impairment of high density lipoprotein resistance to lipid peroxidation and adipose tissue inflammation in obesity complicated by obstructive sleep apnea

Abstract

A Macrophage NBR1-MEKK3 Complex Triggers JNK-Mediated Adipose Tissue Inflammation in Obesity

The c-Jun NH(2)-terminal kinase (JNK) is a critical determinant of obesity-associated inflammation and glucose intolerance. The upstream mechanisms controlling this pathway are still unknown. Here we report that the levels of the PB1 domain-containing adaptor NBR1 correlated with the expression of proinflammatory molecules in adipose tissue from human patients with metabolic syndrome, suggesting that NBR1 plays a key role in adipose-tissue inflammation. We also show that NBR1 inactivation in the myeloid compartment impairs the function, M1 polarization, and chemotactic activity of macrophages; prevents inflammation of adipose tissue; and improves glucose tolerance in obese mice. Furthermore, we demonstrate that an interaction between the PB1 domains of NBR1 and the mitogen-activated kinase kinase 3 (MEKK3) enables the formation of a signaling complex required for the activation of JNK. Together, these discoveries identify an NBR1-MEKK3 complex as a key regulator of JNK signaling and adipose tissue inflammation in obesity.

AB0025 Is Abatacept Successful in Improving Insulin Resistance in A High Fat Diet Model of Obesity?

BackgroundWe previously reported a case of improvement in insulin resistance (IR) in a Rheumatoid Arthritis (RA) patient treated with the fusion protein Abatacept (CTLA-4Ig), selectively designed to modulate the immune...

Endogenous, adipocyte-derived lipids signal the recruitment of proinflammatory immune cells.

Obesity, insulin resistance, and type 2 diabetes have reached epidemic levels in Western countries. Work over the past decade has established that the adipose tissue in obesity is characterized by a state of chronic, low-grade inflammation, which plays a primary role in the development of systemic insulin resistance (1). Critical to the development of adipose tissue inflammation in obesity is the recruitment of specific immune cells, such as proinflammatory macrophages (M1 macrophages) and neutrophils (1–3). However, the precise signals that stimulate the recruitment of these immune cells into the expanding adipose tissue are not well understood. In this issue, Tynan et al. (4) demonstrate that endogenous oils (EOs) isolated from human adipocytes are a potent inflammatory signal. Initially, the authors sought to determine whether EOs derived from human omental adipocytes could enhance antigen-specific immune responses in mice. Remarkably, EOs were as effective as incomplete Freund’s adjuvant at inducing antigen-specific antibody responses. Importantly, fractionation of EOs into lipid and nonlipid components demonstrated that the lipid fraction of EOs were the immunogenic component. To explore their inflammatory properties further, the authors used a model of inflammation in which the recruitment of immune cells was examined following the injection of EOs into the peritoneum of mice. EOs induced a marked shift in the immune cell profile of the peritoneum, characterized by …

Impairment of high-density lipoprotein resistance to lipid peroxidation and adipose tissue inflammation in obesity complicated by obstructive sleep apnea.

Obstructive sleep apnea (OSA) complicates morbid obesity and is associated with increased cardiovascular disease incidence. An increase in the circulating markers of chronic inflammation and dysfunctional high-density lipoprotein (HDL) occur in severe obesity. The objective of the study was to establish whether the effects of obesity on inflammation and HDL dysfunction are more marked when complicated by OSA. Morbidly obese patients (n = 41) were divided into those whose apnea-hypoapnea index (AHI) was more or less than the median value and on the presence of OSA [OSA and no OSA (nOSA) groups]. We studied the antioxidant function of HDL and measured serum paraoxonase 1 (PON1) activity, TNFα, and intercellular adhesion molecule 1 (ICAM-1) levels in these patients. In a subset of 19 patients, we immunostained gluteal sc adipose tissue (SAT) for TNFα, macrophages, and measured adipocyte size. HDL lipid peroxide levels were higher and serum PON1 activity was lower in the high AHI group vs the low AHI group (P < .05 and P < .0001, respectively) and in the OSA group vs the nOSA group (P = .005 and P < .05, respectively). Serum TNFα and ICAM-1 levels and TNFα immunostaining in SAT increased with the severity of OSA. Serum PON1 activity was inversely correlated with AHI (r = -0.41, P < .03) in the OSA group. TNFα expression in SAT directly correlated with AHI (r = 0.53, P < .03) in the subset of 19 patients from whom a biopsy was obtained. Increased serum TNFα, ICAM-1, and TNFα expression in SAT provide a mechanistic basis for enhanced inflammation in patients with OSA. Decreased serum PON1 activity, impaired HDL antioxidant function, and increased adipose tissue inflammation in these patients could be a mechanism for HDL and endothelial dysfunction.

Cyclin-dependent kinase 5 contributes to adipose tissue inflammation in obesity through suppression of Treg differentiation (HUM7P.288)

Abstract Homeostatic imbalance of regulatory and conventional T cells is linked with obesity-induced inflammation and insulin resistance. A decline in the number of both circulating and locally residing T regulatory (Treg) cells in adipose tissues is hallmark of adiposity, inflammation, and glucose intolerance. Recently, the signal transducer and activator of transcription 3 (Stat3) has been implicated as a critical mediator in this process; however, the factors that initiate this inflammatory cascade remain largely unclear. We recently reported a previously unknown function for Cyclin-dependent kinase 5 (Cdk5) in T cells, a kinase renowned otherwise for its role in regulating the development and function of cells in the neuronal lineage. Here we report for the first time that the mice lacking Cdk5 in T cells (Cdk5TKO-mice) are lean compared to their wild type littermates as confirmed by their body mass index, histopathological analysis of adipose tissue as well as complete body MRI scans measuring adiposity index. Moreover we observed a significant increase in circulating CD4+ CD25+ Foxp3+ Treg cells along with a marked decrease in Ser-727 phosphorylation of Stat3. Finally, both pharmacologic and genetic deletion of Cdk5 abrogates the IL6-mediated suppression of TGF-β-induced Treg differentiation. These results further solidify the role of Cdk5 as an essential regulator of T cell function and implicate Cdk5 as a potential therapeutic target for prevention and treatment of obesity.

NanoSOD reduces adipose tissue inflammation in obesity (1107.10)

Obesity‐linked hypertension is resistant to antihypertensive drugs and, adipose tissue (AT) inflammation is considered to mediate the pathological consequences of obesity. The objective of this study is to determine the effectiveness of nanoformulated copper/zinc superoxide dismutase (nanoSOD) in reducing AT inflammation in obesity. Wild type mice were fed a high fat (HF) diet (45% fat) for 8 or 13 wk followed by 2 wk treatment with native or nanoSOD. The drug uptake studies showed that nanoSOD was accumulated in the visceral AT to a greater extent than native SOD in obese mice. The mice fed a HF diet for 8 wk followed by 2 wk treatment with nanoSOD showed an increase in SOD content in stromal vascular cells (SVCs) collected from AT and in the plasma. These mice also showed a reduction in nitrotyrosine level, a marker of superoxide induced oxidative stress, in SVCs. The AT inflammatory markers showed a trend towards a decrease. The serum toxicity markers did not alter in nanoSOD compared to other groups suggesting that the nanoSOD is safe to use in vivo. After 13 wk on a HF diet followed by 2 wk treatment, the SVCs derived from AT of nanoSOD treated mice showed a decrease in the mRNA levels of MCP‐1 and MIP‐1α. Also, the MCP‐1 mRNA and protein levels in total AT was reduced to a greater extent in nanoSOD compared to native SOD treated mice. Our data suggest that nanoSOD may be a potential therapeutic agent to ameliorate obesity‐linked cardiovascular risk factors, in particular, systemic hypertention.Grant Funding Source: National Institutes of Health (NIH‐NIGMS)

Distinct role of complement anaphylatoxin receptors C5aR and C3aR in obese adipose tissue inflammation and insulin resistance

Obese adipose tissue (AT) inflammation contributes critically to development of insulin resistance. It is characterized by the accumulation of macrophages in the obese AT and their polarization into the M1 pro-inflammatory macrophage subtype. The complement system is a major pro-inflammatory system and has been implicated in metabolic disease. Here, we compared the effects of the receptors for complement anaphylatoxins C5a and C3a, C5aR and C3aR, respectively, and their role in obesity, insulin resistance and AT inflammation. We engaged the model of diet-induced obesity by feeding mice a high fat diet (HFD) or a normal diet (ND) and found that expression of C5aR and C3aR was significantly upregulated in the obese AT, as compared to lean AT. Additionally, C5a was present in obese AT in the proximity of macrophage-rich crown-like structures. C5aR- or C3aR-deficiency did not affect weight gain in diet-induced obesity. Obese C5aR-/- as well as C3aR-/- mice displayed improved systemic insulin sensitivity, which was associated with mildly improved glucose tolerance in the C5aR-/- mice. In addition to the improved systemic insulin sensitivity, obese C5aR-/- mice showed improved AT-, but not hepatic insulin sensitivity. In obese C5aR-/- mice, we found reduced accumulation of total and pro-inflammatory M1 macrophages in the obese AT, increased expression of IL-10 and decreased AT fibrosis. These results suggest that C5aR contributes to macrophage accumulation and M1 polarization in the obese AT and that both, C5aR and C3aR contribute to obesity-induced development of systemic insulin resistance.

Inhibitory effects of resveratrol on hypoxia-induced inflammation in 3T3-L1 adipocytes and macrophages

Resveratrol is a natural compound proposed to posses powerful anti-obesity effects. Hypoxia may be an underlying mechanism for adipose tissue dysfunction in obesity. We have previously shown that resveratrol mediates a reduction in hypoxia induced inflammation in whole adipose tissue, thus in this study we investigated whether hypoxia and resveratrol primarily affect adipocytes or the macrophages in the adipose tissue. Hypoxia induced a significant increase in the production of hypoxic key markers in 3T3-L1 adipocytes and THP-1 macrophages, and stimulated gene expression levels of inflammatory markers in macrophages with almost no effect in adipocytes. Resveratrol attenuated the hypoxia induced increase in gene expression levels. These results suggest that the hypoxia-induced inflammatory response in whole adipose tissue mainly is driven by resident macrophages. Moreover, the results suggest that resveratrol could have treatment potential by attenuating adipose tissue inflammation in obesity.

The fractalkine/Cx3CR1 system is implicated in the development of metabolic visceral adipose tissue inflammation in obesity

Diet-induced obesity and related peripheral and central inflammation are major risk factors for metabolic, neurological and psychiatric diseases. The chemokine fractalkine (Cx3CL1) and its receptor Cx3CR1 play a pivotal role in recruitment, infiltration and proinflammatory polarization of leukocytes and micoglial cells, however, the role of fractalkine signaling in the development of metabolic inflammation is not fully resolved. To address this issue, fractalkine receptor deficient (Cx3CR1 gfp/gfp) mice were exposed to normal or fat-enriched diet (FatED) for 10weeks and physiological-, metabolic- and immune parameters were compared to those animals in which the fractalkine signaling is maintained by the presence of one functioning allele (Cx3CR1 +/gfp). Mice with intact fractalkine signaling develop obesity characterized by increased epididymal white fat depots and mild glucose intolerance, recruit leukocytes into the visceral adipose tissue and display increased expression of subset of pro- and anti-inflammatory cytokines when exposed to fat-enriched diet. By contrast, Cx3CR1-deficient (gfp/gfp) mice gain significantly less weight on fat-enriched diet and have smaller amount of white adipose tissue (WAT) in the visceral compartment than heterozygote controls. Furthermore, Cx3CR1 gfp/gfp mice fed a fat-enriched diet do not develop glucose intolerance, recruit proportionally less number of gfp-positive cells and express significantly less MCP-1, IL-1α and TNFα in the WAT than control animals with fat-enriched diet induced obesity. Furthermore, heterozygote obese, but not fractalkine receptor deficient mice express high levels of anti-inflammatory IL-10 and arginase1 markers in the visceral fat. The effect of fat-enriched diet on cytokine expression pattern was specific for the WAT, as we did not detect significant elevation of interleukin-1, tumor necrosis factor-alpha and monocyte chemotacting protein (MCP-1) expression in the liver or in the hypothalamus in either genotype. These results highlight the importance of fractalkine signaling in recruitment and polarization of adipose tissue immune cells and identify fractalkine as a target to fight obesity-induced inflammatory complications.

Docosahexaenoic Acid Decreases Pro-Inflammatory Mediators in an In Vitro Murine Adipocyte Macrophage Co-Culture Model

Paracrine interactions between adipocytes and macrophages contribute to chronic inflammation in obese adipose tissue. Dietary strategies to mitigate such inflammation include long-chain polyunsaturated fatty acids, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, which act through PPARγ-dependent and independent pathways. We utilized an in vitro co-culture model designed to mimic the ratio of macrophages:adipocytes in obese adipose tissue, whereby murine 3T3-L1 adipocytes were cultured with RAW 264.7 macrophages in direct contact, or separated by a trans-well membrane (contact-independent mechanism), with 125 µM of albumin-complexed DHA, EPA, palmitic acid (PA), or albumin alone (control). Thus, we studied the effect of physical cell contact versus the presence of soluble factors, with or without a PPARγ antagonist (T0070907) in order to elucidate putative mechanisms. After 12 hr, DHA was the most anti-inflammatory, decreasing MCP1 and IL-6 secretion in the contact system (−57%, −63%, respectively, p≤0.05) with similar effects in the trans-well system. The trans-well system allowed for isolation of cell types for inflammatory mediator analysis. DHA decreased mRNA expression (p<0.05) of Mcp1 (−7.1 fold) and increased expression of the negative regulator, Mcp1-IP (+1.5 fold). In macrophages, DHA decreased mRNA expression of pro-inflammatory M1 polarization markers (p≤0.05), Nos2 (iNOS; −7 fold), Tnfα (−4.2 fold) and Nfκb (−2.3 fold), while increasing anti-inflammatory Tgfβ1 (+1.7 fold). Interestingly, the PPARγ antagonist co-administered with DHA or EPA in co-culture reduced (p≤0.05) adiponectin cellular protein, without modulating other cytokines (protein or mRNA). Overall, our findings suggest that DHA may lessen the degree of MCP1 and IL-6 secreted from adipocytes, and may reduce the degree of M1 polarization of macrophages recruited to adipose tissue, thereby decreasing the intensity of pro-inflammatory cross-talk between adipocytes and macrophages in obese adipose tissue.