Adipose Tissue Lymphocytes and Macrophages in Obesity and Insulin Resistance

Metabolic syndrome and type 2 diabetes mellitus are associated with an increased number of macrophage cells that infiltrate white adipose tissue (WAT). Previously, we demonstrated that the treatment of subjects with impaired glucose tolerance (IGT) with the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist pioglitazone resulted in a decrease in macrophage number in adipose tissue. Here, adipose tissue samples from IGT subjects treated with pioglitazone were examined for apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. TUNEL-positive cells were identified, and there was a significant 42% increase in TUNEL-positive cells following pioglitazone treatment. Overlay experiments with anti-CD68 antibody demonstrated that most of the TUNEL-positive cells were macrophages. To determine whether macrophage apoptosis was a direct or indirect effect of pioglitazone treatment, human THP1 cells were treated with pioglitazone in vitro, demonstrating increased TUNEL staining in a dose- and time-dependent manner. Furthermore, the appearance of the active proteolytic subunits of caspase-3 and caspase-9 were detected in cell lysate from THP1 cells and also increased in a dose- and time-dependent manner following pioglitazone treatment. Pretreatment with a PPARgamma inhibitor, GW9662, prevented pioglitazone induction of the apoptotic pathway in THP1 cells. Differentiated human adipocytes did not show any significant increase in apoptosis after treatment in vitro with piolgitazone. These findings indicate that PPARgamma has distinct functions in different cell types in WAT, such that pioglitazone reduces macrophage infiltration by inducing apoptotic cell death specifically in macrophages through PPARgamma activation.

Obesity is associated with a significantly increased risk for cancer suggesting that adipose tissue dysfunctions might play a crucial role therein. Macrophages play important roles in adipose tissue as well as in cancers. Here, we studied whether human adipose tissue macrophages (ATM) modulate cancer cell function. Therefore, ATM were isolated and compared with monocyte-derived macrophages (MDM) from the same obese patients. ATM, but not MDM, were found to secrete factors inducing inflammation and lipid accumulation in human T47D and HT-29 cancer cells. Gene expression profile comparison of ATM and MDM revealed overexpression of functional clusters, such as cytokine-cytokine receptor interaction (especially CXC-chemokine) signaling as well as cancer-related pathways, in ATM. Comparison with gene expression profiles of human tumor-associated macrophages showed that ATM, but not MDM resemble tumor-associated macrophages. Indirect co-culture experiments demonstrated that factors secreted by preadipocytes, but not mature adipocytes, confer an ATM-like phenotype to MDM. Finally, the concentrations of ATM-secreted factors related to cancer are elevated in serum of obese subjects. In conclusion, ATM may thus modulate the cancer cell phenotype.

Background: Adipose tissue inflammation may play a critical role in the pathogenesis of insulin resistance (IR) and arteriosclerosis. Previous work has mainly focused on the role of macrophages in human adipose tissue, but little is known about pro-inflammatory T-lymphocytes. Therefore the present study examined the role of CD4-positive lymphocytes in adipose tissue inflammation and IR. Results: Both, CD4-positive lymphocytes and macrophages are present in human visceral adipose tissue as determined by immunohistochemical staining. Most macrophages were HLA-DR positive, reflecting activation through IFNγ, a cytokine released from CD4-positive lymphocytes. Furthermore, SDF-1α, a T-cell chemotactic protein, was also detectable in human adipose tissue. RT-PCR analyses confirmed the expression of IFNγ and SDF1α in visceral adipose tissue. Freshly isolated human adipocytes as well SGBS adipocyte cells express SDF-1α with a down regulation of its expression during adipocyte differentiation in both cell types. In a mouse model of IR, high fat diet induced IR already after 5 weeks which was associated with a marked lymphocyte infiltration in visceral adipose tissue as determined by immunohistochemical staining and RT-PCR. In contrast, macrophages were absent after 5 weeks of diet but could be detected at week 10, suggesting early infiltration of lymphocytes during the development of IR. Conclusion: Pro-inflammatory T-lymphocytes are present in visceral adipose tissue and may contribute to local inflammatory cell activation before the appearance of macrophages. These data suggest that lymphocytes may play an important role in the initiation and perpetuation of adipose tissue inflammation as well as the development of insulin resistance.

Residential macrophages in adipose tissue play a pivotal role in the development of inflammation not only within this tissue, but also affect the proinflammatory status of the whole body. Data on human adipose tissue inflammation and the role of macrophages are rather scarce. We previously documented that the proportion of proinflammatory macrophages in human adipose tissue correlates closely with non-HDL cholesterol concentrations. We hypothesized that this is due to the identical influence of diet on both parameters and decided to analyze the fatty acid spectrum in cell membrane phospholipids of the same individuals as a parameter of the diet consumed. Proinflammatory and anti-inflammatory macrophages were isolated from human adipose tissue (n = 43) and determined by flow cytometry as CD14+CD16+CD36high and CD14+CD16−CD163+, respectively. The spectrum of fatty acids in phospholipids in the cell membranes of specimens of the same adipose tissue was analyzed, and the proportion of proinflammatory macrophage increased with the proportions of palmitic and palmitoleic acids. Contrariwise, these macrophages decreased with increasing alpha-linolenic acid, total n-3 fatty acids, n-3/n-6 ratio, and eicosatetraenoic acid. A mirror picture was documented for the proportion of anti-inflammatory macrophages. The dietary score, obtained using a food frequency questionnaire, documented a positive relation to proinflammatory macrophages in individuals who consumed predominantly vegetable fat and fish, and individuals who consumed diets based on animal fat without fish and nut consumption. he present data support our hypothesis that macrophage polarization in human visceral adipose tissue is related to fatty acid metabolism, cell membrane composition, and diet consumed. It is suggested that fatty acid metabolism might participate also in inflammation and the risk of developing cardiovascular disease.

Diabetes is a growing epidemic, and it has become arguably one of the biggest health challenges of our time. Currently, more than 23 million Americans have diabetes, and the Centers for Disease Control and Prevention estimate that in the last 15 years, the number of people in the United States with diabetes has more than doubled. Diabetes is increasing at an alarming rate in Europe as well, and it is fast becoming a major health threat in developing countries such as India and China. Despite its high prevalence, however, diabetes remains somewhat of a mystery. Although type 1 diabetes mellitus could be attributed to insufficient insulin release by the β-cells of the pancreas, the origins of type 2 diabetes mellitus (which accounts for >90% of the cases of diabetes) remain obscure. Insulin resistance is a cardinal feature of type 2 diabetes mellitus; however, it is not clear how whole-body insulin resistance develops, which specific tissues are affected first and which ones later, and how metabolic changes in individual tissues contribute to the overall development of the disease and its many secondary complications. Article p 538 The origin of diabetes is equally complex. Although diabetes develops in genetically susceptible individuals, it is a complex trait and does not show simple mendelian inheritance. Because the rates of diabetes change with the environment in several population groups, it has been suggested that modifiable environmental factors and lifestyle choices account for more than 90% of adult-onset diabetes.1 Nevertheless, our understanding of the environmental causes of diabetes has remained rather rudimentary, being limited mostly to the impact of physical inactivity or unhealthy dietary choices. In this context, the study by Sun and coworkers published in the present issue of Circulation 2 is interesting because it provides new evidence showing that exposure to particulate air …

A substantial and continually growing body of evidence supports the concept that chronic low-grade inflammation is a central characteristic of obesity contributing to development of insulin resistance in adipose tissue and other insulin target organs including muscle, liver, and the vasculature (recently reviewed in Refs. 1–3). The possibility of a link between inflammation, macrophages, and obesity-associated insulin resistance was first suggested by in vitro observations 20 years ago that the macrophage secretory product TNF impaired insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Shortly thereafter, it was reported that TNF expression was increased in adipose tissue of obese, insulin-resistant rodent models and that systemic neutralization of TNF improved insulin resistance. With the discovery that adipose tissue in obesity contains more macrophages than adipose tissue of normal-weight individuals and evidence that adipose tissue macrophages in obesity are polarized to a more proinflammatory phenotype, the macrophage assumed a central role in the development of adipose tissue inflammation. The field of immunometabolism has now expanded to include studies of the interaction of many types of immune cells with adipocytes (4); however, a number of questions yet remain concerning the function and pathophysiological impact of macrophage infiltration into adipose tissue, particularly in obese humans. For example, it has been difficult to distinguish recently recruited cells from the resident pool of macrophages in human adipose tissue and to unequivocally identify in human adipose samples the polarization of macrophages to a uniquely proinflammatory phenotype, as has been more definitively observed in rodent models (5). In this issue of the JCEM, Krinninger et al (6) address the issue of recruitment of inflammatory cells into adipose tissue. These investigators examined monocytes, the circulating precursors of macrophages, from obese and normal-weight women to determine whether there exist phenotypic differences that could promote accumulation of proinflammatory macrophages in adipose tissue. These investigators find that the total monocyte population in obese women contains a greater percentage of activated, proinflammatory CD14 CD16 monocytes than in normal-weight women. Monocytes from obese women also exhibit increased surface expression of the chemokine receptors CCR2 and CCR5 and an enhanced in vitro migration response to the chemokines MCP-1 and RANTES, the production of which is increased in adipose tissue of obese subjects (3). The authors conclude that circulating monocytes in obese women are in an activated, proinflammatory state, and that they are primed to migrate into adipose tissue in response to enhanced chemotactic signals from the tissue in obesity. Human monocytes are phenotypically heterogeneous and in general are classified according to expression of the surface markers CD14 and CD16 (7). “Classical” CD14 CD16 monocytes constitute approximately 80% of the circulating population. However, in inflammatory situations such as sepsis, rheumatoid arthritis, and infection, there is an increase in monocytes positive for CD16 expression (CD14 CD16 ). These cells exhibit a more macrophage-like phenotype with enhanced antigen-presenting capacity, greater binding affinity for endothelial cells, and increased release of TNF and other proinflammatory cytokines. It should be noted that CD16 monocytes can also be phenotypically heterogeneous, with CD14 expression varying from very low to high (7), a characteristic not studied by Krinninger et al (6). Chemokine receptors (CCR2, CCR3, CCR5) are also variable across the different populations of monocytes, with

Proinflammatory immune cell infiltration in human adipose tissue is associated with the development of insulin resistance. We previously identified, via a gene expression-based genome-wide association study, the cell-surface immune cell receptor CD44 as a functionally important gene associated with type 2 diabetes. We then showed that, compared with controls, Cd44 knockout mice were protected from insulin resistance and adipose tissue inflammation during diet-induced obesity. We thus sought to test whether CD44 is associated with adipose tissue inflammation and insulin resistance in humans. Participants included 58 healthy, overweight/moderately obese white adults who met predetermined criteria for insulin resistance or insulin sensitivity based on the modified insulin-suppression test. Serum was collected from 43 participants to measure circulating concentrations of CD44. Subcutaneous adipose tissue was obtained from 17 participants to compare CD44, its ligand osteopontin (OPN, also known as SPP1) and pro-inflammatory gene expression. CD44 expression on adipose tissue macrophage (ATM) surfaces was determined by flow cytometry. Serum CD44 concentrations were significantly increased in insulin-resistant (IR) participants. CD44 gene expression in subcutaneous adipose tissue was threefold higher in the IR subgroup. The expression of OPN, CD68 and IL6 was also significantly elevated in IR individuals. CD44 gene expression correlated significantly with CD68 and IL6 expression. CD44 density on ATMs was associated with proinflammatory M1 polarisation. CD44 and OPN in human adipose tissue are associated with localised inflammation and systemic insulin resistance. This receptor-ligand pair is worthy of further research as a potentially modifiable contributor to human insulin resistance and type 2 diabetes.

Role of the Gut in Visceral Fat Inflammation and Metabolic Disorders

Background: Studies of changes in adipose tissue (AT) inflammation after weight loss have conflicting results. Our aim was to evaluate the changes in AT macrophages, senescence and AT insulin resistance after weight loss. Methods: Twenty-five participants (18 women) with obesity underwent a lifestyle intervention weight loss program and abdominal-femoral AT biopsies. AT insulin resistance was estimated calculating the insulin concentration that suppresses lipolysis by 50% (IC50) during a hyperinsulinemic-euglycemic clamp. Immunohistochemistry was used to estimate total adipose tissue macrophage (ATM) content (CD68+), pro-inflammatory ATM (CD14+), anti-inflammatory ATM (CD206+). Senescent cells in AT were identified by senescence associated β-galactosidase staining. All parameters were assessed before and after weight loss. Results: Median BMI was 34 kg/m2 (IQR, 32-35). A median percent weight loss of 10.2% (IQR, 6.5-12.1) resulted in a mean IC50 reduction of -6.2 ± 13 µIU/mL, p=0.03. There was no significant change in ATM content in the abdominal or femoral fat depots after weight loss, except for an increase in CD68 ATMs (mean change 4.5 ± 7.1 ATM/100 adipocytes, P=0.02) in the femoral depot. The proportion of senescent pre-adipocytes in abdominal and femoral depots remained unchanged after weight loss. In the abdominal depot, there was no relationship between IC 50 and CD68 or CD206 ATMs after weight loss, R2= 0.11, P=0.15 and R2= 0.08, P =0.22, for CD68 and CD206 ATMs respectively. There was a weak negative relationship between CD14 ATMs and IC 50 (R2=0.2, P =0.04). In the femoral depot, there was a weak negative relationship between IC 50 and CD68 ATMs (R2=0.23, P=0.04), and no relationship between IC 50 and CD14 ATMs (R2= 0.2, P=0.06) or CD206 ATMs after weight loss (R2= 0.06, P=0.33). Conclusion: Moderate weight loss is not associated with reduction in ATM content or senescent pre-adipocytes, despite an improvement in AT insulin resistance. Our results suggest that recruitment of some populations of ATMs increases after weight loss. Disclosure A.E. Espinosa De Ycaza: None. E. Søndergaard: None. M. Morgan-Bathke: None. D.A. Delivanis: None. B.G. Carranza Leon: None. K. Lytle: None. M.D. Jensen: None.

Background and Purpose: Obesity is a leading modifiable contributor to breast cancer mortality due to its association with increased recurrence and decreased overall survival rate. There are over 2.5 million breast cancer survivors, 64% of whom are overweight/obese (BMI >25 kg/m2). A central mechanism by which obesity stimulates cancer progression is through chronic, low-grade inflammation in white adipose tissue, leading to accumulation of adipose tissue macrophages (ATMs), in particular the pro-inflammatory M1 phenotype macrophage. Exercise has been shown to reduce M1 ATMs, and increase the more anti-inflammatory M2 ATMs in obese adults. The purpose of this study was to determine whether a 16-week exercise intervention would positively alter adipose tissue inflammation by changing ATM phenotype and cytokine secretion in obese postmenopausal breast cancer survivors. Experimental Design: Twenty obese postmenopausal breast cancer survivors were recruited from USC and randomized to either the exercise (EX) or control (CON) group. The EX group participated in 16 weeks of supervised exercise sessions 3 times/week. Sessions included total-body resistance training consisting of 8 exercises with a rest period of 45 seconds between each set of resistance exercise followed by 30 minutes of moderate-vigorous intensity (65-80% HRmax) aerobic exercise. The CON group was asked to maintain their current activity levels. Superficial subcutaneous abdominal adipose tissue biopsies were performed at baseline and following the 16-week study period. Adipose tissue samples were analyzed using fluorescence-activated cell sorting (FACS) to characterize ATM characterization (M1 vs M2). Portions (~100 mg) of each biopsy were incubated in media overnight to measure cytokine secretion. A 2x2 (group x time) repeated measures ANOVA was used to evaluate changes in adipose tissue and systemic inflammation. Summary of Results: At baseline, there were no group differences (p>0.05) in age (55.1±5.2 yrs), BMI (34.4±7.5 kg/m2), percent body fat (36.2±4.9%), or ATM M1 (25.4±6.7%) and M2 (4.2±0.9%) levels. EX was associated with a significant decrease in ATM M1 (-18.8±7.3%) and increase in ATM M2 (9.6±1.6%; p<0.01). EX increased adipose tissue secretion of the anti-inflammatory cytokines, adiponectin (14.6±5.2%), IL-12 p40 (2.4±0.8), IL-12 p70 (2.8±1.8%) and decreased secretion of IL-6 (-4.8±2.0%), and TNF-a (-2.0±0.7; p<0.01). The CON group did not experience any changes in ATM characterization and cytokine secretion during the study period (p>0.01). Conclusions: A 16-week aerobic and resistance exercise intervention attenuates adipose tissue inflammation in obese postmenopausal breast cancer survivors. Future large randomized controlled trials are warranted to investigate the impact of exercise-induced reductions in adipose tissue inflammation and breast cancer recurrence. Citation Format: Christina M. Dieli-Conwright, Jean Hughes-Parmentier, Kyuwan Lee, Darcy Spicer, Wendy Mack, Fred Sattler, Steven D. Mittelman. Adipose tissue inflammation in breast cancer survivors: Effects of a 16-week aerobic and resistance exercise intervention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 985. doi:10.1158/1538-7445.AM2017-985

Diet and leukocytes

Macrophage lipid metabolism and inflammatory responses are both regulated by the nuclear receptors PPAR and LXR. Emerging links between inflammation and metabolic disease progression suggest that PPAR and LXR signaling may alter macrophage function and thereby impact systemic metabolism. In this study, the function of macrophage PPAR and LXR in Th1-biased C57BL/6 mice was tested using a bone marrow transplantation approach with PPARγ−/−, PPARδ−/−, PPARγδ−/−, and LXRαβ−/− cells. Despite their inhibitory effects on inflammatory gene expression, loss of PPARs or LXRs in macrophages did not exert major effects on obesity or glucose tolerance induced by a high-fat diet. Treatment with rosiglitazone effectively improved glucose tolerance in mice lacking macrophage PPARγ, suggesting that cell types other than macrophages are the primary mediators of the anti-diabetic effects of PPARγ agonists in our model system. C57BL/6 macrophages lacking PPARs or LXRs exhibited normal expression of most alternative activation gene markers, indicating that macrophage alternative activation is not absolutely dependent on these receptors in the C57BL/6 background under the conditions used here. These studies suggest that genetic background may be an important modifier of nuclear receptor effects in macrophages. Our results do not exclude a contribution of macrophage PPAR and LXR expression to systemic metabolism in certain contexts, but these factors do not appear to be dominant contributors to glucose tolerance in a high-fat-fed Th1-biased bone marrow transplant model.

Metabolic dysfunction is associated with adipose tissue inflammation and macrophage infiltration. PAFR (platelet-activating factor receptor) is expressed in several cell typesand binds to PAF (platelet-activating factor) and oxidized phospholipids. Engagement of PAFR in macrophages drives them towards the anti-inflammatory phenotype. In the present study, we investigated whether genetic deficiency of PAFR affects the phenotype of ATMs (adipose tissue macrophages) and its effect on glucose and insulin metabolism. PARFKO (PAFR-knockout) and WT (wild-type) mice were fed on an SD (standard diet) or an HFD (high-fat diet). Glucose and insulin tolerance tests were performed by blood monitoring. ATMs were evaluated by FACS for phenotypic markers. Gene and protein expression was investigated by real-time reverse transcription-quantitative PCR and Western blotting respectively. Results showed that the epididymal adipose tissue of PAFRKO mice had increased gene expression of Ccr7, Nos2, Il6 and Il12, associated with pro-inflammatory mediators, and reduced expression of the anti-inflammatory Il10. Moreover, the adipose tissue of PAFRKO mice presented more pro-inflammatory macrophages, characterized by an increased frequency of F4/80(+)CD11c(+) cells. Blood monocytes of PAFRKO mice also exhibited a pro-inflammatory phenotype (increased frequency of Ly6C(+) cells) and PAFR ligands were detected in the serum of both PAFRKO and WT mice. Regarding metabolic parameters, compared with WT, PAFRKO mice had: (i) higher weight gain and serum glucose concentration levels; (ii) decreased insulin-stimulated glucose disappearance; (iii) insulin resistance in the liver; (iv) increased expression of Ldlr in the liver. In mice fed on an HFD, some of these changes were potentiated, particularly in the liver. Thus it seems that endogenous ligands of PAFR are responsible for maintaining the anti-inflammatory profile of blood monocytes and ATMs under physiological conditions. In the absence of PAFR signalling, monocytes and macrophages acquire a pro-inflammatory phenotype, resulting in adipose tissue inflammation and metabolic dysfunction.

Therapeutic targeting of adipose tissue macrophages ameliorates liver fibrosis in non-alcoholic fatty liver disease

Adipocyte death has been reported in both obese humans and rodents. However, its role in metabolic disorders, including insulin resistance, hepatic steatosis, and inflammation associated with obesity has not been studied. We now show using real-time reverse transcription-PCR arrays that adipose tissue of obese mice display a pro-apoptotic phenotype. Moreover, caspase activation and adipocyte apoptosis were markedly increased in adipose tissue from both mice with diet-induced obesity and obese humans. These changes were associated with activation of both the extrinsic, death receptor-mediated, and intrinsic, mitochondrial-mediated pathways of apoptosis. Genetic inactivation of Bid, a key pro-apoptotic molecule that serves as a link between these two cell death pathways, significantly reduced caspase activation, adipocyte apoptosis, prevented adipose tissue macrophage infiltration, and protected against the development of systemic insulin resistance and hepatic steatosis independent of body weight. These data strongly suggest that adipocyte apoptosis is a key initial event that contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis associated with obesity in both mice and humans. Inhibition of adipocyte apoptosis may be a new therapeutic strategy for the treatment of obesity-associated metabolic complications.