Lipoxins reduce obesity-induced adipose tissue inflammation in 3D-cultured human adipocytes and explant cultures.

Adipose tissue inflammation drives obesity-related cardiometabolic diseases. Enhancing endogenous resolution mechanisms through administration of lipoxin A4, a specialized pro-resolving lipid mediator, was shown to reduce adipose inflammation and subsequently protects against obesity-induced systemic disease in mice. Here, we demonstrate that lipoxins reduce inflammation in 3D-cultured human adipocytes and adipose tissue explants from obese patients. Approximately 50% of patients responded particularly well to lipoxins by reducing inflammatory cytokines and promoting an anti-inflammatory M2 macrophage phenotype. Responding patients were characterized by elevated systemic levels of C-reactive protein, which causes inflammation in cultured human adipocytes. Responders appeared more prone to producing anti-inflammatory oxylipins and displayed elevated prostaglandin D2 levels, which has been interlinked with transcription of lipoxin-generating enzymes. Using explant cultures, this study provides the first proof-of-concept evidence supporting the therapeutic potential of lipoxins in reducing human adipose tissue inflammation. Our data further indicate that lipoxin treatment may require a tailored personalized-medicine approach.

The immune checkpoint B7-H3 (CD276) regulates adipocyte progenitor metabolism and obesity development.

The immune checkpoint B7-H3 (CD276) is a member of the B7 family that has been studied in the tumor microenvironment and immunotherapy, but its potential role in metabolism remains largely unknown. Here, we show that B7-H3 is highly expressed in mouse and human adipose tissue at steady state, with the highest levels in adipocyte progenitor cells. B7-H3 is rapidly down-regulated upon the initiation of adipocyte differentiation. Combined RNA sequencing and metabolic studies reveal that B7-H3 stimulates glycolytic and mitochondrial activity of adipocyte progenitors. Loss of B7-H3 in progenitors results in impaired oxidative metabolism program and increased lipid accumulation in derived adipocytes. Consistent with these observations, mice knocked out for B7-H3 develop spontaneous obesity, metabolic dysfunction, and adipose tissue inflammation. Our results reveal an unexpected metabolic role for B7-H3 in adipose tissue and open potential new avenues for the treatment of metabolic diseases by targeting the B7-H3 pathway.

Lipoxins modulate neutrophil oxidative burst, integrin expression and lymphatic transmigration differentially in human health and atherosclerosis.

Dysregulated chronic inflammation plays a crucial role in the pathophysiology of atherosclerosis and may be a result of impaired resolution. Thus, restoring levels of specialized pro-resolving mediators (SPMs) to promote the resolution of inflammation has been proposed as a therapeutic strategy for patients with atherosclerosis, in addition to standard clinical care. Herein, we evaluated the effects of the SPM lipids, lipoxin A4 (LXA4 ) and lipoxin B4 (LXB4 ), on neutrophils isolated from patients with atherosclerosis compared with healthy controls. Patients displayed altered endogenous SPM production, and we demonstrated that lipoxin treatment in whole blood from atherosclerosis patients attenuates neutrophil oxidative burst, a key contributor to atherosclerotic development. We found the opposite effect in neutrophils from healthy controls, indicating a potential mechanism whereby lipoxins aid the endogenous neutrophil function in health but reduce its excessive activation in disease. We also demonstrated that lipoxins attenuated upregulation of the high-affinity conformation of the CD11b/CD18 integrin, which plays a central role in clot activation and atherosclerosis. Finally, LXB4 enhanced lymphatic transmigration of human neutrophils isolated from patients with atherosclerosis. This finding is noteworthy, as impaired lymphatic function is now recognized as an important contributor to atherosclerosis. Although both lipoxins modulated neutrophil function, LXB4 displayed more potent effects than LXA4 in humans. This study highlights the therapeutic potential of lipoxins in atherosclerotic disease and demonstrates that the effect of these SPMs may be specifically tailored to the need of the individual.

Healthy Subcutaneous and Omental Adipose Tissue Is Associated with High Expression of Extracellular Matrix Components.

Obesity is associated with extensive expansion and remodeling of the adipose tissue architecture, including its microenvironment and extracellular matrix (ECM). Although obesity has been reported to induce adipose tissue fibrosis, the composition of the ECM under healthy physiological conditions has remained underexplored and debated. Here, we used a combination of three established techniques (picrosirius red staining, a colorimetric hydroxyproline assay, and sensitive gene expression measurements) to evaluate the status of the ECM in metabolically healthy lean (MHL) and metabolically unhealthy obese (MUO) subjects. We investigated ECM deposition in the two major human adipose tissues, namely the omental and subcutaneous depots. Biopsies were obtained from the same anatomic region of respective individuals. We found robust ECM deposition in MHL subjects, which correlated with high expression of collagens and enzymes involved in ECM remodeling. In contrast, MUO individuals showed lower expression of ECM components but elevated levels of ECM cross-linking and adhesion proteins, e.g., lysyl oxidase and thrombospondin. Our data suggests that subcutaneous fat is more prone to express proteins involved in ECM remodeling than omental adipose tissues. We conclude that a more dynamic ability to deposit and remodel ECM may be a key signature of healthy adipose tissue, and that subcutaneous fat may adapt more readily to changing metabolic conditions than omental fat.

Comparative analysis of obesity-related cardiometabolic and renal biomarkers in human plasma and serum.

The search for biomarkers associated with obesity-related diseases is ongoing, but it is not clear whether plasma and serum can be used interchangeably in this process. Here we used high-throughput screening to analyze 358 proteins and 76 lipids, selected because of their relevance to obesity-associated diseases, in plasma and serum from age- and sex-matched lean and obese humans. Most of the proteins/lipids had similar concentrations in plasma and serum, but a subset showed significant differences. Notably, a key marker of cardiovascular disease PAI-1 showed a difference in concentration between the obese and lean groups only in plasma. Furthermore, some biomarkers showed poor correlations between plasma and serum, including PCSK9, an important regulator of cholesterol homeostasis. Collectively, our results show that the choice of biofluid may impact study outcome when screening for obesity-related biomarkers and we identify several markers where this will be the case.

Systems-wide Experimental and Modeling Analysis of Insulin Signaling through Forkhead Box Protein O1 (FOXO1) in Human Adipocytes, Normally and in Type 2 Diabetes.

Insulin resistance is a major aspect of type 2 diabetes (T2D), which results from impaired insulin signaling in target cells. Signaling to regulate forkhead box protein O1 (FOXO1) may be the most important mechanism for insulin to control transcription. Despite this, little is known about how insulin regulates FOXO1 and how FOXO1 may contribute to insulin resistance in adipocytes, which are the most critical cell type in the development of insulin resistance. We report a detailed mechanistic analysis of insulin control of FOXO1 in human adipocytes obtained from non-diabetic subjects and from patients with T2D. We show that FOXO1 is mainly phosphorylated through mTORC2-mediated phosphorylation of protein kinase B at Ser(473) and that this mechanism is unperturbed in T2D. We also demonstrate a cross-talk from the MAPK branch of insulin signaling to stimulate phosphorylation of FOXO1. The cellular abundance and consequently activity of FOXO1 are halved in T2D. Interestingly, inhibition of mTORC1 with rapamycin reduces the abundance of FOXO1 to the levels in T2D. This suggests that the reduction of the concentration of FOXO1 is a consequence of attenuation of mTORC1, which defines much of the diabetic state in human adipocytes. We integrate insulin control of FOXO1 in a network-wide mathematical model of insulin signaling dynamics based on compatible data from human adipocytes. The diabetic state is network-wide explained by attenuation of an mTORC1-to-insulin receptor substrate-1 (IRS1) feedback and reduced abundances of insulin receptor, GLUT4, AS160, ribosomal protein S6, and FOXO1. The model demonstrates that attenuation of the mTORC1-to-IRS1 feedback is a major mechanism of insulin resistance in the diabetic state.

A single mechanism can explain network-wide insulin resistance in adipocytes from obese patients with type 2 diabetes.

The response to insulin is impaired in type 2 diabetes. Much information is available about insulin signaling, but understanding of the cellular mechanisms causing impaired signaling and insulin resistance is hampered by fragmented data, mainly obtained from different cell lines and animals. We have collected quantitative and systems-wide dynamic data on insulin signaling in primary adipocytes and compared cells isolated from healthy and diabetic individuals. Mathematical modeling and experimental verification identified mechanisms of insulin control of the MAPKs ERK1/2. We found that in human adipocytes, insulin stimulates phosphorylation of the ribosomal protein S6 and hence protein synthesis about equally via ERK1/2 and mTORC1. Using mathematical modeling, we examined the signaling network as a whole and show that a single mechanism can explain the insulin resistance of type 2 diabetes throughout the network, involving signaling both through IRS1, PKB, and mTOR and via ERK1/2 to the nuclear transcription factor Elk1. The most important part of the insulin resistance mechanism is an attenuated feedback from the protein kinase mTORC1 to IRS1, which spreads signal attenuation to all parts of the insulin signaling network. Experimental inhibition of mTORC1 using rapamycin in adipocytes from non-diabetic individuals induced and thus confirmed the predicted network-wide insulin resistance.

Phosphorylation of IRS1 at serine 307 in response to insulin in human adipocytes is not likely to be catalyzed by p70 ribosomal S6 kinase.

The insulin receptor substrate-1 (IRS1) is phosphorylated on serine 307 (human sequence, corresponding to murine serine 302) in response to insulin as part of a feedback loop that controls IRS1 phosphorylation on tyrosine residues by the insulin receptor. This in turn directly affects downstream signaling and is in human adipocytes implicated in the pathogenesis of insulin resistance and type 2 diabetes. The phosphorylation is inhibited by rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR) in complex with raptor (mTORC1). The mTORC1-downstream p70 ribosomal protein S6 kinase (S6K1), which is activated by insulin, can phosphorylate IRS1 at serine 307 in vitro and is considered the physiological protein kinase. Because the IRS1 serine 307-kinase catalyzes a critical step in the control of insulin signaling and constitutes a potential target for treatment of insulin resistance, it is important to know whether S6K1 is the physiological serine 307-kinase or not. We report that, by several criteria, S6K1 does not phosphorylate IRS1 at serine 307 in response to insulin in intact human primary adipocytes: (i) The time-courses for phosphorylation of S6K1 and its phosphorylation of S6 are not compatible with the phosphorylation of IRS1 at serine 307; (ii) A dominant-negative construct of S6K1 inhibits the phosphorylation of S6, without effect on the phosphorylation of IRS1 at serine 307; (iii) The specific inhibitor of S6K1 PF-4708671 inhibits the phosphorylation of S6, without effect on phosphorylation of IRS1 at serine 307. mTOR-immunoprecipitates from insulin-stimulated adipocytes contains an unidentified protein kinase specific for phosphorylation of IRS1 at serine 307, but it is not mTOR or S6K1.