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Abstract Background Chronic inflammation may contribute to insulin resistance (IR), metabolic syndrome and atherosclerosis although evidence of causality is lacking in humans. We hypothesized that very low-dose experimental endotoxemia would induce adipose tissue inflammation and systemic IR during a low-grade but asymptomatic inflammatory response and thus provide an experimental model for future tests of pharmacologic and genomic modulation of cardio-metabolic traits in humans. Methods Ten healthy, human volunteers (50% male, 90% Caucasian, mean age 22.7 ± 3.8) were randomized in a double-masked, placebo-controlled, crossover study to separate 36-hour inpatient visits (placebo versus intravenous-LPS 0.6 ng/kg). We measured clinical symptoms via the McGill pain questionnaire and serial vital signs. Plasma and serum were collected for measurement of cytokines, C-reactive protein, insulin and glucose, serial whole blood & subcutaneous adipose tissue mRNA expression were measured by real-time PCR. HOMA-IR, a well-validated measure of IR was calculated to estimate insulin resistance, and frequently sampled intravenous glucose tolerance testing (FSIGTT) was performed to confirm an insulin resistant state. We performed ANOVA and within subject ANOVA to understand the differences in cytokines, adipose tissue inflammation and IR before and after LPS or placebo. Results There was no significant difference between placebo and LPS in clinical responses of symptom scores, body temperature or heart rate. However, low-dose endotoxemia induced a rapid and transient 25-fold induction of plasma TNF-alpha and 100-fold increase in plasma IL-6 (Figure 1B) (p p p = 0.01) increased with MCP-1 (peak 10-fold, F = 5.6, p p p p Conclusions We present a low dose human endotoxemia model of inflammation which induces adipose tissue inflammation and systemic insulin resistance in the absence of overt clinical response. Such a model has the potential for broad and safe application in the study of novel therapeutics and genomic influences in cardio-metabolic disease.

Long intergenic noncoding RNAs (lincRNAs) have emerged as key regulators of cellular functions and physiology. Yet functional lincRNAs often have low, context-specific and tissue-specific expression. We hypothesized that many human monocyte and adipose lincRNAs would be absent in current public annotations due to lincRNA tissue specificity, modest sequencing depth in public data, limitations of transcriptome assembly algorithms, and lack of dynamic physiological contexts. Deep RNA sequencing (RNA-Seq) was performed in peripheral blood CD14+ monocytes (monocytes; average ~247 million reads per sample) and adipose tissue (average ~378 million reads per sample) collected before and after human experimental endotoxemia, an in vivo inflammatory stress, to identify tissue-specific and clinically relevant lincRNAs. Using a stringent filtering pipeline, we identified 109 unannotated lincRNAs in monocytes and 270 unannotated lincRNAs in adipose. Most unannotated lincRNAs are not conserved in rodents and are tissue specific, while many have features of regulated expression and are enriched in transposable elements. Specific subsets have enhancer RNA characteristics or are expressed only during inflammatory stress. A subset of unannotated lincRNAs was validated and replicated for their presence and inflammatory induction in independent human samples and for their monocyte and adipocyte origins. Through interrogation of public genome-wide association data, we also found evidence of specific disease association for selective unannotated lincRNAs. Our findings highlight the critical need to perform deep RNA-Seq in a cell-, tissue-, and context-specific manner to annotate the full repertoire of human lincRNAs for a complete understanding of lincRNA roles in dynamic cell functions and in human disease. [ABSTRACT FROM AUTHOR]

Objectives: This study sought to examine the role of lipoprotein-associated phospholipase A2 (Lp-PLA2/PLA2G7) in human inflammation and coronary atherosclerosis. Background: Lp-PLA2 has emerged as a potential therapeutic target in coronary heart disease. Data supporting Lp-PLA2 are indirect and confounded by species differences; whether Lp-PLA2 is causal in coronary heart disease remains in question. Methods: We examined inflammatory regulation of Lp-PLA2 during experimental endotoxemia in humans, probed the source of Lp-PLA2 in human leukocytes under inflammatory conditions, and assessed the relationship of variation in PLA2G7, the gene encoding Lp-PLA2, with coronary artery calcification. Results: In contrast to circulating tumor necrosis factor-alpha and C-reactive protein, blood and monocyte Lp-PLA2 messenger ribonucleic acid decreased transiently, and plasma Lp-PLA2 mass declined modestly during endotoxemia. In vitro, Lp-PLA2 expression increased dramatically during human monocyte to macrophage differentiation and further in inflammatory macrophages and foamlike cells. Despite only a marginal association of single nucleotide polymorphisms in PLA2G7 with Lp-PLA2 activity or mass, numerous PLA2G7 single nucleotide polymorphisms were associated with coronary artery calcification. In contrast, several single nucleotide polymorphisms in CRP were significantly associated with plasma C-reactive protein levels but had no relation with coronary artery calcification. Conclusions: Circulating Lp-PLA2 did not increase during acute phase response in humans, whereas inflammatory macrophages and foam cells, but not circulating monocytes, are major leukocyte sources of Lp-PLA2. Common genetic variation in PLA2G7 is associated with subclinical coronary atherosclerosis. These data link Lp-PLA2 to atherosclerosis in humans while highlighting the challenge in using circulating Lp-PLA2 as a biomarker of Lp-PLA2 actions in the vasculature. [Copyright &y& Elsevier]

Recent reports demonstrate T-cell infiltration of adipose tissue in early obesity. We hypothesized that interferon (IFN) γ, a major T-cell inflammatory cytokine, would attenuate human adipocyte functions and sought to establish signaling mechanisms. Differentiated human adipocytes were treated with IFNy ± pharmacological inhibitors prior to insulin stimulation. [3H] Glucose uptake and AKT phosphorylation were assessed as markers of insulin sensitivity. IFNγ induced sustained loss of insulin-stimulated glucose uptake in human adipocytes, coincident with reduced Akt phosphorylation and down-regulation of the insulin receptor, insulin receptor substrate-1, and GLUT4. Loss of adipocyte triglyceride storage was observed with IFNγ co-incident with reduced expression of peroxisome proliferator-activated receptor y, adiponectin, perilipin, fatty acid synthase, and lipoprotein lipase. Treatment with IFNγ also blocked differentiation of pre-adipocytes to the mature phenotype. IFNγ-induced robust STAT1 phosphorylation and SOCS1 mRNA expression, with modest, transient STAT3 phosphorylation and SOCS3 induction. Preincubation with a non-selective JAK inhibitor restored glucose uptake and Akt phosphorylation while completely reversing IFNγ suppression of adipogenic mRNAs and adipocyte differentiation. Specific inhibition of JAK2 or JAK3 failed to block IFNγ effects suggesting a predominant role for JAK1-STAT1. We demonstrate that IFNγ attenuates insulin sensitivity and suppresses differentiation in human adipocytes, an effect most likely mediated via sustained JAK- STAT1 pathway activation. [ABSTRACT FROM AUTHOR]

Long intergenic noncoding RNAs (lincRNAs) have emerged as key regulators of cellular functions and physiology. Yet functional lincRNAs often have low, context-specific and tissue-specific expression. We hypothesized that many human monocyte and adipose lincRNAs would be absent in current public annotations due to lincRNA tissue specificity, modest sequencing depth in public data, limitations of transcriptome assembly algorithms, and lack of dynamic physiological contexts. Deep RNA sequencing (RNA-Seq) was performed in peripheral blood CD14 + monocytes (monocytes; average ~247 million reads per sample) and adipose tissue (average ~378 million reads per sample) collected before and after human experimental endotoxemia, an in vivo inflammatory stress, to identify tissue-specific and clinically relevant lincRNAs. Using a stringent filtering pipeline, we identified 109 unannotated lincRNAs in monocytes and 270 unannotated lincRNAs in adipose. Most unannotated lincRNAs are not conserved in rodents and are tissue specific, while many have features of regulated expression and are enriched in transposable elements. Specific subsets have enhancer RNA characteristics or are expressed only during inflammatory stress. A subset of unannotated lincRNAs was validated and replicated for their presence and inflammatory induction in independent human samples and for their monocyte and adipocyte origins. Through interrogation of public genome-wide association data, we also found evidence of specific disease association for selective unannotated lincRNAs. Our findings highlight the critical need to perform deep RNA-Seq in a cell-, tissue-, and context-specific manner to annotate the full repertoire of human lincRNAs for a complete understanding of lincRNA roles in dynamic cell functions and in human disease., (Copyright © 2017 the American Physiological Society.)

Recent reports demonstrate T-cell infiltration of adipose tissue in early obesity. We hypothesized that interferon (IFN) gamma, a major T-cell inflammatory cytokine, would attenuate human adipocyte functions and sought to establish signaling mechanisms. Differentiated human adipocytes were treated with IFNgamma +/- pharmacological inhibitors prior to insulin stimulation. [(3)H]Glucose uptake and AKT phosphorylation were assessed as markers of insulin sensitivity. IFNgamma induced sustained loss of insulin-stimulated glucose uptake in human adipocytes, coincident with reduced Akt phosphorylation and down-regulation of the insulin receptor, insulin receptor substrate-1, and GLUT4. Loss of adipocyte triglyceride storage was observed with IFNgamma co-incident with reduced expression of peroxisome proliferator-activated receptor gamma, adiponectin, perilipin, fatty acid synthase, and lipoprotein lipase. Treatment with IFNgamma also blocked differentiation of pre-adipocytes to the mature phenotype. IFNgamma-induced robust STAT1 phosphorylation and SOCS1 mRNA expression, with modest, transient STAT3 phosphorylation and SOCS3 induction. Preincubation with a non-selective JAK inhibitor restored glucose uptake and Akt phosphorylation while completely reversing IFNgamma suppression of adipogenic mRNAs and adipocyte differentiation. Specific inhibition of JAK2 or JAK3 failed to block IFNgamma effects suggesting a predominant role for JAK1-STAT1. We demonstrate that IFNgamma attenuates insulin sensitivity and suppresses differentiation in human adipocytes, an effect most likely mediated via sustained JAK-STAT1 pathway activation.