Christine Hinkle

Inflammation impairs reverse cholesterol transport in vivo.

Background— Inflammation is proposed to impair reverse cholesterol transport (RCT), a major atheroprotective function of high-density lipoprotein (HDL). The present study presents the first integrated functional evidence that inflammation retards numerous components of RCT. Methods and Results— We used subacute endotoxemia in the rodent macrophage-to-feces RCT model to assess the effects of inflammation on RCT in vivo and performed proof of concept experimental endotoxemia studies in humans. Endotoxemia (3 mg/kg SC) reduced 3H-cholesterol movement from macrophage to plasma and 3H-cholesterol associated with HDL fractions. At 48 hours, bile and fecal counts were markedly reduced consistent with downregulation of hepatic expression of ABCG5, ABCG8, and ABCB11 biliary transporters. Low-dose lipopolysaccharide (0.3 mg/kg SC) also reduced bile and fecal counts, as well as expression of biliary transporters, but in the absence of effects on plasma or liver counts. In vitro, lipopolysaccharide impaired 3H-cholesterol efflux from human macrophages to apolipoprotein A-I and serum coincident with reduced expression of the cholesterol transporter ABCA1. During human (3 ng/kg; n=20) and murine endotoxemia (3 mg/kg SC), ex vivo macrophage cholesterol efflux to acute phase HDL was attenuated. Conclusions— We provide the first in vivo evidence that inflammation impairs RCT at multiple steps in the RCT pathway, particularly cholesterol flux through liver to bile and feces. Attenuation of RCT and HDL efflux function, independent of HDL cholesterol levels, may contribute to atherosclerosis in chronic inflammatory states including obesity, metabolic syndrome, and type 2 diabetes.

Experimental Endotoxemia Induces Adipose Inflammation and Insulin Resistance in Humans

OBJECTIVE An emerging model of metabolic syndrome and type 2 diabetes is of adipose dysfunction with leukocyte recruitment into adipose leading to chronic inflammation and insulin resistance (IR). This study sought to explore potential mechanisms of inflammatory-induced IR in humans with a focus on adipose tissue. RESEARCH DESIGN AND METHODS We performed a 60-h endotoxemia protocol (3 ng/kg intravenous bolus) in healthy adults (n = 20, 50% male, 80% Caucasian, aged 27.3 ± 4.8 years). Before and after endotoxin, whole-blood sampling, subcutaneous adipose biopsies, and frequently sampled intravenous glucose tolerance (FSIGT) testing were performed. The primary outcome was the FSIGT insulin sensitivity index (Si). Secondary measures included inflammatory and metabolic markers and whole-blood and adipose mRNA and protein expression. RESULTS Endotoxemia induced systemic IR as demonstrated by a 35% decrease in Si (3.17 ± 1.66 to 2.06 ± 0.73 × 10−4 [μU · ml−1 · min−1], P < 0.005), while there was no effect on pancreatic β-cell function. In adipose, endotoxemia suppressed insulin receptor substrate-1 and markedly induced suppressor of cytokine signaling proteins (1 and 3) coincident with local activation of innate (interleukin-6, tumor necrosis factor) and adaptive (monocyte chemoattractant protein-1 and CXCL10 chemokines) inflammation. These changes are known to attenuate insulin receptor signaling in model systems. CONCLUSIONS We demonstrate, for the first time in humans, that acute inflammation induces systemic IR following modulation of specific adipose inflammatory and insulin signaling pathways. It also provides a rationale for focused mechanistic studies and a model for human proof-of-concept trials of novel therapeutics targeting adipose inflammation in IR and related consequences in humans.

Interferon γ Attenuates Insulin Signaling, Lipid Storage, and Differentiation in Human Adipocytes via Activation of the JAK/STAT Pathway

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 IFNγ ± 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 γ, 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.

Fractalkine Is a Novel Human Adipochemokine Associated With Type 2 Diabetes

OBJECTIVE Leukocyte infiltration of adipose is a critical determinant of obesity-related metabolic diseases. Fractalkine (CX3CL1) and its receptor (CX3CR1) comprise a chemokine system involved in leukocyte recruitment and adhesion in atherosclerosis, but its role in adipose inflammation and type 2 diabetes is unknown. RESEARCH DESIGN AND METHODS CX3CL1 mRNA and protein were quantified in subcutaneous adipose and blood during experimental human endotoxemia and in lean and obese human adipose. CX3CL1 cellular source was probed in human adipocytes, monocytes, and macrophages, and CX3CL1-blocking antibodies were used to assess its role in monocyte-adipocyte adhesion. The association of genetic variation in CX3CR1 with metabolic traits was determined in a community-based sample. Finally, plasma CX3CL1 levels were measured in a case-control study of type 2 diabetes. RESULTS Endotoxemia induced adipose CX3CL1 mRNA (32.7-fold, P < 1 × 10−5) and protein (43-fold, P = 0.006). Obese subjects had higher CX3CL1 levels in subcutaneous adipose compared with lean (0.420 ± 0.387 vs. 0.228 ± 0.187 ng/mL, P = 0.04). CX3CL1 was expressed and secreted by human adipocytes and stromal vascular cells. Inflammatory cytokine induction of CX3CL1 in human adipocytes (27.5-fold mRNA and threefold protein) was completely attenuated by pretreatment with a peroxisome proliferator–activated receptor-γ agonist. A putative functional nonsynonymous single nucleotide polymorphism (rs3732378) in CX3CR1 was associated with adipose and metabolic traits, and plasma CX3CL1 levels were increased in patients with type 2 diabetes vs. nondiabetics (0.506 ± 0.262 vs. 0.422 ± 0.210 ng/mL, P < 0.0001). CONCLUSIONS CX3CL1-CX3CR1 is a novel inflammatory adipose chemokine system that modulates monocyte adhesion to adipocytes and is associated with obesity, insulin resistance, and type 2 diabetes. These data provide support for CX3CL1 as a diagnostic and therapeutic target in cardiometabolic disease.

Adipocyte Modulation of High-Density Lipoprotein Cholesterol

Background— Adipose harbors a large depot of free cholesterol. However, a role for adipose in cholesterol lipidation of high-density lipoprotein (HDL) in vivo is not established. We present the first evidence that adipocytes support transfer of cholesterol to HDL in vivo as well as in vitro and implicate ATP-binding cassette subfamily A member 1 (ABCA1) and scavenger receptor class B type I (SR-BI), but not ATP-binding cassette subfamily G member 1 (ABCG1), cholesterol transporters in this process. Methods and Results— Cholesterol efflux from wild-type, ABCA1−/−, SR-BI−/−, and ABCG1−/− adipocytes to apolipoprotein A-I (apoA-I) and HDL3 were measured in vitro. 3T3L1 adipocytes, labeled with 3H-cholesterol, were injected intraperitoneally into wild-type, apoA-I transgenic, and apoA-I−/− mice, and tracer movement onto plasma HDL was monitored. Identical studies were performed with labeled wild-type, ABCA1−/−, or SR-BI−/− mouse embryonic fibroblast adipocytes. The effect of tumor necrosis factor-&agr; on transporter expression and cholesterol efflux was monitored during adipocyte differentiation. Cholesterol efflux to apoA-I and HDL3 was impaired in ABCA1−/− and SR-BI−/− adipocytes, respectively, with no effect observed in ABCG1−/− adipocytes. Intraperitoneal injection of labeled 3T3L1 adipocytes resulted in increased HDL-associated 3H-cholesterol in apoA-I transgenic mice but reduced levels in apoA-I−/− animals. Intraperitoneal injection of labeled ABCA1−/− or SR-BI−/− adipocytes reduced plasma counts relative to their respective controls. Tumor necrosis factor-&agr; reduced both ABCA1 and SR-BI expression and impaired cholesterol efflux from partially differentiated adipocytes. Conclusions— These data suggest a novel metabolic function of adipocytes in promoting cholesterol transfer to HDL in vivo and implicate adipocyte SR-BI and ABCA1, but not ABCG1, in this process. Furthermore, adipocyte modulation of HDL may be impaired in adipose inflammatory disease states such as type 2 diabetes mellitus.

Gene Profiling of Human Adipose Tissue During Evoked Inflammation In Vivo

OBJECTIVE Adipose inflammation plays a central role in obesity-related metabolic and cardiovascular complications. However, few human adipose-secreted proteins are known to mediate these processes. We hypothesized that microarray mRNA profiling of human adipose during evoked inflammation could identify novel adipocytokines. RESEARCH DESIGN AND METHODS Healthy human volunteers (n = 14) were treated with intravenous endotoxin (3 ng/kg lipopolysaccharide [LPS]) and underwent subcutaneous adipose biopsies before and after LPS. On Affymetrix U133Plus 2.0 arrays, adipose mRNAs modulated >1.5-fold (with P < 0.00001) were selected. SignalP 3.0 and SecretomeP 2.0 identified genes predicted to encode secreted proteins. Of these, 86 candidates were chosen for validation in adipose from an independent human endotoxemia protocol (N = 7, with 0.6 ng/kg LPS) and for exploration of cellular origin in primary human adipocytes and macrophages in vitro. RESULTS Microarray identified 776 adipose genes modulated by LPS; 298 were predicted to be secreted. Of detectable prioritized genes, 82 of 85 (96% [95% CI 90–99]) were upregulated (fold changes >1.0) during the lower-dose (LPS 0.6 ng/kg) validation study and 51 of 85 (59% [49–70]) were induced greater than 1.5-fold. Treatment of primary adipocytes with LPS and macrophage polarization to M1 proinflammatory phenotype increased expression by 1.5-fold for 58 and 73% of detectable genes, respectively. CONCLUSIONS We demonstrate that evoked inflammation of human adipose in vivo modulated expression of multiple genes likely secreted by adipocytes and monocytes. These included established adipocytokines and chemokines implicated in recruitment and activation of lymphocytes, adhesion molecules, antioxidants, and several novel genes with unknown function. Such candidates may represent biomarkers and therapeutic targets for obesity-related complications.

Translational studies of lipoprotein-associated phospholipase A2 in inflammation and atherosclerosis

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.

Functional Analysis and Transcriptomic Profiling of iPSC-Derived Macrophages and Their Application in Modeling Mendelian Disease

RATIONALE An efficient and reproducible source of genotype-specific human macrophages is essential for study of human macrophage biology and related diseases. OBJECTIVE To perform integrated functional and transcriptome analyses of human induced pluripotent stem cell-derived macrophages (IPSDMs) and their isogenic human peripheral blood mononuclear cell-derived macrophage (HMDM) counterparts and assess the application of IPSDM in modeling macrophage polarization and Mendelian disease. METHODS AND RESULTS We developed an efficient protocol for differentiation of IPSDM, which expressed macrophage-specific markers and took up modified lipoproteins in a similar manner to HMDM. Like HMDM, IPSDM revealed reduction in phagocytosis, increase in cholesterol efflux capacity and characteristic secretion of inflammatory cytokines in response to M1 (lipopolysaccharide+interferon-γ) activation. RNA-Seq revealed that nonpolarized (M0) as well as M1 or M2 (interleukin-4) polarized IPSDM shared transcriptomic profiles with their isogenic HMDM counterparts while also revealing novel markers of macrophage polarization. Relative to IPSDM and HMDM of control individuals, patterns of defective cholesterol efflux to apolipoprotein A-I and high-density lipoprotein-3 were qualitatively and quantitatively similar in IPSDM and HMDM of patients with Tangier disease, an autosomal recessive disorder because of mutations in ATP-binding cassette transporter AI. Tangier disease-IPSDM also revealed novel defects of enhanced proinflammatory response to lipopolysaccharide stimulus. CONCLUSIONS Our protocol-derived IPSDM are comparable with HMDM at phenotypic, functional, and transcriptomic levels. Tangier disease-IPSDM recapitulated hallmark features observed in HMDM and revealed novel inflammatory phenotypes. IPSDMs provide a powerful tool for study of macrophage-specific function in human genetic disorders as well as molecular studies of human macrophage activation and polarization.

A human model of inflammatory cardio-metabolic dysfunction; a double blind placebo-controlled crossover trial.

BackgroundChronic 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.MethodsTen 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.ResultsThere 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 < 0.001 for both) both peaking at two hours, followed by modest inflammation in adipose tissue with increases in mRNA levels of several inflammatory genes known to modulate adipose and systemic insulin resistance. Adipose tissue mRNA levels of IL-6 (peak 6-fold, ANOVA F = 27.5, p < 0.001) and TNF-alpha (peak 1.8-fold, F = 2.9, p = 0.01) increased with MCP-1 (peak 10-fold, F = 5.6, p < 0.01) and fractalkine (CX3CL1) (peak 15-fold, F = 13.3, p < 0.001). Finally, HOMA-IR was 32% higher following LPS compared to placebo (p < 0.01) and insulin sensitivity declined by 21% following LPS compared to placebo (p < 0.05).ConclusionsWe 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.

Metabolic Effects of CX3CR1 Deficiency in Diet-Induced Obese Mice

The fractalkine (CX3CL1-CX3CR1) chemokine system is associated with obesity-related inflammation and type 2 diabetes, but data on effects of Cx3cr1 deficiency on metabolic pathways is contradictory. We examined male C57BL/6 Cx3cr1-/- mice on chow and high-fat diet to determine the metabolic effects of Cx3cr1 deficiency. We found no difference in body weight and fat content or feeding and energy expenditure between Cx3cr1-/- and WT mice. Cx3cr1-/- mice had reduced glucose intolerance assessed by intraperitoneal glucose tolerance tests at chow and high-fat fed states, though there was no difference in glucose-stimulated insulin values. Cx3cr1-/- mice also had improved insulin sensitivity at hyperinsulinemic-euglycemic clamp, with higher glucose infusion rate, rate of disposal, and hepatic glucose production suppression compared to WT mice. Enhanced insulin signaling in response to acute intravenous insulin injection was demonstrated in Cx3cr1-/- by increased liver protein levels of phosphorylated AKT and GSK3β proteins. There were no differences in adipose tissue macrophage populations, circulating inflammatory monocytes, adipokines, lipids, or inflammatory markers. In conclusion, we demonstrate a moderate and reproducible protective effect of Cx3cr1 deficiency on glucose intolerance and insulin resistance.