Robert A. Figler

Links Between Insulin Resistance, Adenosine A2B Receptors, and Inflammatory Markers in Mice and Humans

OBJECTIVE To determine the mechanisms by which blockade of adenosine A2B receptors (A2BRs) reduces insulin resistance. RESEARCH DESIGN AND METHODS We investigated the effects of deleting or blocking the A2BR on insulin sensitivity using glucose tolerance tests (GTTs) and hyperinsulinemic-euglycemic clamps in mouse models of type 2 diabetes. The effects of diabetes on A2BR transcription and signaling were measured in human and mouse macrophages and mouse endothelial cells. In addition, tag single nucleotide polymorphisms (SNPs) in ∼42 kb encompassing the A2BR gene, ADORA2B, were evaluated for associations with markers of diabetes and inflammation. RESULTS Treatment of mice with the nonselective adenosine receptor agonist 5′-N-ethylcarboxamidoadensoine (NECA) increased fasting blood glucose and slowed glucose disposal during GTTs. These responses were inhibited by A2BR deletion or blockade and minimally affected by deletion of A1Rs or A2ARs. During hyperinsulinemic-euglycemic clamp of diabetic KKAY mice, A2BR antagonism increased glucose infusion rate, reduced hepatic glucose production, and increased glucose uptake into skeletal muscle and brown adipose tissue. Diabetes caused a four- to sixfold increase in A2BR mRNA in endothelial cells and macrophages and resulted in enhanced interleukin (IL)-6 production in response to NECA due to activation of protein kinases A and C. Five consecutive tag SNPs in ADORA2B were highly correlated with IL-6 and C-reactive protein (CRP). Diabetes had a highly significant independent effect on variation in inflammatory markers. The strength of associations between several ADORA2B SNPs and inflammatory markers was increased when accounting for diabetes status. CONCLUSIONS Diabetes affects the production of adenosine and the expression of A2BRs that stimulate IL-6 and CRP production, insulin resistance, and the association between ADORA2B SNPs and inflammatory markers. We hypothesize that increased A2BR signaling in diabetes increases insulin resistance in part by elevating proinflammatory mediators. Selective A2BR blockers may be useful to treat insulin resistance.

P-Selectin–Mediated Platelet-Neutrophil Aggregate Formation Activates Neutrophils in Mouse and Human Sickle Cell Disease*

Objective—To determine the role of platelets in stimulating mouse and human neutrophil activation and pulmonary injury in sickle cell disease (SCD). Methods and Results—Both platelet and neutrophil activation occur in SCD, but the interdependence of these events is unknown. Platelet activation and binding to leukocytes were measured in mice and patients with SCD and in controls. Relative to controls, blood obtained from mice or patients with SCD contained significantly elevated platelet-neutrophil aggregates (PNAs). Both platelets and neutrophils found in sickle PNAs were activated. Multispectral imaging (ImageStream) and conventional flow cytometry revealed a subpopulation of activated neutrophils with multiple adhered platelets that expressed significantly more CD11b and exhibited greater oxidative activity than single neutrophils. On average, wild-type and sickle PNAs contained 1.1 and 2.6 platelets per neutrophil, respectively. Hypoxia/reoxygenation induced a further increase in PNAs in mice with SCD and additional activation of both platelets and neutrophils. The pretreatment of mice with SCD with clopidogrel or P-selectin antibody reduced the formation of PNAs and neutrophil activation and decreased lung vascular permeability. Conclusion—Our findings suggest that platelet binding activates neutrophils and contributes to a chronic inflammatory state and pulmonary dysfunction in SCD. The inhibition of platelet activation may be useful to decrease tissue injury in SCD, particularly during the early stages of vaso-occlusive crises.

The A2B Adenosine Receptor Impairs the Maturation and Immunogenicity of Dendritic Cells

The endogenous purine nucleoside adenosine is an important antiinflammatory mediator that contributes to the control of CD4+ T cell responses. While adenosine clearly has direct effects on CD4+ T cells, it remains to be determined whether actions on APC such as dendritic cells (DC) are also important. In this report we characterize DC maturation and function in BMDC stimulated with LPS in the presence or absence of the nonselective adenosine receptor agonist NECA (5′-N-ethylcarboxamidoadenosine). We found that NECA inhibited TNF-α and IL-12 in a concentration-dependent manner, whereas IL-10 production was increased. NECA-treated BMDC also expressed reduced levels of MHC class II and CD86 and were less effective at stimulating CD4+ T cell proliferation and IL-2 production compared with BMDC exposed to vehicle control. Based on real-time RT-PCR, the A2A adenosine receptor (A2AAR) and A2BAR were the predominant adenosine receptors expressed in BMDC. Using adenosine receptor subtype selective antagonists and BMDC derived from A2AAR−/− and A2BAR−/−mice, it was shown that NECA modulates TNF-α, IL-12, IL-10, and CD86 responses predominantly via A2BAR. These data indicate that engagement of A2BAR modifies murine BMDC maturation and suggest that adenosine regulates CD4+ T cell responses by selecting for DC with impaired immunogencity.

The structure and function of A1 and A2B adenosine receptors.

Of the four G protein coupled adenosine receptor (AR) subtypes, the A1 is best suited for studies of reconstitution with G proteins. Recombinant A1 receptors extended with hexahistidine and FLAG have been purified to near homogeneity. In reconstitution assays using pure recombinant G protein subunits, the composition of the gamma subunit influences coupling to purified A1ARs. The least well characterized AR is the A2B. New data indicate that A(2B)ARs can trigger the degranulation of canine and human mast cell lines. Recombinant human A(2B)ARs are blocked by the anti-asthma drugs theophylline and enprofylline at concentrations that are used therapeutically to treat asthma. Although A(2B)ARs have long been known to stimulate adenylyl cyclase, they also can activate phospholipase C and mobilize Ca2+ by signaling through Gq/11. There is great potential for new therapies based on compounds that selectively target individual AR subtypes.

[17] Expression and purification of G-protein α subunits using baculovirus expression system

Publisher Summary This chapter discusses expression and purification of G-protein α subunits using baculovirus expression system. The chapter describes the construction of recombinant baculovirus vectors encoding four G-protein α subunits (G i1 , G i1 , G i3 , and G o ), infection of Spodoptera frugiperda (Sf9) cells with the viruses to generate large amounts of the recombinant protein, and the chromatographic procedures used to purify the α subunits. The production and purification of recombinant baculoviruses is discussed. Recombinant baculoviruses are obtained by transfecting Sf9 cells with a 5 : 1 (mass/mass) mixture of transfer vector and circular wild-type viral DNA using a calcium phosphate precipitation technique modified for insect cells. In summary, using Sf9 cells infected with recombinant baculoviruses expressing three Gi and the Go a subunits, we have been able to obtain milligram amounts of pure a subunits after three column steps. These α subunits are myristoylated as are native α subunits, and they are functional as measured by the ability to interact with angiotensin II receptors 3 and adenosine receptors. The baculovirus/Sf9 insect cell protein expression system appears to offer a number of advantages for the expression of these G-protein α subunits.

Evidence that the acute phase of ischemic preconditioning does not require signaling by the A2B adenosine receptor

Ischemic preconditioning (IPC) is a protective phenomenon in which brief ischemia renders the myocardium resistant to subsequent ischemic insults. Here, we used A(2B)AR gene knock-out (A(2B)KO)/β-galactosidase reporter gene knock-in mice and the A(2B)AR antagonist ATL-801 to investigate the potential involvement of the A(2B)AR in IPC, focusing on the acute phase of protection. Cardioprotection provided by acute IPC elicited by two 3-min occlusion/3-min reperfusion cycles was readily apparent in an isolated, Langendorff-perfused mouse heart model in studies using hearts from A(2B)KO mice. IPC equivalently improved the recovery of contractile function following 20 min of global ischemia and 45 min of reperfusion in both WT and A(2B)KO hearts by ~30-40%, and equivalently decreased the release of cardiac troponin I during the reperfusion period (from 5969 ± 925 to 1595 ± 674 ng/g and 4376 ± 739 to 2278 ± 462 ng/g using WT and A(2B)KO hearts, respectively). Similarly, the infarct size-reducing capacity of acute IPC in an in vivo model of infarction was fully manifested in experiments using A(2B)KO mice, as well as in experiments using rats pretreated with ATL-801. We did observe, however, a marked reduction in infarct size in rats following administration of the selective A(2B)AR agonist BAY 60-6583 (~25% reduction at a dose of 1.0mg/kg). While supportive of its concept as a cardioprotective receptor, these experiments indicate that the mechanism of the early phase of IPC is not dependent on signaling by the A(2B)AR. We present the idea that the A(2B)AR may contribute to the later stages of IPC dependent on the induction of stress-responsive genes.

Development of an in vitro human liver system for interrogating nonalcoholic steatohepatitis.

A barrier to drug development for nonalcoholic steatohepatitis (NASH) is the absence of translational preclinical human-relevant systems. An in vitro liver model was engineered to incorporate hepatic sinusoidal flow, transport, and lipotoxic stress risk factors (glucose, insulin, free fatty acids) with cocultured primary human hepatocytes, hepatic stellate cells (HSCs), and macrophages. Transcriptomic, lipidomic, and functional endpoints were evaluated and compared with clinical data from NASH patient biopsies. The lipotoxic milieu promoted hepatocyte lipid accumulation (4-fold increase, P < 0.01) and a lipidomics signature similar to NASH biopsies. Hepatocyte glucose output increased with decreased insulin sensitivity. These changes were accompanied by increased inflammatory analyte secretion (e.g., IL-6, IL-8, alanine aminotransferase). Fibrogenic activation markers increased with lipotoxic conditions, including secreted TGF-β (>5-fold increase, P < 0.05), extracellular matrix gene expression, and HSC activation. Significant pathway correlation existed between this in vitro model and human biopsies. Consistent with clinical trial data, 0.5 μM obeticholic acid in this model promoted a healthy lipidomic signature, reduced inflammatory and fibrotic secreted factors, but also increased ApoB secretion, suggesting a potential adverse effect on lipoprotein metabolism. Lipotoxic stress activates similar biological signatures observed in NASH patients in this system, which may be relevant for interrogating novel therapeutic approaches to treat NASH.

Effects of adenosine A2A receptor activation and alanyl-glutamine in Clostridium difficile toxin-induced ileitis in rabbits and cecitis in mice

BackgroundSevere Clostridium difficile toxin-induced enteritis is characterized by exuberant intestinal tissue inflammation, epithelial disruption and diarrhea. Adenosine, through its action on the adenosine A2A receptor, prevents neutrophillic adhesion and oxidative burst and inhibits inflammatory cytokine production. Alanyl-glutamine enhances intestinal mucosal repair and decreases apoptosis of enterocytes. This study investigates the protection from enteritis by combination therapy with ATL 370, an adenosine A2A receptor agonist, and alanyl-glutamine in a rabbit and murine intestinal loop models of C. difficile toxin A-induced epithelial injury.MethodsToxin A with or without alanyl-glutamine was administered intraluminally to rabbit ileal or murine cecal loops. Animals were also given either PBS or ATL 370 parenterally. Ileal tissues were examined for secretion, histopathology, apoptosis, Cxcl1/KC and IL-10.ResultsATL 370 decreased ileal secretion and histopathologic changes in loops treated with Toxin A. These effects were reversed by the A2A receptor antagonist, SCH 58261, in a dose-dependent manner. The combination of ATL 370 and alanyl-glutamine significantly further decreased ileal secretion, mucosal injury and apoptosis more than loops treated with either drug alone. ATL 370 and alanyl-glutamine also decreased intestinal tissue KC and IL-10.ConclusionsCombination therapy with an adenosine A2A receptor agonist and alanyl-glutamine is effective in reversing C. difficile toxin A-induced epithelial injury, inflammation, secretion and apoptosis in animals and has therapeutic potential for the management of C. difficile infection.

The Second Extracellular Loop of the Adenosine A1 Receptor Mediates Activity of Allosteric Enhancers

Allosteric enhancers of the adenosine A1 receptor amplify signaling by orthosteric agonists. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied by an agonist, thereby conferring specificity to stressed or injured tissues that produce adenosine. To explore the mechanism of allosteric enhancer activity, we examined their action on several A1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants, and (4) site-specific mutants. These findings were combined with homology modeling of the A1 receptor and in silico screening of an allosteric enhancer library. The binding modes of known docked allosteric enhancers correlated with the known structure-activity relationship, suggesting that these allosteric enhancers bind to a pocket formed by the second extracellular loop, flanked by residues S150 and M162. We propose a model in which this vestibule controls the entry and efflux of agonists from the orthosteric site and agonist binding elicits a conformational change that enables allosteric enhancer binding. This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists.

Contribution of Adenosine A2B Receptors in Clostridium difficile Intoxication and Infection

ABSTRACT Clostridium difficile toxins A (TcdA) and B (TcdB) induce a pronounced systemic and intestinal inflammatory response. A2B adenosine receptors (A2BARs) are the predominant adenosine receptors in the intestinal epithelium. We investigated whether A2BARs are upregulated in human intestinal cells by TcdA or TcdB and whether blockade of A2BARs can ameliorate C. difficile TcdA-induced enteritis and alter the outcome of C. difficile infection (CDI). Adenosine receptor subtype (A1, A2A, A2B, and A3) mRNAs were assayed in HCT-8 cells. Ileal loops from wild-type rabbits and mice and A2BAR−/− mice were treated with TcdA, with or without the selective A2BAR antagonist ATL692 or PSB1115. A murine model of CDI was used to determine the effect of A2BAR deletion or blockade with the orally available agent ATL801, on clinical outcome, histopathology and intestinal interleukin-6 (IL-6) expression from infection. TcdA and TcdB upregulated A2BAR gene expression in HCT-8 cells. ATL692 decreased TcdA-induced secretion and epithelial injury in rabbit ileum. Deletion of A2BARs reduced secretion and histopathology in TcdA-challenged mouse ileum. Deletion or blockade of A2BARs reduced histopathology, IL-6 expression, weight loss, diarrhea, and mortality in C. difficile-infected mice. A2BARs mediate C. difficile toxin-induced enteritis and disease. Inhibition of A2BAR activation may be a potential strategy to limit morbidity and mortality from CDI.