Chanzyme TRPM7 Mediates the Ca2+ Influx Essential for Lipopolysaccharide-Induced Toll-Like Receptor 4 Endocytosis and Macrophage Activation.

Toll-like receptors (TLRs) sense pathogen-associated molecular patterns to activate the production of inflammatory mediators. TLR4 recognizes lipopolysaccharide (LPS) and drives the secretion of inflammatory cytokines, often contributing to sepsis. We report that transient receptor potential melastatin-like 7 (TRPM7), a non-selective but Ca2+-conducting ion channel, mediates the cytosolic Ca2+ elevations essential for LPS-induced macrophage activation. LPS triggered TRPM7-dependent Ca2+ elevations essential for TLR4 endocytosis and the subsequent activation of the transcription factor IRF3. In a parallel pathway, the Ca2+ signaling initiated by TRPM7 was also essential for the nuclear translocation of NFκB. Consequently, TRPM7-deficient macrophages exhibited major deficits in the LPS-induced transcriptional programs in that they failed to produce IL-1ß and other key pro-inflammatory cytokines. In accord with these defects, mice with myeloid-specific deletion of Trpm7 are protected from LPS-induced peritonitis. Our study highlights the importance of Ca2+ signaling in macrophage activation and identifies the ion channel TRPM7 as a central component of TLR4 signaling.

Decreased sarcoplasmic reticulum phospholipids in human skeletal muscle are associated with metabolic syndrome.

Metabolic syndrome affects more than one in three adults and is associated with increased risk of diabetes, cardiovascular disease, and all-cause mortality. Muscle insulin resistance is a major contributor to the development of the metabolic syndrome. Studies in mice have linked skeletal muscle sarcoplasmic reticulum (SR) phospholipid composition to sarcoplasmic/endoplasmic reticulum Ca2+-ATPase activity and insulin sensitivity. To determine if the presence of metabolic syndrome alters specific phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species in human SR, we compared SR phospholipid composition in skeletal muscle from sedentary subjects with metabolic syndrome and sedentary control subjects without metabolic syndrome. Both total PC and total PE were significantly decreased in skeletal muscle SR of sedentary metabolic syndrome patients compared with sedentary controls, particularly in female participants, but there was no difference in the PC:PE ratio between groups. Total SR PC levels, but not total SR PE levels or PC:PE ratio, were significantly negatively correlated with BMI, waist circumference, total fat, visceral adipose tissue, triglycerides, fasting insulin, and homeostatic model assessment for insulin resistance. These findings are consistent with the existence of a relationship between skeletal muscle SR PC content and insulin resistance in humans.

Deficiency of Dab2 (Disabled Homolog 2) in Myeloid Cells Exacerbates Inflammation in Liver and Atherosclerotic Plaques in LDLR (Low-Density Lipoprotein Receptor)-Null Mice-Brief Report.

OBJECTIVE: Inflammatory macrophages promote the development of atherosclerosis. We have identified the adaptor protein Dab2 (disabled homolog 2) as a regulator of phenotypic polarization in macrophages. The absence of Dab2 in myeloid cells promotes an inflammatory phenotype, but the impact of myeloid Dab2 deficiency on atherosclerosis has not been shown. APPROACH AND RESULTS: To determine the role of myeloid Dab2 in atherosclerosis, Ldlr-/- mice were reconstituted with either Dab2-positive or Dab2-deficient bone marrow and fed a western diet. Consistent with our previous finding that Dab2 inhibits NFκB (nuclear factor κ-light-chain-enhancer of activated B cells) signaling in macrophages, Ldlr-/- mice reconstituted with Dab2-deficient bone marrow had increased systemic inflammation as evidenced by increased serum IL-6 (interleukin-6) levels and increased inflammatory cytokine expression levels in liver. Serum lipid levels were significantly lower in Ldlr-/- mice reconstituted with Dab2-deficient bone marrow, and further examination of livers from these mice revealed drastically increased inflammatory tissue damage and massive infiltration of immune cells. Surprisingly, the atherosclerotic lesion burden in Ldlr-/- mice reconstituted with Dab2-deficient bone marrow was decreased compared with Ldlr-/- mice reconstituted with wild-type bone marrow. Further analysis of aortic root sections revealed increased macrophage content and evidence of increased apoptosis in lesions from Ldlr-/- mice reconstituted with Dab2-deficient bone marrow but no difference in collagen or α-smooth muscle actin content. CONCLUSIONS: Dab2 deficiency in myeloid cells promotes inflammation in livers and atherosclerotic plaques in a mouse model of atherosclerosis. Nevertheless, decreased serum lipids as a result of massive inflammatory liver damage may preclude an appreciable increase in atherosclerotic lesion burden in mice reconstituted with Dab2-deficient bone marrow.

Myeloid P2Y2 receptor promotes acute inflammation but is dispensable for chronic high-fat diet-induced metabolic dysfunction.

The purinergic receptor P2Y2 binds ATP to control chemotaxis of myeloid cells, and global P2Y2 receptor knockout mice are protected in models of acute inflammation. Chronic inflammation mediated by macrophages and other immune cells in adipose tissue contributes to the development of insulin resistance. Here, we investigate whether mice lacking P2Y2 receptors on myeloid cells are protected against acute and chronic inflammation. Wild-type mice were transplanted with either wild-type or P2Y2 receptor null bone marrow and treated with a sublethal dose of endotoxin as a model of acute inflammation, or fed a high-fat diet to induce obesity and insulin resistance as a model of chronic inflammation. P2Y2-/- chimeric mice were protected against acute inflammation. However, high-fat diet feeding induced comparable inflammation and insulin resistance in both WT and P2Y2-/- chimeric mice. Of note, confocal microscopy revealed significantly fewer crown-like structures, assemblies of macrophages around adipocytes, in P2Y2-/- chimeric mice compared to WT chimeric mice. We conclude that P2Y2 receptors on myeloid cells are important in mediating acute inflammation but are dispensable for the development of whole body insulin resistance in diet-induced obese mice.

Paracrine Signaling by Pancreatic Islet Cilia.

The primary cilium is a sensory and signaling organelle present on most pancreatic islet endocrine cells, where it receives and interprets a wide range of intra-islet chemical cues including hormones, peptides, and neurotransmitters. The ciliary membrane possesses a molecular composition distinct from the plasma membrane, with enrichment of signaling mediators including G protein-coupled receptors (GPCRs), tyrosine kinase family receptors, membrane transporters and others. When activated, these membrane proteins interact with ion channels and adenylyl cyclases to trigger local Ca2+ and cAMP activity and transmit signals to the cell body. Here we review evidence supporting the emerging model in which primary cilia on pancreatic islet cells play a central role in the intra-islet communication network and discuss how changes in cilia-mediated paracrine function in islet cells might lead to diabetes.

Beta cell primary cilia mediate somatostatin responsiveness via SSTR3.

Somatostatin is a paracrine modulator of insulin secretion and beta cell function with pleotropic effects on glucose homeostasis. The mechanism of somatostatin-mediated communication between delta and beta cells is not well-understood, which we address in this study via the ciliary somatostatin receptor 3 (SSTR3). Primary cilia are membrane organelles that act as signaling hubs in islets by virtue of their subcellular location and enrichment in signaling proteins such as G-protein coupled receptors (GPCRs). We show that SSTR3, a ciliary GPCR, mediates somatostatin suppression of insulin secretion in mouse islets. Quantitative analysis of calcium flux using a mouse model of genetically encoded beta cell-specific GCaMP6f calcium reporter shows that somatostatin signaling alters beta cell calcium flux after physiologic glucose stimulation, an effect that depends on endogenous SSTR3 expression and the presence of intact primary cilia on beta cells. Comparative in vitro studies using SSTR isoform antagonists demonstrate a role for SSTR3 in mediating somatostatin regulation of insulin secretion in mouse islets. Our findings support a model in which ciliary SSTR3 mediates a distinct pathway of delta-to-beta cell regulatory crosstalk and may serve as a target for paracrine modulation.

Homogeneous catalytic system for photoinduced hydrogen production utilizing iridium and rhodium complexes.

An efficient homogeneous catalytic system for the visible-light-induced production of hydrogen from water utilizing cyclometalated iridium(III) and tris-2,2'-bipyridyl rhodium(III) complexes is described. Synthetic modification of the photosensitizer Ir(C--N) 2(N--N) (+) and water reduction catalyst Rh(N--N) 3 (3+) creates a family of catalysts with diverse photophysical and electrochemical properties. Parallel screening of the various catalyst combinations and photoreaction conditions allows the rapid development of an optimized photocatalytic system that achieves over 5000 turnovers with quantum yields ( (1)/ 2 H 2 per photon absorbed) greater than 34%. Photophysical and electrochemical characterization of the optimized system reveals that the reductive quenching pathway provides the necessary driving force for the formation of [Rh(N--N) 2] (0), the active catalytic species for the reduction of water to produce hydrogen. Tests for system poisoning with mercury or CS 2 provide strong evidence that the system is a true homogeneous system for photocatalytic hydrogen production.

Disabled homolog 2 controls macrophage phenotypic polarization and adipose tissue inflammation.

Acute and chronic tissue injury results in the generation of a myriad of environmental cues that macrophages respond to by changing their phenotype and function. This phenotypic regulation is critical for controlling tissue inflammation and resolution. Here, we have identified the adaptor protein disabled homolog 2 (DAB2) as a regulator of phenotypic switching in macrophages. Dab2 expression was upregulated in M2 macrophages and suppressed in M1 macrophages isolated from both mice and humans, and genetic deletion of Dab2 predisposed macrophages to adopt a proinflammatory M1 phenotype. In mice with myeloid cell-specific deletion of Dab2 (Dab2fl/fl Lysm-Cre), treatment with sublethal doses of LPS resulted in increased proinflammatory gene expression and macrophage activation. Moreover, chronic high-fat feeding exacerbated adipose tissue inflammation, M1 polarization of adipose tissue macrophages, and the development of insulin resistance in DAB2-deficient animals compared with controls. Mutational analyses revealed that DAB2 interacts with TNF receptor-associated factor 6 (TRAF6) and attenuates IκB kinase ß-dependent (IKKß-dependent) phosphorylation of Ser536 in the transactivation domain of NF-κB p65. Together, these findings reveal that DAB2 is critical for controlling inflammatory signaling during phenotypic polarization of macrophages and suggest that manipulation of DAB2 expression and function may hold therapeutic potential for the treatment of acute and chronic inflammatory disorders.

Pannexin 1 is required for full activation of insulin-stimulated glucose uptake in adipocytes.

OBJECTIVE: Defective glucose uptake in adipocytes leads to impaired metabolic homeostasis and insulin resistance, hallmarks of type 2 diabetes. Extracellular ATP-derived nucleotides and nucleosides are important regulators of adipocyte function, but the pathway for controlled ATP release from adipocytes is unknown. Here, we investigated whether Pannexin 1 (Panx1) channels control ATP release from adipocytes and contribute to metabolic homeostasis. METHODS: We assessed Panx1 functionality in cultured 3T3-L1 adipocytes and in adipocytes isolated from murine white adipose tissue by measuring ATP release in response to known activators of Panx1 channels. Glucose uptake in cultured 3T3-L1 adipocytes was measured in the presence of Panx1 pharmacologic inhibitors and in adipocytes isolated from white adipose tissue from wildtype (WT) or adipocyte-specific Panx1 knockout (AdipPanx1 KO) mice generated in our laboratory. We performed in vivo glucose uptake studies in chow fed WT and AdipPanx1 KO mice and assessed insulin resistance in WT and AdipPanx1 KO mice fed a high fat diet for 12 weeks. Panx1 channel function was assessed in response to insulin by performing electrophysiologic recordings in a heterologous expression system. Finally, we measured Panx1 mRNA in human visceral adipose tissue samples by qRT-PCR and compared expression levels with glucose levels and HOMA-IR measurements in patients. RESULTS: Our data show that adipocytes express functional Pannexin 1 (Panx1) channels that can be activated to release ATP. Pharmacologic inhibition or selective genetic deletion of Panx1 from adipocytes decreased insulin-induced glucose uptake in vitro and in vivo and exacerbated diet-induced insulin resistance in mice. Further, we identify insulin as a novel activator of Panx1 channels. In obese humans Panx1 expression in adipose tissue is increased and correlates with the degree of insulin resistance. CONCLUSIONS: We show that Panx1 channel activity regulates insulin-stimulated glucose uptake in adipocytes and thus contributes to control of metabolic homeostasis.

The role of pannexin1 in the induction and resolution of inflammation.

Extracellular ATP is an important signaling molecule throughout the inflammatory cascade, serving as a danger signal that causes activation of the inflammasome, enhancement of immune cell infiltration, and fine-tuning of several signaling cascades including those important for the resolution of inflammation. Recent studies demonstrated that ATP can be released from cells in a controlled manner through pannexin (Panx) channels. Panx1-mediated ATP release is involved in inflammasome activation and neutrophil/macrophage chemotaxis, activation of T cells, and a role for Panx1 in inducing and propagating inflammation has been demonstrated in various organs, including lung and the central and peripheral nervous system. The recognition and clearance of dying cells and debris from focal points of inflammation is critical in the resolution of inflammation, and Panx1-mediated ATP release from dying cells has been shown to recruit phagocytes. Moreover, extracellular ATP can be broken down by ectonucleotidases into ADP, AMP, and adenosine, which is critical in the resolution of inflammation. Together, Panx1, ATP, purinergic receptors, and ectonucleotidases contribute to important feedback loops during the inflammatory response, and thus represent promising candidates for new therapies.