Metformin, Empagliflozin, and Their Combination Modulate Ex-Vivo Macrophage Inflammatory Gene Expression.

Type-2 Diabetes Mellitus is a complex, chronic illness characterized by persistent high blood glucose levels. Patients can be prescribed anti-diabetes drugs as single agents or in combination depending on the severity of their condition. Metformin and empagliflozin are two commonly prescribed anti-diabetes drugs which reduce hyperglycemia, however their direct effects on macrophage inflammatory responses alone or in combination are unreported. Here, we show that metformin and empagliflozin elicit proinflammatory responses on mouse bone-marrow-derived macrophages with single agent challenge, which are modulated when added in combination. In silico docking experiments suggested that empagliflozin can interact with both TLR2 and DECTIN1 receptors, and we observed that both empagliflozin and metformin increase expression of Tlr2 and Clec7a. Thus, findings from this study suggest that metformin and empagliflozin as single agents or in combination can directly modulate inflammatory gene expression in macrophages and upregulate the expression of their receptors.

A G1-like state allows HIV-1 to bypass SAMHD1 restriction in macrophages.

An unresolved question is how HIV-1 achieves efficient replication in terminally differentiated macrophages despite the restriction factor SAMHD1. We reveal inducible changes in expression of cell cycle-associated proteins including MCM2 and cyclins A, E, D1/D3 in macrophages, without evidence for DNA synthesis or mitosis. These changes are induced by activation of the Raf/MEK/ERK kinase cascade, culminating in upregulation of CDK1 with subsequent SAMHD1 T592 phosphorylation and deactivation of its antiviral activity. HIV infection is limited to these G1-like phase macrophages at the single-cell level. Depletion of SAMHD1 in macrophages decouples the association between infection and expression of cell cycle-associated proteins, with terminally differentiated macrophages becoming highly susceptible to HIV-1. We observe both embryo-derived and monocyte-derived tissue-resident macrophages in a G1-like phase at frequencies approaching 20%, suggesting how macrophages sustain HIV-1 replication in vivo Finally, we reveal a SAMHD1-dependent antiretroviral activity of histone deacetylase inhibitors acting via p53 activation. These data provide a basis for host-directed therapeutic approaches aimed at limiting HIV-1 burden in macrophages that may contribute to curative interventions.

Selective Enhancement of Insulin Sensitivity in the Endothelium In Vivo Reveals a Novel Proatherosclerotic Signaling Loop.

RATIONALE: In the endothelium, insulin stimulates endothelial NO synthase (eNOS) to generate the antiatherosclerotic signaling radical NO. Insulin-resistant type 2 diabetes mellitus is associated with reduced NO availability and accelerated atherosclerosis. The effect of enhancing endothelial insulin sensitivity on NO availability is unclear. OBJECTIVE: To answer this question, we generated a mouse with endothelial cell (EC)-specific overexpression of the human insulin receptor (hIRECO) using the Tie2 promoter-enhancer. METHODS AND RESULTS: hIRECO demonstrated significant endothelial dysfunction measured by blunted endothelium-dependent vasorelaxation to acetylcholine, which was normalized by a specific Nox2 NADPH oxidase inhibitor. Insulin-stimulated phosphorylation of protein kinase B was increased in hIRECO EC as was Nox2 NADPH oxidase-dependent generation of superoxide, whereas insulin-stimulated and shear stress-stimulated eNOS activations were blunted. Phosphorylation at the inhibitory residue Y657 of eNOS and expression of proline-rich tyrosine kinase 2 that phosphorylates this residue were significantly higher in hIRECO EC. Inhibition of proline-rich tyrosine kinase 2 improved insulin-induced and shear stress-induced eNOS activation in hIRECO EC. CONCLUSIONS: Enhancing insulin sensitivity specifically in EC leads to a paradoxical decline in endothelial function, mediated by increased tyrosine phosphorylation of eNOS and excess Nox2-derived superoxide. Increased EC insulin sensitivity leads to a proatherosclerotic imbalance between NO and superoxide. Inhibition of proline-rich tyrosine kinase 2 restores insulin-induced and shear stress-induced NO production. This study demonstrates for the first time that increased endothelial insulin sensitivity leads to a proatherosclerotic imbalance between NO and superoxide.

Restoring Akt1 activity in outgrowth endothelial cells from South Asian men rescues vascular reparative potential.

Recent data suggest reduced indices of vascular repair in South Asian men, a group at increased risk of cardiovascular events. Outgrowth endothelial cells (OEC) represent an attractive tool to study vascular repair in humans and may offer potential in cell-based repair therapies. We aimed to define and manipulate potential mechanisms of impaired vascular repair in South Asian (SA) men. In vitro and in vivo assays of vascular repair and angiogenesis were performed using OEC derived from SA men and matched European controls, prior defining potentially causal molecular mechanisms. SA OEC exhibited impaired colony formation, migration, and in vitro angiogenesis, associated with decreased expression of the proangiogenic molecules Akt1 and endothelial nitric oxide synthase (eNOS). Transfusion of European OEC into immunodeficient mice after wire-induced femoral artery injury augmented re-endothelialization, in contrast with SA OEC and vehicle; SA OEC also failed to promote angiogenesis after induction of hind limb ischemia. Expression of constitutively active Akt1 (E17KAkt), but not green fluorescent protein control, in SA OEC increased in vitro angiogenesis, which was abrogated by a NOS antagonist. Moreover, E17KAkt expressing SA OEC promoted re-endothelialization of wire-injured femoral arteries, and perfusion recovery of ischemic limbs, to a magnitude comparable with nonmanipulated European OEC. Silencing Akt1 in European OEC recapitulated the functional deficits noted in SA OEC. Reduced signaling via the Akt/eNOS axis is causally linked with impaired OEC-mediated vascular repair in South Asian men. These data prove the principle of rescuing marked reparative dysfunction in OEC derived from these men.

The Gene Ontology knowledgebase in 2023.

The Gene Ontology (GO) knowledgebase (http://geneontology.org) is a comprehensive resource concerning the functions of genes and gene products (proteins and noncoding RNAs). GO annotations cover genes from organisms across the tree of life as well as viruses, though most gene function knowledge currently derives from experiments carried out in a relatively small number of model organisms. Here, we provide an updated overview of the GO knowledgebase, as well as the efforts of the broad, international consortium of scientists that develops, maintains, and updates the GO knowledgebase. The GO knowledgebase consists of three components: (1) the GO-a computational knowledge structure describing the functional characteristics of genes; (2) GO annotations-evidence-supported statements asserting that a specific gene product has a particular functional characteristic; and (3) GO Causal Activity Models (GO-CAMs)-mechanistic models of molecular "pathways" (GO biological processes) created by linking multiple GO annotations using defined relations. Each of these components is continually expanded, revised, and updated in response to newly published discoveries and receives extensive QA checks, reviews, and user feedback. For each of these components, we provide a description of the current contents, recent developments to keep the knowledgebase up to date with new discoveries, and guidance on how users can best make use of the data that we provide. We conclude with future directions for the project.

Liver macrophages and inflammation in physiology and physiopathology of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome, being a common comorbidity of type 2 diabetes and with important links to inflammation and insulin resistance. NAFLD represents a spectrum of liver conditions ranging from steatosis in the form of ectopic lipid storage, to inflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Macrophages that populate the liver play important roles in maintaining liver homeostasis under normal physiology and in promoting inflammation and mediating fibrosis in the progression of NAFLD toward to NASH. Liver macrophages are a heterogenous group of innate immune cells, originating from the yolk sac or from circulating monocytes, that are required to maintain immune tolerance while being exposed portal and pancreatic blood flow rich in nutrients and hormones. Yet, liver macrophages retain a limited capacity to raise the alarm in response to danger signals. We now know that macrophages in the liver play both inflammatory and noninflammatory roles throughout the progression of NAFLD. Macrophage responses are mediated first at the level of cell surface receptors that integrate environmental stimuli, signals are transduced through multiple levels of regulation in the cell, and specific transcriptional programmes dictate effector functions. These effector functions play paramount roles in determining the course of disease in NAFLD and even more so in the progression towards NASH. The current review covers recent reports in the physiological and pathophysiological roles of liver macrophages in NAFLD. We emphasise the responses of liver macrophages to insulin resistance and the transcriptional machinery that dictates liver macrophage function.

Metabolic Hormones Modulate Macrophage Inflammatory Responses.

Macrophages are phagocytotic leukocytes that play an important role in the innate immune response and have established roles in metabolic diseases and cancer progression. Increased adiposity in obese individuals leads to dysregulation of many hormones including those whose functions are to coordinate metabolism. Recent evidence suggests additional roles of these metabolic hormones in modulating macrophage inflammatory responses. In this review, we highlight key metabolic hormones and summarise their influence on the inflammatory response of macrophages and consider how, in turn, these hormones may influence the development of different cancer types through the modulation of macrophage functions.

LXRα Phosphorylation in Cardiometabolic Disease: Insight From Mouse Models.

Posttranslational modifications, such as phosphorylation, are a powerful means by which the activity and function of nuclear receptors such as LXRα can be altered. However, despite the established importance of nuclear receptors in maintaining metabolic homeostasis, our understanding of how phosphorylation affects metabolic diseases is limited. The physiological consequences of LXRα phosphorylation have, until recently, been studied only in vitro or nonspecifically in animal models by pharmacologically or genetically altering the enzymes enhancing or inhibiting these modifications. Here we review recent reports on the physiological consequences of modifying LXRα phosphorylation at serine 196 (S196) in cardiometabolic disease, including nonalcoholic fatty liver disease, atherosclerosis, and obesity. A unifying theme from these studies is that LXRα S196 phosphorylation rewires the LXR-modulated transcriptome, which in turn alters physiological response to environmental signals, and that this is largely distinct from the LXR-ligand-dependent action.

Measuring Apoptotic Cell Engulfment (Efferocytosis) Efficiency.

Efferocytosis is the process of recognizing and removing dead and dying cells, performed by a variety of phagocytic cells including macrophages. It has recently been shown that liver X receptor (LXR) signaling in macrophages regulates the expression of important efferocytosis receptors, bridging and signaling molecules. Here we describe a sensitive yet robust efferocytosis assay, optimized to measure bone marrow-derived macrophage (BMDM) apoptotic cell engulfment capability. This assay can be applied to genetically or pharmacologically altered BMDMs.

Impaired LXRα Phosphorylation Attenuates Progression of Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is a very common indication for liver transplantation. How fat-rich diets promote progression from fatty liver to more damaging inflammatory and fibrotic stages is poorly understood. Here, we show that disrupting phosphorylation at Ser196 (S196A) in the liver X receptor alpha (LXRα, NR1H3) retards NAFLD progression in mice on a high-fat-high-cholesterol diet. Mechanistically, this is explained by key histone acetylation (H3K27) and transcriptional changes in pro-fibrotic and pro-inflammatory genes. Furthermore, S196A-LXRα expression reveals the regulation of novel diet-specific LXRα-responsive genes, including the induction of Ces1f, implicated in the breakdown of hepatic lipids. This involves induced H3K27 acetylation and altered LXR and TBLR1 cofactor occupancy at the Ces1f gene in S196A fatty livers. Overall, impaired Ser196-LXRα phosphorylation acts as a novel nutritional molecular sensor that profoundly alters the hepatic H3K27 acetylome and transcriptome during NAFLD progression placing LXRα phosphorylation as an alternative anti-inflammatory or anti-fibrotic therapeutic target.

Posttranslational Modifications of Lipid-Activated Nuclear Receptors: Focus on Metabolism.

Posttranslational modifications (PTMs) occur to nearly all proteins, are catalyzed by specific enzymes, and are subjected to tight regulation. They have been shown to be a powerful means by which the function of proteins can be modified, resulting in diverse effects. Technological advances such as the increased sensitivity of mass spectrometry-based techniques and availability of mutant animal models have enhanced our understanding of the complexities of their regulation and the effect they have on protein function. However, the role that PTMs have in a pathological context still remains unknown for the most part. PTMs enable the modulation of nuclear receptor function in a rapid and reversible manner in response to varied stimuli, thereby dramatically altering their activity in some cases. This review focuses on acetylation, phosphorylation, SUMOylation, and O-GlcNAcylation, which are the 4 most studied PTMs affecting lipid-regulated nuclear receptor biology, as well as on the implications of such modifications on metabolic pathways under homeostatic and pathological situations. Moreover, we review recent studies on the modulation of PTMs as therapeutic targets for metabolic diseases.

Endothelial SHIP2 Suppresses Nox2 NADPH Oxidase-Dependent Vascular Oxidative Stress, Endothelial Dysfunction, and Systemic Insulin Resistance.

Shc homology 2-containing inositol 5' phosphatase-2 (SHIP2) is a lipid phosphatase that inhibits insulin signaling downstream of phosphatidylinositol 3-kinase (PI3K); its role in vascular function is poorly understood. To examine its role in endothelial cell (EC) biology, we generated mice with catalytic inactivation of one SHIP2 allele selectively in ECs (ECSHIP2Δ/+). Hyperinsulinemic-euglycemic clamping studies revealed that ECSHIP2Δ/+ was resistant to insulin-stimulated glucose uptake in adipose tissue and skeletal muscle compared with littermate controls. ECs from ECSHIP2Δ/+ mice had increased basal expression and activation of PI3K downstream targets, including Akt and endothelial nitric oxide synthase, although incremental activation by insulin and shear stress was impaired. Insulin-mediated vasodilation was blunted in ECSHIP2Δ/+ mice, as was aortic nitric oxide bioavailability. Acetylcholine-induced vasodilation was also impaired in ECSHIP2Δ/+ mice, which was exaggerated in the presence of a superoxide dismutase/catalase mimetic. Superoxide abundance was elevated in ECSHIP2Δ/+ ECs and was suppressed by PI3K and NADPH oxidase 2 inhibitors. These findings were phenocopied in healthy human ECs after SHIP2 silencing. Our data suggest that endothelial SHIP2 is required to maintain normal systemic glucose homeostasis and prevent oxidative stress-induced endothelial dysfunction.

Luciferase Reporter Assays to Assess Liver X Receptor Transcriptional Activity.

Luciferase reporter assays are sensitive and accurate tests that enable the analysis of regulatory sequences, the magnitude of transcriptional activity by transcription factors, and the discovery of gene regulatory elements and small-molecule modulators with high levels of precision. This is made possible through detection of bioluminescence produced by luciferase-coding reporters in a wide range of cellular environments. These assays are routinely used to analyze the activity of transcription factors, including the lipid-activated liver X receptor (LXR), in response to different stimuli as well as for the identification of their ligands. In this chapter we describe in detail the assays performed to investigate LXR activity in a macrophage-like cell line (RAW 267.4). These can be easily adapted to other nuclear receptors and transcription factors.

The nuclear receptor LXR modulates interleukin-18 levels in macrophages through multiple mechanisms.

IL-18 is a member of the IL-1 family involved in innate immunity and inflammation. Deregulated levels of IL-18 are involved in the pathogenesis of multiple disorders including inflammatory and metabolic diseases, yet relatively little is known regarding its regulation. Liver X receptors or LXRs are key modulators of macrophage cholesterol homeostasis and immune responses. Here we show that LXR ligands negatively regulate LPS-induced mRNA and protein expression of IL-18 in bone marrow-derived macrophages. Consistent with this being an LXR-mediated process, inhibition is abolished in the presence of a specific LXR antagonist and in LXR-deficient macrophages. Additionally, IL-18 processing of its precursor inactive form to its bioactive state is inhibited by LXR through negative regulation of both pro-caspase 1 expression and activation. Finally, LXR ligands further modulate IL-18 levels by inducing the expression of IL-18BP, a potent endogenous inhibitor of IL-18. This regulation occurs via the transcription factor IRF8, thus identifying IL-18BP as a novel LXR and IRF8 target gene. In conclusion, LXR activation inhibits IL-18 production through regulation of its transcription and maturation into an active pro-inflammatory cytokine. This novel regulation of IL-18 by LXR could be applied to modulate the severity of IL-18 driven metabolic and inflammatory disorders.

In silico design and biological evaluation of a dual specificity kinase inhibitor targeting cell cycle progression and angiogenesis.

BACKGROUND: Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues. METHODOLOGY: We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) and cyclin-dependent kinase 1 (CDK1). This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A)-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis. CONCLUSIONS: We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.

Endothelium-specific insulin resistance leads to accelerated atherosclerosis in areas with disturbed flow patterns: a role for reactive oxygen species.

OBJECTIVE: Systemic insulin resistance is associated with a portfolio of risk factors for atherosclerosis development. We sought to determine whether insulin resistance specifically at the level of the endothelium promotes atherosclerosis and to examine the potential involvement of reactive oxygen species. METHODS: We cross-bred mice expressing a dominant negative mutant human insulin receptor specifically in the endothelium (ESMIRO) with ApoE(-/-) mice to examine the effect of endothelium-specific insulin resistance on atherosclerosis. RESULTS: ApoE(-/-)/ESMIRO mice had similar blood pressure, plasma lipids and whole-body glucose tolerance, but blunted endothelial insulin signalling, in comparison to ApoE(-/-) mice. Atherosclerosis was significantly increased in ApoE(-/-)/ESMIRO mice at the aortic sinus (226 ± 16 versus 149 ± 24 × 10(3) µm(2), P = 0.01) and lesser curvature of the aortic arch (12.4 ± 1.2% versus 9.4 ± 0.9%, P = 0.035). Relaxation to acetylcholine was blunted in aorta from ApoE(-/-)/ESMIRO mice (Emax 65 ± 41% versus 103 ± 6%, P = 0.02) and was restored by the superoxide dismutase mimetic MnTMPyP (Emax 112 ± 15% versus 65 ± 41%, P = 0.048). Basal generation of superoxide was increased 1.55 fold (P = 0.01) in endothelial cells from ApoE(-/-)/ESMIRO mice and was inhibited by the NADPH oxidase inhibitor gp91ds-tat (-12 ± 0.04%, P = 0.04), the NO synthase inhibitor L-NMMA (-8 ± 0.02%, P = 0.001) and the mitochondrial specific inhibitor rotenone (-23 ± 0.04%, P = 0.006). CONCLUSIONS: Insulin resistance specifically at the level of the endothelium leads to acceleration of atherosclerosis in areas with disturbed flow patterns such as the aortic sinus and the lesser curvature of the aorta. We have identified a potential role for increased generation of reactive oxygen species from multiple enzymatic sources in promoting atherosclerosis in this setting.

Proteomic analysis of IgE-mediated secretion by LAD2 mast cells.

The LAD2 cell line is a relatively recent addition to the range of mast cell analogues and is of particular importance as it is the only human analogue which can be stimulated to degranulate in an IgE-dependent manner. Mast cells are tissue-based effector cells which have historically been shown to play an important role in the adaptive immune response, though there is now gathering evidence of their significance as a component of the innate immune system. These functions can be attributed to the ability of mast cells to regulate secretion of a wide variety of potent biologically active mediators through immediate and delayed responses. This well-orchestrated secretory mechanism of the mast cell makes it an ideal model in which to study this event. In this investigation, two-dimensional electrophoresis was employed as part of the proteomic characterization of the LAD2 human mast cell line, focusing in particular on a global analysis of membrane protein relocation after an IgE-mediated stimulatory event. This investigation has identified six membrane-associated protein spots which became phosphorylated upon IgE-mediated activation, 31 protein spots which displayed consistent recruitment to the membrane fraction, and three which were consistently lost from the soluble fraction. The scenario which emerges reveals a series of substantial changes which affect every compartment of the cell, providing evidence for a coordinated response to a secretory stimulus.

Proteomic screening of a cell line model of esophageal carcinogenesis identifies cathepsin D and aldo-keto reductase 1C2 and 1B10 dysregulation in Barrett's esophagus and esophageal adenocarcinoma.

Esophageal adenocarcinoma (EA) incidence is increasing rapidly and is associated with a poor prognosis. Identifying biomarkers of disease development and progression would be invaluable tools to inform clinical practice. Two-dimensional polyacrylamide gel electrophoresis was used to screen 10 esophageal cell lines representing distinct stages in the development of esophageal cancer. Thirty-three proteins were identified by MALDI-TOF-MS which demonstrated differences in expression across the cell lines. Western blotting and qRT-PCR confirmed increased cathepsin D and aldo-keto reductases 1C2 and 1B10 expression in metaplastic and dysplastic cell lines. Expression of these proteins was further assessed in esophageal epithelium from patients with nonerosive (NERD) and erosive gastro-esophageal reflux disease, Barrett's esophagus (BE) and EA. When compared with normal epithelium of NERD patients, (i) cathepsin D mRNA levels demonstrated a stepwise increase in expression (p<0.05) in erosive, metaplastic and EA tissue; (ii) AKR1B10 expression increased (p<0.05) 3- and 9-fold in erosive and Barrett's epithelium, respectively; and (iii) AKR1C2 levels increased (p<0.05) in erosive and Barrett's epithelium, but were reduced (p<0.05) in EA. These proteins may contribute to disease development via effects on apoptosis, transport of bile acids and retinoid metabolism and should be considered as candidates for further mechanistic and clinical investigations.