Glucose Metabolism In Macrophages Research Articles

Discovery and functional characterization of LncRNAs associated with inflammation and macrophage activation

Long noncoding RNAs (lncRNA) are emerging players in regulation of gene expression and cell signaling and their dysregulation has been implicated in a multitude of human diseases. Recent studies from our laboratory revealed that lncRNAs play critical roles in cytokine regulation, inflammation, and metabolism. We demonstrated that lncRNA HOTAIR, which is a well-known regulator of gene silencing, plays critical roles in modulation of cytokines and proinflammatory genes, and glucose metabolism in macrophages during inflammation. In addition, we recently discovered a series of novel lncRNAs that are closely associated with inflammation and macrophage activation. We termed these as long-noncoding inflammation associated RNAs (LinfRNAs). We are currently engaged in the functional characterization of these hLinfRNAs (human LinfRNAs) with a focus on their roles in inflammation, and we are investigating their potential implications in chronic inflammatory human diseases. Here, we have summarized experimental methods that have been utilized for the discovery and functional characterization of lncRNAs in inflammation and macrophage activation.

Regulation of macrophage polarization and glucose metabolism by the ERK/MAPK-HK1 signaling pathway in paraquat-induced acute lung injury

Acute lung injury is the leading cause of paraquat (PQ) poisoning-related mortality. The mechanism by which macrophages are involved in PQ-induced acute lung injury remains unclear. In recent years, the role of metabolic reprogramming in macrophage functional transformation has received significant attention. The current study aimed to identify the role of altered macrophage glucose metabolism and molecular mechanisms in PQ poisoning-induced acute lung injury. We established a model of acute lung injury in PQ-intoxicated mice via the intraperitoneal injection of PQ. PQ exposure induces macrophage M1 polarization and promotes the release of inflammatory factors, which causes the development of acute lung injury in mice. In vitro analysis revealed that PQ altered glucose metabolism, which could be reversed by siRNA transfection to silence the expression of HK1, a key enzyme in glucose metabolism. RNA sequencing revealed that the ERK/MAPK pathway was the crucial molecular mechanism of PQ pathogenesis. Further, U0126, an ERK inhibitor, could inhibit PQ-induced HK1 activation and macrophage M1 polarization. These findings provide novel insights into the previously unrecognized mechanism of ERK/MAPK-HK1 activation in PQ poisoning.

Hyperandrogenic environment regulates the function of ovarian granulosa cells by modulating macrophage polarization in PCOS.

Polycystic ovary syndrome (PCOS) is a common endocrine-metabolic disorder characterized by oligo-anovulation, hyperandrogenism, and polycystic ovaries, with hyperandrogenism being the most prominent feature of PCOS patients. However, whether excessive androgens also exist in the ovarian microenvironment of patients with PCOS, and their modulatory role on ovarian immune homeostasis and ovarian function, is not clear. Follicular fluid samples from patients participating in their first in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment were collected. Androgen concentration of follicular fluid was assayed by chemiluminescence, and the macrophage M1:M2 ratio was detected by flow cytometry. In an in vitro model, we examined the regulatory effects of different concentrations of androgen on macrophage differentiation and glucose metabolism levels using qRT-PCR, Simple Western and multi-factor flow cytometry assay. In a co-culture model, we assessed the effect of a hyperandrogenic environment in the presence or absence of macrophages on the function of granulosacells using qRT-PCR, Simple Western, EdU assay, cell cycle assay, and multi-factor flow cytometry assay. The results showed that a significantly higher androgen level and M1:M2 ratio in the follicular fluid of PCOS patients with hyperandrogenism. The hyperandrogenic environment promoted the expression of pro-inflammatory and glycolysis-related molecules and inhibited the expression of anti-inflammatory and oxidative phosphorylation-related molecules in macrophages. In the presence of macrophages, a hyperandrogenic environment significantly downregulated the function of granulosa cells. There is a hyperandrogenic microenvironment in the ovary of PCOS patients with hyperandrogenism. Hyperandrogenic microenvironment can promote the activation of ovarian macrophages to M1, which may be associated with the reprogramming of macrophage glucose metabolism. The increased secretion of pro-inflammatory cytokines by macrophages in the hyperandrogenic microenvironment would impair the normal function of granulosa cells and interfere with normal ovarian follicle growth and development.

Temporal changes in glucose metabolism reflect polarization in resident and monocyte-derived macrophages after myocardial infarction.

Metabolic reprogramming from glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle and oxidative phosphorylation may mediate macrophage polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype. We hypothesized that changes in cardiac macrophage glucose metabolism would reflect polarization status after myocardial infarction (MI), ranging from the early inflammatory phase to the later wound healing phase. MI was induced by permanent ligation of the left coronary artery in adult male C57BL/6J mice for 1 (D1), 3 (D3), or 7 (D7) days. Infarct macrophages were subjected to metabolic flux analysis or gene expression analysis. Monocyte versus resident cardiac macrophage metabolism was assessed using mice lacking the Ccr2 gene (CCR2 KO). By flow cytometry and RT-PCR, D1 macrophages exhibited an M1 phenotype while D7 macrophages exhibited an M2 phenotype. Macrophage glycolysis (extracellular acidification rate) was increased at D1 and D3, returning to basal levels at D7. Glucose oxidation (oxygen consumption rate) was decreased at D3, returning to basal levels at D7. At D1, glycolytic genes were elevated (Gapdh, Ldha, Pkm2), while TCA cycle genes were elevated at D3 (Idh1 and Idh2) and D7 (Pdha1, Idh1/2, Sdha/b). Surprisingly, Slc2a1 and Hk1/2 were increased at D7, as well as pentose phosphate pathway (PPP) genes (G6pdx, G6pd2, Pgd, Rpia, Taldo1), indicating increased PPP activity. Macrophages from CCR2 KO mice showed decreased glycolysis and increased glucose oxidation at D3, and decreases in Ldha and Pkm2 expression. Administration of dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, robustly decreased pyruvate dehydrogenase phosphorylation in the non-infarcted remote zone, but did not affect macrophage phenotype or metabolism in the infarct zone. Our results indicate that changes in glucose metabolism and the PPP underlie macrophage polarization following MI, and that metabolic reprogramming is a key feature of monocyte-derived but not resident macrophages.

Long Non-Coding RNAs Nuclear Paraspeckle Assembly Transcript 1 Antagonist Encapsulated by Liposome Nanoparticles Regulates Macrophage Glucose Metabolism Reprogramming in Ulcerative Colitis

This study explored the mechanism by which Long Non-Coding RNA-Nuclear Enriched Abundant Transcript 1 (LncRNA-NEAT1) antagonists encapsulated in liposome nanoparticles regulate reprogramming of macrophage glucose metabolism in ulcerative colitis (UC) patients. Sprague-Dawley (SD) rats were assigned into blank control group, UC model group, LncRNA-NEAT1 antagonist group (antagonist group) and LncRNA-NEAT1 antagonist encapsulated by liposome nanoparticles group (nano+antagonist group). Rats in the blank control group were not given any intervention, while rats in the UC model group were fully anesthetized to establish a UC model. The antagonist group was given LncRNA-NEAT1 antagonists on the basis of the UC model. Combined intervention with LncRNA-NEAT1 antagonists encapsulated by plastid nanoparticles were given to the nano+antagonist group. LncRNA-NEAT1 antagonist encapsulated by liposome nanoparticles reduced colon mucosal damage index and CMDI) score of UC rats, improving gross morphology damage of mucosa, and also reducing the pathological damage of lung tissue in the rats. The LncRNA-NEAT1 antagonist inhibited reprogramming level of glucose metabolism in macrophages, reduced consumption of cellular glucose, and further reduced lactic acid content in the macrophages. In addition, the LncRNA-NEAT1 antagonist further inhibited the expression of glycolysis-related enzymes in macrophages by inhibiting Rho associated coiled-coil containing protein kinase/Myosin streptophilic kinase (ROCK/MLCK) pathway. LncRNA-NEAT1 has a targeting relationship with ROCK/MLCK pathway. In conclusion, down-regulating NEAT1 inhibited the activity of ROCK/MLCK signaling, reducing inflammatory response, thereby repairing the intestinal barrier function, and inhibiting the reprogramming of glucose metabolism in macrophages from the UC rats. In addition, liposome nanoparticle encapsulation has broad prospects in the field of drug delivery, which can achieve the purpose of enhancing drug efficacy.

Glucose Transporter Glut1-Dependent Metabolic Reprogramming Regulates Lipopolysaccharide-Induced Inflammation in RAW264.7 Macrophages.

This study investigated the critical role of Glut1-mediated glucose metabolism in the inflammatory response of macrophages, which are energy-intensive cells within the innate immune system. Inflammation leads to increased Glut1 expression, ensuring sufficient glucose uptake to support macrophage functions. We demonstrated that using siRNA to knock down Glut1 reduces the expression of various pro-inflammatory cytokines and markers, such as IL-6, iNOS, MHC II/CD40, reactive oxygen species, and the hydrogen sulfide (H2S)-producing enzyme cystathionine γ-lyase (CSE). Glut1 activates a pro-inflammatory profile through a nuclear factor (NF)-κB, while silencing Glut1 can prevent lipopolysaccharide (LPS)-induced IκB degradation, blocking NF-κB activation. Glut1's role in autophagy, an essential process for macrophage functions such as antigen presentation, phagocytosis, and cytokine secretion, was also measured. The findings show that LPS stimulation decreases autophagosome formation, but Glut1 knockdown reverses this effect, increasing autophagy beyond control levels. The study highlights Glut1's importance in macrophage immune responses and its regulation of apoptosis during LPS stimulation. Knocking down Glut1 negatively impacts cell viability and mitochondrial intrinsic pathway signaling. These findings collectively suggest that targeting macrophage glucose metabolism through Glut1 could potentially serve as a target for controlling inflammation.

Cystathionine γ-lyase and hydrogen sulfide modulates glucose transporter Glut1 expression via NF-κB and PI3k/Akt in macrophages during inflammation.

Macrophages play a crucial role in inflammation, a defense mechanism of the innate immune system. Metabolic function powered by glucose transporter isoform 1 (Glut1) is necessary for macrophage activity during inflammation. The present study investigated the roles of cystathionine-γ-lyase (CSE) and its byproduct, hydrogen sulfide (H2S), in macrophage glucose metabolism to explore the mechanism by which H2S acts as an inflammatory regulator in lipopolysaccharide- (LPS) induced macrophages. Our results demonstrated that LPS-treated macrophages increased Glut1 expression. LPS-induced Glut1 expression is regulated via nuclear factor (NF)-κB activation and is associated with phosphatidylinositol-3-kinase PI3k activation. Small interfering (si) RNA-mediated silencing of CSE decreased the LPS-induced NF-κB activation and Glut1 expression, suggesting a role for H2S in metabolic function in macrophages during pro-inflammatory response. Confoundingly, treatment with GYY4137, an H2S-donor molecule, also displayed inhibitory effects upon LPS-induced NF-κB activation and Glut1 expression. Moreover, GYY4137 treatment increased Akt activation, suggesting a role in promoting resolution of inflammation. Our study provides evidence that the source of H2S, either endogenous (via CSE) or exogenous (via GYY4137), supports or inhibits the LPS-induced NF-κB activity and Glut1 expression, respectively. Therefore, H2S may influence metabolic programming in immune cells to alter glucose substrate availability that impacts the immune response.

Macrophage Glucose Metabolism Reprogramming and Cardiovascular Complications of Diabetes

Macrophages are the main participants in the pathogenesis of many chronic inflammatory and autoimmune diseases, including diabetes, and cardiovascular complications mediated by abnormal activation of macrophages caused by various reasons are the main causes of morbidity and death in patients with diabetes. Different environmental stimuli can easily lead to glucose metabolism reprogramming in macrophages, resulting in a series of changes in metabolization-related enzymes, metabolites and metabolic pathways. Glucose metabolism reprogramming is closely related to M1 polarization and M2 polarization of macrophages, for example, M1 macrophages tend to anaerobic glycolysis while M2 macrophages tend to aerobic metabolism, and different polarization of macrophages is closely related to various diseases. Therefore, this paper reviews the mechanism and inducing factors of reprogramming of glucose metabolic pathways, as well as the effect of these pathways on macrophage polarization, so as to provide services for clinical research and treatment of the occurrence and development of cardiovascular complications in the context of chronic inflammation of diabetes.

Long noncoding RNAs in regulation of inflammation, immune response, and glucose metabolism

Noncoding RNAs (ncRNAs) are emerging players in cell signaling and metabolism. Studies from our laboratory demonstrate that long noncoding RNA (lncRNA) HOTAIR plays critical roles in inflammation via regulation of NF‐kB activation and cytokine expression. HOTAIR also regulates glucose metabolism in macrophages during inflammation. Importantly, metabolic reprogramming is critically linked to inflammation and macrophage activation. Therefore, our findings showing the HOTAIR mediated regulation of glucose metabolism in macrophages indicate potential roles of lncRNAs in macrophage activation and metabolic reprogramming. In a recent study, we explored potential involvement of other ncRNAs in inflammation and immune signaling. Based on independent RNAseq analyses in mouse and human macrophages, we have discovered a series novel mouse and human lncRNAs which are potential regulators of cytokine expression, inflammation, and immune response. Notably, there appear to be poor conservation among the lncRNAs between mouse and human. Mechanistic studies demonstrated that some of the novel inflammatory lncRNAs are regulated via NF‐kB pathways while others are not, suggesting distinct modes of action of different lncRNAs during inflammatory response in macrophages. Here, we will present the discovery of novel mouse and human lncRNAs that are potential regulators of inflammation and immune signaling and discuss functional roles of selected lncRNAs in cytokine regulation, inflammation, and glucose metabolism in macrophages.

Early secreted antigenic target 6-kDa from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1α

Early secreted antigenic target 6-kDa protein (ESAT6) is an essential virulence factor of Mycobacterium tuberculosis (MTb). However, ESAT6 helped fighting MTb infection according to vaccine studies. It's unclear whether ESAT6 confers protection via enhancing the innate immunity of macrophages, which are the first-line defense against MTb. We profiled the global transcriptional changes and characterized the innate immunity of THP-1 macrophages treated with ESAT6. We found ESAT6 promoted the phagocytosis ability, enhanced reactive oxygen species (ROS) generation and accelerated glucose metabolism in macrophages. Meanwhile, ESAT6 induced a distinctive phenotype of macrophages with a concurrence of pro-inflammatory and anti-inflammatory cytokines and chemokines. ESAT6 increased the expression of HIF1α mRNA and protein. Interfering HIF1α with siRNA defected the capacity of phagocytosis and ROS generation as well as glucose metabolism. Thus, ESAT6 enhanced the protective innate immunity of macrophages partially via HIF1α. This study provided clues for developing therapies against tuberculosis by targeting ESAT6.

Nrf2 exerts mixed inflammation and glucose metabolism regulatory effects on murine RAW264.7 macrophages

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcription factor that mediates a broad range of cellular antioxidative, detoxification and anti-inflammatory effects. However, the precise mechanism by which Nrf2 regulates inflammation and metabolism in macrophages remains controversial and unclear. To further clarify the roles of Nrf2 in inflammation and glucose metabolism regulation, retrovirus-mediated knockdown of Nrf2 was performed in murine RAW264.7 macrophages, and the cells were stimulated with 100 ng/mL lipopolysaccharide for 24 h for M1 activation. qPCR and western blotting results indicated that Nrf2 knockdown significantly enhanced expression of the inflammatory genes Il1a and Il1b in unstimulated macrophages and increased expression of the inflammatory genes Il1a, Il1b, Il6, Il10, Ccl2, Ccl22, and CD38 but decreased that of Tnfa and Tgfb1 in M1 macrophages. Nrf2 knockdown also significantly elevated IL6 and IL10 secretion by M1 macrophages. Western blotting showed that Nrf2 knockdown reduced iNOS protein levels in resting macrophages and enhanced CD38 protein levels in both resting and M1 macrophages. The differential regulation of these macrophage inflammation and polarization markers by Nrf2 reveals multiple roles for Nrf2 in regulating inflammation in macrophages. Moreover, Nrf2 knockdown increased the Glu4 protein level and decreased AKT and GSK3β protein phosphorylation in M1 macrophages, suggesting multiple roles for Nrf2 in regulating glucose metabolism in macrophages. Overall, our results are the first to demonstrate mixed inflammation and glucose metabolism regulatory effects of Nrf2 in macrophages that may occur independent of its classic function in redox regulation. These findings support the potential of Nrf2 as a therapeutic target for the prevention and treatment of inflammation- and obesity-associated syndromes, including diabetes and atherosclerosis.

Science to Practice: Does FDG Differentiate Morphologically Unstable from Stable Atherosclerotic Plaque?

It has been reported that fluorine 18 fluorodeoxyglucose (FDG) positron emission tomography (PET) may detect the inflammatory state and macrophage burden of atherosclerotic plaques and potentially identify vulnerable plaques. However, published reports have been inconsistent in this area. Tavakoli et al ( 1 ) hypothesized that differential regulation of macrophage glucose metabolism by macrophage colony-stimulating factor (M-CSF; inflammation resolving) and granulocyte-M-CSF (GM-CSF; proinflammatory) may contribute to the inconsistency of FDG vessel wall inflammation. After the induction of inflammatory and metabolic profiles, both M-CSF and GM-CSF generated comparable levels of glucose uptake in cultured macrophages and murine atherosclerotic plaques. These findings suggest that although FDG uptake is an indicator of vascular macrophage burden (total number of macrophages), it may not necessarily differentiate morphologically unstable (inflammatory) from stable (noninflammatory) atherosclerotic plaque. Moreover, although atherosclerosis is characterized by macrophage-predominated inflammation, there is a wide range of other vascular diseases in which macrophages and inflammation play an important role in the absence of atherosclerosis. FDG uptake will be indistinguishable in atherosclerosis from large-artery inflammatory vascular disease, such as Takayasu arteritis, chemotherapy- or radiation-induced vascular inflammation, or foreign-body reaction, such as synthetic arterial graft. Because of the nonspecific nature of FDG uptake by any cell (upregulated under hypoxic conditions or other microenvironmental factors), this work calls for a more cautious approach to interpreting vascular FDG uptake as indicative of inflammatory atherosclerosis in the clinical setting.

Differential Regulation of Macrophage Glucose Metabolism by Macrophage Colony-stimulating Factor and Granulocyte-Macrophage Colony-stimulating Factor: Implications for 18F FDG PET Imaging of Vessel Wall Inflammation.

Purpose To determine the divergence of immunometabolic phenotypes of macrophages stimulated with macrophage colony-stimulating factor (M-CSF) and granulocyte-M-CSF (GM-CSF) and its implications for fluorine 18 (18F) fluorodeoxyglucose (FDG) imaging of atherosclerosis. Materials and Methods This study was approved by the animal care committee. Uptake of 2-deoxyglucose and various indexes of oxidative and glycolytic metabolism were evaluated in nonactivated murine peritoneal macrophages (MΦ0) and macrophages stimulated with M-CSF (MΦM-CSF) or GM-CSF (MΦGM-CSF). Intracellular glucose flux was measured by using stable isotope tracing of glycolytic and tricyclic acid intermediary metabolites. 18F-FDG uptake was evaluated in murine atherosclerotic aortas after stimulation with M-CSF or GM-CSF by using quantitative autoradiography. Results Despite inducing distinct activation states, GM-CSF and M-CSF stimulated progressive but similar levels of increased 2-deoxyglucose uptake in macrophages that reached up to sixfold compared with MΦ0. The expression of glucose transporters, oxidative metabolism, and mitochondrial biogenesis were induced to similar levels in MΦM-CSF and MΦGM-CSF. Unexpectedly, there was a 1.7-fold increase in extracellular acidification rate, a 1.4-fold increase in lactate production, and overexpression of several critical glycolytic enzymes in MΦM-CSF compared with MΦGM-CSF with associated increased glucose flux through glycolytic pathway. Quantitative autoradiography demonstrated a 1.6-fold induction of 18F-FDG uptake in murine atherosclerotic plaques by both M-CSF and GM-CSF. Conclusion The proinflammatory and inflammation-resolving activation states of macrophages induced by GM-CSF and M-CSF in either cell culture or atherosclerotic plaques may not be distinguishable by the assessment of glucose uptake. © RSNA, 2016 Online supplemental material is available for this article.

Differential Regulation of Macrophage Glucose Metabolism by M‐CSF and GM‐CSF: Implications for 18F‐FDG PET Imaging of Inflammation

BackgroundThe high glycolytic capacity of macrophages allows for the utilization of 18F‐FDG in the detection of inflammation associated with various diseases. Microenvironmental signals (such as growth factors, cytokines and pathogen‐derived products) have profound effects on determining the activation state of macrophages, which in turn is delicately regulated through reprogramming of cell metabolism. Specifically, glycolysis has been suggested to enhance the pro‐inflammatory macrophage phenotype induced by pathogen‐derived stimuli. Here, we examined whether this phenomenon extends to endogenous microenvironmental activators by investigating the metabolic effects of two critical myeloid growth factors with opposing effects on the macrophage inflammatory state, i.e., macrophage colony‐stimulating factor (M‐CSF) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF).MethodsMurine peritoneal macrophages (Mϕ) were incubated ex vivo for two days in the absence (Mϕ0) or presence of M‐CSF (MϕM‐CSF) or GM‐CSF (MϕGM‐CSF). Glucose uptake was measured using 3H‐2‐deoxyglucose. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), markers of oxidative and glycolytic metabolism, as well as mitochondrial content of the cells and lactate production were quantified. Expression of activation markers, glucose transporters and metabolic enzymes was measured by high‐throughput quantitative reverse‐transcription PCR.ResultsM‐CSF and GM‐CSF stimulated comparable levels of 3H‐2‐deoxyglucose uptake and induced similar expression levels of glucose transporter‐1 and ‐6 in Mϕ. Similarly, mitochondrial biogenesis and oxidative metabolism were induced to the same levels in MϕM‐CSF and MϕGM‐CSF. Unexpectedly, activation of glycolysis was more pronounced in MϕM‐CSF compared to MϕGM‐CSF, as evidenced by higher basal ECAR and ECAR‐to‐OCR ratio, as well as lactate production. A number of critical glycolytic enzymes were also differentially upregulated in MϕM‐CSF compared to MϕGM‐CSF.ConclusionDespite their pro‐inflammatory activation state, MϕGM‐CSF showed the same level of glucose uptake and oxidative metabolism as MϕM‐CSF, which are associated with inflammation resolution. Furthermore, MϕM‐CSF, not the pro‐inflammatory MϕGM‐CSF, demonstrate the higher glycolytic activity. These findings strongly suggest that the reported association between a pro‐inflammatory macrophage phenotype and increased glucose uptake and glycolysis is limited to certain microenvironmental signals, and does not represent a universal phenomenon, calling for a more prudent approach in the interpretation of 18F‐FDG imaging of inflammation.Support or Funding InformationThis work was supported by grants from Radiological Society of North America Grant to S.T. (RR1131) and the National Institute of Health (HL115858) to R.A.

Abstract 175: Lack of Macrophage GLUT1-Mediated Glucose Metabolism Increases Atherosclerotic Lesion Instability

Macrophages play a key role in the pathogenesis of atherosclerosis. Metabolic programs powered by glucose or lipid enable macrophages to elicit pro- or anti-inflammatory responses, respectively. Although the chronic inflammatory feature of atherosclerosis has been well-established, the role of macrophage substrate metabolism in atherogenesis remains unclear. We previously demonstrated that macrophages with elevated GLUT1-mediated glucose metabolism have an increased inflammatory response. Therefore, we created a novel macrophage GLUT1-deficient murine model by crossing GLUT1 floxed to LysM-Cre mice. Recent work suggests that lack of GLUT1 reduces the pro-inflammatory response and the ability for GLUT1-/- macrophages to polarize to the classically activated state in vitro. Therefore, the objective of this study was to examine how macrophage metabolic reprogramming affects the development of atherosclerosis. We hypothesized that macrophages with restricted glucose metabolism due to lack of glucose transporter GLUT1 will have reduced pro-inflammatory activation during atherogenesis. We transplanted bone marrow from Glut1MΦfl/fl or Glut1MΦ-/- mice into Ldlr-/- mice and fed mice a Western diet for 12 weeks. Glut1MΦfl/fl Ldlr-/- or Glut1MΦ-/- Ldlr-/- chimeric mice did not exhibit significant differences in body weight, body composition, blood pressure, fasting blood glucose, or triacylglycerol and LDL and HDL cholesterol. Digital histology analysis of Oil Red O stained slides indicated that deleting macrophage GLUT1 did not affect total lesion area in aortic root; however, mice with blunted glucose metabolism displayed more and larger necrotic cores. Ongoing studies are investigating apoptosis and phagocytic capacity of macrophages with and without GLUT1 to elucidate the roles of macrophage glucose metabolism on the morphology of atherosclerotic lesion. In summary, we observed that mice lacking macrophage GLUT1 developed increased necrotic cores in lesions of the aorta root relative to mice with wild type macrophage GLUT1 which suggests that maintenance of atherosclerotic lesion stability may be regulated by glucose-dependent mechanisms.

Oxidized low-density lipoprotein stimulates macrophage 18F-FDG uptake via hypoxia-inducible factor-1α activation through Nox2-dependent reactive oxygen species generation.

For (18)F-FDG PET to be widely used to monitor atherosclerosis progression and therapeutic response, it is crucial to better understand how macrophage glucose metabolism is influenced by the atherosclerotic microenvironment and to elucidate the molecular mechanisms of this response. Oxidized low-density lipoprotein (oxLDL) is a key player in atherosclerotic inflammation that promotes macrophage recruitment, activation, and foam cell formation. We thus explored the effect of oxLDL on macrophage (18)F-FDG uptake and investigated the underlying molecular mechanism including the roles of hypoxia-inducible factor-1α (HIF-1α) and reactive oxygen species (ROS). RAW264.7 macrophages were stimulated with native LDL, oxLDL, or lipopolysaccharide. Cells were assessed for (18)F-FDG uptake, lactate production, membrane glucose transporter 1 (GLUT1) expression, and hexokinase activity. ROS generation, Nox expression, and HIF-1α activity were also measured. oxLDL (20 μg/mL) induced a 17.5 ± 1.7-fold increase in macrophage (18)F-FDG uptake by 24 h, which was accompanied by increased lactate production, membrane GLUT1 expression, and hexokinase activity. oxLDL-stimulated (18)F-FDG uptake was completely blocked by inhibitors of Src or phosphoinositide 3-kinase. ROS generation was increased to 262.4% ± 17.9% of controls by oxLDL, and N-acetyl-l-cysteine completely abrogated both oxLDL-induced ROS production and (18)F-FDG uptake. oxLDL increased Nox2 expression, and nicotinamide adenine dinucleotide phosphate oxidase inhibition totally blocked increased ROS generation and (18)F-FDG uptake by oxLDL. Finally, there was a clear ROS-dependent increase of HIF-1α accumulation by oxLDL, and silencing of HIF-1α completely abolished the metabolic effect of oxLDL. oxLDL is a strong stimulator of macrophage (18)F-FDG uptake and glycolysis through upregulation of GLUT1 and hexokinase. This metabolic response is mediated by Nox2-dependent ROS generation that promotes HIF-1α activation.

Testing the Role of Myeloid Cell Glucose Flux in Inflammation and Atherosclerosis

Inflammatory activation of myeloid cells is accompanied by increased glycolysis, which is required for the surge in cytokine production. Although in vitro studies suggest that increased macrophage glucose metabolism is sufficient for cytokine induction, the proinflammatory effects of increased myeloid cell glucose flux in vivo and the impact on atherosclerosis, a major complication of diabetes, are unknown. We therefore tested the hypothesis that increased glucose uptake in myeloid cells stimulates cytokine production and atherosclerosis. Overexpression of the glucose transporter GLUT1 in myeloid cells caused increased glycolysis and flux through the pentose phosphate pathway but did not induce cytokines. Moreover, myeloid-cell-specific overexpression of GLUT1 in LDL receptor-deficient mice was ineffective in promoting atherosclerosis. Thus, increased glucose flux is insufficient for inflammatory myeloid cell activation and atherogenesis. If glucose promotes atherosclerosis by increasing cellular glucose flux, myeloid cells do not appear to be the key targets.

HIV-1 Vpr Modulates Macrophage Metabolic Pathways: A SILAC-Based Quantitative Analysis

Human immunodeficiency virus type 1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Furthermore, these macrophages are resistant to cell death and are viral reservoir. However, the impact of Vpr on the macrophage proteome is yet to be comprehended. The goal of the present study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. Cultured human monocytic cells, U937, were differentiated into macrophages and transduced with adenovirus construct harboring the Vpr gene. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. Quantified proteins were selected and clustered by biological functions, pathway and network analysis using Ingenuity computational pathway analysis. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. In addition, the proteomic data demonstrate down regulation of some key mitochondrial enzymes such as glutamate dehydrogenase 2 (GLUD2), adenylate kinase 2 (AK2) and transketolase (TKT). Based on these observations we postulate that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehyrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. Furthermore, dysregulation of mitochondrial glutamate metabolism in macrophages can contribute to neurodegeneration via neuroexcitotoxic mechanisms in the context of NeuroAIDS.

Glucose metabolism is linked to the inflammatory status of macrophages

Background Macrophages infiltrate adipose tissue at the onset of weight gain and directly contribute to adipose inflammation, insulin resistance, and obesity [1]. The type of fuel substrate utilized by macrophages is central to the formation of obesity, a global epidemic [2]. Our goal is to understand the role of macrophage glucose metabolism in the promotion of inflammation and insulin resistance during high fat diet-induced obesity. We hypothesize that macrophages with blunted or elevated glucose metabolism will display limited or exaggerated immune responses, and modulate susceptibility to insulin resistance and obesity, respectively.

The intravenous anesthetic propofol inhibits lipopolysaccharide-induced hypoxia-inducible factor 1 activation and suppresses the glucose metabolism in macrophages

Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor of hypoxia-induced gene expression. Anesthetics and perioperative drugs have been reported to affect HIF-1 activity. However, the effect of propofol on HIF-1 activity is not well documented. In this study, we investigated the effect of propofol on HIF-1 activation using macrophage-differentiated THP-1 cells. Cells were exposed to lipopolysaccharide (LPS) under 20 or 1% O(2) conditions with or without propofol treatment. The cell lysate was subjected to Western blot analysis using anti-HIF-1alpha and HIF-1beta antibodies. HIF-1-dependent gene expression was investigated by quantitative real-time reverse-transcriptase PCR analysis and luciferase assay. The amount of cellular lactate and ATP was assayed. Propofol suppressed HIF-1alpha protein accumulation induced by LPS, but not by hypoxia in the THP-1 cells in a dose-dependent manner by inhibiting the neo-synthesis of HIF-1alpha protein. Induction of the HIF-1 downstream gene expression including glucose transporter 1, enolase 1, lactate dehydrogenase A, pyruvate dehydrogenase kinase-1 and vascular endothelial growth factor was inhibited by propofol. Propofol suppressed LPS-induced lactate accumulation and ATP content in THP-1 cells. Our experimental results indicate that propofol inhibits HIF-1 activation and downstream gene expression induced by LPS and suppressed HIF-1-dependent glucose metabolic reprogramming. HIF-1 suppression by propofol in macrophages may explain molecular mechanisms behind the inhibitory effect of propofol on cellular inflammatory responses.