Diabetes Suppresses Glucose Uptake and Glycolysis in Macrophages
2022; Lippincott Williams & Wilkins; Volume: 130; Issue: 5 Linguagem: Inglês
10.1161/circresaha.121.320060
ISSN1524-4571
AutoresYunosuke Matsuura, Masami Shimizu‐Albergine, Shelley Barnhart, Farah Kramer, Cheng-Chieh Hsu, Vishal Kothari, Jingjing Tang, Sina A. Gharib, Jenny E. Kanter, E. Dale Abel, Rong Tian, Baohai Shao, Karin Bornfeldt,
Tópico(s)Diabetes Management and Research
ResumoHomeCirculation ResearchVol. 130, No. 5Diabetes Suppresses Glucose Uptake and Glycolysis in Macrophages Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBDiabetes Suppresses Glucose Uptake and Glycolysis in Macrophages Yunosuke Matsuura, Masami Shimizu-Albergine, Shelley Barnhart, Farah Kramer, Cheng-Chieh Hsu, Vishal Kothari, Jingjing Tang, Sina A. Gharib, Jenny E. Kanter, E. Dale Abel, Rong Tian, Baohai Shao and Karin E. Bornfeldt Yunosuke MatsuuraYunosuke Matsuura https://orcid.org/0000-0003-2927-7284 Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine (Y.M., M.S.-A., S.B., F.K., C.-C.H., V.K., J.T., J.E.K., B.S., K.E.B.), University of Washington, Seattle. Now with: Division of Cardiovascular Medicine and Nephrology, Department of Internal Medicine, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan (Y.M.). , Masami Shimizu-AlbergineMasami Shimizu-Albergine https://orcid.org/0000-0002-7989-6051 Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine (Y.M., M.S.-A., S.B., F.K., C.-C.H., V.K., J.T., J.E.K., B.S., K.E.B.), University of Washington, Seattle. , Shelley BarnhartShelley Barnhart https://orcid.org/0000-0003-1829-9234 Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine (Y.M., M.S.-A., S.B., F.K., C.-C.H., V.K., J.T., J.E.K., B.S., K.E.B.), University of Washington, Seattle. , Farah KramerFarah Kramer Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine (Y.M., M.S.-A., S.B., F.K., C.-C.H., V.K., J.T., J.E.K., B.S., K.E.B.), University of Washington, Seattle. , Cheng-Chieh HsuCheng-Chieh Hsu https://orcid.org/0000-0002-3423-7159 Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine (Y.M., M.S.-A., S.B., F.K., C.-C.H., V.K., J.T., J.E.K., B.S., K.E.B.), University of Washington, Seattle. , Vishal KothariVishal Kothari https://orcid.org/0000-0002-3612-8810 Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine (Y.M., M.S.-A., S.B., F.K., C.-C.H., V.K., J.T., J.E.K., B.S., K.E.B.), University of Washington, Seattle. , Jingjing TangJingjing Tang https://orcid.org/0000-0001-7132-2777 Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine (Y.M., M.S.-A., S.B., F.K., C.-C.H., V.K., J.T., J.E.K., B.S., K.E.B.), University of Washington, Seattle. , Sina A. GharibSina A. Gharib https://orcid.org/0000-0002-2480-4367 Division of Pulmonary, Critical Care, and Sleep Medicine (S.A.G.), University of Washington, Seattle. , Jenny E. KanterJenny E. Kanter https://orcid.org/0000-0003-3212-772X Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine (Y.M., M.S.-A., S.B., F.K., C.-C.H., V.K., J.T., J.E.K., B.S., K.E.B.), University of Washington, Seattle. , E. Dale AbelE. Dale Abel https://orcid.org/0000-0001-5290-0738 Department of Medicine, David Geffen School of Medicine, University of California Los Angeles (E.D.A.). , Rong TianRong Tian https://orcid.org/0000-0002-3676-3830 Mitochondria and Metabolism Center, Department of Anesthesiology and Pain Medicine (R.T.), University of Washington, Seattle. , Baohai ShaoBaohai Shao https://orcid.org/0000-0001-8832-2845 Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine (Y.M., M.S.-A., S.B., F.K., C.-C.H., V.K., J.T., J.E.K., B.S., K.E.B.), University of Washington, Seattle. and Karin E. BornfeldtKarin E. Bornfeldt Correspondence to: Karin E. Bornfeldt, PhD, Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine, UW Medicine Diabetes Institute, University of Washington School of Medicine, 750 Republican St, Seattle, WA 98109. Email E-mail Address: [email protected] https://orcid.org/0000-0001-9208-6523 Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine (Y.M., M.S.-A., S.B., F.K., C.-C.H., V.K., J.T., J.E.K., B.S., K.E.B.), University of Washington, Seattle. Department of Laboratory Medicine and Pathology (K.E.B.), University of Washington, Seattle. Originally published16 Feb 2022https://doi.org/10.1161/CIRCRESAHA.121.320060Circulation Research. 2022;130:779–781is related toMeet the First AuthorsOther version(s) of this articleYou are viewing the most recent version of this article. Previous versions: February 16, 2022: Ahead of Print Meet the First Author, see p 692A prevailing notion is that increased glucose uptake in response to hyperglycemia in cells involved in atherosclerosis contributes to the increased risk of cardiovascular complications of diabetes. Because macrophages play critical roles in atherosclerosis, numerous studies have focused on how hyperglycemia affects these cells. It is unknown, however, if diabetes indeed increases glucose uptake in macrophages. We, therefore, asked whether diabetes leads to increased glucose uptake and glycolysis in macrophages, using a model of diabetes-accelerated atherosclerosis; LDL (low-density lipoprotein) receptor-deficient (Ldlr−/−) mice in which diabetes was induced by streptozotocin.Diabetic hyperglycemic mice and nondiabetic littermates were fed a low-fat semipurified diet1 for 4 weeks (Figure [A]). Peritoneal macrophages were isolated and sorted by negative selection 4 days after thioglycollate elicitation.2 Measurements were done after an additional 1-hour adhesion purification. Flow cytometry was performed immediately after isolation, identifying macrophages as live CD11B+ F4/80+ cells.Download figureDownload PowerPointFigure. Diabetes suppresses glycolysis in macrophages. A, Diabetes was induced in fasted mice by streptozotocin. Nondiabetic (ND) littermates received vehicle. Thioglycollate-elicited CD11B+F4/80+ macrophages were purified by negative selection. Streptozotocin showed no toxicity before diabetes induction in macrophages by extracellular acidification rate (ECAR) or in plasma by aspartate aminotransferase (AST; Abcam, ab263882) and blood urea nitrogen (BUN; Arbor Assays, K024). B, Macrophage GLUT1 levels measured by flow cytometry (Alexa Fluor 647, #ab195020; 1 µg/mL) and glucose uptake by Glucose Uptake-Glo Assay at 60 min (Promega). ECAR analyzed by Seahorse (Agilent) in XF base medium with 2 mmol/L glutamine. Gene expression by real-time polymerase chain reaction (PCR). C, GLUT1 (Slc2a1)-flox mice (10 generations C57BL/6J) were crossed with Lyz2-Cre mice. Mice heterozygous for Slc2a1 and Lyz2-Cre exhibited reduced GLUT1 but not reduced ECAR. D, Tetramethylrhodamine (TMRM) and MitoTracker Green (MTG; both 100 nmol/L; ThermoFisher) assessed by flow cytometry. CCL2 release by ELISA (Invitrogen, no. 88-7391) 6 h after IFN (interferon)-β stimulation. E, Shotgun proteomics (Orbitrap Lumos; ThermoFisher)1 and pathway analyses identify enriched processes in diabetic vs ND mice (false discovery rate [FDR]<0.05; volcano plot; http://www.webgestalt.org); heatmaps showing differentially abundant proteins nominally significant (P<0.05) calculated by a negative binomial model (http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html) within these pathways (https://uwmdi.org/bornfeldt-data-sharing-portal). F, Diabetic mice treated with an intensive insulin regimen or SGLT2i (sodium-glucose cotransporter 2 inhibitor).1 Blood glucose, oxygen consumption rate (OCR), and ECAR in peritoneal macrophages measured as above. Statistics (GraphPad Prism 9.0.2): 2-tailed unpaired t tests (2 groups, normally distributed data; D'Agostino-Pearson normality tests), Mann-Whitney (2 groups; nonparametric data or small groups), ANOVA (parametric) or Kruskal-Wallis (nonparametric) tests, as indicated. Mean±SEM; n=mice/group as indicated. FCCP indicates carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; and STZ, streptozotocin.Streptozotocin had no detectable toxic effects before development of diabetes (Figure [A]).Rather than increased glucose uptake, CD11B+ F4/80+ macrophages from diabetic mice (Figure [B]) exhibited reduced expression of GLUT1 (glucose transporter 1), the main glucose transporter in these cells, concomitant with reduced glucose uptake and reduced glycolysis estimated by extracellular acidification rate (ECAR). The enzymatic activity of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Abcam; ab204732) was not reduced by diabetes (1.10±0.04-fold of nondiabetic [ND] mice; mean±SD; n=5–9), as has been reported by high glucose in endothelial cells.3 However, phosphofructokinase, platelet type (Pfkp) exhibited reduced gene expression (Figure [B]). The reduced ECAR in macrophages from diabetic mice was not due to the downregulation of GLUT1 because mice with myeloid cell-targeted knockdown of GLUT1 matching the diabetes-induced GLUT1 reduction did not exhibit reduced ECAR (Figure [C]).To investigate if the reduced macrophage ECAR in diabetes is associated with reduced mitochondrial activity, mitochondrial membrane potential was measured by flow cytometry, using tetramethylrhodamine. MitoTracker Green assessed total mitochondrial mass. Macrophages from diabetic mice exhibited reduced mitochondrial membrane potential, without a reduction in mitochondrial mass (Figure [D]).Functionally, macrophages from diabetic mice showed hampered CCL2 (C-C motif chemokine ligand 2) release in response to IFN (interferon)-β (Figure [D]), perhaps due to desensitization.Next, we used global proteomics and pathway analyses to interrogate the overall effect of diabetes on macrophages (Figure [E]). Macrophages from diabetic mice were enriched in processes related to mRNA metabolism, complement and coagulation cascades, and immune and inflammatory responses. In contrast, macrophages from nondiabetic mice were enriched in proteins involved in insulin receptor signaling and recycling, iron uptake, and regulation of intracellular pH via vacuolar ATPases. Thus, diabetes profoundly alters the global macrophage proteome in the absence of increased glucose uptake.We used 2 strategies described previously1 to investigate if the effects of diabetes on macrophage metabolism are mediated by hyperglycemia or by insulin deficiency. Diabetic mice were treated with an intensive insulin regimen or a SGLT2i (sodium-glucose cotransporter 2 inhibitor; dapagliflozin), which suppresses blood glucose levels through insulin-independent urinary glucose excretion. Intensive insulin treatment using subcutaneous insulin pellets and long-acting insulin injections reduced blood glucose levels in diabetic mice over the 4-week study (Figure [F]). Administration of the SGLT2i (25 mg/kg per day; 4 weeks) also suppressed blood glucose levels. At the end of the study, thioglycollate-elicited peritoneal macrophages were isolated as in the Figure [A]. Oxygen consumption rate (OCR) analysis by Seahorse revealed that diabetes suppressed basal OCR by 48±5% (P=0.0004) and maximal OCR by 53±6% (P=0.0005), consistent with the reduced mitochondrial membrane potential (Figure [F]). The SGLT2i was unable to normalize macrophage OCR, whereas OCR in insulin-treated diabetic mice was not significantly different from that in nondiabetic mice (Figure [F]). A similar patten was observed for ATP-linked respiration. Likewise, diabetes suppressed ECAR in macrophages through a mechanism dependent on insulin deficiency rather than hyperglycemia because the SGLT2i failed to restore ECAR in macrophages from diabetic mice.We have previously shown that increased glucose uptake in myeloid cells in nondiabetic mice does not phenocopy the effect of diabetes on atherosclerosis.4 The present study demonstrates that macrophages in diabetic mice exhibit reduced—rather than increased—glucose uptake and glycolysis relative to those from nondiabetic mice.These findings challenge dogma by suggesting that hyperglycemia associated with diabetes does not directly increase macrophage glucose uptake. Instead, our findings support the proposal that lack of insulin associates with dampened macrophage metabolism. We cannot, however, rule out the possibility of accumulation of specific glucose metabolites or that the macrophages we used might not accurately reflect lesion macrophages.Our results cast doubt on increased glucose uptake as a direct contributor to diabetes-induced macrophage phenotypic changes associated with atherosclerosis. This interpretation is consistent with clinical data, which largely implicate risk factors other than hyperglycemia in promoting cardiovascular disease risk in diabetes.5Article InformationSources of FundingThis work was supported by the National Institutes of Health grants R35HL150754, P01HL151328, R01DK121756, P30DK017047, American Diabetes Association #9-18-CVD1-002, American Heart Association #828090, Grants-in-Aid for Scientific Research in Japan (20K17121).DisclosuresNone.FootnotesFor Sources of Funding and Disclosures, see page 781.This manuscript was sent to Joyce Bischoff, Guest Editor, for review by expert referees, editorial decision, and final disposition. Final decisions were approved by Jane Leopold, Guest Editor-in-Chief.Correspondence to: Karin E. Bornfeldt, PhD, Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine, UW Medicine Diabetes Institute, University of Washington School of Medicine, 750 Republican St, Seattle, WA 98109. Email [email protected]eduReferences1. Kanter JE, Shao B, Kramer F, Barnhart S, Shimizu-Albergine M, Vaisar T, Graham MJ, Crooke RM, Manuel CR, Haeusler RA, et al.. Increased apolipoprotein C3 drives cardiovascular risk in type 1 diabetes.J Clin Invest. 2019; 129:4165–4179. doi: 10.1172/JCI127308CrossrefMedlineGoogle Scholar2. Kothari V, Tang J, He Y, Kramer F, Kanter JE, Bornfeldt KE. ADAM17 boosts cholesterol efflux and downstream effects of high-density lipoprotein on inflammatory pathways in macrophages.Arterioscler Thromb Vasc Biol. 2021; 41:1854–1873. doi: 10.1161/ATVBAHA.121.315145LinkGoogle Scholar3. Brownlee M. Biochemistry and molecular cell biology of diabetic complications.Nature. 2001; 414:813–820. doi: 10.1038/414813aCrossrefMedlineGoogle Scholar4. Nishizawa T, Kanter JE, Kramer F, Barnhart S, Shen X, Vivekanandan-Giri A, Wall VZ, Kowitz J, Devaraj S, O'Brien KD, et al.. Testing the role of myeloid cell glucose flux in inflammation and atherosclerosis.Cell Rep. 2014; 7:356–365. doi: 10.1016/j.celrep.2014.03.028CrossrefMedlineGoogle Scholar5. Eckel RH, Bornfeldt KE, Goldberg IJ. Cardiovascular disease in diabetes, beyond glucose.Cell Metab. 2021; 33:1519–1545. doi: 10.1016/j.cmet.2021.07.001CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited ByBornfeldt K (2022) The Remnant Lipoprotein Hypothesis of Diabetes-Associated Cardiovascular Disease, Arteriosclerosis, Thrombosis, and Vascular Biology, 42:7, (819-830), Online publication date: 1-Jul-2022.Related articlesMeet the First AuthorsCirculation Research. 2022;130:692-693 March 4, 2022Vol 130, Issue 5Article InformationMetrics © 2022 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.121.320060PMID: 35170337 Originally publishedFebruary 16, 2022 KeywordsglycolysisglucoseatherosclerosishyperglycemiamacrophagesPDF download Advertisement SubjectsInflammationMetabolism
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