Glutamine Modulates Macrophage Lipotoxicity
"> Figure 1
<p>Glutamine metabolism is increased in activated macrophages. (<b>A</b>) Peritoneal macrophages (pMACs) were treated with control (BSA-PBS), BSA-LPS (100 ng), or palm (250 µM)-LPS (100 ng) for 16 h; and intracellular glutamine levels were quantified by mass spectroscopy; (<b>B</b>) After the indicated stimulations for 16 h, NH<sub>4</sub> release was quantified in the supernatant; (<b>C</b>) pMACs were stimulated in glutamine sufficient (open bars) or glutamine deficient (filled bars) media and NH<sub>4</sub> release into the media was quantified at 16 h. Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. <b>*</b>, <span class="html-italic">p</span> < 0.05 for PBS <span class="html-italic">vs</span>. LPS; #, <span class="html-italic">p</span> < 0.05 glutamine <span class="html-italic">vs</span>. no glutamine.</p> "> Figure 2
<p>Glutamine deficiency protects against lipotoxicity in macrophages. (<b>A</b>,<b>B</b>) pMACs were stimulated with BSA-PBS or palm-LPS in glutamine sufficient (open bars) or glutamine deficient (black bars) media and cell death was assessed at 30 h by annexin-PI (<b>A</b>), or lysosome damage was determined at 24 h by lysotracker red staining (<b>B</b>), both coupled with flow cytometry; (<b>C</b>,<b>D</b>) After the indicated stimulations IL-1β (<b>C</b>) or TNFα (<b>D</b>) release was quantified in the supernatant using ELISA. Bar graphs report the mean ± SE for a minimum of 3 experiments, each performed in triplicate. <b>*</b>, <span class="html-italic">p</span> < 0.05 for PBS <span class="html-italic">vs.</span> LPS; #, <span class="html-italic">p</span> < 0.05 glutamine <span class="html-italic">vs</span>. no glutamine.</p> "> Figure 3
<p>α-ketoglutarate partially restores macrophage lipotoxic phenotypes under glutamine deficient conditions. (<b>A</b>) Macrophages were treated as indicated in glutamine sufficient and deficient media ± 440 nM α-ketoglutarate (α-KG) and cell death (<b>A</b>) or lysotracker low cells (<b>B</b>) were quantified by flow cytometry; (<b>C</b>,<b>D</b>) pMACs were stimulated with palm-LPS in glutamine sufficient and deficient media ± α-KG and IL-1β (<b>C</b>) or TNF α (<b>D</b>) release was quantified by ELISA. Bar graphs report the mean ± SE for a minimum of 3 experiments, each performed in triplicate. <b>*</b>, <span class="html-italic">p</span> < 0.05 for veh <span class="html-italic">vs</span>. α-KG; #, <span class="html-italic">p</span> < 0.05 glutamine <span class="html-italic">vs</span>. no glutamine.</p> "> Figure 4
<p>Inhibition of glutaminolysis partially mimics glutamine deficiency. (<b>A</b>,<b>B</b>) Primary macrophages were stimulated with BSA-PBS in the presece of the glutaminolysis inhibitors BPTES ((<b>A</b>) 10 µM) or C698 ((<b>B</b>) 10 µM); (<b>C</b>) IL-1β release was quantified from pMACs treated with palm-LPS in the presence of BTPES of C968. Bar graphs report the mean ± SE for a minimum of 3 experiments, each performed in triplicate. <b>*</b>, <span class="html-italic">p</span> < 0.05 for veh <span class="html-italic">vs</span>. inhibitor.</p> "> Figure 5
<p>Leucine deficiency does not protect macrophage from lipotoxicity. Macrophages were incubated in complete RPMI media (open bars) or RPMI lacking leucine (gray filled bars) or glutamine (black filled bars), and after the indicated stimulation cell death was determined by annexin-PI flow cytometry. Bar graphs report the mean ± SE for a minimum of 3 experiments, each performed in triplicate. #, <span class="html-italic">p</span> < 0.05 glutamine <span class="html-italic">vs</span>. no glutamine.</p> "> Figure 6
<p>Glutamine deficiency impairs activation of mTORC1. (<b>A</b>) mTORC can assemble in two distinct complexes known as mTORC1 and mTORC2 which lead to the phosphorylation of S6K and AKT, respectively; (<b>B</b>) pMACs were stimulated with BSA-PBS, BSA-LPS, or palm-LPS for 16 h in the presence or absence of glutamine and phosphorlyation of S6K and AKT were assessed by Western blotting. Total S6K and total AKT are shown as control; (<b>C</b>) Macrophages were treated as indicated for 16 h in glutamine sufficient or deficient media ± α-KG (440 nM) and S6K phosphorylation was determined by Western blotting.</p> "> Figure 7
<p>Autophagy is modulated by glutamine deficiency. (<b>A</b>) pMACs were treated with BSA-PBS or palm-LPS in glutamine sufficient or glutamine deficient media ± α-KG (440 nM). The cells were incubated for 16 h followed by 2 h of veh or bafilomycin (BAF; 50 nM). Protein lysates were analyzed for expression of LC3 or p62. Actin is shown as a loading control; (<b>B</b>) Cells were stimulated as indicated for 16 h in media with or without glutamine followed by qRT-PCR assessment of LC3 and p62 mRNA expression. Bar graphs report the mean ± SE for a minimum of 3 experiments, each performed in triplicate. <b>*</b>, <span class="html-italic">p</span> < 0.05 for PBS <span class="html-italic">vs</span>. LPS; #, <span class="html-italic">p</span> < 0.05 glutamine <span class="html-italic">vs</span>. no glutamine.</p> "> Figure 8
<p>Autophagy is dispensable for protective effects of glutamine deficiency. (<b>A</b>) Primary macrophages were isolated from WT (ATG5 flx/flx) or ATG5KO (ATG5 flx/flx-LysM-Cre) followed by stimulation with BSA-PBS or palm-PLS in the presense or absence of glutamine. The levels of LC3 and p62 protein protein expression were assessed by western blotting. Actin is shown as a protein loading control; (<b>B</b>) pMACs from WT and ATG5KO mice were stimulated as indicated and cell death was determinded at 30 h using annexin-PI flow cytometry. The bar graph reports the mean ± SE for a minimum of 3 experiments, each performed in triplicate. *, <span class="html-italic">p</span> < 0.05 for WT <span class="html-italic">vs</span>. ATG5KO.</p> "> Figure 9
<p>Glutamine deficiency alters mitochondrial metabolism. (<b>A</b>–<b>C</b>) pMACs were stimulated for 16 h with BSA-PBS (black), BSA-LPS (red), or palm-LPS (blue) in glutamine sufficient media (<b>A</b>) or glutamine deficient media ± α-KG (<b>B</b>,<b>C</b>) and mitochondrial metabolism was assessed using a Seahorse flux analyzer. Mitochondrial oxygen consumption rate (OCR) was assessed at baseline and after the injection of oligomycin (<b>O</b>), FCCF (<b>F</b>) and rotenone/antamycin (R/A); (<b>D</b>,<b>E</b>) Baesline OCR (<b>D</b>) or extracellular acidification rate (ECAR; (<b>E</b>)) are reported from cells stimulated as in (<b>A</b>–<b>C</b>). Bar graphs report the mean ± SE for a minimum of 3 experiments, each performed in triplicate. *, <span class="html-italic">p</span> < 0.05 for BSA-PBS <span class="html-italic">vs</span>. BSA-LPS; **, <span class="html-italic">p</span> < 0.05 BSA-LPS <span class="html-italic">vs</span>. palm-LPS; #, <span class="html-italic">p</span> < 0.05 glutamine <span class="html-italic">vs</span>. no glutamine.</p> "> Figure 10
<p>Model of macrophage lipotoxicity under glutamine sufficient and deficient conditions. When glutamine is available, TLR4 activation stimulates glutamine uptake, mTORC1 activation, enhanced formation of TCA intermediates, such as α-KG, and increased mitochondrial respiration. The presence of excess fatty acids like palmitate leads to suppression of mitochondrial respiration, which in the setting of LPS activation likely promotes accumulation of toxic metabolites, lipids and/or redox stress. Lysosome dysfunction ensues leading to macrophage cell death and inflammasome activation. In the absence of glutamine, mTORC1 activation and mitochondrial respiration are diminished leading to enhanced rates of autophagy. In this scenario palmitate no longer suppresses mitochondrial function, which appears to protect against lysosome dysfunction, perhaps by decreasing the formation of damaging metabolites and lipids.</p> ">
Abstract
:1. Introduction
2. Experimental Section
2.1. Reagents
2.2. Cell Culture
2.3. Mice
2.4. RNA Isolation and Quantitative RT-PCR
2.5. Western Blotting
2.6. Lysosome Imaging
2.7. Metabolism Assays
2.8. Ammonia Quantification
2.9. Intracellular Glutamine Quantification
3. Results
3.1. Glutamine Deficiency Attenuates Macrophage Lipotoxic Responses
3.2. Oxidative Glutamine Metabolism Is Partially Responsible for the Protection from Lipid Toxicity
3.3. Glutamine Deficiency Is Protective Independent of Leucine
3.4. mTOR Signaling Is Reduced in the Absence of Glutamine
3.5. Glutamine Deficiency Modulates Macrophage Autophagy
3.6. Glutamine Deficiency Prevents the Suppression of Mitochondrial Function by Palmitate
4. Discussion
Acknowledgments
Author Contributions
Conflicts of Interest
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He, L.; Weber, K.J.; Schilling, J.D. Glutamine Modulates Macrophage Lipotoxicity. Nutrients 2016, 8, 215. https://doi.org/10.3390/nu8040215
He L, Weber KJ, Schilling JD. Glutamine Modulates Macrophage Lipotoxicity. Nutrients. 2016; 8(4):215. https://doi.org/10.3390/nu8040215
Chicago/Turabian StyleHe, Li, Kassandra J. Weber, and Joel D. Schilling. 2016. "Glutamine Modulates Macrophage Lipotoxicity" Nutrients 8, no. 4: 215. https://doi.org/10.3390/nu8040215