NAD+-dependent sirtuin 1 and 6 proteins coordinate a switch from glucose to fatty acid oxidation during the acute inflammatory response

Abstract

The early initiation phase of acute inflammation is anabolic and primarily requires glycolysis with reduced mitochondrial glucose oxidation for energy, whereas the later adaptation phase is catabolic and primarily requires fatty acid oxidation for energy. We reported previously that switching from the early to the late acute inflammatory response following TLR4 stimulation depends on NAD(+) activation of deacetylase sirtuin 1 (SirT1). Here, we tested whether NAD(+) sensing by sirtuins couples metabolic polarity with the acute inflammatory response. We found in TLR4-stimulated THP-1 promonocytes that SirT1 and SirT 6 support a switch from increased glycolysis to increased fatty acid oxidation as early inflammation converts to late inflammation. Glycolysis enhancement required hypoxia-inducing factor-1α to up-regulate glucose transporter Glut1, phospho-fructose kinase, and pyruvate dehydrogenase kinase 1, which interrupted pyruvate dehydrogenase and reduced mitochondrial glucose oxidation. The shift to late acute inflammation and elevated fatty acid oxidation required peroxisome proliferator-activated receptor γ coactivators PGC-1α and β to increase external membrane CD36 and fatty acid mitochondrial transporter carnitine palmitoyl transferase 1. Metabolic coupling between early and late responses also required NAD(+) production from nicotinamide phosphoryltransferase (Nampt) and activation of SirT6 to reduce glycolysis and SirT1 to increase fatty oxidation. We confirmed similar shifts in metabolic polarity during the late immunosuppressed stage of human sepsis blood leukocytes and murine sepsis splenocytes. We conclude that NAD(+)-dependent bioenergy shifts link metabolism with the early and late stages of acute inflammation.

FIGURE 1.

Sequential metabolic shifts occur after TLR4 stimulation of THP-1 cells. Cells were assessed during the sequential early initial and late adaptation responses. A, TNF-α is repressed, and IL-10 mRNA is induced during the adaptation stage. B, glucose uptake is increased during the early inflammatory response. C, mitochondrial glucose oxidation is reduced during the early inflammatory response. D, glycolysis is increased during the early inflammatory response. E, palmitic acid uptake is increased during the adaptive response. F, palmitic acid mitochondrial fatty acid oxidation is increased during the hypoinflammatory response. Bar graphs depict mean values ± S.E. of one of three independent experiments.

FIGURE 2.

Dynamics in expression of key metabolic regulators in the THP-1 cell model of sepsis. A, schematic of key regulators in metabolic pathways. B, expression of Glut1 in THP-1 cells during TLR4 stimulation. C, expression of surface Glut1 on THP-1 cells. Cells were stimulated with 1 μg/ml of LPS for different times. Cell surface GluT1 was stained with human GluT1-specific mouse monoclonal antibody followed by rhodamine-conjugated goat anti-mouse IgG1. D, flow cytometry analysis of GluT1 expression in THP-1 cells during TLR4 stimulation. THP-1 cells were cultured for 24 h with or without 1 μg/ml LPS. Cell surface GluT1 was probed with carboxyfluorescein-conjugated human GluT1 monoclonal antibody. E, changes of pyruvate dehydrogenase complex activity during TLR4 stimulation. Cells were stimulated with 1 μg/ml of LPS for different times. Total cell lysates were analyzed by Western blot analysis for expressions of PDHK1, PDHA1, and phospho-PDHA1-Ser-232. F, expression of rate-limiting enzymes in the glucose metabolic pathway during TLR4 stimulation. G, expression of lactate dehydrogenase (LDH) and accumulations of intracellular pyruvate and lactate during TLR4 stimulation. H, expressions of rate-limiting enzymes in the fatty acid metabolic pathway during TLR4 stimulation. Changes of CD36 and CPT-1 were analyzed by Western blot analysis (left panel) and densitometry analysis (right panel). I, expression of external membrane CD36 on THP-1 cells. Cell surface CD36 was probed with FITC-conjugated mouse anti-human CD36. J, flow cytometry analysis of CD36 expression in THP-1 cells during TLR4 stimulation. The cell surface CD36 was stained with FITC-anti-CD36 monoclonal antibody. GluT1, glucose transporter 1. FA, fatty acid.

FIGURE 3.

TLR4-induced metabolic switching of glucose and fatty acid metabolism requires Nampt-dependent NAD⁺ generation. NAD⁺ was depleted by treating cells for 24 h with FK866, and TLR4 was stimulated with LPS for another 24 h. A, schematic of epigenetic gene-selective reprogramming and bioenergy shifts during the acute inflammatory response. B, dynamic changes of cellular NAD⁺ and NADH during TLR4 activation. C, dynamic changes of NAD⁺/NADH ratio during TLR4 activation. D, FK866 reduces mitochondrial glucose oxidation in quiescent cells and restrains its increase at 24 h. E, FK866 inhibits LPS-induced increase in fatty acid oxidation at 24 h. F, FK866 alters key glucose and fatty acid metabolism regulators: PDHK1, PDHA1, p-PDHA1-Ser-232, and CPT-1. Bar graphs represent mean ± S.E. of one of two independent experiments. FK, FK866; med, medium.

FIGURE 4.

NAD⁺-dependent deacetylases SirT6 and SirT1 differentially regulate glucose and fatty acid metabolic switching. THP-1 cells were transfected with gene-specific siRNA for SirT1 and/or SirT6 for 24 h followed by 1 μg/ml LPS stimulation for 24 h. A, SirT1 levels increase and SirT6 levels are sustained after LPS stimulation. B, SirT6 knockdown increases glucose uptake, whereas SirT knockdown has little effect. C, SirT6 knockdown further decreases mitochondrial oxidation, whereas SirT1 has little effect. D, SirT6 knockdown increases glycolysis, whereas SirT1 has little effect. E, SirT1 knockdown reduces palmitic acid uptake, whereas SirT6 has little effect. F, SirT1 knockdown reduces palmitic acid mitochondrial oxidation, whereas SirT6 has little effect. G, SirT6 or SirT1 differentially influence Glut1 and CD36 expression, and SirT1 knockdown diminishes SirT6 (H). Bar graphs represent mean ± S.E. of one of three independent experiments. CTRL, control; R, responsive cells; A, adapted cells.

FIGURE 5.

HIF-1α predominantly regulates glucose metabolism during TLR4 stimulation. THP-1 cells were transfected with HIF-1α-specific siRNA or control siRNA. After 24 h of transfection, cells were stimulated with 1 μg/ml LPS for another 24 h followed by radiolabeling for metabolic analysis. Knockdown HIF-1α significantly decreases uptake of D-[6-¹⁴C]glucose (A) and mitochondrial oxidation of D-[6-¹⁴C]glucose (B) but does not significantly affect uptake of 1-[1⁴C]palmitic acid (C). D, knockdown HIF-1α has no effect on mitochondrial oxidation of 1-[¹⁴C]palmitic acid. Bar graphs depict mean values ± S.E. from two independent experiments. CTRL, control; KD, knockdown.

FIGURE 6.

TLR4-induced fatty acid uptake and oxidation requires PGC-1 induction. THP-1 cells were stimulated with 1 μg/ml of LPS and followed for 24 h. A, TLR4 activation induces expression of PGC-1α and PGC-1β. B, PGC-1α or PGC-1β knockdown reduces CD36 expression, and PGC-1β specifically reduces CPT-1 expression. C, PGC-1 α or β knockdown reduces fatty acid oxidation. Bar graphs depict mean values ± S.E. of one from two independent experiments. CTRL, control; KD, knockdown.

FIGURE 7.

Metabolism and inflammation reprogramming are integrated. Blocking glucose uptake by 2-deoxyglucose (2-DG) decreases TLR4-induced gene transcription of TNF-α (A) and RelB (B). Inhibiting glycolysis regulator HIF-1α DNA binding by echinomycin diminishes transcription of TNF-α (C) and RelB (D). CPT-1 inhibition does not alter TRL4-induced transcription of TNF-α (E) or RelB (F). G, RelB knockdown attenuates TLR4-mediated fatty acid oxidation. Bar graphs represent mean values ± S.E. of one of two independent experiments. CTRL, control.

FIGURE 8.

Glucose and fatty acid oxidation shift in murine and human sepsis. Both murine and human sepsis analyses were performed during the adaptation stage (mice at 24 h after CLP and humans at least 24 h after sepsis onset). A, mitochondrial glucose oxidation is decreased in sepsis murine splenocytes. B, mitochondrial palmitic acid oxidation is increased in sepsis murine splenocytes. C, mitochondrial fatty acid regulators are modified in sepsis murine splenocytes. D, mitochondrial glucose oxidation is decreased in human sepsis leukocytes. E, mitochondrial palmitic acid oxidation is increased in human sepsis leukocytes. F, mitochondrial fatty acid regulators are modified in human sepsis leukocytes. Data are mean values ± S.E. N, normal; P, patients.

FIGURE 9.

Alignment of NAD⁺-dependent metabolic and inflammatory switching after acute TLR4 responses and during sepsis. When sepsis is ignited by TLR responses, the high energy required for the early inflammatory response generates reactive oxygen species (ROS), which stabilizes HIF-1α protein and activates its transcription of glycolysis genes in concert with NF-κB p65 and its activation of proinflammatory genes. HIF-1α increases glycolysis, but inhibits mitochondrial glucose oxidation. As the early inflammatory response changes to late adaptation, glucose metabolism switches to increased fatty acid flux and enhanced fatty acid mitochondrial oxidation. Both the metabolic and adaptive switches require activation of Nampt and NAD⁺ sensors SirT1 and SirT6. SirT6 represses glucose metabolism by epigenetically silencing the HIF-1α path. SirT1 supports fatty acid oxidation by activating the PGC-1 path, which also promotes mitochondrial biogenesis and restores homeostasis in sepsis survivors.