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SIRT1-dependent restoration of NAD+ homeostasis after increased extracellular NAD+ exposure.

ABSTRACT: In the last several years, NAD+ supplementation has emerged as an innovative and safe therapeutic strategy for a wide spectrum of disorders, including diabetes and neuropathy. However, critical questions remain as to how NAD+ and its precursors are taken up by cells, as well as the effects of long-lasting intracellular NAD+ (iNAD+) increases. Here, we investigated the kinetics of iNAD+ levels in different cell types challenged with prolonged exposure to extracellular NAD+ (eNAD+). Surprisingly, we found that after the initial increase, iNAD+ contents decreased back to control levels (iNAD+ resetting). Focusing our attention on HeLa cells, we found that oxygen and ATP consumption occurred with similar temporal kinetics after eNAD+ exposure. Using [³H]NAD+ and [¹⁴C]NAD+, we determined that NAD+ resetting was not due to increased dinucleotide extrusion but rather due to reduced uptake of cleaved NAD+ products. Indeed, eNAD+ exposure reduced the expression of the ecto-5'-nucleotidase CD73, the nicotinamide adenine mononucleotide transporter solute carrier family 12 member 8, and the nicotinamide riboside kinase. Interestingly, silencing the NAD+-sensor enzyme sirtuin 1 prevented eNAD+-dependent transcriptional repression of ecto-5'-nucleotidase, solute carrier family 12 member 8, and nicotinamide riboside kinase, as well as iNAD+ resetting. Our findings provide the first evidence for a sirtuin 1-mediated homeostatic response aimed at maintaining physiological iNAD+ levels in conditions of excess eNAD+ availability. These data may be of relevance for therapies designed to support the NAD+ metabolome via extracellular supplementation of the dinucleotide or its precursors.

SUBMITTER: Buonvicino D

PROVIDER: S-EPMC8233143 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A typical inflammatory response sequentially progresses from pro-inflammatory, immune suppressive to inflammatory repairing phases. Although the physiological inflammatory response resolves in time, severe acute inflammation usually sustains immune tolerance and leads to high mortality, yet the underlying mechanism is not completely understood. Here, using the leukemia-derived THP-1 human monocytes, healthy and septic human peripheral blood mononuclear cells (PBMC), we report that endotoxin dose-dependent switch of nicotinamide adenine dinucleotide (NAD) biosynthesis pathways sustain immune tolerant status. Low dose endotoxin triggered nicotinamide phosphoribosyltransferase (NAMPT)-dependent NAD salvage activity to adapt pro-inflammation. In contrast, high dose endotoxin drove a shift of NAD synthesis pathway from early NAMPT-dependent NAD salvage to late indoleamine 2,3-dioxygenase-1 (IDO1)-dependent NAD de novo biosynthesis, leading to persistent immune suppression. This is resulted from the IDO1-dependent expansion of nuclear NAD pool and nuclear NAD-dependent prolongation of sirtuin1 (SIRT1)-directed epigenetics of immune tolerance. Inhibition of IDO1 activity predominantly decreased nuclear NAD level, which promoted sequential dissociations of immunosuppressive SIRT1 and RelB from the promoter of pro-inflammatory TNF-? gene and broke endotoxin tolerance. Thus, NAMPT-NAD-SIRT1 axis adapts pro-inflammation, but IDO1-NAD-SIRT1-RelB axis sustains endotoxin tolerance during acute inflammatory response. Remarkably, in contrast to the prevention of sepsis death of animal model by IDO1 inhibition before sepsis initiation, we demonstrated that the combination therapy of IDO1 inhibition by 1-methyl-D-tryptophan (1-MT) and tryptophan supplementation rather than 1-MT administration alone after sepsis onset rescued sepsis animals, highlighting the translational significance of tryptophan restoration in IDO1 targeting therapy of severe inflammatory diseases like sepsis.

Project description:BackgroundThe intestine of children with severe malnutrition (SM) shows structural and functional changes that are linked to increased infection and mortality. SM dysregulates the tryptophan-kynurenine pathway, which may impact processes such as SIRT1- and mTORC1-mediated autophagy and mitochondrial homeostasis. Using a mouse and organoid model of SM, we studied the repercussions of these dysregulations on malnutrition enteropathy and the protective capacity of maintaining autophagy activity and mitochondrial health.MethodsSM was induced through feeding male weanling C57BL/6 mice a low protein diet (LPD) for 14-days. Mice were either treated with the NAD+-precursor, nicotinamide; an mTORC1-inhibitor, rapamycin; a SIRT1-activator, resveratrol; or SIRT1-inhibitor, EX-527. Malnutrition enteropathy was induced in enteric organoids through amino-acid deprivation. Features of and pathways to malnutrition enteropathy were examined, including paracellular permeability, nutrient absorption, and autophagic, mitochondrial, and reactive-oxygen-species (ROS) abnormalities.FindingsLPD-feeding and ensuing low-tryptophan availability led to villus atrophy, nutrient malabsorption, and intestinal barrier dysfunction. In LPD-fed mice, nicotinamide-supplementation was linked to SIRT1-mediated activation of mitophagy, which reduced damaged mitochondria, and improved intestinal barrier function. Inhibition of mTORC1 reduced intestinal barrier dysfunction and nutrient malabsorption. Findings were validated and extended using an organoid model, demonstrating that resolution of mitochondrial ROS resolved barrier dysfunction.InterpretationMalnutrition enteropathy arises from a dysregulation of the SIRT1 and mTORC1 pathways, leading to disrupted autophagy, mitochondrial homeostasis, and ROS. Whether nicotinamide-supplementation in children with SM could ameliorate malnutrition enteropathy should be explored in clinical trials.FundingThis work was supported by the Bill and Melinda Gates Foundation, the Sickkids Research Institute, the Canadian Institutes of Health Research, and the University Medical Center Groningen.

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SIRT1-dependent restoration of NAD+ homeostasis after increased extracellular NAD+ exposure.

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