In vivo fluorescence lifetime imaging of macrophage intracellular metabolism during wound responses in zebrafish.

The function of macrophages in vitro is linked to their metabolic rewiring. However, macrophage metabolism remains poorly characterized in situ. Here, we used two-photon intensity and lifetime imaging of autofluorescent metabolic coenzymes, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD), to assess the metabolism of macrophages in the wound microenvironment. Inhibiting glycolysis reduced NAD(P)H mean lifetime and made the intracellular redox state of macrophages more oxidized, as indicated by reduced optical redox ratio. We found that TNFα+ macrophages had lower NAD(P)H mean lifetime and were more oxidized compared to TNFα- macrophages. Both infection and thermal injury induced a macrophage population with a more oxidized redox state in wounded tissues. Kinetic analysis detected temporal changes in the optical redox ratio during tissue repair, revealing a shift toward a more reduced redox state over time. Metformin reduced TNFα+ wound macrophages, made intracellular redox state more reduced and improved tissue repair. By contrast, depletion of STAT6 increased TNFα+ wound macrophages, made redox state more oxidized and impaired regeneration. Our findings suggest that autofluorescence of NAD(P)H and FAD is sensitive to dynamic changes in intracellular metabolism in tissues and can be used to probe the temporal and spatial regulation of macrophage metabolism during tissue damage and repair.

Malonate Promotes Adult Cardiomyocyte Proliferation and Heart Regeneration.

BACKGROUND: Neonatal mouse cardiomyocytes undergo a metabolic switch from glycolysis to oxidative phosphorylation, which results in a significant increase in reactive oxygen species production that induces DNA damage. These cellular changes contribute to cardiomyocyte cell cycle exit and loss of the capacity for cardiac regeneration. The mechanisms that regulate this metabolic switch and the increase in reactive oxygen species production have been relatively unexplored. Current evidence suggests that elevated reactive oxygen species production in ischemic tissues occurs as a result of accumulation of the mitochondrial metabolite succinate during ischemia via succinate dehydrogenase (SDH), and this succinate is rapidly oxidized at reperfusion. Mutations in SDH in familial cancer syndromes have been demonstrated to promote a metabolic shift into glycolytic metabolism, suggesting a potential role for SDH in regulating cellular metabolism. Whether succinate and SDH regulate cardiomyocyte cell cycle activity and the cardiac metabolic state remains unclear. METHODS: Here, we investigated the role of succinate and SDH inhibition in regulation of postnatal cardiomyocyte cell cycle activity and heart regeneration. RESULTS: Our results demonstrate that injection of succinate into neonatal mice results in inhibition of cardiomyocyte proliferation and regeneration. Our evidence also shows that inhibition of SDH by malonate treatment after birth extends the window of cardiomyocyte proliferation and regeneration in juvenile mice. Remarkably, extending malonate treatment to the adult mouse heart after myocardial infarction injury results in a robust regenerative response within 4 weeks after injury via promoting adult cardiomyocyte proliferation and revascularization. Our metabolite analysis after SDH inhibition by malonate induces dynamic changes in adult cardiac metabolism. CONCLUSIONS: Inhibition of SDH by malonate promotes adult cardiomyocyte proliferation, revascularization, and heart regeneration via metabolic reprogramming. These findings support a potentially important new therapeutic approach for human heart failure.

A small molecule G6PD inhibitor reveals immune dependence on pentose phosphate pathway.

Glucose is catabolized by two fundamental pathways, glycolysis to make ATP and the oxidative pentose phosphate pathway to make reduced nicotinamide adenine dinucleotide phosphate (NADPH). The first step of the oxidative pentose phosphate pathway is catalyzed by the enzyme glucose-6-phosphate dehydrogenase (G6PD). Here we develop metabolite reporter and deuterium tracer assays to monitor cellular G6PD activity. Using these, we show that the most widely cited G6PD antagonist, dehydroepiandosterone, does not robustly inhibit G6PD in cells. We then identify a small molecule (G6PDi-1) that more effectively inhibits G6PD. Across a range of cultured cells, G6PDi-1 depletes NADPH most strongly in lymphocytes. In T cells but not macrophages, G6PDi-1 markedly decreases inflammatory cytokine production. In neutrophils, it suppresses respiratory burst. Thus, we provide a cell-active small molecule tool for oxidative pentose phosphate pathway inhibition, and use it to identify G6PD as a pharmacological target for modulating immune response.

Two-stage metabolic remodelling in macrophages in response to lipopolysaccharide and interferon-γ stimulation.

In response to signals associated with infection or tissue damage, macrophages undergo a series of dynamic phenotypic changes. Here we show that during the response to LPS and interferon-γ stimulation, metabolic reprogramming in macrophages is also highly dynamic. Specifically, the TCA cycle undergoes a two-stage remodeling: the early stage is characterized by a transient accumulation of intermediates including succinate and itaconate, while the late stage is marked by the subsidence of these metabolites. The metabolic transition into the late stage is largely driven by the inhibition of pyruvate dehydrogenase complex (PDHC) and oxoglutarate dehydrogenase complex (OGDC), which is controlled by the dynamic changes in lipoylation state of both PDHC and OGDC E2 subunits and phosphorylation of PDHC E1 subunit. This dynamic metabolic reprogramming results in a transient metabolic state that strongly favors HIF-1α stabilization during the early stage, which subsides by the late stage; consistently, HIF-1α levels follow this trend. This study elucidates a dynamic and mechanistic picture of metabolic reprogramming in LPS and interferon-γ stimulated macrophages, and provides insights into how changing metabolism can regulate the functional transitions in macrophages over a course of immune response.

ANGPTL8 promotes the ability of ANGPTL3 to bind and inhibit lipoprotein lipase.

OBJECTIVE: Several members of the angiopoietin-like (ANGPTL) family of proteins, including ANGPTL3 and ANGPTL8, regulate lipoprotein lipase (LPL) activity. Deficiency in either ANGPTL3 or ANGPTL8 reduces plasma triglyceride levels and increases LPL activity, whereas overexpression of either protein does the opposite. Recent studies suggest that ANGPTL8 may functionally interact with ANGPTL3 to alter clearance of plasma triglycerides; however, the nature of this interaction has remained elusive. We tested the hypothesis that ANGPTL8 forms a complex with ANGPTL3 and that this complex is necessary for the inhibition of vascular LPL by ANGPTL3. METHODS: We analyzed the interactions of ANGPTL3 and ANGPTL8 with each other and with LPL using co-immunoprecipitation, western blotting, lipase activity assays, and the NanoBiT split-luciferase system. We also used adenovirus injection to overexpress ANGPTL3 in mice that lacked ANGPTL8. RESULTS: We found that ANGPTL3 or ANGPTL8 alone could only inhibit LPL at concentrations that far exceeded physiological levels, especially when LPL was bound to its endothelial cell receptor/transporter GPIHBP1 (glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1). Physical interaction was observed between ANGPTL3 and ANGPTL8 when the proteins were co-expressed, and co-expression with ANGPTL3 greatly enhanced the secretion of ANGPTL8. Importantly, ANGPTL3-ANGPTL8 complexes had a dramatically increased ability to inhibit LPL compared to either protein alone. Adenovirus experiments showed that 2-fold overexpression of ANGPTL3 significantly increased plasma triglycerides only in the presence of ANGPTL8. Protein interaction assays showed that ANGPTL8 greatly increased the ability of ANGPTL3 to bind LPL. CONCLUSIONS: Together, these data indicate that ANGPTL8 binds to ANGPTL3 and that this complex is necessary for ANGPTL3 to efficiently bind and inhibit LPL.