Metabolic plasticity has been linked to polarized macrophage function, but mechanisms connecting specific fuels to tissue macrophage function remain unresolved. Here we apply a stable isotope ...tracing, mass spectrometry-based untargeted metabolomics approach to reveal the metabolome penetrated by hepatocyte-derived glucose and ketone bodies. In both classically and alternatively polarized macrophages, 13Cacetoacetate (AcAc) labeled ∼200 chemical features, but its reduced form D-13Cβ-hydroxybutyrate (D-βOHB) labeled almost none. 13Cglucose labeled ∼500 features, and while unlabeled AcAc competed with only ∼15% of them, the vast majority required the mitochondrial enzyme succinyl-coenzyme A-oxoacid transferase (SCOT). AcAc carbon labeled metabolites within the cytoplasmic glycosaminoglycan pathway, which regulates tissue fibrogenesis. Accordingly, livers of mice lacking SCOT in macrophages were predisposed to accelerated fibrogenesis. Exogenous AcAc, but not D-βOHB, ameliorated diet-induced hepatic fibrosis. These data support a hepatocyte-macrophage ketone shuttle that segregates AcAc from D-βOHB, coordinating the fibrogenic response to hepatic injury via mitochondrial metabolism in tissue macrophages.
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•Macrophages oxidize acetoacetate (AcAc), but not β-hydroxybutyrate•Metabolism of AcAc in macrophages extends into pathways beyond the TCA cycle•Effective AcAc competition with glucose requires its mitochondrial metabolism•Mitochondrial AcAc metabolism in macrophages protects against liver fibrosis
Puchalska et al. combine stable isotope tracing with untargeted metabolomics to identify the specific roles of the ketone bodies, acetoacetate (AcAc) and D-β-hydroxybutyrate (D-βOHB), in mediating metabolic plasticity in macrophages. They unveil a hepatocyte-macrophage ketone shuttle and show that AcAc protects the liver from high-fat-diet-induced fibrosis.
Disruption of the blood–brain barrier (BBB) is a defining and early feature of multiple sclerosis (MS) that directly damages the central nervous system (CNS), promotes immune cell infiltration, and ...influences clinical outcomes. There is an urgent need for new therapies to protect and restore BBB function, either by strengthening endothelial tight junctions or suppressing endothelial vesicular transcytosis. Although wingless integrated MMTV (Wnt)/β-catenin signaling plays an essential role in BBB formation and maintenance in healthy CNS, its role in BBB repair in neurologic diseases such as MS remains unclear. Using a Wnt/β-catenin reporter mouse and several downstream targets, we demonstrate that the Wnt/β-catenin pathway is up-regulated in CNS endothelial cells in both human MS and the mouse model experimental autoimmune encephalomyelitis (EAE). Increased Wnt/β-catenin activity in CNS blood vessels during EAE progression correlates with up-regulation of neuronal Wnt3 expression, as well as breakdown of endothelial cell junctions. Genetic inhibition of the Wnt/β-catenin pathway in CNS endothelium before disease onset exacerbates the clinical presentation of EAE, CD4⁺ T-cell infiltration into the CNS, and demyelination by increasing expression of vascular cell adhesion molecule-1 and the transcytosis protein Caveolin-1 and promoting endothelial transcytosis. However, Wnt signaling attenuation does not affect the progressive degradation of tight junction proteins or paracellular BBB leakage. These results suggest that reactivation of Wnt/β-catenin signaling in CNS vessels during EAE/MS partially restores functional BBB integrity and limits immune cell infiltration into the CNS.
Throughout the last decade, interest has intensified in intermittent fasting, ketogenic diets, and exogenous ketone therapies as prospective health-promoting, therapeutic, and performance-enhancing ...agents. However, the regulatory roles of ketogenesis and ketone metabolism on liver homeostasis remain unclear. Therefore, we sought to develop a better understanding of the metabolic consequences of hepatic ketone body metabolism by focusing on the redox-dependent interconversion of acetoacetate (AcAc) and D-β-hydroxybutyrate (D-βOHB).
Using targeted and isotope tracing high-resolution liquid chromatography-mass spectrometry, dual stable isotope tracer nuclear magnetic resonance spectroscopy-based metabolic flux modeling, and complementary physiological approaches in novel cell type-specific knockout mice, we quantified the roles of hepatocyte D-β-hydroxybutyrate dehydrogenase (BDH1), a mitochondrial enzyme required for NAD+/NADH-dependent oxidation/reduction of ketone bodies.
Exogenously administered AcAc is reduced to D-βOHB, which increases hepatic NAD+/NADH ratio and reflects hepatic BDH1 activity. Livers of hepatocyte-specific BDH1-deficient mice did not produce D-βOHB, but owing to extrahepatic BDH1, these mice nonetheless remained capable of AcAc/D-βOHB interconversion. Compared to littermate controls, hepatocyte-specific BDH1 deficient mice exhibited diminished liver tricarboxylic acid (TCA) cycle flux and impaired gluconeogenesis, but normal hepatic energy charge overall. Glycemic recovery after acute insulin challenge was impaired in knockout mice, but they were not more susceptible to starvation-induced hypoglycemia.
Ketone bodies influence liver homeostasis. While liver BDH1 is not required for whole body equilibration of AcAc and D-βOHB, loss of the ability to interconvert these ketone bodies in hepatocytes results in impaired TCA cycle flux and glucose production. Therefore, through oxidation/reduction of ketone bodies, BDH1 is a significant contributor to hepatic mitochondrial redox, liver physiology, and organism-wide ketone body homeostasis.
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•Exogenously administered acetoacetate is reduced to D-β-hydroxybutyrate, increasing hepatic NAD+/NADH ratio.•Liver BDH1 is not required for whole body equilibration of acetoacetate and D-β-hydroxybutyrate.•Hepatocyte-specific loss of BDH1 reduces hepatic TCA cycle flux, and TCA-cycle sourced gluconeogenesis.•Hepatocyte-specific loss of BDH1 impairs glycemic recovery without provoking starvation-induced hypoglycemia.
The pathogenesis of human T2D is linked to the progression of nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH). In the normal fed state, impairments of hepatocyte ketogenesis and ...Kupffer cell mitochondrial metabolism of ketone bodies conspire to drive hepatic injury, and lobular fibrosis - key features increasing insulin resistance and T2D. Preliminary data suggest that a local circuit of intrahepatic ketone metabolism may protect the liver from lobular injury, which involves distribution of ketone body carbon into numerous metabolic pathways. Nonetheless, the metabolic products of local hepatic ketone body metabolism that confer protection to the liver, and which are therefore linked to insulin resistance and T2D, remain unknown. Here, we use stable isotope tracing untargeted metabolomics (ITUM) to reveal the chemical space penetrated in vivo by D-13Cβ-hydroxybutyrate (D-βOHB) and its oxidized redox partner 13Cacetoacetate (AcAc). A kinetic study revealed rapid clearance (∼30 min) of both ketones from the bloodstream when introduced as intraperitoneal boluses into wild type mice. Many 13C-labeled metabolites were tissue-specific, particularly distinctly acetylated amino acids. Labeling of TCA cycle intermediates by 13C-labeled ketone bodies also varied by tissue, and by the specific labeled ketone body delivered. Finally, recurrent administration of unlabeled AcAc protected mice from high fat, fibrogenic diet-induced hepatic injury and fibrosis, while unlabeled D-βOHB exacerbated injury and fibrosis.
In conclusion, the two ketone bodies AcAc and D-βOHB, strikingly exhibit distinct metabolic and phenotypic effects in the context of NAFLD/NASH, and insulin resistance.
Disclosure
P. Puchalska: None. J.E. Lengfeld: None. D.B. Stagg: None. P.A. Crawford: Consultant; Spouse/Partner; Medtronic.
Disruption of the blood-brain barrier (BBB) is a defining and early feature of multiple sclerosis (MS) that directly damages the central nervous system (CNS), promotes immune cell infiltration, and ...influences clinical outcomes. There is an urgent need for new therapies to protect and restore BBB function, either by strengthening endothelial tight junctions or suppressing endothelial vesicular transcytosis. Although wingless integrated MMTV (Wnt)/β-catenin signaling plays an essential role in BBB formation and maintenance in healthy CNS, its role in BBB repair in neurologic diseases such as MS remains unclear. Using a Wnt/β-catenin reporter mouse and several downstream targets, we demonstrate that the Wnt/β-catenin pathway is up-regulated in CNS endothelial cells in both human MS and the mouse model experimental autoimmune encephalomyelitis (EAE). Increased Wnt/β-catenin activity in CNS blood vessels during EAE progression correlates with up-regulation of neuronal Wnt3 expression, as well as breakdown of endothelial cell junctions. Genetic inhibition of the Wnt/β-catenin pathway in CNS endothelium before disease onset exacerbates the clinical presentation of EAE, CD4+ T-cell infiltration into the CNS, and demyelination by increasing expression of vascular cell adhesion molecule-1 and the transcytosis protein Caveolin-1 and promoting endothelial transcytosis. However, Wnt signaling attenuation does not affect the progressive degradation of tight junction proteins or paracellular BBB leakage. These results suggest that reactivation of Wnt/β-catenin signaling in CNS vessels during EAE/MS partially restores functional BBB integrity and limits immune cell infiltration into the CNS.
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