Here we report multiple lines of evidence for a comprehensive model of energy metabolism in the vertebrate eye. Metabolic flux, locations of key enzymes, and our finding that glucose enters mouse and ...zebrafish retinas mostly through photoreceptors support a conceptually new model for retinal metabolism. In this model, glucose from the choroidal blood passes through the retinal pigment epithelium to the retina where photoreceptors convert it to lactate. Photoreceptors then export the lactate as fuel for the retinal pigment epithelium and for neighboring Müller glial cells. We used human retinal epithelial cells to show that lactate can suppress consumption of glucose by the retinal pigment epithelium. Suppression of glucose consumption in the retinal pigment epithelium can increase the amount of glucose that reaches the retina. This framework for understanding metabolic relationships in the vertebrate retina provides new insights into the underlying causes of retinal disease and age-related vision loss.
Glycolysis plays a central role in organismal metabolism, but its quantitative inputs across mammalian tissues remain unclear. Here we use 13C-tracing in mice to quantify glycolytic intermediate ...sources: circulating glucose, intra-tissue glycogen, and circulating gluconeogenic precursors. Circulating glucose is the main source of circulating lactate, the primary end product of tissue glycolysis. Yet circulating glucose highly labels glycolytic intermediates in only a few tissues: blood, spleen, diaphragm, and soleus muscle. Most glycolytic intermediates in the bulk of body tissue, including liver and quadriceps muscle, come instead from glycogen. Gluconeogenesis contributes less but also broadly to glycolytic intermediates, and its flux persists with physiologic feeding (but not hyperinsulinemic clamp). Instead of suppressing gluconeogenesis, feeding activates oxidation of circulating glucose and lactate to maintain glucose homeostasis. Thus, the bulk of the body slowly breaks down internally stored glycogen while select tissues rapidly catabolize circulating glucose to lactate for oxidation throughout the body.
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•Most circulating lactate comes from circulating glucose•But most tissue glycolytic intermediates come from glycogen•This discrepancy reflects compartmentalization of glycolysis•Glycolytic flux is concentrated in red muscle and other specialized cell populations
Glycolysis, glycogen metabolism, and gluconeogenesis produce glycolytic intermediates. Here, TeSlaa et al. quantify the sources of glycolytic intermediates in mammalian tissues, finding that the bulk of the body makes glycolytic intermediates mainly using glycogen, but most glycolytic flux comes from circulating glucose and localizes to selected cell types including red muscle.
Tissues derive ATP from two pathways-glycolysis and the tricarboxylic acid (TCA) cycle coupled to the electron transport chain. Most energy in mammals is produced via TCA metabolism
. In tumours, ...however, the absolute rates of these pathways remain unclear. Here we optimize tracer infusion approaches to measure the rates of glycolysis and the TCA cycle in healthy mouse tissues, Kras-mutant solid tumours, metastases and leukaemia. Then, given the rates of these two pathways, we calculate total ATP synthesis rates. We find that TCA cycle flux is suppressed in all five primary solid tumour models examined and is increased in lung metastases of breast cancer relative to primary orthotopic tumours. As expected, glycolysis flux is increased in tumours compared with healthy tissues (the Warburg effect
), but this increase is insufficient to compensate for low TCA flux in terms of ATP production. Thus, instead of being hypermetabolic, as commonly assumed, solid tumours generally produce ATP at a slower than normal rate. In mouse pancreatic cancer, this is accommodated by the downregulation of protein synthesis, one of this tissue's major energy costs. We propose that, as solid tumours develop, cancer cells shed energetically expensive tissue-specific functions, enabling uncontrolled growth despite a limited ability to produce ATP.
Plasma metabolite concentrations and labeling enrichments are common measures of organismal metabolism. In mice, blood is often collected by tail snip sampling. Here, we systematically examined the ...effect of such sampling, relative to gold-standard sampling from an in-dwelling arterial catheter, on plasma metabolomics and stable isotope tracing. We find marked differences between the arterial and tail circulating metabolome, which arise from two major factors: handling stress and sampling site, whose effects were deconvoluted by taking a second arterial sample immediately after tail snip. Pyruvate and lactate were the most stress-sensitive plasma metabolites, rising ~14 and ~5-fold. Both acute handling stress and adrenergic agonists induce extensive, immediate production of lactate, and modest production of many other circulating metabolites, and we provide a reference set of mouse circulatory turnover fluxes with noninvasive arterial sampling to avoid such artifacts. Even in the absence of stress, lactate remains the highest flux circulating metabolite on a molar basis, and most glucose flux into the TCA cycle in fasted mice flows through circulating lactate. Thus, lactate is both a central player in unstressed mammalian metabolism and strongly produced in response to acute stress.
High-dose ascorbate (vitamin C) has shown promising anticancer activity. Two redox mechanisms have been proposed: hydrogen peroxide generation by ascorbate itself or glutathione depletion by ...dehydroascorbate (formed by ascorbate oxidation). Here we show that the metabolic effects and cytotoxicity of high-dose ascorbate in vitro result from hydrogen peroxide independently of dehydroascorbate. These effects were suppressed by selenium through antioxidant selenoenzymes including glutathione peroxidase 1 (GPX1) but not the classic ferroptosis-inhibiting selenoenzyme GPX4. Selenium-mediated protection from ascorbate was powered by NADPH from the pentose phosphate pathway. In vivo, dietary selenium deficiency resulted in significant enhancement of ascorbate activity against glioblastoma xenografts. These data establish selenoproteins as key mediators of cancer redox homeostasis. Cancer sensitivity to free radical-inducing therapies, including ascorbate, may depend on selenium, providing a dietary approach for improving their anticancer efficacy.
Selenium restriction augments ascorbate efficacy and extends lifespan in a mouse xenograft model of glioblastoma, suggesting that targeting selenium-mediated antioxidant defenses merits clinical evaluation in combination with ascorbate and other pro-oxidant therapies.
Detection of small molecule metabolites (SMM), particularly those involved in energy metabolism using MALDI-mass spectrometry imaging (MSI), is challenging due to factors including ion suppression ...from other analytes present (e.g., proteins and lipids). One potential solution to enhance SMM detection is to remove analytes that cause ion suppression from tissue sections before matrix deposition through solvent washes. Here, we systematically investigated solvent treatment conditions to improve SMM signal and preserve metabolite localization. Washing with acidic methanol significantly enhances the detection of phosphate-containing metabolites involved in energy metabolism. The improved detection is due to removing lipids and highly polar metabolites that cause ion suppression and denaturing proteins that release bound phosphate-containing metabolites. Stable isotope infusions of 13C6nicotinamide coupled to MALDI-MSI (“Iso-imaging”) in the kidney reveal patterns that indicate blood vessels, medulla, outer stripe, and cortex. We also observed different ATP:ADP raw signals across mouse kidney regions, consistent with regional differences in glucose metabolism favoring either gluconeogenesis or glycolysis. In mouse muscle, Iso-imaging using 13C6glucose shows high glycolytic flux from infused circulating glucose in type 1 and 2a fibers (soleus) and relatively lower glycolytic flux in type 2b fiber type (gastrocnemius). Thus, improved detection of phosphate-containing metabolites due to acidic methanol treatment combined with isotope tracing provides an improved way to probe energy metabolism with spatial resolution in vivo.
Viruses modulate mitochondrial processes during infection to increase biosynthetic precursors and energy output, fueling virus replication. In a surprising fashion, although it triggers mitochondrial ...fragmentation, the prevalent pathogen human cytomegalovirus (HCMV) increases mitochondrial metabolism through a yet-unknown mechanism. Here, we integrate molecular virology, metabolic assays, quantitative proteomics, and superresolution confocal microscopy to define this mechanism. We establish that the previously uncharacterized viral protein pUL13 is required for productive HCMV replication, targets the mitochondria, and functions to increase oxidative phosphorylation during infection. We demonstrate that pUL13 forms temporally tuned interactions with the mitochondrial contact site and cristae organizing system (MICOS) complex, a critical regulator of cristae architecture and electron transport chain (ETC) function. Stimulated emission depletion superresolution microscopy shows that expression of pUL13 alters cristae architecture. Indeed, using live-cell Seahorse assays, we establish that pUL13 alone is sufficient to increase cellular respiration, not requiring the presence of other viral proteins. Our findings address the outstanding question of how HCMV targets mitochondria to increase bioenergetic output and expands the knowledge of the intricate connection between mitochondrial architecture and ETC function.
Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration, the leading cause of blindness in older adults, with retinal pigment epithelium (RPE) cells playing a key ...role. To better understand the cytotoxic mechanisms underlying oxidative stress, we used cell culture and mouse models of iron overload, as iron can catalyze reactive oxygen species formation in the RPE. Iron-loading of cultured induced pluripotent stem cell-derived RPE cells increased lysosomal abundance, impaired proteolysis and reduced the activity of a subset of lysosomal enzymes, including lysosomal acid lipase (LIPA) and acid sphingomyelinase (SMPD1). In a liver-specific Hepc (Hamp) knockout murine model of systemic iron overload, RPE cells accumulated lipid peroxidation adducts and lysosomes, developed progressive hypertrophy and underwent cell death. Proteomic and lipidomic analyses revealed accumulation of lysosomal proteins, ceramide biosynthetic enzymes and ceramides. The proteolytic enzyme cathepsin D (CTSD) had impaired maturation. A large proportion of lysosomes were galectin-3 (Lgals3) positive, suggesting cytotoxic lysosomal membrane permeabilization. Collectively, these results demonstrate that iron overload induces lysosomal accumulation and impairs lysosomal function, likely due to iron-induced lipid peroxides that can inhibit lysosomal enzymes.
The purpose of this study was to present our hypothesis that aging alters metabolic function in ocular tissues. We tested the hypothesis by measuring metabolism in aged murine tissues alongside ...retinal responses to light.
Scotopic and photopic electroretinogram (ERG) responses in young (3-6 months) and aged (23-26 months) C57Bl/6J mice were recorded. Metabolic flux in retina and eyecup explants was quantified using U-13C-glucose or U-13C-glutamine with gas chromatography-mass spectrometry (GC-MS), O2 consumption rate (OCR) in a perifusion apparatus, and quantifying adenosine triphosphatase (ATP) with a bioluminescence assay.
Scotopic and photopic ERG responses were reduced in aged mice. Glucose metabolism, glutamine metabolism, OCR, and ATP pools in retinal explants were mostly unaffected in aged mice. In eyecups, glutamine usage in the Krebs Cycle decreased while glucose metabolism, OCR, and ATP pools remained stable.
Our examination of metabolism showed negligible impact of age on retina and an impairment of glutamine anaplerosis in eyecups. The metabolic stability of these tissues ex vivo suggests age-related metabolic alterations may not be intrinsic. Future experiments should focus on determining whether external factors including nutrient supply, oxygen availability, or structural changes influence ocular metabolism in vivo.
Pyruvate kinase M2 (PKM2) is a glycolytic enzyme that is expressed in cancer cells. Its role in tumor metabolism is not definitively established, but investigators have suggested that regulation of ...PKM2 activity can cause accumulation of glycolytic intermediates and increase flux through the pentose phosphate pathway. Recent evidence suggests that PKM2 also may have non-metabolic functions, including as a transcriptional co-activator in gene regulation. We reported previously that PKM2 is abundant in photoreceptor cells in mouse retinas. In the present study, we conditionally deleted PKM2 (rod-cre PKM2-KO) in rod photoreceptors and found that the absence of PKM2 causes increased expression of PKM1 in rods. Analysis of metabolic flux from U-
C glucose shows that rod-cre PKM2-KO retinas accumulate glycolytic intermediates, consistent with an overall reduction in the amount of pyruvate kinase activity. Rod-cre PKM2-KO mice also have an increased NADPH availability could favor lipid synthesis, but we found no difference in phospholipid synthesis between rod-cre PKM2 KO and PKM2-positive controls. As rod-cre PKM2-KO mice aged, we observed a significant loss of rod function, reduced thickness of the photoreceptor outer segment layer, and reduced expression of photoreceptor proteins, including PDE6β. The rod-cre PKM2-KO retinas showed greater TUNEL staining than wild-type retinas, indicating a slow retinal degeneration. In vitro analysis showed that PKM2 can regulate transcriptional activity from the PDE6β promoter in vitro. Our findings indicate that both the metabolic and transcriptional regulatory functions of PKM2 may contribute to photoreceptor structure, function, and viability.
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