Publications by authors named "Jianhai Du"

59 Publications

Proline metabolism and transport in retinal health and disease.

Amino Acids 2021 Apr 19. Epub 2021 Apr 19.

Department of Ophthalmology, University of Washington, Seattle, WA, 98109, USA.

The retina is one of the most energy-demanding tissues in the human body. Photoreceptors in the outer retina rely on nutrient support from the neighboring retinal pigment epithelium (RPE), a monolayer of epithelial cells that separate the retina and choroidal blood supply. RPE dysfunction or cell death can result in photoreceptor degeneration, leading to blindness in retinal degenerative diseases including some inherited retinal degenerations and age-related macular degeneration (AMD). In addition to having ready access to rich nutrients from blood, the RPE is also supplied with lactate from adjacent photoreceptors. Moreover, RPE can phagocytose lipid-rich outer segments for degradation and recycling on a daily basis. Recent studies show RPE cells prefer proline as a major metabolic substrate, and they are highly enriched for the proline transporter, SLC6A20. In contrast, dysfunctional or poorly differentiated RPE fails to utilize proline. RPE uses proline to fuel mitochondrial metabolism, synthesize amino acids, build the extracellular matrix, fight against oxidative stress, and sustain differentiation. Remarkably, the neural retina rarely imports proline directly, but it uptakes and utilizes intermediates and amino acids derived from proline catabolism in the RPE. Mutations of genes in proline metabolism are associated with retinal degenerative diseases, and proline supplementation is reported to improve RPE-initiated vision loss. This review will cover proline metabolism in RPE and highlight the importance of proline transport and utilization in maintaining retinal metabolism and health.
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http://dx.doi.org/10.1007/s00726-021-02981-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054134PMC
April 2021

Effect of selectively knocking down key metabolic genes in Müller glia on photoreceptor health.

Glia 2021 Apr 9. Epub 2021 Apr 9.

Discipline of Ophthalmology, Sydney Medical School, The University of Sydney, Save Sight Institute, Sydney, New South Wales, Australia.

The importance of Müller glia for retinal homeostasis suggests that they may have vulnerabilities that lead to retinal disease. Here, we studied the effect of selectively knocking down key metabolic genes in Müller glia on photoreceptor health. Immunostaining indicated that murine Müller glia expressed insulin receptor (IR), hexokinase 2 (HK2) and phosphoglycerate dehydrogenase (PHGDH) but very little pyruvate dehydrogenase E1 alpha 1 (PDH-E1α) and lactate dehydrogenase A (LDH-A). We crossed Müller glial cell-CreER (MC-CreER) mice with transgenic mice carrying a floxed IR, HK2, PDH-E1α, LDH-A, or PHGDH gene to study the effect of selectively knocking down key metabolic genes in Müller glia cells on retinal health. Selectively knocking down IR, HK2, or PHGDH led to photoreceptor degeneration and reduced electroretinographic responses. Supplementing exogenous l-serine prevented photoreceptor degeneration and improved retinal function in MC-PHGDH knockdown mice. We unexpectedly found that the levels of retinal serine and glycine were not reduced but, on the contrary, highly increased in MC-PHGDH knockdown mice. Moreover, dietary serine supplementation, while rescuing the retinal phenotypes caused by genetic deletion of PHGDH in Müller glial cells, restored retinal serine and glycine homeostasis probably through regulation of serine transport. No retinal abnormalities were observed in MC-CreER mice crossed with PDH-E1α- or LDH-A-floxed mice despite Cre expression. Our findings suggest that Müller glia do not complete glycolysis but use glucose to produce serine to support photoreceptors. Supplementation with exogenous serine is effective in preventing photoreceptor degeneration caused by PHGDH deficiency in Müller glia.
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http://dx.doi.org/10.1002/glia.24005DOI Listing
April 2021

Tracing Nitrogen Metabolism in Mouse Tissues with Gas Chromatography-Mass Spectrometry.

Bio Protoc 2021 Feb 20;11(4):e3925. Epub 2021 Feb 20.

Department of Ophthalmology and Visual Sciences, West Virginia University, Morgantown, USA.

Nitrogen-containing metabolites including ammonia, amino acids, and nucleotides, are essential for cell metabolism, growth, and neural transmission. Nitrogen metabolism is tightly coordinated with carbon metabolism in the breakdown and biosynthesis of amino acids and nucleotides. Both nuclear magnetic resonance spectroscopy and mass spectrometry including gas chromatography-mass spectrometry (GC MS) and liquid chromatography (LC MS) have been used to measure nitrogen metabolism. Here we describe a protocol to trace nitrogen metabolism in multiple mouse tissues using N-ammonia coupled with GC MS. This protocol includes detailed procedures in tracer injection, tissue preparation, metabolite extraction, GC MS analysis and natural abundance corrections. This protocol will provide a useful tool to study tissue-specific nitrogen in metabolically active tissues such as the retina, brain, liver, and tumor.
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http://dx.doi.org/10.21769/BioProtoc.3925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952953PMC
February 2021

Mutant NMNAT1 leads to a retina-specific decrease of NAD+ accompanied by increased poly(ADP-ribose) in a mouse model of NMNAT1-associated retinal degeneration.

Hum Mol Genet 2021 Mar 11. Epub 2021 Mar 11.

Department of Ophthalmology and Visual Sciences, West Virginia University, Morgantown, WV, 26506, USA.

Nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) is required for nuclear NAD+ biosynthesis in all nucleated cells, and despite its functional ubiquity, mutations in this gene lead to an isolated retinal degeneration. The mechanisms underlying how mutant NMNAT1 causes disease are not well understood, nor is the reason why the pathology is confined to the retina. Using a mouse model of NMNAT1-associated retinal degeneration that harbors the p.Val9Met mutation, we tested the hypothesis that decreased function of mutant NMNAT1 has a greater effect on the levels of NAD+ in the retina than elsewhere in the body. Measurements by liquid chromatography with tandem mass spectrometry (LC-MS/MS) showed an early and sustained decrease of NAD+ in mutant retinas that was not observed in other tissues. To understand how consumers of nuclear NAD+ are affected by the reduced availability of NAD+ in mutant retinas, poly(ADP-ribose) polymerase (PARP) and nuclear sirtuin activity were evaluated. PARP activity was elevated during disease progression, as evidenced by overproduction of poly(ADP-ribose) (PAR) in photoreceptors, whereas histone deacetylation (HDAC) activity of nuclear sirtuins was not altered. We hypothesized that PARP could be activated because of elevated levels of oxidative stress; however, we did not observe oxidative DNA damage, lipid peroxidation, or a low glutathione (GSH) to oxidized glutathione (GSSG) ratio. TUNEL staining revealed that photoreceptors appear to ultimately die by apoptosis, although the low NAD+ levels and overproduction of PAR suggests that cell death may include aspects of the parthanatos cell death pathway.
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http://dx.doi.org/10.1093/hmg/ddab070DOI Listing
March 2021

Absence of retbindin blocks glycolytic flux, disrupts metabolic homeostasis, and leads to photoreceptor degeneration.

Proc Natl Acad Sci U S A 2021 Feb;118(6)

Department of Biomedical Engineering, University of Houston, Houston, TX 77204;

We previously reported a model of progressive retinal degeneration resulting from the knockout of the retina-specific riboflavin binding protein, retbindin ( ). We also demonstrated a reduction in neural retinal flavins as a result of the elimination of RTBDN. Given the role of flavins in metabolism, herein we investigated the underlying mechanism of this retinal degeneration by performing metabolomic analyses on predegeneration at postnatal day (P) 45 and at the onset of functional degeneration in the P120 retinas. Metabolomics of hydrophilic metabolites revealed that individual glycolytic products accumulated in the P45 neural retinas along with the elevation of pentose phosphate pathway, while TCA cycle intermediates remained unchanged. This was confirmed by using C-labeled flux measurements and immunoblotting, revealing that the key regulatory step of phosphoenolpyruvate to pyruvate was inhibited via down-regulation of the tetrameric pyruvate kinase M2 (PKM2). Separate metabolite assessments revealed that almost all intermediates of acylcarnitine fatty acid oxidation, ceramides, sphingomyelins, and multiple toxic metabolites were significantly elevated in the predegeneration neural retina. Our data show that lack of RTBDN, and hence reduction in flavins, forced the neural retina into repurposing glucose for free-radical mitigation over ATP production. However, such sustained metabolic reprogramming resulted in an eventual metabolic collapse leading to neurodegeneration.
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http://dx.doi.org/10.1073/pnas.2018956118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8017963PMC
February 2021

Xanthine Oxidase Drives Hemolysis and Vascular Malfunction in Sickle Cell Disease.

Arterioscler Thromb Vasc Biol 2021 Feb 3;41(2):769-782. Epub 2020 Dec 3.

Department of Pharmacology and Chemical Biology (H.M.S., D.A.V., A.C.S.), University of Pittsburgh, PA.

Objective: Chronic hemolysis is a hallmark of sickle cell disease (SCD) and a driver of vasculopathy; however, the mechanisms contributing to hemolysis remain incompletely understood. Although XO (xanthine oxidase) activity has been shown to be elevated in SCD, its role remains unknown. XO binds endothelium and generates oxidants as a byproduct of hypoxanthine and xanthine catabolism. We hypothesized that XO inhibition decreases oxidant production leading to less hemolysis. Approach and Results: Wild-type mice were bone marrow transplanted with control (AA) or sickle (SS) Townes bone marrow. After 12 weeks, mice were treated with 10 mg/kg per day of febuxostat (Uloric), Food and Drug Administration-approved XO inhibitor, for 10 weeks. Hematologic analysis demonstrated increased hematocrit, cellular hemoglobin, and red blood cells, with no change in reticulocyte percentage. Significant decreases in cell-free hemoglobin and increases in haptoglobin suggest XO inhibition decreased hemolysis. Myographic studies demonstrated improved pulmonary vascular dilation and blunted constriction, indicating improved pulmonary vasoreactivity, whereas pulmonary pressure and cardiac function were unaffected. The role of hepatic XO in SCD was evaluated by bone marrow transplanting hepatocyte-specific XO knockout mice with SS Townes bone marrow. However, hepatocyte-specific XO knockout, which results in >50% diminution in circulating XO, did not affect hemolysis levels or vascular function, suggesting hepatocyte-derived elevation of circulating XO is not the driver of hemolysis in SCD.

Conclusions: Ten weeks of febuxostat treatment significantly decreased hemolysis and improved pulmonary vasoreactivity in a mouse model of SCD. Although hepatic XO accounts for >50% of circulating XO, it is not the source of XO driving hemolysis in SCD.
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http://dx.doi.org/10.1161/ATVBAHA.120.315081DOI Listing
February 2021

Metabolic Features of Mouse and Human Retinas: Rods versus Cones, Macula versus Periphery, Retina versus RPE.

iScience 2020 Nov 14;23(11):101672. Epub 2020 Oct 14.

Departments of Ophthalmology and Biochemistry, West Virginia University, WVU Eye Institute, One Medical Center Dr, PO Box 9193, Morgantown, WV 26506, USA.

Photoreceptors, especially cones, which are enriched in the human macula, have high energy demands, making them vulnerable to metabolic stress. Metabolic dysfunction of photoreceptors and their supporting retinal pigment epithelium (RPE) is an important underlying cause of degenerative retinal diseases. However, how cones and the macula support their exorbitant metabolic demand and communicate with RPE is unclear. By profiling metabolite uptake and release and analyzing metabolic genes, we have found cone-rich retinas and human macula share specific metabolic features with upregulated pathways in pyruvate metabolism, mitochondrial TCA cycle, and lipid synthesis. Human neural retina and RPE have distinct but complementary metabolic features. Retinal metabolism centers on NADH production and neurotransmitter biosynthesis. The retina needs aspartate to sustain its aerobic glycolysis and mitochondrial metabolism. RPE metabolism is directed toward NADPH production and biosynthesis of acetyl-rich metabolites, serine, and others. RPE consumes multiple nutrients, including proline, to produce metabolites for the retina.
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http://dx.doi.org/10.1016/j.isci.2020.101672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644940PMC
November 2020

Selective knockdown of hexokinase 2 in rods leads to age-related photoreceptor degeneration and retinal metabolic remodeling.

Cell Death Dis 2020 10 20;11(10):885. Epub 2020 Oct 20.

Save Sight Institute, Discipline of Ophthalmology, Sydney Medical School, University of Sydney, Sydney, NSW, 2000, Australia.

Photoreceptors, the primary site of phototransduction in the retina, require energy and metabolites to constantly renew their outer segments. They preferentially consume most glucose through aerobic glycolysis despite possessing abundant mitochondria and enzymes for oxidative phosphorylation (OXPHOS). Exactly how photoreceptors balance aerobic glycolysis and mitochondrial OXPHOS to regulate their survival is still unclear. We crossed rhodopsin-Cre mice with hexokinase 2 (HK2)-floxed mice to study the effect of knocking down HK2, the first rate-limiting enzyme in glycolysis, on retinal health and metabolic remodeling. Immunohistochemistry and Western blots were performed to study changes in photoreceptor-specific proteins and key enzymes in glycolysis and the tricarboxylic acid (TCA) cycle. Changes in retinal structure and function were studied by optical coherence tomography and electroretinography. Mass spectrometry was performed to profile changes in C-glucose-derived metabolites in glycolysis and the TCA cycle. We found that knocking down HK2 in rods led to age-related photoreceptor degeneration, evidenced by reduced expression of photoreceptor-specific proteins, age-related reductions of the outer nuclear layer, photoreceptor inner and outer segments and impaired electroretinographic responses. Loss of HK2 in rods led to upregulation of HK1, phosphorylation of pyruvate kinase muscle isozyme 2, mitochondrial stress proteins and enzymes in the TCA cycle. Mass spectrometry found that the deletion of HK2 in rods resulted in accumulation of C-glucose along with decreased pyruvate and increased metabolites in the TCA cycle. Our data suggest that HK2-mediated aerobic glycolysis is indispensable for the maintenance of photoreceptor structure and function and that long-term inhibition of glycolysis leads to photoreceptor degeneration.
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http://dx.doi.org/10.1038/s41419-020-03103-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576789PMC
October 2020

Inhibition of Mitochondrial Respiration Impairs Nutrient Consumption and Metabolite Transport in Human Retinal Pigment Epithelium.

J Proteome Res 2021 01 25;20(1):909-922. Epub 2020 Sep 25.

Department of Ophthalmology, West Virginia University, Morgantown, West Virginia 26506, United States.

Mitochondrial respiration in mammalian cells not only generates ATP to meet their own energy needs but also couples with biosynthetic pathways to produce metabolites that can be exported to support neighboring cells. However, how defects in mitochondrial respiration influence these biosynthetic and exporting pathways remains poorly understood. Mitochondrial dysfunction in retinal pigment epithelium (RPE) cells is an emerging contributor to the death of their neighboring photoreceptors in degenerative retinal diseases including age-related macular degeneration. In this study, we used targeted-metabolomics and C tracing to investigate how inhibition of mitochondrial respiration influences the intracellular and extracellular metabolome. We found inhibition of mitochondrial respiration strikingly influenced both the intracellular and extracellular metabolome in primary RPE cells. Intriguingly, the extracellular metabolic changes sensitively reflected the intracellular changes. These changes included substantially enhanced glucose consumption and lactate production; reduced release of pyruvate, citrate, and ketone bodies; and massive accumulation of multiple amino acids and nucleosides. In conclusion, these findings reveal a metabolic signature of nutrient consumption and release in mitochondrial dysfunction in RPE cells. Testing medium metabolites provides a sensitive and noninvasive method to assess mitochondrial function in nutrient utilization and transport.
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http://dx.doi.org/10.1021/acs.jproteome.0c00690DOI Listing
January 2021

Metabolic signature of eyelid basal cell carcinoma.

Exp Eye Res 2020 09 7;198:108140. Epub 2020 Jul 7.

Department of Ophthalmology, West Virginia University, Morgantown, WV, 26506, USA; Department of Biochemistry, West Virginia University, Morgantown, WV, 26506, USA. Electronic address:

Purpose: Eyelid basal cell carcinoma (BCC) is the most common eyelid malignancy. Metabolic reprogramming is critical in tumorigenesis, but the metabolic feature of eyelid BCC remains elusive. In this study, we aim to reveal the metabolic profile in eyelid BCC using targeted metabolomics. Eyelid samples were collected from patients who had removal of BCC and from control patients who underwent blepharoplasty. Multivariate analysis of metabolomics data distinguished the two groups, indicating that eyelid BCC has significantly different metabolome than the healthy tissue. We found 16 increased and 11 decreased metabolites in the BCC tissues. These metabolites were highly enriched in the metabolism of nicotinamide adenine dinucleotide (NAD), glutathione metabolism, polyamine metabolism, and the metabolism of glycine, serine, threonine, arginine and proline. amino acid metabolism. Metabolites from NAD metabolism (Nicotinamide; Nicotinamide riboside; N1-Methylnicotinamide) had the highest sensitivity, specificity, and prediction accuracy in a prediction model for eyelid BCC. In conclusion, eyelid BCC has a signature change of cell metabolome. Metabolites in NAD metabolic pathways could potentially be biomarkers or therapeutic targets for eyelid BCC.
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http://dx.doi.org/10.1016/j.exer.2020.108140DOI Listing
September 2020

The retina and retinal pigment epithelium differ in nitrogen metabolism and are metabolically connected.

J Biol Chem 2020 02 17;295(8):2324-2335. Epub 2020 Jan 17.

Department of Ophthalmology, West Virginia University, Morgantown, West Virginia 26506; Department of Biochemistry, West Virginia University, Morgantown, West Virginia 26506. Electronic address:

Defects in energy metabolism in either the retina or the immediately adjacent retinal pigment epithelium (RPE) underlie retinal degeneration, but the metabolic dependence between retina and RPE remains unclear. Nitrogen-containing metabolites such as amino acids are essential for energy metabolism. Here, we found that N-labeled ammonium is predominantly assimilated into glutamine in both the retina and RPE/choroid [N]Ammonium tracing show that, like the brain, the retina can synthesize asparagine from ammonium, but RPE/choroid and the liver cannot. However, unless present at toxic concentrations, ammonium cannot be recycled into glutamate in the retina and RPE/choroid. Tracing with N-labeled amino acids show that the retina predominantly uses aspartate transaminase for synthesis of glutamate, glutamine, and aspartate, whereas RPE uses multiple transaminases to utilize and synthesize amino acids. Retina consumes more leucine than RPE, but little leucine is catabolized. The synthesis of serine and glycine is active in RPE but limited in the retina. RPE, but not the retina, uses alanine as mitochondrial substrates through mitochondrial pyruvate carrier. However, when the mitochondrial pyruvate carrier is inhibited, alanine may directly enter the retinal mitochondria but not those of RPE. In conclusion, our results demonstrate that the retina and RPE differ in nitrogen metabolism and highlight that the RPE supports retinal metabolism through active amino acid metabolism.
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http://dx.doi.org/10.1074/jbc.RA119.011727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039560PMC
February 2020

Metabolic Deregulation of the Blood-Outer Retinal Barrier in Retinitis Pigmentosa.

Cell Rep 2019 07;28(5):1323-1334.e4

Department of Ophthalmology and Visual Sciences, University of Louisville Health Sciences Center, Louisville, KY 40202, USA; James Graham Brown Cancer Center, University of Louisville Health Sciences Center, Louisville, KY 40202, USA; Birth Defects Center, University of Louisville Health Sciences Center, Louisville, KY 40202, USA. Electronic address:

Retinitis pigmentosa (RP) initiates with diminished rod photoreceptor function, causing peripheral and night-time vision loss. However, subsequent loss of cone function and high-resolution daylight and color vision is most debilitating. Visual pigment-rich photoreceptor outer segments (OS) undergo phagocytosis by the retinal pigment epithelium (RPE), and the RPE also acts as a blood-outer retinal barrier transporting nutrients, including glucose, to photoreceptors. We provide evidence that contact between externalized phosphatidylserine (PS) on OS tips and apical RPE receptors activates Akt, linking phagocytosis with glucose transport to photoreceptors for new OS synthesis. As abundant mutant rod OS tips shorten in RP, Akt activation is lost, and onset of glucose metabolism in the RPE and diminished glucose transport combine to cause photoreceptor starvation and accompanying retinal metabolome changes. Subretinal injection of OS tip mimetics displaying PS restores Akt activation, glucose transport, and cone function in end-stage RP after rods are lost.
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http://dx.doi.org/10.1016/j.celrep.2019.06.093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693665PMC
July 2019

Proline mediates metabolic communication between retinal pigment epithelial cells and the retina.

J Biol Chem 2019 06 19;294(26):10278-10289. Epub 2019 May 19.

From the Departments of Ophthalmology and

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells between the choroid and the retina. RPE dysfunction underlies many retinal degenerative diseases, including age-related macular degeneration, the leading cause of age-related blindness. To perform its various functions in nutrient transport, phagocytosis of the outer segment, and cytokine secretion, the RPE relies on an active energy metabolism. We previously reported that human RPE cells prefer proline as a nutrient and transport proline-derived metabolites to the apical, or retinal, side. In this study, we investigated how RPE utilizes proline and why proline is a preferred substrate. By using [C]proline labeling both and , we found that the retina rarely uses proline directly, whereas the RPE utilizes it at a high rate, exporting proline-derived mitochondrial intermediates for use by the retina. We observed that in primary human RPE cell culture, proline is the only amino acid whose uptake increases with cellular maturity. In human RPE, proline was sufficient to stimulate serine synthesis, increase reductive carboxylation, and protect against oxidative damage. Blocking proline catabolism in RPE impaired glucose metabolism and GSH production. Notably, in an acute model of RPE-induced retinal degeneration, dietary proline improved visual function. In conclusion, proline is an important nutrient that supports RPE metabolism and the metabolic demand of the retina.
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http://dx.doi.org/10.1074/jbc.RA119.007983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6664195PMC
June 2019

Human macular Müller cells rely more on serine biosynthesis to combat oxidative stress than those from the periphery.

Elife 2019 04 30;8. Epub 2019 Apr 30.

Save Sight Institute, Sydney Medical School, Faculty of Medicine and Health, The University of Sydney, Sydney, Australia.

The human macula is more susceptible than the peripheral retina to developing blinding conditions such as age-related macular degeneration, diabetic retinopathy. A key difference between them may be the nature of their Müller cells. We found primary cultured Müller cells from macula and peripheral retina display significant morphological and transcriptomic differences. Macular Müller cells expressed more phosphoglycerate dehydrogenase (PHGDH, a rate-limiting enzyme in serine synthesis) than peripheral Müller cells. The serine synthesis, glycolytic and mitochondrial function were more activated in macular than peripheral Müller cells. Serine biosynthesis is critical in defending against oxidative stress. Intracellular reactive oxygen species and glutathione levels were increased in primary cultured macular Müller cells which were more susceptible to oxidative stress after inhibition of PHGDH. Our findings indicate serine biosynthesis is a critical part of the macular defence against oxidative stress and suggest dysregulation of this pathway as a potential cause of macular pathology.
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http://dx.doi.org/10.7554/eLife.43598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533082PMC
April 2019

Hepatocyte-Specific Ablation or Whole-Body Inhibition of Xanthine Oxidoreductase in Mice Corrects Obesity-Induced Systemic Hyperuricemia Without Improving Metabolic Abnormalities.

Diabetes 2019 06 1;68(6):1221-1229. Epub 2019 Apr 1.

Department of Physiology and Pharmacology, Health Sciences Center, West Virginia University, Morgantown, WV

Systemic hyperuricemia (HyUA) in obesity/type 2 diabetes facilitated by elevated activity of xanthine oxidoreductase (XOR), which is the sole source of uric acid (UA) in mammals, has been proposed to contribute to the pathogenesis of insulin resistance/dyslipidemia in obesity. Here, the effects of hepatocyte-specific ablation of , the gene encoding XOR (HXO), and whole-body pharmacologic inhibition of XOR (febuxostat) on obesity-induced insulin resistance/dyslipidemia were assessed. Deletion of hepatocyte substantially lowered liver and plasma UA concentration. When exposed to an obesogenic diet, HXO and control floxed (FLX) mice became equally obese, but systemic HyUA was absent in HXO mice. Despite this, obese HXO mice became as insulin resistant and dyslipidemic as obese FLX mice. Similarly, febuxostat dramatically lowered plasma and tissue UA and XOR activity in obese wild-type mice without altering obesity-associated insulin resistance/dyslipidemia. These data demonstrate that hepatocyte XOR activity is a critical determinant of systemic UA homeostasis, that deletion of hepatocyte is sufficient to prevent systemic HyUA of obesity, and that neither prevention nor correction of HyUA improves insulin resistance/dyslipidemia in obesity. Thus, systemic HyUA, although clearly a biomarker of the metabolic abnormalities of obesity, does not appear to be causative.
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http://dx.doi.org/10.2337/db18-1198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610025PMC
June 2019

Abnormal mTORC1 signaling leads to retinal pigment epithelium degeneration.

Theranostics 2019 30;9(4):1170-1180. Epub 2019 Jan 30.

Eye Institute, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, China.

Retinal pigment epithelial (RPE) degeneration is potentially involved in the pathogenesis of several retinal degenerative diseases. mTORC1 signaling is shown as a crucial regulator of many biological processes and disease progression. In this study, we aimed at investigating the role of mTORC1 signaling in RPE degeneration. : Western blots were conducted to detect mTORC1 expression pattern during RPE degeneration. Cre-loxP system was used to generate RPE-specific mTORC1 activation mice. Fundus, immunofluorescence staining, transmission electron microscopy, and targeted metabolomic analysis were conducted to determine the effects of mTORC1 activation on RPE degeneration Electroretinography, spectral-domain optical coherence tomography, and histological experiments were conducted to determine the effects of mTORC1 activation on choroidal and retinal function . : RPE-specific activation of mTORC1 led to RPE degeneration as shown by the loss of RPE-specific marker, compromised cell junction integrity, and intracellular accumulation of lipid droplets. RPE degeneration further led to abnormal choroidal and retinal function. The inhibition of mTORC1 signaling with rapamycin could partially reverse RPE degeneration. Targeted metabolomics analysis further revealed that mTORC1 activation affected the metabolism of purine, carboxylic acid, and niacin in RPE. : This study revealed that abnormal activation of mTORC1 signaling leads to RPE degeneration, which could provide a promising target for the treatment of RPE dysfunction-related diseases.
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http://dx.doi.org/10.7150/thno.26281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401408PMC
January 2020

Loss of MPC1 reprograms retinal metabolism to impair visual function.

Proc Natl Acad Sci U S A 2019 02 11;116(9):3530-3535. Epub 2019 Feb 11.

Department of Ophthalmology, West Virginia University, Morgantown, WV 26506;

Glucose metabolism in vertebrate retinas is dominated by aerobic glycolysis (the "Warburg Effect"), which allows only a small fraction of glucose-derived pyruvate to enter mitochondria. Here, we report evidence that the small fraction of pyruvate in photoreceptors that does get oxidized by their mitochondria is required for visual function, photoreceptor structure and viability, normal neuron-glial interaction, and homeostasis of retinal metabolism. The mitochondrial pyruvate carrier (MPC) links glycolysis and mitochondrial metabolism. Retina-specific deletion of MPC1 results in progressive retinal degeneration and decline of visual function in both rod and cone photoreceptors. Using targeted-metabolomics and C tracers, we found that MPC1 is required for cytosolic reducing power maintenance, glutamine/glutamate metabolism, and flexibility in fuel utilization.
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http://dx.doi.org/10.1073/pnas.1812941116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397593PMC
February 2019

Modulating GLUT1 expression in retinal pigment epithelium decreases glucose levels in the retina: impact on photoreceptors and Müller glial cells.

Am J Physiol Cell Physiol 2019 01 21;316(1):C121-C133. Epub 2018 Nov 21.

Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University , Philadelphia, Pennsylvania.

The retina is one of the most metabolically active tissues in the body and utilizes glucose to produce energy and intermediates required for daily renewal of photoreceptor cell outer segments. Glucose transporter 1 (GLUT1) facilitates glucose transport across outer blood retinal barrier (BRB) formed by the retinal pigment epithelium (RPE) and the inner BRB formed by the endothelium. We used conditional knockout mice to study the impact of reducing glucose transport across the RPE on photoreceptor and Müller glial cells. Transgenic mice expressing Cre recombinase under control of the Bestrophin1 ( Best1) promoter were bred with Glut1 mice to generate Tg-Best1-Cre:Glut1 mice ( RPEΔGlut1). The RPEΔGlut1 mice displayed a mosaic pattern of Cre expression within the RPE that allowed us to analyze mice with ~50% ( RPEΔGlut1) recombination and mice with >70% ( RPEΔGlut1) recombination separately. Deletion of GLUT1 from the RPE did not affect its carrier or barrier functions, indicating that the RPE utilizes other substrates to support its metabolic needs thereby sparing glucose for the outer retina. RPEΔGlut1 mice had normal retinal morphology, function, and no cell death; however, where GLUT1 was absent from a span of RPE greater than 100 µm, there was shortening of the photoreceptor cell outer segments. RPEΔGlut1 mice showed outer segment shortening, cell death of photoreceptors, and activation of Müller glial cells. The severe phenotype seen in RPEΔGlut1 mice indicates that glucose transport via the GLUT1 transporter in the RPE is required to meet the anabolic and catabolic requirements of photoreceptors and maintain Müller glial cells in a quiescent state.
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http://dx.doi.org/10.1152/ajpcell.00410.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383144PMC
January 2019

Flavin homeostasis in the mouse retina during aging and degeneration.

J Nutr Biochem 2018 12 15;62:123-133. Epub 2018 Sep 15.

Department of Biomedical Engineering, University of Houston, Houston, TX 77204. Electronic address:

Involvement of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in cellular homeostasis has been well established for tissues other than the retina. Here, we present an optimized method to effectively extract and quantify FAD and FMN from a single neural retina and its corresponding retinal pigment epithelium (RPE). Optimizations led to detection efficiency of 0.1 pmol for FAD and FMN while 0.01 pmol for riboflavin. Interestingly, levels of FAD and FMN in the RPE were found to be 1.7- and 12.5-fold higher than their levels in the retina, respectively. Both FAD and FMN levels in the RPE and retina gradually decline with age and preceded the age-dependent drop in the functional competence of the retina as measured by electroretinography. Further, quantifications of retinal levels of FAD and FMN in different mouse models of retinal degeneration revealed differential metabolic requirements of these two factors in relation to the rate and degree of photoreceptor degeneration. We also found twofold reductions in retinal levels of FAD and FMN in two mouse models of diabetic retinopathy. Altogether, our results suggest that retinal levels of FAD and FMN can be used as potential markers to determine state of health of the retina in general and more specifically the photoreceptors.
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http://dx.doi.org/10.1016/j.jnutbio.2018.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162609PMC
December 2018

Metabolic signature of the aging eye in mice.

Neurobiol Aging 2018 11 7;71:223-233. Epub 2018 Aug 7.

Department of Ophthalmology, West Virginia University, Morgantown, WV, USA; Department of Biochemistry, West Virginia University, Morgantown, WV, USA. Electronic address:

Aging is a major risk factor for age-related ocular diseases including age-related macular degeneration in the retina and retinal pigment epithelium (RPE), cataracts in the lens, glaucoma in the optic nerve, and dry eye syndrome in the cornea. We used targeted metabolomics to analyze metabolites from young (6 weeks) and old (73 weeks) eyes in C57 BL6/J mice. Old mice had diminished electroretinogram responses and decreased number of photoreceptors in their retinas. Among the 297 detected metabolites, 45-114 metabolites are significantly altered in aged eye tissues, mostly in the neuronal tissues (retina and optic nerve) and less in cornea, RPE/choroid, and lens. We noted that changes of metabolites in mitochondrial metabolism and glucose metabolism are common features in the aged retina, RPE/choroid, and optic nerve. The aging retina, cornea, and optic nerve also share similar changes in Nicotinamide adenine dinucleotide (NAD), 1-methylnicotinamides, 3-methylhistidine, and other methylated metabolites. Metabolites in taurine metabolism are strikingly influenced by aging in the cornea and lens. In conclusion, the aging eye has both common and tissue-specific metabolic signatures. These changes may be attributed to dysregulated mitochondrial metabolism, reprogrammed glucose metabolism and impaired methylation in the aging eye. Our findings provide biochemical insights into the mechanisms of age-related ocular changes.
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http://dx.doi.org/10.1016/j.neurobiolaging.2018.07.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162115PMC
November 2018

Impact of euthanasia, dissection and postmortem delay on metabolic profile in mouse retina and RPE/choroid.

Exp Eye Res 2018 09 1;174:113-120. Epub 2018 Jun 1.

Department of Ophthalmology, West Virginia University, Morgantown, WV 26506, USA; Department of Biochemistry, West Virginia University, Morgantown, WV 26506, USA. Electronic address:

Metabolomics studies in the retina and retinal pigment epithelium (RPE) in animal models or postmortem donors are essential to understanding the retinal metabolism and to revealing the underlying mechanisms of retinal degenerative diseases. We have studied how different methods of euthanasia (CO or cervical dislocation) different isolation procedures and postmortem delay affect metabolites in mouse retina and RPE/choroid using LC MS/MS and GC MS. Compared with cervical dislocation, CO exposure for 5 min dramatically degrades ATP and GTP into purine metabolites in the retina while raising intermediates in glucose metabolism and amino acids in the RPE/choroid. Isolation in cold buffer containing glucose has the least change in metabolites. Postmortem delay time-dependently and differentially impacts metabolites in the retina and RPE/choroid. In the postmortem retina, 18% of metabolites were changed at 0.5 h (h), 41% at 4 h and 51% at 8 h. However, only 6% of metabolites were changed in the postmortem RPE/choroid and it steadily increased to 20% at 8 h. Notably, both postmortem retina and RPE/choroid tissue showed increased purine metabolites. Storage of eyes in cold nutrient-rich medium substantially blocked the postmortem change in the retina and RPE/choroid. In conclusion, our study provides optimized methods to prepare fresh or postmortem retina and RPE/choroid tissue for metabolomics studies.
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http://dx.doi.org/10.1016/j.exer.2018.05.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110973PMC
September 2018

How Excessive cGMP Impacts Metabolic Proteins in Retinas at the Onset of Degeneration.

Adv Exp Med Biol 2018 ;1074:289-295

Department of Ophthalmology, University of Washington, Seattle, WA, USA.

Aryl-hydrocarbon receptor interacting protein-like 1 (AIPL1) is essential to stabilize cGMP phosphodiesterase 6 (PDE6) in rod photoreceptors. Mutation of AIPL1 leads to loss of PDE6, accumulation of intracellular cGMP, and rapid degeneration of rods. To understand the metabolic basis for the photoreceptor degeneration caused by excessive cGMP, we performed proteomics and phosphoproteomics analyses on retinas from AIPL1-/- mice at the onset of rod cell death. AIPL1-/- retinas have about 18 times less than normal PDE6a and no detectable PDE6b. We identified twelve other proteins and thirty-nine phosphorylated proteins related to cell metabolism that are significantly altered preceding the massive degeneration of rods. They include transporters, kinases, phosphatases, transferases, and proteins involved in mitochondrial bioenergetics and metabolism of glucose, lipids, amino acids, nucleotides, and RNA. In AIPLI-/- retinas mTOR and proteins involved in mitochondrial energy production and lipid synthesis are more dephosphorylated, but glycolysis proteins and proteins involved in leucine catabolism are more phosphorylated than in normal retinas. Our findings indicate that elevating cGMP rewires cellular metabolism prior to photoreceptor degeneration and that targeting metabolism may be a productive strategy to prevent or slow retinal degeneration.
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http://dx.doi.org/10.1007/978-3-319-75402-4_35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6005689PMC
May 2019

Deletion of GLUT1 in mouse lens epithelium leads to cataract formation.

Exp Eye Res 2018 07 28;172:45-53. Epub 2018 Mar 28.

Department of Pathology, Anatomy & Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA. Electronic address:

The primary energy substrate of the lens is glucose and uptake of glucose from the aqueous humor is dependent on glucose transporters. GLUT1, the facilitated glucose transporter encoded by Slc2a1 is expressed in the epithelium of bovine, human and rat lenses. In the current study, we examined the expression of GLUT1 in the mouse lens and determined its role in maintaining lens transparency by studying effects of postnatal deletion of Slc2a1. In situ hybridization and immunofluorescence labeling were used to determine the expression and subcellular distribution of GLUT1 in the lens. Slc2a1 was knocked out of the lens epithelium by crossing transgenic mice expressing Cre recombinase under control of the GFAP promoter with Slc2a1 mice to generate Slc2a1;GFAP-Cre (LensΔGlut1) mice. LensΔGlut1 mice developed visible lens opacities by around 3 months of age, which corresponded temporally with the total loss of detectable GLUT1 expression in the lens. Spectral domain optical coherence tomography (SD-OCT) imaging was used to monitor the formation of cataracts over time. SD-OCT imaging revealed that small nuclear cataracts were first apparent in the lenses of LensΔGlut1 mice beginning at about 2.7 months of age. Longitudinal SD-OCT imaging of LensΔGlut1 mice revealed disruption of mature secondary fiber cells after 3 months of age. Histological sections of eyes from LensΔGlut1 mice confirmed the disruption of the secondary fiber cells. The structural changes were most pronounced in fiber cells that had lost their organelles. In contrast, the histology of the lens epithelium in these mice appeared normal. Lactate and ATP were measured in lenses from LensΔGlut1 and control mice at 2 and 3 months of age. At 2 months of age, when GLUT1 was still detectable in the lens epithelium, albeit at low levels, the amount of lactate and ATP were not significantly different from controls. However, in lenses isolated from 3-month-old LensΔGlut1 mice, when GLUT1 was no longer detectable, levels of lactate and ATP were 50% lower than controls. Our findings demonstrate that in vivo, the transparency of mature lens fiber cells was dependent on glycolysis for ATP and the loss of GLUT1 transporters led to cataract formation. In contrast, lens epithelium and cortical fiber cells have mitochondria and could utilize other substrates to support their anabolic and catabolic needs.
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http://dx.doi.org/10.1016/j.exer.2018.03.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716165PMC
July 2018

Disruption of De Novo Serine Synthesis in Müller Cells Induced Mitochondrial Dysfunction and Aggravated Oxidative Damage.

Mol Neurobiol 2018 Aug 30;55(8):7025-7037. Epub 2018 Jan 30.

Save Sight Institute, The University of Sydney, 8 Macquarie Street, Sydney, NSW, 2000, Australia.

De novo serine synthesis plays important roles in normal mitochondrial function and cellular anti-oxidative capacity. It is reported to be mainly activated in glial cells of the central nervous system, but its role in retinal Müller glia remains unclear. In this study, we inhibited de novo serine synthesis using CBR-5884, a specific inhibitor of phosphoglycerate dehydrogenase (PHGDH, a rate limiting enzyme in de novo serine metabolism) in MIO-M1 cells (immortalized human Müller cells) and huPMCs (human primary Müller cells) under mild oxidative stress. Alamar blue and LDH (lactate dehydrogenase) assays showed significantly reduced metabolic activities and increased cellular damage of Müller cells, when exposed to CBR-5884 accompanied by mild oxidative stress; however, CBR-5884 alone had little effect. The increased cellular damage was partially reversed by supplementation with exogenous serine/glycine. HSP72 (an oxidative stress marker) and reactive oxygen species (ROS) levels were significantly increased; glutathione and NADPH/NADP levels were pronouncedly reduced under PHGDH inhibition accompanied by oxidative stress. JC-1 staining and Seahorse respiration experiments showed that inhibition of de novo serine synthesis in Müller cells can also increase mitochondrial stress and decrease mitochondrial ATP production. qPCR and Western blot demonstrated an increased expression of HSP60 (a key mitochondrial stress-related gene), and this was further validated in human retinal explants. Our study suggests that de novo serine synthesis is important for Müller cell survival, particularly when they are exposed to mild oxidative stress, possibly by maintaining mitochondrial function and generating glutathione and NADPH to counteract ROS.
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http://dx.doi.org/10.1007/s12035-017-0840-8DOI Listing
August 2018

Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye.

Elife 2017 09 13;6. Epub 2017 Sep 13.

Department of Biochemistry, University of Washington, Seattle, United States.

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.
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http://dx.doi.org/10.7554/eLife.28899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617631PMC
September 2017

Human retinal pigment epithelial cells prefer proline as a nutrient and transport metabolic intermediates to the retinal side.

J Biol Chem 2017 08 14;292(31):12895-12905. Epub 2017 Jun 14.

Department of Ophthalmology, West Virginia University, Morgantown, West Virginia 26506; Department of Biochemistry, West Virginia University, Morgantown, West Virginia 26506. Electronic address:

Metabolite transport is a major function of the retinal pigment epithelium (RPE) to support the neural retina. RPE dysfunction plays a significant role in retinal degenerative diseases. We have used mass spectrometry with C tracers to systematically study nutrient consumption and metabolite transport in cultured human fetal RPE. LC/MS-MS detected 120 metabolites in the medium from either the apical or basal side. Surprisingly, more proline is consumed than any other nutrient, including glucose, taurine, lipids, vitamins, or other amino acids. Besides being oxidized through the Krebs cycle, proline is used to make citrate via reductive carboxylation. Citrate, made either from C proline or from C glucose, is preferentially exported to the apical side and is taken up by the retina. In conclusion, RPE cells consume multiple nutrients, including glucose and taurine, but prefer proline, and they actively synthesize and export metabolic intermediates to the apical side to nourish the outer retina.
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http://dx.doi.org/10.1074/jbc.M117.788422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5546030PMC
August 2017

Reductive carboxylation is a major metabolic pathway in the retinal pigment epithelium.

Proc Natl Acad Sci U S A 2016 12 1;113(51):14710-14715. Epub 2016 Dec 1.

Department of Ophthalmology, University of Washington, Seattle, WA 98109;

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells that requires an active metabolism to maintain outer retinal homeostasis and compensate for oxidative stress. Using C metabolic flux analysis in human RPE cells, we found that RPE has an exceptionally high capacity for reductive carboxylation, a metabolic pathway that has recently garnered significant interest because of its role in cancer cell survival. The capacity for reductive carboxylation in RPE exceeds that of all other cells tested, including retina, neural tissue, glial cells, and a cancer cell line. Loss of reductive carboxylation disrupts redox balance and increases RPE sensitivity to oxidative damage, suggesting that deficiencies of reductive carboxylation may contribute to RPE cell death. Supporting reductive carboxylation by supplementation with an NAD precursor or its substrate α-ketoglutarate or treatment with a poly(ADP ribose) polymerase inhibitor protects reductive carboxylation and RPE viability from excessive oxidative stress. The ability of these treatments to rescue RPE could be the basis for an effective strategy to treat blinding diseases caused by RPE dysfunction.
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http://dx.doi.org/10.1073/pnas.1604572113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5187684PMC
December 2016

Quantitative Method to Investigate the Balance between Metabolism and Proteome Biomass: Starting from Glycine.

Angew Chem Int Ed Engl 2016 12 15;55(50):15646-15650. Epub 2016 Nov 15.

Northwest Metabolomics Research Center, Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, WA, 98109, USA.

The balance between metabolism and biomass is very important in biological systems; however, to date there has been no quantitative method to characterize the balance. In this methodological study, we propose to use the distribution of amino acids in different domains to investigate this balance. It is well known that endogenous or exogenous amino acids in a biological system are either metabolized or incorporated into free amino acids (FAAs) or proteome amino acids (PAAs). Using glycine (Gly) as an example, we demonstrate a novel method to accurately determine the amounts of amino acids in various domains using serum, urine, and cell samples. As expected, serum and urine had very different distributions of FAA- and PAA-Gly. Using Tet21N human neuroblastoma cells, we also found that Myc(oncogene)-induced metabolic reprogramming included a higher rate of metabolizing Gly, which provides additional evidence that the metabolism of proliferating cells is adapted to facilitate producing new cells. It is therefore anticipated that our method will be very valuable for further studies of the metabolism and biomass balance that will lead to a better understanding of human cancers.
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http://dx.doi.org/10.1002/anie.201609236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6238948PMC
December 2016

Reprogramming metabolism by targeting sirtuin 6 attenuates retinal degeneration.

J Clin Invest 2016 12 14;126(12):4659-4673. Epub 2016 Nov 14.

Retinitis pigmentosa (RP) encompasses a diverse group of Mendelian disorders leading to progressive degeneration of rods and then cones. For reasons that remain unclear, diseased RP photoreceptors begin to deteriorate, eventually leading to cell death and, consequently, loss of vision. Here, we have hypothesized that RP associated with mutations in phosphodiesterase-6 (PDE6) provokes a metabolic aberration in rod cells that promotes the pathological consequences of elevated cGMP and Ca2+, which are induced by the Pde6 mutation. Inhibition of sirtuin 6 (SIRT6), a histone deacetylase repressor of glycolytic flux, reprogrammed rods into perpetual glycolysis, thereby driving the accumulation of biosynthetic intermediates, improving outer segment (OS) length, enhancing photoreceptor survival, and preserving vision. In mouse retinae lacking Sirt6, effectors of glycolytic flux were dramatically increased, leading to upregulation of key intermediates in glycolysis, TCA cycle, and glutaminolysis. Both transgenic and AAV2/8 gene therapy-mediated ablation of Sirt6 in rods provided electrophysiological and anatomic rescue of both rod and cone photoreceptors in a preclinical model of RP. Due to the extensive network of downstream effectors of Sirt6, this study motivates further research into the role that these pathways play in retinal degeneration. Because reprogramming metabolism by enhancing glycolysis is not gene specific, this strategy may be applicable to a wide range of neurodegenerative disorders.
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http://dx.doi.org/10.1172/JCI86905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127684PMC
December 2016

Deficient glucose and glutamine metabolism in knockout mice contributes to altered visual function.

Mol Vis 2016 12;22:1198-1212. Epub 2016 Oct 12.

Department of Systems Biology, University of Alcalá, Alcalá de Henares, Spain.

Purpose: To characterize the vision phenotype of mice lacking , the mitochondrial aspartate-glutamate carrier mutated in global cerebral hypomyelination (OMIM 612949).

Methods: We tested overnight dark-adapted control and aralar-deficient mice for the standard full electroretinogram (ERG) response. The metabolic stress of dark-adaptation was reduced by 5 min illumination after which the ERG response was monitored in darkness. We used the electrical response to two identical saturating light flashes (paired-flash stimulation) to isolate the inner retina and photoreceptor responses. Retinal morphology was examined with hematoxylin and eosin staining, immunohistochemistry of antibodies against retinal cells, and 4',6-diamidino-2-phenylindole (DAPI) labeling.

Results: Aralar plays a pivotal role in retina metabolism as aralar provides de novo synthesis pathway for glutamine, protects glutamate from oxidation, and is required for efficient glucose oxidative metabolism. Aralar-deficient mice are not blind as their retinas have light-evoked activity. However, we report an approximate 50% decrease in the ERG amplitude response in the light-evoked activity of dark-adapted retinas from aralar-deficient mice, in spite of normal retina histology. The defective response is partly reversed by exposure to a brief illumination period, which lowers the metabolic stress of dark-adaptation. The metabolic stress and ERG alteration takes place primarily in photoreceptors, but the response to two flashes applied in fast succession also revealed an alteration in synaptic transmission consistent with an imbalance of glutamate and an energy deficit in the inner retina neurons.

Conclusions: We propose that compromised glucose oxidation and altered glutamine and glutamate metabolism in the absence of aralar are responsible for the phenotype reported.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063090PMC
January 2018