Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyMethods: Mouse retina, cerebellum, hippocampus and olfactory bulbwere isolated and cultured in Krebs-Ringer bicarbonate (KRB)buffer. The retina and other neurons were incubated with 5 mM of avariety of 13C labeled fuel sources. Rat retinas were isolated andincubated in KRB buffer and sectioned at 50 micron thickness.Metabolites from both the tissue and the medium were extracted andmeasured by GC-MS. Results were corrected for the naturalabundance of isotopes. The overall enrichment and the distribution oflabeled carbons in each metabolite were calculated.Results: Retinas converted glucose to lactate and pyruvate andreleased them into the medium ~4 times higher than other neurons.Unlike other neurons retinas did not release glutamate. Retina had thehighest rate of 13C enrichment of the mitochondrial TCA cycleintermediates from glucose among all the neurons. By incubatingretinas and other neurons with the same concentration of 13Cglucose, 13C glutamine, 13C pyruvate or 13C lactate for 2 hours, wefound the 13C glutamine produced the highest 13C enrichment ofsuccinate, fumarate and malate. In the retina, glutamine robustlycompeted with glucose for enrichment of all mitochondrialintermediates except citrate. By incubating with 12C glucose togetherwith 13C glutamine, we found that retinas had the highest rate of 13Cenrichment in mitochondrial intermediates of all the types of neuronswe tested. Furthermore, rat retina sections showed that glutaminelabeling was localized primarily in the mitochondrial intermediates inthe photoreceptor layer.Conclusions: The retina has much higher metabolic rate from bothglucose and glutamine than other neurons. The retina, particularly thephotoreceptor layer, selectively uses glutamine over glucose,pyruvate and lactate to fuel mitochondria.Commercial Relationships: Jianhai Du, None; Whitney M.Cleghorn, None; Guy C. Chan, None; Jonathan Linton, None;Martin Sadilek, None; Viren Govindaraju, None; James B.Hurley, NoneSupport: NIH EY017863Program Number: 692 Poster Board Number: D0249Presentation Time: 10:30 AM - 12:15 PMUnique expression and regulation of glycolytic enzyme PKM2 inPhotoreceptor cells and the role of enzymatic activity modulatingmetabolism of the retinaKen Lindsay, Jonathon Linton, James B. Hurley. Biochemistry,University of Washington, Seattle, WA.Purpose: The photoreceptor cell is one of the most energydemanding cells in the body. While being a post-mitotic cell, thedaily turnover of Rod Outer Segment Discs imposes a need forprotein and lipid synthesis that rivals that of a rapidly dividing cell.This intense metabolic demand suggests that the expression ofglycolytic enzymes associated with increases in anabolism, typicallyassociated with tumors and other rapidly dividing cells may beexpressed in the retina and be regulated to fuel these processes. Initialobservations demonstrated that the retina expresses a form ofpyruvate kinase, PKM2, previously thought to be primarilyassociated with cancer cells. The purpose of this study was to analyzethe localization and possible regulation of PKM2 in retinas.Methods: We analyzed the distribution of PKM2, by enzymaticactivity assays and immunoblotting of serial sections of light anddark adapted rat retinas. Western Blot analyses of serial sections usedRhodopsin, Recoverin and Synaptotagmin as landmarks fororientation of enzymes throughout the retina. An antibody specific toPKM2 was used to localize this isoform in the retina.Results: Serial Sectioning revealed that PKM2 is present only in thephotoreceptor layer of the retina. While total expression of PKM2 isinsensitive to light, the fraction of enzymatic activity in thephotoreceptor layer increased significantly in Dark Adapted retinas (n= 7). Immunoblot analyses showed that phosphorylation of Y105 onPKM2, typically associated with decreased PKM2 enzyme activity,occurs independently of light.Conclusions: Our findings show that the PKM2 isoform of pyruvatekinase is present specifically in the photoreceptor layer of rat retinas.We also found that the fraction of pyruvate kinase activity that is inthe outer retina is diminished by light adaptation. Additional studieswill address how PKM2 helps photoreceptors to meet their unusualmetabolic demands.Commercial Relationships: Ken Lindsay, None; Jonathon Linton,None; James B. Hurley, NoneSupport: EY017863Program Number: 693 Poster Board Number: D0250Presentation Time: 10:30 AM - 12:15 PMMetabolic pathways of photoreceptors in light and darkWhitney M. Cleghorn 1 , Jianhai Du 1 , Martin Sadilek 2 , VirenGovindaraju 1 , James B. Hurley 1 . 1 Biochemistry, University ofWashington, Seattle, WA; 2 Chemistry, University of Washington,Seattle, WA.Purpose: Rod and cone photoreceptors (PRs) need to produce bothATP and anabolic precursors to survive. The metabolic demands ofPRs are different in light and dark: dark adapted PRs use energy tofuel ion exchange, while PRs in the light undergo anabolicmetabolism to regenerate rhodopsin and synthesize new membranes.The purpose of this study is to understand how the metabolicpathways differ in PRs under light and dark conditions.Methods: Retinas were isolated from C57BL mice (6-8 weeks old)and cultured in 2 ml KRB buffer with either 5 mM 13C glucose or 5mM 13C glutamine in 37oC CO2 incubator. The metabolites wereanalyzed by GC-MS (Agilent 5973 MSD/6890 GC). Mass spectrawere collected and isotopomer distributions were corrected fornaturally-occurring heavy isotopes using the software, Isocor. Themean enrichment (ME) of each metabolite was calculated.Results: Carbons from uniformly labeled 13C glucose areincorporated rapidly into glycolytic intermediates and into citrate, butare incorporated more slowly into other TCA cycle intermediates.Interestingly, two of the TCA cycle metabolites, succinate andfumarate, show lower incorporation than surrounding intermediates,suggesting the carbons enter from multiple points into the TCA cycle.The overall incorporation of glucose is qualitatively similar in thedark, however accumulation of labeled intermediates happens faster.Incorporation of 13C labeled glutamine is well isolated fromglycolytic intermediates. Carbons from glutamine are incorporatedinto TCA cycle intermediates at the same rate as glucose, and showhigher incorporation in the dark. Interestingly, malate shows thebiggest increase in 13C in dark adapted retinas, regardless of the fuelused. In addition, our experiments show that the overall redox state ofthe retina is stable in glucose but not glutamine, but the reducingpower (lac/pyr ratio) is used up faster in the dark.Conclusions: Glutamine is the major source for formation of TCAcycle intermediates, whereas glucose provides cytosolic reducingpower for the photoreceptors. Generally each of these fuels is usedsimilarly in light and dark, but all of the metabolic reactions occurfaster in darkness than in light. In particular, pyruvate carboxylaseand malic enzyme activity appear to be faster in darkness.Commercial Relationships: Whitney M. Cleghorn, None; JianhaiDu, None; Martin Sadilek, None; Viren Govindaraju, None;James B. Hurley, NoneSupport: NEI grants EY017863 and EY06641Program Number: 694 Poster Board Number: D0251©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.