期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 290, 期 13, 页码 8121-8132出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M114.631549
关键词
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资金
- National Institutes of Health [R33DK070291]
- Case Mouse Metabolic and Phenotyping Center (MMPC) Grant [U24DK76174]
- American Heart Association [12GRNT12050453]
- Cleveland Mt. Sinai Health Care Foundation
We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (approximate to citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [C-13(2),H-2(3)]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA ( e. g. [C-13(6)] glucose or [1,2-C-13(2)] palmitate) or/and M1 acetyl-CoA ( e. g. [1-13C] octanoate). The total acetylCoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions ( glucose +/- palmitate +/- insulin +/- dichloroacetate). The data revealed ( i) substrate cycling between glycogen and glucose- 6-P and between glucose-6-P and triose phosphates, ( ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and ( iii) the channeling of mitochondrial acetylCoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA.
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