Contribution of leucine to oxidative metabolism of the rat medullary thick ascending limb |
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Authors: | Marie-Marcelle Trinh-Trang-Tan Olivier Levillain Lise Bankir |
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Institution: | (1) INSERM Unité 90, Hôpital Necker, F-75743 Paris Cedex 15, France |
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Abstract: | It has recently been reported that branched-chain amino acid aminotransferase (BCAATase) is inhomogeneously distributed in the kidney. BCAATase activity is several-fold higher in the medullary thick ascending limb (MTAL) than in other nephron segments. The present work was designed to determine whether leucine, a branched-chain amino acid (AA), is used as metabolic fuel by this nephron segment. MTAL were isolated from the inner stripe of the outer medulla of adult Sprague Dawley rats by mild enzymatic digestion and appropriate sieving. Leucine aminotransferase activity measured in homogenates of MTAL was 653±52 pmol -ketoglutarate formed/ g protein per hour, a value threefold higher than that observed in the renal cortex or muscle in the same rats. Substrate oxidation was assessed by measuring14CO2 production from tracer amounts of uniformly labeled14C-amino acids or glucose in isolated MTAL incubated in modified Earle balanced salt solution. When each substrate was offered at a concentration of 1 mM, leucine oxidation was much higher than that of unbranched AA, but fivefold lower than that of glucose. With 1 mM glucose and 1 mM leucine in the medium, leucine oxidation was close to that of glucose (123±8 versus 177±15 pmol CO2/ g protein per hour), probably because glucose contributed to the formation of -ketoglutarate, a cosubstrate for leucine transamination. Inhibition of salt transport by furosemide (0.1 mM) decreased oxidation of both substrates by 60–70%. Inhibition of salt transport by ouabain (1 mM) decreased glucose oxidation markedly. However, it doubled leucine oxidation when glucose was absent from the medium and decreased leucine oxidation by only 28% when glucose was present. This might be due to an ouabain-dependent alteration in membrane permeability to AA. These findings show that leucine is oxidized in rat MTAL and may contribute to supporting active transport in this segment. This contribution could be important after a protein meal or on high protein diet, situations in which plasma level of branched-chain AA is elevated. |
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Keywords: | Branched-chain amino acid aminotransferase Amino acid oxidation Glucose oxidation Furosemide Ouabain Thick ascending limb Renal metabolism |
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