Estimation of gluconeogenesis and glycogenolysis in vivo using tritiated water.
Estimation of gluconeogenesis and glycogenolysis in vivo using tritiated water.
复制标题
使用氚化水估计体内糖异生和糖原分解。
DOI：
10.1042/bj2790911
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Rognstad,R
中科院分区：
文献类型：
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作者：
Rognstad,R
在禁食的不同阶段，通过包括预充连续输注[6- 3 H]葡萄糖至稳态的公认程序，可以很容易地估计肝脏葡萄糖输出量。在肝糖原基本耗尽的长期禁食状态下，估计肝再生没有困难，因为这等于内源性葡萄糖输出。然而，随着禁食时间的缩短，肝葡萄糖输出将来自糖原分解和糖原异生。基于早期的3 HHO体外研究[1]，我描述了一种方法，该方法涉及在体内将氚掺入葡萄糖的C-6，以估计在禁食状态下肝再生和糖原分解对肝脏葡萄糖输出的相对贡献。在先前使用分离的肝细胞的实验中，在含有3 HHO的培养基中，当L-乳酸盐、丙酮酸盐或L-谷氨酰胺为所用的致凋亡底物时，氚被广泛掺入到所形成的葡萄糖的所有六个碳上[1]。葡萄糖C-6上两个位置的氚掺入比活度接近于3 HHO培养基的900%。然而，当在3 HHO培养基中由糖原分解形成葡萄糖时，在厌氧条件下，为了防止任何异生，在葡萄糖的C-6上的氚掺入可以忽略不计，尽管如预期的那样，在C-2上发现了广泛的掺入[2]。在从L-乳酸的产氢过程中，氚也被广泛地掺入到碳2、3、4和5上，并且当从在磷酸丙糖水平上进入产氢途径的底物（如果糖和二羟基丙酮）发生产氢时也是如此。当在禁食大鼠肝细胞中由果糖形成葡萄糖时，葡萄糖C-6的每个氢上的氚比放射性约为培养基3 HHO中的氚比放射性的8%[1]，当加入磷酸烯醇式丙酮酸羧激酶抑制剂巯基吡啶甲酸盐时，这种掺入减少到约2%（我未发表的结果）。因此，当果糖是产酶底物时，果糖形成的乳酸和一定程度的产酶可能产生了大量的C-6标记。因此，氚在C-6上的掺入很可能主要局限于那些在丙酮酸水平或在克雷布斯循环中间体水平进入途径的产氚底物。由脂肪分解产生的甘油也将为肝脏生成提供碳，尽管据估计该来源不到总肝葡萄糖产量的10%[3，4]。由于甘油在磷酸丙糖水平上进入产氢途径，我们假设在体内来自该来源的葡萄糖的C-6上的氚掺入可以忽略不计。甘油生成必须独立于14 C标记甘油的稳态输注[3，4]。净肝葡萄糖输出和甘油生成之间的差异可以划分为糖原分解葡萄糖生成和非甘油生成。我忽略了戊糖循环通量，它应该是在禁食，非脂肪生成和代谢状态下的碳的小净流出，其中循环的化学计量是1葡萄糖6-磷酸-。6二氧化碳[5]。为了确定氚掺入的程度，
Hepatic glucose output can be readily estimated at different stages of fasting by accepted procedures involving primed continuous infusion of [6-3H] glucose to steady state. In the longterm fasted state in which liver glycogen is essentially depleted, there is no difficulty in estimating gluconeogenesis, since this is equal to the endogenous glucose output. However, with shorter fasting periods, hepatic glucose output will be derived both from glycogenolysis and gluconeogenesis. Based on earlier studies in vitro with 3HHO [1], I describe an approach involving tritium incorporation onto C-6 of glucose in vivo to estimate relative contributions of gluconeogenesis and glycogenolysis to hepatic glucose output in the fasted state. In previous experiments with isolated hepatocytes, in a medium containing 3HHO, tritium was incorporated extensively on to all six carbons of the glucose formed, when L-lactate, pyruvate or L-glutamine was the gluconeogenic substrate employed [1]. Tritium incorporation on the two positions on C-6 of glucose was nearly 900% of the specific activity of the medium 3HHO. However, when glucose was formed in a 3HHO medium from glyco-genolysis, under anaerobic conditionsto prevent any gluconeogenesis, there was negligible tritium incorporation on C-6 of glucose, although extensive incorporation on to C-2 was found, as expected [2]. During gluconeogenesis from L-lactate, tritium was also extensively incorporated onto carbons 2, 3, 4 and 5, and this was true also when gluconeogenesis occurred from substrates that entered the gluconeogenic pathway at the triose-phosphate level, substrates such as fructose and dihydroxyacetone. When glucose was formed from fructose in hepatocytes from fasted rats, the tritium specific radioactivity on each hydrogen of C-6 of glucose was about 8% of that of the medium 3HHO [1], and this incorporation was reduced to about2% when thephosphoenolpyruvate carboxykinase inhibitor, mercaptopicolinate, was added (my unpublished results). Thus lactate formation from fructose and a degree of gluconeogenesis from this lactate may have produced much of this C-6 labelling when fructose was the gluconeogenic substrate. It seems likely, therefore, that tritium incorporation on C-6 is largely confinedto those gluconeogenic substrates that enter the pathway at the pyruvate level, or at the level of a Krebs cycle intermediate. Glycerol produced from lipolysis will also contribute carbon for gluconeogenesis, al-though this source has been estimated to beless than 10% of the total hepatic glucoseproduction [3, 4]. As glycerol enters the gluconeogenic pathway at the triose-phosphate level, we assume negligible tritium incorporation on C-6 of glucose from this source in vivo. Glycerol gluconeogenesis must be independently determined from steady state infusion of 14C-labelled glycerol [3, 4].The difference between net hepatic glucose outputand glycerol gluconeogenesis can bepartitioned between glycogenolytic glu-cose production and non-glycerol gluconeogenesis. I am neglecting pentose cycle flux, which should be a small net outflow of carbon in the fasted, non-lipogenic and gluconeogenic state, where the stoichiometry of the cycle is 1 glucose 6-phosphate-. 6 CO2 [5]. To determine the extent of tritium incorporation from