Fasting hyperglycemia in diabetes mellitus is caused by unregulated glucagon secretion that activates gluconeogenesis (GNG) and increases the use of pyruvate, lactate, amino acids, and glycerol. Studies of GNG in hepatocytes, however, tend to test a limited number of substrates at non-physiologic concentrations. Therefore, we treated cultured primary hepatocytes with three identical substrate mixtures of pyruvate/lactate, glutamine, and glycerol at serum fasting concentrations, where a different U-¹³C or 2-¹³C labeled substrate was substituted in each mix. In the absence of glucagon stimulation, 80% of glucose produced in primary hepatocytes incorporated either one or two ¹³C-labeled glycerol molecules in a 1:1 ratio, reflecting the high overall activity of this pathway. In contrast, glucose produced from ¹³C-labeled pyruvate/lactate or glutamine rarely incorporated two labeled molecules. While glucagon increased glycerol and pyruvate/lactate contribution to glucose carbon by 1.6- and 1.8-fold, respectively, glutamine contribution to glucose carbon was increased 6.4-fold in primary hepatocytes. To account for substrate ¹³C carbon loss during metabolism, we also performed a metabolic flux analysis, which confirmed that the majority of glucose carbon produced by primary hepatocytes was from glycerol. In vivo studies using a PKA-activation mouse model that represents elevated glucagon activity confirmed that most circulating lactate carbons originated from glycerol, but very little glycerol was derived from lactate carbons, reflecting glycerol’s importance as a carbon donor to GNG. Given diverse entry points for GNG substrates, hepatic glucagon action is unlikely to be due to a single mechanism.

糖尿病中的空腹高血糖是由胰高血糖素分泌不受调节引起的，胰高血糖素分泌会激活糖异生 (GNG) 并增加丙酮酸、乳酸、氨基酸和甘油的使用。然而，肝细胞中 GNG 的研究倾向于在非生理浓度下测试有限数量的底物。因此，我们用空腹血清浓度的丙酮酸/乳酸、谷氨酰胺和甘油的三种相同底物混合物处理培养的原代肝细胞，其中在每种混合物中替换不同的U- ¹³ C或2- ¹³ C标记底物。在没有胰高血糖素刺激的情况下，原代肝细胞中产生的 80% 的葡萄糖以 1:1 的比例掺入一个或两个¹³ C 标记的甘油分子，反映了该途径的高整体活性。相比之下，由¹³ C 标记的丙酮酸/乳酸或谷氨酰胺产生的葡萄糖很少掺入两个标记分子。虽然胰高血糖素使甘油和丙酮酸/乳酸对葡萄糖碳的贡献分别增加了1.6倍和1.8倍，但在原代肝细胞中谷氨酰胺对葡萄糖碳的贡献增加了6.4倍。为了解释代谢过程中底物¹³ C 碳的损失，我们还进行了代谢通量分析，证实原代肝细胞产生的大部分葡萄糖碳来自甘油。使用代表胰高血糖素活性升高的 PKA 激活小鼠模型的体内研究证实，大多数循环乳酸碳源自甘油，但很少有甘油源自乳酸碳，这反映了甘油作为 GNG 碳供体的重要性。 鉴于 GNG 底物的不同进入点，肝胰高血糖素的作用不太可能是由单一机制引起的。