Glycerol not lactate is the major net carbon source for gluconeogenesis in mice during both short and prolonged fasting

Fasting results in major metabolic changes including a switch from glycogenolysis to gluconeogenesis to maintain glucose homeostasis. However, the relationship between the length of fasting and the relative contribution of gluconeogenic substrates remains unclear. We investigated the relative contribution of glycogen, lactate, and glycerol in glucose production of male C57BL/6 J-albino mice after 6, 12, and 18 h of fasting. We used non-perturbative infusions of 13C3 lactate, 13C3 glycerol, and 13C6 glucose combined with liquid chromatography mass spectrometry and metabolic flux analysis to study the contribution of substrates in gluconeogenesis (GNG). During infusion studies, both lactate and glycerol significantly label about 60% and 30–50% glucose carbon, respectively, but glucose labels much more lactate (∼90%) than glycerol carbon (∼10%). Our analyses indicate that lactate, but not glycerol is largely recycled during all fasting periods such that lactate is the largest direct contributor to GNG via the Cori cycle but a minor source of new glucose carbon (overall contribution). In contrast, glycerol is not only a significant direct contributor to GNG but also the largest overall contributor to GNG regardless of fasting length. Prolonged fasting decreases both the whole body turnover rate of glucose and lactate but increases that of glycerol, indicating that the usage of glycerol in GNG become more significant with longer fasting. Collectively, these findings suggest that glycerol is the dominant overall contributor of net glucose carbon in GNG during both short and prolonged fasting. •Prolonged fasting significantly decreases the turnover rate of glucose and lactate but increases the glycerol turnover rate in mice.•In both short and prolonged fasting, lactate is the largest direct contributor to gluconeogenesis but a minor source of new carbon entry.•Glycerol is the second largest direct contributor to gluconeogenesis and the dominant overall carbon contributor during both short and prolonged fasting.