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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

Gluconeogenesis (GNG) is de novo production of glucose from endogenous carbon sources. Although it is a commonly studied pathway, particularly in disease, there is a lack of consensus about substrate preference. Moreover, primary hepatocytes are the current gold standard for in vitro liver studies, but no direct comparison of substrate preference at physiological fasting concentrations has been performed. We show that mouse primary hepatocytes prefer glycerol to pyruvate/lactate in glucose production assays and 13C isotope tracing studies at the high concentrations commonly used in the literature, as well as at more relevant fasting, physiological concentrations. In addition, when glycerol, pyruvate/lactate, and glutamine are all present, glycerol is responsible for over 75% of all glucose carbons labeled. We also found that glycerol can induce a rate-limiting enzyme of GNG, glucose-6-phosphatase. Lastly, we suggest that glycerol is a better substrate than pyruvate to test in vivo production of glucose in fasting mice. In conclusion, glycerol is the major carbon source for GNG in vitro and in vivo and should be compared with other substrates when studying GNG in the context of metabolic disease states.