Figure 4.

Glutamine metabolism in primary cryopreserved human hepatocytes from donors genotyped to be homozygous GLS L581L/L and heterozygous L581L/P. (a) Glucose production in the presence of glucagon or PBS vehicle for 1 hour with extracellular glutamine, lactate, and pyruvate substrates. Values are from 5 (L581L/L) and 6 (L581L/P) donors, each representing the mean of 3 technical replicates. *P < 0.05, by two way anova. b–h. Hepatocytes from GLS L581L/L and L581L/P donors were given [¹³C₅]glutamine and unlabeled lactate and pyruvate in kinetic flux studies. Intracellular (b) [¹³C₅]glutamine, (c) [¹³C₅]glutamate, (d) [¹³C₅]a-ketoglutarate, (e) [¹³C₄]malate, (f) [¹³C₄]aspartate, (g) [¹³C₆]UDP-glucose, and (h) [¹³C₆] hexose was measured. Values represent the mean of 6 biological replicates, each from a unique biological hepatocyte donor, for each group and time point, with each biological replicate representative of 3 technical replicates. Error bars represent SEM. The steady state (3 hour time point) data from this experiment was replicated in a separate study (data not shown). *P < 0.05, two-tailed Student’s t-test. (i) Intracellular glutamate level was measured in immortalized human hepatocytes expressing either GLS2(WT) or GLS2(L581P), with cells transduced with lentivirus empty vector as a control. Values represent 4 biological replicates. **P < 0.01, two-tailed Student’s t-test. (j) A model summarizing the proposed effects of glucagon on mitochondrial fluxes. IDH: isocitrate dehydrogenase; SCoASynth: succinate-CoA synthetase.