Previs S F, Hallowell P T, Neimanis K D, David F, Brunengraber H
Department of Nutrition, Case Western Reserve University, Cleveland, Ohio 44106, USA.
J Biol Chem. 1998 Jul 3;273(27):16853-9. doi: 10.1074/jbc.273.27.16853.
We previously reported (Previs, S. F., Fernandez, C. A., Yang, D., Soloviev, M. V., David, F., and Brunengraber, H. (1995) J. Biol. Chem. 270, 19806-19815) that glucose made in isolated livers from starved rats perfused with physiological concentrations of lactate, pyruvate, and either [2-13C]- or [U-13C3]glycerol had a mass isotopomer distribution incompatible with glucose being made from a homogeneously labeled pool of triose phosphates. Similar data were obtained in live rats infused with [U-13C3]glycerol. We ascribed the labeling heterogeneity to major decreases in glycerol concentration and enrichment across the liver. We concluded that [13C]glycerol is unsuitable for tracing the contribution of gluconeogenesis to total glucose production. We now report isotopic heterogeneity of gluconeogenesis in hepatocytes, even when all cells are in contact with identical concentrations and enrichments of gluconeogenic substrates. Total rat hepatocytes were incubated with concentrations of glycerol, lactate, and pyruvate that were kept constant by substrate infusions. To modulate competition between substrates, the (glycerol)/(lactate + pyruvate) infusion ratio ranged from 0.23 to 3. 60. Metabolic and isotopic steady states were achieved in all cases. The apparent contribution of gluconeogenesis to glucose production (f) was calculated from the mass isotopomer distribution of glucose. When all substrates were 13C-labeled, f was 97%, as expected in glycogen-deprived hepatocytes. As the infusion ratio ([13C]glycerol)/(lactate + pyruvate) increased, f increased from 73% to 94%. In contrast, as the infusion ratio (glycerol)/([13C]lactate + [13C]pyruvate) increased, f decreased from 93% to 76%. In all cases, f increased with the rate of supply of the substrate that was labeled. Variations in f show that the 13C labeling of triose phosphates was not equal in all hepatocytes, even when exposed to the same substrate concentrations and enrichments. We also showed that zonation of glycerol kinase activity is minor in rat liver. We conclude that zonation of other processes than glycerol phosphorylation contributes to the heterogeneity of triose phosphate labeling from glycerol in rat liver.
我们之前报道过（Previs, S. F., Fernandez, C. A., Yang, D., Soloviev, M. V., David, F., and Brunengraber, H. (1995) J. Biol. Chem. 270, 19806 - 19815），在用生理浓度的乳酸、丙酮酸以及[2 - 13C]-或[U - 13C3]甘油灌注饥饿大鼠的分离肝脏中产生的葡萄糖，其质量同位素异构体分布与由均匀标记的磷酸丙糖池生成葡萄糖不相符。在输注[U - 13C3]甘油的活体大鼠中也获得了类似的数据。我们将标记的异质性归因于肝脏中甘油浓度和丰度的大幅下降。我们得出结论，[13C]甘油不适用于追踪糖异生对总葡萄糖生成的贡献。我们现在报道，即使所有细胞都接触相同浓度和丰度的糖异生底物，肝细胞中糖异生也存在同位素异质性。将大鼠原代肝细胞与通过底物输注保持恒定浓度的甘油、乳酸和丙酮酸一起孵育。为了调节底物之间的竞争，（甘油）/（乳酸 + 丙酮酸）输注比例范围为0.23至3.60。在所有情况下均达到了代谢和同位素稳态。根据葡萄糖的质量同位素异构体分布计算糖异生对葡萄糖生成的表观贡献（f）。当所有底物都用13C标记时，f为97%，这在糖原缺乏的肝细胞中是预期的。随着输注比例（[13C]甘油）/（乳酸 + 丙酮酸）增加，f从73%增加到94%。相反，随着输注比例（甘油）/（[13C]乳酸 + [13C]丙酮酸）增加，f从93%下降到76%。在所有情况下，f随着标记底物的供应速率增加而增加。f的变化表明，即使暴露于相同的底物浓度和丰度，磷酸丙糖的13C标记在所有肝细胞中也不相等。我们还表明，大鼠肝脏中甘油激酶活性的区域化程度较小。我们得出结论，除甘油磷酸化之外的其他过程的区域化导致了大鼠肝脏中甘油生成的磷酸丙糖标记的异质性。
