The Occurrence and Mechanism of the Hexose Monophosphate Shunt in Rat Liver Slices

It was recently shown by Bloom et al. (1,2) that, in liver, the 1st carbon of glucose is converted to CO2 to a larger extent than is the 6th carbon, and that the Cl402 derived from glucose-l-Cl4 exceeds that from glucose evenly labeled with CY4.These workers suggested that the hexose monophosphate shunt plays a significant rale in glucose metabolism in liver.A previous report from this laboratory dealt with equations for estimating the extent of the hexose monophosphate shunt in that tissue (3).In the present report, those equations have been extended, and the assumptions upon which they are based have been examined.The operation of this shunt in liver slices was studied, and some phases of its mechanism have been elucidated. Materials and MethodsPreparation of Labeled Substrates-Glucose evenly labeled with Cl4 (glucose-E-C14) w&s prepared photosynthetically as described by Putman and Hassid (4).Glucose-l-Cl4 and ribose-i-Cl4 were purchased from the National Bureau of Standards.Glucose-6-Cl4 was prepared by the method of Sowden (5) as modified by Roseman (6).All sugars were checked chromatographically for purity.Incubation Procedure-Long-Evis rats weighing from 200 to 300 gm. were fed a high carbohydrate diet (7) for 3 days before they were sacrificed.The preparation of liver slices and the incubation techniques were described in previous reports (8).Isolation of C14-Glucose from hcubation Mixture and Determinution of Cl4 Content of Its Individual Carbon Atoms-Glucose-Cl4 was isolated chromat.ographically on paper (8) and eluted with water.Carrier glucose was added, and the solutions were concentrated.The C14-glucose was degraded by fermentation with Leuconostoc mesenteroides which yields COz from carbon 1, ethanol from carbons 2 and 3, and lactate from carbons 4, 5, and 6 (9).The organisms were grown for 12 hours and harvested essentially as described by Gunsalus and Gibbs (9).The organisms were washed three times and tested for activity before use.