Determination of pathways of glycogen synthesis and the dilution of the three-carbon pool with [U-13C] The enrichment of 13C and the specific activity of 14C in glycogen formed by the indirect path were 20-25% of glycogen formed directly from glucose. The dilution is of two kinds: (i) an exchange of labeled carbon with unlabeled carbon in the tricarboxylic acid cycle and (ii) dilution by unlabeled nonglucose carbon. Methods to calculate the two types of dilution are presented. In control rats the dilution factor by exchange in the tricarboxylic acid cycle is 1.4, and the dilution by unlabeled carbon is 2.5-to 3.0-fold, with the overall dilution about 4-fold. In rats preinjected with glucagon, the dilution through the tricarboxylic acid cycle was unaffected but that by nonglucose carbon was decreased.It is now generally realized that only a part of liver glycogen is derived from intact glucose (1). However, there still prevails confusion as to what the direct path represents and how its contribution to glycogen may be determined. Very little attention has been paid to the indirect path, the synthesis of glycogen from three-carbon compounds. We have previously described (2, 3) the application of gas chromatography/mass spectroscopy (GC/MS) with [U-13C] and three rats were not treated. At the end of the infusion, portal and arterial blood was sampled and liver was homogenized in 6% (vol/vol) perchloric acid, and glucose and glycogen were isolated. Arterial blood glucose at sacrifice (165 min after start of infusion) averaged 6.5 ,umol/g of blood, and portal blood glucose was 9.5 ,tmol/g of blood, but '3C enrichment and specific activities in portal and arterial blood were virtually the same. Liver glycogen content averaged 85Armol/g. Glycogen content of fasted rats was very low, from 0.7 to 1.3 ,umol/g (n = 3), and no correction for initial glycogen content was applied. GC/MS analysis and corrections for natural abundance and 12C in [U-13C]glucose were as described (2-4). Spectrograms of the infusate, blood glucose, and glycogen have been published elsewhere (3).The total infused dose of [14C]-and [3H]glucose was 120 X 106 cpm each. Blood and glycogen glucose were degraded to obtain the specific activity of 14C in C-1 and C-6 of glucose. C-1 of glucose was released enzymatically as CO2. The incubation mixture was 0.1 M Hepes'NaOH (pH 8), 8 mM NADP, 5 mM ATP, 10 mM MgCl2, hexokinase (5 units/ml), glucose-6-phosphate dehydrogenase (10 units/ml), and 0.6 unit of phosphogluconate dehydrogenase. Total volume was 5 ml containing 10 ,mol ofglucose. Incubation was overnight at room temperature. CO2 was released by acidification with H2SO4 and was trapped in 2 ml of 50% (wt/vol) phenethylamine. The activity of C-6 of glucose after periodate oxidation was obtained as the dimedon derivative of formaldehyde as described by Bloom (5 [U-_3C]glucose is 600%.) The conventional term for enrichment is atom percent excess (APE), equal to 61 mn/6 for glucose, a term not used by us. The term relative molar enrichment (or relative enric...
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