Determination of the glycogen synthesis pathway by 13C nuclear magnetic resonance analysis

Abstract

The level of hepatic glycogen synthesized directly from glucose was measured in rats with [1- 13C]glucose. The nuclear magnetic resonance (NMR) spectrum of glucose was used to measure the distribution of the 13C label from C 1 to the other carbons. Female Sprague-Dawley rats were surgically implanted with catheters in the left carotid artery and the right jugular vein, followed by a 3-day recovery period and a 24-hour fast to deplete liver glycogen. A 2-hour infusion of the fasted animal with [1- 13C]glucose was immediately followed by the removal of blood and liver tissue. The liver was divided into the right, left, caudate, and medial lobes, and then freeze-clamped in liquid nitrogen and stored at −80°C. The 13C NMR glucose spectra were obtained from glycogen that was isolated from each liver lobe and hydrolyzed to glucose with amyloglucosidase. Spectra were obtained at 50.3 MHz in a narrow-bore Gemini 200-MHz NMR spectrometer (Varian, Palo Alto, CA). The distribution of 13C onto glucose carbons was measured from these spectra, and the percent direct pathway was calculated to be 29% ± 2.5%. Metabolic variation for the synthesis of glycogen within the liver was determined by measuring the direct pathway contribution in each of the four liver lobes. Percent direct pathway values were similar ( P > .05) in right (35% ± 4.9%), left (26% ± 5.1%), medial (25% ± 4.9%), and caudate (27% ± 5.6%) lobes. For some of the animals, the direct pathway was determined by infusion with [6- 13C]glucose. These results were then compared with the results of C 1-labeled glucose to measure the loss of C 1 from the infused glucose as CO 2 in the pentose phosphate pathway (PPP). The percent direct pathway determined from [1- 13C]glucose compared with [6- 13C]glucose indicates negligible PPP activity from infused glucose. Finally, labeled carbon was found on C 3 and C 4, indicating a flow of glucose through the citric acid cycle (CAC).