¹³ C NMR studies of gluconeogenesis in rat liver cells: Utilization of labeled glycerol by cells from euthyroid and hyperthyroid rats

Abstract

The gluconeogenic pathway from [1,3-'3C]glycerol has been followed in suspensions of isolated rat hepatocytes at 250C by 13C NMR at 90.5 MHz. The flow of label through the major pathway from glycerol to Lglycerol 3-phosphate and into glucose was Rollowed in cells from control and triiodothyronine-treated rats. Treatment increased the rates of glucose formation and glycerol consumption 2-fold and decreased the aGP level to 40%. We calculate that t60% of the flux is through the mitochondrial glycerol phosphate dehydrogenase in cells from triiodothyronine-treated rats, compared with~15% in cells from the controls. Equal distribution of label between the trioses of glucose was obtained and, because the C3-C4 spin-spin coupling gives the distribution of labeled carbons in the same molecule, it was possible to measure the amount of triose from unlabeled fructose incorporated into the glucose labeled at carbons 1, 3,4, and 6. About 10% of the hexoses had flowed through the pentose cycle and back into the hexose pathway in cells from fasted rats. From the distribution of label at glucose carbons not labeled via the major pathway and from the carbon spin-spin splitting patterns observed, we conclude that transketolase is reversible whereas transaldolase is essentially irreversible in the nonoxidative pentose branch.Recently it has become possible to study metabolism in cellular suspensions by 31P (1) and '3C (2) high-resolution NMR. In recent 31P high-resolution NMR studies of suspensions of rat liver cells we have determined the cytosolic pH and the mitochondrial pH simultaneously from the positions of the two resolved resonances from inorganic phosphate (3). A preliminary account of 13C NMR studies of intermediates and end products of gluconeogenesis in rat liver cells from a '3C-labeled alanine substrate has been reported (4).The present communication illustrates the ability of 13C NMR to follow the gluconeogenic pathway from glycerol in liver cells obtained from euthyroid and hyperthyroid iats and to obtain, very simply, simultaneous quantitative measurements of the contributions of different pathways. This process has been intensively studied in perfused liver (5, 6), freeze-clamped liver (7), and isolated hepatocytes (8,9). The early measurements by Freedland and Krebs (5) of the rate of glucose production from glycerol in perfused rat liver showed that the rate was appreciably increased in thyroxine-treated rats. Even earlier measurements by Lardy and coworkers (10) demonstrated a large increase in the rate of a-glycerophosphate oxidation by liver mitochondria from thyroid-fed rats that was due to the increased activity of the mitochondrial aGP dehydrogenase. METHODS AND MATERIALSLiver parenchymal cells were isolated from male SpragueDawley rats (190-240 g) that had been starved for 24 hr prior to sacrifice. In weight-matched groups, the animals received intraperitoneal injections of either L-3,3',5-triiothyronine (T3; Calbiochem, B grade) dissolved in isotonic saline at pH 9.8 (T3 treated, hyperthyroid)...

Mechanism of liver glycogen repletion in vivo by nuclear magnetic resonance spectroscopy.

“…With respect to assumption c, most studies have shown that there is a rapid and essentially complete equilibration between dihydroxy-acetone phosphate and glyceraldehyde-3-phosphate at the triose isomerase reaction (21,22). Furthermore, our own data support the assumption in that the enrichment of C-2 was equal to the enrichment of C-5.…”

Substrate cycling between gluconeogenesis and glycolysis in euthyroid, hypothyroid, and hyperthyroid man.

“…Any activity of this cycle will cause additional loss of deuterium from the 2-position of glucose and lead to an overestimation of the SCR between glucose and glucose-6-phosphate. It has been calculated that -8% of glucose is metabolized by the hexose monophosphate pathway in man (31) and that this activity was not altered in hepatocytes taken from T3-treated rats (32).…”

Elucidation of the role of fructose 2,6-bisphosphate in the regulation of glucose fluxes in mice using in vivo13C NMR measurements of hepatic carbohydrate metabolism

“…Body temperature was maintained at 37.0 ± 0.5°C with a warm water circulation system based on a feedback obtained from a temperature probe placed on the abdomen of the mice (Cole Parmer, Vernon Hills, IL, USA). 99% enriched [1][2][3][4][5][6][7][8][9][10][11][12][13] C]D-glucose (20% w/v solution, Isotec Inc., Miamisburg, OH) was infused either intravenously through the tail vein (n ¼ 9) or intraperitoneally (n ¼ 3) with an initial bolus of approximately 50 mgAEkg )1…”

“…First, this is the first study to implement and use full threedimensional localization of 13 C NMR signals of glycogen in the intact liver in vivo. Second, during infusion of [1][2][3][4][5][6][7][8][9][10][11][12][13] C]glucose, label incorporation was observed not only into the C1 of glycogen but also the C6, which can only occur by label scrambling at the level of the trioses. In addition, the signals of glycogen C2-C5 were detected, possibly reflecting changes in natural abundance glycogen (i.e.…”

“…To examine the role of Fru-2,6-P 2 in the regulation of hepatic carbohydrate metabolism in vivo, Fru-2,6-P 2 levels were elevated in ADM mice with adenovirus-mediated overexpression of a double mutant bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (n ¼ 6), in comparison to normal control mice (control, n ¼ 6). The rates of hepatic glycogen synthesis in the ADM and control mouse liver in vivo were measured using new advances in 13 C NMR including 3D localization in conjunction with [1][2][3][4][5][6][7][8][9][10][11][12][13] C]glucose infusion. In addition to glycogen C1, the C6 and C2-C5 signals were measured simultaneously for the first time in vivo, which provide the basis for the estimation of direct and indirect synthesis of glycogen in the liver.…”