13C NMR study of gluconeogenesis from labeled alanine in hepatocytes from euthyroid and hyperthyroid rats.

Abstract

Metabolism of [3-'3C]alanine in the presence and absence of 3-hydroxybutyrate or ethanol has been followed at 250C by 13C NMR at 90.5 MHz in primary hepatocytes from untreated rats and rats treated with triiodothyronine and not allowed to eat for 24 hr. The phosphoenolpyruvate/pyruvate futile cycle was followed in situ by comparing the concentration of 13C at the scrambled alanine C2 position with that at glucose C5. In the absence of ethanol, the flux through pyruvate kinase was 60% of the gluconeogenic flux in hepatocytes from hyperthyroid rats, compared with 25% in the controls. Incubation with ethanol reduced the pyruvate kinase flux in the hyperthyroid state to that measured in the controls. Under all conditions, the relative concentration of label at the aspartate C2 and C3 sites was 1:2, whereas at the corresponding carbons in glutamate, randomization was almost complete. These observations, which require flux of unscrambled label into aspartate, are consistent with intramitochondrial synthesis of aspartate only if there is incomplete mixing of the intramitochondrial oxaloacetate pool. The 13C enrichment measured in the ketone bodies is increased by the presence of exogenous f3-hydroxybutyrate. The greater labeling that we observe at C2 of f3-hydroxybutyrate compared with C4 under this condition is explained by the flow through 3-hydroxy-3-methylglutaryl-coenzyme A synthase.In a recent '3C NMR study of suspensions of isolated hepatocytes from euthyroid and hyperthyroid rats, we measured the flux through the gluconeogenic pathway from labeled glycerol into glucose, determined the pentose cycle activity, and examined the pathway through the nonoxidative pentose branch (1). In another study, in which we followed the metabolism of 13C-labeled alanine in perfused mouse liver, the competition between ethanol and alanine into the tricarboxylic acid cycle was investigated, and it was also suggested that the 13C NMR spectra provided a unique probe ofthe in situ flux through pyruvate kinase (2).The present 13C study shows the effect of thyroid hormone treatment upon the flux through pyruvate kinase during gluconeogenesis from labeled alanine in primary rat hepatocytes. The results confirm our previous studies in perfused mouse liver of the competition between ethanol and alanine into the tricarboxylic acid cycle. METHODS AND MATERUILSLiver parenchymal cells were isolated from male Sprague-Dawley rats (190-240 g) that had not eaten for 24 hr. The animals received intraperitoneal injections of either L-3,3',5-triiodothyronine (T3; Calbiochem, B grade) dissolved in isotonic saline at pH 9.8, 8 jig of T3 per 100 g of body weight per day (T3-treated, hyperthyroid), or isotonic saline alone (or no injections) (euthyroid control) for 4 days prior to cell preparation.The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.The liver cell preparation, the resuspens...

Response to Burgess

“…1,5,6 are incompatible with our human data 2 and inconsistent with animal studies that have directly measured this flux in vitro 3,8,9 and in vivo 4 . This difference may arise because [U- 13 C]propionate, the tracer used by Burgess et al 5,6 , probably promotes pyruvate cycling anaplerosis, and gluconeogenesis by mass action effects and/or by its rapid conversion to propionyl-CoA and subsequent activation of pyruvate carboxylase 10 .…”

Measurement of gluconeogenesis and pyruvate recycling in the rat liver: a simple analysis of glucose and glutamate isotopomers during metabolism of [1,2,3‐13C3]propionate

“…Numerous techniques have been used for such measurements, including GC-mass spectrometry [1][2][3] NMR [4][5][6], and radiotracers [7,8]. One key control point that connects these two pathways is the interconversion of PEP and pyruvate, catalyzed in the forward direction by pyruvate kinase and in the reverse direction by the combined action of pyruvate carboxylase and phosphoenolpyruvate carboxykinase.…”

Metabolism ofD- andL-[13C]alanine in rat liver detected by1H and13C NMR spectroscopyin vivo andin vitro

“…It has been employed in the study of the metabolism of [3-13 C]alanines in different physiological states; [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] Stromski et al 17 studied in vivo hepatic gluconeogenesis, and Shalwitz et al 18 hepatic glycogen synthesis from [3-13 C]-L-alanine in rats. The effect of the peroxisome proliferator perfluoro-n-decanoic acid on hepatic gluconeogenesis and glycogenesis was investigated after infusion of [3][4][5][6][7][8][9][10][11][12][13] C]-L-alanine. 19 To our knowledge, hepatic metabolism after infusion of D-alanine or [1-13 C]alanine has so far not been studied in perfused livers or in livers in vivo by 13 C NMR spectroscopy.…”