Rate-Limiting Steps of Gluconeogenesis in Liver Cells as Determined with the Aid of Fluoro-Dicarboxylic Acids

Abstract

Fluoromalate (an inhibitor of malate dehydrogenase and the malate carrier) or difluorooxaloacetate (an inhibitor of aspartate aminotransferase) inhibited gluconeogenesis from both pyruvate and lactate, but had no effect on glucose formation from fructose or endogenous substrates. When cells were incubated with both fluorocarboxylic acids simultaneously, an additive effect on the inhibition of glucose formation from lactate was observed, but the inhibition was not additive when pyruvate was the glucogenic precursor. Pyruvate removal was reduced I5 O/ , , , by difluorooxaloacetate and 44O/, by fluoromalate. Lactate production from pyruvate was inhbited 36O/, by fluoromalate but was almost unaffected by ditluorooxaloacetate.The results suggest that both malate-oxaloacetate and glutamate-aspartate shuttles participate, but to different extents, in glucose formation from either pyruvate or lactate. The malateoxaloacetate shuttle predominates during gluconeogenesis from pyurvate whereas the glutamateaspartate shuttle is of greater importance when lactate is the gluconeogenic precursor. However, the inhibition by fluoromalate of glucose formation from lactate indicates that not all the reducing equivalents arising during lactate oxidation are directly available for the reductive step of gluconeogenesis.Neither of the two shuttles seems to compensate for inhibition of the alternative system. This lack of compensation implies that each shuttle is working close to or a t its maximal capacity. Hence, transfer processes between mitochondria and cytoplasm must be considered as potential rate-limiting and regulatory sites for gluconeogenesis. The failure of fluoromalate to inhibit ethanol oxidation suggests that alternatives to the malate-oxaloacetate shuttle exist for the transfer to the mitochondria of reducing equivalents generated in the cytoplasmic compartment.

Regulatory Role of Reducing‐Equivalent Transfer from Substrate to Oxygen in the Hepatic Metabolism of Glycerol and Sorbitol

“…The animals were fasted for 18 h to deplete hepatic glycogen. Suspensions of isolated parenchymal cells were obtained as described previously [13,14] and used in amounts of 120--220mg, wet weight, for each incubation. Incubations were carried out in a differential respirometer a t 37 "C with a gas phase of air, and oxygen uptake was measured by standard manometric techniques.…”

“…In the present experiments the strategy of approach has been similar to the one used in a previous study [13]. Suspensions of intact isolated liver cells [I41 have been used in conjunction with specific enzyme inhibitors to assess the relative activities of the various potential shuttle systems during hepatic metabolism of glycerol and sorbitol.…”

Stimulatory Effects of Thyroxine Administration on Reducing‐Equivalent Transfer from Substrate to Oxygen during Hepatic Metabolism of Sorbitol and Glycerol

“…After appropriate correction, the rates of substrate uptake are equivalent to the rates of hydrogen flux from these substrates to 0, [l]. As in previous studies [1,3,12] the dry weight of the cells was obtained after precipitation with trichloracetic acid and a conversion factor of 3.77 [12] …”

“…After perfusion for 15 to 25 min, the softened liver was dispersed in 60 ml of the same medium and the dispersion transferred to two 250-ml conical flasks, gassed with 95O/, O,, 50/, CO, and shaken a t 120 cycles/min for 12 min a t 37 "C to break up cell clumps and to digest debris. The cells were filtered through nylon mesh of 210 pm pore size (Tobler, Ernst and Traber Inc., New York, U.S.A.), washed three times in ice-cold enzyme-free medium, devoid of added bicarbonate, and use immediately for metabolic studies, as described previously [1,3,12]. The cells (100-160 mg wet weight) were incubated for 40min in a final volume of 4 m l a t 37 "C with a gas phase of air.…”

The Role of Hydrogen Translocating Shuttles during Ethanol Oxidation in Hepatocytes from Euthyroid and Hyperthyroid Rats

“…In liver mitochondria from euthyroid rats the maximal rate of glycerol-3-phosphate oxidation was 13 nmol 0 2 x (mg mitochondi-ial protein)-' x min-'. Since 1 g wet weight of liver contains ;ihoiiI 60 mg mitochondrial protein [42], and since Z 2 dry weight of cells is equal to 3.77 g wet weight [43,44], the capacity of the glycerol-3-phosphate cycle is 13 x 2 x 60 x 60 x 3.77 x = 353 pmol x (g dry weight of cells)-' x h-'. The K , value of glycerol 3-phosphate for mitochondrial oxidation was 1.05 m M ; this is equivalent to 2.1 pmol/g dry weight of cells, assuming that 1 g dry weight of cells contains 2 ml of cytosolic water [44].…”