Fructose 1,6-bisphosphatase in rat liver cytosol: Activation after glucagon treatment in vivo and inhibition by fructose 2,6-bisphosphate in vitro

Mörikofer‐Zwez, Stéphanie; Stoecklin, Franziska B.; Walter, Paul

doi:10.1016/s0006-291x(81)80016-2

Cited by 23 publications

(5 citation statements)

References 17 publications

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“…A possible resolution of this problem comes from the recent discovery of fructose 2,6-bisphosphate, a potent regulator of both phosphofructokinase and fructose bisphosphatase, whose concentration in hepatocytes can be changed by a number of effectors of metabolism, including glucagon (Hers & Van Schaftingen, 1982). A glucagon-induced fall in fructose 2,6-bisphosphate levels (Richards & Uyeda, 1980) would be expected to reduce phosphofructokinase activity and increase fructose bisphosphatase activity to an extent dependent on the phosphorylation states of these enzymes, which may be changed by glucagon (Furuya & Uyeda, 1980;Brand & Soling, 1982;Morikofer-Zwez et al, 1981). The situation is further complicated by the fact that many of the effects of phosphorylation and of fructose 2,6-bisphosphate are changes in substrate affinity and/or affinity for allosteric activators and inhibitors, whose cellular levels can also vary.…”

Section: Substrate Cyclementioning

confidence: 99%

Quantitative analysis of intermediary metabolism in hepatocytes incubated in the presence and absence of glucagon with a substrate mixture containing glucose, ribose, fructose, alanine and acetate

Rabkin¹,

Blum²

1985

Biochemical Journal

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Hepatocytes were isolated from the livers of fed rats and incubated, in the presence and absence of 100 nM-glucagon, with a substrate mixture containing glucose (10 mM), fructose (4 mM), alanine (3.5 mM), acetate (1.25 mM), and ribose (1 mM). In any given incubation one substrate was labelled with 14C. Incorporation of 14C into glucose, glycogen, CO2, lactate, alanine, glutamate, lipid glycerol and fatty acids was measured after 20 and 40 min of incubation under quasi-steady-state conditions [Borowitz, Stein & Blum (1977) J. Biol. Chem. 252, 1589-1605]. These data and the measured O2 consumption were analysed with the aid of a structural metabolic model incorporating all reactions of the glycolytic, gluconeogenic, and pentose phosphate pathways, and associated mitochondrial and cytosolic reactions. A considerable excess of experimental measurements over independent flux parameters and a number of independent measurements of changes in metabolite concentrations allowed for a stringent test of the model. A satisfactory fit to the data was obtained for each condition. Significant findings included: control cells were glycogenic and glucagon-treated cells glycogenolytic during the second interval; an ordered (last in, first out) model of glycogen degradation [Devos & Hers (1979) Eur. J. Biochem. 99, 161-167] was required in order to fit the experimental data; the pentose shunt contributed approx. 15% of the carbon for gluconeogenesis in both control and glucagon-treated cells; net flux through the lower Embden-Meyerhof pathway was in the glycolytic direction except during the 20-40 min interval in glucagon-treated cells; the increased gluconeogenesis in response to glucagon was correlated with a decreased pyruvate kinase flux and lactate output; fluxes through pyruvate kinase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase were not coordinately controlled; Krebs cycle activity did not change with glucagon treatment; flux through the malic enzyme was towards pyruvate formation except for control cells during interval II; and 'futile' cycling at each of the five substrate cycles examined (including a previously undescribed cycle at acetate/acetyl-CoA) consumed about 26% of cellular ATP production in control hepatocytes and 21% in glucagon-treated cells.

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Section: Substrate Cyclementioning

confidence: 99%

Quantitative analysis of intermediary metabolism in hepatocytes incubated in the presence and absence of glucagon with a substrate mixture containing glucose, ribose, fructose, alanine and acetate

Rabkin¹,

Blum²

1985

Biochemical Journal

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“…Because an elevation of hepatic fructose-2,6-P2 by 2,5-AMol could explain the observed inhibition of gluconeogenesis and stimulation of glycolysis (30)(31)(32), direct measurements of fructose-2,6-P2 were performed. At concentrations as low as 0.1 mM, 2,5-AM-ol blocks most of the dihydroxvacetone-induced increase of fructose-2,6-P2 concentrations and significantly reduces the increase caused by glucose or by fructose (Table 1).…”

Section: Effects Of 25-am-ol On Gluconeogenesis and Glycolysis Inmentioning

confidence: 99%

Regulation of carbohydrate metabolism by 2,5-anhydro-D-mannitol.

Riquelme¹,

Wernette-Hammond²,

Kneer³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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In hepatocytes isolated from fasted rats, 2,5-anhydromannitol inhibits gluconeogenesis from lactate plus pyruvate and from substrates that enter the gluconeogenic pathway as triose phosphate. This fructose analog has no effect, however, on gluconeogenesis from xylitol, a substrate that enters the pathway primarily as fructose 6-phosphate. The sensitivity of gluconeogenesis to 2,5-anhydromannitol depends on the substrate metabolized; concentrations of 2,5-anhydromannitol required for 50% inhibition increase in the order lactate plus pyruvate < dihydroxyacetone < glycerol < sorbitol < fructose. The inhibition by 2,5-anhydromannitol of gluconeogenesis from dihydroxyacetone is accompanied by an increase in lactate formation and by two distinct crossovers in gluconeogenic-glycolytic metabolite patterns i.e., increases in pyruvate concentrations with decreases in phosphoenolpyruvate and increases in fructose-1,6-bisphosphate concentrations with little change in fructose 6-phosphate. In addition, 2,5-anhydromannitol blocks the ability of glucagon to stimulate gluconeogenesis and inhibit lactate production from dihydroxyacetone. 2,5-Anhydromannitol decreases cellular fructose 2,6-bisphosphate content in hepatocytes; therefore the effects of the fructose analog are not mediated by fructose 2,6-bisphosphate, a naturally occurring allosteric regulator. 2,5-Anhydromannitol also inhibits gluconeogenesis in hepatocytes isolated from fasted diabetic rats, but higher concentrations of the analog are required.2,5-Anhydro-D-mannitol (2,5-AM-ol), an analog of P-D-fructose locked in the furan ring structure, is phosphorylated by fructokinase to form 2,5-AM-ol-l-P (1, 2).Because 2,5-AM-ol is symmetrical, the monophosphate product can be considered an analog of both fructose-i-P and fructose-6-P (3). Because of the stability of its ring structure, 2,5-AMI-ol monophosphate cannot be cleaved bv aldolase in a manner similar to that of fructose-l-P, nor can it act as a substrate for phosphoglucoisomerase and be converted to glucose-6-P in a manner similar to that of fructose-6-P. 2,5-AM-ol monophosphate is a substrate for phosphofructokinase 1 (4, 5). The resulting product, 2,5-AM-ol bisphosphate, is an analog of 8-fructose-1,6-P2 rather than a-fructose-1,6-P2 and, as such, is not hydrolyzed readily by fructose-1,6-bisphosphatase, which prefers the a anomer (6). The bisphosphate compound, thus, should accumulate within the cell. In vitro experiments have shown that 2,5-AM-ol bisphosphate is a competitive inhibitor of fructose-1,6-bisphosphatase (7,8). In view of the described findings, the potential of 2,5-AM-ol to act as a regulator of gluconeogenesis and glycolysis was examined in isolated rat hepatocytes.METHODS AND MATERIALS Synthesis of 2,5-AM-ol. 2,5-AM-ol was prepared as in ref.9 except the crystallization step was omitted. The crude 2,5-AM1-ol, in 5 mM ammonium borate (pH 9), was purified on Dowex-I-X-8 (borate) (10). The unretained material, after deionization and repeated concentration by evaporation of methanol, showe...

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“…The structure of this compound was first put forth as fructose 2,6-bisphosphate by Van Schaftingen and Hers (1) and subsequently confirmed by us spectroscopically and by 13C NMR (5). Fructose 2,6-bisphosphate is an allosteric activator of 6-phosphofructo-l-kinase (1,2,(4)(5)(6)(7) and an inhibitor of fructose 1,6-bisphosphatase (8)(9)(10). The level of fructose 2,6-bisphosphate has been shown to vary widely depending on the hormonal and dietary state (2,(11)(12)(13).…”

mentioning

confidence: 93%

Regulation of 6-phosphofructo-2-kinase activity by cyclic AMP-dependent phosphorylation.

el-Maghrabi¹,

Claus²,

Pilkis³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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Addition of glucagon to isolated rat hepatocytes resulted in inhibition of 6-phosphofructo-2-kinase (ATP:D-fructose-6-phosphate 2-phosphotransferase) activity in extracts of the cells and in a decrease in the intracellular level of fructose 2,6-bisphosphate. The effect on 6-phosphofructo-2-kinase was characterized by a decrease in the affinity of the enzyme for fructose 6-phosphate. To investigate the mechanism of action of glucagon, 6-phosphofructo-2-kinase from rat liver was partially purified by polyethylene glycol precipitation, DEAE-cellulose chromatography, (NH4)2SO4 fractionation, Sephacryl S-200 gel filtration, DEAE-Sephadex chromatography, and Sephadex G-100 gel filtration. Incubation of the purified enzyme with the catalytic subunit ofthe cyclic AMP-dependent protein kinase from rat liver and [y-32P]ATP resulted in 32p incorporation into a protein with a subunit Mr of 49,000 as determined by NaDodSO4 disc gel electrophoresis. Associated with this phosphorylation was an inhibition of 6-phosphofructo-2-kinase activity that was also characterized by a decrease in the affinity of the enzyme for fructose 6-phosphate. Both the phosphorylation and the inhibition of the purified 6-phosphofructo-2-kinase were blocked by addition of the heatstable protein kinase inhibitor. It is concluded that the glucagoninduced decrease in fructose 2,6-bisphosphate levels observed in isolated hepatocytes is due, at least in part, to cyclic AMP-dependent phosphorylation and inhibition of 6-phosphofructo-2-ldnase.A number of groups have presented evidence for the existence in liver of a novel effector of 6-phosphofructo-1-kinase (1-7). The structure of this compound was first put forth as fructose 2,6-bisphosphate by Van Schaftingen and Hers (1) and subsequently confirmed by us spectroscopically and by 13C NMR (5). Fructose 2,6-bisphosphate is an allosteric activator of 6-phosphofructo-l-kinase (1, 2, 4-7) and an inhibitor of fructose 1,6-bisphosphatase (8-10). The level of fructose 2,6-bisphosphate has been shown to vary widely depending on the hormonal and dietary state (2, 11-13). For example, glucagon addition to isolated hepatocytes results in a 90% decrease in its level within minutes (2,4,11,12). These results suggest that the enzyme responsible for the synthesis or degradation (or both) offructose 2,6-bisphosphate is regulated by a cyclic AMP (cAMP)-dependent phosphorylation mechanism (11). Recently, the enzyme responsible for fructose 2,6-bisphosphate synthesis was isolated and shown to catalyze the transfer of the -y phosphate of ATP to the C-2 position of fructose 6-phosphate (14-16). The goal of the present study was to determine whether rat liver 6-phosphofructo-2-kinase is regulated by cAMP-dependent protein kinase-catalyzed phosphorylation and to characterize this regulation. MATERIALS AND METHODSPreparation and Incubation of Isolated Hepatocytes. Isolated rat hepatocytes were prepared from fed rats (male, Sprague-Dawley, 175-225 g) as described (3). The cells were suspended (final concentration, 50 mg o...

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Fructose 1,6-bisphosphatase in rat liver cytosol: Activation after glucagon treatment in vivo and inhibition by fructose 2,6-bisphosphate in vitro

Cited by 23 publications

References 17 publications

Quantitative analysis of intermediary metabolism in hepatocytes incubated in the presence and absence of glucagon with a substrate mixture containing glucose, ribose, fructose, alanine and acetate

Quantitative analysis of intermediary metabolism in hepatocytes incubated in the presence and absence of glucagon with a substrate mixture containing glucose, ribose, fructose, alanine and acetate

Regulation of carbohydrate metabolism by 2,5-anhydro-D-mannitol.

Regulation of 6-phosphofructo-2-kinase activity by cyclic AMP-dependent phosphorylation.

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