Inhibitor Of Phosphoenolpyruvate Carboxykinase Research Articles

Control of gluconeogenesis by phosphoenolpyruvate carboxykinase in cotyledons of Cucurbita pepo L.

3-Mercaptopicolinic acid, a non-competitive inhibitor of phosphoenolpyruvate carboxykinase (EC 4.1.1.19) was used to study the control of gluconeogenesis by this enzyme in germinating marrow (Cucurbita pepo) cotyledons. In vitro, phosphoenolpyruvate carboxykinase was inhibited by 3-mercaptopicolinic acid, with a Ki of 5.9 μM. At 25°C the inhibitor caused an increase in the label incorporated from [2-14C]acetate into CO2, and a decrease in the label incorporated into the insoluble and neutral fractions. Phosphoenolpyruvate carboxykinase had a flux control coefficient for gluconeogenesis (CPEPCKJ) of between 0.7 and 1.0. 3-Mercaptopicolinic acid was a less effective inhibitor of phosphoenolpyruvate carboxykinase at lower temperatures (Ki = 8.6 μM at 17°C, 13.3 μM at 10°C) and had similar effects on the metabolism of [2-14C]acetate by marrow cotyledons when the temperature was reduced to 17°C and 10°C. The control coefficient for this enzyme did not change with temperature, indicating that phosphoenolpyruvate carboxykinase exerts a high degree of control over gluconeogenesis at all temperatures examined.

Glycogen synthesis from glucose by direct and indirect pathways in hepatocyte cultures from different nutritional states

The conversion of glucose to glycogen by direct and indirect pathways was determined from the incorporation of [6- 3H,U- 14C]glucose into glycogen in hepatocyte cultures isolated from fed, fasted or fasted-refed rats. Mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase (PEPCK) was used to determine the extent by which 6-tritium is lost by mechanisms not involving flux through PEPCK. Glucose conversion to glycogen was lower in hepatocytes from fasted and higher in hepatocytes from fasted-refed rats than in hepatocytes from rats fed ad libitum. Insulin increased glycogen synthesis in hepatocytes from all nutritional states, and it decreased the 3 H 14 C ratio incorporated into glycogen. This increased loss of 6-tritium was only in part mercaptopicolinate-sensitive. Lactate and pyruvate (2 mM + 0.2 mM) increased glycogen deposition, largely by stimulation of glucose conversion to glycogen by the direct pathway. Insulin-induced glucokinase mRNA expression was higher in hepatocytes from fed than from fasted or refed rats whereas PEPCK mRNA expression was lowest in hepatocytes from fasted-refed rats. Hepatocyte cultures derived from different nutritional states express differences in glycogen synthesis from glucose by direct and indirect pathways as well as differences in the extent by which pyruvate cycling accounts for loss of 6-tritium.

Glutamine metabolism in rat small intestine: synthesis of three-carbon products in isolated enterocytes

Glutamine is a major respiratory fuel for enterocytes but the extent of glutamine decarboxylation in these cells is not certain. The metabolism of differentially labeled L-[ 14C]glutamine was studied in enterocytes isolated from fed rats. The results indicate that glutatime undergoes two decarboxylations and yields a three carbon end product. The first decarboxylation is presumably at α-ketoglutarate dehydrogenase but the identity of the second reaction is not clear. The addition of 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, was without effect on either the rate of glutamine metabolism or the extent of decarboxylation. Labeled glutamine carbon was recovered in three carbon products primarily as alanine with lesser amounts as lactate. The addition of glucose to the incubation medium did not change the rate of glutamine metabolism, or decarboxylation, but lactate became the major labeled three carbon end product. The results show that the fate, alanine or lactate, of glutamine derived pyruvate in enterocytes depends on the relative rate of flux through pyruvate and indicates that one cytosolic pool of pyruvate exists in these cells. The limited oxidation of glutamine in enterocytes ensures that the gluconeogenic potential of glutamine is conserved within the body.

Inhibition of glucose-6-phosphate phosphohydrolase by 3-mercaptopicolinate and two analogs is metabolically directive.

3-Mercaptopicolinae (3-MP) blocks gluconeogenesis from lactate, pyruvate, alanine, and other substrates through its inhibition of phosphoenolpyruvate carboxykinase. Nevertheless, we observed increased glycogenesis, net glucose uptake, and glucose-6-P levels in livers perfused with glucose in the presence of 3-MP. In perfusions with 20 mM dihydroxyacetone, increased glycogenesis and decreased glucose production were observed with 3-MP. These metabolic effects suggested additional site(s) of action of 3-MP. Further studies showed that 3-MP inhibits glucose-6-P phosphohydrolase activity of intact liver microsomes. Several compounds with structural similarities to 3-MP (2-mercaptonicotinic acid, picolinic acid, cysteine, reduced glutathione, nicotinic acid, quinolinic acid, tryptophan, and pyridine) were tested for their effect on glucose-6-P phosphohydrolase activity. Two of these compounds, 2-mercaptonicotinic acid and picolinic acid, were found to inhibit. In perfusions including 7.5 mM fructose, the addition of 3-MP, 2-mercaptonicotinic acid, or picolinic acid increased glycogenesis, decreased glucose production, and increased hepatic glucose-6-P concentrations. These observations indicate that the inhibition of glucose-6-P phosphohydrolase may play a role in enhanced glycogenesis from glucose, dihydroxyacetone, and fructose in isolated livers from 48-h fasted rats perfused with 3-MP or certain sulfhydryl-containing and sulfhydryl-devoid analogs.

The effect of treatment of the rat with bacterial endotoxin on gluconeogenesis and pyruvate metabolism in subsequently isolated hepatocytes

The effect of treatment of rats with bacterial endotoxin on gluconeogenesis and the flux through pyruvate kinase, phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase and pyruvate dehydrogenase (PDH) was measured in isolated hepatocytes, prepared from animals starved for 18 h, incubated in the presence of 1 mM pyruvate. The lipopolysaccharide reduced gluconeogenesis by 50% and lowered the flux through pyruvate kinase, PEPCK and pyruvate carboxylase by comparable amounts. There was no effect of endotoxaemia on PDH flux, indicating that the lowered rate of gluconeogenesis is not the result of a redistribution of pyruvate metabolism between oxidation and carboxylation. The results confirm that a stimulation of pyruvate kinase activity following treatment with lipopolysaccharide is not involved in the inhibition of gluconeogenesis, but that the effect resides at the level of phosphoenolpyruvate formation. The most favoured mechanism for the inhibition of glucose synthesis is via an inhibition of PEPCK and subsequent feedback inhibition of pyruvate carboxylase, although a secondary effect at the level of the mitochondria and pyruvate carboxylase cannot be excluded.

The contribution of pyruvate cycling to loss of [6-3H]glucose during conversion of glucose to glycogen in hepatocytes: effects of insulin, glucose and acinar origin of hepatocytes

1. During conversion of [6-3H,U-14C]glucose to glycogen in liver, loss of 6-3H can occur either by cycling via pyruvate (between glycolysis and gluconeogenesis) or by other mechanisms. We used mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, to determine the extent to which pyruvate cycling contributes to loss of 6-3H during glucose conversion to glycogen in hepatocytes. 2. Mercaptopicolinate increased the 3H/14C ratio in glycogen during incubation of rat, guinea pig, pig and human hepatocytes with [6-3H,U-14C]glucose. The increase in the 3H/14C ratio in glycogen caused by mercaptopicolinate was greater in periportal than in perivenous rat hepatocytes, indicating that cycling of glucose via pyruvate is more prominent in cells with a higher gluconeogenic relative to glycolytic capacity. 3. The effect of mercaptopicolinate on the 3H/14C ratio in glycogen was observed both in the absence and in the presence of insulin, indicating that stimulation of glycogen synthesis by insulin is not associated with inhibition of pyruvate cycling. In rat and guinea pig but not in pig hepatocytes, the effects of mercaptopicolinate on the 3H/14C ratio in glycogen were greater at 10-15 mM glucose than at 30 mM glucose, suggesting diminished cycling via pyruvate at high glucose concentrations. 4. Insulin increased the loss of 6-3H during stimulation of conversion of glucose to glycogen in hepatocytes from all species. This was due in part to an increase in pyruvate cycling and in part to other mechanisms that are not inhibited by mercaptopicolinate. 5. These results suggest that pyruvate cycling is a significant, but not exclusive, component of the loss of 6-3H in the hepatocyte during glucose conversion to glycogen. The extent of pyruvate cycling is dependent on the acinar origin of the hepatocytes and on the glucose concentration and presence of insulin.

The indirect pathway of hepatic glycogen synthesis and reduction of food intake by metabolic inhibitors

The increasingly recognized role of the indirect pathway (glycolysis followed by hepatic gluconeogenesis) for glucose utilization and glycogen synthesis by the liver led us to administer 3-mercaptopicolinate (3MP), an inhibitor of phosphoenolpyruvate-carboxykinase, in an attempt to assess the role of liver glycogen or hexose-phosphates in the food-intake reducing effects of (-)hydroxy-citrate. Administration of (-)hydroxy-citrate increased hepatic glycogen content in i.v. glucose refed rats. Using the glucuronide probe technique, the mechanism of increased glycogen deposition was shown to be prolongation of indirect pathway (recycled) input. Daily (-)hydroxy-citrate significantly reduced food intake (from 12.0 ± 2.3 to 6.4 ± g/day, p < 0.05) and had no chronic effect on hepatic glycogen content in rats trained to a single daily meal (meal-fed). Administration of 3MP completely suppressed hepatic glycogen synthesis (< 0.5 mg/g) when give alone or with (-)hydroxy-citrate. Isotopi studies confirmed inhibitation of the indirect pathway of UDP-glucose synthesis. 3MP accentuated rather than prevented the (-)hydroxy-citrate reduction in food intake in meal-fed rats (intake 2.7 ± 2.4 g/day). When given alone, 3MP also reduced intake (6.1 ± 3.6 g/day). Severe hypoglycemia was observed (glucose < 20 mg/dl) in several meal-fed rats given repeated daily doses of 3MP, yet food intake did not occur despite food availability. Neither 3MP nor (-)hydroxy-citrate had any effects when given after the daily meal. We conclude that the role of the indirect glycogen synthesis pathway must be considered in any theory of regulation of food intake by hepatic metabolites and that, if the effects of these metabolic inhibitors can be shown not to be toxic or non-specific, neither hepatic glycogen nor hexose-phosphates are involved in the food-intake suppressive effects of (-)hydroxycitrate.

The role of insulin and corticosterone in the toxicity of dioxins

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) alters the homeostasis of numerous hormones among them that of corticosterone and insulin. Hormonal status in turn affects toxicity of TCDD as shown by rapid exitus in response to nontoxic doses of insulin and a shifting of the dose response curve to the left in adrenalectomized rats. These two hormones are key regulators of PEPCK (phosphoenolpyruvate carboxykinase), which is the rate determining enzyme of gluconeogenesis. PEPCK end hence gluconeogenesis is dose-dependently inhibited by TCDD and its penta-, hexa- and hepta-chloro homologues as well as their mixture revealing an essentially perfect structure/activity relationship and strict additivity in their inhibitory effect. This structure/activity relationship shows a highly significant correlation with the structure/activity relationship of acute toxicity suggesting that inhibition of PEPCK is the key lesion in the toxicity of dioxins and of all other compounds acting by this mechanism. Inhibition of PEPCK is a very early effect beginning 4 to 8 hrs after an oral dose of TCDD and it is at maximum by 16 hrs after dosing. Amount of PEPCK-protein (Western blot) and PEPCK-mRNA (Northern blot) reflect the decreased activity of PEPCK suggesting that TCDD reduces the transcription of the PEPCK gene. Hormonal changes (insulin and corticosterone) are not apparent until 4 to 8 days after dosing indicating that they are secondary to an earlier lesion and as such they represent a response of the organism to the primary insult. Moreover, hormonal changes (decreased insulin and increased corticosterone) are consistent with maximum stimulation of PEPCK (as in the case in pair-fed controls) and yet PEPCK is dose-dependently inhibited. Therefore, we suggest that the mechanism of action of dioxins consists of an interaction with the PEPCK gene which renders the insulin- and glucocorticoid-responsive regions nonresponsive to endogenous stimuli.

Inhibition of phosphoenolpyruvate carboxykinase by 6-phosphogluconate in rat liver

Various concentrations of 6-phosphogluconate inhibit rat liver phosphoenolpyruvate carboxykinase activity. 0.04 mM 6-phosphogluconate, which is the concentration found in vivo, caused a 50% inhibition of 6-phosphoenolpyruvate carboxykinase activity. 6-Phosphogluconate lowered the Vmax of the enzyme and increased the concentration of phosphoenolpyruvate required to achieve one-half of the maximum velocity. The role of 6-phosphogluconate as a regulator of the coordination of fluxes through three metabolic pathways is discussed.

Stimulation of glycogen synthesis in hepatocytes by added amino acids is related to the total intracellular content of amino acids

Katz et al. [Katz, J., Golden, S. & Wals, P.A. (1976) Proc. Natl Acad. Sci. USA 73, 3433-3437] were the first to report that in hepatocytes isolated from fasted rats and incubated with either dihydroxyacetone, glucose or other sugars, glycogen synthesis was greatly accelerated by addition of amino acids. We have looked for possible mediators responsible for this effect and have tested the effect of alanine, proline, asparagine, glutamine or a combination of ammonia with either pyruvate or lactate in activating glycogen synthesis from dihydroxyacetone. The following observations were made. 1. Stimulation of glycogen synthesis by alanine, proline or asparagine does not require production of glutamine since the effect also occurs in periportal hepatocytes which lack glutamine synthetase. 2. Under various conditions, stimulation of glycogen synthesis by added amino acids directly correlated with increases in the intracellular content of amino acids, expressed in osmotic equivalents. 3. 3-Mercaptopicolinic acid, the inhibitor of phosphoenolpyruvate carboxykinase, further enhances stimulation of glycogen synthesis by amino acids because it increases the intracellular accumulation of aspartate and glutamate. 4. The previously reported enhancement by leucine of the stimulation of glycogen synthesis by glutamine [Chen. K. S. & Lardy, H. A. (1985) J. Biol. Chem. 260, 14683-14688] can be ascribed to inhibition of urea synthesis by leucine which results in accumulation of glutamate and of ammonia, the essential activator of glutaminase. It is concluded that activation of glycogen synthesis by added amino acids is due to an increase in intracellular osmolarity following their uptake and the accumulation of intracellular catabolites. This results in an increase in hepatic volume which stimulates glycogen synthesis [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955-959].

Glutamine synthesis from aspartate in guinea-pig renal cortex

1. Glutamine was found to be the main carbon and nitrogen product of the metabolism of aspartate in isolated guinea-pig kidney-cortex tubules. Glutamate, ammonia and alanine were only minor products. 2. Carbon-balance calculations and the release of 14CO2 from [U-14C]aspartate indicate that oxidation of the aspartate carbon skeleton occurred. 3. A pathway involving aspartate aminotransferase, glutamate dehydrogenase, glutamine synthetase, phosphoenolpyruvate carboxykinase, pyruvate kinase, pyruvate dehydrogenase and enzymes of the tricarboxylic acid cycle is proposed for the conversion of aspartate into glutamine. 4. Evidence for this pathway was obtained by: (i) inhibiting aspartate removal by amino-oxyacetate, an inhibitor of transaminases, (ii) the use of methionine sulphoximine, an inhibitor of glutamine synthetase, which induced a large increase in ammonia release from aspartate, (iii) the use of quinolinate, an inhibitor of phosphoenolpyruvate carboxykinase, which inhibited glutamine synthesis from aspartate, (iv) the use of alpha-cyano-4-hydroxycinnamate, an inhibitor of the mitochondrial transport of pyruvate, which caused an accumulation of pyruvate from aspartate, and (v) the use of fluoroacetate, an inhibitor of aconitase, which inhibited glutamine synthesis with concomitant accumulation of citrate from aspartate.

Glycogenesis from lactate in rabbit skeletal muscle fiber types

The path of glycogen synthesis from three-carbon precursors was studied via single-pass perfusions in three distinct rabbit skeletal muscle preparations, i.e., glycolytic (greater than 99% type IIb), oxidative (greater than 97% type I), and mixed (type I, IIa, and IIb). The extent of interaction between the Krebs cycle and glycogenesis was assessed utilizing [1-14C]- or [2-14C]lactate at basal (1.1 +/- 0.1 mM) and elevated (8.1 +/- 0.3 mM) lactate concentrations (protocols 1 and 2). Under conditions in which the net balance of glucose and lactate, [14C]lactate removal, and venous lactate-specific activity were similar, the yields of 14CO2 and [14C]glycogen were not significantly influenced by position of the label. Additional perfusions were performed with lactate (8.0 +/- 0.1 mM) and acetate (1.0 +/- 0.1 mM) as sole substrates and either [U-14C]lactate or [2-14C]acetate as the tracer. Under conditions of net glycogen synthesis, the incorporation of [14C]lactate into glycogen [in disintegrations/min (dpm).g-1.2 h-1] was 40,940 +/- 3,320, 1,540 +/- 320, and 32,600 +/- 4,100 in the glycolytic, oxidative, and mixed preparations, respectively. However, no incorporation of [2-14C]acetate into glycogen was observed in any preparation, despite a significant yield of 14CO2. Mercaptopicolinic acid, a potent inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), demonstrated no significant effect on net substrate balance, tracer uptake, net glycogen synthesis, incorporation of [14C]lactate and [3H]-glucose into glycogen, or 14CO2 yield. Current results suggest an extramitochondrial route for net glycogen synthesis from three-carbon precursors, exclusive of PEPCK, that is consistent across all mammalian skeletal muscle fiber types.

Absence of a stimulatory effect of the corpus cardiacum on gluconeogenesis in cockroach ( Periplaneta americana) fat body

The haemolymph alanine pool was labelled with [ 14C]alanine to measure conversion of the amino acid to trehalose. The haemolymph alanine concentration was increased approx. 10% by this procedure. Although incorporation of label into trehalose was linear for 2 h the rate of incorporation was very low, accounting for only 1% of the injected alanine during this period. Injection of corpus cardiacum extract into intact cockroaches increased haemolymph trehalose by more than 100% 1 and 2 h after injection. The same concentration of extract did not alter the rate of conversion of[ 14C]alanine to trehalose in vivo. In vivo, the total incorporation of 14C from [ 14C]alanine, [ 14C]glutamic acid and [ 14C]glycine into fat body glycogen was not affected by corpus cardiacum extract. Neither basal trehalose synthesis nor that stimulated by corpus cardiacum extract was altered by amino(oxy)acetic acid, an inhibitor of aminotransferases or mercaptopicolinic acid, an inhibitor of phosphoenolpyruvate carboxykinase. The results indicate that the gluconeogenic pathway is not of major importance for the synthesis of trehalose and glycogen. Furthermore, no evidence was obtained which would support the proposal that the pathway might be regulated by hormonal factors from the corpus cardiacum.

Contribution of purine nucleotide cycle to intranephron ammoniagenesis in rats.

To evaluate the contribution of the purine nucleotide cycle (PNC) in renal ammoniagenesis, ammonia production (AP) in cortical tubular suspensions and microdissected nephron segments of control and acidotic rats was determined using various amino acids, including glutamine (Gln) and aspartate (Asp). In the cortical tubular suspensions, the best substrate for ammoniagenesis was Gln (153.1 +/- 19.4 nmol.mg protein-1.15 min-1) followed by Asp (70.9 +/- 11.4). Metabolic acidosis resulted in a significant increase of AP only from Gln (316.5 +/- 36.1 nmol.mg protein-1.15 min-1, P less than 0.01 vs. control). Intranephron distribution of AP (pmol/mm or a glomerulus/15 min) from Gln showed that the first segment of the proximal tubule (S1) was highest in control (95.7 +/- 9.0), and its AP markedly increased in acidosis (221.6 +/- 8.3, P less than 0.001 vs. control). The most interesting and striking finding was that with Asp as a substrate, AP was maximal in S1 (165.0 +/- 32.8), with a value exceeding that from Gln. An adenylosuccinase inhibitor, 6-mercaptopurine (0.1 mM), significantly inhibited AP from Asp in S1 and S3, and from Gln in S1. On the contrary, a specific inhibitor of phosphoenolpyruvate carboxykinase, 3-mercaptopicolinate (0.1 mM), caused a significant decrease of AP from Gln, but not from Asp, in S1. From these results it could be concluded that AP via PNC can occur at high rates, especially in S1, only when Asp is present at high concentrations.

Synthesis of malate from phospho enolpyruvate by rabbit liver mitochondria: implications for lipogenesis

(1) Rabbit liver mitochondria can convert exogenous phospho enolpyruvate to malate. (2) Malate production is dependent on phospho enolpyruvate and HCO 3 − and is stimulated by CN − or malonate alone and especially in combination. (3) Malate production is inhibited 70% by 3-mercaptopicolinate, a specific inhibitor of phospho enolpyruvate carboxykinase, and 50–60% by 1,2,3-benzenetricarboxylate, an inhibitor of the tricarboxylate tranporter. (4) Rat liver mitochondria incubated with phospho enolpyruvate under identical conditions do not produce malate. (5) Malate production from phospho enolpyruvate is stimulated by exogenous GDP or IDP but not by ADP. (6) Data support the conclusion that malate is being produced from oxalacetate generated by reversal of mitochondrial phospho enolpyruvate carboxykinase. A possible role of this enzyme in hepatic lipogenesis is suggested.

Photosynthesis in phosphoenolpyruvate carboxykinase-type C 4 plants: Photosynthetic activities of isolated bundle sheath cells from Urochloa panicoides

A method has been developed for rapidly preparing bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase-type C 4 plant. These cells catalyzed both HCO − 3- and oxaloacetate-dependent oxygen evolution; oxaloacetate-dependent oxygen evolution was stimulated by ATP. For this activity oxaloacetate could be replaced by aspartate plus 2-oxoglutarate. Both oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution were accompanied by PEP production and both were inhibited by 3-mercaptopicolinic acid, an inhibitor of PEP carboxykinase. The ATP requirement for oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution could be replaced by ADP plus malate. The increased oxygen evolution observed when malate plus ADP was added with oxaloacetate was accompanied by pyruvate production. These results are consistent with oxaloacetate being decarboxylated via PEP carboxykinase. We suggest that the ATP required for oxaloacetate decarboxylation via PEP carboxykinase may be derived by phosphorylation coupled to malate oxidation in mitochondria. These bundle sheath cells apparently contain diffusion paths for the rapid transfer of compounds as large as adenine nucleotides.

Gluconeogenesis from fructose predominates in periportal regions of the liver lobule.

Gluconeogenesis from fructose was studied in periportal and pericentral regions of the liver lobule in perfused livers from fasted, phenobarbital-treated rats. When fructose was infused in increasing concentrations from 0.25 to 4 mM, corresponding stepwise increases in glucose formation by the perfused liver were observed as expected. Rates of glucose and lactate production from 4 mM fructose were around 100 and 75 mumol/g/h, respectively. Rates of fructose uptake were around 190 mumol/g/h when 4 mM fructose was infused. 3-Mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, decreased glucose formation from fructose maximally by 20% suggesting that a fraction of the lactate formed from fructose is used for glucose synthesis. A good correlation (r = 0.92) between extra oxygen consumed and glucose produced from fructose was observed. At low fructose concentrations (less than 0.5 mM), the extra oxygen uptake was much greater than could be accounted for by glucose synthesis possibly reflecting fructose 1-phosphate accumulation. Furthermore, fructose diminished ATP/ADP ratios from about 4.0 to 2.0 in periportal and pericentral regions of the liver lobule indicating that the initial phosphorylation of fructose via fructokinase occurs in both regions of the liver lobule. Basal rates of oxygen uptake measured with miniature oxygen electrodes were 2- to 3-fold higher in periportal than in pericentral regions of the liver lobule during perfusions in the anterograde direction. Infusion of fructose increased oxygen uptake by 65 mumol/g/h in periportal areas but had no effect in pericentral regions of the liver lobule indicating higher local rates of gluconeogenesis in hepatocytes located around the portal vein. When perfusion was in the retrograde direction, however, glucose was synthesized nearly exclusively from fructose in upstream, pericentral regions. Thus, gluconeogenesis from fructose is confined to oxygen-rich upstream regions of the liver lobule in the perfused liver.

A nuclear magnetic resonance study of the role of phosphoenol pyruvate carboxykinase (PEPCK) in the glucose metabolism of Dipetalonema viteae

13C-NMR has been applied to the study of the metabolism of [1- 13C]glucose by macrofilariae of Dipetalonema viteae under conditions of restricted glucose supply. In a medium buffered with 13C-labelled bicarbonate, succinate labelled in the carboxyl position is formed in good yield. Quinolinic acid, a known inhibitor of phosphoenol pyruvate carboxykinase (PEPCK) has been shown to suppress the formation of labelled succinate from [1- 13C]glucose. Both sets of experiments support the formation of succinate through the PEPCK-mediated carboxylation of phosphoenol pyruvate, followed by the operation of a partial tricarboxylic acid cycle.

The inhibition of phosphoenolpyruvate carboxykinase (guanosine triphosphate) gene expression by insulin is not mediated by protein kinase C.

The role protein kinase C plays in the regulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression by insulin and phorbol esters was studied in H4IIE hepatoma cells (ATCC CRL 1548). The combined effects of phorbol 12-myristate 13-acetate (PMA) and insulin on the suppression of mRNA coding for PEPCK (mRNAPEPCK) synthesis were additive. A potent inhibitor of both cyclic nucleotide-dependent protein kinases and protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, inhibited the cAMP and PMA-mediated regulation of mRNAPEPCK synthesis, but did not affect the action of insulin. Desensitization of the protein kinase C pathway by exposure to PMA for 16 h abolished the subsequent action of the phorbol ester, but did not affect insulin- or cAMP-mediated regulation of PEPCK gene expression. We conclude that insulin suppresses PEPCK gene expression independently from the protein kinase C-mediated pathway used by phorbol esters.

Gluconeogenesis in Rhizobium leguminosarum MNF3841

Summary: Gluconeogenesis in Rhizobium leguminosarum MNF3841 takes place via the derepressible enzymes phosphoenolpyruvate carboxykinase (PEPCK) and fructose bisphosphate aldolase. A Tn5-induced PEPCK deficient mutant (MNF3085) fails to grow on a wide range of simple carbon sources. PEPCK and fructose bisphosphate aldolase are rapidly derepressed when cells are transferred from a sugar to an organic acid as the sole carbon source. The addition of 0·1 mM-sucrose causes an 80% inhibition of PEPCK and fructose bisphosphate aldolase synthesis, and 0·4 mM-sucrose causes a complete inhibition of PEPCK synthesis. Pea bacteroids of MNF3841 purified on a Percoll gradient contain low levels of PEPCK and fructose bisphosphate aldolase. This bacteroid-associated PEPCK activity is clearly of bacterial not plant origin because of its nucleotide requirement, and the fact that bacteroids of MNF3085 contain no PEPCK activity at all. Although the mutant lacks PEPCK it is still able to nodulate and fix N2 as effectively as the parent. The capacity to synthesize sugars via gluconeogenesis is not required for an effective symbiosis. Furthermore, these data suggest that pea bacteroids receive sufficient sugar to compensate for the gluconeogenic defect in strain MNF3085, but insufficient to completely repress the synthesis of PEPCK in the wild-type.