[13C]bicarbonate labelled from hyperpolarized [1-13C]pyruvate is an in vivo marker of hepatic gluconeogenesis in fasted state

AbstractHyperpolarized [1-13C]pyruvate enables direct in vivo assessment of real-time liver enzymatic activities by 13C magnetic resonance. However, the technique usually requires the injection of a highly supraphysiological dose of pyruvate. We herein demonstrate that liver metabolism can be measured in vivo with hyperpolarized [1-13C]pyruvate administered at two- to three-fold the basal plasma concentration. The flux through pyruvate dehydrogenase, assessed by 13C-labeling of bicarbonate in the fed condition, was found to be saturated or partially inhibited by supraphysiological doses of hyperpolarized [1-13C]pyruvate. The [13C]bicarbonate signal detected in the liver of fasted rats nearly vanished after treatment with a phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, indicating that the signal originates from the flux through PEPCK. In addition, the normalized [13C]bicarbonate signal in fasted untreated animals is dose independent across a 10-fold range, highlighting that PEPCK and pyruvate carboxylase are not saturated and that hepatic gluconeogenesis can be directly probed in vivo with hyperpolarized [1-13C]pyruvate.

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Evidence that the stimulation of lipogenesis in the mammary glands of starved lactating rats re-fed with a chow diet is dependent on continued hepatic gluconeogenesis during the absorptive period. Effects of a gluconeogenic inhibitory, mercaptopicolinic acid, in vivo

Biochemical Journal ◽

10.1042/bj2280727 ◽

1985 ◽

Vol 228 (3) ◽

pp. 727-733 ◽

Cited By ~ 8

Author(s):

D H Williamson ◽

V Ilic ◽

R G Jones

Keyword(s):

Mammary Gland ◽

Phosphoenolpyruvate Carboxykinase ◽

Mammary Glands ◽

Hepatic Glycogen ◽

Chow Diet ◽

Hepatic Gluconeogenesis ◽

Rapid Stimulation ◽

Stimulation Of

The rapid stimulation of lipogenesis in mammary gland that occurs on re-feeding starved lactating rats with a chow diet was decreased (60%) by injection of mercaptopicolinic acid, an inhibitor of hepatic gluconeogenesis at the phosphoenolpyruvate carboxykinase step. Mercaptopicolinate had no effect on lipogenesis in mammary glands of fed lactating rats. The inhibition of lipogenesis persisted in vitro when acini from mammary glands of re-fed rats treated with mercaptopicolinate were incubated with [1-14C]glucose. Mercaptopicolinate added in vitro had no significant effect on lipogenesis in acini from starved-re-fed lactating rats. Mercaptopicolinate prevented the deposition of glycogen and increased the rate of lipogenesis in livers of starved-re-fed lactating rats, whereas it had no significant effect on livers of fed lactating rats. Administration of intraperitoneal glucose restored the rate of mammary-gland lipogenesis in re-fed rats treated with mercaptopicolinate to the values for re-fed rats. Hepatic glycogen deposition was also restored, and the rate of hepatic lipogenesis was stimulated 5-fold. It is concluded that stimulation of mammary-gland lipogenesis on re-feeding with a chow diet after a period of starvation is in part dependent on continued hepatic gluconeogenesis during the absorptive period. Possible sources of the glucose precursors are discussed.

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Transcriptional repression of the gluconeogenic gene PEPCK by the orphan nuclear receptor SHP through inhibitory interaction with C/EBPα

Biochemical Journal ◽

10.1042/bj20061549 ◽

2007 ◽

Vol 402 (3) ◽

pp. 567-574 ◽

Cited By ~ 28

Author(s):

Min Jung Park ◽

Hee Jeong Kong ◽

Hye Young Kim ◽

Hyeong Hoe Kim ◽

Joon Hong Kim ◽

...

Keyword(s):

Nuclear Receptor ◽

Transcriptional Repression ◽

Phosphoenolpyruvate Carboxykinase ◽

Regulatory Element ◽

Direct Interaction ◽

Orphan Nuclear Receptor ◽

Hepatic Gluconeogenesis ◽

Inhibitory Interaction

SHP (short heterodimer partner) is an orphan nuclear receptor that plays an important role in regulating glucose and lipid metabolism. A variety of transcription factors are known to regulate transcription of the PEPCK (phosphoenolpyruvate carboxykinase) gene, which encodes a rate-determining enzyme in hepatic gluconeogenesis. Previous reports identified glucocorticoid receptor and Foxo1 as novel downstream targets regulating SHP inhibition [Borgius, Steffensen, Gustafsson and Treuter (2002) J. Biol. Chem. 277, 49761–49796; Yamagata, Daitoku, Shimamoto, Matsuzaki, Hirota, Ishida and Fukamizu (2004) J. Biol. Chem. 279, 23158–23165]. In the present paper, we show a new molecular mechanism of SHP-mediated inhibition of PEPCK transcription. We also show that the CRE1 (cAMP regulatory element 1; −99 to −76 bp relative to the transcription start site) of the PEPCK promoter is also required for the inhibitory regulation by SHP. SHP repressed C/EBPα (CCAAT/enhancer-binding protein α)-driven transcription of PEPCK through direct interaction with C/EBPα protein both in vitro and in vivo. The formation of an active transcriptional complex of C/EBPα and its binding to DNA was inhibited by SHP, resulting in the inhibition of PEPCK gene transcription. Taken together, these results suggest that SHP might regulate a level of hepatic gluconeogenesis driven by C/EBPα activation.

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Changes in the hypothalamic contents of LHRH-I and -II and in pituitary responsiveness to synthetic chicken LHRH-I and -II during the progesterone-induced surge of LH in the laying hen

Journal of Endocrinology ◽

10.1677/joe.0.1270487 ◽

1990 ◽

Vol 127 (3) ◽

pp. 487-496 ◽

Cited By ~ 9

Author(s):

S. C. Wilson ◽

R. A. Chairil ◽

F. J. Cunningham ◽

R. T. Gladwell

Keyword(s):

Plasma Concentration ◽

Pituitary Gland ◽

Laying Hens ◽

Plasma Concentrations ◽

Posterior Hypothalamus ◽

Anterior Hypothalamus ◽

Significant Fall ◽

Basal Plasma

ABSTRACT The contents of LHRH-I and -II in the anterior hypothalamus and posterior hypothalamus (including the mediobasal hypothalamus and median eminence) were measured at 90, 180 and 360 min after the i.m. injection of laying hens with progesterone. Whilst no changes were observed in the content of LHRH-I in the anterior hypothalamus, LHRH-I in the posterior hypothalamus tended to fall at 90 and 180 min after injection of progesterone in hens maintained on 16 h light:8 h darkness (16L:8D) and 8L:16D respectively. Pretreatment of laying hens with tamoxifen significantly increased the hypothalamic contents of LHRH-I and -II, raised the basal plasma concentration of LH and modified the LH response to progesterone injection. In hens in which tamoxifen prevented an increase in the plasma concentration of LH after progesterone injection, the content of LHRH-I in the posterior hypothalamus remained unchanged. In contrast, in hens in which progesterone stimulated a steep increase in LH within 90 min, there was a pronounced and significant fall in LHRH-I content of the posterior hypothalamus. No change in the hypothalamic content of LHRH-II was observed during the progesterone-induced surge of LH until plasma concentrations had attained maximal values or started to decline. Then, in hens maintained on 16L:8D, a significant fall in the content of LHRH-II in the anterior hypothalamus was found at both 180 and 360 min after injection with progesterone. Tests in vitro and in vivo of the responsiveness of the pituitary gland to synthetic LHRH-I and -II revealed no change at 90 min after injection of laying hens with progesterone, when plasma concentrations of LH were increasing, but a pronounced reduction when plasma LH concentrations were maximal or falling. These results suggest that LHRH-I mediates in the progesterone-induced increase in the plasma concentration of LH. Although the subsequent decline in plasma LH was associated with a reduced responsiveness of the pituitary gland to LHRH, a significant correlation between the contents of LHRH-I and -II in the anterior hypothalamus and a fall in the hypothalamic content of LHRH-II when plasma LH was maximal or declining allows the possibility of an involvement of this peptide in the neuroendocrine events preceding ovulation. Journal of Endocrinology (1990) 127, 487–496

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Measurement of Metabolic Fluxes through Pyruvate Kinase, Phosphoenolpyruvate Carboxykinase, Pyruvate Dehydrogenase, and Pyruvate Carboxylase in Hepatocytes of Different Acinar Origin

Archives of Biochemistry and Biophysics ◽

10.1006/abbi.1996.0066 ◽

1996 ◽

Vol 326 (2) ◽

pp. 202-206 ◽

Cited By ~ 14

Author(s):

Claire G. Jones ◽

Michael A. Titheradge

Keyword(s):

Pyruvate Kinase ◽

Pyruvate Dehydrogenase ◽

Pyruvate Carboxylase ◽

Phosphoenolpyruvate Carboxykinase ◽

Metabolic Fluxes

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Effect of dexamethasone on gluconeogenesis, pyruvate kinase, pyruvate carboxylase and pyruvate dehydrogenase flux in isolated hepatocytes

Biochemical Journal ◽

10.1042/bj2890821 ◽

1993 ◽

Vol 289 (3) ◽

pp. 821-828 ◽

Cited By ~ 20

Author(s):

C G Jones ◽

S K Hothi ◽

M A Titheradge

Keyword(s):

Pyruvate Kinase ◽

Pyruvate Dehydrogenase ◽

Pyruvate Carboxylase ◽

Phosphoenolpyruvate Carboxykinase ◽

Tricarboxylic Acid Cycle ◽

Acute Effect ◽

Isolated Hepatocytes ◽

Pyruvate Oxidation ◽

Parallel Increase ◽

Glucose Output

Treatment of 18 h-starved rats with dexamethasone and subsequent isolation and incubation of the hepatocytes in the presence of the steroid increased gluconeogenic flux with both 1.0 mM pyruvate and 1.0 mM lactate plus 0.2 mM pyruvate as the substrate. The magnitude of stimulation was comparable with both substrates. The increase in glucose output was accompanied by an increased flux through pyruvate carboxylase, although the absolute flux and magnitude were considerably less in the presence of the more reduced substrate. The effect of the steroid on the flux through pyruvate dehydrogenase was substrate-dependent, an inhibition occurring with the more oxidized substrate. There was no effect of steroid treatment on [1-14C]lactate or pyruvate oxidation or on tricarboxylic-acid-cycle flux as measured by [3-14C]pyruvate oxidation. Dexamethasone treatment resulted in a parallel increase in both pyruvate kinase flux and glucose synthesis with both substrates employed, indicating that the steroid had no effect on the partitioning of phosphoenolpyruvate between pyruvate and lactate formation and gluconeogenesis. Similarly there was no effect of the steroid on either the activity ratio or the total pyruvate kinase activity in the cells. It is suggested that the acute effect of the dexamethasone to increase gluconeogenesis resides at the level of phosphoenolpyruvate formation, i.e. pyruvate carboxylase and possibly phosphoenolpyruvate carboxykinase.

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Assessment of Aspartate and Bicarbonate Produced From Hyperpolarized [1-13C]Pyruvate as Markers of Renal Gluconeogenesis

Frontiers in Physiology ◽

10.3389/fphys.2021.792769 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hikari A. I. Yoshihara ◽

Arnaud Comment ◽

Juerg Schwitter

Keyword(s):

Phosphoenolpyruvate Carboxykinase ◽

Left Kidney ◽

The Body ◽

Spin Labels ◽

Resonance Spectroscopy ◽

Fasted State ◽

Renal Gluconeogenesis ◽

Invasive Techniques ◽

Fasted Rats ◽

Marked Drop

As both a consumer and producer of glucose, the kidney plays a significant role in glucose homeostasis. Measuring renal gluconeogenesis requires invasive techniques, and less invasive methods would allow renal gluconeogenesis to be measured more routinely. Magnetic resonance spectroscopy and imaging of infused substrates bearing hyperpolarized carbon-13 spin labels allows metabolism to be detected within the body with excellent sensitivity. Conversion of hyperpolarized 1-13C pyruvate in the fasted rat liver is associated with gluconeogenic flux through phosphoenolpyruvate carboxykinase (PEPCK) rather than pyruvate dehydrogenase (PDH), and this study tested whether this was also the case in the kidney. The left kidney was scanned in fed and overnight-fasted rats either with or without prior treatment by the PEPCK inhibitor 3-mercaptopicolinic acid (3-MPA) following infusion of hyperpolarized 1-13C pyruvate. The 13C-bicarbonate signal normalized to the total metabolite signal was 3.2-fold lower in fasted rats (p = 0.00073) and was not significantly affected by 3-MPA treatment in either nutritional state. By contrast, the normalized [1-13C]aspartate signal was on average 2.2-fold higher in the fasted state (p = 0.038), and following 3-MPA treatment it was 2.8-fold lower in fed rats and 15-fold lower in fasted rats (p = 0.001). These results confirm that, unlike in the liver, most of the pyruvate-to-bicarbonate conversion in the fasted kidney results from PDH flux. The higher conversion to aspartate in fasted kidney and the marked drop following PEPCK inhibition demonstrate the potential of this metabolite as a marker of renal gluconeogenesis.

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Inhibitory effects of histidine and their reversal. The roles of pyruvate carboxylase and N10-formyltetrahydrofolate dehydrogenase

Biochemical Journal ◽

10.1042/bj1770833 ◽

1979 ◽

Vol 177 (3) ◽

pp. 833-846 ◽

Cited By ~ 31

Author(s):

M C Scrutton ◽

I Beis

Keyword(s):

Rat Liver ◽

Pyruvate Carboxylase ◽

Phosphoenolpyruvate Carboxykinase ◽

Specific Activity ◽

Competitive Inhibitor ◽

Product Inhibition ◽

Detectable Effect ◽

Formyltetrahydrofolate Dehydrogenase ◽

Hydrolysis Of

1. N10-Formyltetrahydrofolate dehydrogenase was purified to homogeneity from rat liver with a specific activity of 0.7–0.8 unit/mg at 25 degrees C. The enzyme is a tetramer (Mw = 413,000) composed of four similar, if not identical, substrate addition and give the Km values as 4.5 micron [(-)-N10-formyltetrahydrofolate] and 0.92 micron (NADP+) at pH 7.0. Tetrahydrofolate acts as a potent product inhibitor [Ki = 7 micron for the (-)-isomer] which is competitive with respect to N10-formyltetrahydrofolate and non-competitive with respect to NADP+. 3. Product inhibition by NADPH could not be demonstrated. This coenzyme activates N10-formyltetrahydrofolate dehydrogenase when added at concentrations, and in a ratio with NADP+, consistent with those present in rat liver in vivo. No effect of methionine, ethionine or their S-adenosyl derivatives could be demonstrated on the activity of the enzyme. 4. Hydrolysis of N10-formyltetrahydrofolate is catalysed by rat liver N10-formyltetrahydrofolate dehydrogenase at 21% of the rate of CO2 formation based on comparison of apparent Vmax. values. The Km for (-)-N10-folate is a non-competitive inhibitor of this reaction with respect to N10-formyltetrahydrofolate, with a mean Ki of 21.5 micron for the (-)-isomer. NAD+ increases the maximal rate of N10-formyltetrahydrofolate hydrolysis without affecting the Km for this substrate and decreases inhibition by tetrahydrofolate. The activator constant for NAD+ is obtained as 0.35 mM. 5. Formiminoglutamate, a product of liver histidine metabolism which accumulates in conditions of excess histidine load, is a potent inhibitor of rat liver pyruvate carboxylase, with 50% inhibition being observed at a concentration of 2.8 mM, but has no detectable effect on the activity of rat liver cytosol phosphoenolpyruvate carboxykinase measured in the direction of oxaloacetate synthesis. We propose that the observed inhibition of pyruvate carboxylase by formiminoglutamate may account in part for the toxic effect of excess histidine.

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Activities of the enzymes of hepatic gluconeogenesis in periparturient dairy cows with induced fatty liver

Journal of Dairy Research ◽

10.1017/s0022029904000020 ◽

2004 ◽

Vol 71 (2) ◽

pp. 129-134 ◽

Cited By ~ 26

Author(s):

Absolom Murondoti ◽

Ruurd Jorritsma ◽

Anton C Beynen ◽

Theo Wensing ◽

Math JH Geelen

Keyword(s):

Fatty Liver ◽

Dairy Cows ◽

Pyruvate Carboxylase ◽

Phosphoenolpyruvate Carboxykinase ◽

Post Partum ◽

Sampling Period ◽

Negative Energy ◽

Hepatic Gluconeogenesis ◽

Gluconeogenic Enzymes ◽

Blood Glucose Concentrations

The objective was to measure the activities of all the enzymes essential for hepatic gluconeogenesis in dairy cows with induced fatty liver. We aimed to induce severe fatty liver in ten experimental cows by overfeeding them during the dry period while seven control cows were maintained on a restricted diet. To induce a marked negative energy balance, the experimental cows were deprived of feed for 8 h immediately after parturition. In addition, the experimental cows were given a restricted amount of diet during the first 5 d of lactation. Liver samples were collected 1 week before and 1, 2 and 4 weeks after parturition. Before parturition, liver triacylglycerol concentrations did not differ between the two groups. After parturition, the experimental cows developed marked fatty liver as indicated by a higher level of triacylglycerols in the liver compared with the control cows.Before parturition, all gluconeogenic enzymes in the liver were lower in experimental cows than in control cows. Phosphoenolpyruvate carboxykinase, pyruvate carboxylase and propionyl-CoA carboxylase were significantly lower and fructose 1,6-bisphosphatase and glucose 6-phosphatase tended to be lower in the experimental cows. The activities of two crucial enzymes for gluconeogenesis in ruminants, i.e., phosphoenolpyruvate carboxykinase and propionyl-CoA carboxylase, remained low throughout the sampling period post partum. Activities of pyruvate carboxylase and glucose 6-phosphatase in the experimental cows post partum were upgraded to values similar to those of the control cows. The results showed that the capacity for hepatic gluconeogenesis before parturition was lower in cows with induced fatty liver than in control cows. After parturition, the low activities of crucial gluconeogenic enzymes indicated insufficient production of glucose. It is suggested that the low gluconeogenic capacity leads successively to low blood glucose concentrations, low insulin levels and high rates of mobilization of fatty acid, causing severe hepatic lipidosis.

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[14C]bicarbonate fixation into glucose and other metabolites in the liver of the starved rat under halothane anaesthesia. Metabolic channelling of mitochondrial oxaloacetate

Biochemical Journal ◽

10.1042/bj2270851 ◽

1985 ◽

Vol 227 (3) ◽

pp. 851-865 ◽

Cited By ~ 16

Author(s):

D F Heath ◽

J G Rose

Keyword(s):

Pyruvate Carboxylase ◽

Phosphoenolpyruvate Carboxykinase ◽

Co2 Exchange ◽

Published Data ◽

Citrate Cycle ◽

Incomplete Mixing ◽

Metabolic Channelling ◽

Glucose Output

Previous attempts to account for the labelling in vivo of liver metabolites associated with the citrate cycle and gluconeogenesis have foundered because proper allowance was not made for the heterogeneity of the liver. In the basal state (anaesthetized after 24h starvation) this heterogeneity is minimal, and we show that labelling by [14C]bicarbonate can be interpreted unambiguously. [14C]Bicarbonate was infused to an isotopic steady state, and measurements were made of specific radioactivities of blood bicarbonate, alanine, glycerol and lactate, of liver alanine and lactate, and of individual carbon atoms in blood glucose and liver aspartate, citrate and malate. (Existing methods for several of these measurements were extensively modified.) The results were combined with published rates of gluconeogenesis, uptake of gluconeogenic precursors by the liver, and citrate-cycle flux, all measured under similar conditions, and with estimates of other rates made from published data. To interpret the results, three ancillary measurements were made: the rate of CO2 exchange by phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) under conditions that simulated those in vivo; the 14C isotope effect in the pyruvate carboxylase (EC 6.4.1.1) reaction (14C/12C = 0.992 +/- 0.008; S.E.M., n = 8); the ratio of labelling by [2-14C]- to that by [1-14C]-pyruvate of liver glutamate 1.5 min after injection. This ratio, 3.38, is a measure of the disequilibrium in the mitochondria between malate and oxaloacetate. The data were analysed with due regard to experimental variance, uncertainties in values of fluxes measured in vitro, hepatic heterogeneity and renal glucose output. The following conclusions were reached. The results could not be explained if CO2 fixation was confined to pyruvate carboxylase and there was only one, well-mixed, pool of oxaloacetate in the mitochondria. Addition of the other carboxylation reactions, those of PEPCK, isocitrate dehydrogenase (EC 1.1.1.42) and malic enzyme (EC 1.1.1.40), was not enough. Incomplete mixing of mitochondrial oxaloacetate had to be assumed, i.e. that there was metabolic channelling of oxaloacetate formed from pyruvate towards gluconeogenesis. There was some evidence that malate exchange across the mitochondrial membrane might also be channelled, with incomplete mixing with that in the citrate cycle. Calculated rates of exchange of CO2 by PEPCK were in agreement with those measured in vitro, with little or no activation by Fe2+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)

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