Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis.

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THE REGULATION OF HEPATIC GLUCONEOGENESIS AND GLYCOLYSIS S. J. Pilkis Department of Physiology and Biophysics, State University of New York, Stony

Brook,

New

York

11794

D. K. Granner

Department of Mol ec ular Physiol ogy a nd Nashville, Tennessee 37232 KEY WORDS:

Biophysics, Vanderbilt University,

substrate cycles, gene regulation, hormone action, glucose homeostasis

INTRODUCTION

Glucose is a major energy source for all mammalian cells, and it is the principal source of energy for the brain. A constant supply of glucose must be provided in order to ensure against hypoglycemia and the potentially cata strophic effect this can have on cells of the nervous system. Continual ingestion of a carbohydrate-rich diet can provide the glucose, but prolonged periods of fasting, as in sleep or protracted exercise, can place the organism at risk. Also, restraint on hyperglycemia, which has its own set of deleterious consequences, must be exerted. An intricate mechanism for maintaining the blood glucose within a relatively narrow range has evolved to accomplish these purposes. This involves the production of glucose by the liver, from glycogenolysis and gluconeogenesis, and the peripheral clearance of glucose by tissues such as the skeletal muscle, adipose tissue, and the splanchnic bed, 885

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PILKIS & GRANNER

including the liver. This chapter deals with the homeostatic role the liver plays as a producer/consumer, of glucose. Hepatic glucose metabolism begins and ends with the movement of glucose into or out of the hepatocyte through one member of a family of glucose transporters. This transporter, GLUT2, is discussed in the chapter by Pessin & Bell (this volume). The hepatocyte can

also store glucose in the form of glycogen. The mechanisms of hepatic

glycogen synthesis and degradation are beyond the scope of this chapter. We restrict our comments to the acute and chronic regulation of hepatic glu

Annu. Rev. Physiol. 1992.54:885-909. Downloaded from www.annualreviews.org Access provided by Kaohsiung Medical University on 04/26/18. For personal use only.

coneogenesis or glycolysis, per se.

HEPATIC SUBSTRATE CYCLES The fact that hepatocytes contain rate-controlling enzymes specific for glu coneogenesis ([phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6bisphosphatase Fru-I,6-P2ase), and glucose-6-phosphatase (Glu-6-Pase)] and glycolysis [pyruvate kinase (PK), 6-phosphofructo I-kinase (6-PF-I-K), and glucokinase (GK)] makes a sensitive control system possible. Cycling of the substrates and products of these opposing reactions can be governed in rate and direction of net flux by changes in allosteric effectors, by changes in the concentration of the enzymes involved in the cycles, andlor by covalent modification of these enzymes (51, 99). According to this view, three sub strate cycles, each of which is driven by enzymes acting in opposite direc tions, determine whether the hepatocyte produces or utilizes glucose (Figure

1). The direction and magnitude of net pathway flux through these three substrate cycles depends on the relative activity of the seven enzymes illus trated in Figure 1. Although many other enzymes are involved in these processes, they either catalyze equilibrium reactions, and therefore are not rate-controlling, or they catalyze reactions that are not quantitatively impor tant. Flux through the enzymes of these cycles is modulated by short-term (seconds to minutes) and long-term (minutes to hours) regulatory mechanisms (51, 99). An endogenous source of glucose is required when animals are starved or fed a low carbohydrate diet, or subjected to prolonged exercise. These conditions result in high plasma levels of glucagon, glucocorticoids, and catecholamines which, in tum, result in increased activity of PEPCK, Fru-l,6-P2ase and G-6-Pase and a coordinate decrease of PK, 6-PF-I-K and GK (51, 99, 100). These changes drive the three substrate cycles in the direction of gluconeogenesis. Reciprocal changes of the activity of these enzymes also occur when animals are fed a diet rich in carbohydrate, particu larly after a prolonged fast. In this situation, the plasma insulin concentration increases, levels of the counter-regulatory hormones decrease, and glycolytic flux and glycogen synthesis predominate. The short-term effects of glucagon,

REGULATION OF SUBSTRATE CYCLE ENZYMES

887

",GLUCOSE,


r--G,u- -

--' e 1

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8

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� ·

....

(±)

� I PK .

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t PEP

PEPC ,

OAA

..

�

/

�

LAC ALA Figure 1

Substrate cycles in the glycolytic/gluconeogenic pathway . The enzymes of the three

hepatic substrate cycles, which are subject to short-term and long-term regulation by hormones, are L-type pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK), fructose 1 ,6bisphosphatase

(Fru-l,6-Pzase),

6-phosphofructo-l-kinase

(6-PF-I-K),

6-phosphofructo-2-

kinase/fructose 2,6-bisphosphatase (6-PF-2-K/Fru 2,6-P2ase), glucokinase (OK), and glucose 6-phosphatase (Olu-6-Pase). Fructose-2,6-bisphosphate (Fru-2,6-P2) is an activator of 6-PF-I-K and an inhbitor of Fru-! ,6-P2 ase.


888

PILKIS & GRANNER

insulin, and catecholamines on substrate flux are mediated via changes in the level of cAMP with concomitant changes in the phosphorylation of several substrate cycle enzymes and/or by changes in allosteric effectors (52, 99, 100). These effectors act on a limited number of the enzymes, i.e. PK, 6-PF-I-K, and Fru-l,6-P2ase. The long-term effects of hormones on the expression of genes that encode the substrate cycle enzymes are also mediated by changes in cAMP as well as by cAMP-independent mechanisms, including the actions of insulin and glucocorticoids (SO). In the following sections we summarize the molecular mechanisms whereby the coordinated activity of the seven key regulatory substrate cycle enzymes, established by the interaction of these several hormones, determines whether the hepatocyte is a consumer or producer of glucose. SHORT-TERM REGULATION OF SUBSTRATE CYCLE ENZYME ACTIVITY Regulation at the Fru-6-PIFru-J,6-P2 Cycle

While there is no evidence for short-term hormonal regulation of the enzymes of Glu/Glu-6-P cycle, there is complex and important short-term regulation of the Fru-6-P/Fru-l,6-P2 substrate cycle (l4, 51, 98-100). This latter cycle is a pivotal crossroad for glycolytic/gluconeogenic flux in liver. Fru-l,6-P2, by modulating pyruvate kinase activity, affects substrate cycling at the pyruvatel PEP substrate cycle (13, 14, 98-100). Fru-l,6-P2 levels are controlled by the activities of the gluconeogenic enzyme Fru-l , 6-P2ase and by 6-PF-l-K, the opposing glycolytic enzyme. The activity of these enzymes and net flux through the Fru-6-P/Fru 1,6-P2 cycle are modulated by hormones and dietary status (11, 12, 62, 66, 133, 134). For example, starvation and glucagon both decrease flux through 6-PF-I-K by inhibiting the enzyme, while increasing Fru- l ,6-P2ase activity and, ipso facto, the rate of gluconeogenesis. Both enzymes are substrates for cAMP-dependent protein kinase, and it was first thought that this was the mechanism whereby these activities were altered (7, 15, 25, 26, 61, 101, 109, 116). However in vitro phosphorylation by cAMP-dependent protein kinase had little effect on the activity of these enzymes (see References 94 and 98 for review). Furthermore, the inhibition of 6-PF-I-K activity seen in crude extracts of hepatocytes incubated with glucagon disappeared on partial purification of the enzyme (16). It was subsequently shown that a low molecular weight effector, Fru-2, 6-Pz, was involved in the regulation of both 6-PF-I-K and Fru- l ,6-P2ase by cAMP (14, ,52, 92, 100). Fru-2,6-P2 is a potent allosteric activator of 6-PF-I-K and is a competitive inhibitor of Fru-l,6-Pzase (14, 51, 52, 63, 92, 98-100). The importance of the effects of Fru-2,6-Pz on these two enzymes vis-a-vis regulation of hepatic ,



889

gluconeogenesis is underscored by the observation that this effector is subject to both nutritional and hormonal regulation (14, 92, 93, 131). When the level of Fru-2,6-P2 is low, the rates of gluconeogenesis are high (starvation and diabetes). When the level of Fru-2,6-P2 is high, the rate of gluconeogenesis is low (refeeding and insulin administration). Synthesis of Fru-2,6-P2 involves a 6-PF-2-K reaction (Fru-6-P + ATP -'? Fru-2,6-P2 + ADP), whereas its degradation is catalyzed by a specific Fru-2,6-P2ase reaction (Fru-2,6-P2 -'? Fru-6-P + P). A unique bifunctional enzyme, 6-PF-2-KlFru-2,6-P2ase, catalyzes both of these reactions (14,28,32,79, 95). Phosphorylation of this enzyme by cAMP-dependent protein kinase results in inhibition of the kinase and activation of the bisphosphatase, whereas dephosphorylation results in opposite changes (99, 100). These changes explain the rapid modulation of Fru-2,6-P2 that occurs when l3-adrenergic agonists, glucagon or insulin, are added to isolated hepatocytes (14, 46, 93, 100). The increase in Fru-2,6-P2 that occurs when a-adrenergic agonists are added to hepatocytes isolated from fed rats is not due to changes in phosphorylation state of the enzyme ( 14, 46), but rather to enhanced glycogenolysis and the provision of more Fru-6-P for the enzyme 6-PF-2-K (55). Rat liver 6-PF-2-KlFru-2,6-P2ase is a homodimer whose subunit (55 kd) is constructed of three domains: (a) an NHz-terminal regulatory domain (resi dues 1-32), which contains the cAMP-dependent phosphorylation site (Ser32); (b) the kinase domain (residues 33-249); and (c) the bisphosphatase domain (residue 250--470) (4). The kinase and bisphosphatase domains have been expressed separately in heterologous expression systems, and they are independent catalytic domains (126, 128). The kinase and bisphosphatase halves of the bifunctional enzyme are structurally similar to the glycolytic enzymes 6-PF-I-K and phosphoglycerate mutase, respectively (4). Com puter-assisted modeling of the C-terminal bisphosphatase domain reveals a hydrophobic core and active site residue constellation equivalent to that found in yeast phosphoglycerate mutase (4, 30, 100). Experiments involving site directed mutagenesis and analysis of substrate specificity confirm the hypoth esis that the bisphosphate domain is structurally and functionally related to the phosphoglycerate mutase family ( 125, 127). Sequence patterns derived from the structural alignment of mutases and the bisphosphatases reveal a signifi cant similarity to a family of acid phosphatases (4, 100). Fru-2,6-P2ase and the phosphoglycerate mutase/acid phosphatase all catalyze their respective reactions via a phosphohistidine. intermediate (4, 100, 132). The N-terminal kinase domain, in turn, forms a nucleotide-binding fold that is analogous to a segment of 6-PF- 1-K, which suggests that both 6-PF-I-K and 6-PF-2-K bind Fru-6-P and ATP with similar geometry (4). The bifunctional enzyme is probably the product of a gene fusion involving the kinase and the mutase/ phosphatase catalytic units (4, 100). It is possible that the regulatory region,

890

PILKIS &

GRANNER

with its cAMP-dependent phosphorylation site, was added later to provide the mechanism whereby this enzyme acts as a switching system to modulate glycolytic/gluconeogenic flux. Cyclic AMP-dependent protein kinase is the only protein kinase known to catalyze the phosphorylation of hepatic 6-PF-2-KlFru-2,6-P2ase, while de phosphorylation of the enzyme is catalyzed primarily by protein phosphatase 2A (79, 89). It was originally believed that modulation of the phosphorylation


state of the bifunctional enzyme was mediated only by changes in the extent of cAMP-dependent phosphorylation, but recent evidence suggests that de phosphorylation is also regulated. Insulin stimulates the dephosphorylation of 6-PF-2-KlFru-2,6-P2ase and PK under conditions in which cAMP levels were unchanged (3). The postulation that insulin promotes the activity of one or more phosphatases is consistent with the small increase in phosphatase 2A activity seen when insulin is added to isolated hepatocytes (40).

Regulation at the Pyruvate/PEP Cycle Several studies suggest that pyruvate kinase (PK) is an important site of

hormonal regulation of gluconeogenesis (51, 99). Some PEP is recycled to pyruvate during gluconeogenesis in perfused liver and isolated hepatocytes

(42-44, 112), and carbon flux through PK is affected by the nutritional state and hormones (112-114). Glucagon and cAMP strongly inhibit this flux, whereas epinephrine is only marginally effective (38, 96, 104, 113). Insulin relieves the inhibition of PK flux and activity caused by glucagon (6, 97, 104). The complex regulation of PK is probably related to its role as a glycolytic enzyme. Liver-type PK, an allosteric enzyme, exhibits sigmoidal kinetics with regard to its substrate, PEP. PK is allosterically activated by Fru-l,6-P2 and is allosterically inhibited by alanine and ATP. At physiological con centrations of alanine, ATP, and PEP, the enzyme would be completely inhibited unless it were activated by Fru-l ,6-P2 (41). Thus, regulation of the Fru-6-PlFru-I,6-P2 and pyruvate/PEP cycles are linked by Fru-I,6-P2 (14, 98-100). Rat liver PK can be phosphorylated in vitro by cAMP-dependent protein kinase (27, 36, 73). Phosphorylation increases the apparent Km of the enzyme for PEP, but has no effect on activity in the presence of a saturating concentra tion of substrate or Fru-l,6-P2 (6, 27, 36, 73, 96, 110, 111). The phosphory lated enzyme is more readily inhibited by alanine and ATP than is the nonphosphorylated enzyme, but it is less readily activated by Fru-l,6-P2 than is the nonphosphorylated enzyme (27, 36, 73). Allosteric effectors of PK affect activity and also modulate phosphorylation of the enzyme by cAMP-dependent protein kinase (29, 39, 73). For example,

?

both Fru-1, -P2 and PEP inhibit the rate of phosphorylation by cAMP-



891

dependent protein kinase, while alanine relieves the inhibition by physiolog ical concentrations of either Fru-1,6-P2 or PEP. Thus PK is a better substrate for cAMP-dependent protein kinase when the enzyme is inhibited than when it is activated. 2 Hepatic PK can also be phosphorylated in vitro by Ca + -calmodulin dependent protein kinase on the same seryl residue as that phosphorylated by cAMP-dependent protein kinase. It is also phosphorylated on a threonyl residue located five residues COOH-terminal from the cAMP-dependent phosphorylation site (119, 123). Phosphorylation of either or both sites results in decreased affinity for PEP and the inhibition of enzyme activity (119). The effects of cAMP-dependent phosphorylation on PK have also been observed in vivo and in isolated liver cell systems. The addition of glucagon to isolated hepatocytes or perfused liver, or its administration in vivo, results in increased phosphate content of PK and, concomitantly, inhibition of enzyme activity and flux (6,13,38,42,45,57,72,110,111,124). Insulin opposes the action of glucagon on PK by virtue of its ability to decrease cAMP levels (13). In addition, the ability of glucagon to increase the phosphorylation of the enzyme in hepatocytes is modulated by Fru-1,6-P2 or alanine in a manner consistent with their effects on cAMP-dependent protein z . kinase-catalyzed phosphorylation of the enzyme in vitro (13, 42). Ca + calmodulin-dependent phosphorylation probably accounts for the small effect a-adrenergic agonists have on PK activity and flux in hepatocytes (8, 45, 124). Insulin also suppresses the effect of a-adrenergic agonists on PK activity by a cAMP-independent mechanism (13), perhaps by stimulating dephosphorylation of the enzyme (3). Coordinate Regulation at the Fru 6-P/Fru 1,6-P2 and Pyruvate/PEP Substrate Cycles

Substantial data now support the hypothesis that the short-term effect of insulin, glucagon, and adrenergic agents on glycolysis and gluconeogenesis involves modulation of PK by a phosphorylation mechanism and of the enzymes of the Fru-6-P/Fru-1,6-Pz cycle via changes in Fru-2,6-P2 (Figure 2). Under physiologic conditions, however, the rate of gluconeogenesis is limited by reactions at the pyruvate/PEP cycle and not by reactions at the Fru-6-P/Fru 1,6-P2 cycle since the rate of glucose synthesis is much greater for substrates that enter the pathway at the triose phosphate level than for substrates such as lactate and alanine (37). The importance of modulation of Fru-2,6-P2 levels for regulation of gluconeogenesis is that it provides a mechanism for control of Fru- l ,6-P2• For example, glucagon lowers hepatic Fru-l ,6-P2, and this amplifies the inhibition of PK by phosphorylation and contributes to increased gluconeogenesis (99, 100). Conversely, Fru-2,6-P2 also acts as an important signal molecule for glycolysis in liver since glycoly-

892

PILKIS & GRANNER


tic flux occurs only when Fru-2,6-P2 is elevated ( l 00). Therefore, regulation at the Fru-6-P/Fru- l ,6-P2 and pyruvate/PEP substrate cycles can be thought of as being coordinated by the cAMP-dependent protein kinase-catalyzed phosphorylation of PK and of 6-PF-2-KlFru-2,6-P2ase, and by alterations of Fru-l ,6-P2 brought about by changes in the phosphorylation state of the bifunctional enzyme (Figure 2).

LONG-TERM REGULATION OF SUBSTRATE CYCLE ENZYME ACTIVITY In the following section we briefly summarize the current knowledge of the mechanism(s) whereby hormones control expression of the genes that encode key regulatory enzymes in the glycolytic/gluconeogenic pathway. The GluIGlu-6-Pase Cycle GLUCOKINASE Upon entering the hepatocyte through the GLUT2 transpor ter, glucose is converted to Glu-6-P by the enzyme glucokinase (GK). GK is an unusual member of the hexokinase (HK) family. Its Km for glucose is >5 mM, whereas the Km for HK I-III (GK is HK IV) varies between 20 and 120 /-tM (see References 75 and 139, for review). In the hepatocyte, glucose is efficiently converted to Glu-6-P because GK, unlike HK I-III, is not subject to feedback inhibition by the product, Glu-6-P (139). Since GLUT2 operates by facilitated diffusion, it is the activity of GK, which keeps intracellular glucose concentrations very low, that determines glucose clearance by hepatocytes. GK activity is not altered by covalent modification so changes in its activity are entirely due to changes in the amount of the protein. Gene transcription, mRNAGK levels, and GK activity are decreased when plasma glucagon is

Figure 2

Coordinate regulation at the Fru-6-P/Fru-I,6-P2 and pyruvate/PEP substrate cycles. A.

Sites of action of stimulatory hormones on hepatic gluconeogenesis in the rat. Glucagon and {3and a-adrenergic agonists interact with their respective receptors in the plasma membrane. Glucagon and the {3-adrenergic agonists act to enhance adenylate cyclase activity. which leads to elevated cAMP, increased cAMP-dependent protein kinase, and phosphorylation of pyruvate kinase and the bifunctional enzyme at seryl residues. This leads directly and indirectly to diminished PEP�Pyr flux, enhanced Fru-l ,6-P2�Fru-6-P flux, and reduced Fru-6-P�Fru- l ,6P2 flux, and to decreased Fru- I ,6-P2, which leads to increased glucose synthesis. The much 2+ smaller effect of Ca2+ -linked hormones on gluconeogenesis is mediated by Ca -calmodulin dependent protein kinase-catalyzed phosphorylation of pyruvate kinase at both seryl and threonyl residues. B. Sites of insulin action to reverse the effect of stimulatory hormones on glu coneogenesis in the rat. Insulin binds to its tyrosine-kinase receptor resulting in enhanced low K", phosphodiesterase activity, which reduces cAMP levels and cAMP actions on pyruvate kinase and the bifunctional enzyme. In addition, insulin-receptor interaction opposes the Ca2+_


893

A

o o o I \


'..... -.---------"FI,S-P2 ------' ... '\ PEP.... \

'S:'

I'

• PK

OAA

t / 'PYR" .-

ER-P·!

LAC

tcA-P.K·

PM t p-AGONIST B

C(

AGONIST

GLUCOSE

•

calmodulin-dependent phosphorylation of pyruvate kinase by an as yet unknown mechanism. Possibilities include suppression of changes in Ca2+ flux, insulin-mediated effects on protein kinases or phosphatases, or as yet unidentified tyrosine-protein kinase phosphorylation events. Regulation at the two substrate cycles is coordinated by changes in the level of Fru-1,6-P2• In panel A, Fru-I,6-P2 is decreased , which amplifies the inhibitory effect of cAMP-dependent and calmodulin-dependent phosphorylation of pyruvate kinase. In panel B, the level of Fru-l,6-P2 is elevated, compared to that in panel A, and pyruvate kinase activity and flux are enhanced.


894

PILKIS & GRANNER

high and plasma insulin is low (e.g. fasting or diabetes) (59, 76, 81, 91, 120, 139). Refeeding a diet high in carbohydrate increases plasma insulin and decreases plasma glucagon, and these changes have profound and rapid effects on transcription of the GK gene. A 20--30-fold increase in transcription occurs within 30-60 min after the injection of insulin into a diabetic rat, or after its addition to primary cultures of hep�tocytes (2, 58, 59, 76, 122). GK mRNA increases accordingly (58, 59), and a significant increase of GK activity and attendant flux from glucose to Glu-6-P follows (9, 58, 59, 81, 139). The inhibitory effect of glucagon (or cAMP, its intracellular messenger) on GK gene transcription is dominant over the stimulatory effect of insulin since in cultured cell systems, cAMP blocks the effect of insulin at all concentrations of insulin (58, 59). These hormone effects are not dependent on the presence of glucose in the medium (58, 59). The GK gene in hepatic and pancreatic (3 cells uses different first exons (75, 76). These different exons, IH and 1.8, are separated by �12 kb in the rat gene. This means that different transcription initiation sites, promoters, and regulatory elements are functional in these cells and that the alternative splicing results in different primary transcripts (75). Although some success has been achieved in mapping the basal promoter elements of the hepatic GK gene (83), attempts to identify hormone response elements have been un successful, in part because of the lack of a tissue culture cell line in which the endogenous gene (and transgene genes) are regulated. GLUCOSE-6-PHOSPHATASE Glu-6-Pase, a multi-subunit microsomal en zyme, has proven to be refractory to purification and cDNA cloning, therefore nothing is known about the regulation of its mRNA or the gene, nor is there any evidence for covalent modification of the enzyme. Hepatic glucose 6-phosphatase activity is increased by starvation and in diabetic rats (84, 85). These conditions favor glycogenolysis which, when coupled with elevated Glu-6-Pase levels and decreased GK activity (and decreased glycolysis), results in net carbon flux towards glucose and its export from the hepatocyte. It will not be surprising if insulin reduces, and cAMP increases, Glu-6-Pase gene transcription, just as occurs with PEPCK and Fru- l ,6-P2ase, the other gluconeogenic enzymes.

The Fru 6 PIFru J 6 P2 Cycle -

-

-

,

-

As with GK, hepatic 6-PF-I-K activity is reduced in fasting and diabetes and restored to normal levels by refeeding and insulin administration, respectively (24). Hepatic 6-PF-I-K mRNA increases when fasted animals are refed a high carbohydrate diet (47). Dibutyryl cAMP blunts this induction, thus the inhibitory effect of cAMP may be dominant in 6-PF-I-K regulation, as it is in the case of GK. The mRNA also increases in

6-PHOSPHOFRUCTO-I-KINASE


REGULAnON OF SUBSTRATE CYCLE ENZYMES

895

livers of diabetic animals treated with insulin (47), which suggests that 6-PF-I-K gene transcription, like that of PEPCK, GK, and 6-PF-2-KlFru-2,6P2ase, is under reciprocal control by insulin and cAMP (50). A recent report confirms this hypothesis (115). Human tissues contain three 6-PF-I-K isozymes, each encoded by a sepa rate gene (138). The human muscle gene contains at least two alternative promoters and produces three different mRNAs (80). The mouse liver 6-PF-l K gene has recently been isolated (115), but its expression has not been studied in a cultured cell system, consequently nothing is known about hormone response elements. The structure of the human gene has not been reported. FRUCTOSE 1,6-BISPHOSPHATASE Fru-l ,6-P2ase, which catalyzes the hy drolysis of Fru-l ,6-P2 to Fru-6-P, is induced by diabetes and starvation, as are its companion gluconeogenic enzymes. The tenfold increase of hepatic Fru1,6-P2ase mRNA that occurs in diabetic rats is reduced to basal levels by insulin (34). The addition of cAMP increases Fur-l ,6-P2ase mRNA in cul tured hepatocytes (33), and insulin decreases it. There are three consensus cAMP regulatory elements (CRE) in the 5' -flanking region of the gene. Expression of a Fru-l ,6-P2ase promoter-driven luciferase gene in kidney cells was activated by cAMP, which is consistent with the function of one or more of these elements (33). This suggests that cAMP partially acts by enhancing the rate of transcription of the gene. Insulin opposed the action of cAMP on reporter gene activity, which suggests that insulin also modulates gene transcription (35). Although the regulation of this gene has not yet been studied extensively, it is reasonable to predict that it will closely resemble that documented for the PEPCK gene.

The syn thesis and degradation of Fru-2,6-P2 constitutes an important subcycle within the Fru-6-P/Fru 1,6-P2 cycle. Given that Fru-2,6-P2 serves as a switch between gluconeogenesis and glycolysis, it is not surprising that the bifunc tional enzyme is subject to a complex pattern of regulation. The amount of this bifunctional enzyme is decreased during starvation and in diabetes and is restored by refeeding a high carbohydrate diet or by insulin administration, respectively (19, 20, 102). The increase in mRNA that occurs with refeeding or insulin administration correlates with the increase in the amount of enzyme protein. A more complex situation occurs in starvation or diabetes where the amount of protein is decreased without a corresponding decrease of mRNA. This may indicate decreased mRNA translation and/or enhanced protein degradation (19, 20, 102). The amount of 6-PF-2-K/Fru-2, P2ase and its cognate mRNA is reduced in

6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE 2, 6-BISPHOSPHATASE

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PILKIS & GRANNER

adrenalectomized rats (77, 102). The administration of glucocorticoids to such animals increases 6-PF-2-KlFru-2,6-P2ase mRNA by increasing the rate of transcription of the gene. Olucocorticoids also prevent the loss of bifunc tional enzyme mRNA that occurs when hepatocytes are placed into primary culture, which results in lOa-fold induction of this mRNA (67, 69). Insulin or dexamethasone result in a l 0-20-fold increase of bifunctional enzyme mRNA in rat hepatoma (FfO-2B) cells, and these effects are completely blocked by the addition of cAMP (10). Insulin and dexamethasone increase gene transcription in proportion to their effects on mRNA, so alterations of mRNA stability are apparently not involved. The effect of insulin requires the presence of glucose, which suggests that glucose, or a metabolite of glucose, modulates gene expression, as is the case with pyruvate kinase (see below). Thus there is complex control of 6-PF-2-KlFru-2,6-P2ase gene expression, which is particularly evident in the fact that both insulin and glucocorticoids, which are usually metabolic antagonists, induce the enzyme. At least two genes encode isozymes of the rat bifunctional enzyme (70). One of these genes, expressed only in heart tissue, encodes an isozyme with NHr and COOH-terminal regions that are different from the liver enzyme (31, 65, 108, 117). The other bifunctional enzyme gene encodes at least two isozymes in a tissue-specific manner. Alternative splicing from two promoters is responsible for the two isozymes (19, 21, 22, 69). A muscle-specific transcript is initiated at an upstream promoter and is processed to mRNA by incorporating exon l a and splicing out exon Ib (21). A liver-specific tran script is initiated at a promoter five kb downstream that incorporates exon lb. Exons 2-14 are common to both mRNAs. This pattern is reminiscent of the case with glucokinase (see above). Because expression of the 6-PF-2-KlFru2,6-P2ase gene is regulated by insulin, glucocorticoids, and cAMP, the 5' -flanking region of the gene probably contains the respective hormone response elements. Recently, a complex glucocorticoid response element has been localized to the first intron of the liver/skeletal muscle gene (71).


�

The Pyruvate/PEP Cycle

The chronic regulation of hepatic PK (PK-L) by hormones and dietary factors is extremely complex. PK-L activity and mRNA decrease in starvation and diabetes, and they are restored to normal by refeeding a high carbohydrate diet or by insulin administration (78, 105). Whereas the effects of insulin are direct in the case of OK and PEPCK, the situation appears to be much more complex with PK. The stimulatory effect of insulin is slow in onset, and it requires ongoing protein synthesis, which suggests that the induction of another gene product may be a prerequisite (82). PK-L gene expression is stimulated by the combination of glucose and insulin in primary cultures of adult rat hepatocytes, provided thyroid hormone and L-TYPE PYRUVATE KINASE



897

glucocorticoids are present (23). Neither glucose nor insulin work by them selves. Glucagon, acting via cAMP, inhibits the synthesis of PK-L mRNA by decreasing gene transcription in cultured hepatocytes (23) and in vivo (136). Glucagon increases the rate of degradation of PK mRNA (82, 135). Carbohy drates also stimulate PK-L mRNA accumulation (23). This effect appears to involve transcription and mRNA stabilization, perhaps in conjunction with insulin, and the permissive action of thyroid hormones and glucocorticoids may be required (23). The DNA sequence necessary for glucose-stimulated expression of the PK-L gene was localized to the region between -197 and -96 base pairs from the transcription start site (128a). Consensus CRE sequences are present in the PK-L gene, but are located far upstream (-2. 3 kb) and downstream (+ 5.8 kb) from the initiation site (17). A sequence resembling a glucocorticoid-responsive element was also identi fied (17). These sequence homologies have not yet been supported by studies on localization of functional hormone response elements in the hepatic L-type PK promoter. Work has centered on defining the DNA elements and associ ated binding proteins involved in basal and tissue-specific expression (48, 130, 135-137, 140). PHOSPHOENOLPYRUVATE CARBOXYKINASE The PEPCK gene was iso lated before any of the other genes discussed herein and is the most ex tensively studied (5). In contrast to PK, its glycolytic counterpart, hepatic PEPCK activity is markedly increased in fasted or diabetic animals and is reduced in carbohydrate-fed animals (129). The increased plasma insulin that follows a carbohydrate meal results in a decreased rate of PEPCK synthesis (1, 49). This is the direct result of a decrease in PEPCK mRNA which, in tum, is due to the fact that insulin rapidly inhibits transcription of the PEPCK gene (49, 118). This effect, studied most extensively in H4IIE hepatoma cells, occurs in minutes, is mediated through the insulin receptor, is promptly reversed upon removal of insulin from the culture medium, and does not require ongoing protein synthesis (86). The elevated plasma glucagon (or intracellular cAMP) characteristic of the fasting condition induces PEPCK synthesis. This too is related to enhanced transcription of the PEPCK gene (49,68, 118), although cAMP also stabilizes PEPCK mRNA against degrada tion (54). Glucocortieoids also increase PEPCK by stimulating transcription of the gene and by stabilizing PEPCK mRNA (90). Glucocorticoid and cAMP-induced stabilization of PEPCK mRNA are mediated by nucleotide domains contained within the 3' region of the mRNA (53, 90). The effects of glucocorticoids and cAMP on transcription are additive, and insulin exerts a dominant inhibitory effect on transcription, whether these positive effectors are added individually or in combination (118).


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The basal PEPCK gene promoter consists of three major elements, a CAAT box, a combined basal enhancer element and cAMP response element (CRE), and a TATA box (107). Other elements, most notably ones that bind the transcription factors ClEBP, HNF-1 and HNF-4, may be involved in basal transcription and may modulate hormonal effects (88). The PEPCK gene CRE, used to formulate the first consensus sequence for a CRE, mediates most, but not all, of the effect cAMP has on PEPCK gene transcription (88, 106, 107, 121). Glucocorticoid hormones stimulate PEPCK gene transcription through a uniquely complex glucocorticoid response unit (GRU) (56). This GRU spans 110 base pairs (from -465 to - 355), and it consists of a tandem array of two accessory factor-binding sites (AF1 and AF2) and two glucocorticoid receptor-binding sites (GR1 and GR2) (56). The entire complex is required for a maximal response to glucocorticoids. The mechanism of action of these accessory factor sites is not clear, but it is interesting that a combined genetic and functional analysis shows that AF1 also serves as a retinoic acid response element (retinoic acid induces transcription of the PEPCK gene) (74) and is a binding site for HNF-4. A similar analysis places one insulin-responsive element within the AF2 sequence at -416 to -402 (87). The coincident location of an element that acts as a part of the GRU and as an insulin responsive sequence could account for the dominant inhibitory effect insulin has on the glucocorticoid response. An explanation for the dominant effect of insulin on the cAMP response has not yet been presented. THE MOLECULAR PHYSIOLOGY OF GLUCONEOGENESIS AND GLYCOLYSIS The molecular physiology of gluconeogenesis and glycolysis involves a complex interplay of nutrition and hormones to regulate the phosphorylation state and gene expression of substrate cycle enzymes via short-term and long-term effects, respectively (Table 1). In fed animals, the high activity of GK, 6-PF-1-K and PK favors net flux toward pyruvate. The activity of PEPCK is low, as is that of other gluconeogeneic enzymes, thus flux toward glucose is low. As an animal initiates a fast, plasma insulin levels begin to fall. This relieves the dominant inhibition insulin has on the synthesis of the gluconeogenic enzyme PEPCK and allows hormones such as glucagon and f3-adrenergic agonists to stimulate adenylate cyclase and increase cAMP levels. The increase of cAMP also results in a phosphorylation-induced inactivation of 6-PF-2-K and activation of Fru-2,6-P2ase, with a resultant decrease of Fru-2,6-P2. This leads to decreased activity of 6-PF-I-K and an activation of Fru-l,6-Pase. cAMP mediated phosphorylation inhibits PK activity, as does the decreased level of

REGULATION OF SUBSTRATE CYCLE ENZYMES Table 1

Regulation of hepatic glucose metabolism Enzyme activity

Gene expression Ins

Enzyme

t

GK 6PF-I-K PK 6PF-2-KlFru-2,6-P2ase

PEPCK


899

i i' t s ig tg i

t o

0

Fru- I ,6-P2ase

?

Glu-6-Pase

cA o

t

0

o

ts

0

its i ?

Gc

Ph

Allosteric

1$

No

No

?

No

P

Yes Yes

Yes Yes Yes

No

No

No

Yes

i i ts ? ?

No

Summary of long-term and short-term regulation of the genes involved in hepatic glucose homeostasis. Long-term gene expression regulation: t, gene transcription measured; g, effect

requires glucose; , dominant effect; P, permissive requirement; affected;

?,

no

information

available.

Short-term

/1:,., no change; s, mRNA stability

regulation:

Ph,

phosphorylation!

dephosphorylation reactions; allosteric, regulation by allosteric effectors. Other abbreviations

include Ins, insulin; cA, cyclic AMP; Gc, glucocorticoids.

Fru-l,6-P2, which results from the activation of Fru-l,6-Pase. The elevated cAMP activates PEPCK gene transcription, which leads to a two to threefold increase in PEPCK activity. All of this results in decreased cycling of PEP to pyruvate and of Fru-6-P to Fru-l ,6-P2, and the net result is an increased rate of gluconeogenesis. After 24 hr of starvation, hepatic cAMP levels and the rate of glu coneogenesis are elevated, whereas the level of Fru-2,6-Pz is reduced to 10% of that in livers of fed animals. Substrate cycling is diminished because of increased flux through Fru-l,6-Pzase and PEPCK and diminished flux through 6-PF-I-K and PK. Short-term regulation by glucagon or catechola mines provides little additional stimulation of gluconeogenesis because the activity of enzymes, acutely responsive to these hormones in the livers of fed animals (PK, 6-PF-2-K, and Fru-! ,6-P2ase), has already been altered by phosphorylation mechanisms. The rate of gluconeogenesis after prolonged starvation, or in diabetes, is further elevated by increased substrate supply, and most importantly, by changes in the concentration of various enzymes. The combination of an elevated intracellular cAMP and decreased plasma insulin activates transcrip tion of the genes that encode the activities of the gluconeogenic enzymes PEPCK, Fru-l,6-Pzase and probably G-6-Pase. Conversely, transcription of the genes that encode the glycolytic enzymes GK, 6-PF-I-K, and PK is reduced, as is that of the bifunctional enzyme. During long-term starvation, or in diabetes, the enzymes responsive to glucagon and catecholamines are already phosphorylated, hence acute regula tion of gluconeogenic flux superimposed on the existing rate is not observed. Restoration of the short-term modulation of the phosphorylation state and

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activity of PK and 6-PF-2-KlFru2,6-Pzase by refeeding or insulin administra tion, respectively, takes many hours to achieve. The elevated levels of cAMP must first decrease and the level of Fru-2,6-P2 must increase. Full restoration also requires that insulin inhibits expression of the PEPCK and Fru-l ,6-P2ase genes and induces the mRNAs for OK, PK, and 6-PF-2-K/Fru-2,6-P2ase. It is clear that the extent of carbon flux in the gluconeogeneic/glycolytic pathway depends on numerous, complex factors whose contribution varies depending on the nutritional and hormonal status of the animal.

SUMMARY Understanding the regulation of hepatic glucose metabolism had its founda tion in the elucidation of several pathways, but recent advances have come from the application of molecular genetics. Five years ago little was known about the primary structure of the key regulatory enzymes. Since then, the primary sequence of liver OK, 6-PF-I-K, Fru-l ,6-P2ase, PK, PEPCK, and 6-PF-2-KlFru-2,6-Pzase have been derived from cDNA sequences and/or determined by direct protein sequencing. This has provided new insights into the molecular mechanisms of catalysis and the regulation of these enzymes by covalent modification. Isolation of the cDNAs for these enzymes also has allowed for the quantitation of specific mRNAs and permitted analysis of hormonal control of specific gene expression. The genes for these enzymes have been isolated and sequenced, and their promoter regions are being identified and characterized. Hormone response elements have been de lineated in several of the promoters. The promoter regions for 6-PF-2-KlFru2,6-P2ase and Fru-l,6-P2ase have also been identified, and future research will focus on the elucidation of the mechanisms whereby hormones regulate the expression of these genes. A number of generalizations can be made about the regulation of gene expression of glycolytic/gluconeogenic enzymes (Table 1). First, there is coordinate hormonal regulation of gene expression and these effects are consonant with their physiologic actions. Insulin induces the mRNAs that encode glycolytic enzymes and represses the mRNAs that encode gluconeo genic enzymes; cAMP has opposite effects. Both can increase or decrease transcription. Whereas insulin and cAMP affect all of these mRNAs, gluco corticoids appear to have a more restricted action. Second, transcriptional and posttranscriptional regulatory mechanisms are involved. The synthesis of all of the mRNAs discussed is regulated by hormones. Relatively little is known about how mRNA stability is regulated in general, but it is clear that PEPCK mRNA is stabilized by agents that increase the rate of transcription of the gene. Under appropriate metabolic signals this dual control of mRNA synthesis and stability provides for a



901

long-term increase in PEPCK mRNA and protein. Studies with PK mRNA are less direct, but suggest a similar dual mechanism. It will be interesting to see whether multilevel regulation is restricted to these two mRNAs, both of which are involved in the same substrate cycle, or whether the stability of other mRNAs involved in hepatic glucose metabolism is also affected. Third, glucose appears to be important in the regulation of these hepatic genes. Glucose is required for the effects of insulin on the PK and bifunctional enzyme genes. Since these mRNAs encode enzymes that catalyze in termediate or distal reactions in the glycolytic pathway, and since the regula tion of GK gene transcription by insulin is independent of glucose, it is possible that a glucose metabolite is the active agent and that it is generated as a consequence of the stimulation of GK gene transcription by insulin. In creased catabolism of glucose could account for the fact that insulin is necessary, but not sufficient, for the induction of PK and the bifunctional enzyme. Cis-acting DNA sequences required for regulation by glucose have been identified for the yeast enolase gene (18), the hepatic S 14 gene (60), and perhaps for the L-type pyruvate kinase gene (128a). Trans-acting proteins have not been identified in any of these examples. Whether regulation of gene expression of gluconeogenic enzymes by insulin is dependent on glucose is uncertain, although this does not seem to be the case for PEPCK (50). Fourth, it appears that negative regulation is dominant for many of these genes. Dominance, as used here, implies that one agonist overrides the effect of a saturating concentration of a second agonist. Studies of the PEPCK gene showed that inhibition of transcription by insulin was dominant over the stimulatory effects of cAMP and glucocorticoids. Dominance is not a univer sal feature of insulin action, however, since the stimulation of transcription of glycolytic enzyme genes by insulin is overridden by the inhibitory effect of cAMP. This restraint of gluconeogenesis and glycolysis by the action of whichever hormone exerts the negative effect may be of central importance, but the molecular mechanisms for such dominance are unknown. Fifth, analysis of the organization of a number of genes reveals that alternate exon use allows for special types of regulation. Cell-specific regula tion of expression is accomplished, in part, by the fact that alternate first exons provide unique mRNAs for PK, GK, and the bifunctional enzyme in different tissues. Therefore, different promoter regions and control elements are employed, and an enzyme that catalyzes a specific reaction can serve a different physiologic purpose in two different tissues (e.g. 6-PF-2-K/Fru-2, 6P2ase in the hepatocyte and the muscle cell; GK in the hepatocyte and pancreatic f3 cell). Alternate exon use appears to be employed by the hepatic glycolytic enzyme genes (GK, 6-PF-I-K, 6-PF-2-KlFru 2, 6-P2ase, PK), but not by the gluconeogenic enzyme genes PEPCK and Fru- l ,6-P2ase. The importance of this distinction awaits further analysis of the function of these' promoters.

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FUTURE DIRECTIONS The current challenge is to determine the molecular mechanisms whereby coordinate regulation of transcription, mRNA stability, glucose-dependent hormonal regulation of gene expression, dominance, and alternate exon use occur. Hormonal regulation of gene expression is presumably accomplished by trans-acting protein factors that bind to cis-acting DNA elements. One must first isolate and identify these trans-acting factors, then determine how they are regulated (e.g. by covalent modification, allosteric effectors, changes in gene expression). Some formidable challenges are in sight. Little is known about how a hormone exerts positive and negative effects upon separate genes, and the fact that these can occur simultaneously within a cell is a complicating issue. The cis/trans model of gene regulation has been suc cessfully applied to an analysis of the gluconeogenic enzyme gene promoters such as PEPCK and Fru-I,6-P2ase, but with little success to glycolytic enzyme genes such as PK, GK, and the bifunctional enzyme. Confounding this issue is the fact that many of these genes are not expressed or regulated in standard tissue culture cell lines. Even when suitable cell lines are available, there are instances where the fusion gene/transfection approach has not worked. If this continues to be true, especially as more of these promoters are analyzed, the possibility that there is something different about the hormonal regulation of these glycolytic enzyme genes must be considered. Glu-6-Pase represents another fertile area of future research. It is the only key regUlatory enzyme in the pathway that has not been cloned. Much new information will be forthcoming once cDNAs are obtained for its catalytic subunit and the putative regulatory subunits. For example, the catalytic subunit of mammalian Glu-6-Pase catalyzes its reaction via a phosphohisti. dine enzyme intermediate ( lISa). Elucidation of the structure of this subunit will probably reveal that it is a member of the Fru-2,6-P2ase/phosphoglycer ate mutase/acid phosphatase enzyme family. The regulation of the glu cose-6·phosphatase gene remains unresolved, but it is likely to resemble that of the other gluconeogenic enzyme genes. Despite major advances in knowledge of the structure and function of hepatic regulatory enzymes, and the control of metabolism via phosphoryla tion dephosphorylation mechanisms, there has been a decline of interest in metabolic regulation in recent years. This may have 'occurred because the paucity of approaches for analyzing the control of pathway flux, but the recent advances in DNA/RNA recombinant technology have opened exciting new avenues. Por example, it is possible to transfect a cell derived from the liver with a gene encoding a modified form of an enzyme, for example, 6-PF-2-K/ Fru-2, 6-P2ase, which lacks phosphorylation sites. When expressed, one can , determine the effect this modification has on metabolic flux. This will involve



903

engineering chimeric genes with potent regulatable promoters so that in creased amounts of these enzymes can be produced at will in transfected cells. Metabolic pathway engineering should allow investigators to sort out the complex interplay that occurs between short-term, acute regulation of enzyme activity via covalent modification and/or allosteric effectors, and the long term effects on gene expression. In addition, the use of transgenic animals has great potential for the analysis of metabolic pathways under various physi ological conditions. Homologous recombination is another powerful an alytical tool. This approach can be used to inactivate genes such as those encoding the key regulatory substrate cycle enzymes. In situ structure/ function analysis can be conducted by substituting genes that express an enzyme altered by site-directed mutagenesis. Thus stable cell lines can be created with targeted mutations and the effects of such changes on metabolic cycling can be assessed. The question of how the liver regulates the utilization and synthesis of glucose has been studied intensively by physiologists and biochemists for decades. Much has been learned, but many questions remain unanswered. The tools to approach these problems successfully are growing in number and incisiveness, and their use in the future should yield rich rewards. Literature Cited 1 . Andreone, T. L . , Beale, E. G . , Bar, R. S . , Grann er , D . K . 1 982. Insulin de creases phosphoenolpyruvate carboxyki nase (GTP) mRNA activity by a recep tor-mediated process. J. BioI. Chern. 257:35-38 2 . Andreone, T. L . , Printz, R. L . , Pilkis, S. J . , Magnuson, M. A . , Granner, D. K. 1 989. The amino acid sequence of rat liver glucokinase deduced from cloned eDNA. 1. Bioi. Chern. 264:3 63-69 3 . Assimacopoulaus, J. F . , Jeanrenaud, B . 1 990. Insulin activates 6-phosphofructo2-kinase and pyruvate kinase in liver: Indirect evidence for action via a phos phatase. 1. Bioi. Chern. 265 :7202-6 4. Bazan, F . , Fletterick, R . , Pilkis, S. J . 1 989. Evolution o f a bifunctional en zyme: 6-phosphofructo-2-kinase/fruc tose 2 , 6-bi sphosphatase . Proc. Natl. Acad. Sci. USA 86:9642-46 5 . Beale, E. G . , Chrapkiewicz, N . B . , Scobie, H . A . , Metz, R . J . , Quick, D . P . • et al. 1 98 5 . Rat cy tosolic phos phoen olpyruvate c arboxykinase: Struc tures of the protein, mRNA, and gene. J. BioI. Chern. 260 : 10748-60 6. Blair, 1. B . , Cimbala, M. A . , Foster, 1. L . , Morgan, R. A . 1 976. Hepatic pyru vate kinase: regulation by g luc agon,

cyclic adensine 3 ' ,5 ' -monophosphate and insulin in the perfused rat liver. 1. Bioi. Chern. 25 1 : 3756--62 7. Castano, L. G . , Nieto, A . , Feliu, 1. E. 1 979. Inactivation of phosphofructoki nase by glucagon in rat hepatocytes. 1 . Bioi. Chern. 254:5576--79 8. Chan, T. M . , Exton, J. H . 1 97 8 . Studies un a-adrenergic activation of hepatic glucose output: studies on a-adenergic

inhibition of hepatic pyruvate kinase and activation of gluconeogensis. 1. Bioi. Chern. 253:6396--400 9. Christ, B . , Probst, I . , lungermann , K.

1 986. Antigonistic regulation of the glu

cose/g luco se 6-phosphate cyc le by in sulin and glucagon in cultured hepato c yte s . Biochem . 1. 238: 1 85-9 1 1 0 . Cifuentes, M. , Espinet, c . , Lange, A . J . , Pilkis, S . J . , Hod, Y. 1 99 1 . Hormon al control of 6-phosphofructo 2 ,6-ki nase/fructose 2,6-bisphosphatase gene expression in rat FTO 2B cells. 1. BioI. Chern . 266: 1 55 7-63 I I . Clark, M. G . , Bloxham, D. P . , Holland, P. c . , Lardy, H. A. 1 974. Estimation of the fructose I , 6-diphosphatase-phospho fructokinase substrate cycle and its rel ationship to gluconeogeneses in rat liver in vivo . 1. Bioi. Chern. 249:279-90


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1 2 . Clark, M. G . , Kneer, N. M . , Bosch, A. L., Lardy , H . A. 1974. The fructose 1 ,6-disphosphatase-phosphofructokinase substrate cycle: a site of regulation of hepatic gluconeogenesis by glucagon. 1. Bioi. Chern. 249:5695-703 1 3 . Claus, T. H . , EI-M aghrabi, M. R . , Pil kis, S . J. 1 979. Modulation of the phosphorylation state of rat liver pyru vate kinase by allosteric effectors and insulin. 1. Bioi. Chern. 254:7855-64 1 4 . Claus, T. H . , EI-Maghrabi, M . R . , Re gen, D. M . , Stewart, B . , McGrane, M . , e t al. 1984. Role o f fructose 2 ,6-bis phosphatate in the regulation of hepatic metabolism. Curro Top . Cell Regul. 23:57-86 1 5 . Claus, T. H . , Schlumpf, J. R . , EI Maghrabi, M. R . , McGrane, M . , Pilkis, S. J. 1 98 1 . Glucagon stimulation of fructose 1 ,6-bisphosphatase phosphory lation in rat hepatocytes . Biochern. Bio phys. Res. Cornrnun. 100:7 1 6-23 1 6 . C lau s, T. H . , Schlumpf, J . R . , EI Maghrabi, M. R . , Pilkis, J . , Pilkis, S. J . 1 980. Mechanism of action o f glucagon on hepatocyte phosphofructokinase ac tivity. Proc. Natl. Acad. Sci. USA 77: 6501-5 1 7 . Cognet, M. , Yu, C . L . , Vaulont, S . , Kahn, A. , Marie, J . 1987. Structure of the rat L-type pyruvate kinase gene. 1. Mol. Bioi. 196: 1 1-25 1 8 . Cohen, R. , Holland, J. P . , Yokoi, T . , Holland, M . J . 1 986. Identification o f a regulatory region that mediates glucose dependent induction of the Saccharomy ces cerevisiae enolase gene EN02 . Mol. Cell. Bioi. 6:2287-97 1 9 . Colosia, A. D . , Marker, A. J . , Lange, A . J . , EI-Maghrabi , M. R . , Granner, D . K . , et a l . 1 9 8 8 . Induction o f rat liver 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase mRNA by refeeding and insulin. 1. Bioi. Chem. 263 : 1 8669-77 20. Crepin, K. M . , Darville, M. I . , Hue, L . , Rousseau, G . G . 1988. Starvation o r di abetes decreases the content but not the mRNA of 6-phosphofructo-2-kinase in rat liver. FEBS Lett. 227 : 1 36-40 2 1 . Crepin, K. M . , Darville, M. I . , Hue, L . , Rousseau, G . G . 1989. Characterization of distinct mRNAs coding for putative isozymes of 6-phosphofructo-2-kinasel fructose 2,6-bisphosphatase. Eur. 1. Biochem. 1 83 : 433-40 2 2. Darville, M. I . , Crepin, K. M . , Hue, L . , Rousseau, G . G . 1989. 5 ' -Flanking se quence and structure of the gene encod ing rat 6-phosphotiucto-2-kinase/fruc tose 2 ,6-bisphosphatase. Proc. Natl. Acad. Sci. USA 86:6543-47 23. Decaux, J . -F . , Antoine , B . , Kahn, A .

24.

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35. 36.

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44.

45 .

46.

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Regulation of hepatic energy metabolism and gluconeogenesis by BAD.

Physiologic significance of glucocorticoids and insulin in the regulation of hepatic gluconeogenesis during starvation in rats.

CREB and FoxO1: two transcription factors for the regulation of hepatic gluconeogenesis.

A role for mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) in the regulation of hepatic gluconeogenesis.

Genetic variation in activity of the enzymes of glycolysis and gluconeogenesis between inbred strains of mice.

Inhibition of hepatic gluconeogenesis by dichloroacetate.

Inhibition of hepatic gluconeogenesis by phenethylhydrazine (phenelzine).

Dietary iron, circadian clock, and hepatic gluconeogenesis.

On the mechanism of glucagon stimulation of hepatic gluconeogenesis.

Silencing of FGF-21 expression promotes hepatic gluconeogenesis and glycogenolysis by regulation of the STAT3-SOCS3 signal.

Ca2(+)-fatty acid interaction in the control of hepatic gluconeogenesis.

Regulation of glycolysis and level of the Crassulacean acid metabolism.

Retinoic acid-related orphan receptor γ (RORγ): a novel participant in the diurnal regulation of hepatic gluconeogenesis and insulin sensitivity.

Development of enzymes of glycolysis and gluconeogenesis in human fetal liver.

Effects of hypothalamic stimulation on gluconeogenesis and glycolysis in rat liver.

Hepatic p38α regulates gluconeogenesis by suppressing AMPK.

Switch of glycolysis to gluconeogenesis by dexamethasone for treatment of hepatocarcinoma.

Simultaneous stimulation of glycolysis and gluconeogenesis by feeding in the anterior intestine of the omnivorous GIFT tilapia, Oreochromis niloticus.

gluconeogenesis in regenerating rat liver.

Molecular physiology of the dynamic regulation of carbon catabolite repression in Escherichia coli.

Hepatic Mitochondrial Pyruvate Carrier 1 Is Required for Efficient Regulation of Gluconeogenesis and Whole-Body Glucose Homeostasis.

Glucagon stimulation of hepatic gluconeogenesis in neonatal pigs.

Interrelationship between hepatic ureagenesis and gluconeogenesis in early sepsis.