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Molecular Endocrinology 18(5):1051–1063
Copyright © 2004 by The Endocrine Society
doi: 10.1210/me.2003-0357
MINIREVIEW
Sweet Changes: Glucose Homeostasis Can Be
Altered by Manipulating Genes Controlling Hepatic
Glucose Metabolism
JAMES J. COLLIER
AND
DONALD K. SCOTT
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences
Center, New Orleans, Louisiana 70112
The liver is responsible for glucose synthesis in the
fasting state, and glucose uptake, storage, and
utilization in the fed state. A phenotypic switch,
normally initiated by insulin or glucagon, controls
the transition between the two states, which includes transcriptional alterations that regulate
metabolic enzyme abundance for multiple metabolic pathways in a coordinated manner. A network of transcription factors, coactivators, and
corepressors direct these changes, thus acting as
transcriptional sensors of the nutritional status of
an organism. The inability of the hepatocyte to
undergo this metabolic reprogramming is characteristic of diabetes mellitus. Modulations that con-
trol the amount of individual metabolic enzymes or
transcription factors can initiate the fasting-to-fed
transition of the hepatocyte in an insulin-independent manner. Alternatively, overexpression of key
regulators of metabolism can lock hepatocytes in
the fasted state. These manipulations alter hepatic
glucose flux, leading to either amelioration or induction of diabetes mellitus. These maneuvers reveal the complexity of the coordinated mechanisms used by the liver to alter its phenotype and
provide evidence for the control strength of metabolic signaling. (Molecular Endocrinology 18:
1051–1063, 2004)
T
phatase (PFK2), acetyl-coenzyme A carboxylase, and
fatty acid synthase (FAS) (Refs 1–3 and Fig. 1). Conversely, genes that are activated during the fasted
state, such as phosphoenolpyruvate carboxykinase
(PEPCK), fructose-1,6-bisphosphatase, and carnitine
palmitoyl transferase (CPT) I and II are down-regulated
(1–3). This phenotypic switch requires genes to be
stimulated or repressed by a coordinated process.
Normally, various hormones contribute significantly
both to the regulation of these metabolic genes and to
modifications in specific proteins that control hepatic
metabolic processes. However, by simply overexpressing key metabolic enzymes, or the factors that
regulate their abundance, these alterations in gene
transcription can be achieved regardless of hormonal
status (1, 4–9).
Historically, insulin was found to alter the expression
of a number of hepatic metabolic enzyme genes, but in
many cases glucose was required in the medium for
this effect (3, 10). By experimentally separating glucose flux from insulin signaling in tissue culture, a
condition that would not normally occur in vivo, several genes were found to be primarily responsive to
glucose rather than to insulin (11–15). Table 1 lists
representative genes that are regulated by either insulin, glucose, or both in an additive or synergistic
fashion. What is evident from Table 1 is that glucose is
HE LIVER IS strategically positioned to influence
glucose homeostasis through a delicate balance
between hepatic glucose uptake and utilization (HGU)
and hepatic glucose production (HGP). Physiologically, insulin and glucose act jointly to influence the
fasted-to-fed transition in the liver by promoting expression of genes normally induced in the fed state,
including glucokinase (GK), liver pyruvate kinase (LPK), 6-phosphofructo-2-kinase/fructose-2,6-bisphos-
Abbreviations: ChREBP, Carbohydrate response element
binding protein; CPT, carnitine palmitoyl transferase; DM
PFK2, double-mutant PFK2; FAS, fatty acid synthase; F-2,6P2, fructose-2,6-bisphosphate; GFAT, glutamine fructose-6phosphate amidotransferase; GK, glucokinase; GKRP, GKregulatory protein; GlcNaC, N-acetyl glucosamine; G6Pase,
glucose 6-phosphatase; HGP, hepatic glucose production;
HGU, hepatic glucose uptake and utilization; L-PK, liver pyruvate kinase; PEPCK, phosphoenolpyruvate carboxykinase;
PGC-1␣, PPAR-␥ coactivator-1␣; PKA, protein kinase A;
PKF1 and -2, 6-phosphofructo-1-kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; PPAR, peroxisomal proliferator-activated receptor; PP1 and PP2A, protein
phosphatases 1 and 2A; SREBP, sterol-regulatory elementbinding protein; STZ, streptozotocin; Xu-5-P, xylulose
5-phosphate.
Molecular Endocrinology is published monthly by The
Endocrine Society (http://www.endo-society.org), the
foremost professional society serving the endocrine
community.
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1052 Mol Endocrinol, May 2004, 18(5):1051–1063
Collier and Scott • Minireview
Table 1. Both Insulin and Glucose Contribute to the Transcriptional Fasted-to-Fed Transition in the Liver
2 Gluconeogenesis
Effector
Insulin
Glucose
Representative Genes
PEPCK and G6Pase
PEPCK
1 Glycolysis and lipogenesis
Refs.
3
4, 14, 15
Effector
Representative
Genes
Insulin
Glucose
Insulin and glucose
GK
PK
FAS
Refs.
59, 60
6, 7
115
can either promote or prevent the development of
diabetes in a hormone-independent fashion, and
these manipulations are considered on an individual
basis in this review (Fig. 2). The effects of insulin and
glucagon on hepatic glucose metabolism are well documented and have been reviewed elsewhere (20–26).
The present discussion will focus on how alterations of
glucose metabolism potently affect hepatocyte function. During this process, two main questions will be
addressed: 1) How can altering metabolic enzyme
abundance influence both the phenotype of the hepatocyte and whole-body glucose homeostasis? 2) What
are the coordinating mechanisms required to mediate
this process?
Fig. 1. Coordinated Regulation of Hepatic Gene Transcription by Insulin and Glucose
Insulin, via a signaling cascade, promotes the hepatic expression of GK and the induction or modification of various
transcription factors that participate in the phenotypic switch
in the liver. Increases in GK abundance lead to enhanced
glucose phosphorylation. The signals afforded by glucose
metabolism, in conjunction with the insulin signal, promote an
increase in glycolytic and lipogenic enzyme genes while repressing expression of gluconeogenic and ketogenic enzyme
genes. These changes are coordinated by multiple transcription factors, coactivators, and corepressors.
capable of promoting the fasted-to-fed transition in
the absence of insulin provided that GK is expressed
(4, 13–15). GK activity is rate controlling for increased
glucose flux in the liver, and its expression requires
insulin (16, 17). Once glucose flux is increased, it can
simultaneously promote glycolysis and lipogenesis,
and decrease gluconeogenesis by inhibiting PEPCK.
Note that both glucose and insulin can inhibit PEPCK
expression in the absence of the other effector (14,
15). By revealing the cooperative and overlapping
partnership between insulin and glucose in the liver, an
important question arose: if one could promote the
hepatic fasting-to-fed transition by artificially increasing glucose flux in the absence of insulin, what would
be the effect of these manipulations in whole animals
or in a diabetic model system? In the last decade it has
become apparent that modulations of hepatic enzyme
abundance, termed “metabolic engineering” (18, 19),
HEPATIC ENZYME ELEVATIONS LEADING
TO DIABETES
PEPCK
The synthesis of glucose by the liver is critical when
there is a paucity of carbohydrates in the diet. This is
particularly crucial for tissues such as the brain and
red blood cells, which can not use fatty acids as a fuel
source. The breakdown of glycogen can only maintain
euglycemia for a short period. Consequently, the net
synthesis of glucose from carbon sources such as
lactate, glycerol, and amino acids plays a key role in
maintaining euglycemia during a prolonged fast.
PEPCK is central to this process because it catalyzes
the rate-controlling reaction of the gluconeogenic
pathway by converting oxaloacetate into phosphoenolpyruvate. The abundance of the cytosolic form
of this enzyme is mainly controlled by the rate of
transcription of its gene (27). The PEPCK gene is activated by cAMP and glucocorticoids and suppressed
by insulin and glucose (14, 28, 29). The inhibition of
PEPCK by insulin is the most important physiological
mechanism to inhibit the synthesis of this rate-controlling enzyme at the end of a fast. However, the
metabolism of glucose is sufficient to override the
combined stimulatory effects of dexamethasone and
cAMP on PEPCK gene transcription. This effect by
glucose can be insulin independent, provided that GK
is expressed by some artificial means, and clearly
demonstrates the potential strength of the GK-mediated, glucose-derived signal (14, 15).
Collier and Scott • Minireview
Fig. 2. Pathways of Hepatic Glucose Metabolism
Flux through various hepatic pathways of glucose metabolism control overall glucose homeostasis. The asterisk (*)
and the green color indicate that an increased expression
prevents or offsets metabolic perturbations associated with
either type 1 or type 2 diabetes mellitus, or sometimes both.
The exclamation point (!) and the red color indicate that an
increased abundance leads to a phenotype of insulin resistance and/or diabetes.
Dysregulation of PEPCK is a contributing factor to
the pathogenesis of both type 1 and type 2 diabetes
mellitus (30, 31). Not surprisingly, transgenic mice
overexpressing PEPCK in the liver display increased
HGP (30, 32). These animals have fasting hyperglycemia, decreased glycogen storage, and glucose intolerance, suggesting insulin resistance (32). Further
analysis reveals that PEPCK-overexpressing mice
have higher blood glucose and insulin levels relative to
control animals in both the fasted and fed states.
Glucose 6-phosphatase (G6Pase) mRNA levels are
enhanced, concomitant with a decrease in the levels
of the insulin receptor substrate 2 protein (30). Thus,
increasing hepatic gluconeogenesis by PEPCK overexpression leads to insulin resistance and symptoms
resembling type 2 diabetes mellitus.
G6Pase
Also vital to glucose production is the enzyme
G6Pase, which catalyzes the dephosphorylation of
glucose 6-phosphate to free glucose as the terminal
step in gluconeogenesis and glycogenolysis. This process is carried out in the lumen of the endoplasmic
reticulum and requires several other proteins in addition to the catalytic subunit. These include transporters for glucose 6-phosphate, glucose, and inorganic
phosphate (33). The end result of this reaction allows
glucose to be transported out of the hepatocyte and
into the circulation. The gene encoding the G6Pase
catalytic subunit is stimulated by cAMP and repressed
by insulin in a manner similar to that for PEPCK (34–
Mol Endocrinol, May 2004, 18(5):1051–1063
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36). In contrast to the PEPCK gene, the G6Pase gene
is stimulated by glucose, an effect that is not well
understood (37, 38). If the balance involving glucose
phosphorylation and glucose 6-phosphate hydrolysis
is perturbed, producing a high G6Pase to GK ratio,
abnormal hepatic glucose metabolism results and is
manifested physiologically as a metabolic disturbance
resembling type 2 diabetes mellitus (39). Thus, a delicate balance exists where GK and G6Pase compete
to control the direction of glucose flux and ultimately
the metabolic signaling system that promotes the fasting-to-fed transition of the liver.
Obese rodents have increased G6Pase activity and
exhibit hepatic insulin resistance when compared with
lean controls (39). Direct evidence linking G6Pase to
the development of insulin resistance was gained from
experiments wherein the catalytic subunit of G6Pase
was overexpressed via adenoviral vectors in primary
hepatocyte cultures and in vivo in rats. In primary
hepatocytes, G6Pase overexpression augments activity of this enzyme and is sufficient to increase glucose
6-phosphate hydrolysis (40). These cells also display
decreased lactate production and glucose usage relative to cells treated with a control adenovirus. These
studies demonstrate that G6Pase overexpression impairs HGU, while simultaneously enhancing HGP. In
similar studies, adenovirus-mediated overexpression
of G6Pase in the liver results in decreased glycogen
storage, hyperinsulinemia, and glucose intolerance
(39). This is a phenotype remarkably similar to type 2
diabetes mellitus and is yet another example of how
the overexpression of one key enzyme can perturb the
phenotype of the hepatocyte and overall glucose homeostasis. Thus, increased flux through gluconeogenesis, whether via PEPCK or G6Pase overexpression,
results in decreased HGU and increased HGP. These
modulations have deleterious effects on whole-animal
glucose homeostasis and may explain, at least in part,
the metabolic defects observed in both type 1 and
type 2 diabetes.
Peroxisomal Proliferator-Activated Receptor
(PPAR)-␥ Coactivator-1/Foxo1
PPAR-␥ coactivator-1␣ (PGC-1␣), originally discovered as a protein induced by adaptive thermogenesis,
interacts with the nuclear hormone receptor PPAR-␥
to facilitate gene activation (41). PGC-1␣ promotes the
expression of uncoupling protein 1, an inner mitochondrial membrane protein that dissociates fuel oxidation
from ATP production, thereby generating heat. The
activity of PGC-1␣ is dependent on a physical interaction with specific transcription factors via a leucinerich (LxxLL) domain; it was subsequently shown that
this motif mediates contact with several nuclear hormone receptors, including the estrogen and glucocorticoid receptors (42).
Recently, Spiegelman and colleagues (8) made the
observation that hepatic PGC-1␣ is a coactivator of
several gluconeogenic genes, including G6Pase and
1054 Mol Endocrinol, May 2004, 18(5):1051–1063
PEPCK. This coactivator protein is induced by cAMP
and glucocorticoids in hepatocytes and is elevated in
the livers of fasting and diabetic animals. Adenoviral
overexpression of PGC-1␣ increases glucose production in primary hepatocytes and activates gluconeogenesis in vivo by increasing the transcription of key
enzymes involved in glucose synthesis, such as
PEPCK and G6Pase (8). Additionally, the cAMP response element binding protein, activated by phosphorylation in response to a variety of signals, regulates hepatic gluconeogenesis in part by up-regulating
PGC-1␣ (43). Signals that represent the fasted state,
including cAMP and glucocorticoids, promote glucose
synthesis while inhibiting glucose utilization. Mice with
a heterozygous deletion in cAMP response element
binding protein exhibit fasting hypoglycemia that is
corrected by hepatic overexpression of a PGC-1␣encoding adenovirus, but not by a control adenovirus.
Thus, a pathological enhancement of PGC-1␣, such
as occurs during diabetes, may amplify the glucocorticoid and cAMP signal to the PEPCK gene, resulting
in uncontrolled HGP (43). Furthermore, the transcription factor Foxo1, which is regulated by exclusion from
the nucleus upon insulin signaling through phosphorylation by protein kinase B (Akt), also interacts with
PGC-1␣ to stimulate gluconeogenesis (36). Indeed,
when a constitutively active version of Foxo1 is overexpressed in mice, there is a corresponding increase
in the expression of the PEPCK and G6Pase genes,
resulting in elevated blood glucose concentrations
(44). Conversely, when a Foxo1 dominant-negative
mutant, generated by deletion of a carboxyl domain
fragment, is infused into db/db mice, there is a significant lowering of blood glucose levels (45). These observations define Foxo1 as a critical intermediate in
the insulin-mediated regulation of gluconeogenesis.
Thus, the direction of glucose flux in the hepatocyte
affects the organism as a whole, such that increasing
flux through anabolic, glucose-producing pathways
interferes with the ability of the liver to transit from the
fasting to the fed state. As a result, the control mechanisms that maintain euglycemia, including those that
regulate gene transcription, are interrupted. This
makes the interaction between PGC-1␣ and transcription factors that are responsible for activating genes
involved in glucose production, such as Foxo1, potentially novel therapeutic targets for diabetes drug
design.
Glutamine Fructose-6-Phosphate
Amidotransferase
So far the discussion has been restricted to manipulations that increase HGP and their roles in contributing to the disruption of the fasted-to-fed phenotypic
switch in the hepatocyte. Several different pathways in
the hepatocyte contribute to the breakdown and storage of glucose, including glycolysis, the hexose
monophosphate shunt, and glycogen and hexosamine
biosynthesis. As we will discuss below, the activation
Collier and Scott • Minireview
of glucose-utilizing pathways usually promotes the hepatic fasting-to-fed transition via a poorly understood
signaling system (4, 46, 47), which prevents the onset
of type 1 or type 2 diabetes mellitus, or both. By
contrast, the hexosamine biosynthetic pathway, which
involves glucose catabolism, can actually lead to insulin resistance (48). Marshall and co-workers (49) first
characterized this pathway as a mediator of glucoseinduced desensitization of the insulin-stimulated adipocyte glucose transport system. The rate-controlling
enzyme is glutamine-fructose-6-phosphate amidotransferase (GFAT). This enzyme catalyzes the reaction: fructose 6-phosphate ⫹ glutamine 3 glucosamine 6-phosphate ⫹ glutamate. The final
products of the pathway are nucleotide hexosamines,
such as uridine diphosphate-N-acetyl glucosamine
(GlcNAc), that are substrates for O-linked GlcNAc
transferase. O-linked GlcNAc transferase catalyzes
the glycosylation of serine and threonine residues for a
number of nuclear and cytosolic proteins, including
glycogen synthase, c-Myc, and RNA polymerase II
(50–53). This mechanism may allow cells to sense fuel
availability and thus coordinately regulate their metabolism according to nutrient status.
Nutrient excess can promote insulin resistance via
accumulation of hexosamine biosynthetic pathway
end products. Several studies have confirmed the role
of GFAT in establishing insulin resistance in multiple
tissues, including the liver. Transgenic mouse models
established by McClain and colleagues (54–56) illustrate that moderate GFAT overactivity results in a phenotype resembling type 2 diabetes. Further, infusing
glucosamine into animals leads to a marked accumulation of hexosamine biosynthetic pathway end products that precedes the onset of insulin resistance (57).
To our knowledge, these are the only known examples
whereby a modest enhancement in the levels of a
rate-controlling enzyme in a catabolic, glucose-utilizing pathway, or infusing the end products of this pathway, increase the likelihood of developing insulin
resistance.
HEPATIC ENZYME ELEVATIONS AMELIORATING
OR PREVENTING DIABETES
GK
The liver removes a substantial portion of glucose from
the blood and thus is specially suited to metabolize
large amounts of carbohydrates (58). The breakdown
of glucose proceeds with phosphorylation of the hexose and its subsequent metabolism through various
pathways (Fig. 2). GK (hexokinase IV) is the major
glucose-phosphorylating enzyme in the liver; its abundance is regulated at a variety of levels, including
transcriptionally by insulin and glucagon, and posttranslationally by the GK regulatory protein (GKRP)
(59, 60). Insulin is required for GK expression and may
therefore be viewed as permissive for the ability of
Collier and Scott • Minireview
glucose to impact hepatic transcriptional patterns.
Thus, if GK activity is enhanced by alternative means,
such as with plasmid transfection, adenoviral transduction, or transgenic overexpression, insulin is no
longer required to initiate the metabolic reprogramming that occurs during the fasted-to-fed transition (4,
13, 14).
This principle can be illustrated in hepatoma cells,
which in contrast to primary hepatocytes, typically
lack GK activity. When hepatoma cells are stimulated
with high levels of glucose (15–30 mM), there are no
appreciable effects on glucose flux and utilization as
measured by lactate production in the cell culture
media or glycogen accumulation in the cell (17). Furthermore, glucose-responsive genes, such as L-type
pyruvate kinase (L-PK) and PEPCK, are unaffected by
high-glucose treatment in the absence of GK. Thus,
these cells remain, metabolically and phenotypically,
in the fasted state. However, when GK is expressed in
hepatoma cells, glucose metabolism generates signals that stimulate or repress specific genes in a coordinated manner (14, 38). Conversely, blocking GK
activity with mannoheptulose or glucosamine, competitive inhibitors of mammalian hexokinases, effectively reduces the ability of glucose to initiate this
phenotypic switch (14, 61). One may argue that a lack
of the glucose transporter 2 would possibly limit glucose flux into and out of the cells, even if GK was
present. However, in the studies we present in this
review, this does not seem to be the case because
overexpression of GK is sufficient to restore glucose
flux and gene expression patterns to those resembling
the fed state (4, 17, 62).
GK activity is highly correlated with blood glucose
concentration and is thus considered to be a hepatic
“glucose-sensor” (63). Thus, extinguishing the signal
provided by GK-derived glucose metabolism would
theoretically impede the ability of the hepatocyte to
undergo the fasting-to-fed transition. Indeed, liverspecific GK knockout mice exhibit a 40% increase in
blood glucose concentrations (64), presumably due to
a lack of glucose signaling and therefore an inability to
initiate the correct metabolic reprogramming.
Hepatic GK mRNA and protein levels are very low in
both human and rodent diabetes; insulin treatment
rapidly restores GK activity to the hepatocyte (65).
Because of these observations, hepatic replacement
of this enzyme provides a possible therapeutic strategy to treat diabetes, and this idea has received considerable attention experimentally. Ferre et al. (4) generated transgenic mice wherein a portion of the
PEPCK promoter was used to drive liver-specific GK
expression. These mice have a 2-fold increase in GK
activity relative to control animals, which results in
increased hepatic glycogen storage. This modest GK
overexpression in the liver protects against streptozotocin (STZ)-induced diabetes. STZ destroys pancreatic ␤-cells, which normally secrete insulin to prevent
hyperglycemia. Insulin deficiency is a hallmark of type
1 diabetes, and because insulin is markedly reduced
Mol Endocrinol, May 2004, 18(5):1051–1063
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or absent in the STZ-animal, the fasted-to-fed transition of the liver is impaired. However, in GK-expressing transgenic mice treated with STZ, the phenotypic
switch promoting HGU and decreasing HGP is carried
out solely based on signals derived from glucose metabolism. This metabolic reprogramming by the glucose-derived signals allows the fed pattern of liver
gene expression to be established in the absence of
insulin. For example, the expression of the PFK2 and
L-PK genes are increased, whereas expression of the
PEPCK, CPT I, and CPT II genes are decreased (4).
This reflects a switch from glucose production to glucose uptake and utilization that promotes the lowering
of serum glucose, triglycerides, free fatty acids, and
ketone bodies in GK-overexpressing transgenics relative to control mice (4). This study demonstrates that
a modest augmentation of GK activity in the liver enhances glucose metabolism and promotes overall glucose homeostasis.
In a different set of studies, involving adenoviral GK
infusion into the liver of normal rats, an approximate
5-fold increase in glycogen storage is achieved in the
fasted state (66). These authors noted that relatively
low (⬃3-fold) overexpression of GK does not significantly alter the plasma levels of triglycerides, free fatty
acids, glucose, or insulin in these rats. However, this
moderate increase in GK activity is sufficient to promote the fed patterns of gene expression in the fasting
state of these animals (67), and although not tested,
one would predict improved glucose tolerance. In contrast, a 6-fold overexpression of GK increases circulating triglycerides approximately 3-fold and free fatty
acids about 4-fold. Although this level of GK activity
did promote a 38% decrease in blood glucose levels
concomitant with a 3-fold decrease in circulating insulin levels, it also raised the concern that GK overexpression leads to hyperlipidemia (66).
In other models of elevated GK activity, dyslipidemia
is not observed. For example, overexpression of the
human GK gene in transgenic mice using a liver-specific apolipoprotein A-I gene enhancer does not promote increased plasma triglycerides, but does improve glucose tolerance (68). Furthermore, transgenic
mice with an extra GK gene locus display increased
hepatic glucose metabolism, are protected from dietinduced diabetes, and do not develop hyperlipidemia
(62, 69). One common feature of GK-overexpressing
animals that do not display dyslipidemia is a modest
(2- to 3-fold) increase in GK activity. Clearly, increases
of 6-fold and above, although protective against diabetes mellitus, are deleterious in terms of lipid accumulation (66, 70).
Taken together, these studies imply that increasing
GK activity may be a rational intervention to treat both
major forms of diabetes mellitus. The logical argument
is that glucose metabolism provides signals that coordinately regulate hepatic gene transcription patterns
(Fig. 1); these signals drive the hepatocyte toward the
fed state by inducing the appropriate metabolic enzymes, leading to increases in glycolysis, glycogen
1056 Mol Endocrinol, May 2004, 18(5):1051–1063
storage, and fatty acid synthesis. Simultaneously, glucose and ketone production are inhibited. This phenotypic switch occurs in hepatocytes irrespective of
islet hormones, provided that GK is present. However,
the exact signals and the factors required have not
been completely characterized. It is important to emphasize that the dissociation of glucose signaling from
insulin signaling is likely to occur only in pathological
conditions in which therapeutic interventions have
been made, as these two effectors are closely associated under normal (nondiabetic) conditions.
GKRP
The GK gene is regulated transcriptionally by insulin,
glucagon, and other hormones (59, 60). Another level
of control is maintained through the actions of GKRP.
This 68-kDa protein sequesters GK in the nucleus
during the fasting state (71). Binding of GK to GKRP is
favored when fructose 6-phosphate levels are high,
whereas elevated fructose 1-phosphate levels promote the release of GK from GKRP (71, 72). Physiologically, a nuclear pool of GK provides a readily available source of the enzyme during the fasted-to-fed
transition. Indeed, GK contains a nuclear export sequence that ensures its proper return to the cytosol
after release from the GKRP (73, 74). The importance
of this mechanism is apparent in mice that have the
GKRP gene ablated (75, 76). Farrelly et al. (75) noted
that GK activity is approximately 33% and 83% lower
in mice heterozygous and homozygous, respectively,
for the GKRP knockout when compared with control
mice. GK mRNA levels remain essentially unchanged
in these animals. In a different study, Grimsby and
colleagues (76) noted a 16% and 41% decrease in GK
activity in heterozygous and homozygous GKRP
knockout mice, respectively, when compared with
wild-type animals. Both sets of studies confirmed that
plasma insulin levels remain normal in the mutant
mice. However, GKRP⫺/⫺ animals have a marked reduction in glucose clearance rates compared with
wild-type controls (75, 76). Thus, it is noteworthy that
the GKRP has at least three theoretical functions: 1)
sequestering GK in the nucleus to prevent futile cycling (glucose 3 glucose ⫺6-phosphate 3 glucose) in
the fasting state; 2) protecting GK against proteolytic
degradation; and 3) maintaining a nuclear reserve of
GK that can be quickly released after a meal.
With these functions in mind, it is not surprising that
GKRP overexpression protects against the development of diet-induced diabetes (77). Mice infused with
an adenovirus expressing the GKRP gene display improved glucose tolerance and lower fasting blood glucose and insulin levels when compared with animals
receiving a control adenovirus (77). This decrease in
blood glucose and insulin may be indicative of an
increased sensitivity to insulin. One further explanation
for these results is that increasing the abundance of
the GKRP extends GK half-life by protecting the enzyme from degradation (77). This mechanism allows
Collier and Scott • Minireview
for the release of GK from a nuclear pool, where it is
transported into the cytoplasm after a meal. Thus,
promoting a GK reserve ensures that the signal
afforded by glucose metabolism will be present to
help promote the fasting-to-fed transition of the
hepatocyte.
Glycogen Targeting Subunits
Numerous proteins that are modified by hormones
and metabolites control the regulation of glycogen
synthesis and breakdown (78–80). Glycogen degradation helps maintain euglycemia between meals,
whereas glycogen synthesis helps remove glucose
from the blood via storage in muscle, fat, and liver. The
glucose used as a substrate for glycogen synthesis
originates from two distinct sources. Glucose phosphorylation by GK facilitates direct entry into the glycogen synthase reaction and therefore is termed the
“direct” pathway. Glucose derived from other precursors, such as lactate, glycerol, and amino acids,
through gluconeogenesis is called the “indirect” pathway. Both sources of glucose, however, culminate in
the production of glucose 6-phosphate, activation of
glycogen synthase, and thus promotion of hepatic
glycogen storage (81).
GK overexpression enhances glycogen storage via
the direct pathway by increasing glucose phosphorylation in the hepatocyte (4, 66). An alternative method
of promoting glycogen storage is to increase the abundance of the glycogen targeting subunits, which bind
to both the glycogen particle and to the enzymes that
regulate its synthesis and degradation. The targeting
of protein phosphatase 1 (PP1) to glycogen appears to
be one mechanism by which the enzymes of glycogen
metabolism are regulated. For example, enhancing the
availability of scaffolding proteins that target PP1 to
the glycogen molecule increases the activity of glycogen synthase and decreases the activity of phosphorylase, thereby enhancing hepatic glycogen accumulation. This enhancement ultimately arises from the
glucose 6-phosphate that is produced by both the
direct and indirect pathways of glycogen synthesis
(47). There are several different isoforms of the glycogen targeting subunit proteins, and they are encoded
by four different genes in the same family (79). PTG
(protein targeting to glycogen) and PPPR6 are ubiquitously distributed; GL is expressed primarily in liver;
Gm/RGl is found predominately in muscle. These isoforms, although similar in that they all bind to glycogen
and protein phosphatase-1, have different binding affinities for glycogen synthase, glycogen phosphorylase, and phosphorylase kinase. They also promote
differential levels of glycogen synthesis when overexpressed in cultured hepatocytes (82).
Newgard and colleagues (79) have established a
variant of the Gm isoform, termed Gm⌬C, which is
generated by truncation of a 735-amino acid, sarcoplasmic reticulum-binding domain found in the
COOH-terminal region of the protein. This mutated
Collier and Scott • Minireview
protein exhibits the full capacity to respond to glycogenolytic signals, which is in contrast to GL or PTG (82).
Indeed, maneuvers that increase the amount of glycogen
storage in the liver are able to reverse glucose intolerance in high-fat fed rats (83). Importantly, this manipulation also functions successfully in a rodent model of type
1 diabetes by preventing both hyperglycemia and the
other metabolic disturbances generated by STZ treatment (84). It is important to note that maintaining the
ability of the hepatocyte to break down glycogen is critical to the success of this strategy (47, 84).
The data from Gm⌬C overexpression in STZ-treated
animals are quite striking. First, glycogen storage decreases G6Pase mRNA levels, but not PEPCK or pyruvate carboxylase mRNA levels (47). Second, blood
triglycerides, free fatty acids, and ketone bodies are all
normalized in the GM⌬C mice relative to control STZtreated mice (84). Remarkably, the lowering of blood
glucose and other parameters in STZ-injected animals
treated with GM⌬C occurs despite GK protein levels
being undetectable by Western blot analysis (84). Finally, food intake is decreased in STZ-treated GM⌬Cexpressing mice relative to controls. STZ injection and
thus insulin depletion cause hyperphagia, yet storage
of liver glycogen in GM⌬C-treated mice prevents it.
The underlying mechanisms of these effects are unclear. However, it is of interest to note that we are not
aware of any other data demonstrating that blood
glucose levels are normalized in a type 1 model of
diabetes in the absence of liver GK. Therefore, several
explanations may be put forth based on the available
data: 1) it is possible that precursors of glucose (e.g.
lactate and glycerol), generated by glucose metabolism and fatty acid breakdown in extrahepatic tissues,
are removed from the blood stream by the liver and
subsequently stored as glycogen through the indirect
pathway; 2) lowering the expression of the G6Pase
gene, a phenotype observed when glycogen storage is
enhanced by glycogen targeting subunit overexpression (47), prevents glucose export from the liver into
the circulation; 3) hepatic glycogen storage promotes
a satiety signal that prevents the hyperphagia that
would otherwise exacerbate the hyperglycemia in an
STZ-treated (and thus insulin-deficient) rodent. It is
possible, even likely, that combinations of these possibilities account for the observations seen in GM⌬Coverexpressing animals. Also, it is interesting that the
protective mechanisms of GK and GM⌬C, in the prevention of diabetes, may be different. Importantly, the
common link between these two different treatments
is increased HGU and possibly reduced hepatic glucose output, resulting in the lowering of blood glucose.
6-Phosphofructo-2-Kinase/Fructose-2,6Bisphosphatase (PFK2)
The pace of glycolysis is set predominately by the
enzyme 6-phosphofructo-1-kinase (PFK1), which catalyzes the reaction: fructose-6-phosphate ⫹ ATP 3
fructose-1,6-bisphosphate ⫹ ADP (58). This enzyme is
Mol Endocrinol, May 2004, 18(5):1051–1063
1057
allosterically regulated by ATP in a negative fashion
and AMP and fructose-2,6-bisphosphate (F-2,6-P2) in
a positive fashion. The enzyme PFK2 (or bifunctional
enzyme) controls both the synthesis and degradation
of F-2,6-P2, the most important allosteric regulator of
PFK1 (85). PFK2 has two distinct active sites; the
activity of each depends on the nutritional and hormonal state of the hepatocyte. For example, hormonal
regulation by glucagon, through cAMP activation of
the protein kinase A (PKA), promotes phosphorylation
at serine 32 of PFK2 that renders the kinase portion of
the bifunctional enzyme essentially inactive through
conformational changes. Thus, the phosphatase domain predominates under these conditions, effectively
reducing F-2,6-P2 levels and promoting gluconeogenesis while concomitantly decreasing flux through glycolysis. Lange and co-workers have taken advantage
of this knowledge to generate an enzyme that has a
high kinase to bisphosphatase activity ratio. By making S32A (removes the PKA phosphorylation site) and
H258A (reduces bisphosphatase enzyme activity) mutations, a double-mutant enzyme is produced that is a
much more potent generator of F-2,6-P2 than the wildtype enzyme (85).
The double-mutant PFK2 (DM PFK2), which constitutively increases F-2,6-P2 levels, effectively lowers
blood glucose levels in both STZ-treated and genetically insulin-resistant mice (86, 87). In the STZ-treated
mice, an approximate 30-fold increase in the concentration of F-2,6-P2 is generated by infusion of an adenovirus expressing the DM PFK2 vs. a control adenovirus (86). The subsequent increase in HGU results
in a lowering of blood glucose from 22 mM (STZcontrol) to 11 mM (STZ-DM PFK2) and coincides with
comparable decreases in plasma free fatty acids and
triglycerides (86). In genetically insulin-resistant mice,
which have a phenotype that mimics type 2 diabetes,
a similar correction of metabolic perturbations is observed. Blood glucose levels are normalized in the DM
PFK2 treated-animals whereas control animals remain
hyperglycemic. Also, there is lowering of serum lipids
in the DM PFK2 mice relative to control mice. Another
interesting observation is that increased levels of hepatic F-2,6-P2 down-regulate the expression of the
G6Pase gene and concomitantly increase the expression of the GK gene (87). These changes promote the
transition of the hepatocyte from the fasting to the fed
state. The mechanism underlying these results remains undetermined at the present time. It should be
noted that these effects are typically associated with
insulin signaling. However, it is becoming increasingly
clear that glucose metabolism, in this case by augmenting F-2,6-P2 levels, has effects that are equally
potent.
Sterol-Regulatory Element-Binding Protein
(SREBP) 1-c
As mentioned in the introduction and diagrammed in
Fig. 1, insulin and glucose act in concert to influence
1058 Mol Endocrinol, May 2004, 18(5):1051–1063
hepatic gene transcription patterns. One transcription
factor that has been suggested as a mediator of the
insulin signal is SREBP-1c (88, 89). The SREBP family
is encoded by two genes that produce three proteins.
The SREBP-1 gene, through alternative splicing and
the use of alternative promoters, produces two distinct transcription factors, termed SREBP-1a and
SREBP-1c (90–92). The SREBP-2 gene product is a
single protein with approximately 50% homology
when compared with the products of the SREBP-1
gene. Each of these three transcription factors shares
a similar modular structure that consists of: 1) an
amino-terminal basic helix-loop-helix leucine zipper
domain; 2) a central hydrophobic fragment that contains transmembrane segments; and 3) a carboxyterminal stretch of approximately 590 amino acids that
has regulatory functions (91, 92). These proteins are
inserted into the cytosolic face of the endoplasmic
reticulum membrane. Specific signals can promote
proteolytic cleavage and release of the mature, active
basic helix-loop-helix-LZ-containing transcription factor, which is then transported into the nucleus to activate target genes (93).
There is evidence that SREBP 1-c is an insulinmediated transcription factor that stimulates GK expression. Foretz and colleagues (94) demonstrated
that a mutated, mature form of SREBP 1-c, which is
directly imported into the nucleus, functions as a dominant-positive transcription factor and induces GK
gene transcription without a need for insulin. Further,
these investigators found that a dominant-negative
form of the transcription factor, which dimerizes with
the endogenous protein but does not bind DNA, prevents the insulin induction of the GK gene (94). Interestingly, and in contrast to the above study, Stoeckman and Towle (95) found that a dominant-positive
form of SREBP 1-c, controlled by a tetracycline-inducible adenoviral vector, only induced GK mRNA levels
to 2% of that seen with insulin treatment in primary
hepatocytes, while simultaneously increasing the expression of FAS.
Nonetheless, insulin induces SREBP 1-c in hepatocytes and restores expression of this factor in STZtreated mice (96, 97). Therefore, hepatic adenoviral
replacement of SREBP 1-c was used to examine
whether this factor could correct STZ-induced diabetes in mice (9). STZ treatment completely ablates the
expression of GK, FAS, and the low-density lipoprotein receptor, whereas PEPCK is strongly up-regulated due to the lack of insulin signaling. In contrast,
mice treated with an adenovirus expressing the dominant-positive form of SREBP 1-c display robust induction of the GK and FAS genes, whereas expression
of the PEPCK gene is strongly repressed (9). These
effects at the gene expression level correlate with an
increase in liver glycogen and triglycerides in SREBP
1-c-overexpressing mice; blood glucose also decreased significantly in SREBP 1-c-treated mice relative to mice receiving a control adenovirus (9). These
data are consistent with those obtained by Hanson
Collier and Scott • Minireview
and colleagues (98), who found that SREBP-1c overexpression in primary hepatocytes represses the
cAMP stimulation of the PEPCK gene, whereas a
dominant-negative form of the transcription factor
promotes PEPCK mRNA accumulation. Therefore, it
appears that the hypoglycemic effects of hepatic overexpression of SREBP 1-c arise by its ability to mimic
the action(s) of insulin.
c-Myc
c-Myc is a transcription factor that is involved in many
diverse cellular processes, including growth, differentiation, apoptosis, and metabolism (99, 100). This factor, a protooncogene, has been studied largely in the
context of cancer biology and recently was suggested
to have a role in contributing to the Warburg effect,
whereby tumor cells increase their glycolytic rate to
provide energy for various growth processes (101).
c-Myc interacts with another basic helix-loop-helix
factor, Max, and the resulting heterodimer binds to
E-box elements (consensus ⫽ CACGTG) to activate
gene transcription (102, 103). Carbohydrate response
elements, first described in the glucose-responsive
L-PK and S14 genes, contain sequences very similar
to sites favored by Myc-Max heterodimers (12, 104).
Studies by Bosch and colleagues (5, 105, 106) have
demonstrated that there is a connection between carbohydrate metabolism and c-Myc expression levels. A
3-fold hepatic overexpression of c-Myc, under control
of the PEPCK promoter in mice, leads to increased
hepatic glucose metabolism and improved glucose
disposal (105). These animals display increases in
both GK and L-PK mRNA; activity of these glycolytic
enzymes is also enhanced relative to control animals
(105). Further, this modest augmentation of c-Myc
abundance protects against the development of STZand diet-induced diabetes (5, 106). STZ-treated mice
with a 3-fold increase in c-Myc levels have an increased hepatic expression of the GK, PFK2, and
L-PK genes, concomitant with a decrease in the expression of the PEPCK gene, all relative to STZtreated control mice (5). Moreover, c-Myc overexpression restores the abundance of SREBP-1c in the
hepatocyte (107).
Blood glucose levels in the c-Myc-expressing transgenics are normalized, presumably due to the ability of
c-Myc to promote the fasting-to-fed transition of the
liver. This genetic reprogramming promotes appropriate metabolic enzyme gene induction, with the end
result of glucose removal from the blood via enhancement of hepatic glucose storage and utilization. Further, blood ketone levels are normalized in STZtreated Myc overexpressing mice relative to control
mice, probably because of the decrease in the expression of the CPT I, CPT II, and 3-hydroxy-3-methylglutaryl coenzyme A synthase genes (5). Moreover, these
c-Myc-induced regulatory mechanisms also prevent
obesity and development of insulin resistance (106).
All of these changes take place in the absence of
Collier and Scott • Minireview
insulin action, demonstrating the ability of elevated
c-Myc levels to coordinately regulate hepatic gene
transcription patterns. Questions that remain unaddressed are whether c-Myc directly transactivates the
GK, PFK2, or SREBP-1c genes, all three genes, or
induces their expression via an indirect mechanism.
We have shown recently that reducing the abundance of c-Myc or interfering with Myc-Max DNA
binding in hepatoma cells and primary hepatocytes
decreases the glucose-stimulated expression of the
L-PK and G6Pase genes (108). Reducing c-Myc levels
by 50%, using a recombinant adenovirus expressing
antisense c-myc mRNA, inhibits glucose-stimulated
L-PK gene expression by approximately 60% and
completely blocks the glucose induction of the
G6Pase gene. In contrast, the glucose repression of
hormone-activated PEPCK promoter activity is unaffected by the reduction in c-Myc abundance (108). We
interpret these data to indicate that multiple glucosesignaling pathways exist in the hepatocyte and that
there are c-Myc-dependent and -independent mechanisms of glucose-mediated transcriptional regulation. These findings corroborate those of Bosch and
co-workers (5, 105–107) and suggest that c-Myc may
be a key factor in the regulation of hepatic metabolic
enzyme genes. An alternative view is that c-Myc is
primarily involved in cell proliferation and contributes
to the changes in gene expression in a manner independent of the normal insulin- and glucose-signaling
pathways. However, the transcription factor network
required for the glucose-mediated reprogramming of
metabolic genes is still under investigation.
Carbohydrate Response Element Binding
Protein (ChREBP)
Recently, Uyeda and co-workers (109) discovered a
novel hepatic transcription factor that participates in
the regulation of the L-PK gene by glucose, which they
named ChREBP. ChREBP is an 864-amino acid (⬃95
kDa) protein that contains a nuclear localization signal
and a basic helix-loop-helix leucine zipper motif (110).
This transcription factor also contains several phosphorylation sites for PKA as well as one recognized by
the AMP-activated kinase. Indeed, nuclear import and
export are controlled by glucose and cAMP in an
antagonistic fashion that is dependent on protein
phosphorylation.
ChREBP resides in the cytosol when Ser196, which
is a site recognized by PKA near the nuclear localization signal, is phosphorylated upon cAMP stimulation
(111). Glucose promotes dephosphorylation by activation of PP2A (112). This phosphatase is activated by
xylulose 5-phosphate (Xu-5-P), a metabolite generated by the nonoxidative branch of the pentose phosphate pathway (113). Further, Ser626 and Thr666 are
phosphorylated by PKA, modifications that prevent
DNA binding; activation of PP2A by Xu-5-P removes
the phosphoryl group and increases affinity of
ChREBP for DNA (110). Thus, increased glucose flux
Mol Endocrinol, May 2004, 18(5):1051–1063
1059
and metabolism, possibly via a pentose phosphate
pathway metabolite, promote the ability of at least one
factor to regulate metabolic enzyme gene expression.
Based on these observations, we speculate that
overexpressing glucose 6-phosphate dehydrogenase
(G6PDH), the rate-controlling enzyme of the pentose
phosphate pathway, may be able to generate a phenotypic switch in the liver by driving flux through the
pathway and generating Xu-5-P and perhaps other
signaling metabolites (Fig. 2). Xu-5-P alleviates cAMPstimulated PKA phosphorylation of ChREBP (and possibly other factors), thereby promoting increased activity of this regulator. Moreover, a logical hypothesis
is that a dominant-positive form of ChREBP (containing S196A, T666A, and S626A mutations) would be
sufficient to activate the phenotypic switch of the liver,
similar to the mechanisms discussed throughout this
review. One might predict, then, that overexpression
of G6PDH, a gene normally induced by insulin (114),
or overexpression of a dominant-positive ChREBP
would be able to prevent STZ-induced diabetes, dietinduced diabetes, or both (Fig. 2). Further studies are
required to confirm these hypotheses.
CONCLUSION
The common link among the studies discussed herein
is that molecular manipulations that alter hepatic glucose metabolism regulate the fasted-to-fed transition
in hepatocytes in a hormone-independent manner.
This metabolic reprogramming can result in a shift
from HGP to HGU, or vice versa. These changes affect
overall glucose homeostasis. Modulations that increase HGP either induce or exacerbate insulin resistance, leading to diabetes. Conversely, increasing glucose uptake, utilization, and storage in the liver
protects against the development of hyperglycemia
and overt diabetes. The reciprocal regulation of glucose producing and utilizing enzymes requires that
multiple transcription factors, coactivators, and corepressors function in a coordinated fashion. Finally, the
ability of glucose to modulate hepatic gene transcription patterns and the capability of individual metabolic
pathways to influence overall glucose homeostasis
provide new targets for therapeutic intervention and is
a testament to the hormone-like control strength of
metabolic signaling.
Note Added in Proof
Since the submission of this manuscript, further studies supporting the ideas discussed here have become
available. Lange and colleagues (116) demonstrated
that increasing hepatic F-2,6-P2 levels promotes Akt
phosphorylation independently of insulin action, providing a potential link between glucose metabolism
1060 Mol Endocrinol, May 2004, 18(5):1051–1063
and insulin signaling. Work by Montminy’s group has
revealed two novel proteins that participate in controlling the hepatic fasted-to-fed transition. Hairy Enhancer of Split, a transcriptional repressor that is upregulated during the fasting state, prevents PPAR-␥induced transcriptional activity, also promoting the
fasting state phenotype (17). TRB3, which binds to
nonphosphorylated Akt and thus prevents its phosphorylation, leads to hyperglycemia when overexpressed in mice by promoting HGP (118).
Acknowledgments
We thank members of the Scott and Claycomb laboratories for critical reading of the manuscript. We also thank
David T. Eckert for graphic assistance. We apologize to any
of our colleagues whose work was omitted due to space
limitations.
Received September 15, 2003. Accepted December 15,
2003.
Address all correspondence and requests for reprints to:
Donald K. Scott, Department of Biochemistry and Molecular
Biology, Louisiana State University Health Sciences Center,
1901 Perdido Street, New Orleans, Louisiana 70112. E-mail:
[email protected].
This work was supported by a Career Development Award
from the American Diabetes Association (to D.K.S.).
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