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Molecular Physiology of Mammalian Glucokinase

September 2008
Cellular and Molecular Life Sciences 66(1):27-42

September 2008
66(1):27-42

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PubMed

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CC BY-NC 2.0

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The glucokinase (GCK) gene was one of the first candidate genes to be identified as a human "diabetes gene". Subsequently, important advances were made in understanding the impact of GCK in the regulation of glucose metabolism. Structure elucidation by crystallography provided insight into the kinetic properties of GCK. Protein interaction partners of GCK were discovered. Gene expression studies revealed new facets of the tissue distribution of GCK, including in the brain, and its regulation by insulin in the liver. Metabolic control analysis coupled to gene overexpression and knockout experiments highlighted the unique impact of GCK as a regulator of glucose metabolism. Human GCK mutants were studied biochemically to understand disease mechanisms. Drug development programs identified small molecule activators of GCK as potential antidiabetics. These advances are summarized here, with the aim of offering an integrated view of the role of GCK in the molecular physiology and medicine of glucose homeostasis.

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Review

Molecular Physiology of Mammalian Glucokinase

P. B. Iynedjian

Department of Cell Physiolgy and Metabolism, University of Geneva School of Medicine,

CMU 1 Rue Michel-Servet, 1211 Geneva 4 (Switzerland), Fax: +41 22/379 55 43,

e-mail: patrick.iynedjian@medecine.unige.ch

Received 11 June 2008; received after revision 18 July 2008; accepted 30 July 2008

Online First 26 August 2008

Abstract. The glucokinase (GCK) gene was one of the

first candidate genes to be identified as a human

“diabetes gene”. Subsequently, important advances

were made in understanding the impact of GCK in the

regulation of glucose metabolism. Structure elucida-

tion by crystallography provided insight into the

kinetic properties of GCK. Protein interaction part-

ners of GCK were discovered. Gene expression

studies revealed new facets of the tissue distribution

of GCK, including in the brain, and its regulation by

insulin in the liver. Metabolic control analysis coupled

to gene overexpression and knockout experiments

highlighted the unique impact of GCK as a regulator

of glucose metabolism. Human GCK mutants were

studied biochemically to understand disease mecha-

nisms. Drug development programs identified small

molecule activators of GCK as potential antidiabetics.

These advances are summarized here, with the aim of

offering an integrated view of the role of GCK in the

molecular physiology and medicine of glucose homeo-

stasis.

Keywords. Glucokinase, diabetes, glucose metabolism, insulin, hexokinase, islet of Langerhans, hepatocyte,

MODY.

Introduction

This review focuses on the enzyme known as mamma-

lian glucokinase. Somewhat ironically, this enzyme is

actually not a glucokinase in the strict sense, denoting

a glucose phosphorylating enzyme with stringent

substrate specificity for glucose, such as exists in

lower organisms. Mammalian glucokinase is able to

phosphorylate hexoses like mannose or fructose in

addition to glucose, a property shared with three other

hexokinases present at various levels and in various

combinations in human, rat, and mouse tissues [1].

The four mammalian isoenzymes display extensive

sequence identities, leaving no doubt about a close

phylogenetic relationship [2], and arguing for the

terminology hexokinases I– IV (or A– D), in which

hexokinase IV (or D) designates mammalian gluco-

kinase. In spite of these considerations, the term

glucokinase has become entrenched in tradition,

probably because it conveys a sense of the very unique

role of this enzyme in the regulation of glucose

homeostasis. This tradition is followed in the current

article, with the understanding that glucokinase

(GCK) stands for hexokinase IV (or D). Whether

the name should be reconsidered in the future is an

open question, because a gene encoding a functional

ADP-dependent glucokinase reminiscent of primitive

organisms was recently identified in the mouse and

human genomes [3]. The level of activity and physio-

logical role of this enzyme in mammalian tissues are

currently unknown.

Glucokinase was identified as an enzyme of rat liver in

1962. In 1992, two studies using the candidate gene

approach reported a genetic linkage between a form

Cell. Mol. Life Sci. 66 (2009) 27 – 42

1420-682X/09/010027-16

DOI 10.1007/s00018-008-8322-9

 Birkhuser Verlag, Basel, 2008

Cellular and Molecular Life Sciences

of maturity onset diabetes of the young, named

MODY 2, and the GCK gene [4, 5] . This discovery

came as the culmination of three decades of research

in biochemistry, physiology and molecular biology,

which collectively demonstrated the preferential ex-

pression of GCK in hepatocytes and b-cells of the

pancreas, and suggested a key role in glucose metab-

olism. These investigations, which had conferred to

GCK the rank of prime candidate as a “diabetes

gene”, were the subject of two reviews published in

1993 [6, 7].

In recent years our perception of the pivotal role of

GCK in the regulation of glucose metabolism has

been reinforced by important results in a variety of

experimental areas. Structural investigations by X-

ray crystallography shed new light on the very

special enzyme kinetics of GCK. Gene expression

studies revealed novel facets of the tissue distribu-

tion of GCK, notably in brain, and of the insulin

regulation of expression in the liver. Genetic

manipulations in cells and mice led to major

advances in our understanding of the glucose sensor

function of GCK at the cellular level and its impact

on whole body glucose homeostasis. This article was

written to assemble knowledge from very diverse

lines of investigation, with the aim of offering an

integrated view of the unique place of GCK in

physiology and medicine.

Three-dimensional structure of GCK: cooperative

glucose kinetics explained

Glucose taken up by mammalian cells has to be

converted into glucose 6-phosphate as a prelude to

further utilization for glycolysis, the pentose phos-

phate pathway or glycogen synthesis. Although some

contribution to overall glucose phosphorylation by the

recently identified ADP-dependent glucokinase [3]

cannot be ruled out, the bulk of glucose phosphor-

ylation occurs at the expense of ATP in the generic

reaction catalyzed by the hexokinases: hexose +ATP

!hexose 6-phosphate +ADP [8].

Among the hexokinases, GCK displays unique en-

zyme kinetics in two respects. First, GCK has an

affinity for glucose (S0.5 ﬃ6 mM) more than 20 times

lower than that of the next ranking hexokinase,

hexokinase II. Second, the rate of the GCK reaction

displays a sigmoid rather than hyperbolic dependence

on glucose concentration [1]. These two kinetic

properties, low affinity and cooperativity with respect

to glucose, allow GCK to operate as an ultrasensitive

physiological glucose sensor in cells endowed with

non-limiting glucose transport activity at the plasma

membrane (see below).

The cooperativity of GCK with glucose cannot be

explained by the classical mechanisms of allostery for

multisubunit proteins, because GCK is catalytically

active in the monomeric state [9]. Moreover, GCK

harbours a single glucose binding site (the active site)

[10], excluding the possibility of multisite allostery in

which two molecules of glucose would bind simulta-

neously to the enzyme. Deviation from hyperbolic

kinetics is sometimes noted for monomeric enzymes

in case of random addition of substrates, but this is not

the case for the GCK catalytic cycle, which involves an

ordered addition of substrates in which glucose binds

first and ATP second [11]. A mechanism of positive

cooperativity applicable to monomeric enzymes,

named the mnemonic model, was proposed for GCK

on the basis of early enzymological investigations [12] .

Predictions from the mnemonic model were con-

firmed by recent studies using pre-steady-state meas-

urements of the intrinsic fluorescence of GCK during

binding of glucose and other ligands [13, 14]. The

mnemonic mechanism of cooperativity includes the

following features: i) equilibrium between two con-

formational states of GCK with vastly different

glucose affinities; ii) large predominance of the low

affinity conformation in the absence of glucose; iii)

slow interconversion between the conformational

states for glucose-free GCK; iv) conversion from the

low affinity to the high affinity conformational state

strongly accelerated upon glucose binding to GCK ; v)

only the high affinity form of the enzyme is catalyti-

cally competent, and the rate of the catalytic step(s) is

very fast compared to the conformational transition

rate.

Explicit support for the model came recently from

structural data on human GCK obtained by Kamata

and colleagues, who solved the structure of GCK by

X-ray crystallography [15]. Crystals were obtained

from GCK with short truncations at the NH2-terminal

end (11 or 15 amino acids) under two distinct

crystallization conditions: in the presence of glucose

plus a small-molecule activator of GCK (see below),

or in the absence of any ligand. The overall structure

of the protein was comprised of a large and a small

globular domain connected by a hinge made up of

three flexible loops. With glucose and the activator

present, the space between the two domains assumed

the shape of a narrow, deep cleft containing the

glucose binding pocket. The binding site for the

activator was also localized in the hinge region, at a

distance from the glucose binding pocket. The struc-

ture was very similar to that of the C-terminal half of

hexokinase I crystallized in the presence of glucose,

called the closed form of hexokinase, and was there-

fore designated the “closed” form of GCK. By

contrast, GCK crystals formed without ligands dis-

28 P. B. Iynedjian Molecular physiology of glucokinase

played the so-called “super-open” conformation. In

this configuration, the cleft space between the two

domains of GCK was much broader, as the result of

two conformational changes compared to the closed

form: tilt and rotation of the small domain with

respect to the large domain, and extensive rearrange-

ment of the secondary structural elements of the small

domain itself [15].

The two X-ray structures of GCK could readily be

fitted into the mnemonic model of enzyme mecha-

nism. The super-open conformation represents the

low glucose affinity, catalytically inactive form of

enzyme. The closed conformation corresponds to the

high affinity form of GCK with glucose and ATP

bound at the active site during catalysis. An additional

conformation of GCK, intermediary between the

super-open and the closed states, was postulated for

the high affinity form of the enzyme free of substrates

or products. This putative conformation was desig-

nated the “open” form. The crystal structure of GCK

in the open form remains to be established. However,

X-ray diffraction studies of crystals of the C-terminal

half of hexokinase I formed in the absence of glucose

can serve as a model for the open form. Compared to

the closed form, the open form displayed a wider

interdomain cleft due to slight twist of the small

domain relative to the large domain, without change

in the internal topology of the small domain core [16] .

The transition between open and closed conforma-

tions would be very fast and easily reversible, in

contrast to the more complex molecular reorganiza-

tion involved in the slow transition from the super-

open to the open conformations. Indeed, the predic-

tion of an intermediary open state of GCK was borne

out in a recent study using targeted molecular

dynamics simulation to dissect the transition from

the closed to the super-open configurations of GCK

[17].

Regulation of glucokinase activity by protein-protein

interaction

Interaction with hepatic glucokinase regulatory pro-

tein. The best understood mechanism of rapid regu-

lation of GCK activity relies on protein-protein

interaction between GCK and a partner termed

GCK regulatory protein (GCKR). The association

between the two proteins is ligand-dependent and

inhibitory to GCK activity. Ligands for GCKR are

fructose-6-phosphate and fructose-1-phosphate,

which are mutual competitors. Binding of fructose 6-

phosphate to GCKR favors the GCKR-GCK inter-

action with a negative effect on enzyme activity, while

binding of fructose-1-phosphate weakens the GCKR-

GCK interaction and releases active GCK. Intra-

hepatic fructose-1-phosphate rises postprandially

after intestinal absorption of fructose and its con-

version to fructose-1-phosphate by liver fructokinase.

Conversely, inhibition of GCK by fructose-6-phos-

phate liganded to GCKR would provide a mechanism

for indirect negative feedback, since fructose 6-

phosphate is in equilbrium with glucose 6-phosphate

(the product of the GCK reaction) through the

phosphohexose isomerase step of glycolysis [18].

The conformational state of GCK preferred for

binding to GCKR is the super-open form. This was

suggested by experiments in a cell-free assay with

purified proteins, in which the association of GCK

with GCKR was estimated by co-immunoprecipita-

tion. The amount of GCK associated with GCKR was

reduced under conditions favoring the closed con-

formational state of GCK, that is during incubation at

elevated glucose concentrations in the presence of

ATP, or in the presence of glucose together with a

small molecule activator of GCK. This suggested that

the interface for binding to GCKR would be disrupted

by the conformational transition from the super-open

to the closed forms of GCK [19].

The reversible association of GCK with GCKR does

more than simply regulate the catalytic activity of

GCK, it also appears to underlie the intracellular

trafficking of GCK between cytoplasm and nucleus. In

transfected rat hepatocytes, GCK tagged with green

fluorescent protein was localized in the nucleus at low

glucose and was released to the cytoplasm at high

glucose, but GCK mutants with reduced affinity for

GCKR were predominantly cytoplasmic at low as well

as high glucose [20]. In cell lines devoid of GCK and

GCKR, such as COS-1, HeLa and HEK293T cells,

forced expression of GCK alone resulted in cytoplas-

mic localization, whereas co-expression with GCKR

resulted in nuclear accumulation of both proteins [20 –

22]. In a rat islet b-cell line with no endogenous

GCKR, doxycycline-inducible expression of GCKR

resulted in nuclear accumulation of GCKR together

with translocation of endogenous GCK from an

extranuclear localization to the nuclei [22].

A model (Fig. 1) to account for these results proposes

that GCKR in the free state would shuttle between

nucleus and cytoplasm. Under low glucose conditions,

the assembly of GCKR-GCK complex would result in

nuclear import and trapping of both proteins by

molecular mechanisms not yet completely elucidated.

Masking of a nuclear export sequence (NES) between

amino acids 300 and 310 of GCK sequence would be

part of this mechanism [21]. In metabolic states

accompanied by high glucose or fructose-1-phosphate

concentrations, and sufficient ATP levels [23], the

GCK-GCKR complex would dissociate and the NES

Cell. Mol. Life Sci. Vol.66, 2009 Review Article 29

of GCK would become functional, allowing for the

rapid export of GCK from nuclei. In the isolated

perfused liver system and in primary cultures of rat

hepatocytes, GCK and GCKR were indeed mainly

localized to the nuclei at glucose concentrations

around 5 mM. Higher glucose levels or fructose

addition to the medium caused a rapid translocation

of GCK to a predominant cytoplasmic localization

[24, 25]. This was accompanied to a lesser degree by

redistribution of GCKR to the cytoplasm [26, 27]. As

predicted by this model, mice with homozygous

inactivation of the GCKR gene had an extranuclear

localization of hepatic GCK under all metabolic

conditions [28, 29]. Interestingly, the steady state

amount of GCK in the liver was reduced about 50 % in

the GCKR knock-out animals, suggesting the possi-

bility that the interaction with GCKR might stabilize

GCK against degradation.

Recently, a minor fraction of GCK and GCKR in

hepatocytes was reported to be associated with the

mitochondrial fraction, raising the suggestion of

distinct pools of cytoplasmic GCK with specialized

metabolic functions [30]. However, this notion should

be considered with caution, because other authors

failed to detect GCK activity or immunoreactivity in

isolated mouse liver mitochondria [31].

Interaction with other proteins. The impact of the

GCKR model led several investigators to search for

new protein partners that might affect GCK activity or

localization, notably in the islet b-cells in which

GCKR is essentially absent. A list of GCK interacting

proteins with their major properties is given in

Table 1. For virtually all of these proteins other than

GCKR, additional investigations are necessary for

definitive proof that the proposed interaction with

GCK has an impact on GCK activity and/or cell

function.

The bifunctional enzyme 6-phosphofructo-2-kinase/

fructose-2,6-bisphosphatase (PFKFB) is responsible

for the regulated formation and degradation of

fructose 2,6-bisphosphate, a key allosteric activator

of the enzyme 6-phosphofructo-1-kinase, and thereby

of glycolysis. An interaction of the fructose-2,6-bi-

sphosphatase domain of PFKBP with GCK was

demonstrated in a yeast two-hybrid system [34].

Overexpression of PFKBP in insulinoma b-cells

resulted in increased GCK specific activity without

increase in the cellular content of GCK protein [35,

36]. Although provocative, the published studies did

not directly demonstrate an association of GCK with

PFKFB in mammalian cells, nor did they provide

insight into a possible mechanism for the apparent

increase in GCK activity in cells co-expressing

PFKBP. These issues should be further investigated

before a conclusive model of GCK regulation by

association with PFKBP becomes widely accepted.

A model in which neuronal nitric oxide synthase

(NOS) would serve to anchor GCK to the periphery of

insulin secretory granules in the basal state in bTC3

insulinoma cells was proposed by Rizzo and Piston

[37]. Autocrine activation of NOS by insulin during

Figure 1. Regulation of GCK activity and subcellular localization by interaction with GCKR. The scheme depicts hepatocytes after an

overnight fast (postabsorptive) and during the ingestive phase after a carbohydrate containing meal (postprandial). The nuclear

compartment (circle) communicates with the cytoplasmic space by nuclear pores. The blue and cyan ovals represent the large and small

lobes of GCK respectively. After carbohydrate ingestion, the glucose concentration in plasma and hepatocytes rises, as does the

concentration of fructose-1-phosphate. As a result,the GCKR (red)-GCK interaction is loosened, allowing GCK to bind glucose, adopt the

closed (catalytically active) conformation and exit from the nucleus to generate glucose-6-phosphate for glycogen synthesis and glycolysis.

30 P. B. Iynedjian Molecular physiology of glucokinase

glucose stimulated secretion would stimulate the

nitrosylation of GCK on a specific cysteine residue,

and its release from the granules to the cytosol. The

general validity of this model remains uncertain,

however, because glucose-induced translocation of

GCK from the secretory granules to the cytosol was

not confirmed in MIN6 insulinoma cells [38].

Human GCK was recently found to be polyubiquiti-

nated at multiple Lys residues in a cell-free system.

Additionally, a sequence motif compatible with an

ubiquitin interacting consensus was identified in the

C-terminal a-helix of human GCK. This motif was the

target for non-covalent binding of free pentaubiquitin

chains in a cell-free system. Ubiquitin chains added to

a GCK enzyme assay mixture resulted in an increased

Kcat [39]. The occurrence and physiological implica-

tion of this interaction in living cells were not

investigated.

Multiprotein assembly at the outer mitochondrial

membrane. In investigations of the function of the

BH3-only proapoptotic protein BAD, Danial and

colleagues described a large multimolecular assembly

comprising BAD, GCK and other proteins in a highly

purified mitochondrial fraction from the mouse liver

[40]. Analysis of the supramolecular complex using

immunochemistry and LC-tandem MS peptide anal-

ysis revealed that the complex contained GCK and

BAD, as well as the Wiskott–Aldrich family member

WAVE, the catalytic subunits of protein phospatase 1

(PP1) and of protein kinase A (PKA). The essential

protein in nucleating the complex appeared to be

BAD, since the complex was absent in BAD knock-

out (BAD-/-) mice. The oxidative metabolism of

glucose was reduced in hepatocytes from BAD-/-

mice, suggesting a function of the complex in meta-

bolic regulation. However, caution is warranted,

because GCK associated with mitochondria was a

very small fraction of the total [40], if present at all

[31]. Complicating the matter further, the role and

regulation of the protein kinase and phosphatase

associated with the complex remain elusive.

The relevance of the Danial complex was recently

extended to the b-cells of the islets of Langerhans [41] .

Perifused islets from BAD-/- mice displayed glucose

unresponsive insulin secretion. Total assayable GCK

activity in islet homogenates from BAD-/- mice was

reduced to less than 30% of the wild-type level.

Glucose-sensitive insulin release was restored after

treatment of islets from BAD-/- animals with cell-

permeable synthetic peptides corresponding in se-

quence to a BH3 a-helix of BAD. Peptides harboring a

phosphomimetic Ser 155 to Asp mutation were active,

while a peptide with an Ala mutation was ineffective.

Peptides active in the insulin secretion assay also

induced strong increases in GCK activity when added

to INS-1 insulinoma cells in culture. Furthermore,

photoactivatable BAD peptides were shown to cross-

link with GCK in homogenates of INS-1 cells. It is

worth pointing out that a much larger fraction of

cellular GCK appeared to be under BAD control in

the islets than in the liver. Whether most or all of the

GCK interacting with BAD resided at the outer

mitochondrial membrane in islet cells was not exper-

imentally demonstrated.

Table 1. Protein interaction partners of glucokinase.

Name Method of identification Biochemical

effects Cellular effects References

glucokinase regulatory

protein (GCKR) protein purification from liver extract inhibits GCK

activity regulates GCK activity and

localization in hepatocytes reviewed

in [18]

glucokinase-associated-

phosphatase yeast two-hybrid system increases GCK

activty unknown [32]

propionyl-CoA

carboxylase b-subunit yeast two-hybrid system increases GCK

affinity and

activity

unknown [33]

6-phosphofructo-2-

kinase/fructose-2,6-

bisphosphatase

screening of random peptide display phage

library for binding to GCK no effect in GCK

activity assay overexpression in insulinoma

cells increases GCK activity [34 – 36]

neuronal nitric oxide

synthase co-immunoprecipitation nitrosylates

GCK anchors GCK to the periphery

of insulin secretory granules [37]

pentaubiquitin identification of ubiquitin interacting motif

in GCK increases GCK

affinity and

activity

unknown [39]

bad peptide analysis of multiprotein complex in

liver mitochondrial fraction,

immunochemistry

increases GCK

activity regulates GCK activity and

localization in hepatocytes and

b-cells

[40, 41]

Cell. Mol. Life Sci. Vol.66, 2009 Review Article 31

Regulation of glucokinase gene expression

Tissue-specific promoters. A defining feature of the

GCK gene in mammalian species is the presence of

two alternative promoters. The promoters are sepa-

rated by approximately 30 kbp of genomic DNA and

responsible for transcription initiation in a mutually

exclusive manner in distinct tissues. The upstream

promoter and adjacent leader exon drive the synthesis

of GCK mRNA in non-hepatic tissues, that is islet

cells, enteroendocrine cells, specialized glucose-sensi-

tive neurons in the central nervous system (CNS), and

additional scattered cells in various tissues [42]. This

promoter is denominated the neuroendocrine pro-

moter. By contrast, the downstream so-called liver

promoter and its associated leader exon serve for

transcription initiation in hepatocytes only. Each of

the leader exons specifies the 5 untranslated regions

of the mRNAs as well as the first 15 codons for the

GCK protein sequences, which therefore differ at

their NH2-terminal ends in liver and other tissues.

During splicing of the primary transcripts, the donor

splice sites at the end of the neuroendocrine or liver

leader exons are ligated to the acceptor site of a

common exon 2 and further splicing incorporates

common exons 3– 10 in the two types of mRNAs.

Thus, the GCK enzymes in neuroendocrine cells and

hepatocytes have a common amino acid sequence

over most of their length. There is currently no known

functional difference in the GCK enzymes arising

from the short distinct leader sequences.

Alternative promoters allow for the control of tran-

scription initiation by distinct sets of cis-acting regu-

latory elements in different tissues. In early experi-

ments, Magnuson and colleagues showed that a 294 bp

rat DNA fragment of the neuroendocrine promoter

activated the expression of luciferase in insulinoma

cells, but not 3T3-preadipocytes [43]. In transgenic

mice, the same DNA fragment drove the expression of

a human growth hormone reporter gene in pancreatic

islet cells, intestinal cells and brain cells [42]. Two

major functional cis-acting elements were delineated

in this promoter fragment [43]. One element bound

proteins which did not match known transcription

factors [44], whereas the other element was identified

as a binding site for Beta2/NeuroD, a basic helix-loop-

helix zipper (bHLHz) transcriptional activator im-

portant in the ontogeny of the endocrine pancreas

[45]. Another key transcription factor of early pan-

creas development, Pdx-1, was proposed to activate

the neuroendocrine GCK promoter [46], but this

notion was subsequently dispelled [47].

There are no published investigations using transgenic

mice to define the regulatory sequences responsible

for the hepatocyte-specific activity of the liver GCK

promoter. In transient transfection assays, a 200 bp

liver promoter fragment from the rat gene was

competent to stimulate the expression of a luciferase

reporter in primary hepatocytes and hepatoma culture

cells, but not in insulinoma cells [48, 49]. More

importantly, a 300 bp enhancer active only in primary

hepatocytes was mapped between nucleotides -1000

and -700 with respect to the start of transcription of the

rat gene and at a similar position in the human gene.

This sequence exhibited 6 DNAse I protection foot-

prints, but the identity of the binding proteins was not

elucidated [48].

Transcriptional induction of gene expression by

insulin in liver. In the liver, expression of GCK is

strictly dependent on the presence of insulin. Thus,

GCK mRNA and protein disappear from the livers of

insulino-deficient rats and are restored after insulin

treatment [50]. In cultured rat hepatocytes, the effect

of insulin as inducer of GCK was shown to be

primarily at a transcriptional level, triggering a 15-

to 30-fold increase in GCK mRNA in three hours, and

taking place in glucose-free as well as glucose-

containing medium without any synergy by addition

of glucose [6]. Induction of GCK by insulin and

repression by glucagon were also documented in

hepatocytes isolated from the human liver [51].

Insulin induction of GCK in hepatocytes was shown to

be suppressed by the inhibitors of phosphoinositide 3-

kinase (PI3-kinase) wortmannin and LY294002, im-

plying that activation of PI3-kinase following insulin

binding to its receptor was essential. Protein kinase B

(PKB, also called Akt) is a major protein kinase

activated as a result of PI3-kinase stimulation. To test

for a possible role of PKB in mediating GCK

induction, hepatocytes were transduced for expres-

sion of an estrogen receptor-PKB chimeric protein

that could be conditionally activated by tamoxifen.

The transduced hepatocytes reponded to the addition

of tamoxifen by a rise of GCK mRNA, mimicking the

effect of insulin [52]. In separate experiments, insulin

induction of GCK mRNAwas abrogated by inhibitors

of PKB activation [53, 54] . Additionally, GCK mRNA

and protein were induced in the livers of streptozo-

tocin diabetic mice following in vivo transduction of

adenoviruses encoding kinase active versions of

PFKFB, a maneuver that unexpectedly triggered the

activation of PKB [55]. A role of PKB in insulin

induction of GCK was questioned by Matsumoto and

colleagues [56], who observed that they could inhibit

insulin-mediated induction of GCK mRNA in hep-

atocytes by expressing a dominant negative NH2-

terminal fragment of insulin receptor substrate-1

(IRS-1), while PKB activation apparently remained

unaffected. An explanation for this negative result

32 P. B. Iynedjian Molecular physiology of glucokinase

might be that GCK induction is mediated by a minor

pool of PKB functionally associated with IRS-1,

making a small contribution in assays of total cellular

PKB activity and therefore difficult to quantify.

The transcriptional regulators which respond to the

insulin activation of PKB (and perhaps of accessory

signaling cascades) and stimulate transcription at the

liver GCK promoter remain elusive. The sterol-

regulatory element binding protein SREBP1c, a

master regulator of lipogenic enzymes such as fatty

acid synthase (FAS) and acetyl-CoA carboxylase

(ACC), was proposed to be a mediator of insulin

induction of GCK. Briefly, SREBP1c itself was shown

to be induced at the transcriptional level by insulin in

hepatocytes [57, 58]. Overexpression of SREBP1c in

primary hepatocytes was accompanied by an elevation

of GCK mRNA and, conversely, a dominant negative

mutant of SREBP1c inhibited insulin-dependent in-

duction of GCK mRNA [59]. More recently, inves-

tigators identified a tandem DNA element around

position -200 in the rat liver GCK promoter, which

could bind bacterially expressed mature SREBP1c in

DNAse protection assay and electrophoretic mobility

shift assay (EMSA) [60]. However, in other inves-

tigations, inducible expression of mature SREBP1c in

hepatocytes failed to produce a significant increase of

GCK mRNA [61]. In SREBP1c ko mice, GCK

mRNA was induced normally during the fasting-

refeeding transition [62]. Moreover, liver transduc-

tion of adenovirus encoding active PKB (i.e. mimick-

ing insulin signaling) resulted in strong induction of

hepatic GCK mRNA in normal as well as SREBP1 ko

mice [63]. In our own experiments in insulin stimu-

lated hepatocytes, SREBP1c failed to bind to the liver

GCK promoter, while its binding to the FAS promoter

was readily discerned by chromatin immunoprecipi-

tation [64]. Also, insulin produced a more than 10-fold

augmentation of GCK mRNA amount in a time frame

in which there was strictly no increase in transcrip-

tionally mature SREBP1c protein. Vice-versa, activa-

tion of the nuclear receptor liver X receptor (LXR)

with a synthetic ligand produced a strong induction of

SREBP1c and its target gene FAS without any rise of

GCK mRNA [64]. Together, these results seem to

virtually rule out a determinant role of SREBP1c in

the rapid induction of liver GCK by insulin in rat

hepatocytes.

Binding sites for the transcriptional activators hep-

atocyte nuclear factor-4 (HNF-4) and hypoxia-indu-

cible factor-1a (HIF-1a) were identified within the

first 100 bp of the liver GCK promoter [65, 66]. Co-

expression of HNF-4 and HIF-1awith GCK reporter

plasmids in primary rat hepatocytes resulted in

additive transactivation of the GCK promoter,

which was further augmented by expression of the

co-activator p300. More importantly, co-transfection

of plasmids encoding constitutively active PI3K or

PKB stimulated GCK promoter activity, as long as an

intact binding site for HIF-1awas present in the

promoter DNA fragment. Together, these findings

suggested that co-operation between HNF-4, HIF-1a

and p300 at the liver GCK promoter might be

important for induction of the gene in response to

acute insulin signaling [66]. The proposal that HIF-1a

might be a mediator in insulin signaling is significant,

because hypoxia and insulin were previously shown to

synergize in inducing GCK gene expression in cul-

tured hepatocytes [67]. This type of synergy might in

part account for the enrichment of insulin inducible

enzymes such as GCK in the underoxygenated

perivenous region of the liver lobule, compared to

the periportal region [68]. Based on findings in

another system, insulin might hypothetically induce

HIF-1atarget genes by relieving HIF-1a from co-

repression by a forkhead box class O (FOXO) factor,

through a mechanism involving PKB-mediated phos-

phorylation and functional inactivation of FOXO

[69]. However, arguing against this possibility, levels

of hepatic GCK mRNA in adult mice with liver

specific knockout of FOXO1 were similar to those of

control mice in both the fasted and fed states [70].

Hormone and nutrient regulation of glucokinase gene

expression in islets of Langerhans. Glucose refeeding

after a fast is an experimental paradigm often used to

study acute hyperinsulinemia in the intact animal.

Consistent with data in cultured hepatocytes, GCK

mRNA was virtually absent from the livers of fasted

rats and accumulated massively within hours of

feeding a glucose-enriched diet [71]. By contrast,

GCK mRNA and protein levels in islets of Langer-

hans were not significantly depressed in fasted rats

compared to freely fed animals and, more importantly,

were not acutely induced during the fasting-refeeding

transition [72]. Thus, constitutive expression of GCK

in the pancreatic islets contrasted with inducible GCK

gene expression in the liver.

In the MIN6 insulinoma cell line, GCK mRNA was

higher during culture at 30 mM than at 3 mM glucose

concentration, or following the addition of subnano-

molar concentrations of insulin to the medium [73].

However, luciferase reporter plasmids driven by the

neuroendocrine GCK promoter were unaffected after

a rise in glucose or insulin concentrations [74],

suggesting that, in insulinoma cells, the major effects

of glucose or insulin might be to stabilize GCK mRNA

against degradation rather than stimulate transcrip-

tion from the neuroendocrine promoter. In rat INS-1

insulinoma cells, GCK mRNA and protein levels were

increased about 50% after 24 hours of culture in the

Cell. Mol. Life Sci. Vol.66, 2009 Review Article 33

presence of insulin-like growth factor-1 (IGF-1),

consistent with a modest transcriptional effect of

IGF-1. An inhibitory cis-acting DNA element target-

ed by FOXO1 was identified at position 500 in the

neuroendocrine promoter. Thus, FOXO1 might act in

this system as a repressor, itself negatively regulated

by IGF-1 [75].

Metabolic role of hepatic glucokinase

Regulatory role of glucokinase at the hepatocellular

level. The liver is equipped with an insulin-independ-

ent facilitative glucose transport system (now known

to be mostly by GLUT2) with apparent affinity

constant of 17 mM and high transport rate, allowing

extremely rapid equilibration of the glucose concen-

tration across the hepatocyte plasma membrane [76].

Unlimiting glucose transport, and the unique glucose

affinity of GCK (which is the predominant hexokinase

isoenzyme in the liver), ensure that fluctuations of the

plasma glucose concentration are rapidly translated

into changes in the rate of glucose phosphorylation

inside the hepatocyte. If, in turn, the GCK reaction

controls the substrate flux along the entire length of

the pathways from glucose to final products, the

overall metabolic rate in these pathways will be

regulated autonomously in response to changes in

the plasma glucose concentration.

In hepatocytes with adenovirus mediated overexpres-

sion of GCK, compared to hepatocytes transduced

with control vectors, rates of glycogen synthesis were

augmented in proportion to GCK activity [77, 78].

This effect was attributed to an increase in the cellular

content of glucose-6-phosphate, a product of the GCK

reaction and an allosteric activator of glycogen

synthase [79]. Overexpression of GCK also resulted

in proportionate stimulation of glycolysis and glucose

oxidation [80, 81]. As stated above, the fact that GCK

exerts dominant control on glycogen synthesis and

glycolysis has a crucial physiological implication: it

confers to hepatocytes the ability for cell autonomous

regulation of glucose metabolism in response to

fluctuations of the plasma glucose.

Regulatory role of liver glucokinase at the organismal

level. The impact of altering liver glucokinase activity

on whole body glucose homeostasis was illustrated in

transgenic mice with an extracopy of the GCK gene

locus. These animals had an approximately 50%

increase in hepatic GCK activity over the wild-type

level and a 25% reduction in plasma glucose in the

postabsorptive state, reflecting increased glucose

clearance. During an hyperglycemic clamp, overall

glucose utilization was similar in control and trans-

genic mice, in spite of plasma insulin levels that were

40 % lower in the transgenics. Liver glycogen synthesis

over the period of hyperglycemia was increased about

3.5 times in transgenic mice compared to wild-type

[82]. The level of immunodetectable GCK was lower

in the pancreatic islets of transgenic mice with addi-

tional GCK gene copies than in wild-type animals,

perhaps owing to destabilization of the GCK protein

in islets at low glycemic levels.

Another mouse model with overexpression of liver

GCK was produced by transgenesis of GCK cDNA

fused to the phosphoenolpyruvate carboxykinase

(PEPCK) promoter. This construct allowed for high

GCK activity to be maintained in the livers of trans-

genic mice made insulino-deficient by streptozotocin

treatment, while virtual suppression of hepatic GCK

was noted in nontransgenic littermates [83]. Transgenic

mice were largely protected against streptozotocin-

induced hyperglycemia. They maintained normal levels

of liver glycogen and lactate, compared to severe

depletion of these metabolites in the livers of non-

transgenic diabetic mice, consistent with a determinant

role of GCK in hepatic glycogen synthesis and glycol-

ysis.

The effects of loss of hepatic GCK function on glucose

homeostasis were investigated using a gene targeting

system allowing for liver-specific inactivation of the

GCK gene [84]. Mice with homozygous GCK gene

deletion in the hepatocytes survived to adult age with

virtually complete absence of GCK enzyme activity in

liver tissue. In the freely fed state, the mice exhibited

modest hyperglycemia with relative hyperinsulinemia

compared to controls, indicative of an insulin resistant

state. Liver glycogen in the freely fed state was

normal, implying that gluconeogenesis (and perhaps

high affinity hexokinases) produced sufficient glu-

cose-6-phosphate in the basal state to ensure glycogen

formation and compensate for the ablation of GCK.

However, during a hyperglycemic clamp, the rate of

liver glycogen synthesis failed to rise and whole body

glucose turnover was lower than in control animals.

Interestingly, glucose-induced insulin secretion during

hyperglycemia was markedly diminished in liver

knockout mice, possibly reflecting functional damage

to the b-cells of the islets of Langerhans due to chronic

hyperglycemia and secretory overstimulation [84].

The role of liver GCK in restraining hepatic glucose

output in response to hyperglycemia was also high-

lighted in mice with global heterozygous inactivation

of the GCK gene [85]. These mice exhibited a 50%

reduction of GCK activity in the livers, and presum-

ably in islets and other neuroendocrine cells as well.

Glucose metabolism was investigated by the glucose

clamp technique combined with pancreatic clamping

with somatostatin, in order to maintain circulating

34 P. B. Iynedjian Molecular physiology of glucokinase

insulin concentrations at the same fixed level in

knockout and wild-type mice. Hyperglycemia caused

a 10% reduction of the rate of hepatic glucose

production in knockout mice, compared to 40% in

controls, demonstrating that liver GCK is instrumen-

tal in insulin-independent inhibition of hepatic glu-

cose production in response to high glucose [85]. Since

GCK gene inactivation was ubiquitous in these mice,

it cannot be excluded that decreased GCK activity at

extrahepatic sites, for example in the hypothalamus

(see below), might have compromised a neurally-

mediated mechanism contributing to the suppression

of hepatic glucose production during hyperglycemia.

Metabolic role of islet b-cell glucokinase

Regulatory role of glucokinase in the b-cell. Insulin

secretion elicited by hyperglycemia requires stimula-

tion of glucose metabolism in the b-cells. Coupling

between metabolism and secretion relies on the

generation of metabolic products, most prominently

ATP generated by glycolysis and glucose oxidation,

which act as upstream signals in cascades leading to

the stimulation of the exocytic machinery [86, 87]. For

instance, a rise in the cellular ATP/AMP ratio causes

the inhibition of ATP-sensitive K+channels in the

plasma membrane, leading to membrane depolariza-

tion and activation of voltage-sensitive Ca++ channels.

The influx of extracellular Ca++ ions then somehow

promotes the exocytosis of insulin secretory granules.

The ability of the b-cells to increase their rate of

glucose metabolism in response to a rise in the

extracellular glucose concentration is basic to the

regulation of insulin secretion. The biochemical

properties which allow glucose to regulate its own

metabolism, and thereby insulin secretion, are the

same in the b-cells and in hepatocytes. The first

property is unrestricted glucose transport to ensure

rapid glucose equilibration across the plasma mem-

brane [88, 89]. The second aspect is that glucose

phosphorylation is predominantly catalyzed by GCK

[90], which provides for concentration-dependent

adjustment of the rate of the initial reaction of glucose

metabolism. The third property is a metabolic organ-

ization in which flux along the entire pathways of

glycolysis and glucose oxidation is mostly controlled

at the initial reaction, that catalyzed by GCK.

The extent to which glucose phosphorylation does

control the rate of glycolysis in insulin secreting cells

was investigated in INS-1 derived insulinoma cells

that had been engineered for doxycycline inducible

GCK expression. Moderate increases in GCK activity

elicited by small doses of doxycycline were accom-

panied by quantitatively comparable fractional in-

creases in glycolytic rate at physiologically relevant

extracellular glucose concentration. The fact that the

ratio of fractional increase in glycolytic flux on

fractional increase in GCK activity was close to

unity showed that GCK exerted overwhelming con-

trol over the glycolytic rate [91]. Not surprisingly, at

very high levels of GCK overexpression and especially

at high glucose levels, a limit to the increase in

glycolytic flux was attained, indicating that one or

several steps in the lower part of the glycolytic

pathway became limiting [91, 92]. The glyceralde-

hyde-phosphate dehydrogenase reaction was incrimi-

nated as such a step, due to NAD+depletion [93].

Regulatory role of b-cell glucokinase at the organ-

ismal level. A strategy targeting the neuroendocrine

promoter region of the GCK gene was used to

generate mice with GCK deficiency in the b-cells of

the islets of Langerhans (and other cell-types utilizing

the same promoter) [94]. Mice homozygous for the

deleted allele developed severe diabetes and died

during the first week after birth. Glucose-stimulated

insulin secretion was completely absent in islets from

newborn null mice. Mice heterozygous for the deleted

allele survived to adulthood with slight hyperglyce-

mia, intolerance to a glucose load and reduced

glucose-stimulated hyperinsulinemia. As expected,

GCK activity in islets was reduced to about 50% of

the wild-type level in the heterozygotes. Isolated islets

form heterozygous mice exhibited impaired glucose

stimulated insulin release, most apparent at a glucose

concentration around 10 mM [94]. Similar findings

were reported in mice made deficient in islet GCK

activity by a LoxP strategy using Cre recombinase

expressed from the rat insulin II gene promoter. The

mice with heterozygous inactivation of b-cell GCK

were markedly impaired in ability to raise plasma

insulin during a hyperglycemic clamp in comparison

to controls [84]. Collectively, these data underlined

the role of islet GCK in glucose-induced insulin

secretion and maintenance of glucose homeostasis in

the whole animal. Whether the ablation of GCK in

other neuroendocrine or insulin expressing cells might

have contributed to the observed phenotype in the

above experiments remains to be determined.

Glucokinase in the brain

Transgenic mice harboring a chimeric human growth

hormone (hGH) structural gene transcribed from the

rat neuroendocrine GCK promoter had abundant

hGH in their pancreatic islets as expected, and more

surprisingly, also displayed strong immunoreactivity

to hGH in isolated hypothalamic cells [42]. Following

Cell. Mol. Life Sci. Vol.66, 2009 Review Article 35

this discovery, Jetton and colleagues detected GCK

mRNA by reverse transcription-polymerase chain

reaction (RT-PCR) in several regions of the rat brain

and localized GCK transcripts as well as immunor-

eactive protein in situ in the hypothalamus [42].

Subsequently, the neuroendocrine form of GCK

mRNA was selectively identified by RT-PCR assay

as well as in situ hybridization in the ventromedial and

arcuate nuclei of the rat hypothalamus [95]. At the

protein level, immunoblotting and enzyme assays

confirmed the presence of GCK in rat hypothalamic

extracts [96].

In the ventromedial hypothalamus, GCK mRNA was

shown to be expressed in neurons synthesizing pre-

proopiomelanocortin (POMC) and neuropeptide Y

(NPY)/agouti-related peptide (AgRP), which play

critical roles in neural pathways involved in the

regulation of food intake and energy expenditure.

Experiments using primary cultures of rat hypothala-

mic neurons incubated with metabolic inhibitors such

as mannoheptulose or alloxan suggested that GCK

activity was instrumental for the glucose response of

glucose-excited and glucose-inhibited neurons as well

[97]. Transcript profiling at the single cell level

confirmed the presence of GCK mRNA in approx-

imately 50 % of glucose-excited and glucose-inhibited

neurons in the ventromedial area of the hypothal-

amus. Some of these neurons also contained mRNAs

for subunits of ATP-sensitive K+channels [98]. In

primary cultures of neurons from the rat ventromedial

hypothalamus, treatment with specific small interfer-

ing RNA (siRNA) to knock-down GCK resulted in

the virtual disappearance of excitatory as well as

inhibitory responses to glucose elevation [99]. Col-

lectively, these findings supported the idea that the

glucose sensor of at least a fraction of ventromedial

hypothalamic neurons was GCK, functioning as the

rate determining enzyme of glucose metabolism and

controlling neuronal excitability. However, this pro-

posal raises puzzling questions. First, the interstitial

glucose concentration in brain in the postabsorptive

state is around 1.5 mM, well below the optimal range

of concentrations where GCK displays ultrasensitive

kinetics with respect to glucose concentration. The

second key issue is that of coupling between glucose

metabolism and neuronal activity. Many neurons are

equipped with ATP-sensitive K+channels, and the

excitability of such neurons might then be controlled

by the rate of glucose metabolism and the attending

fluctuations in cellular ATP content, in ways reminis-

cent of the islet b-cells [100]. Other glucose-derived

metabolites with a signaling role might be intermedi-

ates of the lipogenic pathway [101].

Molecular medicine of glucokinase

Maturity onset diabetes of the young (MODY2).

Approximately 200 mutations of the human GCK

gene, including missense, non-sense, and splice site

mutations, were described in the literature. The

majority of these mutations cause a form of familial

hyperglycemia or mild diabetes, transmitted as an

autosomal dominant trait and often diagnosed before

25 years of age. Based on these characteristics,

diabetes associated with GCK mutations was included

in the group of maturity onset diabetes of the young

(MODY), and was named MODY2. Clinically,

MODY2 was defined as a syndrome of impaired

fasting glucose level or mild diabetes, with hyper-

glycemia maintained at stable level for many years,

little propensity for late neurovascular complications

and often adequate glycemic control with diet alone

[102, 103] . Several cases of MODY2 were diagnosed in

women with a history of gestational diabetes [104].

Recently, a new nomenclature was proposed for the

MODY syndromes, MODY2 being replaced by the

descriptive name “familial mild fasting hyperglyce-

mia” [105]. Rare cases of infants inheriting GCK

mutations from two heterozygous parents were de-

scribed in the literature. They presented an autosomal

recessive form of permanent neonatal diabetes melli-

tus (PNDM) [106].

Clinical investigations in patients with MODY2 re-

vealed disorders of liver metabolism and pancreatic

islet function predicted from basic studies in cells and

animals. Net synthesis of glycogen in the liver after

each of three daily meals was decreased by about 50 %

in patients compared to controls [107]. Additionally,

the suppression of hepatic glucose output by hyper-

glycemia was deficient in patients with a GCK

mutation [108]. Patients with a variety of missense

or nonsense GCK mutations exhibited postprandial

plasma insulin levels comparable to controls, although

glucose concentrations were higher in the patients, a

finding compatible with a loss of sensitivity of the b-

cells to glucose. More specifically, during a ramping

glucose infusion, the glucose dose-response curve for

insulin secretion was found to be shifted to the right in

the MODY2 patients [109].

The biochemical properties of many GCK mutants

identified in MODY2 individuals were investigated

using bacterially produced protein. A wide range of

abnormalities affecting the turn-over number of the

enzyme, affinity for the substrates, and cooperativity

with respect to glucose were described [110 – 112].

Mathematical models based on the kinetics properties

were used to predict the threshold of glucose-stimu-

lated insulin secretion by b-cells harboring a given

heterozygous mutation. On average, prediction for a

36 P. B. Iynedjian Molecular physiology of glucokinase

number of mutants was in agreement with the level of

fasting hyperglycemia noted in cohorts of MODY2

patients [111]. Some mutants were found to have

unaltered kinetic properties compared to the wild-

type enzyme, but to display accelerated temperature-

dependent loss of activity in the test tube, in one case

correlating with decreased protein half-life in trans-

duced cells [111, 113].

Several mouse lines with heritable hyperglycemia

were produced through phenotype-driven mutagene-

sis programs using the potent mutagen N-ethyl-N-

nitrosourea (ENU). Interestingly, the majority of

hyperglycemic lines that were genetically character-

ized so far harbored mutations in the GCK gene [114,

115], several of which corresponded to mutations that

had been described in pedigrees with MODY2 or in

one case in PNDM [116]. These mice displayed

spontaneous hyperglycemia in the freely fed state,

and glucose intolerance accompanied by relative

hypoinsulinemia after an oral glucose load, similar

to abnormalities found in human MODY2 patients

[116].

Persistent hyperinsulinemic hypoglycemia of infancy

(PHHI). A small number of heterozygous missense

GCK mutations were identified as probable cause of

hyperinsulinism in infancy, a rare syndrome charac-

terized by chronic low fasting plasma glucose and

bouts of symptomatic hypoglycemia [117]. Kinetic

studies of the mutant enzymes revealed an increase in

glucose affinity with or without increased turnover

number (kcat) [118– 120] . A gene knock-in strategy

was used to produce mice with a GCK allele encoding

a hyperactive mutant GCK with increased affinity for

glucose. The mice were hyperinsulinemic relative to

their plasma glucose levels. However, the severity of

hypoglycemia was less than expected, suggesting a

compensatory mechanism in the liver. Paradoxically,

liver GCK activity and protein content in these mice

were lower than normal, suggesting the possibility of a

shorter half-life of the mutant enzyme due to impaired

interaction with GCKR [121].

Small-molecule activators of glucokinase as potential

antidiabetic drugs

Type 2 diabetes melltus (T2D) is a common disease of

multifactorial etiology, in which both a genetic pre-

disposition determined by multiple genes, and lifestyle

factors such as excess calorie intake and low physical

activity, play causative roles. The increasing preva-

lence of T2D, and its serious consequences on health

at both the individual and public levels, create

incentives for the development of novel antidiabetic

drugs. Physiopathologically, T2D consists of defective

insulin action in target tissue, including reduced

insulin suppression of liver glucose production, and

defective insulin secretion and other functions in the

b-cells of the islets of Langerhans [122]. The central

role of GCK in facilitating glucose disposal by the liver

on the one hand, and insulin secretion by the islets of

Langerhans on the other hand, provided a strong

rationale for the search of small molecule activators of

GCK in drug discovery programs aimed at developing

a new class of antidiabetic drugs. Such programs were

implemented by several major drug companies, and a

limited number of molecules acting as GCK activators

are currently in early stages of clinical trials.

Small molecule activators of GCK emerged from a

high throughput screening strategy based on the

capacity of chemicals to increase the activity of

recombinant GCK in an enzyme assay mixture con-

taining GCKR. The first promising compound was

described in 2003 by Grippo and colleagues [123] and

shown actually to be a direct activator of GCK in the

absence of GCKR. This compound, designated RO-

28-1675, had the dual effects of increasing both the

affinity for glucose and the turnover number of GCK,

that is, it shifted the glucose S0.5 of GCK from 8.5 mM

to 0.8 mM and increased the kcat by approximately

80% at maximal concentration. When added to

isolated rat islets of Langerhans, RO-28-1675 stimu-

lated the rate of glycolysis, lowered the threshold for

glucose-stimulated insulin secretion and increased the

maximal secretory response. A single oral adminis-

tration of RO-28-1675 to normal or obese mice had a

clear-cut hypoglycemic effect accompanied by hyper-

insulinemia. Treated mice displayed improved glucose

tolerance after an oral glucose load. Endogenous

glucose production was reduced during a hyperglyce-

mic hyperinsulinemic clamp in RO-28-1675-treated

rats compared to controls, consistent with an effect of

the drug on hepatic GCK activity [123]. A variety of

chemically distinct small molecule activators of GCK

were subsequently discovered in several laboratories.

The biochemical and cellular effects, as well as

biological actions in experimental animals, were

essentially similar to those of the prototype RO-28-

1675 [19, 124– 126]. An expert review of the current

status of GCK activators in drug development was

recently published [127].

An implicit hope in developing activators of the

physiological glucose sensor was the idea of restoring

“physiological” glucose-sensing, particularly in the

islet b-cells. However, the strong increase in the

glucose affinity of GCK induced by all activators

known to date might drastically lower the threshold

for insulin release and cause maximal insulin secretion

at low plasma glucose levels. Whether the design of

Cell. Mol. Life Sci. Vol.66, 2009 Review Article 37

drug preparations without liability of inducing serious

hypoglycemia is possible remains to be seen. Another

concern with GCK activators is related to the activa-

tion of hepatic lipogenesis leading to hepatosteatosis

(and associated insulin resistance), as was noted in

transgenic mice with hepatic overexpression of GCK

[128, 129]. Finally, in a more general vein, the long

term benefit of using insulinotropic agents in the

treatment of T2D (at least early in the disease) was

recently questioned, because of concerns about the

possibility of precipitating islet failure through hyper-

secretory activity [130].

Conclusions and perspectives

The evidence presented in this review should leave

little doubt about the status of GCK as an eminent

player in the regulation of glucose metabolism and

homeostasis. This status relies on the mechanism

whereby GCK operates as an ultrasensitive glucose

sensor, recently illuminated by structural data. The

localization of such a sensor inside, rather than on the

surface, of cells is striking. It is increasingly clear that

the traffic of GCK between distinct subcellular sites,

regulated by reversible interactions with diverse

scaffold proteins, might contribute to shape the

metabolic activity of cells, but by which exact molec-

ular mechanisms remains to be explained, notably in

the pancreatic b-cells. In hypothalamic nuclei and

other brain areas, it will be important to resolve the

apparent paradox of GCK operating under less than

optimal glucose concentrations for substrate regula-

tion of the enzyme reaction. Some of the most

intriguing unanswered questions pertain to the regu-

lation of GCK gene expression in hepatocytes. The

transcriptional induction of GCK is a dramatic effect

of insulin in the liver, perhaps the best example of

induction of any gene in any cell-type by insulin.

However, details about the regulatory proteins of

transcription which mediate this effect are still a black

box. The burstlike pattern of regulation of GCK

transcription in rat liver with very large, rapid and

transient inductive effects of insulin for a protein with

relatively slow turnover raises puzzling questions with

respect to the biological advantage that might arise

from this type of regulation. In the diabetes field, the

development of GCK activators through the preclin-

ical phase has generated exciting results, but also

illustrated some liabilities. If these can be overcome,

drugs with a novel mechanism of action will become

available as an additional option in the panoply of oral

antidiabetics.

Acknowledgements. The long term support of research in the

authors laboratory by the Swiss National Science Foundation is

gratefully acknowledged.

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To access this journal online:

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42 P. B. Iynedjian Molecular physiology of glucokinase

Identification of Structural Motifs Critical for Human G6PC2 FunctionInformed by Sequence Analysis and an AlphaFold2-Predicted Model

Article

Full-text available

Dec 2023

G6PC2 encodes a glucose-6-phosphatase (G6Pase) catalytic subunit, primarily expressed in pancreatic islet beta cells, that modulates the sensitivity of insulin secretion to glucose and thereby regulates fasting blood glucose (FBG). Mutational analyses were conducted to validate an AlphaFold2 (AF2)-predicted structure of human G6PC2 in conjunction with a novel method to solubilize and purify human G6PC2 from a heterologous expression system. These analyses show that residues forming a predicted intramolecular disulfide bond are essential for G6PC2 expression and that residues forming part of a type 2 phosphatidic acid phosphatase (PAP2) motif are critical for enzyme activity. Additional mutagenesis shows that residues forming a predicted substrate cavity modulate enzyme activity and substrate specificity and residues forming a putative cholesterol recognition amino acid consensus (CRAC) motif influence protein expression or enzyme activity. This CRAC motif begins at residue 219, the site of a common G6PC2 non-synonymous single nucleotide polymorphism (SNP), rs492594 (Val219Leu), though the functional impact of this SNP is disputed. In microsomal membrane preparations, the L219 variant has greater activity than the V219 variant, but this difference disappears when G6PC2 is purified in detergent micelles. We hypothesize that this was due to a differential association of the two variants with cholesterol. This concept was supported by the observation that the addition of cholesteryl hemi-succinate to the purified enzymes decreased the Vmax of the V219 and L219 variants ~8-fold and ~3 fold, respectively. We anticipate that these observations should support the rational development of G6PC2 inhibitors designed to lower FBG.

Biochemical evidence that the whole compartment activity behavior of GAPDH differs between the cytoplasm and nucleus

Article

Full-text available

Aug 2023
PLOS ONE

Some metabolic enzymes normally occur in the nucleus and cytoplasm. These compartments differ in molecular composition. Since post-translational modification and interaction with allosteric effectors can tune enzyme activity, it follows that the behavior of an enzyme as a catalyst may differ between the cytoplasm and nucleus. We explored this possibility for the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Homogenates of pristine nuclei and cytoplasms isolated from Xenopus laevis oocytes were used for whole compartment activity profiling in a near-physiological buffer. Titrations of NAD+ revealed similar whole compartment activity profiles for GAPDH in nuclear and cytoplasmic homogenates. Surprisingly however GAPDH in these compartments did not have the same behavior in assays of the dependence of initial velocity (v0) on G3P concentration. First, the peak v0 for nuclear GAPDH was up to 2.5-fold higher than the peak for cytoplasmic GAPDH. Second, while Michaelis Menten-like behavior was observed in all assays of cytoplasm, the v0 versus [G3P] plots for nuclear GAPDH typically exhibited a non-Michaelis Menten (sigmoidal) profile. Apparent Km and Vmax (G3P) values for nuclear GAPDH activity were highly variable, even between replicates of the same sample. Possible sources of this variability include in vitro processing of a metabolite that allosterically regulates GAPDH, turnover of a post-translational modification of the enzyme, and fluctuation of the state of interaction of GAPDH with other proteins. Collectively these findings are consistent with the hypothesis that the environment of the nucleus is distinct from the environment of the cytoplasm with regard to GAPDH activity and its modulation. This finding warrants further comparison of the regulation of nuclear and cytoplasmic GAPDH, as well as whole compartment activity profiling of other enzymes of metabolism with cytosolic and nuclear pools.

Hepatic selective insulin resistance at the intersection of insulin signaling and metabolic dysfunction-associated steatotic liver disease

Article

May 2024
CELL METAB

Clinical investigation of glucokinase activators for the restoration of glucose homeostasis in diabetes

Article

May 2024

As a sensor, glucokinase (GK) controls glucose homeostasis, which progressively declines in patients with diabetes. GK maintains the equilibrium of glucose levels and regulates the homeostatic system set points. Endocrine and hepatic cells can both respond to glucose cooperatively when GK is activated. GK has been under study as a therapeutic target for decades due to the possibility that cellular GK expression and function can be recovered, hence restoring glucose homeostasis in patients with type 2 diabetes. Five therapeutic compounds targeting GK are being investigated globally at the moment. They all have distinctive molecular structures and have been clinically shown to have strong antihyperglycemia effects. The mechanics, classification, and clinical development of GK activators are illustrated in this review. With the recent approval and marketing of the first GK activator (GKA), dorzagliatin, GKA's critical role in treating glucose homeostasis disorder and its long‐term benefits in diabetes will eventually become clear. image

(Re-)Viewing Role of Intracellular Glucose Beyond Extracellular Regulation of Glucose-Stimulated Insulin Secretion by Pancreatic Cells

Article

Full-text available

Feb 2024

For glucose-stimulated-insulin-secretion (GSIS) by pancreatic β-cells in animals, it is believed that ATP generated from glucose metabolism is primarily responsible for GSIS. However, this ignores two well established aspects in literature: (a) intracellular ATP generation from other sources resulting in an overall pool of ATP, regardless of the original source, and (b) that intracellular glucose transport is 10 to 100-fold higher than intracellular glucose phosphorylation in β-cells. The latter especially provides an earlier unaddressed, but highly appealing, observation pertaining to (at least transient) presence of intracellular glucose molecules. Could these intracellular glucose molecules be responsible for specificity of GSIS to glucose (instead of the widely believed ATP production from its metabolism). In this work, we provide a comprehensive compilation of literature on glucose and GSIS using various cellular systems - all studies focus only on extracellular role of glucose in GSIS. Further, we carried out a comprehensive analysis of differential gene expression in Mouse Insulinoma 6 (Min6) cells, exposed to low and high extracellular-glucose concentrations (EGC), from the existing whole transcriptome data. Expression of other genes involved in Glycolysis, Krebs cycle, and electron transport chain were found to be unaffected by EGC except Gapdh, Atp6v0a4 & Cox20. Remarkably 3 upregulated genes (Atp6v0a4, Cacnb4, Kif11) in high EGC were identified to have an association with cellular secretion. Using glucose as a possible ligand for the 3 proteins, computational investigations were carried out (that will require future 'wet validation', both in vitro and in vivo, e.g., using primary islets and animal models). The glucose-affinity/binding scores (in kcal/mol) obtained were also compared with glucose binding scores for positive controls (GCK and GLUT2), along with negative controls (RPA1, KU70-80, POLA1, ACAA1A, POLR1A). Binding affinity scores of glucose molecules for the 3 proteins were found to be closer to positive controls. Therefore, we report glucose binding ability of 3 secretion-related proteins, and a possible direct role of intracellular glucose molecules in GSIS.

The Impact of Blood Glucose and Antidiabetic Medications on Osteoarthritis: A Mendelian Randomization Study

Preprint

Full-text available

Feb 2024

Objective Blood glucose metabolism is a crucial risk factor for the onset of osteoarthritis (OA). Our aim is to assess the causal relationship between blood glucose levels and the incidence of osteoarthritis, as well as the impact of antidiabetic medications on osteoarthritis outcomes, using Mendelian randomization (MR) methods. Methods This is a two-sample Mendelian randomization design. Osteoarthritis was chosen as the outcome, and causal relationships with three exposures—glycated hemoglobin (HbA1c), fasting blood glucose (FG), and postprandial two-hour blood glucose (2hGlu)—were evaluated. Additionally, this study examined the relationship between common antidiabetic medication target genes (GCK, HFE, HK1, HKDC1, TCF7L2, and INS) and osteoarthritis. The inverse variance-weighted method (IVW) is primarily employed for effect estimation. Results HbA1c levels were negatively correlated with hip OA (OR 0.725, 95% CI 0.558–0.944) but not associated with knee OA or OA at any site. FG levels were negatively correlated with hip OA (OR 0.675, 95% CI 0.547–0.834) and OA at any site (OR 0.889, 95% CI 0.801–0.986). Gene predictions revealed a negative correlation between GCK and hip OA (OR 0.359, 95% CI 0.238–0.542), HFE and hip OA(OR 0.332, 95% CI 0.181–0.607), HK1 and knee OA (OR 0.735, 95% CI 0.565–0.955) and OA at any site (OR 0.743, 95% CI 0.636–0.868), HKDC1 and knee OA(OR 0.731, 95% CI 0.555–0.963) and OA at any site (OR 0.771, 95% CI 0.655–0.908), while INS shows a positive correlation with knee OA (OR 17.277, 95% CI 4.408–67.722) and OA at any site (OR 4.168, 95% CI 1.844–9.422). Conclusion MR analysis revealed a correlation between reduced HbA1c levels and an increased risk of hip OA, and FG was associated with an increased risk of hip OA and OA at any site. Gene predictions suggest that activating GCK, HFE, HK1, and HKDC1 genes with antidiabetic medications may reduce the risk of developing osteoarthritis. Consideration of antidiabetic medications for treating osteoarthritis is plausible, but clinical validation is necessary.

Impaired Physiological Regulation of ß Cells: Recent Findings from Type 2 Diabetic Patients

Chapter

Full-text available

Jan 2024

Recent studies have emphasized the multiple aspects of non-coding micro-RNAs in the regulation of pancreatic ß cells in type 2 diabetic patients. Thus, highlighting the significance of non-coding regions of the genome in regulating pancreatic endocrine cells. Functional dysregulation of pancreatic endocrine cells increases the incidence of metabolic disorders in otherwise healthy individuals. A precise understanding of the molecular biology of metabolic dysregulation is important from cellular and clinical perspectives. The current chapter will highlight the important recent findings from type 2 diabetic human patients and aims to enhance our current understanding of ß cell pathophysiology from a clinical perspective for the development of novel therapeutic approaches to control this global incidence.

Tetrahydropalmatine from medicinal plants activates human glucokinase to regulate glucose homeostasis

Article

Nov 2023
BIOTECHNOL APPL BIOC

Many synthetic glucokinase activators (GKAs), modulating glucokinase (GK), an important therapeutic target in diabetes have failed to clear clinical trials. In this study, an in silico structural similarity search with differing scaffolds of reference GKAs have been used to identify derivatives from natural product databases. Ten molecules with good binding score and similar interactions to that in the co‐crystallized GK as well good activation against recombinant human GK experimentally were identified. Tetrahydropalmatine, an alkaloid present in formulations and drugs from medicinal plants, has not been explored as an antidiabetic agent and no information regarding its mechanism of action or GK activation exists. Tetrahydropalmatine activates GK with EC 50 value of 71.7 ± 17.9 μM while lowering the S 0.5 (7.1 mM) and increasing V max (9.22 μM/min) as compared to control without activator ( S 0.5 = 10.37 mM; V max = 4.8 μM/min). Kinetic data ( α and β values) suggests it to act as mixed, nonessential type activator. Using microscale thermophoresis, K d values of 3.8 μM suggests a good affinity for GK. In HepG2 cell line, the compound potentiated the uptake of glucose and maintained glucose homeostasis by increasing the expression of GK, glycogen synthase, and insulin receptor genes and lowering the expression of glucokinase regulatory protein (GKRP) and glucagon. Tetrahydropalmatine at low concentrations could elicit a good response by reducing expression of GKRP, increasing expression of GK while also activating it. Thus, it could be used alone or in combination as therapeutic drug as it could effectively modulate GK and alter glucose homeostasis.

Mitochondrial regulation in human pluripotent stem cells during reprogramming and β cell differentiation

Article

Full-text available

Oct 2023

Mexico Mitochondria are the powerhouse of the cell and dynamically control fundamental biological processes including cell reprogramming, pluripotency, and lineage specification. Although remarkable progress in induced pluripotent stem cell (iPSC)-derived cell therapies has been made, very little is known about the role of mitochondria and the mechanisms involved in somatic cell reprogramming into iPSC and directed reprogramming of iPSCs in terminally differentiated cells. Reprogramming requires changes in cellular characteristics, genomic and epigenetic regulation, as well as major mitochondrial metabolic changes to sustain iPSC self-renewal, pluripotency, and proliferation. Differentiation of autologous iPSC into terminally differentiated β-like cells requires further metabolic adaptation. Many studies have characterized these alterations in signaling pathways required for the generation and differentiation of iPSC; however, very little is known regarding the metabolic shifts that govern pluripotency transition to tissue-specific lineage differentiation. Understanding such metabolic transitions and how to modulate them is essential for the optimization of differentiation processes to ensure safe iPSC-derived cell therapies. In this review, we summarize the current understanding of mitochondrial metabolism during somatic cell reprogramming to iPSCs and the metabolic shift that occurs during directed differentiation into pancreatic β-like cells.

Comparative study of carbohydrate levels on growth, oxidative stress and glucolipid metabolism of hybrid fish between Megalobrama amblycephala (♀) × Culter alburnus (♂) and Culter alburnus

Article

Sep 2023

Distinct roles for insulin and insulin-like growth factor-1 receptors in pancreatic β-cell glucose sensing revealed by RNA silencing

Article

Full-text available

Nov 2004

The importance of the insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF-lR) for glucose-regulated insulin secretion and gene expression in pancreatic islet,beta-cells is at present unresolved. Here, we have used small interfering RNAs (siRNAs) to silence the expression of each receptor selectively in clonal MIN6 beta-cells. Reduction of IR levels by > 90% completely inhibited glucose (30 mM compared with 3 mM)-induced insulin secretion, but had no effect on depolarization-stimulated secretion. IR depletion also blocked the accumulation of preproinsulin (PPI), pancreatic duodenum homoeobox-1 (PDX-1) and glucokinase (GK) mRNAs at elevated glucose concentrations, as assessed by quantitative real-time PCR analysis (TaqMan((R))). Similarly, depletion of IGF-1R inhibited glucose-induced insulin secretion but, in contrast with the effects of IR silencing, had little impact on the regulation of gene expression by glucose. More-over, loss of IGF-i=lR, but not IR, markedly inhibited glucose-stimulated increases in cytosolic and mitochondrial ATP, suggesting a role for IGF-1R in the maintenance of oxidative metabolism and in the generation of mitochondrial coupling factors. RNA silencing thus represents a useful tool for the efficient and selective inactivation of receptor tyrosine kinases in isolated,B-cells. By inhibiting glucose-stimulated insulin secretion through the inactivation of IGF-1R, this approach also demonstrates the existence of insulin-independent mechanisms whereby elevated glucose concentrations regulate PPI, PDX-1 and GK gene expression in beta-cells.

Characterization of Glucokinase-binding Protein Epitopes by a Phage-displayed Peptide Library

Article

Full-text available

Nov 2001

The low affinity glucose-phosphorylating enzyme glucokinase shows the phenomenon of intracellular translocation in beta cells of the pancreas and the liver. To identify potential binding partners of glucokinase by a systematic strategy, human beta cell glucokinase was screened by a 12-mer random peptide library displayed by the M13 phage. This panning procedure revealed two consensus motifs with a high binding affinity for glucokinase. The first consensus motif, LSAXXVAG, corresponded to the glucokinase regulatory protein of the liver. The second consensus motif, SLKVWT, showed a complete homology to the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which acts as a key regulator of glucose metabolism. Through yeast two-hybrid analysis it became evident that the binding of glucokinase to PFK-2/FBPase-2 is conferred by the bisphosphatase domain, whereas the kinase domain is responsible for dimerization. 5′-Rapid amplification of cDNA ends analysis and Northern blot analysis revealed that rat pancreatic islets express the brain isoform of PFK-2/FBPase-2. A minor portion of the islet PFK-2/FBPase-2 cDNA clones comprised a novel splice variant with 8 additional amino acids in the kinase domain. The binding of the islet/brain PFK-2/ FBPase-2 isoform to glucokinase was comparable with that of the liver isoform. The interaction between glucokinase and PFK-2/FBPase-2 may provide the rationale for recent observations of a fructose-2,6-bisphosphate level-dependent partial channeling of glycolytic intermediates between glucokinase and glycolytic enzymes. In pancreatic beta cells this interaction may have a regulatory function for the metabolic stimulus-secretion coupling. Changes in fructose-2,6-bisphosphate levels and modulation of PFK-2/FBPase-2 activities may participate in the physiological regulation of glucokinase-mediated glucose-induced insulin secretion.

Permanent Neonatal Diabetes Caused by Glucokinase Deficiency: Inborn Error of the Glucose-Insulin Signaling Pathway

Article

Full-text available

Nov 2003
DIABETES

Neonatal diabetes can be either permanent or transient. We have recently shown that permanent neonatal diabetes can result from complete deficiency of glucokinase activity. Here we report three new cases of glucokinase-related permanent neonatal diabetes. The probands had intrauterine growth retardation (birth weight <1,900 g) and insulin-treated diabetes from birth (diagnosis within the first week of life). One of the subjects was homozygous for the missense mutation Ala378Val (A378V), which is an inactivating mutation with an activity index of only 0.2% of wild-type glucokinase activity. The second subject was homozygous for a mutation in the splice donor site of exon 8 (intervening sequence 8 [IVS8] + 2T-->G), which is predicted to lead to the synthesis of an inactive protein. The third subject (second cousin of subject 2) was a compound heterozygote with one allele having the splice-site mutation IVS8 + 2T-->G and the other the missense mutation Gly264Ser (G264S), a mutation with an activity index of 86% of normal activity. The five subjects with permanent neonatal diabetes due to glucokinase deficiency identified to date are characterized by intrauterine growth retardation, permanent insulin-requiring diabetes from the first day of life, and hyperglycemia in both parents. Autosomal recessive inheritance and enzyme deficiency are features typical for an inborn error of metabolism, which occurred in the glucose-insulin signaling pathway in these subjects.

Kinetic evidence for a 'Mnemonical' mechanism for rat liver glucokinase

Article

Full-text available

Jul 1977

Inhibition studies of glucokinase were carried out with the products of the reaction, glucose 6-phosphate and MgADP-, as well as with ADP3-, Mg2+ and ATP4-. The results of these, together with those of kinetic studies of the uninhibited reaction described previously [Storer & Cornish-Bowden (1976) Biochem. J. 159, 7-14], indicate that the enzyme obeys a 'mnemonical' mechanism. This implies that the co-operativity observed with glucose as substrate arises because glucose binds differentially to two forms of the free enzyme that are not in equilibrium under steady-state conditions. The mechanism predicts the decrease in glucose co-operativity observed at low concentrations of MgATP2-. The product-inhibition results suggest that glucose 6-phosphate is released first and that it is possibly displaced by MgATP2- in a concerted reaction.

Close linkage of glucokinase locus on chromosome 7p to early-onset non-insulin-dependent diabetes mellitus

Article

Jun 1992

Nature 356, 162-164 (1992) THE following was omitted from the Acknowledgements section of this Letter: "This work was supported in part by a NIH grant to M.A.P. Y.T. was a recipient of a mentor based fellowship award of the American Diabetes Association."

Transport and metabolism of glucose in an insulin-secreting cell line, .beta.TC-1

Article

Dec 1991

Kinetic characteristics of glucose transport and glucose phosphorylation were studied in the islet cell line beta-TC-1 to explore the roles of these processes in determining the dependence of glucose metabolism and insulin secretion on external glucose. The predominant glucose transporter present was the rat brain/erythrocyte type (Glut1), as determined by RNA and immunoblot analysis. The liver/islet glucose transporter (Glut2) RNA was not detected. The functional parameters of zero-trans glucose entry were K(m) = 9.5 +/- 2 mM and V(max) = 15.2 +/- 2 nmol min-1 (mu-L of cell water)-1. Phosphorylation kinetics of two hexokinase activities were characterized in situ. A low-K(m) (0.036 mM) hexokinase with a V(max) of 0.40 nmol min-1 (mu-L of cell water)-1 was present along with a high-K(m) (10 mM) hexokinase, which appeared to conform to a cooperative model with a Hill coefficient of about 1.4 and a V(max) of 0.3 nmol min-1 (mu-L of cell water)-1. Intracellular glucose at steady state was about 80% of the extracellular glucose from 3 to 15 mM, and transport did not limit metabolism in this range. In this static (nonperifusion) system, 2-3 times more immunoreactive insulin was secreted into the medium at 15 mM glucose than at 3 mM. The dependence of insulin secretion on external glucose roughly paralleled the dependence of glucose metabolism on external glucose. Simulations with a model demonstrated the degree to which changes in transport activity would affect intracellular glucose levels and the rate of the high-K(m) hexokinase (with the potential to affect insulin release).

Functional Glucokinase Isoforms Are Expressed in Rat Brain

Article

May 2000
J NEUROCHEM

Recently, the description of glucokinase mRNA in certain neuroendocrine cells has opened new ways to characterize this enzyme in the rat brain. In this study, we found glucokinase mRNA and a similar RNA splicing pattern of the glucokinase gene product in rat hypothalamus and pancreatic islets; the mRNA that codes for B1 isoform was the most abundant, with minor amounts of those coding for the B2, P1, P2, P1/B2, and P2/B2 isoforms. Glucokinase gene expression in rat brain gave rise to a protein of 52 kDa with a high apparent Km for glucose and no product inhibition by glucose 6-phosphate, with a contribution to the total glucose phosphorylating activity of between 40 and 14%; the hypothalamus and cerebral cortex were the regions of maximal activity. Low and high Km hexokinases were characterized by several criteria. Also, using RT-PCR analysis we found a glucokinase regulatory protein mRNA similar to that previously reported in liver. These findings indicate that the glucokinase present in rat brain should facilitate the adaptation of this organ to fluctuations in blood glucose concentrations, and the expression of glucokinase and GLUT-2 in the same hypothalamic neurons suggests a role in glucose sensing.

Fundamental Metabolic Differences between Hepatocytes and Islet β-cells Revealed by Glucokinase Overexpression †

Article

Mar 1998

Adenovirus-mediated overexpression of the glucose phosphorylating enzyme glucokinase causes large changes in glycolytic flux and glucose storage in isolated rat hepatocytes, but not in pancreatic islets. We have used the well-differentiated insulinoma cell line INS-1 to investigate the basis for these apparent cell-type specific differences. We find that 2- or 5-[3H]glucose usage is increased at low (</=5 mM) but not high glucose concentrations in INS-1 cells treated with a recombinant adenovirus containing the glucokinase cDNA (AdCMV-GKI), while glucose usage is increased at both low and high glucose concentrations in similarly treated hepatocytes. Utilization of 2-[3H]glucose in INS-1 cells is suppressed in glucokinase overexpressing INS-1 cells in a rapid, glucose concentration-dependent, and reversible fashion, while such regulation is largely absent in hepatocytes. Levels of hexose phosphates (glucose-6-phosphate, fructose-6-phosphate, and fructose-1,6-bisphosphate) were profoundly and rapidly elevated following the switch to high glucose in either AdCMV-GKI-treated INS-1 cells or hepatocytes relative to controls. In contrast, triose phosphate levels (glyceraldehyde-3-phosphate + dihydroxyacetone phosphate) were much higher in AdCMV-GKI-treated INS-1 cells than in similarly treated hepatocytes, suggesting limited flux throught the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) step in the former cells. Hepatocytes were found to contain approximately 62 times more lactate dehydrogenase (LDH) activity than INS-1 cells, and this was reflected in a 3-fold increase in lactate production in AdCMV-GKI-treated hepatocytes relative to similarly treated INS-1 cells. Since the amounts of G3PDH activity in INS-1 and hepatocyte extracts are similar, we suggest that flux through this step in INS-1 cells is limited by failure to regenerate NAD in the LDH reaction and that a fundamental difference between hepatocytes and islet beta-cells is the limited capacity of the latter to metabolize glycolytic intermediates beyond the G3PDH step.

Maintenance of the monomeric structure of glucokinase under reacting conditions

Article

Oct 1978

Glucokinase is a monomeric protein under native and denaturating conditions yet presents a sigmoidal saturation function for glucose. These peculiarities suggested the possibility that polymerization occurs under assay conditions. Thus the apparent molecular weight of glucokinase was determined by gel filtration at 4 °C and at 30 °C in the presence of substrates and products, singly and in combination, creating during the filtration similar conditions as used in the assay. Gel filtration was performed also in the presence of N-acetylglucosamine, which is a competitive inhibitor and shifts to an hyperbole the saturation function for glucose. The same elution behavior, that is, a single symmetrical peak, was observed in every system used. This persistent monomeric form of glucokinase excludes the possibility that the sigmoidal function is the result of the interaction of different subunits. The possibility of an association-dissociation equilibrium in which the kinetic properties of the enzyme depend on the particular molecular weight species may also be rejected.

Linkage of Type 2 diabetes to the glucokinase gene

Article

Jun 1992

Maturity-onset diabetes of the young (MODY) is a subtype of type 2 diabetes that presents from the second decade and has an autosomal dominant mode of inheritance. We have investigated the glucokinase gene, a candidate gene for diabetes, in two MODY pedigrees. In a large 5-generation pedigree (BX) with 15 diabetic members, use of a microsatellite polymorphism revealed linkage of diabetes to the glucokinase locus on chromosome 7p. A peak lod score of 4.60 was obtained at a recombination fraction (theta) of zero. This finding suggests that a defective glucokinase gene contributes to the diabetes phenotype in this pedigree. This is not universal in MODY since linkage to the glucokinase locus was excluded in a second pedigree M (lod score = -7.36 at theta = 0). The affected members in pedigree BX were diagnosed either when young (in pregnancy or on screening) or when they presented symptomatically in middle and old age; most of them were treated by diet alone. Defects in the glucokinase gene may play an important part in the pathogenesis of type 2 diabetes.

Molecular Physiology of Mammalian Glucokinase

Abstract

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