9. Melo, L. G., A. T. Veress, U. Ackermann, S. C. Pang, T. G. Flynn, and H.
Sonnenberg. Chronic hypertension in ANP knockout mice: contribution of
peripheral resistance. Regul. Pept. 79: 109–115, 1999.
10. Melo, L. G., A. T. Veress, U. Ackermann, and H. Sonnenberg. Chronic regulation of arterial blood pressure by ANP: role of endogenous vasoactive
endothelial factors. Am. J. Physiol. Heart Circ. Physiol. 275: H1826–H1833,
1998.
11. Melo, L. G., A. T. Veress, U. Ackermann, M. E. Steinhelper, S. C. Pang, Y.
Tse, and H. Sonnenberg. Chronic regulation of arterial blood pressure in
ANP transgenic and knockout mice: role of cardiovascular sympathetic
tone. Cardiovasc. Res. 43: 437–444, 1999.
12. Melo, L. G., A. T. Veress, C. K. Chong, U. Ackermann, and H. Sonnenberg.
Salt-sensitive hypertension in ANP knockout mice is prevented by AT-1
receptor antagonist losartan. Am. J. Physiol. Regulatory Integrative Comp.
Physiol. 277: R624–R630, 1999.
13. Melo, L. G., A. T. Veress, C. K. Chong, S. C. Pang, T. G. Flynn, and H. Sonnenberg. Salt-sensitive hypertension in ANP knockout mice: potential role
of abnormal plasma renin activity. Am. J. Physiol. Regulatory Integrative
Comp. Physiol. 274: R255–R261, 1998.
14. Oliver, P. M., J. E. Fox, R. Kim, H. A. Rockman, H.-S. Kim, R. L. Reddick,
K. N. Pandey, S. L. Milgram, O. Smithies, and N. Maeda. Hypertension,
cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A. Proc. Natl. Acad. Sci. USA 94: 14730–14735, 1997.
15. Veress, A. T., C. K. Chong, L. J. Field, and H. Sonnenberg. Blood pressure and
fluid-electrolyte balance in ANF-transgenic mice on high- and low-salt diets.
Am. J. Physiol. Regulatory Integrative Comp. Physiol. 269: R186–R192, 1995.
Regulation of Mammalian Gene Expression by Glucose
Recent data suggest that cells from species as diverse as yeast and mammals may use similar mechanisms
to detect changes in nutrient concentration. Here we review recent advances in understanding how
glucose regulates gene transcription in mammals.
R
ecognizing nutrients is a fundamental problem for all living
cells. For simple organisms, such as bacteria and yeast, it’s
a question of life and death. For example, when their preferred
carbon source, glucose, is unavailable, bacteria activate genes
that enable them to use lactose through the familiar lac
operon. Similarly, germinating plant seeds must switch from
stored fuel sources to utilizing carbohydrates synthesized as
soon as the seedling is capable of photosynthesis. In complex
animals, including mammals, the job of sensing changes in
nutrient concentration in the blood and responding with the
expression of genes involved in storing the sugar is performed
by highly specialized cell types. These fall into two groups. The
first major cell/tissue group comprises those that must accumulate and store glucose when the concentration in the blood
rises—namely liver, skeletal muscle, and adipose tissue. The
second group is made up of specialized neuroendocrine cells
that provide hormonal cues to other cells. The most important
of these “command and control” cells are located in the pancreas in specialized microorgans called islets of Langerhans.
Islet β-cells secrete insulin when blood glucose rises, whereas
islet α-cells release the stress hormone glucagon when it falls.
Understanding how islet cells sense changes in the concentration of blood glucose is an important question for human
health since defective glucose-stimulated insulin release is a
contributory factor in non-insulin-dependent diabetes mellitus
(NIDDM). Possibly providing an even higher level of control
are glucose-sensitive neurons in the ventromedial hypothalamus, which may then modulate β-cell function through neuronal means as well as affect feeding behavior.
Until recently, research into the intracellular signaling
mechanisms whereby glucose regulates genes in mammalian
cells has been undertaken largely without reference to sim-
G. A. Rutter, J. M. Tavaré, and D. G. Palmer are in the Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.
0886-1714/99 5.00 © 2000 Int. Union Physiol. Sci./Am.Physiol. Soc.
pler organisms. However, it is becoming apparent that similar mechanisms may be involved in both cases and may have
been conserved through evolution for a billion or more years.
In principle, glucose could act on target cells either by binding to the receptor at the cell surface or through its metabolism. In bacteria and mammals, both systems are involved,
though in mammals only one mechanism is used by any
given cell. Although taste bud cells on the tongue sense glucose through a cell surface receptor, glucose sensing by islet,
liver, adipose, and muscle cells almost certainly involves
metabolism of the sugar. Here we will survey those genes in
animals that are transcriptionally regulated by glucose
through its intracellular metabolism.
Classes of nutrient-regulated genes
Glucose increases the expression of genes whose protein
products catalyze important regulatory steps in the pathways
of lipid synthesis (Table 1) (3, 15). In the liver, the glucokinase
gene is transcriptionally activated by insulin through a
“direct” intracellular signaling mechanism that is independent
of extracellular glucose. Similarly, induction by insulin of
sterol regulatory element binding protein/adipocyte determination differentiation dependent factor 1 (SREBP/ADD1; see
below), an important transcription factor in the regulation of
the fatty acid synthase (FAS) gene, is independent of external
glucose. We will refer to these as “purely insulin-responsive”
(pIR) genes. In contrast, several genes appear to be responsive
to both insulin and to glucose (GIR genes). For example, the
phospho-enol pyruvate carboxykinase (PEPCK) and glucose
6-phosphatase genes are repressed by insulin both through
glucose-dependent and -independent mechanisms.
The next group of genes is common to liver, adipose tissue,
and (in the case of two of them) to islet β-cells. These encode
FAS, the liver isoform of pyruvate kinase (L-PK) and acetyl-CoA
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Guy A. Rutter, Jeremy M. Tavaré, and D. Gail Palmer
Table 1.
Glucose-responsive genes in mammals
Purely Insulin-Responsive
Genes
Gene
Location
Glucokinase
Liver
Sterol regulatory
Adipose, liver
element binding
protein
Glucose/Insulin Responsive
Genes
Gene
Location
L-Pyruvate
kinase
Acetyl-CoA carboxylase
Spot-14
Fatty acid synthase
Glyceraldehyde phosphate
dehydrogenase
Phospho-enol pyruvate carboxykinase
Glucose 6-phosphatase
Phosphofructokinase-1
Regulation of L-PK and S14 genes
The critical regulatory regions of both of these genes are
similar and located approximately –1440 bp upstream of the
transcriptional start point in the S14 gene and at –160 bp in
150
News Physiol. Sci. • Volume 15 • June 2000
adipose, β-cells Insulin (?)
adipose, β-cells
adipose
adipose
muscle
β-cells
Liver, kidney
Liver, kidney
Liver, muscle
the L-PK gene. DNA fingerprinting reveals no change in the
occupancy of the L-PK promoter during stimulation of liver
cells by glucose (A. Kahn, personal communication). Each of
these promoter elements includes a tandem repeat of a socalled E (enhancer) box. These 6-bp palindromic sequences
with the structure 5’-C(C/A)NG(T/G)G are based on the Herpes simplex virus major-late transcription factor binding site
(CACGTG). In the L-PK and S14 promoters, the two E boxes
(together called the glucose or carbohydrate response element) are separated by 5 bp. Although the sequence of this
region is unimportant, deviations from 5-bp spacing dramatically decrease the responsiveness to glucose, presumably
indicating that transcription factors bound at each E box must
interact and be located on the same face of the DNA. Adjacent to this E box region of both promoters is a binding site
for an ancillary factor, which in the case of the L-PK gene is
likely to be hepatic nuclear factor-4 (HNF4).
The ubiquitous and abundant transcription factor upstream
stimulatory factor (USF) binds efficiently to oligonucleotides
derived from the L-PK and S14 E boxes. USF is encoded by two
genes (USF1 and USF2) and is a member of the basic/helixloop-helix/leucine zipper (b/HLH/LZ) family. To investigate the
in vivo role of USF in binding and transactivation via the L-PK
and S14 E box regions, the groups of A. Kahn in Paris and H. C.
Towle in Minneapolis have used similar approaches, yet with
apparently conflicting conclusions. Thus A. Kahn’s group
found that expression of truncated USF constructs, capable of
heterodimerization but lacking a DNA binding domain,
blocked L-PK induction by glucose in hepatocytes. Similarly,
targeted gene disruption by homologous recombination has
implied crucial roles for the USF gene products USF1 and
USF2a/b (a and b being splice variants). However, long periods
of incubation (3 days) are required for the effects of dominantnegative USF on hepatoma cells, whereas nonconditional
knockouts always bear the risk of developmental alterations
indirectly related to the disrupted gene. Indeed, H. C. Towle’s
group observed that dominant-negative versions of USF had no
effect on glucose induction of L-PK in primary cultured hepatocytes, when much shorter post-transfection periods were
used. However, Kennedy et al (6) used direct microinjection
and imaging techniques (12) to demonstrate that anti-USF1 or
-USF2 antibodies strongly and specifically inhibited L-PK
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carboxylase (ACC). The product of a further gene in this
group, called Spot-14 (S14), may be involved in lipogenesis.
In the liver, these genes are all activated by external glucose
in the physiological concentration range (4–16 mM) provided
that insulin is present (5) The requirement for insulin is
explained by the need for the prior induction of the glucokinase gene. Glucose, which quickly equilibrates across the
liver cell plasma membrane via the high-Michaelis-Menten
constant (Km) glucose transporter GLUT2 can then be phosphorylated to glucose 6-phosphate (G6P), which may provide the signal for the regulation of these genes. In adipose
tissue, insulin is required to stimulate the insertion of the glucose transporter GLUT4 into the plasma membrane, thereby
promoting the uptake of glucose into the adipocyte. Low-Km
hexokinases (types I–III) then rapidly phosphorylate glucose
to G6P. These ideas are summarized in Fig. 1.
Detection of changes in blood glucose concentration by
these tissues is ultimately dependent, therefore, on the sensing
of glucose by the insulin-producing islet β-cells. In these cells,
gene expression must therefore be controlled directly by the
sugar (although secreted insulin may then exert further control;
see below). The ACC and L-PK genes are also strongly induced
by elevated concentrations of glucose in the islet, but here the
effect is entirely independent of insulin. This is because in
β-cells, glucokinase is constitutively expressed from a distinct
β-cell-specific promoter. The physiological role for increased
L-PK and ACC gene expression in the islet is somewhat puzzling, since the FAS gene is largely inactive in these cells.
Enhanced metabolic flux through glycolysis and the synthesis
of malonyl-CoA (the substrate of FAS) may instead be required
to provide tight control of fatty acid oxidation. A model in
which malonyl-CoA contributes more centrally to β-cell glucose sensing has recently lost some support. Finally, glucose
and insulin also control the expression of certain glycolytic
enzymes, notably glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) and phosphofructokinase-1 (PFK-1) (14).
Liver,
Liver,
Liver,
Liver,
Liver,
Purely GlucoseResponsive Genes
Gene
Location
promoter activation by elevated glucose, providing strong evidence for USF2 involvement in islet β-cells.
Emerging data now suggests that another factor, chick ovalbumin upstream promoter-transcription factor II (COUP-TFII),
may be involved in regulating the L-PK gene. Using a yeast
“one-hybrid” screen to identify proteins capable of binding
the L-PK E box, A. Kahn’s group have demonstrated that
COUP-TFII, a zinc-finger binding protein, is a likely binding
protein at this site (Kahn et al., personal communication).
Although USF was also identified in this screen, another factor recently implicated in the regulation of glucose-sensitive
gene expression, SREBP (see below), was not. Overexpression
of COUP-TFII reversed the trans-activation of the L-PK promoter by USF2 and, in the model proposed by Kahn, may
bind to the most 5’ E box in the L-PK promoter region.
The FAS and ACC genes
The FAS gene carries only one E box element, centered
approximately –60 bp upstream of the transcriptional start
point. This site appears to confer responsiveness to insulin and
glucose (10), whereas the role of a second, canonical E box in
the first intron (centered at +290 bp) is unclear. As well as
binding USF, consensus E boxes are able to bind the transcription factor called SREBP or ADD. There are two SREBP genes
in mammals, SREBP-1 and SREBP-2. SREBP-1 gives rise to two
alternatively spliced forms. SREBP-1c is expressed in liver and
adipose tissue, and SREBP-1a is expressed in hepatoma cell
lines. Transgenic animals overexpressing SREBP-1 suffer from a
dramatic increase in hepatic triacylglycerol deposition. SREBP
binds a consensus response element, 5’-ATCACCCCAC, present in genes responsible for sterol biosynthesis, as well as to
consensus E boxes (CACGTG). Indeed, it is conceivable that
SREBP could interact with the distal E box of the S14 gene.
However, it is uncertain whether SREBP is capable of binding
to the L-PK E box (CACGGG) or proximal S14 E box
(CACAGG), which differ at least at one position from the consensus E box sequence (CACGTG).
SREBP activity is regulated in a complex manner. Low cholesterol levels provoke the release from the endoplasmic reticulum of the active NH2-terminal fragment of SREBP via proteolytic cleavage of a precursor form. The released fragment,
which contains the b/HLH/LZ domain responsible for DNA
binding, then migrates to the nucleus, leading to transcriptional activation of target genes. Using a Tyr→Ala mutant that
fails to bind DNA but is able to dimerize with endogenous
SREBP, P. Ferré’s group report a powerful inhibition of FAS,
ACC, S14, and L-PK gene expression in hepatoma cells. H. C.
Towle’s group replicate these data with a truncated SREBP
entirely lacking the DNA binding domain—but with a twist:
wild-type, unmutated SREBP acts identically to the truncated
protein. This may imply that overexpression is titrating an
SREBP binding protein, which may be acting as a receptor for
the glucose-derived signal.
In summary, there is good evidence for the occupancy of
the FAS E box by SREBP but less for a role of this factor in
interacting with the S14 and L-PK genes. Mice in which
SREBP-1 has been ablated by targeted disruption may provide
clues, although this model is limited by the fact that ablation
of SREBP-1 results in a compensatory increase of SREBP-2 and
no clear phenotype. It is thus possible that a number of transcription factors may bind to the different carbohydrate
response elements but that another ancillary protein represents the true “receptor” of the glucose-generated signal.
The ACC gene is expressed from distinct promoters in a tissue-specific manner. Promoter II is controlled by glucose,
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FIGURE 1. Likely signaling pathways involved in the regulation of glucose-sensitive genes. In the β-cell, glucose enters cell via GLUT2 transporter and is metabolized by
glucokinase (GK) to glucose 6-phosphate (G6P)/xylulose 5-phosphate (X5P), leading to activation of glucose/insulin responsive (GIR) genes. In liver, activation of insulin
receptor initiates expression of purely insulin-responsive (pIR) genes such as GK, which is required for metabolism of glucose. In adipose tissues, insulin stimulates translocation of GLUT4 transporter to plasma membrane, which promotes uptake of glucose into cell. In adipocytes, glucose is metabolized to G6P/X5P by hexokinase (HK).
believed to act not via an E box but instead through a GC-rich
hexanucleotide site that binds another ubiquitously-expressed
transcription factor, Sp1.
How are the transcription factors regulated—is there a
soluble “mediator”?
“The insulin gene, expressed only in islet β-cells,
is regulated transcriptionally by glucose….”
complements the metabolic function of the yeast HK but not
the ability of HK substrates to regulate gene expression. Thus
HK itself, perhaps through an increase in the level of a phosphorylated intermediate, may initiate a signaling cascade. This
model is analogous to the role of the phospho-enol pyruvatecarbohydrate phosphotransferase system in bacteria. Here
glucose controls the levels of the phosphorylated form of one
component of this system, called IIAGlc, by accepting the
phosphoryl group from this protein. Since phospho-IIAGlc
maintains adenylate cyclase activity, decreased levels of phospho-IIAGlc in response to glucose diminish intracellular cAMP
concentrations and inhibit cAMP-activated genes. A perplexing feature of any equivalent mammalian system is that glucokinase activity predominates in the liver and in islet β-cells,
whereas low-Km HK isoforms are operative in adipose tissue.
Kinase cascades and the role of AMP kinase
Inhibition of protein phosphatases 1 and 2A with okadaic
acid completely blocks the ability of glucose to induce the
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Regulation of the insulin gene by glucose
The insulin gene, expressed only in islet β-cells, is regulated
transcriptionally by glucose (1). Important cis-acting elements,
located within 350 bp of the transcriptional start site, have been
defined by deletion analysis. Particularly important are four
sites, A1, 2, 3, and 5, that bind the homeodomain transcription
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The simplest mechanism would initially involve a small,
diffusible molecule derived from glucose metabolism. Subsequently, regulation of glucose-sensitive genes may then
involve changes in the phosphorylation state of key proteins.
G6P or the pentose phosphate pathway intermediate xylulose
5-phosphate (X5P) may be the key triggering intermediate
(3, 15). Xylitol, the precursor of X5P, activates L-PK gene in
hepatoma cells and suppresses PEPCK expression in vivo. X5P
activates protein phosphatase 2A, potentially providing a link
with a requirement for a protein dephosphorylation event.
Evidence for a role of G6P stems from the observation that
2-deoxyglucose (DOG), which is metabolized to DOG6P but
no further, induces FAS expression in adipocytes. By contrast,
3-orthomethyl-glucose, which enters liver and fat cells but is
not metabolized, is ineffective. P. Ferré’s group have reported
a close correlation between G6P concentration and FAS
mRNA in cultured hepatocytes and adipocytes but none with
X5P. Unambiguous answers to these questions may well
require microinjection of nonmetabolizable forms of these
putative coupling molecules (e.g., DOG6P), and single cell
analysis of gene expression (6, 12).
However, analogy with plants and yeast suggests an alternative mechanism. In plants, hexokinase (HK) acts not just to
catalyze metabolic flux but also, by a mechanism that is not
yet defined, to generate a signal. In genetic studies, plant HK
FAS gene in liver cells (2, 8). The search is therefore on for the
protein kinases and/or phosphatases involved. One possible
candidate is AMP-activated protein kinase (AMPK), a metabolic stress-activated enzyme and serine/threonine-specific
kinase, which is stimulated by AMP. AMPK is also activated
through phosphorylation by an upstream kinase (AMPKK),
which is itself activated by AMP. Since intracellular AMP concentrations increase steeply as ATP levels fall, AMP provides
a sensitive detector of metabolic status. Incubation of liver
cells with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranose (AICAR) prompts the inactivation
of L-PK, S14, and FAS genes, suggesting that a decrease in
AMPK activity could be involved in the effects of glucose.
Active AMPK is a heterotrimer of α-, β-, and γ-subunits, with
catalytic activity possibly residing in the α-subunit (4). Molecular cloning of the AMPK subunits has revealed striking similarity to the products of the Saccharomyces cerevisiae Snf (for
sucrose nonfermenting) gene family—AMPK subunits α1 and
α2 of Rattus norvegicus correspond to Snf1p of S. cerevisiae; the
β-subunits (β1 and β2) are similar to Sip1p, 2p, and Gal83p;
subunits γ1, γ2, and γ3 show similarities to Snf4p. The activity
of the yeast Snf1 kinase is increased by at least an order of magnitude during the switch from glucose-containing to sucrosecontaining medium. During this transition, there is a transient
but dramatic increase in intracellular AMP concentration,
which recovers after the induction of genes that allow growth
on sucrose (in particular the invertase gene). Snf1 phosphorylates the Tip1p subunit of the nuclear repressor of invertase (and
other genes), termed mig1 (for mannose-insensitive gene).
Phosphorylation of this subunit causes its export from the
nucleus, prompting transcription of glucose-repressed genes.
Although regulation of AMPK under metabolic stress is
well documented in animal cells, changes in AMPK activity
in response to hormones and glucose have been less easy to
detect. Early studies in which the activity of the AMPK substrate 3-hydroxy-3-methylglutaryl-CoA reductase was followed in the liver demonstrated diurnal changes, implying
AMPK activity fluctuations. However, direct enzymatic assay
failed to reveal differences in liver AMPK activity, which also
failed to materialize after stimulation of liver cells with
insulin or glucose (D. G. Hardie, personal communication).
However, Salt et al. (13) have recently reported that, in β-cellderived INS-1 and HIT-T15 cells, AMPK activation and AMP
levels are both suppressed since the glucose concentration
rises over the range that activates insulin secretion. These
assays were performed by immunoprecipitation and using a
specific peptide based around a Ser-Ala-Met-Ser (SAMS)
motif of ACC. Given the difficulty of assaying AMPK activity,
it seems possible that closer inspection may reveal that
AMPK is indeed under the control of glucose and insulin.
factor pancreatic/duodenum/Xenopus 1 (PDX-1, also called
IPF-1, IUF-1, STF-1, or IDX-1), whereas sites binding the factors RIPE3B, USF, and E47 may also be important. Systematic
mutagenesis suggests that the A3 site is important for induction
by glucose. Increased occupancy of the A3 site is observed
with elevated glucose. Both A1 and A3 sites include a
TAATG/C motif, the canonical recognition sites for homeodomain proteins. This observation led to the eventual cloning
of PDX-1, a factor expressed strongly only in islet β-cells, some
gut cells, and (much more weakly) in pancreatic exocrine cells
(J. Egan, personal communication). Human mutations in this
factor cause a hereditary form of maturity-onset diabetes of the
young (MODY4), and transgenic mice heterozygous for PDX1 expression (pdx-1 +/–) develop diabetes. Homozygous disruption or mutation of the gene causes pancreatic agenesis
both in mice and humans. At the molecular level, overexpressed myc-epitope-tagged PDX-1 migrates from the cytosol
to the nucleus of glucose-stimulated β-cells (11). This may be
associated with an apparent change in molecular mass from
31 to 46 kDa (the calculated relative mass of the full-length
283-amino acid protein is 34 kDa). However, convincing
immunocytochemical evidence for the activated translocation
of the endogenous protein, at normal levels of expression, is
presently unavailable. Intriguingly, chimaeras between PDX-1
and GFP are located exclusively within the nucleus and are
unresponsive to glucose (11). This suggests that addition of the
bulky (27-kDa) fluorescent protein at either end of PDX-1 may
disrupt an interaction with a binding protein involved in the
cytosol-to-nucleus translocation.
How may glucose transmit a signal to activate PDX-1?
Data from K. Docherty’s group suggest that the stress-activated members of the mitogen activated protein (MAP) family of protein kinases may be critical. One of these is termed
variously p38, reactivating kinase (RK) or stress-activated protein kinase-2 (SAPK2) and is implicated from the use of the
inhibitor SB-203580, which blocks the effect of glucose on
PDX-1 DNA binding. Although direct data reporting the activation of SAPK have been lacking, a kinase believed to lie
immediately downstream, MAPKAPK2, is activated, consistent with SAPK stimulation. Data from K. Docherty’s group,
and that of G. A. Rutter, also indicate that glucose may exert
these effects via phosphatidylinositol 3-kinase (PI3K). With
the use of in-gel kinase assays, we have now also found in rat
islets a protein kinase with apparent molecular mass of 63
kDa that is capable of phosphorylating the SAPK substrate
ATF-2 (a transcription factor) is activated by elevated glucose
concentrations (16 vs. 3 mM). Intriguingly, all of the above
data suggest that elevated glucose concentrations may act to
increase one type of cellular stress (osmotic→SAPKs) and
decrease another (metabolic→AMPK).
Could insulin gene activation by glucose be mediated by
released insulin? Since insulin secretion requires increases in
intracellular Ca2+ concentration, whereas activated insulin
gene expression may not, this argues against a role for the
released hormone. However, others have suggested that
increases in intracellular Ca2+ concentration are important for
insulin gene expression. As suggested by recent data from I.
Leibiger et al. in Stockholm (9), Docherty in Aberdeen, and
ourselves (G. A. Rutter and H. J. Kennedy, unpublished data),
exogenous insulin may activate insulin gene expression
directly. Even more dramatically, β-cell-specific ablation of the
insulin receptor (7) produces mice that are glucose intolerant
and show a drastically reduced first phase of glucose-induced
insulin release—a phenotype strikingly similar to NIDDM.
These observations also provide an explanation for the impact
on β-cell function of mutations in the insulin receptor substrate
IRS-2. These ideas are summarized in Fig. 2.
Conclusions and perspectives
Comparison of the mechanisms whereby glucose regulates
genes in simple organisms is providing fascinating new insights
into how the sugar acts in mammalian cells. With the availability of the complete human genome, it should soon be possible
to see just how well conserved these signaling pathways really
are. This in turn may lead to novel treatments for NIDDM.
We thank Dr. I. Leclerc for critical reading of the manuscript.
Work in our laboratories is supported by the Medical Research Council
(UK), the Wellcome Trust, The British Diabetic Association (BDA), The Biotechnology and Biological Sciences Research Council, The European Community,
and the Royal Society of London.
J. M. Tavaré is a BDA Senior Research Fellow.
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FIGURE 2. Potential signaling pathways involved in transcriptional regulation of insulin gene (j) in β-cell. WM, wortmannin. In β-cell, glucose is metabolized to G6P
(which may act as a signaling metabolite), prompting an increase in intracellular ATP concentration, which then inhibits an ATP-sensitive K+ channel in plasma membrane. Subsequent lowering of membrane potential promotes uptake of Ca2+ into cell via a voltage-sensitive Ca2+ channel, and this in turn stimulates secretion of insulin
from intracellular stores. Binding of insulin to its receptor in plasma membrane may then initiate a signaling cascade ultimately activating expression of insulin gene.
References
Stimulatory and Inhibitory Functions of the R Domain
on CFTR Chloride Channel
Jianjie Ma
CFTR is a chloride channel whose gating process involves coordinated interactions among the
regulatory (R) domain and the nucleotide-binding folds (NBFs). Protein kinase A phosphorylation of serine residues renders the R domain from inhibitory to stimulatory and enables
ATP binding and hydrolysis at the NBFs, which in turn control opening and closing of the
chloride channel.
I
n 1989, the gene responsible for cystic fibrosis was isolated,
and the protein product of this gene was named cystic fibrosis transmembrane conductance regulator (CFTR; Ref. 12).
The amino acid sequence of CFTR was used to predict a
structure, which consists of two sets of six membrane-spanning domains (MSDs), two nucleotide-binding folds (NBFs),
and an intracellular regulatory (R) domain (Fig. 1A). This
structure is similar to that of the ATP-binding cassette (ABC)
family of transporters, but the R domain is unique to CFTR.
Research during the last decade has identified CFTR as a
multifunctional protein, which provides the pore of a linear
conductance chloride channel and also functions to regulate
other membrane proteins. Mutations in CFTR leading to
defective regulation or transport of chloride ions across the
apical surface of epithelial cells are the primary cause of the
genetic disease cystic fibrosis (15).
As a chloride channel, CFTR is regulated by two cytosolic
pathways. For the channel to open, the protein must first be
phosphorylated by a cAMP-dependent protein kinase (PKA),
and then intracellular ATP must bind to the NBFs and subseJ. Ma is an Associate Professor in the Department of Physiology and Biophysics at Case Western Reserve University School of Medicine, 10900
Euclid Avenue, Cleveland, OH 44106.
154
News Physiol. Sci. • Volume 15 • June 2000
quently be hydrolyzed. The use of compounds that alter the
ATP hydrolysis cycle of CFTR, such as AMP-PNP, pyrophosphate, and vandate, provides evidence that hydrolysis of ATP
is required for both channel opening and closing transitions.
Structure-function studies have suggested that ATP hydrolysis
at NBF1 is responsible for opening of the chloride channel
and that ATP hydrolysis at NBF2 terminates a burst of open
events (5). Since cells normally contain high intracellular
levels of ATP, regulation in the intact cell of CFTR is by
phosphorylation. The R domain contains the consensus
phosphorylation sites for PKA that are the basis for physiological regulation of channel opening (12).
Patch-clamp technique vs. lipid bilayer reconstitution of
CFTR channel
The CFTR chloride channels are located in the apical
membrane of epithelial cells. To study the function and regulation of the CFTR channel, two electrophysiological methods are commonly used: the patch-clamp technique and
lipid bilayer reconstitution. These studies are carried out
with either the primary or immortal cultures of epithelial
cells expressing the endogenous CFTR proteins or the heterologous cell lines that either stably or transiently express
0886-1714/99 5.00 © 2000 Int. Union Physiol. Sci./Am.Physiol. Soc.
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