Clark, A. R. Nutrient regulation of insulin gene expres- sion. FASEBJ. 8: 20-27;. 1994. Key Words: transcription islets of Langerhans. ISLETS. OF. LANGERHANS.
.-sI_
VlIVV
Nutrient KEVIN
regulation DOCIIERTY5
Department
of Medicine,
of insulin AND
ANDREW
University
of Birmingham,
The /3 cell of the islets of Langerhans contributes along with other factors to glucose homeostasis by sensing changes in the plasma glucose concentrations and adjusting the rate of insulin production and release. Over short periods of time, insulin production is controlled principally through translation of pre-existing mRNA. Over longer periods, insulin mRNA levels are modulated through effects on the rate of transcription of the insulin gene, and also through changes in the rate of decay of insulin mRNA. These long-term effects may be important in allowing the i3 cell to adapt to changes in diet or periods of fasting. Several mechanisms involved in the control of the rate of translation of insulin mRNA have been described. Effects of glucose metabolism on the turnover of insulin mRNA have yet to be characterized in detail. At the level of transcription, cis-acting DNA elements and trans-acting factors involved in the transient response of the insulin gene to changes in intracellular CAMP levels, or to signals generated as a result of glucose metabolism, have been identifIed.Docherty, K., Clark, A. R. Nutrient regulation of insulin gene expression. FASEBJ. 8: 20-27; 1994.
ISLETS
OF
transcription
LANGERHANS
islets of Langerhans
CONTAIN
PHENOTYPICALLY
distinct
cells, which somatostatin
produce insulin (/3 cells),2 glucagon (a cells), (D cells), and pancreatic polypeptide cells (PP cells). The release of insulin from the pancreatic /3 cells plays an important role, along with other factors, in maintaining normoglycemia. It has been known for many years that insulin secretion and biosynthesis are sensitive to circulating glucose levels. It has also become clear that the level of insulin mRNA in pancreatic /3 cells is modulated by glucose, and that this results from effects both on transcription of the insulin gene and on the rate of turnover of insulin mRNA. More recently, the DNA sequences and transcription factors involved in the regulation of insulin gene transcription have been characterized, This review will focus on the transient response of the insulin gene to nutrient and hormonal stimuli. However, these events are best described within the context of the large body of published work on the control of insulin secretion and biosynthesis. One major goal is to ascertain to what extent the intracellular signaling events controlling insulin secretion, biosynthesis, and gene expression are interrelated.
GLUCOSE
SENSING
IN THE
Queen
Elizabeth
Hospital,
Birmingham,
B15 2TH,
U.K.
secretion (Fig. 1) (1). Glucose uptake into the /3 cell is facilitated by a high Km glucose transporter (GLUT-2), which corresponds to the transporter isoform in hepatocytes (2). Transport of glucose is not rate-limiting for glucose utilization because the cellular glucose concentration rapidly equilibrates with changes in blood glucose concentration. Once inside the /3 cell, glucose is phosphorylated by glucokinase to generate glucose-6-phosphate. Glucokinase exhibits a high Km for glucose (12 mM), which allows it to function as a glucose sensor, adjusting the rate of metabolic flux through glycolysis to the extracellular glucose concentration (3, 4). Metabolism of glucose within the /3 cell is thought to generate a discrete signal (or signals), which interacts with distal signals that are common to many different cell types (5). The nature of the proximal metabolic signal (or signals) is unknown. Processing of glucose-6-phosphate through glycolysis and the Krebs cycle is required for the full insulin secretory response to be observed. However, a signal for secretion may arise during glycolysis because glyceraldehyde, which enters the glycolytic pathway as a triose phosphate, is a potent secretagogue whereas pyruvate, which is metabolized in the mitochodrion, does not stimulate insulin release, although it can enhance the secretory response to glucose. On the other hand, the stimulatory effect of leucine is mediated through mitochondrial metabolism of its deamination product, 2-ketisocaproate. A detailed account of nutrient metabolism in the /3 cell is beyond the scope of this article, and readers are directed to a recent review on this topic (6). glucose-mediated changes in metabolites within /3 cell that can be linked to increased insulin secretion have been recorded. Thus, glucose metabolism results in an increase in the ratio of ATP/ADP, changes in the redox state (conversion of NAD(P) to NAD(P)H), changes in intracellular pH, an increase in malonyl CoA, and increased phospholipid metabolism. It has been proposed that a localized increase in the ATP/ADP ratio leads to closure of ATPdependent K channels (KATP), membrane depolarization, and the opening of voltage-dependent Ca2 channels (Fig. I (7)). The influx of extracellular Ca2 sets in motion the events leading to the exocytotic release of the contents of the insulin granule (Fig. 1 (8 + )). The increased intracellular free Ca2 concentration may also act on mitochondrial dehydrogenases to sustain the elevated ATP/ADP ratio. Glucose-induced insulin secretion is also subject to stimulatory and inhibitory modulation by hormones and Certain
the
/3 CELL
The most important physiological regulator of insulin secretion is glucose. Other nutrient secretagogues include mannose, leucine, and ketone bodies. It is now well established that glucose metabolism is important for its effects on insulin
20
expression
R. CLARK
ABSTRACT
Key Words:
gene
tTo whom of Medicine, U.K.
correspondence should be addressed, at: Department Queen Elizabeth Hospital, Birmingham, B15 2TH,
0892-6638/94/0008-0020/$01
.50. © FASEB
REVIEWS Hormones
Glucose
clase, causing an increase in the cellular CAMP concentration. Glucose itself evokes a modest increase in /3 cell cAMP levels (9). However, the role of CAMP in insulin secretion is not a primary one. Protein kinase A activation potentiates but does not initiate insulin secretion (10). Hormonal mediators such as glucagon and GLP-l may induce glucose competence in /3 cells. Thus glucagon may maintain the /3 cell glucose signaling system in a functionally competent state during periods of fasting, while GLP-1 may confer glucose competence during the fed state when GLP-1 concentrations rise with the postprandial increase in the concentration of blood glucose (11). How CAMP might prime the /3 cell is not understood, although possible mechanisms include phosphorylation of voltage-dependent Ca2 channels or some as yet unspecified component of the secretory pathway (Fig. 1
(6+)). NUTRIENT BIOSYNTHESIS
secretion Figure 1. Scheme depicting mechanisms whereby nutrients (glucose and leucine) and hormones stimulate insulin production and secretion in /3 cells. Metabolism of glucose and leucine by way of glycolysis and the Krebs cycle generates a signal (or signals) of unknown properties. This signal has multiple effects on insulin
production.
It increases
the rate of transcription
of the insulin
gene
(1+), the half-life of insulin mRNA by decreasing the rate of breakdown (2-) and the efficiency of translation of insulin mRNA (3+). Hormones (see text) affect insulin production through cAMPmediated activation of protein kinase A (PKA). PKA increases the rate of transcription of the insulin gene (4+) and decreases the rate
of breakdown secretion phorylation
of insulin
mRNA
(6+) by unknown of membrane
(5-).
mechanisms ion channels
PKA
also potentiates
that might include or some component
insulin phosof the
secretory pathway. A consequence of aerobic metabolism is an elevation in intracellular ATP levels. ATP inhibits the ATP-sensitive K channel (7-) causing membrane depolarization, which in turn opens the voltage-sensitive Ca channel allowing extracellular Ca2 to enter the cell. Ca2 has many effects on (3 cell activity, including stimulation of insulin secretion (8+), activation of adenylate cyclase (not shown), and activation of mitochondrial dehydrogenases (not shown). Abbreviations: PKA, protein kinase A; ER, endoplasmic reticulum; NTPs, nucleotide triphosphates.
neurotransmitters, including glucagon, glucagon-like peptide-1(7-37) (GLP-1),2 gastric inhibitory polypeptide (GIP), vasoactive intestinal polypeptide (VIP), cholecystokinm (CC K), arginine vasopressin (AVP), acetylcholine, epinephrine, somatostatin, gastrin-releasing polypeptide (GRP), and galanin (8). Some of these such as GIP, glucagon, and GLP-1 act through the activation of adenylate cy-
NUTRIENT
REGULATION
OF INSULIN
GENE EXPRESSION
CONTROL
OF
PROINSULIN
Insulin is synthesized as a larger precursor, preproinsulin, which undergoes post-translational proteolysis. The NH2terminal signal peptide is removed cotranslationally by a signal peptidase located within the endoplasmic reticulum (ER) to generate proinsulin. Conversion of proinsulin to insulin occurs at a later stage in the secretory pathway. These events have been reviewed elsewhere (12). This section will focus on the control of proinsulin biosynthesis. Translational effects on proinsulin biosynthesis occur within approximately 20 mm after addition of glucose and are unaffected by inhibitors of RNA synthesis (13). Welsh and co-workers (14) have performed extensive investigations of the translational control of proinsulin biosynthesis, and concluded that at least three mechanisms are involved. At concentrations of 3.3 mM and more, glucose stimulates the rate of initiation of translation (Fig. 1 (3+)), as demonstrated by an increased transfer of cytoplasmic insulin mRNA to subcellular fractions containing ribosomes and larger polysomes (14). Glucose also increases the transfer of initiated insulin mRNA from free to membrane-bound ribosomes, a process involving signal recognition particle (SRP)-mediated transfer of ribosomes bearing nascent preproinsulin from the cytoplasm to the ER (Fig. 1 (3 +)). This redistribution of insulin mRNA may occur as a result of glucose stimulating the interaction of the SRP initiation complex with the SRP receptor on the ER, possibly through a modification of the SRP complex. In addition, studies of the distribution of translating ribosomes on insulin mRNA have demonstrated an increase in ribosomal pausing, under conditions of low glucose, after approximately 70 amino acids of preproinsulin have been synthesized (15). This is consistent with SRPmediated control of translation or it may reflect the effect of glucose on the interaction of proteins with RNA stem loop structures within the 5’ untranslated region of the insulin mRNA (16). The third mechanism involves increased elongation of the nascent preproinsulin (Fig. 1 (3+)). This was
2Abbreviations: GLP-1, glucagon-like peptide-l(7-37); GIP, gastric inhibitory polypeptide; VIP, vasoactive intestinal polypeptide; CCK, cholecystokinin; AVP, arginine vasopressin; GRP, gastrinreleasing polypeptide; SRP, signal recognition particle; IBMX, isobutylmethylxanthine; IAPP, islet amyloid polypeptide; Gi, inhibitory GT subunit; HLH, helix-loop-helix; IEF-l, insulin enhancer factor 1; CAT, chloramphenicol acetyl transferase; CRE, CAMP response element; CREB, CRE-binding protein; PMA, phorbol-12-myristate 13 acetate.
21
flLV
IVV
demonstrated in isolated islets incubated in the presence of low concentrations of cycloheximide, under which conditions elongation is the rate-limiting step for translation. In this experiment the rate of proinsulin biosynthesis was stimulated by glucose at concentrations of up to 5.6 mM (14). The observation that glucose stimulates the biosynthesis of growth hormone in islets isolated from transgenic mice carrying a metallothionein-growth hormone gene fusion is consistent with the hypothesis that an SRP-mediated mechanism may be involved in regulating the translational efficiency of secreted proteins in islets (17). However, in addition to proinsulin, glucose stimulates the synthesis of at least 100 other islet proteins (18). Of these proteins, not all that are cotranslationally inserted into the lumen of the RER respond in a similar manner to glucose, whereas some cytoplasmic proteins that lack signal sequences exhibit a large stimulation. This suggests that release from SRPmediated arrest may not be the only mechanism involved in the response to glucose. The biosynthesis of proinsulin is coupled to secretion insofar as many insulin secretagogues also stimulate proinsulin biosynthesis. However, the transducing systems differ in some respects (19). First, glucose-stimulated insulin release is inhibited in Ca2 free medium, whereas biosynthesis is still activated. Second, tolbutamide and phosphodiesterase inhibitors such as isobutylmethyixanthine (IBMX) potentiate glucose-stimulated insulin release by increasing islet cAMP levels, but do not affect proinsulin biosynthesis. Third, the threshold for glucose-stimulated insulin secretion (4.2-5.6 mM) is higher than that for proinsulin synthesis (2.5-3.9 mM). In the HIT T15 /3 cell line, glucose control of insulin secretion and biosynthesis are uncoupled in that secretion of insulin is markedly stimulated by glucose over the range 2 to 11 mM, with no effect on proinsulin synthesis (20). However, the inability to stimulate proinsulin biosynthesis in these cells may be due to the high basal level of translation of the relatively small amount of insulin mRNA in the HIT T15 cells; translation is at a maximal level at low concentrations and cannot be further stimulated when the glucose concentration is increased. The dose-response curve of glucose-induced proinsulin biosynthesis is sigmoidal in purified rat /3 cell preparations (21). This could be interpreted as a progressively increasing activity of functionally homogeneous cells or a heterogeneous responsiveness of the /3 cell population. Autoradiographic analysis of glucose-stimulated [3H]leucine incorporation by individual cells supported the latter conclusion, showing that the sigmoidal dose-response curve reflects a dose-dependent recruitment of pancreatic /3 cells to biosynthetic activity. This heterogeneity is also seen with respect to glucokinase (22). Thus, glucokinase immunoreactivity varies considerably within various /3 cells in an isolated rat islet preparation, suggesting that glucose phosphorylation is the enzymatic control point for this functional diversity.
NUTRIENT INSULIN
AND HORMONAL mRNA LEVELS
EFFECTS
ON
Although regulation of insulin production occurs in the short term (over periods of minutes and hours) at the level of translation of pre-existing mRNA, it is also clear that longer-term control of insulin production is mediated through changes in the level of insulin mRNA. In this way the /3 cell can rapidly replenish insulin stores, enabling it to respond to changes in blood glucose levels throughout the day while at the same time having the capacity to adapt to more long-term dietary
22
Vol. 8
January
1994
changes or to periods bf fasting. Thus, fasting of rats for up to 4 days results in decreased islet insulin mRNA levels, which can be rapidly restored to normal upon refeeding or injection of glucose (23, 24). In further long-term in vivo experiments, insulin mRNA levels were shown to increase when the demand for insulin increased in corticosteroidinduced insulin resistance and after pancreatectomy (25, 26). These changes in insulin mRNA levels correlate closely with the capacity of the islets to synthesize insulin, thus stressing the physiological significance of these changes in insulin mRNA levels. Although these modulatory effects on insulin mRNA levels operate over relatively long periods, the /3 cell can respond by increasing insulin mRNA levels over short time periods when an extremely large demand for insulin secretion is placed on the cell. In perfused rat pancreas, high glucose alone has no effect on insulin mRNA levels over short periods (2 h), while a combination of glucose and arginine induce a significant increase in insulin mRNA over the same time period (27). Insulin and islet amyloid polypeptide (IAPP) mRNA levels are coregulated during chronic adaptive changes in glucose-stimulated insulin secretion (28). This may reflect common transcriptional control mechanisms shared by the insulin and IAPP genes (29) or common mechanisms regulating the turnover of insulin and IAPP mRNA (see below). Mechanisms involved in the modulation of insulin mRNA levels have been studied in in vitro culture systems. In one of the first studies, incubation of mouse islets for several days in high glucose concentrations resulted in a dramatic increase in insulin mRNA levels (30). Although no effects on insulin mRNA levels were observed in mouse islets after I h incubation in high glucose (31), glucose-induced increases in insulin mRNA were seen after 4 h in isolated rat (32) or human islets (33) and in the HIT T15/3 cell line (34). These effects could be mimicked to some extent by analogs of cAMP or by phorbol esters that activate the protein kmnase C signaling pathway (34). Other nutrients and hormones modulate the levels of insulin mRNA. L-Leucine and its metabolic product 2-ketoisocaproate both increase insulin mRNA levels in isolated rat islets, emphasizing the role of mitochondrial metabolism in generating signals that control insulin gene transcription or mRNA breakdown (35). Polyamines, the level of which increases in /3 cells in parallel with glucose-mediated increases in insulin mRNA, may be involved in controlling insulin production. Thus attenuation of the increase in polyamines prevents the increase in insulin mRNA (36). Glucose stimulates an increase in insulin mRNA levels through effects on its rate of synthesis (Fig. 1 (1+)) and degradation (Fig. 1 (2-)). Transcriptional effects were observed by measuring the rate of incorporation of [3H]uridine into insulin mRNA in isolated islets (37) or by transcription run-on assays performed on nuclei prepared from 3 cell lines (see below). The rate of degradation of insulin mRNA was studied by measuring the rate of decay of [3Hluridinelabeled insulin mRNA in islets incubated in the presence of the transcriptional inhibitor actinomycin D. Insulin mRNA was found to be relatively stable, turning over slowly with a half-life of about 30 h. Its half-life was almost three-fold longer in islets cultured in 17 mM glucose compared with 3.3 mM glucose (38). Cholera toxin mimicked this effect in the RINm5F 3 cell line, suggesting that CAMP may be involved in these events (Fig. 1 (5-)). Somatostatin, which is one of the most potent inhibitors of insulin secretion, causes a decrease in insulin mRNA levels in the 3 cell line HIT T15 by destabilizing insulin mRNA. The half-life of insulin mRNA, as measured by actinomycin D decay curves, was 24
The FASEB Journal
DOCHERTY
AND
CLARK
REVIEWS h in the absence and 16 h in the presence of somatostatin (39). The glucocorticoid, dexamethasone, by destabilizing insulin mRNA, also negatively regulates insulin gene expression in HIT T15 /3 cells and islet preparations that have been dispersed into cells (40, 41). However, this effect is lost in reaggregated or intact islets (41), which led to the conclusion that insulin gene regulation is influenced by cell-to-cell contact within islets. Moreover, the inhibitory effects of dexamethasone on insulin mRNA levels are prevented by cAMP analogs, suggesting that CAMP may have a dominant stimulatory effect over glucocorticoid-mediated negative effects and that this is lost in single cell preparations that normally exhibit reduced cAMP levels. The mechanisms whereby cAMP or other nutrient-induced signals might stabilize insulin mRNA are not known. One possibility is that insulin mRNA contains discrete sequences within 5’ or 3’ untranslated regions that interact with RNA-binding proteins (16). Phosphorylation of such trans-acting proteins could influence
their
binding
activity,
and
hence
the
stability
of the
trolling transcription lin gene transcription acting
elements
of the insulin is dependent (i.e.,
sequences
of DNA as the structural tors. These sequences enhancers, although Promoters site, are
are
gene. Regulation on the interaction located
located
proximal
to
tion
start
site.
This
the initiation
sequence
site. The
binds
insulin
effect
on islet
insulin
the
stretch
transcription
factor
discrete sequence elements that interact tory proteins to modulate the activity positive or negative manner (47). Most of the published work on insulin
within
negative
the
start
lin
gene.
Because
thoroughly
this rat
the
rat
characterized,
gene. insulin
tween
-333
gene
rat)
the
gene
I
has
sections
of regulatory in Fig. 2. fused
to
the
that
three
distinct
are insu-
been will
most on in the
focus
sequences
(48) showed
has
human
CAT reporter sequences be-
+51 of the rat insulin I gene were in transfected /3 cell lines. This region in detail by a sensitive block mutagenesis
(49). It was found that deleterious effects upon
of
(there
and
for expression ther mapped
sets
expression
II genes
and
following
Using insulin gene fragments gene, Walker and co-workers
and
is composed
with nuclear regulaof the promoter in a
I and
insulin the
The arrangement I gene is shown
TFIID
gene enhancer
levels,
a dominant
same
typically 100 base pairs in length, and represent the site of assembly of the initiation complex, which comprises RNA polymerase II and some general transcription factors. Like most other promoters, the insulin promoter possesses a conserved sequence motif, TATAA (the TATA box), around 20 to 30 nucleotides upstream (-20 to -30) of the transcrip-
come from studies of the rat insulin two nonallelic insulin genes in the
has
the
gene) with trans-acting protein fachave been classified as promoters or this distinction is often blurred.
mRNA. GLP-1, a potent stimulator of insulin secretion, increases the rate of transcription of the insulin gene by mechanisms involving cAMP (Fig. 1 (4+)). This stimulating effect of GLP-! is inhibited by the neuropeptide hormone galanin, which is released by sympathetic stimulation of nerve terminals in the endocrine pancreas. Galanin is thought to inhibit insulin secretion via a receptor-linked pertussis toxinsensitive (Gi) signaling pathway. Thus, the inhibitory effect of galanin on insulin gene expression may be mediated by inhibition of GLP-1 activation of adenylate cyclase (42). The insulin gene also appears to be responsive to serum insulin levels. In the presence of hyperinsulinemia and high glucose insulin
on
of insuof cis-
sufficient was furstudy
several mutations had moderate transcription, whereas mutations
regions
decreased
the
rate
of transcrip-
mRNA levels in the rat (43). It is not known whether this effect is on the half-life of insulin mRNA or on transcription of the insulin gene. There exists well-documented effects of the proliferative state of cells in culture on insulin gene expression. This may reflect the constant tussle between signals that induce the cell to proliferate or retain a differentiated state. Sodium butyrate treatment increases the rate of transcription of insulin and glucagon genes in Rin 1056A cells (44). However, sodium butyrate was subsequently shown to slow cell growth and increase the number of cells that stained immunoposi-
The first of these was the TATA box (-20 to -30); the other two coincided with identical 8 base pair sequence motifs GCCATCTG at -105 and -231. These
tive
helix (HLH) forming two
for
glucagon
and
insulin,
suggesting
that
its
primary
effect is to induce a more differentiated state. Similarly, RINm5F cells exhibit a slower growth rate and higher insulin mRNA levels when grown on a defined protein matrix (45).
Thus
related;
hormone
production
and
cell growth
are
inversely
rapid
cell growth may prevent normal expression of the insulin gene. Insulin gene expression in cultured cells may also be influenced by the phase of the cell cycle (46). RIN 5AH.8 cells exhibit a dramatic decrease in insulin mRNA levels as they approach proliferation arrest, whereas cells undergoing cell cycle arrest after treatment with sodium butyrate maintain normal levels of insulin mRNA. This emphasizes the difficulties in interpreting modulatory effects on insulin mRNA levels in cultured cell lines.
CIS-ACTING ELEMENTS FACTORS CONTROLLING TRANSCRIPTION
AND TRANS-ACTING INSULIN GENE
REGULATION
OF INSULIN
motifs
fivefold
or
have
more.
been
termed
GENE EXPRESSION
the
IEBI
(or
NIR)
box
and
the
IEB2
(or FAR) box, respectively (Fig. 2). A double mutation of the IEB1 and IEB2 motifs effectively abolishes transcription. The known
IEB sequence as E boxes,
belongs which
to a class of regulatory sites, have the consensus sequence
CANNTG and are implicated in the tissue-specific tion of genes in muscle, pancreas, and lymphoid boxes
ture,
bind
and
a family
of protein
proteins. amphipathic
there
factors
known
regulatissue. E
as helix-loop-
They possess a domain capable of helices separated by a ioop struc-
is usually
an
adjacent
basic
domain.
The
helix-loop-helix HLH proteins,
domain is involved in dimerization between whereas the basic domain mediates DNA binding. The IEB sequences bind a factor, IEF-1 (insulin enhancer factor 1), which in addition to /3 cells is present in a cells and pituitary cells, but not in nonendocrine cells. By screening expression clones for IEB-binding
libraries factors
with IEB sequences, have been obtained
cDNA from
mouse, hamster, single gene, E2A, ternative splicing. Park and Walker was a heterodimeric
and rat. All the clones are derived from a which generates at least two proteins by alUsing an in vitro subunit exchange assay, (50) obtained data indicating that IEF-1 HLH complex composed of an E2A gene product and IESF-1 (insulin enhancer specific factor 1), an HLH protein of Mr 25 kDa (50). As the E2A proteins are expressed in a wide variety of cells, the restricted expression of the heterodimer pression of IESF-1.
Before describing the nutrient control of insulin gene transcription, the following section will briefly summarize our current understanding of the mechanisms involved in con-
NUTRIENT
tion
IEF-1
may
be due
to restricted
tissue
ex-
In this case IESF-1 represents an important /3 cell transcription factor, and its isolation and characterization represent an important goal. However, sequences in addition
to the
IEB
sites
are
also
important
in controlling
23
REVIEWS IEF-2
Cl IUFI? lsl I Imx-I cdx-3 HNFI a IEF-I
CREB
IEF-l
-l77
-l05
-198
-23l
-350
IPF-l IUFI ?
ttE1t
-l
TGACGTCC (CRE) TFAATAATCTAATTA GCCATCTG CANNTG TAAT Box E Box (FLAT0rE2) (IEBI orNIR)
GCCATCTG
CANNTG E Box (IEB2 or FAR)
-76
CTFAATG TAAT Box (P1)
can be seen at the protein level. The E2A protein, E47, which comprises part of the IEF1 heterodimer binding at the FAR box, does not activate the FF mini-enhancer on its own. However, when transfected into fibroblasts expressing lmx-I, E47 causes a dramatic synergistic activation of the minienhancer. These experiments emphasize the potential importance of lmx-1; it has restricted tissue expression and will interact with E47 to activate transcription of an FF minienhancer construct (55). Other sequences that may play a role in regulating transcription of the rat insulin I gene include; the GAGA box (-57 to -40), which binds a widely distributed zinc finger protein (Pur-1), and an enhancer core sequence at (-285 to -332), which is also known as the El footprint. There also exists a cAMP response element (CRE) at -184 to -177 in the rat insulin I gene and at equivalent positions in the rat insulin II and human insulin genes. The role of this element in the nutrient and hormonal control of insulin gene transcription will be described in the following section.
FF mini-enhancer Figure 2. Arrangement of regulatory elements and their binding proteins in the rat insulin I gene. The scale (-1 to -350) denotes nucleotides upstream of the transcription start site. Sequence elements are described below the scheme and the regulatory proteins
above. The factor IUF1 binds to a related
sequence
in the human
insulin gene enhancer. This factor has not been shown to bind at the indicated sites in the rat insulin I gene, but it is related to IPF-l and factor Cl, and thus is likely to bind at the indicated TAAT box
elements.
Further
details
are provided
in the text.
transcription of the gene, as combinations of IEB1 and IEB2 fail to confer tissue-specific gene expression in transgenic mice
(51).
An important additional regulatory sequence contains a TAAT motif (29). In the rat insulin I gene a TAAT box is located at -77. This promoter-proximal TAAT box binds a factor, IPF-l (insulin promoter factor 1), which is present in /3 cells but not in a cells (52). In addition, a highly AT-rich tract around position -198 (the FAR linked AT rich or FLAT element) contains four copies of the sequence TAATthree on the upper strand and one on the lower strand (Fig. 2) (53). This region can be subdivided into two domains that contain the sequence TAATTA (FLAT-E) and TAATAAT (FLAT-F). There is some form of negative interaction between these domains, such that mutation of the FLAT-E motif leads to a substantial increase in the rate of transcription. The FLAT region binds different proteins (Fig. 2), several of which have been characterized after isolation of their corresponding cDNAs and shown to contain homeodomains. These proteins include: 1) Isl-l (54), a factor related to homeodomain proteins of the nematode Caenorhabditis elegans, which contains, in addition to its homeodomain, a Cys/His-rich region (LIM domain); 2) cdx-3 (55) (caudallike box 3); 3) lmx-l (LIM box 1) (55), which also contains an LIM domain; and 4) the liver-enriched protein HNF1a (hepatocyte nuclear factor la) (56). Other factors that have yet to be fully characterized include IEF-2 (57) and IUF-l (insulin upstream factor 1), which are present in /3 cells but not in a cells, and factor Cl, which may be important in the transient response of the insulin gene to glucose stimulation (see below). The E (FAR) box and the TAAT (FLAT) box function synergistically. Neither element can function alone but both are required for enhancer activity, constituting what has been termed the FF (FAR/FLAT) mini-enhancer. This synergism
24
Vol. 8
January
1994
NUTRIENT INSULIN
AND HORMONAL GENE TRANSCRIPTION
CONTROL
OF
Direct effects of glucose on insulin gene transcription have been observed by using transcription run-on assays (58), or transfection of plasmid constructs containing insulin gene fragments joined to a reporter gene encoding chloramphenicol acetyl transferase (CAT) or firefly luciferase (59-61). Glucose metabolism is necessary for its stimulatory effect on insulin gene transcription. Thus glucose stimulation of CAT activity driven by a rat insulin I gene fragment (-345 to +1) was inhibited by mannoheptulose in transfected HIT cells, whereas leucine and its metabolite 4-methyl-2-oxopentanoate (which is further metabolized in mitochondria) stimulated CAT activity (59). Cotransfection of islet /3 cells with insulin gene reporter constructs, along with cDNAs encoding enzymes involved in glucose metabolism, indicated that, as for insulin secretion, anaerobic glycolysis could generate a signal (or signals) mediating transcriptional control of the insulin gene (62). The nature of this proximal signal (or signals) is unknown. Other studies have looked specifically at the role of cAMP-, Ca2-, and phorbol ester-stimulated signaling pathways as a clue to defining the link between glucose metabolism and the increase in insulin gene transcription. As mentioned previously, analogs of cAMP have been shown to stimulate insulin mRNA levels in RINm5F (33, 34) and HIT /3 cell lines (34), and also in rat and human islet preparations (33). Direct effects of CAMP on insulin gene transcription in HIT cells have been observed using transcription run-on assays (63) and transfected insulin gene/reporter constructs (59, 60, 63). In the mouse /3FC cell line, however, no effect of cAMP on insulin gene transcription was observed (58); the reason for this discrepancy is not obvious, although it may reflect the difficulties in obtaining reproducible results with transcription run-on assays. Using transfected reporter gene constructs, the cAMP effect in HIT cells was mapped to an 8 base pair sequence of the rat insulin 1 gene (-177 to -184) that exhibited a close match (TGACGTCC) to the well-characterized cAMP response element (C RE). Deletion or mutagenesis of this sequence abolished the CAMP stimulatory effect (63). A role for this sequence element might be incorporated into a model whereby increases in /3 cell cAMP levels would result in dissociation of the catalytic subunit of protein kinase A from its regulatory subunit. The free active catalytic subunit would then translocate to the nucleus where it is thought to phos-
The FASEB Journal
DOCHERTY
AND
CLARK
REVIEWS phorylate a 43-kDa CRE-binding protein (CREB), and increase its ability to activate transcription (64). The demonstration that a 43-kDa protein present in HIT cell nuclear extracts could bind to the insulin gene CRE suggests that this pathway of CREB activation may be involved in the control of insulin gene transcription (Fig. 3) (63). The role of Ca2 in the control of insulin gene transcription is controversial. In HIT T15 /3 cells transfected with a rat insulin I gene/CAT construct (-345 to + 1), coincubation with the Ca2 channel blocker verapamil did not significantly decrease the effect of glucose on CAT activity, whereas raising extracellular Ca2 decreased CAT activity in response to glucose (59). In transfected fetal rat islets, however, verapamil reduced the response of a similar rat insulin I gene fragment (-410 to +1) (60), while the calcium channel blocker D600 inhibited glucose induced insulin gene transcription as measured by transcription run-on assays in /3TC cells (58). The phorbol ester, phorbol-l2-myristate 13 acetate (PMA), has been shown to stimulate insulin mRNA levels in HIT T15 /3 cells (34) (but not in human islets (33)), and to stimulate CAT activity in HIT T15 cells transfected with a rat insulin I gene/CAT construct (59). Phorbol esters affect gene transcription through the binding of a complex, AP1, to a phorbol ester-responsive element. AP1 consists of a collection of structurally related transcription factors that be-
Hormone stimulation
Adenylate cyclase
cAMP
Glucose metabolism Protein kinase A P
Signal
-350
-198
-231
FLAT
IEBI/FAR
(E box)
-177
CRE
(TAAT box)
-76
-105
IEBI/NIR (C
-I
Pt
box) (TAAT box)
Figure 3. Effect of glucose metabolism and hormonal stimulation on transcription of the rat insulin I gene. A speculative model depicting how nutrients and hormones might control transcription of the insulin gene. Glucose metabolism generates a signal (or signals) that stimulates the binding of factor Cl to its binding Site within the FLAT element. Hormone stimulation activates adenylate cyclase, resulting in an increase in cAMP levels and activation of protein kinase A. Protein kinase A phosphorylates the CAMP response element (CRE) binding protein (CREB) and stimulates CREB binding to its cognate site within the enhancer region. The
scheme studies,
attempts to pull together the results of currently available and suggests that there are two pathways controlling the
transcriptional activity of the gene. This, however, is likely to represent an over-simplification, as cAMP has numerous additional effects on transcription. For example, it has been shown to inhibit the activity of myogenic HLH proteins (70), raising the possibility that it might also affect the interaction of the HLH heterodimer, IEF-1, at the IEB2/FAR or IEB1/NIR sites.
NUTRIENT
REGULATION
OF INSULIN
GENE EXPRESSION
long to the Jun and Fos family. The Jun proteins bind DNA as either homodimers orJun-Fos heterodimers, whereas Fos proteins must heterodimerize with Jun proteins. Phosphorylation of c-Jun at COOH-terminal sites adjacent to the DNA binding domain inhibits DNA binding. Phorbol esters are thought to act by stimulating protein kinase C to phosphorylate an uncharacterized phosphatase that then dephosphorylates and activates c-Jun. c-Jun can also form heterodimers with
members
CREs may
of the
(64). allow
the
other, and stimulated human
This
CREB
proteins
may CAT
insulin
family
overlapping
of proteins
specificity
to antagonize
in part activity
explain in HIT
gene/CAT
that
bind
of AP1 and
or synergize
to
CREB
with
each
the inhibition of cAMPcells cotransfected with a
construct
and
a plasmid
bearing
a
c-Jun cDNA (65). Although current transient metabolic ditional sequences in
high
glucose
transfected
with
data support the role of the CRE in the control of insulin gene transcription, adhave been implicated. Thus, incubation stimulated CAT activity in fetal rat islets a construct containing the rat insulin I gene -196 to -247, which lack the CRE (60).
sequences from The glucose response region -193 to -227 3),
and
was
shown
element
was
that contained
to be active
(61). A factor (Cl factor) that identified. The DNA binding
further
mapped
to
element
(Fig.
human
islets
the FLAT
in adult
rat
and
a
within this region was of Cl was modulated by extracellular glucose with a fourto fivefold increase in binding activity observed when rat islets were incubated for 1 to 3 h in 20 mM glucose compared with 2 mM glucose. The Cl factor resembles in several respects a factor, IUF-l, that binds to a similar sequence in the human insulin gene enhancer (66, 67). IUF-l may in turn be similar to the factor IPF-l (see Fig. 2). Like IPF-l, expression of IUF-1 is restricted to /3 cells (68). Our own data confirm that there is a substantial reduction in the activity of IUF-l in isolated rat islets incubated for 3 h in 3 mM compared with 20 mM glucose (W. MacFarlane and K. Docherty, unpublished results). This may explain the progressive decrease in insulin mRNA and insulin content during a 24-h period after isolation and culture of islets (30-32). Preliminary studies from our laboratory indicate that phosphorylation can modulate the binding of IUF-1 to its DNA recognition sequence in the insulin gene (M. L. Read and K. Docherty, unpublished results). Further support for a role for IUF-I in the nutrient control of insulin gene transcription comes from the finding that chronically culturing the HITT’15 /3 cell line in media containing high glucose concentrations leads to a decrease in insulin mRNA levels, and a concomitant decrease in IUF-l binding activity (69). These results may explain the mechanisms whereby chronic hyperglycemia can impair /3 cell function in patients with type 2 diabetes mellitus.
CONCLUDING
bound activity
REMARKS
Progress has been made in our understanding of the transcriptional control of insulin gene expression. Regulatory sequences have been identified and cDNAs encoding important regulatory proteins have been cloned and sequenced. In terms of the mechanisms involved in the developmental timing or tissue specificity of insulin gene expression, we may only
be
scratching
at the
surface
of the
problem.
However,
the identification of sequences that mediate a transcriptional response to glucose and the preliminary characterization of protein factors that bind to these sequences in a glucosedependent manner represent exciting progress. It is likely
25
REVIEWS that transient metabolic effects on insulin gene transcription may be mediated through post-transcriptional modification of transcription factors. The availability of antibodies raised against these and other gene regulatory proteins will enable investigators to close the loop between nutrientand hormone-stimulated /3 cell signaling events, the phosphorylation status of nuclear transacting factors, and the transcriptional activity of the insulin gene. The identification of factors and RNA sequences involved in the modulatory effects of nutrients and hormones on the stability of insulin mRNA represents another important area of research. It is clear that a combination of biochemical and molecular biology skills will be required to resolve these problems. Supported supported
by a grant by
from
a fellowship
from
the the
Wellcome British
Trust. Diabetic
A. R. C. was
cose and arginine on the preproinsulin in the perfused rat pancreas: comparison
messenger ribonucleic with insulin secretion.
acid level Endocri-
nology 124, 707-711 28. Koryani, L., Bourey, R., Turk, J., Mueckler, M., and Permutt, (1992) Differential expression of rat pancreatic islet beta-cell
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27
