Nutrient regulation of insulin gene expression. - The FASEB Journal

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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

1. Ashcroft, S. J. H. (1980) Glucoreceptor mechanisms and the control of insulin release and biosynthesis. Diabetologia 18, 5-15 2. Johnson, N. H., Newgard, C. B., Milburn, J. L., Lodish, H. F., and Thorens, B. (1990) The high K,,, glucose transporter of islets of Langerhans is functionally similar to the low affinity transporter of liver has an identical primary sequence. J. Biol. Chem. 265, 6548-6551

4. Randle, P. J. (1993) Glucokinase and candidate genes for type 2 (noninsulin-dependent) diabetes mellitus. Diabetologia 36, 269-275 5. MacDonald, M. J. (1990) Elusive proximal signals of 13-cells for insulin secretion. Diabetes 39, 1461-1466

F. M., and Ashcroft, S. J. H. (1992) Mechanisms of insulin In Insulin, Molecular Biology to Pathology (Ashcroft, F. M., and Ashcroft, S. J. H., eds) pp. 97-150, Oxford University Press, Oxford, New York, Toronto 7. Cook, D. L., and Hales, C. N. (1984) Intracellular ATP directly blocks K channels in pancreatic /3-cells. Nature (London) 311, 269-271 8. HoIst, J. J. (1992) Role of classical and peptidergic neurotransmitters in insulin secretion. In Nutrient Regulation of Insulin Secretion (Flatt, P. R., ed) 6. Ashcroft, secretion.

pp. 23-39, Portland Press, 9. Charles, M. A., Lawecki, secretion. Interrelationships Che,n. 250, 6134-6140

J.,

London and Chapel Hill Pictet, R., and Grodsky, of glucose, cAMP and

G. (1975) calcium.

Translational control of insulin biosynthesis. Evidence for regulation of elongation, initiation and signal-recognition.particle-mediated translational arrest by glucose. Biochem. J. 235, 13590-13594 15. Wolin, S. L., and Walter, P. (1988) Ribosome pausing and stacking during translation of a eukaryotic mRNA. EMBO]. 7, 3559-3569 16. Knight, S. W., and Docherty, K. (1992) RNA-protein interactions in the 5’ untranslated region of preproinsulin mRNA. J. Mol. Endocrinol. 8,

R. E., Brinster, R. L., and Steiner, D. F. (1986) hormone synthesis by glucose in islets of Langetransgenic mice. j Biol. Chem. 261, 12915-12917 E. M., Rutherford, N. G., and Hutton, J. C. (1991)

rhans isolated from 18. Guest, P. C., Bailyes, Insulin secretory granule biogenesis: co-ordinate regulation of the biosynthesis of the majority of constituent proteins. Biochem. J. 274, 431-437 19. Guest, P. C., and Hutton, J. C. (1992) Biosynthesis of insulin secretory granule proteins. In Nutrient Regulation of Insulin Secretion (Flatt, P. R., ed) pp. 59-82, Portland Press, London and Chapel Hill 20. Wang, S. Y. (1989) The acute effects of glucose on the insulin biosynthetic-secretory pathway in simian virus 40-transformed hamster pancreatic islet /3 cell line. Endocrinology 124, 1980-1987 21. Schuit, R. C., In’tVeld, P. A., and Pipeleers, D. G. (1988) Glucose stimu-

January

1994

29.

proinsulin

and

islet

amyloid

polypeptide

fasting, insulin-induced hypoglycaemia Diabetologia 35, 1125-1132

Clark, A. R., and Docherty, K. (1993) Cell-specific the islets of Langerhans: E boxes and TAAT boxes.

M. A. glucose genes

and dexamethagene expression in Biochem. Soc. Trans.

21, 154-159 30. Brundstedt, J., and Chan, S. J. (1982) Direct effect of glucose on the preproinsulin mRNA level in isolated pancreatic islets. Biochem. Biophys. Res. Commun. 106, 1383-1389 31.

Itoh, N., and Okamoto, H. (1980) Translational control synthesis by glucose. Nature (London) 283, 100-102

of proinsulin

32. Giddings, S. J., Chirgwin, J. M., and Permutt, M. A. (1985) Glucose regulated insulin biosynthesis in isolated rat pancreatic islets is accompanied by changes in proinsulin mRNA. Diabetes Res. 2, 71-75 33. Hammonds, P., Schofield, P. N., Ashcroft, S. J. H., Sutton, R., and Gray, D. W. R. (1987) Regulation and specificity of glucose-stimulated insulin gene expression in human islets of Langerhans. FEBSLeLts. 223, 131-137 34. Hammonds, P., Schofield, P. N., and Ashcroft, S. J. H. (1987) Glucose regulates

simian 35. Welsh,

Insulin

perimental Pharmacology. Vol. 92, Insulin (Cuatrecasas, P., and Jacobs, S., eds) pp. 68-92, Springer Verlag, Berlin, Heidelberg, New York 13. Permutt, M. A., and Kipnis, D. M. (1972) Insulin biosynthesis. I. On the mechanism of glucose stimulation. J. Biol. Chem. 247, 1194-1199 14. Welsh, M., Schrerberg, N., Gilmore, R., and Steiner, D. F. (1986)

(GLUT2),

after prolonged sone treatment.

preproinsulin

messenger

RNA

levels

in a clonal

cell

line

of

virus 40-transformed B cells. FEBS Lett. 213, 149-154 N., Welsh, M., Steiner, D. F., and Hellerstr#{246}m,C. (1987)

Mechanisms of leucineand sis in isolated rat pancreatic

theophylline-stimulated insulin islets. Biochem. j 246, 245-248 for polyamines in glucose-stimulated

J. Biol.

10. Hughes, S. J., and Ashcroft, S. J. H. (1992) CAMP, protein phosphorylation and insulin secretion. In Nutrient Regulation of Insulin Secretion (Flatt, P. R., ed) pp. 271-288, Portland Press, London and Chapel Hill II. HoIz, G. G., and Habener, J. F. (1992) Signal transduction crosstalk in the endocrine system: pancreatic 13-cells and the glucose competence concept. TIBS 17, 388-393 12. Steiner, D. F. (1990) The biosynthesis of insulin. In Handbook of Ex-

225-234 17. Welsh, M., Hammer, Stimulation of growth

transporter

and

3. Matschinsky, F. M. (1990) Glucokinase as glucose sensor and metabolic signal generator in pancreatic 3 cells and hepatocytes. Diabetes 39, 647-652

Vol. 8

of pan-

22. Jetton, T. L., and Magnuson, M. A. (1992) Heterogeneous expression of glucokinase among pancreatic j3 cells. Proc. Nat!. Acad. &i. USA 89, 2619-2623 23. Giddings, S. J., Chirgwin, J., and Permutt, M. A. (1981) The effect of fasting and feeding on preproinsulin messenger RNA in rats.]. Clin. Invest. 67, 952-960 24. Giddings, S. J., Chirgwin, J., and Permutt, M. A. (1982) Effects of glucose on proinsulin messenger RNA in rats in vivo. Diabetes 31, 624-629 25. Giddings, S. J., Orland, M. J., Weir, G. C., Bonner-Weir, S., and Permutt, M. A. (1985) Impaired insulin biosynthetic capacity in a rat model for noninsulin-dependent diabetes: studies with dexamethasone. Diabetes 34, 235-240 26. Orland, M. J., Chyn, R., and Permutt, M. A. (1985) Modulation of proinsulin messenger RNA after partial pancreatectomy in rats: relationships to glucose homeostasis. J. Clin. Invest. 75, 2047-2055 27. Koh, G., Seino, Y., Takeda, J., Fukumoto, H., Kurose, T., Tsuji, K., Tsuda, K., Taminato, T., and Imura, H. (1989) Short term effects of glu-

Association.

REFERENCES

26

lates proinsulin biosynthesis by a dose-dependent recruitment creatic beta cells. Proc. NatI. Acad. Sci. USA 85, 3865-3869

biosynthe-

36. Welsh, N. (1990) A role insulin gene expression. Biochem. J. 271, 393-397 37. Nielsen, D. A., Welsh, M., Casadaban, M. J., and Steiner, D. F. (1985) Control of insulin gene expression in pancreatic /3 cells and in an insulin-secreting cell line, RIN-5F cells. I. Effects of glucose and cyclic AMP on the transcription of insulin mRNA. j Biol. Chem. 260, 38.

13585-13589 Welsh, M., Nielsen, D. A., MacKreIl, Control of insulin gene expression insulin-secreting cell line, RIN-5F mRNA stability. J. Biol. Chein. 260,

A. J., and Steiner, C. F. (1985) in pancreatic (3 cells and in an cells. II. Regulation of insulin

13590-13594 39. Philippe, J. (1993) Somatostatin inhibits insulin through a posttranscriptional mechanism in a hamster abetes 42, 244-249 40. Philippe, J., and Missotten, M. (1990) Dexamethasone biosynthesis by destabilising insulin ster insulinoma cells. Endocrinology

41. Philippe,

J., Giordano,

E., Gjinovci,

gene expression islet cell line. Di-

messenger ribonucleic 127, 1640-1645

A., and Meda,

inhibits

insulin

acid

in ham-

P. (1992) Cyclic

adenosine monophosphate prevents the glucocorticoid-mediated inhibition of insulin gene expression in rodent islet cells. j Clin. Invest. 90,

2228-2233 42. Fehmann, H-C., and Habener, J. F. (1992) Galanin inhibits proinsulin gene expression stimulated by the insulinotropic hormone glucagon-like peptide 1 (7-37) in mouse insulinoma /3TC.I cells. Endocrinology 130, 2890-2896 43.

Koranyi,

L., James,

D. E., Kraegen,

E. W., and

Permutt,

M. A. (1992)

Feedback inhibition of insulin gene expression by insulin. j Gun. Invest. 89, 432-436 44. Powers, A. C., Philippe, J., Hermann, H., and Habener, J. F. (1988) Sodium butyrate increases glucagon and insulin gene expression by recruiting immunocytochemically negative cells to produce hormone. Diabetes 37, 1405-1420 45. Muschel, R., Khoury, G., and Reid, L. M. (1986) Regulation of insulin mRNA abundance and adenylation: dependence on hormones and matrix substrata. Mol. CelL Biol. 6, 337-341

The FASEB Journal

DOCHERTY

AND

CLARK

REVIEWS 46.

Karlsen5

mark,

A. E., Fujimoto,

W. Y., Rabinovitch,

A. (1991) Effects of sodium

insulin

gene

butyrate

P., Dube, S., and Lernon proliferation-dependent

expression

and insulin release in glucose-sensitive RIN-5AH cells. j Biol. Chem. 266, 7 542-7548 47. Clark, A. R., and Docherty, K. (1992) The insulin gene. In Insulin, Molecular Biology to Pathology (Ashcroft, F. M., and Ashcroft, S. J., eds) pp. 37-63, Oxford University Press, Oxford

48. Walker, M. D., Edlund,

T., Boulet,

0.,

Edlund,

T., Moss,

M. D. (1987) A mutational control region: expression

of insulin

and 61.

W. J., and

L. B., Rutter,

analysis of the insulin in beta cells is dependent

Walker, gene transcription on two related se-

quences 50.

51.

within the enhancer. Proc. Nat!. A.cad. Sci. USA 84, 8819-8823 C. W., and Walker, M. D. (1992) Subunit structure of cell-specific

Park, E box-binding bility shift. Dandoy-Dron,

proteins analysed Chem. 267,

J. Biol.

by quantitation 15642-15649

induces insulin gene transcription in a murine pancreatic /3-cell line.]. Biol. Chem. 266, 11141-11143 59. Goodison, S., Kenna, S., and Ashcroft, S. J. H. (1992) Control of insulin gene expression by glucose. Biochein. J. 285, 563-568 60. German, M. S., Moss, L. G., and Rutter, W. J. (1990) Regulation of in-

W. J. (1983) Cell-

A. M., and Rutter,

specific expression controlled by the 5’ flanking region chymotrypsin genes. Nature (London) .306, 557-561

49. Karlsson,

and /3-cells. Mo!. Encocrinol. 5, 897-903 58. Efrat, S., Surana, M., and Fleischer, N. (1991) Glucose

of electrophoretic

mo-

F., Monthioux, E., Jami, J., and Bucchini, D. (1991) regions of rat insulin I gene necessary for expression in transgenic mice. Nucleic Acids Res. 18, 4925-4930 Aronheim, A., Ohlsson, H., Park, C. W., Edlund, T, and Walker, M. D. (1991) Distribution and characterisation of helix-loop-helix

3865-3869

62. German, M. 5. (1993) Glucose sensing in pancreatic islet beta cells: the key role of glucokinase and glycolytic intermediates. Proc. NatI. Acad. Sci. USA 90, 1781-1785 63. Philippe, J., and Missotten, M. (1990) Functional characterization of a cAMP-responsive 1465-1469

Regulatory 52.

enhancer-binding Nucleic Acids Res.

proteins from 19, 3893-3899

pancreatic

/3

cells

and

Synergistic activation of the insulin gene by a LIM-homeodomain protein: building a functional insulin minienhancer complex. Genes & Dcv. 6, 2165-2176 56. Emens, L. A., Landers, D. W., and Moss, L. G. (1992) Hepatocyte nuclear factor Ia is expressed in a hamster insulinoma line and transactivates the rat insulin I gene. Proc. Nat!. Acad. Sci. USA 89, 7300-7304 57. Ohlsson, H., Thor, S., and Edlund, T. (1991) Novel insulin promoterand enhancer-binding proteins that discriminate between pancreatic a-

NUTRIENT

REGULATION

OF INSULIN

GENE

EXPRESSION

element

of the rat insulin

I gene.]

Biol.

Chem.

265,

64. Karin, 65.

lymphocytes.

53. German, M. S., Moss, L. G., Wang, J., and Rutter, W. J. (1992) The insulin and amyloid polypeptide genes contain similar cell-specific promoter elements that bind identical 13-cell nuclear complexes. Mol. Cell. BioL 12, 1777-1788 54. Karlsson, 0., Thor, S., Norberg, T., Ohisson, H., and Edlund, T. (1990) Insulin gene enhancer binding protein Isl-l is a member of a novel class of proteins containing both a homeoand Cys-His domain. Nature (London) 344, 879-882 55. German, M. S., Wang, J., Chadwick, R. B., and Rutter, W. J. (1992)

sulin gene expression by glucose and calcium in transfected primary islet cultures. j Biol. Chem. 265, 22063-22066 Melloul, D., Ben-Neriah, Y., and Cerasi, E. (1993) Glucose modulates the binding of an islet-specific factor to a conserved sequence within the rat I and human insulin promoters. Proc. NatI. Acad. Sci. USA 90,

M., and Smeal, T. (1992) Control of transcription factors by signal transduction pathways: the beginning of the end. TIBS 17, 418-422 Inagaki, N., Maekawa, T., Sudo, T., Ishii, S., Seino, Y., and Imura, H. (1992) c-Jun represses the human insulin promoter activity that depends on multiple

66.

cAMP

response

elements.

Proc. NatL Acad. Sci. USA 89,

1045-1049 Boam, D. S. W., and Docherty, K. (1989) A tissue specific nuclear factor binds to multiple sites in the human insulin gene enhancer. Biochem. J.

264, 233-239 67. Scott, V., Clark, A. R., Hutton, J. C., and Docherty, K. (1991) Two proteins acts as the IUFI insulin gene enhancer binding factor. FEBS Lett.

290, 68.

27-30 Clark, A. R., Petersen, H. V., Read, M. L., Scott, V., Michelsen, B., and Docherty, K. (1993) Human insulin gene enhancer binding proteins in pancreatic a and /3 cell lines. FEBS Lett. 329, 139-143

69. Olson,

K. L., Redmon,J.

B., Towle, H. C., and Robertson,

Chronic exposure of HIT cells to high glucose concentrations cally decreases insulin gene transcription and alters binding gene regulatory protein. j Clin. Invest. 92, 514-519

R. P. (1993) paradoxiof insulin

70. Li, L., Heller-Harrison, R., Czech, M., and Olson, E. N. (1992) Cyclic AMP-dependent protein kinase inhibits the activity of myogenic helixloop-helix proteins. Mol. Cell. Biol. 12, 4478-4485

27