Biochem. J. (2008) 415, 1–10 (Printed in Great Britain) doi:10.1042/BJ20081029 1

REVIEW ARTICLEGlucose regulation of insulin gene expression in pancreatic β-cellsSreenath S. ANDRALI1, Megan L. SAMPLEY1, Nathan L. VANDERFORD1 and Sabire OZCAN2

Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, 741 South Limestone, Lexington, KY 40536, U.S.A.

Production and secretion of insulin from the β-cells of thepancreas is very crucial in maintaining normoglycaemia. This isachieved by tight regulation of insulin synthesis and exocytosisfrom the β-cells in response to changes in blood glucose levels.The synthesis of insulin is regulated by blood glucose levels at thetranscriptional and post-transcriptional levels. Althoughmany transcription factors have been implicated in the regul-ation of insulin gene transcription, three β-cell-specific tran-scriptional regulators, Pdx-1 (pancreatic and duodenal homeobox-1), NeuroD1 (neurogenic differentiation 1) and MafA (V-mafmusculoaponeurotic fibrosarcoma oncogene homologue A),have been demonstrated to play a crucial role in glucoseinduction of insulin gene transcription and pancreatic β-cellfunction. These three transcription factors activate insulin geneexpression in a co-ordinated and synergistic manner in responseto increasing glucose levels. It has been shown that changes inglucose concentrations modulate the function of these β-cell

transcription factors at multiple levels. These include changes inexpression levels, subcellular localization, DNA-binding activity,transactivation capability and interaction with other proteins.Furthermore, all three transcription factors are able to induceinsulin gene expression when expressed in non-β-cells, includingliver and intestinal cells. The present review summarizes the recentfindings on how glucose modulates the function of the β-celltranscription factors Pdx-1, NeuroD1 and MafA, and therebytightly regulates insulin synthesis in accordance with bloodglucose levels.

Key words: diabetes, glucose, insulin, NeuroD1 (neurogenicdifferentiation 1), pancreatic and duodenal homeobox-1 (Pdx-1),V-maf musculoaponeurotic fibrosarcoma oncogene homologue A(MafA).

INTRODUCTION

Increases in blood glucose levels stimulate insulin genetranscription and insulin secretion [1,2]. Insulin gene transcriptionis mainly controlled by a 340 bp promoter region upstreamof the transcription start site of the insulin gene. The insulinpromoter is organized in a complex arrangement of discretecis-acting sequence motifs, which serve as binding sites forboth ubiquitous and β-cell-specific transcription factors [3,4].The co-ordinated interaction between cis-elements and trans-acting factors at the promoter contributes to both the β-cell-specific expression of the insulin gene and the regulation ofexpression in response to glucose, calcium levels, nutrientavailability and hormone signalling [5,6]. Much of the glucoseresponsiveness inherent to the insulin promoter is conferred bythe A3, E1 and C1 sites, which are bound by the transcriptionfactors Pdx-1 (pancreatic and duodenal homeobox-1) [7–14],NeuroD1 (neurogenic differentiation 1)/Beta2 [15–17] and MafA(V-maf musculoaponeurotic fibrosarcoma oncogene homologueA) [12,18,19] respectively. These three transcription factors actin a co-ordinated and synergistic manner to stimulate insulingene expression in response to increased glucose levels [20–22](Figure 1).

Compared with humans, rodents (rat and mouse) have two in-sulin genes, of which the insulin II gene and its promoter is mostlysimilar to that of humans [23]. On the basis of the analysis oftranscription factor-binding sites and spacing of the cis-regulatoryelements, there are significant differences between rodent andhuman insulin gene promoters [24]. However, a detailed com-parative analysis of the insulin promoters from various speciessuggest that all three regulatory elements (A3, E1 and C1), im-portant for glucose regulation of insulin gene expression, are con-served between species with the A-box (A3) to which Pdx-1 bindsbeing the most highly conserved one [24].

Although Pdx-1, NeuroD1 and MafA have been shown to becrucial for glucose regulation of insulin gene transcription, theexact mechanisms by which glucose modulates the function ofthese transcription factors remains to be established. Studies fromseveral laboratories, including ours, suggest that glucose regulatesthe function of these transcription factors by different mechan-isms. In the case of Pdx-1, it has been proposed that glucosemodulates the function of Pdx-1 by regulating the localization,DNA-binding activity and interaction of Pdx-1 with multipleproteins [25–31]. Several lines of evidence suggest that NeuroD1changes its localization in response to changing glucose levels.Under normal glucose conditions, NeuroD1 is mainly cytosolic,

Abbreviations used: bHLH, basic helix–loop–helix; CAMKII, Ca2+/calmodulin-dependent protein kinase II; CBP, CREB (cAMP-response-element-binding protein)-binding protein; CHOP, C/EBP (CCAAT/enhancer-binding protein)-homologous protein; E, embryonic day; ERK, extracellular-signal-regulated kinase; Fox, Forkhead box; GLP1, glucagon-like peptide 1; GLUT2, glucose transporter 2; GSK3, glycogen synthase kinase 3; HAT, histoneacetyltransferase; HBP, hexosamine biosynthetic pathway; HDAC, histone deacetylase; MafA, V-maf musculoaponeurotic fibrosarcoma oncogenehomologue A; MAPK, mitogen-activated protein kinase; MARE, Maf recognition element; MODY, maturity-onset diabetes of the young; NeuroD1,neurogenic differentiation 1; O-GlcNAcase; O-linked N-acetylglucosaminidase; OGT, O-linked GlcNAc transferase; PAS, Per-Arnt-Sim; P/CAF, p300/CBP-associated factor; PCIF-1, Pdx-1 C-terminus-interacting factor-1; Pdx-1, pancreatic and duodenal homeobox-1; PI3K, phosphoinositide 3-kinase; pol II,RNA polymerase II; SHP, small heterodimer partner.

1 These authors made an equal contribution to this review.2 To whom correspondence should be addressed (email sozcan@uky.edu).

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Figure 1 Co-ordinated and synergistic activation of insulin gene expressionby Pdx-1, NeuroD1 and MafA

Pdx-1 binds to the A-boxes, NeuroD1 to the E-boxes and MafA to the C1 element within the400 bp region of the insulin promoter and activate insulin gene expression in a co-ordinatedand synergistic manner in the presence of elevated blood glucose levels. All three elements areconserved within the insulin promoters of various species, including human and rodent insulinpromoters. NeuroD1 binds to the E1 element as a heterodimer with the ubiquitous transcriptionfactor E47.

and exposure to high glucose causes its translocation into thenucleus [32,33]. MafA expression itself requires high glucose. Al-though expression of MafA is very low under normal glucose con-ditions, MafA transcription increases drastically in response tohigh glucose [34–37]. The major focus of the present review is onthe recent findings on glucose regulation of Pdx-1, NeuroD1 andMafA in pancreatic β-cells and the implication of these findingson insulin synthesis and pancreatic β-cell function. Furthermore,the mechanisms by which these β-cell-specific transcriptionfactors activate insulin gene transcription will also be discussed.

ROLE OF PDX-1 IN GLUCOSE REGULATION OF INSULINGENE EXPRESSION

Role of Pdx-1 in pancreatic β-cell function

Pdx-1 {also known as STF-1 (somatostatin transactivating factor1) [38], IPF-1 (insulin promoter factor 1) [39], IUF-1 (insulinupstream factor 1) [40], IDX-1 (islet and duodenal homeobox1) [41] and GSF (glucose-sensitive factor) [42]} is the majorregulator of glucose-stimulated insulin gene transcription andis essential for embryonic development of the pancreas [8–12,14,43,44]. The homeodomain transcription factor Pdx-1 is firstexpressed in the primitive gut tube during embryonic developmentof the pancreas around E (embryonic day) 8.5. Pdx-1 homozygousknockout mice fail to develop a pancreas and die shortly after birth,because of a lack of insulin [45–47]. Heterozygous Pdx-1 micehave a normal pancreas, but are hyperglycaemic, which is causedby decreased insulin production. Pancreatic agenesis is alsoobserved in a patient carrying a homozygous single nucleotidedeletion in the PDX1 gene [48]. β-Cell-specific disruption of thePDX1 gene results in diabetes with aging and suggests that Pdx-1is essential for maintenance of β-cell identity [49]. These findingsindicate that Pdx-1 is required for early development of thepancreas as well as for pancreatic β-cell maturation and function.

In the fully developed pancreas, Pdx-1 expression is mainlyrestricted to the insulin-producing β-cells and somatostatin-producing δ-cells within the pancreatic islets. However, Pdx-1expression also has been observed in the epithelial cells of theduodenum [38,41]. Pdx-1 primarily acts in the β-cell toup-regulate the transcription of several β-cell-specific genes,including insulin, GLUT2 (glucose transporter-2) [50], gluco-kinase [51], somatostatin [38,52], islet amyloid polypeptide [52]and MafA [53], as well as auto-regulating its own expression. Italso has been reported that Pdx-1 functions as a transcriptionalrepressor for glucagon [49,54], cytokeratin K19 [55] and c-Myc[56]. The repressor activity of Pdx-1 appears to be important inmaintenance of β-cell identity by inhibiting the expression ofgenes specific for α-cells (glucagon) and pancreatic ductal cells(cytokeratin K19).

The transactivation domain of Pdx-1 is at the N-terminus, andthis domain also mediates unique protein–protein interactionswith other transcription factors such as NeuroD1 and transc-riptional co-regulators, including p300 [22,29,57–61]. Pdx-1 alsocontains a central DNA-binding homeodomain with a novelnuclear localization signal [62,63]. Activation of insulin genetranscription by Pdx-1 occurs via binding to the A-elements ofthe insulin promoter. Various point mutations in the PDX1 genehave been shown to result in pancreatic agenesis, impaired glucosetolerance and/or diabetes in humans. Specific point mutations inPDX1 also are associated with MODY (maturity-onset diabetesof the young) 4 and late-onset Type 2 diabetes, characterized bya decline in β-cell function [48,64,65].

Molecular mechanisms of Pdx-1-mediated activation of insulingene transcription

Recent findings suggest that one of the mechanisms by whichPdx-1 promotes insulin gene transcription is by mediating histonemodifications at the insulin promoter. Pdx-1 has been demons-trated to recruit the HAT (histone acetyltransferase) p300 to theinsulin promoter only at high levels of glucose (10–30 mM),which leads to increased acetylation of histones [57,58,66].The histone methyltransferase Set9 has also been shown to berecruited to the insulin promoter via Pdx-1, leading todimethylation of histone H3 Lys4 [67]. These histone acetylationand methylation events lead to changes in the chromatin structurethat promote insulin gene transcription [67–70]. Binding ofPdx-1 to the insulin promoter is required for pol II (RNApolymerase II) to adopt the elongation isoform for active trans-cription. In the absence of Pdx-1, pol II appears to be unableto switch from the initiation (pSer5) to the elongation (pSer2)isoform, which is required for active transcriptional elongation[67,71]. These observations suggest that Pdx-1 regulates insulingene transcription by promoting a transcriptionally activechromatin structure, which enhances elongation by pol II.

There is also evidence that Pdx-1 participates in repression ofgene expression and mediates transcriptional repression of gluca-gon [54], cytokeratin K19 and c-Myc [55,56]. However, it is notknown whether the ability of Pdx-1 to repress transcription isrelated to changes in chromatin structure. Interestingly, Pdx-1 hasbeen shown to recruit HDAC (histone deacetylase)-1 and -2 to theinsulin promoter in response to low glucose levels (1–3 mM) todown-regulate insulin gene transcription [29]. It is therefore likelythat Pdx-1 mediates repression of gene expression by recruitingco-repressors. Pdx-1 is also reported to interact with PCIF-1 (Pdx-1 C-terminus-interacting factor-1), a TRAF (tumour-necrosis-factor-receptor-associated factor) and POZ domain-containing nuclear factor that inhibits Pdx-1-transactivationpotential independent of histone co-repressor/HDAC recruitment[72,73]. However, the exact mechanism by which PCIF-1 inhibitstransactivation by Pdx-1 remains to be established. Bridge-1(PSMD9) is another protein that has been shown to interact withPdx-1 and stimulate Pdx-1 transcriptional activity. The co-regulator Bridge-1 is a PDZ-like domain protein, and its abilityto activate transcription appears to be dependent on its interactionwith the HAT p300 [60,74]. In summary, Pdx-1 appears to regulategene transcription by promoting the assembly of multiproteincomplexes at the insulin promoter.

Regulation of Pdx-1 function by glucose and othersignalling pathways

Glucose, the physiological regulator of insulin gene transcription,modulates Pdx-1 function in pancreatic β-cells by multiple mech-anisms. We have discovered that glucose regulates the interaction

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Glucose regulation of β-cell transcription 3

of Pdx-1 with various co-regulators in a phosphorylation-dependent manner in the mouse insulinoma cell line MIN6. Underlow or normal glucose conditions (1–3 mM), Pdx-1 is mainlyassociated with HDAC-1 and HDAC-2 to down-regulate insulingene expression [29]. Increases in glucose levels (10–30 mM)disrupt the interaction of Pdx-1 with HDACs and promote itsassociation with the HAT p300, which leads to hyperacetylationof histone H4 and induction of insulin gene transcription [58,66].This switch in Pdx-1 interaction in response to high glucoserequires a phosphorylation event that causes changes in Pdx-1localization [58]. On low glucose, Pdx-1 is localized mainly tothe nuclear periphery and interacts with HDAC-1 and HDAC-2, whereas on high glucose, Pdx-1 interacts with p300 and islocalized throughout the nucleus. Blocking of Pdx-1 localizationto the nuclear periphery on low glucose by treatment with thephosphatase inhibitor okadaic acid disrupts the ability of Pdx-1 tointeract with HDAC-1 and HDAC-2 and promotes its interactionwith the HAT p300 [29,58].

Changes in glucose levels regulate the subcellular localizationof Pdx-1. On low glucose (1–3 mM), Pdx-1 is mainly localizedto the nuclear periphery and, in response to glucose stimulation,Pdx-1 becomes phosphorylated and shuttles to the nucleoplasmand is associated with target gene promoters [27,75]. Severalsignalling pathways including the p38/SAPK (stress-activatedprotein kinase) [75,76], PI3K (phosphoinositide 3-kinase) [77],atypical PKC (protein kinase C) [78] and MAPK (mitogen-activated protein kinase) pathways [79,80], as well as PAS (Per-Arnt-Sim) kinase [81] have been implicated in Pdx-1 phospho-rylation, nucleocytoplasmic shuttling, DNA binding andtransactivation potential. Ser61 and Ser66 of Pdx-1 have beendemonstrated to be phosphorylated in response to p38, MAPKand PI3K signalling. Thr152 has been shown to be phosphorylatedby PAS kinase and to regulate the nucleocytoplasmic shuttlingand transactivation potential of Pdx-1 [81]. However, the exactresidues that become phosphorylated by the various kinases andtheir physiological significance still remain to be determined.

Various studies indicate that modification by phosphorylationalso regulates the stability of the Pdx-1 protein. The DNA-dependent protein kinase has been shown to phosphorylate Thr11

in Pdx-1 in response to radiation-induced DNA damage to pro-mote Pdx-1 protein degradation [30]. Another study has demons-trated that GSK3 (glycogen synthase kinase 3) phosphorylatesPdx-1 on Ser61 and/or Ser66 in response to oxidative stress, whichalso results in degradation of the Pdx-1 protein [82,83]. Thesestudies provide a possible mechanism for previous observationswhere Pdx-1 protein levels have been shown to be reduced underglucotoxic and oxidative stress conditions [26,84–86]. However,other studies have shown that oxidative stress inhibits Pdx-1nuclear localization and DNA binding through activation ofthe JNK (c-Jun N-terminal kinase) pathway [87]. The nuclearexclusion of Pdx-1 would also explain the decreases in insulingene transcription observed under oxidative stress conditions. Theinterplay of Pdx-1 protein stability and intracellular localizationrequires further investigation to elucidate fully the role of Pdx-1in β-cell dysfunction under oxidative stress conditions.

Several studies suggest that Pdx-1 may be subject to a numberof other post-translational modifications, including SUMOylation[88] and O-linked glycosylation [28]. Although SUMOylation ofPdx-1 appears to modulate Pdx-1 localization and stability [88],glycosylation of Pdx-1 has been reported to regulate Pdx-1 DNA-binding activity [28]. Exposure of pancreatic islets to elevatedlevels of fatty acids such as palmitate has been shown to reducethe nuclear localization and expression levels of Pdx-1 [89–91]. Other factors that regulate Pdx-1 levels include the GLP1(glucagon-like peptide 1), which is produced in the intestine and

Figure 2 Glucose regulates the subcellular localization and interaction ofPdx-1 with co-regulators

Under low-glucose conditions, Pdx-1 is mainly localized to the nuclear periphery. Resultsfrom our laboratory suggest that Pdx-1 interacts with co-repressors (HDACs) underlow-glucose conditions (1–3 mM) to down-regulate insulin gene expression. Elevated levels ofglucose (10–30 mM) mediate the phosphorylation of Pdx-1, which disrupts the localization ofPdx-1 to the nuclear periphery and enables its interaction with co-activators such as the HATp300 and the histone methyltransferase Set7/9 in order to activate insulin gene transcription.Symbols: A, A-box; P, phospho group; ∼ insulin mRNA.

promotes glucose-regulated transcription of insulin [92,93]. GLP1has been shown to increase Pdx-1 levels, as well as enhance thetransactivation domain and DNA-binding activity of Pdx-1 and itsnuclear localization [92,94,95]. Expression of Pdx-1 in pancreaticβ-cells appears to be independent of the glucose concentration;however, glucotoxic conditions lead to reduction in Pdx-1 levels.

In summary, changes in glucose levels have been shown toregulate the nuclear localization, DNA binding and transactivationcapacity of Pdx-1 and its interaction with co-regulators. Nuclearlocalization and Pdx-1 interaction with co-activators appears to bedependent on phosphorylation of Pdx-1 (Figure 2). On the basis ofthe published results, Pdx-1 is mainly in the nuclear peripheryunder low or normal glucose conditions and interacts withHDACs. In response to high glucose, Pdx-1 becomes phos-phorylated and translocates into the nucleoplasm and interactswith the HAT p300 to stimulate insulin gene expression (Figure 2).

FUNCTION OF NeuroD1 IN GLUCOSE-INDUCTION OF INSULIN GENEEXPRESSION

Role of NeuroD1 in pancreatic β-cell function

NeuroD1, also known as Beta2, belongs to the bHLH (basichelix–loop–helix) family of transcription factors and functions ina complex with the ubiquitously expressed E47 protein [16,96].The NeuroD1 protein consists of 355 amino acids with the bHLHDNA-binding domain located at residues 100–155. Defects inNeuroD1 function have been associated with many diseases,including ataxia, deafness, and Type 1 and Type 2 diabetes [15].

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4 S. S. Andrali and others

NeuroD1 is required for terminal neuronal differentiation andectopic expression of NeuroD1 in Xenopus embryos mediates theconversion of epidermal cells into neurons [96–98]. NeuroD1 isexpressed widely throughout the developing CNS (central nervoussystem) and the auditory and vestibular systems [99].

In addition to neuronal cells, NeuroD1 is also expressed inboth the developing and adult pancreas [16,17]. In the developingpancreas, NeuroD1 is detected as early as E9.5 in the pancreaticprimordia, indicating that NeuroD1 is present in early precursorcells. At E17.5, NeuroD1 is mainly expressed in small clusters ofendocrine cells. NeuroD1 is present in all mature β-cells and inapprox. 2% of α-cells. NeuroD1 has been implicated in regulationof insulin and glucagon gene expression [16,100]. HomozygousNeuroD1−/− newborn mice are hyperglycaemic and haveketonuria, and die within 5 days of birth [101]. Further analysissuggests that the severe diabetes observed in these mice is dueto a significant reduction in the number of islet cells, which isdue to a failure of endocrine cells to aggregate into mature islets.However, the viability of the homozygous NeuroD1 mice appearsto be dependent on genetic background [102]. Specific mutationsin NeuroD1 have been linked to the MODY6 locus [103,104].A polymorphism in Ala45 to threonine has been associated withType 1 diabetes in some populations [105,106].

NeuroD1 is also implicated in regulation of insulin secretionby glucose via modulation of the expression of genes such asneuronatin (Nna), which is an ion channel transporter [107],and Sur1, which forms potassium channels with Kir6.2 [108].In a recent study, introduction of NeuroD1 into a human fetalepithelial cell line induced the expression of several genesrequired for vesicular trafficking and exocytosis, includingSec24D, SNAP25 (25 kDa synaptosome-associated protein),syntaxin1 and Munc18 [109]. This finding suggests thatNeuroD1 may regulate insulin exocytosis in pancreatic β-cells by directly inducing the expression of genes involved inexocytosis. Other targets of NeuroD1 include IGRP (islet-specificglucose-6-phophatase catalytic-subunit-related protein) [110] andglucokinase [111].

Regulation of insulin gene transcription by NeuroD1

NeuroD1-directed transcription of insulin has been shownto occur via a DNA consensus sequence known as E-box(CANNTG) within the insulin promoter [57]. NeuroD1 itselfis unable to bind DNA and associates with DNA as a hetero-dimer with another ubiquitously expressed bHLH protein E47[112,113]. The ability of NeuroD1 to activate transcriptionis enhanced by its interaction with the co-activator p300 orCBP [CREB (cAMP-response-element-binding protein)-bindingprotein] [57,114,115]. NeuroD1 also has been shown to interactwith SHP (small heterodimer partner), an orphan nuclear receptorthat functions as a repressor of transcription [116]. SHP hasbeen demonstrated to compete for p300 binding to NeuroD1and therefore functions as a co-repressor. NeuroD1 interactionwith Pdx-1 is essential to synergistically activate insulin genetranscription in β-cells [57,113].

Modulation of NeuroD1 function by glucose

Glucose has been shown to regulate the nuclear localizationas well as transactivation capacity of NeuroD1 via post-transcriptional modification. NeuroD1 is modified by phospho-rylation by ERK1/2 (extracellular-signal-regulated kinase 1/2)[80] and GSK3β [117], by acetylation by p300 [118] and byO-linked glycosylation by OGT (O-linked GlcNAc transferase)

[32]. The MAPKs ERK1/2 have been implicated in regulation ofinsulin gene transcription in pancreatic β-cells, and the activityof ERK1/2 is increased in response to glucose [79,80,119]. Inthe presence of high glucose, NeuroD1 becomes phosphorylatedby ERK2 at multiple sites within its transactivation domain,which enhances the transactivation capacity of NeuroD1 [80].Regulation of insulin gene transcription by ERK1/2 is mediatedby phosphorylation of Pdx-1, NeuroD1 and E47.

Glucose-mediated phosphorylation has also been proposed toregulate NeuroD1 nuclear localization. In low-glucose-incubatedMIN6 cells, NeuroD1 has been shown to be mainly localized inthe cytosol, whereas exposure to high glucose caused NeuroD1translocation into the nucleus [33]. Treatment of pancreatic β-cells with the MEK (MAPK/ERK kinase) inhibitor PD98059blocked the nuclear translocation of NeuroD1 in pancreatic β-cells. Furthermore, a serine-to-alanine mutation of NeuroD1 atSer274 interfered with NeuroD1 nuclear translocation, dimeriza-tion with E47 and DNA-binding activity [33]. Another kinase thathas been implicated in regulation of NeuroD1 function is GSK3β.Phosphorylation of NeuroD1 by GSK3β has been shown to inhibitNeuroD1-induced ectopic neurogenesis [117]. Phosphorylationof NeuroD1 by CaMKII (Ca2+/calmodulin-dependent proteinkinase II) at Ser336 in primary neurons has been demons-trated to be important for induction of dendritic morphogenesis[120]. However, the role of GSK3β and CaMKII in regulationof NeuroD1 function in pancreatic β-cells remains unknown.Acetylation of NeuroD1 by P/CAF (p300/CBP-associated factor)appears to regulate the DNA-binding and transactivation capacityof NeuroD1 in insulinoma cell lines [118].

Recent findings from our laboratory suggest that increasedflux via the HBP (hexosamine biosynthetic pathway) (caused byhigh levels of glucose) mediates the O-GlcNAc modification ofNeuroD1 [32] (Figure 3). Under high-glucose conditions (10–30 mM), NeuroD1 interacts with OGT and becomes O-GlcNAc-modified. This modification causes NeuroD1 to translocate fromthe cytosol into the nucleus (Figure 4). On the other hand,under low-glucose conditions (1–3 mM), NeuroD1 becomesdeglycosylated by interacting with the O-GlcNAcase (O-linkedN-acetylglucosaminidase), which causes its exit from the nucleus(Figure 4). Since phosphorylation has also been implicated inNeuroD1 nuclear translocation, it is possible that there is aYin–Yang relationship between phosphorylation and O-GlcNAcmodification of NeuroD1. Indeed, Ser274, which has been shown tobe phosphorylated by MAPKs, is predicted to be also O-GlcNAc-modified [32,33].

REGULATION OF INSULIN GENE TRANSCRIPTION BY MafA

Role of MafA in β-cell function

MafA is a basic leucine zipper transcription factor belongingto the large Maf family of transcription factors. Large Maftranscription factors have an N-terminal transactivation domain, aleucine zipper domain (responsible for homodimerization) and aDNA-recognition region (responsible for binding DNA elementstermed Maf recognition elements or MAREs) [121,122]. MafA isexpressed in pancreatic β-cells, as well as in the eye, and has beenimplicated in lens development [123,124]. In pancreatic β-cells,MafA plays an important role in glucose regulation of insulin geneexpression [34,35,37,125]. Beyond insulin expression, MafA alsoappears to play a role in mediating the expression of a numberof other genes, including granuphilin [126], adiponectin [127],GLUT2 [20,128,129], Nkx6.1 (NK6 homeobox 1) [128], Pdx-1[128,130], pyruvate carboxylase [128], prohormone convertase1/3 [128,131] and CHOP [C/EBP (CCAAT/enhancer-binding

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Glucose regulation of β-cell transcription 5

Figure 3 UDP-GlcNAc synthesis via the HBP and O-linked glycosylation of proteins by OGT

Flux through the HBP leads to the synthesis of UDP-GlcNAc, which is used as the substrate for OGT. O-GlcNAcase is responsible for removing O-GlcNAc from modified proteins. AGM1,N-acetylglucosamine phosphomutase 1; DON, 6-diazo-5-oxo-L-norleucine; GFAT, glutamine:fructose-6-phosphate amidotransferase; GNA, glucosamine-6-phosphate acetyltransferase; PUGNAc,O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate; UAP, UDP-N-acetylglucosamine pyrophosphorylase.

Figure 4 High levels of glucose mediate the O-GlcNAc modification ofNeuroD1 and its translocation into the nucleus

In the presence of low glucose (1–3 mM), NeuroD1 is mainly localized to the cytosol. Inresponse to high glucose (10–30 mM), it becomes O-GlcNAc-modified by OGT in the cytosol,which causes its translocation into the nucleus. Once in the nucleus, NeuroD1 heterodimerizeswith E47 and activates insulin gene expression by recruiting co-activators such as p300.Phosphorylation of NeuroD1 by ERK has also been shown to mediate its translocation into thenucleus. E47, which heterodimerizes with NeuroD1 and binds to the E1 element within the insulinpromoter, is also phosphorylated by ERK, and phosphorylated E47 translocates into the nucleus.Symbols: Ac, acetyl group; G, GlcNAc; NPC, nuclear pore complex; P, phospho group.

protein)-homologous protein] [132]. However, it is unclearwhether many of these genes are bona fide MafA target genesor whether their regulation by MafA is indirect.

Expression of MafA is observed at later stages of β-celldevelopment around E13.5, suggesting a role for MafA inβ-cell maintenance [125]. MafA-knockout mice are viable, butdevelop diabetes with age as the result of decreased expression ofβ-cell-specific genes, including insulin, reduced insulin secretion,decreased β-cell number and altered islet architecture [129]. TheMafA-knockout animals do not have as severe a phenotype as the

Pdx-1 [46] or NeuroD1 [101] -knockout animals. The reason forthis could be that MafA expression is restricted to the β-cells ofthe pancreas [37,125], unlike Pdx-1 and NeuroD1, or because theother large Maf family members, MafB or c-Maf, may compensatefor the loss of MafA. MafB and c-Maf have been shown to beexpressed in insulin-positive cells and can activate the insulinpromoter in in vitro experiments [37,133,134]. Furthermore,recent findings suggest that, during β-cell development, MafBis expressed before MafA expression and is replaced by MafAafter insulin expression is initiated [133,135]. These data suggestthat, although MafB is important for differentiation of α- and β-cells, MafA plays a role in β-cell function after birth, which isconsistent with the finding that MafA-knockout mice have normalislet structure at birth. Gene-transfer experiments indicate thatMafA is capable of inducing insulin gene expression in liver cells[21,136,137] and in intestinal cells [20,131]. As in β-cells, MafAsynergizes with Pdx-1 and NeuroD1 to enhance the synthesis ofinsulin in non-β-cells [20,21,136,137].

Regulation of insulin gene expression by MafA

MafA binds to the C1 element within the insulin promoter,which was originally designated as the RIPE3b1 element anddemonstrated to be important for expression of the rat insulinII gene [138,139]. Earlier studies demonstrated that the C1element is important for the β-cell-specific expression of insulin[138,140–142] and for the glucose-dependent regulation of insulinexpression [139,143,144]. Later studies indicated that a β-cell-specific transcription factor(s) is bound to the C1 element in aglucose- and phosphorylation-dependent manner [141,143–146].

Like other Maf-binding elements, the C1 element containsa MARE, which is required for MafA binding [34,125].The binding of MafA to the C1 element is a crucial eventleading to transcriptional up-regulation of insulin both invitro [34,35,37,125,143,147,148] and in vivo [127,129]. Themechanism by which MafA participates in insulin transcriptionincludes binding to and synergizing with Pdx-1, NeuroD1 andNFAT (nuclear factor of activated T-cells) [148–150].

Regulation of MafA levels by glucose and other signals

MafA binds to the C1 element of the insulin promoter in a glucose-dependent manner [143,144], and this is due to accumulationof MafA in response to high glucose (10–25 mM) conditions.Both MafA mRNA and protein levels increase in response to highglucose [34,36,147,148]. We have determined recently that thehigh glucose-dependent up-regulation of MafA is dependent onthe HBP and O-linked glycosylation of an unknown protein(s)

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Figure 5 Glucose regulation of MafA transcription requires O-GlcNAcmodification of proteins

Our recent findings suggest that low-glucose conditions (1–3 mM) down-regulate MafA levelsby decreasing MafA transcription. High concentrations of glucose (10–25 mM) stimulate MafAtranscription via O-GlcNAc of unknown transcription factor(s) (X) and/or other signallingproteins. Once MafA levels increase, a portion of it is ubiquitinated and degraded in aproteasome-dependent manner. The remaining MafA translocates to the nucleus and participatesin the glucose-dependent transcription of the insulin gene. Symbols: G, GlcNAc; ∼, insulinmRNA.

[36] (Figures 3 and 5). Our findings suggest that a transcriptionfactor regulating MafA expression may be O-GlcNAc-modifiedand thereby induces MafA expression [36] (Figure 5). Recently,biochemical studies revealed that Fox (Forkhead box) O1, FoxA2,Nkx2.2 (NK2 homeobox 2) and Pdx-1 bind directly to theMafA promoter and mediate MafA transcription [53,151,152].Bioinformatics analysis indicates that NeuroD1 may also bind tothe promoter of MafA [53]. Of these factors, Pdx-1 and NeuroD1are known to be O-GlcNAc-modified, which promotes theirbinding to the insulin promoter [28,32]. Previous findings suggestthat FoxO1, which has been shown to regulate MafA expression[152], is also O-GlcNAc-modified [153,154]. Therefore Pdx-1, NeuroD1 and FoxO1 may be crucial factors in regulatingthe high-glucose-dependent expression of MafA via O-linkedglycosylation.

MafA levels in β-cells also appear to be regulated by post-transcriptional mechanisms. Recently, GSK3 was demonstratedto be responsible for constitutively phosphorylating MafA atmultiple residues, leading to its destabilization and degradation bythe proteasome [155,156]. Phosphorylation of Ser61, Thr57, Thr53

and Ser49 by GSK3 causes MafA ubiquitination and degradation[156].

In addition to regulating MafA protein levels, phosphorylationhas been implicated in modulating the binding of MafA tothe insulin promoter [145,146] and to the CHOP promoterin pancreatic β-cells [132]. Furthermore, phosphorylation mayregulate the transactivation potential of MafA via recruitment ofthe co-activator P/CAF [156]. In the neuroretina, phosphorylation

of Ser14 and Ser65 within the transactivation domain of MafA hasbeen shown to be critical for activation of crystallin genes byMafA [157,158]. The importance of Ser14 and Ser65 within MafAfor induction of transcription from a MARE-containing reportergene was confirmed in another independent study [159]. TheMAPK p38 also has been shown to phosphorylate MafA at Thr57

and Thr113 within the transactivation domain. Furthermore, usingMS, Ser272 located in the C-terminus of MafA was demonstratedto be a substrate for p38 [160]. A mutant MafA protein lackingall three p38 phosphorylation sites was unable to induce thelens differentiation programme [160]. However, the exact roleof phosphorylation in regulating the transactivation capacity ofMafA in pancreatic β-cell remains to be determined.

The current findings suggest that enhanced production ofMafA under high-glucose conditions may regulate the glucose-dependent insulin gene transcription, whereas decreased pro-duction and proteasomal degradation of MafA probably allowsfor rapid inhibition of insulin transcription under low-glucoseconditions (Figure 5). This model assumes that β-cells mustup-regulate MafA expression before the induction of insulintranscription. However, it is currently unknown whether MafAexpression and MafA binding to the insulin promoter occursbefore the induction of glucose-dependent insulin transcription.

Chronic hyperglycaemia or glucotoxicity is a hallmark ofdiabetes, which leads to β-cell failure, including a loss of insulinexpression [84,86,161–163]. Several in vitro studies indicatethat one of the major factors contributing to decreased insulinexpression during glucotoxicity is decreased expression of MafAfollowed by its reduced binding to the insulin promoter [84–86,89,162–166]. These findings indicate that the loss of MafAmay cause or enhance the glucotoxic effects in β-cells duringhyperglycaemia. Consistent with this idea, MafA expression islost or reduced in several diabetic animal models, includingFoxO307 mice [152,167], insulin receptor L2 mutant mice[152,168], βDKO (β-cell double-knockout) mice lacking both theinsulin and IGF-1 (insulin-like growth factor 1) receptors [169],Pdx-1PBHNF6 [transgenic mice in which HNF6 (hepatic nuclearfactor 6) expression is maintained in postnatal islets] mice [170]and the NZO (New Zealand obese) mice [171]. Moreover, theMafA-knockout animals themselves also develop diabetes [129].Chronically increased levels of lipids (lipotoxicity) have also beenshown to reduce MafA levels by inhibiting MafA gene expression[89,161]. Exposure to pro-inflammatory cytokines also causesdown-regulation of MafA levels [172,173]. Down-regulation ofMafA levels is observed under various disease conditions andmay contribute to the development of diabetes.

CONCLUSIONS AND PERSPECTIVES

The transcription factors Pdx-1, NeuroD1 and MafA are majorregulators of insulin gene transcription and β-cell function ingeneral. These β-cell-specific transcription factors interact witheach other to tightly control insulin gene expression in a co-ordinated and synergistic manner (Figure 1). Although glucosemodulates the function of all three of these transcription factors,the mechanisms by which glucose regulates their function isdistinctive. In the case of Pdx-1, glucose appears to mainlyregulate the interaction of Pdx-1 with various proteins such asco-activators and co-repressors in a phosphorylation-dependentmanner in order to control β-cell-specific gene transcription(Figure 2). Nuclear localization as well as the transactivationcapability of Pdx-1 has been shown to be regulated by glucose(Figure 2). Glucose regulation of NeuroD1 involves changes inNeuroD1 subcellular localization. Although NeuroD1 is mainly

c© The Authors Journal compilation c© 2008 Biochemical Society

Glucose regulation of β-cell transcription 7

cytosolic on low glucose, exposure to high glucose causes O-linked glycosylation and phosphorylation of NeuroD1 and itstranslocation into the nucleus, where it can activate transcriptionin a co-ordinated manner (Figure 4). Interestingly, the expressionof MafA itself is responsive to changes in glucose levels.Transcription of MafA is only induced when high glucoseis present, whereas MafA transcription is down-regulated inresponse to low glucose (Figure 5). All three of these transcriptionfactors have been demonstrated to induce the expression of thenormally silent insulin gene in liver and other non-β-cells and aretherefore of therapeutic interest. Because of the major importanceof Pdx-1, NeuroD1 and MafA in pancreatic β-cell function, it isnot surprising that the mechanisms by which glucose regulatestheir function is complex and involves several pathways andsignals in addition to glucose. Detailed understanding of themechanisms by which changes in glucose levels modulatethe function of these β-cell-specific transcription factors maycontribute to the development of novel strategies to treat andprevent diabetes and its associated complications.

M. L. S. and N. L. V. are supported by an American Heart Association Predoctoral Fellowshipfrom Great Rivers Affiliate. S. O. is supported by Grant Number R01DK067581 from NIDDK(National Institute of Diabetes and Digestive and Kidney Disease) and 1-05-CD-15 fromthe American Diabetes Association. We apologize to all authors whose original work wewere unable to cite owing to space constraints.

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90 Hagman, D. K., Latour, M. G., Chakrabarti, S. K., Fontes, G., Amyot, J., Tremblay, C.,Semache, M., Lausier, J. A., Roskens, V., Mirmira, R. G. et al. (2008) Cyclical andalternating infusions of glucose and intralipid in rats inhibit insulin gene expression andPdx-1 binding in islets. Diabetes 57, 424–431

91 Yoshikawa, H., Tajiri, Y., Sako, Y., Hashimoto, T., Umeda, F. and Nawata, H. (2001)Effects of free fatty acids on β-cell functions: a possible involvement of peroxisomeproliferator-activated receptors α or pancreatic/duodenal homeobox. Metab. Clin. Exp.50, 613–618

92 Hussain, M. A. and Habener, J. F. (2000) Glucagon-like peptide 1 increasesglucose-dependent activity of the homeoprotein IDX-1 transactivating domain inpancreatic β-cells. Biochem. Biophys. Res. Commun. 274, 616–619

93 Stoffers, D. A., Kieffer, T. J., Hussain, M. A., Drucker, D. J., Bonner-Weir, S., Habener,J. F. and Egan, J. M. (2000) Insulinotropic glucagon-like peptide 1 agonists stimulateexpression of homeodomain protein IDX-1 and increase islet size in mouse pancreas.Diabetes 49, 741–748

94 Wang, X., Zhou, J., Doyle, M. E. and Egan, J. M. (2001) Glucagon-like peptide-1 causespancreatic duodenal homeobox-1 protein translocation from the cytoplasm to thenucleus of pancreatic β-cells by a cyclic adenosine monophosphate/protein kinaseA-dependent mechanism. Endocrinology 142, 1820–1827

95 Buteau, J., Roduit, R., Susini, S. and Prentki, M. (1999) Glucagon-like peptide-1promotes DNA synthesis, activates phosphatidylinositol 3-kinase and increasestranscription factor pancreatic and duodenal homeobox gene 1 (PDX-1) DNA bindingactivity in β (INS-1)-cells. Diabetologia 42, 856–864

96 Lee, J. E., Hollenberg, S. M., Snider, L., Turner, D. L., Lipnick, N. and Weintraub, H.(1995) Conversion of Xenopus ectoderm into neurons by NeuroD, a basichelix–loop–helix protein. Science 268, 836–844

97 Miyata, T., Maeda, T. and Lee, J. E. (1999) NeuroD is required for differentiation of thegranule cells in the cerebellum and hippocampus. Genes Dev. 13, 1647–1652

98 Schwab, M. H., Bartholomae, A., Heimrich, B., Feldmeyer, D., Druffel-Augustin, S.,Goebbels, S., Naya, F. J., Zhao, S., Frotscher, M., Tsai, M. J. and Nave, K. A. (2000)Neuronal basic helix–loop–helix proteins (NEX and BETA2/Neuro D) regulate terminalgranule cell differentiation in the hippocampus. J. Neurosci. 20, 3714–3724

99 Kim, W. Y., Fritzsch, B., Serls, A., Bakel, L. A., Huang, E. J., Reichardt, L. F., Barth, D. S.and Lee, J. E. (2001) NeuroD-null mice are deaf due to a severe loss of the inner earsensory neurons during development. Development 128, 417–426

100 Dumonteil, E., Laser, B., Constant, I. and Philippe, J. (1998) Differential regulation of theglucagon and insulin I gene promoters by the basic helix–loop–helix transcriptionfactors E47 and BETA2. J. Biol. Chem. 273, 19945–19954

101 Naya, F. J., Huang, H. P., Qiu, Y., Mutoh, H., DeMayo, F. J., Leiter, A. B. and Tsai, M. J.(1997) Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrinedifferentiation in BETA2/neuroD-deficient mice. Genes Dev. 11, 2323–2334

102 Huang, H. P., Chu, K., Nemoz-Gaillard, E., Elberg, D. and Tsai, M. J. (2002) Neogenesisof β-cells in adult BETA2/NeuroD-deficient mice. Mol. Endocrinol. 16, 541–551

103 Fajans, S. S., Bell, G. I. and Polonsky, K. S. (2001) Molecular mechanisms and clinicalpathophysiology of maturity-onset diabetes of the young. N. Engl. J. Med. 345,971–980

104 Malecki, M. T., Jhala, U. S., Antonellis, A., Fields, L., Doria, A., Orban, T., Saad, M.,Warram, J. H., Montminy, M. and Krolewski, A. S. (1999) Mutations in NEUROD1 areassociated with the development of type 2 diabetes mellitus. Nat. Genet. 23, 323–328

105 Cinek, O., Drevinek, P., Sumnik, Z., Bendlova, B., Sedlakova, P., Kolouskova, S.,Snajderova, M. and Vavrinec, J. (2003) NEUROD polymorphism Ala45Thr is associatedwith Type 1 diabetes mellitus in Czech children. Diabetes Res. Clin. Pract. 60, 49–56

106 Mochizuki, M., Amemiya, S., Kobayashi, K., Kobayashi, K., Ishihara, T., Aya, M., Kato,K., Kasuga, A. and Nakazawa, S. (2002) The association of Ala45Thr polymorphism inNeuroD with child-onset Type 1a diabetes in Japanese. Diabetes Res. Clin. Pract. 55,11–17

107 Chu, K. and Tsai, M. J. (2005) Neuronatin, a downstream target of BETA2/NeuroD1 in thepancreas, is involved in glucose-mediated insulin secretion. Diabetes 54, 1064–1073

108 Kim, J. W., Seghers, V., Cho, J. H., Kang, Y., Kim, S., Ryu, Y., Baek, K., Aguilar-Bryan, L.,Lee, Y. D., Bryan, J. and Suh-Kim, H. (2002) Transactivation of the mouse sulfonylureareceptor I gene by BETA2/NeuroD. Mol. Endocrinol. 16, 1097–1107

109 Ishizuka, N., Minami, K., Okumachi, A., Okuno, M. and Seino, S. (2007) Induction byNeuroD of the components required for regulated exocytosis. Biochem. Biophys.Res. Commun. 354, 271–277

110 Martin, C. C., Svitek, C. A., Oeser, J. K., Henderson, E., Stein, R. and O’Brien, R. M.(2003) Upstream stimulatory factor (USF) and neurogenic differentiation/β-cell E boxtransactivator 2 (NeuroD/BETA2) contribute to islet-specific glucose-6-phosphatasecatalytic-subunit-related protein (IGRP) gene expression. Biochem. J. 371, 675–686

111 Moates, J. M., Nanda, S., Cissell, M. A., Tsai, M. J. and Stein, R. (2003) BETA2 activatestranscription from the upstream glucokinase gene promoter in islet β-cells and gutendocrine cells. Diabetes 52, 403–408

112 Mutoh, H., Fung, B. P., Naya, F. J., Tsai, M. J., Nishitani, J. and Leiter, A. B. (1997) Thebasic helix–loop–helix transcription factor BETA2/NeuroD is expressed in mammalianenteroendocrine cells and activates secretin gene expression. Proc. Natl. Acad.Sci. U.S.A. 94, 3560–3564

113 Glick, E., Leshkowitz, D. and Walker, M. D. (2000) Transcription factor BETA2 actscooperatively with E2A and PDX1 to activate the insulin gene promoter. J. Biol. Chem.275, 2199–2204

114 Qiu, Y., Sharma, A. and Stein, R. (1998) p300 mediates transcriptional stimulation by thebasic helix–loop–helix activators of the insulin gene. Mol. Cell. Biol. 18, 2957–2964

115 Sharma, A., Moore, M., Marcora, E., Lee, J. E., Qiu, Y., Samaras, S. and Stein, R. (1999)The NeuroD1/BETA2 sequences essential for insulin gene transcription colocalizewith those necessary for neurogenesis and p300/CREB binding protein binding.Mol. Cell. Biol. 19, 704–713

116 Kim, J. Y., Chu, K., Kim, H. J., Seong, H. A., Park, K. C., Sanyal, S., Takeda, J., Ha, H.,Shong, M., Tsai, M. J. and Choi, H. S. (2004) Orphan nuclear receptor smallheterodimer partner, a novel corepressor for a basic helix–loop–helix transcription factorBETA2/neuroD. Mol. Endocrinol. 18, 776–790

117 Marcus, E. A., Kintner, C. and Harris, W. (1998) The role of GSK3β in regulatingneuronal differentiation in Xenopus laevis. Mol. Cell. Neurosci. 12, 269–280

118 Qiu, Y., Guo, M., Huang, S. and Stein, R. (2004) Acetylation of the BETA2 transcriptionfactor by p300-associated factor is important in insulin gene expression. J. Biol. Chem.279, 9796–9802

119 Lawrence, M., Shao, C., Duan, L., McGlynn, K. and Cobb, M. H. (2008) The proteinkinases ERK1/2 and their roles in pancreatic β cells. Acta Physiol. 192, 11–17

120 Gaudilliere, B., Konishi, Y., de la Iglesia, N., Yao, G. and Bonni, A. (2004) ACaMKII-NeuroD signaling pathway specifies dendritic morphogenesis. Neuron 41,229–241

121 Blank, V. and Andrews, N. C. (1997) The Maf transcription factors: regulators ofdifferentiation. Trends Biochem. Sci. 22, 437–441

122 Motohashi, H., Shavit, J. A., Igarashi, K., Yamamoto, M. and Engel, J. D. (1997) Theworld according to Maf. Nucleic Acids Res. 25, 2953–2959

123 Ogino, H. and Yasuda, K. (1998) Induction of lens differentiation by activation of a bZIPtranscription factor, L-Maf. Science 280, 115–118

124 Reza, H. M. and Yasuda, K. (2004) Roles of Maf family proteins in lens development.Dev. Dyn. 229, 440–448

125 Matsuoka, T. A., Artner, I., Henderson, E., Means, A., Sander, M. and Stein, R. (2004)The MafA transcription factor appears to be responsible for tissue-specific expression ofinsulin. Proc. Natl. Acad. Sci. U.S.A. 101, 2930–2933

126 Kato, T., Shimano, H., Yamamoto, T., Yokoo, T., Endo, Y., Ishikawa, M., Matsuzaka, T.,Nakagawa, Y., Kumadaki, S., Yahagi, N. et al. (2006) Granuphilin is activated bySREBP-1c and involved in impaired insulin secretion in diabetic mice. Cell Metab. 4,143–154

127 Tsuchiya, M., Yoshida, T., Taniguchi, S., Yasuda, K., Maeda, A., Hayashi, A., Tanaka, J.,Shigemoto, M., Nitta, K. and Tsuchiya, K. (2007) In vivo suppression of mafA mRNAwith siRNA and analysis of the resulting alteration of the gene expression profile inmouse pancreas by the microarray method. Biochem. Biophys. Res. Commun. 356,129–135

128 Wang, H., Brun, T., Kataoka, K., Sharma, A. J. and Wollheim, C. B. (2007) MAFA controlsgenes implicated in insulin biosynthesis and secretion. Diabetologia 50, 348–358

129 Zhang, C., Moriguchi, T., Kajihara, M., Esaki, R., Harada, A., Shimohata, H., Oishi, H.,Hamada, M., Morito, N., Hasegawa, K. et al. (2005) MafA is a key regulator ofglucose-stimulated insulin secretion. Mol. Cell. Biol. 25, 4969–4976

130 Samaras, S. E., Zhao, L., Means, A., Henderson, E., Matsuoka, T. A. and Stein, R. (2003)The islet β cell-enriched RIPE3b1/Maf transcription factor regulates pdx-1 expression.J. Biol. Chem. 278, 12263–12270

131 Nomura, S., Nakamura, T., Hashimoto, T., Nishio, Y., Maegawa, H., Kudo, M. andKashiwagi, A. (2006) MafA differentiates rat intestinal cells into insulin-producing cells.Biochem. Biophys. Res. Commun. 349, 136–143

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10 S. S. Andrali and others

132 Lawrence, M. C., McGlynn, K., Naziruddin, B., Levy, M. F. and Cobb, M. H. (2007)Differential regulation of CHOP-10/GADD153 gene expression by MAPK signaling inpancreatic β-cells. Proc. Natl. Acad. Sci. U.S.A. 104, 11518–11525

133 Nishimura, W., Kondo, T., Salameh, T., El Khattabi, I., Dodge, R., Bonner-Weir, S. andSharma, A. (2006) A switch from MafB to MafA expression accompanies differentiationto pancreatic β-cells. Dev. Biol. 293, 526–539

134 Tsuchiya, M., Taniguchi, S., Yasuda, K., Nitta, K., Maeda, A., Shigemoto, M. andTsuchiya, K. (2006) Potential roles of large mafs in cell lineages and developingpancreas. Pancreas 32, 408–416

135 Artner, I., Blanchi, B., Raum, J. C., Guo, M., Kaneko, T., Cordes, S., Sieweke, M. andStein, R. (2007) MafB is required for islet β cell maturation. Proc. Natl. Acad. Sci. U.S.A.104, 3853–3858

136 Kaneto, H., Matsuoka, T. A., Nakatani, Y., Miyatsuka, T., Matsuhisa, M., Hori, M. andYamasaki, Y. (2005) A crucial role of MafA as a novel therapeutic target for diabetes.J. Biol. Chem. 280, 15047–15052

137 Muniappan, L. and Ozcan, S. (2007) Induction of insulin secretion in engineered livercells by nitric oxide. BMC Physiol. 7, 11

138 Crowe, D. T. and Tsai, M. J. (1989) Mutagenesis of the rat insulin II 5′-flanking regiondefines sequences important for expression in HIT cells. Mol. Cell. Biol. 9, 1784–1789

139 German, M. S. and Wang, J. (1994) The insulin gene contains multiple transcriptionalelements that respond to glucose. Mol. Cell. Biol. 14, 4067–4075

140 Hwung, Y. P., Gu, Y. Z. and Tsai, M. J. (1990) Cooperativity of sequence elementsmediates tissue specificity of the rat insulin II gene. Mol. Cell. Biol. 10, 1784–1788

141 Shieh, S. Y. and Tsai, M. J. (1991) Cell-specific and ubiquitous factors are responsiblefor the enhancer activity of the rat insulin II gene. J. Biol. Chem. 266, 16708–16714

142 Whelan, J., Poon, D., Weil, P. A. and Stein, R. (1989) Pancreatic β-cell-type-specificexpression of the rat insulin II gene is controlled by positive and negative cellulartranscriptional elements. Mol. Cell. Biol. 9, 3253–3259

143 Sharma, A., Fusco-DeMane, D., Henderson, E., Efrat, S. and Stein, R. (1995) The role ofthe insulin control element and RIPE3b1 activators in glucose-stimulated transcriptionof the insulin gene. Mol. Endocrinol. 9, 1468–1476

144 Sharma, A. and Stein, R. (1994) Glucose-induced transcription of the insulin gene ismediated by factors required for β-cell-type-specific expression. Mol. Cell. Biol. 14,871–879

145 Zhao, L., Cissell, M. A., Henderson, E., Colbran, R. and Stein, R. (2000) The RIPE3b1activator of the insulin gene is composed of a protein(s) of approximately 43 kDa, whoseDNA binding activity is inhibited by protein phosphatase treatment. J. Biol. Chem. 275,10532–10537

146 Matsuoka, T., Zhao, L. and Stein, R. (2001) The DNA binding activity of the RIPE3b1transcription factor of insulin appears to be influenced by tyrosine phosphorylation.J. Biol. Chem. 276, 22071–22076

147 Kajihara, M., Sone, H., Amemiya, M., Katoh, Y., Isogai, M., Shimano, H., Yamada, N. andTakahashi, S. (2003) Mouse MafA, homologue of zebrafish somite Maf 1, contributes tothe specific transcriptional activity through the insulin promoter. Biochem. Biophys.Res. Commun. 312, 831–842

148 Zhao, L., Guo, M., Matsuoka, T. A., Hagman, D. K., Parazzoli, S. D., Poitout, V. andStein, R. (2005) The islet β cell-enriched MafA activator is a key regulator of insulingene transcription. J. Biol. Chem. 280, 11887–11894

149 Aramata, S., Han, S. I., Yasuda, K. and Kataoka, K. (2005) Synergistic activation of theinsulin gene promoter by the β-cell enriched transcription factors MafA, Beta2, andPdx1. Biochim. Biophys. Acta 1730, 41–46

150 Lawrence, M. C., McGlynn, K., Park, B. H. and Cobb, M. H. (2005) ERK1/2-dependentactivation of transcription factors required for acute and chronic effects of glucose on theinsulin gene promoter. J. Biol. Chem. 280, 26751–26759

151 Doyle, M. J., Loomis, Z. L. and Sussel, L. (2007) Nkx2.2-repressor activity is sufficientto specify α-cells and a small number of β-cells in the pancreatic islet. Development134, 515–523

152 Kitamura, Y. I., Kitamura, T., Kruse, J. P., Raum, J. C., Stein, R., Gu, W. and Accili, D.(2005) FoxO1 protects against pancreatic β cell failure through NeuroD and MafAinduction. Cell Metab. 2, 153–163

153 Kuo, M., Zilberfarb, V., Gangneux, N., Christeff, N. and Issad, T. (2008) O-GlcNAcmodification of FoxO1 increases its transcriptional activity: a role in the glucotoxicityphenomenon? Biochimie 90, 679–685

154 Housley, M. P., Rodgers, J. T., Udeshi, N. D., Kelly, T. J., Shabanowitz, J., Hunt, D. F.,Puigserver, P. and Hart, G. W. (2008) O-GlcNAc regulates FoxO activation in response toglucose. J. Biol. Chem. 283, 16283–16292

155 Han, S. I., Aramata, S., Yasuda, K. and Kataoka, K. (2007) MafA stability in pancreatic β

cells is regulated by glucose and is dependent on its constitutive phosphorylation atmultiple sites by glycogen synthase kinase 3. Mol. Cell. Biol. 27, 6593–6605

156 Rocques, N., Abou Zeid, N., Sii-Felice, K., Lecoin, L., Felder-Schmittbuhl, M. P.,Eychene, A. and Pouponnot, C. (2007) GSK-3-mediated phosphorylation enhancesMaf-transforming activity. Mol. Cell. 28, 584–597

157 Benkhelifa, S., Provot, S., Nabais, E., Eychene, A., Calothy, G. and Felder-Schmittbuhl,M. P. (2001) Phosphorylation of MafA is essential for its transcriptional and biologicalproperties. Mol. Cell. Biol. 21, 4441–4452

158 Benkhelifa, S., Provot, S., Lecoq, O., Pouponnot, C., Calothy, G. andFelder-Schmittbuhl, M. P. (1998) mafA, a novel member of the maf proto-oncogenefamily, displays developmental regulation and mitogenic capacity in avian neuroretinacells. Oncogene. 17, 247–254

159 Nishizawa, M., Kataoka, K. and Vogt, P. K. (2003) MafA has strong cell transformingability but is a weak transactivator. Oncogene 22, 7882–7890

160 Sii-Felice, K., Pouponnot, C., Gillet, S., Lecoin, L., Girault, J. A., Eychene, A. andFelder-Schmittbuhl, M. P. (2005) MafA transcription factor is phosphorylated by p38MAP kinase. FEBS Lett. 579, 3547–3554

161 Poitout, V., Hagman, D., Stein, R., Artner, I., Robertson, R. P. and Harmon, J. S. (2006)Regulation of the insulin gene by glucose and fatty acids. J. Nutr. 136, 873–876

162 Robertson, R., Zhou, H., Zhang, T. and Harmon, J. S. (2007) Chronic oxidativestress as a mechanism for glucose toxicity of the β cell in type 2 diabetes.Cell. Biochem. Biophys. 48, 139–146

163 Robertson, R. P., Harmon, J., Tran, P. O., Tanaka, Y. and Takahashi, H. (2003) Glucosetoxicity in β-cells: type 2 diabetes, good radicals gone bad, and the glutathioneconnection. Diabetes 52, 581–587

164 Harmon, J. S., Stein, R. and Robertson, R. P. (2005) Oxidative stress-mediated,post-translational loss of MafA protein as a contributing mechanism to loss of insulingene expression in glucotoxic β cells. J. Biol. Chem. 280, 11107–11113

165 Olson, L. K., Sharma, A., Peshavaria, M., Wright, C. V., Towle, H. C., Rodertson, R. P.and Stein, R. (1995) Reduction of insulin gene transcription in HIT-T15 β cellschronically exposed to a supraphysiologic glucose concentration is associated with lossof STF-1 transcription factor expression. Proc. Natl. Acad. Sci. U.S.A. 92, 9127–9131

166 Park, K. G., Lee, K. M., Seo, H. Y., Suh, J. H., Kim, H. S., Wang, L., Won, K. C., Lee,H. W., Park, J. Y., Lee, K. U. et al. (2007) Glucotoxicity in the INS-1 rat insulinoma cellline is mediated by the orphan nuclear receptor small heterodimer partner. Diabetes 56,431–437

167 Nakae, J., Biggs, 3rd, W. H., Kitamura, T., Cavenee, W. K., Wright, C. V., Arden, K. C. andAccili, D. (2002) Regulation of insulin action and pancreatic β-cell function by mutatedalleles of the gene encoding forkhead transcription factor Foxo1. Nat. Genet. 32,245–253

168 Okamoto, H., Nakae, J., Kitamura, T., Park, B. C., Dragatsis, I. and Accili, D. (2004)Transgenic rescue of insulin receptor-deficient mice. J. Clin. Invest. 114, 214–223

169 Ueki, K., Okada, T., Hu, J., Liew, C. W., Assmann, A., Dahlgren, G. M., Peters, J. L.,Shackman, J. G., Zhang, M., Artner, I. et al. (2006) Total insulin and IGF-I resistance inpancreatic β cells causes overt diabetes. Nat. Genet. 38, 583–588

170 Tweedie, E., Artner, I., Crawford, L., Poffenberger, G., Thorens, B., Stein, R., Powers,A. C. and Gannon, M. (2006) Maintenance of hepatic nuclear factor 6 in postnatal isletsimpairs terminal differentiation and function of β-cells. Diabetes 55, 3264–3270

171 Jurgens, H. S., Neschen, S., Ortmann, S., Scherneck, S., Schmolz, K., Schuler, G.,Schmidt, S., Bluher, M., Klaus, S., Perez-Tilve, D. et al. (2007) Development of diabetesin obese, insulin-resistant mice: essential role of dietary carbohydrate in β celldestruction. Diabetologia 50, 1481–1489

172 Chin-Chance, C. V., Newman, M. V., Aronovitz, A., Blomeier, H., Kruger, J., Lee, E. J. andLowe, Jr, W. L. (2006) Role of the mitogen-activated protein kinases incytokine-mediated inhibition of insulin gene expression. J. Invest. Med. 54, 132–142

173 Oetjen, E., Blume, R., Cierny, I., Schlag, C., Kutschenko, A., Kratzner, R., Stein, R. andKnepel, W. (2007) Inhibition of MafA transcriptional activity and human insulin genetranscription by interleukin-1β and mitogen-activated protein kinase kinase kinase inpancreatic islet β cells. Diabetologia 50, 1678–1687

Received 23 May 2008/19 June 2008; accepted 2 July 2008Published on the Internet 12 September 2008, doi:10.1042/BJ20081029

c© The Authors Journal compilation c© 2008 Biochemical Society