Selective Insulin Signaling through A and B Insulin Receptors Regulates Transcription of Insulin and Glucokinase Genes in Pancreatic β Cells

Molecular Cell, Vol. 7, 559–570, March, 2001, Copyright 2001 by Cell Press

Selective Insulin Signaling through A and B InsulinReceptors Regulates Transcription of Insulinand Glucokinase Genes in Pancreatic b Cells

ceptors as the primary target, include signaling via mito-gen-activated protein (MAP) kinases and phosphoinosi-tol-3 kinase (PI3K). The insulin receptor (IR), the firststep in these cascades, exists in two isoforms as a resultof alternative mRNA splicing of the 11th exon of the insulin

Barbara Leibiger,*§ Ingo B. Leibiger,*§‖Tilo Moede,* Sabine Kemper,*Rohit N. Kulkarni,† C. Ronald Kahn,†Lina Moitoso de Vargas,‡ and Per-Olof Berggren**The Rolf Luft Center for Diabetes ResearchDepartment of Molecular Medicine proreceptor transcript (Seino et al., 1989). The A type

(IR-A), or Ex112 (Ullrich et al., 1985), lacks whereas theKarolinska InstitutetS-171 76 Stockholm B type (IR-B), or Ex111 (Ebina et al., 1985), contains

the respective sequence coding for 12 amino acids inSweden†Research Division the C terminus of the a chain of the receptor. To date,

no insulin-induced effect has been reported that dis-Joslin Diabetes Center andDepartment of Medicine criminates signaling via A- and B-type receptors. In fact,

the functional significance of these IR isoforms remainsHarvard Medical SchoolBoston, Massachusetts 02215 unclear.

Recent studies have shown that the insulin-producing‡Department of MedicineNew England Medical Center and pancreatic b cell is a target for insulin action, with insulin

effects on transcription, translation, Ca21 flux, and exo-Tufts University School of MedicineBoston, Massachusetts 02111 cytosis (Leibiger et al., 1998a, 2000; Xu and Rothenberg,

1998; Xu et al., 1998; Aspinwall et al., 1999; Kulkarni etal., 1999a). In an animal model with a b cell–specificknockout for IR, there is a decrease in glucose-stimu-Summarylated insulin release and a decrease in the insulin contentof the cell (Kulkarni et al., 1999a). In addition, disruptionInsulin signaling is mediated by a complex network ofof insulin signaling in the b cell at the level of insulindiverging and converging pathways, with alternativereceptor substrate (IRS)-1 (Kulkarni et al., 1999b) orproteins and isoforms at almost every step in the pro-IRS-2 (Withers et al., 1998) leads to altered growth andcess. We show here that insulin activates the tran-function of the b cell. Consequently, insulin resistancescription of its own gene and that of the b cell glucoki-may not only affect the function of the “classical” insulinnase gene (bGK) by different mechanisms. Whereastarget tissues muscle, fat and liver, but also apply toinsulin gene transcription is promoted by signalingthe pancreatic b cell and thereby affect b cell function.through insulin receptor A type (Ex112), PI3K class

In the present study, we show selective insulin signal-Ia, and p70s6k, insulin stimulates the bGK gene bying via the two isoforms of the insulin receptor (i.e., IR-Asignaling via insulin receptor B type (Ex111), PI3Kand IR-B) in the pancreatic b cell. Insulin that is secretedclass II–like activity, and PKB (c-Akt). Our data provideby b cells upon glucose stimulation up-regulates tran-evidence for selectivity in insulin action via the twoscription of its own gene as well as that of the b cellisoforms of the insulin receptor, the molecular basistranscription unit of the glucokinase (bGK) gene in anbeing preferential signaling through different PI3K andautocrine feedback loop. More interestingly, while theprotein kinases.insulin gene is activated by insulin signaling via IR-Ainvolving PI3K class Ia, p70 s6 kinase (p70s6k), andIntroductionCa21/calmodulin dependent kinases, insulin-stimulatedbGK transcription occurs via IR-B, PI3K class II–likeUnderstanding selectivity in signal transduction is oneactivity, and protein kinase B (PKB/c-Akt). These resultsof the most challenging tasks in current cell biology.provide evidence that signaling via either IR-A or IR-BOver the years, insulin signaling has served as one ofand the subsequent activation of different classes ofthe model examples in hormone-induced signal trans-PI3K and protein kinases (i.e., p70s6k and PKB) repre-duction. Malfunction of insulin signaling, referred to assent a mechanism for selective insulin action. We fur-insulin resistance, is one of the major causes of type 2thermore show a preferential activation of p70s6k anddiabetes mellitus (non-insulin-dependent diabetes mel-PKB as a result of insulin signaling via IR-A and IR-B,litus), the most common metabolic disorder in man.respectively, in insulin-producing and non-insulin-pro-Insulin has been shown to exhibit pleiotropic effectsducing cells.involving mitogenic and/or metabolic events. Moreover,

the effect of insulin is tissue as well as developmentdependent. The fact that insulin may transduce its signal Results and Discussionthrough a variety of pathways has been discussed inextensive detail (White and Kahn, 1994). The two major Glucose Activates Glucokinase Gene Transcription

via Secreted Insulinpathways described to date, which employ insulin re-Insulin, secreted upon glucose stimulation, is a key fac-tor in the up-regulation of insulin gene transcription (Lei-‖ To whom correspondence should be addressed (e-mail: ingo@biger et al., 1998a). The promoters of both the insulinenk.ks.se).

§These authors contributed equally to this work. gene and the bGK gene contain many similar cis ele-

Molecular Cell560

ments (Shelton et al., 1992; Leibiger et al., 1994a, 1994b; insulin gene, the addition of 20 mU per ml was required toWatada et al., 1996). To test whether transcription of gain an effect on bGK promoter activation (Figure 2E).bGK is regulated by similar mechanisms as the insulin Stimulation with 5 mU of insulin per ml of culture mediumgene, we studied the role of glucose and insulin in regu- for 5 min led to an bGK promoter–driven increase inlation of bGK mRNA steady-state levels. Stimulation of GFP fluorescence in isolated primary pancreatic b cellscultured islets (Figure 1A) or insulin-producing HIT-T15 (Figure 2C), HIT cells, and intact pancreatic islets (datacells with 16.7 mM glucose led to an increase in bGK not shown).mRNA levels 60 min following start of stimulation. This is Thus, our data support the view that the insulin genesimilar in time course to the effect of glucose to stimulate and the bGK gene are both stimulated by insulin se-insulin mRNA levels (Leibiger et al., 1998a, 1998b). creted in response to glucose. Interestingly, a higher

To define in more detail the dynamics of bGK mRNA, concentration of insulin is needed to activate bGK tran-we analyzed the half-life time, stability, and transcrip- scription when compared with the insulin gene.tional rate of the bGK mRNA pool. As shown in Figure1B, the half-life time of bGK mRNA was z60 min and Insulin-Stimulated Glucokinase Gene Transcriptionwas not changed in the presence or absence of glucose. Utilizes Signal Transduction, which Is DifferentOn the other hand, stimulation of HIT cells with 16.7 mM from that of the Insulin Geneglucose led to an increase in bGK gene transcripts as Our studies on insulin-stimulated insulin gene transcrip-early as 15 min and reached a maximum of transcrip- tion have shown the involvement of PI3K, p70s6k, andtional activity at 30 min in a nuclear run-off assay (Figure Ca21/calmodulin-dependent kinase(s) in the signaling1C). This effect of glucose on bGK transcription initiation cascade (Leibiger et al., 1998a). Because previous datawas also observed in normal pancreatic islets (Figure from others and our laboratory suggest that insulin- and1D). To further corroborate these data, we established bGK-promoters can bind the same transcription factorsa reporter gene assay using the bGK promoter coupled (Shelton et al., 1992; Leibiger et al., 1994a, 1994b; Wa-to the green fluorescent protein (GFP) (prbGK.GFP). We tada et al., 1996) and both genes respond positively toused the rat bGK promoter fragment up to nucleotide many of the same stimuli (glucose, insulin, secreta-2278, since this has been shown to contain all cis ele- gogs) at the level of transcription, we questioned whetherments responsible for both glucose-dependent and cell- both genes might be regulated by the same signalingtype-specific transcriptional control (Jetton et al., 1994, pathway.1998). Stimulation with 16.7 mM glucose led to an in- To test whether the same protein kinases that arecrease in bGK promoter–driven GFP fluorescence in HIT involved in insulin-triggered insulin gene transcriptioncells, isolated primary pancreatic b cells, and intact pan- contribute to insulin-triggered transcription of bGK, wecreatic islets (Figure 1E). As with the nuclear run-off studied the effect of pharmacological inhibitors on insu-assay, the dynamics of the activation of bGK promoter– lin-stimulated bGK promoter activity (Figure 3). We com-driven GFP expression were similar, if not identical, to bined insulin stimulation (5 mU/ml for 5 min at substimu-those of the glucose-stimulated insulin gene promoter latory glucose concentrations) with the cotreatment of(Leibiger et al., 1998a, 1998b).

islet cells and HIT cells with inhibitors of protein kinaseTo determine whether glucose metabolism per se or

C (PKC; 150 nM bisindolylmalemide I [BIM]), PI3K (25secreted insulin is a requirement for the up-regulation

mM LY294002 [LY]), p70s6k (10 nM rapamycin [rap]),of bGK transcription, we investigated the effect of insulin

MAP kinases Erk1/2 (20 mM PD98059 [PD9]) and p38/secretagogues on bGK mRNA steady-state levels andRK/SAPK2a 1 SAPK1/JNK (10 mM PD169316 [PD1]), IR

bGK promoter–driven GFP expression. Insulin secreta-tyrosine kinase (100 mM HNMPA-(AM)3 [HNMPA]), andgogues, like KCl or the sulfonylurea compound gliben-Ca21/calmodulin-dependent kinase II (CaMKII; 400 nMclamide, stimulate insulin secretion by depolarizing theautocamtide-2 related inhibitory peptide [AC]). The effi-b cell plasma membrane and provoking influx of extra-ciency of these inhibitors was verified by the respectivecellular Ca21 through voltage-gated L-type Ca21 chan-protein kinase assays in cell lysates of inhibitor-treatednels (reviewed in Berggren and Larsson, 1994). Asand nontreated cells following glucose/insulin stimula-shown in Figure 2, stimulation with either 50 mM KCl ortion (data not shown). In agreement with the data on1 mM glibenclamide for 5 min, at substimulatory glucoseinsulin-stimulated insulin gene transcription, insulin-concentrations, led to an increase in bGK mRNA steady-stimulated bGK transcription was not sensitive to inhibi-state levels (Figure 2A) and to an elevation in bGK pro-tion of PKC or MAP kinases Erk1/2 and p38 but wasmoter–driven GFP expression (Figure 2C). Alternatively,sensitive to inhibition of IR tyrosine kinase by HNMPA-preventing stimulus-induced insulin secretion by block-(AM)3 (Figure 3A). However, to our surprise, insulin-stim-ing L-type Ca21 channels using nifedipine abolished up-ulated bGK transcription was not inhibited by LY294002,regulation of bGK mRNA levels (Figure 2B).rapamycin, or autocamtide-2 related inhibitory peptide,We next studied the effect of exogenously adminis-suggesting that signaling via PI3K/p70s6k and via CaM-tered insulin on bGK mRNA steady-state levels and bGKKII, respectively, is not involved (Figures 3A and 3B).promoter–driven GFP expression at substimulatory glu-To further confirm that insulin stimulates insulin genecose concentrations (Figures 2D and 2E). Addition oftranscription and bGK transcription using different signal-only 50 mU of insulin per ml to fully supplemented cultureing pathways, we established a technique that allowedmedium was sufficient to evoke bGK mRNA levels inmonitoring of insulin and bGK promoter activities simul-pancreatic islets (Figure 2D). Interestingly, a more care-taneously in the same cell. In addition to prbGK.GFP,ful comparison of the necessary amounts of exogenouswe generated an expression construct where the ratinsulin to trigger promoter activities revealed that in-

stead of 5–10 mU of insulin per ml, as is the case with the insulin I promoter (2410/11 bp) controlled the expres-

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Figure 1. Effect of Glucose on bGK mRNA Steady-State Levels, Transcription Initiation, and mRNA Stability

(A) Elevation of bGK mRNA steady-state levels in isolated islets after stimulation with 16.7 mM glucose (15 min).(B) Dynamics of bGK mRNA stability in islet cells at 3 mM glucose (closed squares) and after stimulation with 16.7 mM glucose for 15 min(open squares). Actinomycin D (5 mg/ml) was present all the time under nonstimulatory conditions (closed squares), whereas in the case ofstimulation (open squares) the inhibitor was added 45 min after start of stimulation. In (A) and (B), bGK mRNA values are presented aspercentages of mRNA levels of the nonstimulated control at minute 0 (given as 100%).(C and D) Dynamics of bGK transcription initiation in response to glucose stimulation in HIT cells (C) and isolated islets (D). Transcriptioninitiation was studied by nuclear run-off analysis. Elevation of RNA levels in stimulated cells is shown as the percentage of RNA levels of thenonstimulated control (given as 100%). In (A)–(D), all data are shown as mean values 6 S.E. (n 5 3).(E) On-line monitoring of glucose-stimulated bGK promoter–driven GFP expression in transfected HIT-T15 cells, islet cells, and whole islets.Representative images of HIT cells (n 5 40), islet cells (n 5 40), and islets (n 5 3) are shown 60 and 240 min after start of glucose stimulation.The pseudo-color images were created by converting the original “gray-scale” data using Isee software; the fluorescence increases from blueto red. Scale bars, 10 mm.

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Figure 2. Effect of Secretagogues, Voltage-Dependent L-Type Ca21 Channel Blockers,and Exogenous Insulin on Endogenous bGKmRNA Levels and bGK Promoter–Driven GFPExpression

(A) Elevation of endogenous bGK mRNA lev-els in cultured pancreatic islets in responseto stimulation for 5 min with either 50 mM KCl(KCl) or 1 mM glibenclamide (glib) at 3 mMglucose.(B) Elevation of endogenous bGK mRNA lev-els in islet cells in response to stimulation for15 min with 16.7 mM glucose with or without10 mM nifedipine (nif). In (A) and (B), data areshown as mean values 6 S.E. (n 5 3), andamounts of bGK mRNA are presented as thepercentage of mRNA levels of the nonstimu-lated control (given as 100%).(C) On-line monitoring of bGK promoter–driven GFP expression in islet cells. Islet cellswere transfected with prbGK.GFP (bGK) orwith pRcCMV.GFP (CMV) as control and incu-bated with 16.7 mM glucose, 50 mM KCl, 1mM glibenclamide (glib) or 5 mU/ml insulin(ins). Data are shown as mean values 6 S.E.(n 5 8).(D and E) Effect of increasing concentrationsof insulin added to the culture medium for 5min, on (D) endogenous bGK mRNA levelsin isolated pancreatic islets and on (E) bGKpromoter–driven GFP expression and insulinpromoter–driven DsRed expression in trans-fected HIT cells. In (D), amounts of bGKmRNA are presented as the percentage ofmRNA levels of nonstimulated control (givenas 100%), and data are shown as meanvalues 6 S.E. (n 5 3). In (E), HIT cells werecotransfected with prbGK.GFP (open bars)and prIns1.DsRed (closed bars) and stimu-lated for 5 min with the indicated amounts ofexogenous insulin. On-line monitoring dataare presented as the ratio of fluorescenceobtained at minutes 240 and 60 and representmean values 6 S.E. (n 5 7).

sion of the red fluorescent protein DsRed (Matz et al., ploying a signaling pathway that is different from thatutilized by the insulin promoter.1999), prIns1.DsRed. Because of their different excita-

tion and emission profiles, the signals generated by the Besides signaling via the MAP kinase and the PI3K/mTOR/p70s6k pathways, insulin has been shown to ex-two fluorescent proteins can be measured directly in

the same cell. Following cotransfection of islet cells and ert its effect via the activation of PKB(c-Akt) (Coffer etal., 1998). To test whether stimulation with either glucoseHIT cells with prbGK.GFP and prIns1.DsRed, insulin-

stimulated insulin and bGK promoter activities were or insulin leads to the activation of PKB in pancreatic bcells, we studied PKB activity following stimulation withmonitored as DsRed and GFP fluorescence, respec-

tively. As shown in Figure 3C, stimulation with 5 mU/ either 16.7 mM glucose or 5 mU of insulin/ml. As shownin Figure 4, PKB activation was observed 5 min followingml insulin led to an elevation in both DsRed and GFP

fluorescence, as expected. This increase in fluores- stimulation with 16.7 mM glucose (Figure 4A) and 2 minfollowing stimulation with 5 mU of insulin/ml, at substim-cence was abolished when the cells were treated with

HNMPA-(AM)3, thereby blocking the tyrosine kinase ac- ulatory glucose concentrations (Figure 4B). Preventionof glucose-induced insulin secretion by treatment oftivity of IRs. By combining insulin stimulation with phar-

macological inhibitors of either PI3K (LY294002), p70s6k insulin-producing cells with the L-type Ca21 channelblocker nifedipine abolished glucose-induced activation(rapamycin), or CaMKII (autocamtide-2 related inhibitory

peptide), we show that activation of the insulin promoter of PKB, as did inhibition of insulin signaling by HNMPA-(AM)3 (data not shown). These data suggest that PKBis abolished (no increase in DsRed fluorescence). On

the other hand, no effect on insulin-stimulated bGK pro- is activated in response to glucose-stimulated insulinsecretion. Because of the lack of a selective pharmaco-moter activity (increase in GFP fluorescence) was ob-

served in the same cell (Figure 3C). logical inhibitor of PKB, we tested its involvement ininsulin-stimulated bGK gene transcription by transientlyThus, insulin activates the bGK promoter by em-

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Figure 3. Effect of Various Protein Kinase Inhibitors on Insulin-Stimulated Insulin and bGK Promoter Activity and Endogenous bGK mRNALevels

(A) On-line monitoring of insulin promoter–driven (closed bars) and bGK promoter–driven (open bars) GFP expression in transfected islet cells.Data are presented as the ratio of fluorescence obtained at minutes 240 and 60 and represent mean values 6 S.E. (n 5 10).(B) Amounts of bGK mRNA are presented as the percentage of mRNA levels of nonstimulated control (given as 100%). Data are shown asmean values 6 S.E. (n 5 3).(C) HIT cells were cotransfected with prbGK.GFP (open bars) and prIns1.DsRed (closed bars). On-line monitoring data are presented as theratio of fluorescence obtained at minutes 240 and 60 and represent mean values 6 S.E. (n 5 13).

overexpressing PKBa/c-Akt1. Whereas overexpression whereas 150 nM wortmannin was necessary to blockinsulin-stimulated bGK promoter activity (Figure 4E).of PKBa had no effect on insulin-stimulated insulin gene

transcription, it led to a more pronounced effect on insu- These data indicate that insulin-stimulated bGK genetranscription occurs by signaling via PDK1/PKB, whereaslin-stimulated bGK promoter–driven GFP expression

(Figure 4C). According to the current view, insulin-stimu- insulin-stimulated insulin gene transcription is mediatedvia PI3K/p70s6k and CaMKII.lated PKB activation involves the phosphorylation of

PKB by the phosphoinositol-dependent kinase 1, PDK1(Vanhaesebroeck and Alessi, 2000). Indeed, transient Insulin Signaling via IR-A Activates Insulin Gene

Promoter Whereas Signaling via IR-Boverexpression of PDK1 led to a pronounced stimulationof insulin-triggered bGK promoter activity, whereas over- Activates bGK Promoter

Previous data on insulin-stimulated insulin gene tran-expression of the antisense transcript of PDK1 abol-ished the stimulatory effect of insulin on insulin-trig- scription (Leibiger et al., 1998a) favored signaling via IR

but did not exclude the signaling via IGF-I receptors orgered bGK promoter activity (Figure 4C). Noteworthily,transient overexpression of either PKBa or PDK1 did possible hybrids of insulin- and IGF-I receptors. The

loss of insulin effect when treating cells with HNMPA-not lead per se to an increased basal insulin or bGKpromoter activity (data not shown). (AM)3, an inhibitor of the IR tyrosine kinase (Saperstein

et al., 1989), supported the idea that signaling via IR isInterestingly, the activation of PKB has so far beenshown to be dependent on the activity of PI3K (Vanhaese- crucial. Consequently, we examined whether the ex-

pression of IR per se is an absolute requirement forbroeck and Alessi, 2000) and therefore to be sensitive tothe independent pharmacological inhibitors wortmannin insulin-stimulated insulin and bGK gene expression.

Therefore, we analyzed insulin and bGK mRNA levels inand LY294002. Whereas treatment of insulin-producingcells with 25 mM LY294002 clearly abolished insulin- response to glucose/insulin stimulation in isolated islets

from bIRKO mice, a knockout model that lacks the ex-stimulated rat insulin I gene promoter activity, it didnot block insulin-stimulated rat bGK promoter activity pression of IR specifically in the pancreatic b cell (Kul-

karni et al., 1999a). Stimulation with either 16.7 mM glu-(Figure 3). When analyzing the effect of LY294002on insulin-stimulated insulin and bGK promoter activity cose or 5 mU of insulin/ml led to an increase in both

endogenous insulin and bGK mRNA levels in islets ofin a dose-dependent manner in cells cotransfectedwith prbGK.GFP and prIns1.DsRed, we observed that wild-type mice, whereas no increase in insulin and bGK

mRNA levels was observed in islets prepared fromLY294002 inhibited the two promoters at different con-centrations. Whereas 25 mM LY294002 blocked insulin- bIRKO mice (Figure 5A). These data suggest that the

expression of the IR in pancreatic b cells is an absolutestimulated insulin promoter activity, 100 mM LY294002was needed to completely abolish insulin-stimulated requirement to gain the stimulatory effect by insulin on

both insulin and bGK gene expression and that signalingbGK promoter activity (Figure 4D). The effect of wort-mannin was similarly concentration dependent. Treat- via IGF-I receptors is unlikely to be involved. This is

consistent with the finding that activation of IGF-I recep-ment of cells with 50 nM wortmannin was sufficientto inhibit insulin-stimulated insulin promoter activity, tors by stimulation with 2.6 nM IGF-I did not activate

Molecular Cell564

Figure 4. Role of PKB and PI3K in Insulin-Stimulated Insulin Gene and bGK Tran-scription

(A and B) Dynamics of PKB activities follow-ing stimulation with 16.7 mM glucose (A) or5 mU of insulin per ml in HIT cells (B). Activi-ties of PKB are presented as the percentageof the activity of the nonstimulated control(given as 100%). Data are shown as meanvalues 6 S.E. (n 5 3).(C) On-line monitoring of HIT cells cotrans-fected with prbGK.GFP (open bars), prIns1.D-sRed (closed bars), and either kinase-inactivemutant of PKBa, i.e., PKBaD308/437 (mock),wild-type PKBa (PKB), wild-type PDK1 (PDK1),or an antisense construct of PDK1 (PDK1anti-sense). Data are presented as the ratio offluorescence obtained at minutes 240 and 60and represent mean values 6 S.E. (n 5 8).(D and E) Effect of different concentrations ofPI3K inhibitors LY294002 (D) and wortmannin(E) on bGK promoter–driven GFP expression(open bars) and insulin promoter–drivenDsRed expression (closed bars) in cotrans-fected HIT cells. Data are presented as theratio of fluorescence obtained at minutes 240and 60 and represent mean values 6 S.E.(n 5 7).

insulin promoter– or bGK promoter–driven reporter gene Employing coexpression of prbGK.GFP and prIns1.D-sRed in the same cell, we observed that treatment withexpression in insulin-producing cells (data not shown).

Employing RT–PCR with subsequent DNA sequence the B-type receptor-specific antibody (aIR-B) abolishedinsulin-stimulated prbGK.GFP expression, whereas itanalysis, we have previously observed that insulin-pro-

ducing cells express both IR-A and IR-B (Leibiger et did not affect insulin-stimulated prIns1.DsRed expres-sion (Figure 5C). In addition, treatment of insulin-produc-al., 1998a). Transient overexpression of IR-A led to a

pronounced effect of insulin stimulation on insulin pro- ing cells with aIR-B abolished elevation of bGK mRNAlevels following stimulation with either 16.7 mM glucosemoter activity, whereas overexpression of IR-B had no

effect. To test a similar effect for insulin-stimulated bGK or 5 mU of insulin/ml at substimulatory glucose concen-trations (data not shown). As expected, treatment oftranscription, we cotransfected islet cells and HIT cells

with prbGK.GFP and prIns1.DsRed in combination with transfected cells with an antibody that blocks insulinsignaling via both receptor isoforms (aIR-AB) sup-either IR-A or IR-B. To our surprise, we found that over-

expression of IR-B led to a pronounced activation of pressed insulin-stimulated activation of bGK- and insu-lin-promoters (Figure 5C). Accordingly, treatment of in-the bGK promoter while overexpression of IR-A had

no effect (Figure 5B). Moreover, overexpression of an sulin-producing cells with this antibody also abolishedinsulin-stimulated elevation of bGK mRNA steady-stateinactive IR-B mutant (IR-Bm), described as M1153I

(Levy-Toledano et al., 1994), did not lead to a further levels (data not shown). By contrast, treatment of trans-fected cells with an antibody that blocks signaling viaeffect on rbGK promoter–driven GFP expression (Figure

6B). To test the involvement of IR-B in insulin-stimulated IGF-I receptors (aIGF-1R) did not affect insulin-stimu-lated activation of bGK promoter– and insulin promoter–bGK promoter activation in more detail, we treated islet

cells and HIT cells, prior to glucose/insulin stimulation, driven reporter gene expression (Figure 5C).These data indicate that insulin stimulates the insulinwith an anti-IR-B antibody. This antibody selectively

binds to the a chain of IR-B and therefore selectively gene promoter through IR-A, whereas it stimulates thebGK gene promoter via IR-B.blocks insulin binding to IR-B and signaling via IR-B.

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Figure 5. The Role of Insulin Receptors

(A) Effect of stimulation with either 16.7 mM glucose (15 min) or 5 mU of insulin per ml (5 min) on endogenous bGK mRNA steady-state levelsin isolated pancreatic islets obtained from bIRKO mice (bIRKO) or control animals (wild type). The values of bGK mRNA are presented aspercentages of mRNA levels of the nonstimulated islets (given as 100%). Data are shown as mean values 6 S.E. (n 5 3).(B) On-line monitoring of bGK promoter–driven GFP expression (open bars) and insulin promoter–driven DsRed expression (closed bars) incotransfected islet cells. Cells were cotransfected with prbGK.GFP and prIns1.DsRed and either expression constructs for wild-type isoformsof IR-A, IR-B, or the M1153I mutant of the respective receptor isoform, i.e., IR-Am and IR-Bm, respectively. Data are presented as the ratioof fluorescence obtained at minutes 240 and 60 and represent mean values 6 S.E. (n 5 10).(C) Effect of antibodies that block signaling through IR-A and IR-B (aIR-AB), through IR-B (aIR-B), and through IGF-I receptors (aIGF-1R) oninsulin-stimulated bGK promoter–driven GFP expression (open bars) and insulin promoter–driven DsRed expression (closed bars) in cotrans-fected islet cells. Cells were incubated with a 0.67 mg/ml concentration of the respective antibodies 30 min prior to stimulation and throughoutstimulation. Data are presented as the ratio of fluorescence obtained at minutes 240 and 60 and represent mean values 6 S.E. (n 5 10).

To start to understand the molecular mechanisms that receptor isoforms with GFP and DsRed at the C terminusof the b subunit. Tagging both IR isoforms did not inter-underlie the selectivity in insulin signaling via the two

IR isoforms, we aimed to explain the different sensitivity fere with their physiological function (e.g., overexpres-sion of the tagged IR isoform led to a pronounced insu-for PI3K inhibitors we observed between insulin-stimu-

lated insulin- and bGK-promoter activation (Figures 4D lin effect on the respective promoter activity to thesame extent as the untagged IR) (data not shown).and 4E). One possible interpretation would be that the

same PI3K is involved in the transcription of both genes Whereas transient coexpression of the same, but differ-ently tagged (IR-AzDsRed/IR-AzGFP and IR-BzGFP/but that a lower PI3K activity is sufficient to trigger the

cascade that activates bGK gene transcription via PKB. IR-BzDsRed), IR isoform led to a complete colocaliza-tion (data not shown), coexpression of the differentlyIf this is the case, inhibition of PI3K-mediated bGK tran-

scription should require higher concentrations of wort- tagged IR-A and IR-B in either combination (IR-AzDsRed/IR-BzGFP and IR-AzGFP/IR-BzDsRed) clearlymannin or LY294002 to be fully effectuated. This should

also be reflected upon by an inverse relationship be- showed IR isoforms that are not colocalized. This pat-tern of distinct IR isoform distribution was observed intween required inhibitor concentration and sensitivity of

the respective promoter activity to insulin. If the same insulin-producing cells HIT (Figure 6A), INS1, and MIN6,as well as in non-insulin-producing cells HEK293 andPI3K is sufficient to lead to the activation of both insulin-

and bGK-promoters, then the promoter activity that is COS7 (data not shown). To test whether the two IRisoforms do utilize different classes of PI3K, we overex-least sensitive to the inhibitors (i.e., bGK) would be ex-

pected to require less insulin to become stimulated. As pressed either IR-AzGFP or IR-BzGFP in HIT cells andstudied the sensitivity of PI3K activity to wortmannin indemonstrated in Figure 2E, this is not the case. On the

contrary, more insulin is needed to stimulate bGK pro- vitro following immunoprecipitation with GFP antibod-ies. Whereas the PI3K activity in the IR-A immunoprecip-moter–driven GFP expression. Another interpretation is

that the different sensitivity in vivo could be due to a itate was inhibited by wortmannin in the lower nanomo-lar range, as typical for PI3K class I and III, the PI3Kdifferent accessibility of the inhibitor for the same type

of PI3K as a result of a different distribution/localization activity in the IR-B immunoprecipitate was only inhibitedat higher concentrations (Figure 6B), as described forof the two IR isoforms, or it could be due to the involve-

ment of different classes of PI3K, exhibiting a different PI3K class II (see Fruman et al., 1998). To test whetherinsulin-stimulated insulin gene transcription involvessensitivity to wortmannin and LY294002 as described

for PI3K classes I and III versus class II (reviewed in IR-A-mediated insulin signaling via PI3K class Ia, wecombined insulin stimulation with the transient over-Fruman et al., 1998). To test whether IR-A and IR-B

exhibit a distinct distribution in vivo, we tagged both expression of the dominant-negative form of the PI3K

Molecular Cell566

Figure 6. Molecular Mechanisms Involved in the Selective Insulin Signaling via IR-A and IR-B

(A) Distribution of IR-AzGFP (green) and IR-BzDsRed (red) in HIT cells obtained by laser scanning confocal microscopy. Areas in yellowindicate colocalization of the two IR isoforms. This is a representative image out of a total of 25.(B) PI3K activity in GFP immunoprecipitates obtained from insulin-stimulated (150 nM insulin for 5 min) HIT cells overexpressing either IR-AzGFP (closed bars) or IR-BzGFP (open bars). The amount of wortmannin included in the in vitro assay is indicated. Data are presented asmean values 6 S.E. (n 5 3).(C) Effect of overexpression of dominant-negative p85 PI3K subunit, Dp85, on insulin-stimulated bGK promoter–driven GFP expression (openbars) and insulin promoter–driven DsRed expression (closed bars) in cotransfected HIT cells. Data are presented as the ratio of fluorescenceobtained at minutes 240 and 60 and represent mean values 6 S.E. (n 5 10).(D and E) Analysis of insulin-stimulated p70 s6 kinase and PKB activities in HIT (D) and HEK293 (E) cells following transfection with IR-A orIR-B. Cells were stimulated with insulin for 10 min and lysed after a further 10 min. Data are represented as percentages of the nonstimulated,mock-transfected control, set as 100%, and presented as mean values 6 S.E. (n 5 3).

class Ia adaptor protein p85 (i.e., Dp85). Whereas tran- through PI3K class Ia and p70s6k on the one hand, andvia IR-B through a different PI3K activity, very similar tosient overexpression of Dp85 totally abolished insulin-

stimulated insulin promoter activity, this approach had that of class II, and PKB on the other.When separately overexpressing IR isoforms in HITno effect on insulin-stimulated bGK promoter activation

(Figure 6C). cells, we observed a more pronounced activation ofp70s6k in cells overexpressing IR-A in response to insu-Taken together, these data suggest a selectivity in

insulin signaling in insulin-producing cells via IR-A lin stimulation, while cells overexpressing IR-B showed

Selective Signaling via A and B Insulin Receptors567

Figure 7. Selective Activation of Insulin andGlucokinase Gene Transcription by SelectiveInsulin Signaling via A- and B-Type InsulinReceptors

The scheme illustrates the coupling betweeninsulin exocytosis and the activation of tran-scription of insulin and glucokinase genes.

a trend toward a higher PKB activity (Figure 6D). To test al., 2000; Michael et al., 2000) to the failure in IR-B orIR-A function, respectively, a direct proof of the involve-whether this effect is purely specific for the pancreatic

b cell, we analyzed both PKB and p70s6k activities in ment of IR isoforms remains to be shown in IR isoform–specific knockout models. Attempts to correlate tissue-non-insulin-producing HEK293 cells that were tran-

siently overexpressing either IR-A or IR-B. Most interest- specific expression of IR isoforms with diabetes mellitushas generated conflicting results (Mosthaf et al., 1991;ingly, insulin-stimulated p70s6k activity here was also

more pronounced in cells overexpressing IR-A, whereas Benecke et al., 1992; Norgren et al., 1993) that do notclarify the functional role of either isoform.insulin-stimulated PKB activity seemed to be more

tightly coupled with the overexpression of IR-B (Fig- Little is known about selective insulin signaling via A-and B-type IR. Besides the affinity for insulin, differencesure 6E).in their kinase activity (Kellerer et al., 1992) as well asinternalization and recycling (Vogt et al., 1991; Yama-Conclusionguchi et al., 1991) have been described. These data haveSelectivity in insulin signaling is currently discussed asimplied differences in the function of either IR isoform,the result of the activation of specific signal transductionbut no isoform-specific insulin-induced effect has beenpathways. This selectivity may be gained by activatingreported so far. In the present paper, we provide a “read-specific adaptor proteins (i.e., IRS and Shc proteins)out” system for discriminating selective signaling viathat “channel” the insulin signal in a more defined waythe two IR isoforms. We have demonstrated that theby specifically interacting with downstream located ef-molecular basis for this selectivity could be provided byfector proteins (Myers and White, 1996; Virkamaki etthe different localization of the two IR isoforms in theal., 1999). Whereas the importance of IRS proteins inplasma membrane and their different sensitivity for insu-achieving insulin effects in different tissues is currentlylin. Mechanistically, this enables preferential activationunder extensive investigation, the possibility of selectiveof IR-A/PI3K Ia/p70s6k in pancreatic b cell glucose/insulin signaling via the two isoforms of the IR has beeninsulin–stimulated insulin gene transcription and IR-B/neglected. Studies on general and tissue-specific IRPI3K class II–like/PKB in glucose/insulin–stimulatedknockout models have demonstrated that a defect IR-bGK transcription (Figure 7). That this specificity in sig-mediated insulin signaling leads to a type 2 diabetes–likenaling has a wider implication than to the pancreatic bphenotype (reviewed in Taylor, 1999). However, thesecell is demonstrated by the data obtained in non-insulin-knockouts do not discriminate between the two IR iso-producing HEK293 cells.forms. This is of importance, since earlier studies clearly

Thus, our data clearly demonstrate that selectivity ofestablished differences in tissue-specific IR isoform ex-insulin signaling can be gained by signaling throughpression as well as in their activation profile. Whereasthe two IR isoforms and reinforce the concept of theIR-B, which shows a 2-fold lesser affinity for insulin inpancreatic b cell as a target for positive insulin action.comparison to IR-A (Mosthaf et al., 1990; Yamaguchi et

al., 1991; McClain, 1991), is predominantly expressedExperimental Proceduresin liver and muscle, IR-A is mainly expressed in brain

(Moller et al., 1989; Seino and Bell, 1989; Mosthaf et al., Materials1990). Although it is tempting to link the phenotypes of Bisindolylmalemide I, PD98059, wortmannin, LY294002, rapamycin,

HNMPA-(AM)3, autocamtide-2 related inhibitory peptide, and nifedi-the liver- and brain-specific IR knockouts (Bruning et

Molecular Cell568

pine were purchased from Calbiochem. Actinomycin D was from RNA AnalysisLevels of bGK mRNA were analyzed by comparative RT–PCR asSigma. Microcystin-LR and PD169316 were purchased from Alexis

Biochemicals. Rabbit anti-Insulin Receptor a and Rabbit anti-Insulin described in Leibiger et al. (1998b) using primers 59-GTTCCTACTGGAGTATGACC-39 and 59-CCTCCTCTGATTCGATGAAG-39 for char-Receptor B antibodies were from Biodesign. Mouse monoclonal

IGF-IRa was from Pharmingen. Oligonucleotides were synthesized acterizing bGK mRNA in HIT cells, and primers 59-TGGATGACAGAGCCAGGATGG-39 and 59-ACTTCTGAGCCTTCTGGGGTG-39 forat Genset (France).bGK mRNA in rat and mouse pancreatic islets and islet cells. Levelsof b actin mRNA were analyzed by RT–PCR using primers 59-AACTGGExpression ConstructsAACGGTGAAGGCGA-39 and 59-AACGGTCTCACGTCAGTGTA-39.The construction of prIns1.GFP has been described earlier (LeibigerPCR conditions were chosen that guaranteed the amplification ofet al., 1998a). prIns1.DsRed was generated by exchanging the GFPbGK and actin fragments within the linear range, as verified byexpression cassette versus the DsRed-expression cassette fromtesting various numbers of amplification cycles (10–35). PCR prod-pDsRed1–1 (Clontech). prbGK.GFP was generated by exchangingucts were separated on a 6% polyacrylamide sequencing gel andthe CAT expression cassette of prbGK-278.CAT (Leibiger et al., 1994a)analyzed by phosphorimaging. Quantification was performed withversus the GFP-expression cassette obtained from pRcCMVi.GFPTINA software 2.07d (Raytest). Values of bGK mRNA were normal-(Moede et al., 1999). Constructs for expression of the human IR-Aized by b actin values.and -B types, pRcCMVi.hIR(A) and pRcCMVi.hIR(B), were generated

by subcloning the respective IR-A and IR-B sequences frompCMVHIR(A) and pCMVHIR(B) (kindly provided by A. Ullrich, MPI Analysis of PKB Activityfor Biochemistry, Martinsried, Germany) into pRcCMVi. GFP- and Analysis of PKB activity was performed employing the Akt1/PKBaDsRed-tagged variants were generated by introducing a ClaI-site Immunoprecipitation Kinase Assay Kit (Upstate Biotech.) accordinginto the IRb subunit coding sequence 69 nucleotides in front of the to the manufacturer’s instructions.stop codon and subcloning of the GFP or DsRed cDNA, respectively,in-frame. Site-directed mutagenesis to introduce the M1153I muta-

Analysis of p70s6k Activitytion into the IR cDNA was performed by employing the Quik-p70 s6 kinase from cell lysates was immunoprecipitated using aChange Mutagenesis Kit (Stratagene). Adenovirus-based vectorp70s6k antibody (Upstate Biotech.). Analysis of p70s6k activity wasAd.rbGK.GFP was constructed by subcloning the rbGK.GFP cas-performed employing the S6 Kinase Assay Kit (Upstate Biotech.)sette into pAC.CMV.pLpA and performing homologous recom-according to the manufacturer’s instructions.bination as described in Moitoso de Vargas et al. (1997). All

vector constructions were verified by DNA sequence analysis. Plas-mids pCMV5.PKBa, pCMV5.PKBaD308/437, pCMV5.PDK1, and Analysis of PI3K ActivitypCMV5.PDK1 antisense were kindly provided by D.R. Alessi (MRC HIT cells were transfected with either pRcCMVi.hIR(A)zGFP orPhosphorylation Unit, University of Dundee, UK), and pcDNA3- pRcCMVi.hIR(B)zGFP. Cell lysates containing 2 mg of protein wereZeo.Dp85 was a gift from C.P. Downes (Department of Biochemistry, subjected to immunoprecipitation using anti-GFP antibody A-11122University of Dundee, UK). (Molecular Probes). PI3K activity was analyzed in the GFP immuno-

precipitates as described in Krook et al. (1997) using L-a-phosphati-Cell Culture and Transfection dylinositol from Avanti Polar-Lipids, Inc.Isolation of pancreatic islets, the culture of islets, islet cells, andHIT-T15 cells, and their transfection have been described in Leibiger

On-Line Monitoring of GFP and DsRed Expressionet al. (1998a; 1998b). Following transfection, HIT cells were culturedand Detection of Fluorescencefor 12–18 hr in RPMI 1640 medium containing 0.1 mM glucose, 10%Detection of fluorescence by digital imaging fluorescence micros-fetal calf serum and supplemented with 100 U/ml penicillin, 100copy was performed as described previously (Leibiger et al., 1998a;mg/ml streptomycin, 2 mM glutamine at 5% CO2 and 378C. Before1998b). The following filter settings were used: for GFPS65T: excitationstimulation, islets and islet cells were incubated for 2 hr in RPMIat 485 nm, a 505 nm dicroic mirror, and an emission band-pass filter1640 medium supplemented as above but containing 3 mM glucose.of 500–530 nm; for DsRed: excitation at 558 nm, a 565 nm dicroicHEK293 cells were grown in DMEM containing 5.5 mM glucose,mirror, and a 580 nm long-pass filter for emission. On-line monitoring10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin,was initiated 60 min following the start of stimulation and the 602 mM glutamine at 5% CO2 and 378C, and were transfected by themin value of GFP or DsRed fluorescence was set to 1.0. Cells to becalcium phosphate/coprecipitation technique as described earliermonitored were chosen randomly at minute 60 from six fields of(Leibiger et al., 1994a). Islets were transduced with Ad.rbGK.GFPvision, and fluorescence was monitored up to minute 240. By over-as described in Moitoso de Vargas et al. (1997) and analyzed bylaying the fluorescence and phase-contrast images, the positionslaser scanning confocal microscopy.of cells on the coverslip could be checked. Fluorescence intensityCultured islets, islet cells, and HIT cells were stimulated with eitherwas calculated by using the Isee software for UNIX (Inovision Corpo-16.7 mM glucose for 15 min or 50 mM KCl, 1 mM glibenclamide orration).with various concentrations of insulin for 5 min at substimulatory

Laser scanning confocal microscopy was performed using aglucose concentrations (3 mM for islets/islet cells and 0.1 mM forLEICA TCS SP2 (Leica Lasertechnik GmbH) with the following set-HIT cells). All experiments were performed in RPMI 1640 medium,tings: Leica HCX PL APO 633/1.20/0.17 UV objective lens, excitationsupplemented as above. Protein kinase inhibitors, antibodies, orwavelength 488 nm (Ar Laser) and 543 nm (HeNe laser), a 488/543nifedipine were added to the culture medium 30 min prior to stimula-double dichroic mirror, and detection of GFP at 505–525 nm andtion and were kept in the medium throughout stimulation. AfterDsRed at 605–670 nm. Laser scanning confocal microscopy of trans-stimulation, islets, islet cells, and HIT cells were cultured further atduced islets was performed as described in Leibiger et al. (1998b).substimulatory glucose concentrations. For RNA analysis, islets and

cells were harvested 60 min after start of stimulation, if not indicatedotherwise. For analysis of protein kinase activities or tyrosine phos- Acknowledgmentsphorylation, cells were harvested immediately following stimulation.

The authors wish to thank Drs. A.M. Bertorello, K. Michelsen, T.Schwarz-Romond, X. Wang, and N. Welsh for sharing material andNuclear Run-Off Analysis

Nuclear run-off analysis was performed as described previously unpublished data. This work was supported by funds from Karolin-ska Institutet; by grants from Juvenile Diabetes Foundation Interna-(Leibiger et al., 1998b), except that labeled RNA was hybridized to

2.5 mg of cDNA of glucokinase, insulin, b actin, and control pBlue- tional (JDFI), the Novo Nordisk Foundation, the Nordic Insulin Foun-dation Committee, EC Biotechnology Project (BIO4-CT98-0286), thescript DNA, which were immobilized on nitrocellulose filters. For

nuclear run-off analysis on islets, nuclei from 2000 islets per experi- Swedish Diabetes Association, and the Swedish Medical ResearchCouncil (72X-12594, 03X-13394, 72X-00034, 72XS-12708, 72X-ment were used. Data were analyzed by phosphorimaging. Values

obtained for bGK mRNA were normalized by b actin mRNA values. 09890); and in part by NIH GRASP Center grant DK34928 and NIH

Selective Signaling via A and B Insulin Receptors569

grants DK31036 and DK09825-02. L. M.V. is funded by a Career insulin gene transcription by glucose. Proc. Natl. Acad. Sci. USA95, 9307–9312.Development Award from JDFI.

Leibiger, B., Wahlander, K., Berggren, P.O., and Leibiger, I.B. (2000).Received August 21, 2000; revised January 8, 2001. Glucose-stimulated insulin biosynthesis depends on insulin-stimu-

lated insulin gene transcription. J. Biol. Chem. 275, 30153–30156.

References Levy-Toledano, R., Caro, L.H.P., Accili, D., and Taylor, S.I. (1994).Investigation of the mechanism of the dominant negative effect of

Aspinwall, C.A., Lakey, J.R.T., and Kennedy, R.T. (1999). Insulin- mutations in the tyrosine kinase domain of the insulin receptor.stimulated insulin secretion in single pancreatic beta cells. J. Biol. EMBO J. 13, 835–842.Chem. 274, 6360–6365. Matz, M.V., Fradkov, A.F., Labas, Y.A., Savitsky, A.P., Zaraisky, A.G.,Benecke, H., Flier, J.S., and Moller, D.E. (1992). Alternatively spliced Markelov, M.L., and Lukyanov, S.A. (1999). Fluorescent proteinsvariants of the insulin receptor protein. Expression in normal and from nonbioluminescent Anthozoa species. Nat. Biotech. 17,diabetic human tissues. J. Clin. Invest. 89, 2066–2070. 969–973.

McClain, D.A. (1991). Different ligand affinities of the two insulinBerggren, P.O., and Larsson, O. (1994). Ca21 and pancreatic B-cellfunction. Biochem. Soc. Trans. 22, 12–18. receptor splice variants are reflected in parallel changes in sensitiv-

ity for insulin action. Mol. Endocrinol. 5, 734–739.Bruning, J.C., Gautam, D., Burks, D.J., Gillette, J., Schubert, M.,Michael, M.D., Kulkarni, R.N., Postic, C., Previs, S.F., Shulman, G.I.,Orban, P.C., Klein, R., Krone, W., Muller-Wieland, D., and Kahn, C.R.Magnuson, M.A., and Kahn, C.R. (2000). Loss of insulin signaling(2000). Role of brain insulin receptor in control of body weight andin hepatocytes leads to severe insulin resistance and progressivereproduction. Science 289, 2122–2125.hepatic dysfunction. Mol. Cell 6, 87–97.Coffer, P.J., Jin, J., and Woodgett, J.R. (1998). Protein kinase BMoede, T., Leibiger, B., Pour, H.G., Berggren, P.O., and Leibiger,(c-Akt): a multifunctional mediator of phosphoinositol 3-kinase acti-I.B. (1999). Identification of a nuclear localization signal, RRMKWKK,vation. Biochem. J. 335, 1–13.in the homeodomain transcription factor PDX-1. FEBS Lett. 461,Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., Ou,229–234.J.H., Masiarz, F., Kan, Y.W., Goldfine, I.D., et al. (1985). The humanMoitoso de Vargas, L., Sobolewski, J., Siegel, R., and Moss, L.G.insulin receptor cDNA: the structural basis for hormone-activated(1997). Individual b cells within the intact islet differently respondtransmembrane signalling. Cell 40, 747–758.to glucose. J. Biol. Chem. 272, 26573–26577.Fruman, D.A., Myers, R.A., and Cantley, L.C. (1998). Phosphoinosi-Moller, D.E., Yokota, A., Caro, J.F., and Flier, J.S. (1989). Tissue-tide kinases. Annu. Rev. Biochem. 67, 481–507.specific expression of two alternatively spliced insulin receptorJetton, T.L., Liang, Y., Pettepher, C.C., Zimmerman, E.C., Cox, F.G.,mRNA in man. Mol. Endocrinol. 3, 1263–1269.Horvath, K., Matschinsky, F.M., and Magnuson, M.A. (1994). AnalysisMosthaf, L., Grako, K., Dull, T.J., Coussens, L., Ullrich, A., andof upstream glucokinase promoter activity in transgenic mice andMcClain, D.A. (1990). Functionally distinct insulin receptors gener-identification of glucokinase in rare neuroendocrine cells in the brainated by tissue-specific alternative splicing. EMBO J. 9, 2409–2413.and gut. J. Biol. Chem. 269, 3641–3654.Mosthaf, L., Vogt, B., Haring, H., and Ullrich, A. (1991). Altered ex-Jetton, T.L., Moates, J.M., Lindner, J., Wright, C.V.E., and Magnuson,pression of insulin receptor types A and B in the skeletal muscle ofM.A. (1998). Targeted oncogenesis of hormone-negative pancreaticnon-insulin-dependent diabetes mellitus patients. Proc. Natl. Acad.islet progenitor cells. Proc. Natl. Acad. Sci. USA 95, 8654–8659.Sci. USA 88, 4728–4730.

Kellerer, M., Lammers, R., Ermel, B., Tippmer, S., Vogt, B., Ober-Myers, M.G., Jr., and White, M.F. (1996). Insulin signal transductionmaier-Kusser, B., Ullrich, A., and Haring, H.U. (1992). Distinctand the IRS proteins. Annu. Rev. Pharmacol. Toxicol. 36, 615–658.a-subunit structures of human insulin receptor A and B variants

determine differences in tyrosine kinase activities. Biochemistry 31, Norgren, S., Zierath, J., Galuska, D., Wallberg-Henriksson, H., andLuthman, H. (1993). Differences in the ratio of mRNA encoding two4588–4596.isoformes of the insulin receptor between control and NIDDM pa-Krook, A., Whitehead, J.P., Dobson, S.P., Griffiths, M.R., Ouwens,tients: the RNA variant with exon 11 predominates in both groups.M., Baker, C., Hayward, A.C., Sen, S.K., Maassen, J.A., Siddle, K.,Diabetes 42, 675–681.et al. (1997). Two naturally occuring insulin receptor tyrosine kinaseSaperstein, R., Vicario, P.P., Strout, H.V., Brady, E., Slater, E.E.,domain mutants provide evidence that phosphoinositide 3-kinaseGreenlee, W.J., Ondeyka, D.L., Patchett, A.A., and Hangauer, D.G.activation alone is not sufficient for the mediation of insulin’s meta-(1989). Design of a selective insulin receptor tyrosine kinase inhibitorbolic and mitogenic effects. J. Biol. Chem. 272, 30208–30214.and its effect on glucose uptake and metabolism in intact cells.Kulkarni, R.N., Bruning, J.C., Winnay, J.N., Postic, C., Magnuson,Biochemistry 28, 5694–5701.M.A., and Kahn, C.R. (1999a). Tissue-specific knockout of the insulinSeino, S., and Bell, G.I. (1989). Alternative splicing of human insulinreceptor in pancreatic b cells creates an insulin secretory defectreceptor messenger RNA. Biochem. Biophys. Res. Comm. 159,similar to that of type 2 diabetes. Cell 96, 329–339.312–316.Kulkarni, R.N., Winnay, J.N., Daniels, M., Bruning, J.C., Flier, S.N.,Seino, S., Seino, M., Nishi, S., and Bell, G.I. (1989). Structure of theHanahan, D., and Kahn, C.R. (1999b). Altered function of insulinhuman insulin receptor gene and characterization of its promoter.receptor substrate-1-deficient mouse islets and cultured b-cell lines.Proc. Natl. Acad. Sci. USA 86, 114–118.J. Clin. Invest. 104, R69–R75.Shelton, K.D., Franklin, A.J., Khoor, A., Beechem, J., and Magnuson,Leibiger, I.B., Walther, R., Pett, U., and Leibiger, B. (1994a). PositiveM.A. (1992). Multiple elements in the upstream glucokinase pro-and negative regulatory elements are involved in transcriptional con-moter contribute to transcription in insulinoma cells. Mol. Cell. Biol.trol of the rat glucokinase gene in the insulin producing cell line HIT12, 4578–4589.M2.2.2. FEBS Lett. 337, 161–166.Taylor, S.I. (1999). Deconstructing type 2 diabetes. Cell 97, 9–12.Leibiger, B., Walther, R., and Leibiger, I.B. (1994b). The role of the

proximal CTAAT-box of the rat glucokinase upstream promoter in Ullrich, A., Bell, J.R., Chen, E.Y., Herrera, R., Petruzzelli, L.M., Dull,transcriptional control in insulin-producing cells. Biol. Chem. T.J., Gray, A., Coussens, L., Liao, Y.C., Tsubokawa, M., et al. (1985).Hoppe-Seyler 375, 93–98. Human insulin receptor and its relationship to the tyrosine kinase

family of oncogenes. Nature 313, 756–761.Leibiger, I.B., Leibiger, B., Moede, T., and Berggren, P.O. (1998a).Exocytosis of insulin promotes insulin gene transcription via the Vanhaesebroeck, B., and Alessi, D.R. (2000). The PI3K–PDK1 con-insulin receptor/PI-3 kinase/p70 s6 kinase and CaM kinase path- nection: more than just a road to PKB. Biochem. J. 346, 561–576.ways. Mol. Cell 1, 933–938. Virkamaki, A., Ueki, K., and Kahn, C.R. (1999). Protein–protein inter-

action in insulin signaling and the molecular mechanism of insulinLeibiger, B., Moede, T., Schwarz, T., Brown, G.R., Kohler, M., Lei-biger, I.B., and Berggren, P.O. (1998b). Short-term regulation of resistance. J. Clin. Invest. 103, 931–943.

Molecular Cell570

Vogt, B., Carrascosa, J.M., Ermel, B., Ullrich, A., and Haring, H.U.(1991). The two isotypes of the human insulin receptor (HIR-A andHIR-B) follow different internalization kinetics. Biochem. Biophys.Res. Commun. 177, 1013–1018.

Watada, H., Kajimoto, Y., Umayahara, Y., Matsuoka, T., Kaneto, H.,Fujitani, Y., Kamada, T., Kawamori, R., and Yamasaki, Y. (1996). Thehuman glucokinase gene b-cell-type promoter: an essential role ofinsulin promoter factor 1/PDX-1 in its activation in HIT-T15 cells.Diabetes 45, 1478–1488.

White, M.F., and Kahn, C.R. (1994). The insulin signaling system. J.Biol. Chem. 269, 1–4.

Withers, D.J., Sanchez-Gutierrez, J.C., Towery, H., Ren, J.M., Burks,D.J., Previs, S., Zhang, Y., Bernal, D., Shulman, G.I., Bonner-Weir, S.,and White, M.F. (1998). Disruption of IRS-2 causes type 2 diabetes inmice. Nature 391, 900–903.

Xu, G.G., and Rothenberg, P.L. (1998). Insulin receptor signaling inthe b-cell influences insulin gene expression and insulin content.Evidence for autocrine b-cell regulation. Diabetes 47, 1243–1252.

Xu, G., Kwon, G., Marshall, C.A., Lin, T.A., Lawrence, J.C., Jr., andMcDaniel, M.L. (1998). Branched-chain amino acids are essential inthe regulation of PHAS-I and p70 s6 kinase by pancreatic b-cells.A possible role in protein translation and mitogen signaling. J. Biol.Chem. 273, 28178–28184.

Yamaguchi, Y., Flier, J.S., Yokota, A., Benecke, H., Backer, J.M., andMoller, D.E. (1991). Functional properties of two naturally occurringisoforms of the human insulin receptor in Chinese hamster ovarycells. Endocrinology 129, 2058–2066.