Selective Insulin Signaling Through a and B Insulin Receptors Regulates Transcription of Insulin and Glucokinase Genes in Pancreatic ␤ Cells

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Molecular Cell, Vol. 7, 559–570, March, 2001, Copyright 2001 by Cell Press Selective Insulin Signaling through A and B Insulin Receptors Regulates Transcription of Insulin and Glucokinase Genes in Pancreatic ␤ Cells

Barbara Leibiger,*§ Ingo B. Leibiger,*§k ceptors as the primary target, include signaling via mito- Tilo Moede,* Sabine Kemper,* gen-activated protein (MAP) kinases and phosphoinosi- Rohit N. Kulkarni,† C. Ronald Kahn,† tol-3 kinase (PI3K). The insulin receptor (IR), the first Lina Moitoso de Vargas,‡ and Per-Olof Berggren* step in these cascades, exists in two isoforms as a result *The Rolf Luft Center for Diabetes Research of alternative mRNA splicing of the 11th exon of the insulin Department of Molecular Medicine proreceptor transcript (Seino et al., 1989). The A type Karolinska Institutet (IR-A), or Ex11Ϫ (Ullrich et al., 1985), lacks whereas the S-171 76 Stockholm B type (IR-B), or Ex11ϩ (Ebina et al., 1985), contains Sweden the respective sequence coding for 12 amino acids in † Research Division the C terminus of the ␣ chain of the receptor. To date, Joslin Diabetes Center and no insulin-induced effect has been reported that dis- Department of Medicine criminates signaling via A- and B-type receptors. In fact, Harvard Medical School the functional significance of these IR isoforms remains Boston, Massachusetts 02215 unclear. ‡ Department of Medicine Recent studies have shown that the insulin-producing New England Medical Center and pancreatic ␤ cell is a target for insulin action, with insulin Tufts University School of Medicine effects on transcription, translation, Ca2ϩ flux, and exo- Boston, Massachusetts 02111 cytosis (Leibiger et al., 1998a, 2000; Xu and Rothenberg, 1998; Xu et al., 1998; Aspinwall et al., 1999; Kulkarni et al., 1999a). In an animal model with a ␤ cell–specific Summary knockout for IR, there is a decrease in glucose-stimulated insulin release and a decrease in the insulin content Insulin signaling is mediated by a complex network of of the cell (Kulkarni et al., 1999a). In addition, disruption diverging and converging pathways, with alternative of insulin signaling in the ␤ cell at the level of insulin proteins and isoforms at almost every step in the proreceptor substrate (IRS)-1 (Kulkarni et al., 1999b) or cess. We show here that insulin activates the tran- IRS-2 (Withers et al., 1998) leads to altered growth and scription of its own gene and that of the ␤ cell glucoki- function of the ␤ cell. Consequently, insulin resistance nase gene (␤GK) by different mechanisms. Whereas may not only affect the function of the “classical” insulin insulin gene transcription is promoted by signaling target tissues muscle, fat and liver, but also apply to through insulin receptor A type (Ex11Ϫ), PI3K class the pancreatic ␤ cell and thereby affect ␤ cell function. Ia, and p70s6k, insulin stimulates the ␤GK gene by In the present study, we show selective insulin signal- signaling via insulin receptor B type (Ex11ϩ), PI3K ing via the two isoforms of the insulin receptor (i.e., IR-A class II–like activity, and PKB (c-Akt). Our data provide and IR-B) in the pancreatic ␤ cell. Insulin that is secreted evidence for selectivity in insulin action via the two by ␤ cells upon glucose stimulation up-regulates tran- isoforms of the insulin receptor, the molecular basis scription of its own gene as well as that of the ␤ cell being preferential signaling through different PI3K and transcription unit of the glucokinase (␤GK) gene in an protein kinases. autocrine feedback loop. More interestingly, while the insulin gene is activated by insulin signaling via IR-A Introduction involving PI3K class Ia, p70 s6 kinase (p70s6k), and Ca2ϩ/calmodulin dependent kinases, insulin-stimulated Understanding selectivity in signal transduction is one ␤GK transcription occurs via IR-B, PI3K class II–like of the most challenging tasks in current cell biology. activity, and protein kinase B (PKB/c-Akt). These results Over the years, insulin signaling has served as one of provide evidence that signaling via either IR-A or IR-B the model examples in hormone-induced signal trans- and the subsequent activation of different classes of duction. Malfunction of insulin signaling, referred to as PI3K and protein kinases (i.e., p70s6k and PKB) repre- insulin resistance, is one of the major causes of type 2 sent a mechanism for selective insulin action. We fur- diabetes mellitus (non-insulin-dependent diabetes mel- thermore show a preferential activation of p70s6k and litus), the most common metabolic disorder in man. PKB as a result of insulin signaling via IR-A and IR-B, Insulin has been shown to exhibit pleiotropic effects respectively, in insulin-producing and non-insulin-pro- involving mitogenic and/or metabolic events. Moreover, ducing cells. the effect of insulin is tissue as well as development dependent. The fact that insulin may transduce its signal Results and Discussion through a variety of pathways has been discussed in extensive detail (White and Kahn, 1994). The two major Glucose Activates Glucokinase Gene Transcription pathways described to date, which employ insulin re- via Secreted Insulin Insulin, secreted upon glucose stimulation, is a key fac- k To whom correspondence should be addressed (e-mail: ingo@ tor in the up-regulation of insulin gene transcription (Lei- enk.ks.se). biger et al., 1998a). The promoters of both the insulin §These authors contributed equally to this work. gene and the ␤GK gene contain many similar cis ele- Molecular Cell 560

ments (Shelton et al., 1992; Leibiger et al., 1994a, 1994b; insulin gene, the addition of 20 ␮U per ml was required to Watada et al., 1996). To test whether transcription of gain an effect on ␤GK promoter activation (Figure 2E). ␤GK is regulated by similar mechanisms as the insulin Stimulation with 5 mU of insulin per ml of culture medium gene, we studied the role of glucose and insulin in regu- for 5 min led to an ␤GK promoter–driven increase in lation of ␤GK mRNA steady-state levels. Stimulation of GFP fluorescence in isolated primary pancreatic ␤ cells cultured islets (Figure 1A) or insulin-producing HIT-T15 (Figure 2C), HIT cells, and intact pancreatic islets (data cells with 16.7 mM glucose led to an increase in ␤GK not shown). mRNA levels 60 min following start of stimulation. This is Thus, our data support the view that the insulin gene similar in time course to the effect of glucose to stimulate and the ␤GK 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 ␤GK mRNA, concentration of insulin is needed to activate ␤GK tran- we analyzed the half-life time, stability, and transcrip- scription when compared with the insulin gene. tional rate of the ␤GK mRNA pool. As shown in Figure ف ␤ 1B, the half-life time of GK mRNA was 60 min and Insulin-Stimulated Glucokinase Gene Transcription was not changed in the presence or absence of glucose. Utilizes Signal Transduction, which Is Different On the other hand, stimulation of HIT cells with 16.7 mM from that of the Insulin Gene ␤ glucose led to an increase in GK gene transcripts as Our studies on insulin-stimulated insulin gene transcrip- early as 15 min and reached a maximum of transcription have shown the involvement of PI3K, p70s6k, and tional activity at 30 min in a nuclear run-off assay (Figure Ca2ϩ/calmodulin-dependent kinase(s) in the signaling ␤ 1C). This effect of glucose on GK transcription initiation cascade (Leibiger et al., 1998a). Because previous data was also observed in normal pancreatic islets (Figure from others and our laboratory suggest that insulin- and 1D). To further corroborate these data, we established ␤GK-promoters can bind the same transcription factors ␤ a reporter gene assay using the GK promoter coupled (Shelton et al., 1992; Leibiger et al., 1994a, 1994b; Wa- ␤ to the green fluorescent protein (GFP) (pr GK.GFP). We tada et al., 1996) and both genes respond positively to ␤ used the rat GK promoter fragment up to nucleotide many of the same stimuli (glucose, insulin, secreta- Ϫ 278, since this has been shown to contain all cis ele- gogs) at the level of transcription, we questioned whether ments responsible for both glucose-dependent and cell- both genes might be regulated by the same signaling type-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 are crease in ␤GK promoter–driven GFP fluorescence in HIT involved in insulin-triggered insulin gene transcription cells, isolated primary pancreatic ␤ cells, and intact pan- contribute to insulin-triggered transcription of ␤GK, we creatic islets (Figure 1E). As with the nuclear run-off studied the effect of pharmacological inhibitors on insu- assay, the dynamics of the activation of ␤GK promoter– lin-stimulated ␤GK 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 kinase To determine whether glucose metabolism per se or C (PKC; 150 nM bisindolylmalemide I [BIM]), PI3K (25 secreted insulin is a requirement for the up-regulation ␮M LY294002 [LY]), p70s6k (10 nM rapamycin [rap]), of ␤GK transcription, we investigated the effect of insulin MAP kinases Erk1/2 (20 ␮M PD98059 [PD9]) and p38/ secretagogues on ␤GK mRNA steady-state levels and RK/SAPK2a ϩ SAPK1/JNK (10 ␮M PD169316 [PD1]), IR ␤GK promoter–driven GFP expression. Insulin secreta- tyrosine kinase (100 ␮M HNMPA-(AM)3 [HNMPA]), and gogues, like KCl or the sulfonylurea compound gliben- 2ϩ clamide, stimulate insulin secretion by depolarizing the Ca /calmodulin-dependent kinase II (CaMKII; 400 nM ␤ cell plasma membrane and provoking influx of extra- autocamtide-2 related inhibitory peptide [AC]). The effi- cellular Ca2ϩ through voltage-gated L-type Ca2ϩ chan- ciency of these inhibitors was verified by the respective nels (reviewed in Berggren and Larsson, 1994). As protein kinase assays in cell lysates of inhibitor-treated shown in Figure 2, stimulation with either 50 mM KCl or and nontreated cells following glucose/insulin stimula- 1 ␮M glibenclamide for 5 min, at substimulatory glucose tion (data not shown). In agreement with the data on concentrations, led to an increase in ␤GK mRNA steady- insulin-stimulated insulin gene transcription, insulin- ␤ state levels (Figure 2A) and to an elevation in ␤GK pro- stimulated GK transcription was not sensitive to inhibi- moter–driven GFP expression (Figure 2C). Alternatively, tion of PKC or MAP kinases Erk1/2 and p38 but was preventing stimulus-induced insulin secretion by block- sensitive to inhibition of IR tyrosine kinase by HNMPA- ϩ ing L-type Ca2 channels using nifedipine abolished up- (AM)3 (Figure 3A). However, to our surprise, insulin-stim- regulation of ␤GK mRNA levels (Figure 2B). ulated ␤GK transcription was not inhibited by LY294002, We next studied the effect of exogenously adminis- rapamycin, or autocamtide-2 related inhibitory peptide, tered insulin on ␤GK mRNA steady-state levels and ␤GK suggesting that signaling via PI3K/p70s6k and via CaM- promoter–driven GFP expression at substimulatory glu- KII, respectively, is not involved (Figures 3A and 3B). cose concentrations (Figures 2D and 2E). Addition of To further confirm that insulin stimulates insulin gene only 50 ␮U of insulin per ml to fully supplemented culture transcription and ␤GK transcription using different signal- medium was sufficient to evoke ␤GK mRNA levels in ing pathways, we established a technique that allowed pancreatic islets (Figure 2D). Interestingly, a more care- monitoring of insulin and ␤GK promoter activities simul- ful comparison of the necessary amounts of exogenous taneously in the same cell. In addition to pr␤GK.GFP, insulin to trigger promoter activities revealed that in- we generated an expression construct where the rat stead of 5–10 ␮U of insulin per ml, as is the case with the insulin I promoter (Ϫ410/ϩ1 bp) controlled the expres- Selective Signaling via A and B Insulin Receptors 561

Figure 1. Effect of Glucose on ␤GK mRNA Steady-State Levels, Transcription Initiation, and mRNA Stability (A) Elevation of ␤GK mRNA steady-state levels in isolated islets after stimulation with 16.7 mM glucose (15 min). (B) Dynamics of ␤GK 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 ␮g/ml) was present all the time under nonstimulatory conditions (closed squares), whereas in the case of stimulation (open squares) the inhibitor was added 45 min after start of stimulation. In (A) and (B), ␤GK mRNA values are presented as percentages of mRNA levels of the nonstimulated control at minute 0 (given as 100%). (C and D) Dynamics of ␤GK transcription initiation in response to glucose stimulation in HIT cells (C) and isolated islets (D). Transcription initiation was studied by nuclear run-off analysis. Elevation of RNA levels in stimulated cells is shown as the percentage of RNA levels of the nonstimulated control (given as 100%). In (A)–(D), all data are shown as mean values Ϯ S.E. (n ϭ 3). (E) On-line monitoring of glucose-stimulated ␤GK promoter–driven GFP expression in transfected HIT-T15 cells, islet cells, and whole islets. Representative images of HIT cells (n ϭ 40), islet cells (n ϭ 40), and islets (n ϭ 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 blue to red. Scale bars, 10 ␮m. Molecular Cell 562

Figure 2. Effect of Secretagogues, Voltage- Dependent L-Type Ca2ϩ Channel Blockers, and Exogenous Insulin on Endogenous ␤GK mRNA Levels and ␤GK Promoter–Driven GFP Expression (A) Elevation of endogenous ␤GK mRNA levels in cultured pancreatic islets in response to stimulation for 5 min with either 50 mM KCl (KCl) or 1 ␮M glibenclamide (glib) at 3 mM glucose. (B) Elevation of endogenous ␤GK mRNA levels in islet cells in response to stimulation for 15 min with 16.7 mM glucose with or without 10 ␮M nifedipine (nif). In (A) and (B), data are shown as mean values Ϯ S.E. (n ϭ 3), and amounts of ␤GK mRNA are presented as the percentage of mRNA levels of the nonstimulated control (given as 100%). (C) On-line monitoring of ␤GK promoter– driven GFP expression in islet cells. Islet cells were transfected with pr␤GK.GFP (␤GK) or with pRcCMV.GFP (CMV) as control and incubated with 16.7 mM glucose, 50 mM KCl, 1 ␮M glibenclamide (glib) or 5 mU/ml insulin (ins). Data are shown as mean values Ϯ S.E. (n ϭ 8). (D and E) Effect of increasing concentrations of insulin added to the culture medium for 5 min, on (D) endogenous ␤GK mRNA levels in isolated pancreatic islets and on (E) ␤GK promoter–driven GFP expression and insulin promoter–driven DsRed expression in transfected HIT cells. In (D), amounts of ␤GK mRNA are presented as the percentage of mRNA levels of nonstimulated control (given as 100%), and data are shown as mean values Ϯ S.E. (n ϭ 3). In (E), HIT cells were cotransfected with pr␤GK.GFP (open bars) and prIns1.DsRed (closed bars) and stimulated for 5 min with the indicated amounts of exogenous insulin. On-line monitoring data are presented as the ratio of fluorescence obtained at minutes 240 and 60 and represent mean values Ϯ S.E. (n ϭ 7).

sion of the red fluorescent protein DsRed (Matz et al., ploying a signaling pathway that is different from that 1999), prIns1.DsRed. Because of their different excita- utilized by the insulin promoter. tion and emission profiles, the signals generated by the Besides signaling via the MAP kinase and the PI3K/ two fluorescent proteins can be measured directly in mTOR/p70s6k pathways, insulin has been shown to ex- the same cell. Following cotransfection of islet cells and ert its effect via the activation of PKB(c-Akt) (Coffer et HIT cells with pr␤GK.GFP and prIns1.DsRed, insulin- al., 1998). To test whether stimulation with either glucose stimulated insulin and ␤GK promoter activities were or insulin leads to the activation of PKB in pancreatic ␤ monitored as DsRed and GFP fluorescence, respec- cells, we studied PKB activity following stimulation with tively. As shown in Figure 3C, stimulation with 5 mU/ either 16.7 mM glucose or 5 mU of insulin/ml. As shown ml insulin led to an elevation in both DsRed and GFP in Figure 4, PKB activation was observed 5 min following fluorescence, as expected. This increase in fluores- stimulation with 16.7 mM glucose (Figure 4A) and 2 min cence was abolished when the cells were treated with following stimulation with 5 mU of insulin/ml, at substim-

HNMPA-(AM)3, thereby blocking the tyrosine kinase ac- ulatory glucose concentrations (Figure 4B). Prevention tivity of IRs. By combining insulin stimulation with phar- of glucose-induced insulin secretion by treatment of macological inhibitors of either PI3K (LY294002), p70s6k insulin-producing cells with the L-type Ca2ϩ channel (rapamycin), or CaMKII (autocamtide-2 related inhibitory blocker nifedipine abolished glucose-induced activation peptide), we show that activation of the insulin promoter of PKB, as did inhibition of insulin signaling by HNMPA- is abolished (no increase in DsRed fluorescence). On (AM)3 (data not shown). These data suggest that PKB the other hand, no effect on insulin-stimulated ␤GK pro- is activated in response to glucose-stimulated insulin moter activity (increase in GFP fluorescence) was ob- secretion. Because of the lack of a selective pharmaco- served in the same cell (Figure 3C). logical inhibitor of PKB, we tested its involvement in Thus, insulin activates the ␤GK promoter by em- insulin-stimulated ␤GK gene transcription by transiently Selective Signaling via A and B Insulin Receptors 563

Figure 3. Effect of Various Protein Kinase Inhibitors on Insulin-Stimulated Insulin and ␤GK Promoter Activity and Endogenous ␤GK mRNA Levels (A) On-line monitoring of insulin promoter–driven (closed bars) and ␤GK 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 Ϯ S.E. (n ϭ 10). (B) Amounts of ␤GK mRNA are presented as the percentage of mRNA levels of nonstimulated control (given as 100%). Data are shown as mean values Ϯ S.E. (n ϭ 3). (C) HIT cells were cotransfected with pr␤GK.GFP (open bars) and prIns1.DsRed (closed bars). On-line monitoring data are presented as the ratio of fluorescence obtained at minutes 240 and 60 and represent mean values Ϯ S.E. (n ϭ 13). overexpressing PKB␣/c-Akt1. Whereas overexpression whereas 150 nM wortmannin was necessary to block of PKB␣ had no effect on insulin-stimulated insulin gene insulin-stimulated ␤GK promoter activity (Figure 4E). transcription, it led to a more pronounced effect on insu- These data indicate that insulin-stimulated ␤GK gene lin-stimulated ␤GK promoter–driven GFP expression transcription occurs by signaling via PDK1/PKB, whereas (Figure 4C). According to the current view, insulin-stimu- insulin-stimulated insulin gene transcription is mediated lated PKB activation involves the phosphorylation of via PI3K/p70s6k and CaMKII. PKB by the phosphoinositol-dependent kinase 1, PDK1 (Vanhaesebroeck and Alessi, 2000). Indeed, transient Insulin Signaling via IR-A Activates Insulin Gene overexpression of PDK1 led to a pronounced stimulation Promoter Whereas Signaling via IR-B of insulin-triggered ␤GK promoter activity, whereas over- Activates ␤GK Promoter expression of the antisense transcript of PDK1 abol- Previous data on insulin-stimulated insulin gene tran- ished the stimulatory effect of insulin on insulin-trig- scription (Leibiger et al., 1998a) favored signaling via IR gered ␤GK promoter activity (Figure 4C). Noteworthily, but did not exclude the signaling via IGF-I receptors or transient overexpression of either PKB␣ or PDK1 did possible hybrids of insulin- and IGF-I receptors. The not lead per se to an increased basal insulin or ␤GK loss of insulin effect when treating cells with HNMPA- promoter activity (data not shown). (AM)3, an inhibitor of the IR tyrosine kinase (Saperstein Interestingly, the activation of PKB has so far been et al., 1989), supported the idea that signaling via IR is shown to be dependent on the activity of PI3K (Vanhaese- crucial. Consequently, we examined whether the ex- broeck and Alessi, 2000) and therefore to be sensitive to pression of IR per se is an absolute requirement for the independent pharmacological inhibitors wortmannin insulin-stimulated insulin and ␤GK gene expression. and LY294002. Whereas treatment of insulin-producing Therefore, we analyzed insulin and ␤GK mRNA levels in cells with 25 ␮M LY294002 clearly abolished insulin- response to glucose/insulin stimulation in isolated islets stimulated rat insulin I gene promoter activity, it did from ␤IRKO mice, a knockout model that lacks the ex- not block insulin-stimulated rat ␤GK promoter activity pression of IR specifically in the pancreatic ␤ cell (Kul- (Figure 3). When analyzing the effect of LY294002 karni et al., 1999a). Stimulation with either 16.7 mM glu- on insulin-stimulated insulin and ␤GK promoter activity cose or 5 mU of insulin/ml led to an increase in both in a dose-dependent manner in cells cotransfected endogenous insulin and ␤GK mRNA levels in islets of with pr␤GK.GFP and prIns1.DsRed, we observed that wild-type mice, whereas no increase in insulin and ␤GK LY294002 inhibited the two promoters at different con- mRNA levels was observed in islets prepared from centrations. Whereas 25 ␮M LY294002 blocked insulin- ␤IRKO mice (Figure 5A). These data suggest that the stimulated insulin promoter activity, 100 ␮M LY294002 expression of the IR in pancreatic ␤ cells is an absolute was needed to completely abolish insulin-stimulated requirement to gain the stimulatory effect by insulin on ␤GK promoter activity (Figure 4D). The effect of wort- both insulin and ␤GK gene expression and that signaling mannin was similarly concentration dependent. Treat- via IGF-I receptors is unlikely to be involved. This is ment of cells with 50 nM wortmannin was sufficient consistent with the finding that activation of IGF-I recep- to inhibit insulin-stimulated insulin promoter activity, tors by stimulation with 2.6 nM IGF-I did not activate Molecular Cell 564

Figure 4. Role of PKB and PI3K in Insulin- Stimulated Insulin Gene and ␤GK Tran- scription (A and B) Dynamics of PKB activities following stimulation with 16.7 mM glucose (A) or 5 mU of insulin per ml in HIT cells (B). Activi- ties of PKB are presented as the percentage of the activity of the nonstimulated control (given as 100%). Data are shown as mean values Ϯ S.E. (n ϭ 3). (C) On-line monitoring of HIT cells cotransfected with pr␤GK.GFP (open bars), prIns1.D- sRed (closed bars), and either kinase-inactive mutant of PKB␣, i.e., PKB␣⌬308/437 (mock), wild-type PKB␣ (PKB), wild-type PDK1 (PDK1), or an antisense construct of PDK1 (PDK1anti- sense). Data are presented as the ratio of fluorescence obtained at minutes 240 and 60 and represent mean values Ϯ S.E. (n ϭ 8). (D and E) Effect of different concentrations of PI3K inhibitors LY294002 (D) and wortmannin (E) on ␤GK promoter–driven GFP expression (open bars) and insulin promoter–driven DsRed expression (closed bars) in cotransfected HIT cells. Data are presented as the ratio of fluorescence obtained at minutes 240 and 60 and represent mean values Ϯ S.E. (n ϭ 7).

insulin promoter– or ␤GK promoter–driven reporter gene Employing coexpression of pr␤GK.GFP and prIns1.D- expression in insulin-producing cells (data not shown). sRed in the same cell, we observed that treatment with Employing RT–PCR with subsequent DNA sequence the B-type receptor-specific antibody (␣IR-B) abolished analysis, we have previously observed that insulin-pro- insulin-stimulated pr␤GK.GFP expression, whereas it ducing cells express both IR-A and IR-B (Leibiger et did not affect insulin-stimulated prIns1.DsRed expres- al., 1998a). Transient overexpression of IR-A led to a sion (Figure 5C). In addition, treatment of insulin-produc- pronounced effect of insulin stimulation on insulin pro- ing cells with ␣IR-B abolished elevation of ␤GK mRNA moter activity, whereas overexpression of IR-B had no levels following stimulation with either 16.7 mM glucose effect. To test a similar effect for insulin-stimulated ␤GK or 5 mU of insulin/ml at substimulatory glucose concen- transcription, we cotransfected islet cells and HIT cells trations (data not shown). As expected, treatment of with pr␤GK.GFP and prIns1.DsRed in combination with transfected cells with an antibody that blocks insulin either IR-A or IR-B. To our surprise, we found that over- signaling via both receptor isoforms (␣IR-AB) sup- expression of IR-B led to a pronounced activation of pressed insulin-stimulated activation of ␤GK- and insu- the ␤GK promoter while overexpression of IR-A had lin-promoters (Figure 5C). Accordingly, treatment of in- no effect (Figure 5B). Moreover, overexpression of an sulin-producing cells with this antibody also abolished inactive IR-B mutant (IR-Bm), described as M1153I insulin-stimulated elevation of ␤GK mRNA steady-state (Levy-Toledano et al., 1994), did not lead to a further levels (data not shown). By contrast, treatment of trans- effect on r␤GK promoter–driven GFP expression (Figure fected cells with an antibody that blocks signaling via 6B). To test the involvement of IR-B in insulin-stimulated IGF-I receptors (␣IGF-1R) did not affect insulin-stimu- ␤GK promoter activation in more detail, we treated islet lated activation of ␤GK promoter– and insulin promoter– cells and HIT cells, prior to glucose/insulin stimulation, driven reporter gene expression (Figure 5C). with an anti-IR-B antibody. This antibody selectively These data indicate that insulin stimulates the insulin binds to the ␣ chain of IR-B and therefore selectively gene promoter through IR-A, whereas it stimulates the blocks insulin binding to IR-B and signaling via IR-B. ␤GK gene promoter via IR-B. Selective Signaling via A and B Insulin Receptors 565

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 ␤GK mRNA steady-state levels in isolated pancreatic islets obtained from ␤IRKO mice (␤IRKO) or control animals (wild type). The values of ␤GK mRNA are presented as percentages of mRNA levels of the nonstimulated islets (given as 100%). Data are shown as mean values Ϯ S.E. (n ϭ 3). (B) On-line monitoring of ␤GK promoter–driven GFP expression (open bars) and insulin promoter–driven DsRed expression (closed bars) in cotransfected islet cells. Cells were cotransfected with pr␤GK.GFP and prIns1.DsRed and either expression constructs for wild-type isoforms of 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 ratio of fluorescence obtained at minutes 240 and 60 and represent mean values Ϯ S.E. (n ϭ 10). (C) Effect of antibodies that block signaling through IR-A and IR-B (␣IR-AB), through IR-B (␣IR-B), and through IGF-I receptors (␣IGF-1R) on insulin-stimulated ␤GK promoter–driven GFP expression (open bars) and insulin promoter–driven DsRed expression (closed bars) in cotransfected islet cells. Cells were incubated with a 0.67 ␮g/ml concentration of the respective antibodies 30 min prior to stimulation and throughout stimulation. Data are presented as the ratio of fluorescence obtained at minutes 240 and 60 and represent mean values Ϯ S.E. (n ϭ 10).

To start to understand the molecular mechanisms that receptor isoforms with GFP and DsRed at the C terminus underlie the selectivity in insulin signaling via the two of the ␤ subunit. Tagging both IR isoforms did not inter- IR isoforms, we aimed to explain the different sensitivity fere with their physiological function (e.g., overexpres- for PI3K inhibitors we observed between insulin-stimu- sion of the tagged IR isoform led to a pronounced insu- lated insulin- and ␤GK-promoter activation (Figures 4D lin effect on the respective promoter activity to the and 4E). One possible interpretation would be that the same extent as the untagged IR) (data not shown). same PI3K is involved in the transcription of both genes Whereas transient coexpression of the same, but differ- /GFPفGFP and IR-BفDsRed/IR-Aفbut that a lower PI3K activity is sufficient to trigger the ently tagged (IR-A -DsRed), IR isoform led to a complete colocalizaفcascade that activates ␤GK gene transcription via PKB. IR-B If this is the case, inhibition of PI3K-mediated ␤GK tran- tion (data not shown), coexpression of the differently فscription should require higher concentrations of wort- tagged IR-A and IR-B in either combination (IR-A DsRed) clearlyفGFP/IR-BفGFP and IR-Aفmannin or LY294002 to be fully effectuated. This should DsRed/IR-B also be reflected upon by an inverse relationship be- showed IR isoforms that are not colocalized. This pat- tween required inhibitor concentration and sensitivity of tern of distinct IR isoform distribution was observed in the respective promoter activity to insulin. If the same insulin-producing cells HIT (Figure 6A), INS1, and MIN6, PI3K is sufficient to lead to the activation of both insulin- as well as in non-insulin-producing cells HEK293 and and ␤GK-promoters, then the promoter activity that is COS7 (data not shown). To test whether the two IR least sensitive to the inhibitors (i.e., ␤GK) would be ex- isoforms do utilize different classes of PI3K, we overex- GFP in HIT cells andفGFP or IR-Bفpected to require less insulin to become stimulated. As pressed either IR-A demonstrated in Figure 2E, this is not the case. On the studied the sensitivity of PI3K activity to wortmannin in contrary, more insulin is needed to stimulate ␤GK pro- vitro following immunoprecipitation with GFP antibod- moter–driven GFP expression. Another interpretation is ies. Whereas the PI3K activity in the IR-A immunoprecip- that the different sensitivity in vivo could be due to a itate was inhibited by wortmannin in the lower nanomo- different accessibility of the inhibitor for the same type lar range, as typical for PI3K class I and III, the PI3K of PI3K as a result of a different distribution/localization activity in the IR-B immunoprecipitate was only inhibited of the two IR isoforms, or it could be due to the involve- at higher concentrations (Figure 6B), as described for ment of different classes of PI3K, exhibiting a different PI3K class II (see Fruman et al., 1998). To test whether sensitivity to wortmannin and LY294002 as described insulin-stimulated insulin gene transcription involves for PI3K classes I and III versus class II (reviewed in IR-A-mediated insulin signaling via PI3K class Ia, we Fruman et al., 1998). To test whether IR-A and IR-B combined insulin stimulation with the transient over- exhibit a distinct distribution in vivo, we tagged both expression of the dominant-negative form of the PI3K Molecular Cell 566

Figure 6. Molecular Mechanisms Involved in the Selective Insulin Signaling via IR-A and IR-B DsRed (red) in HIT cells obtained by laser scanning confocal microscopy. Areas in yellowفGFP (green) and IR-BفA) Distribution of IR-A) indicate 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- GFP (open bars). The amount of wortmannin included in the in vitro assay is indicated. Data are presented asفGFP (closed bars) or IR-BفA mean values Ϯ S.E. (n ϭ 3). (C) Effect of overexpression of dominant-negative p85 PI3K subunit, ⌬p85, on insulin-stimulated ␤GK promoter–driven GFP expression (open bars) and insulin promoter–driven DsRed expression (closed bars) in cotransfected HIT cells. Data are presented as the ratio of fluorescence obtained at minutes 240 and 60 and represent mean values Ϯ S.E. (n ϭ 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 or IR-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 Ϯ S.E. (n ϭ 3). class Ia adaptor protein p85 (i.e., ⌬p85). Whereas tran- through PI3K class Ia and p70s6k on the one hand, and sient overexpression of ⌬p85 totally abolished insulin- via IR-B through a different PI3K activity, very similar to stimulated insulin promoter activity, this approach had that of class II, and PKB on the other. no effect on insulin-stimulated ␤GK promoter activation When separately overexpressing IR isoforms in HIT (Figure 6C). cells, we observed a more pronounced activation of Taken together, these data suggest a selectivity in p70s6k in cells overexpressing IR-A in response to insu- insulin signaling in insulin-producing cells via IR-A lin stimulation, while cells overexpressing IR-B showed Selective Signaling via A and B Insulin Receptors 567

Figure 7. Selective Activation of Insulin and Glucokinase Gene Transcription by Selective Insulin Signaling via A- and B-Type Insulin Receptors The scheme illustrates the coupling between insulin exocytosis and the activation of transcription 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 or whether this effect is purely specific for the pancreatic IR-A function, respectively, a direct proof of the involve- ␤ cell, we analyzed both PKB and p70s6k activities in ment of IR isoforms remains to be shown in IR isoform– non-insulin-producing HEK293 cells that were tran- specific knockout models. Attempts to correlate tissue- siently overexpressing either IR-A or IR-B. Most interest- specific expression of IR isoforms with diabetes mellitus ingly, insulin-stimulated p70s6k activity here was also has generated conflicting results (Mosthaf et al., 1991; more pronounced in cells overexpressing IR-A, whereas Benecke et al., 1992; Norgren et al., 1993) that do not insulin-stimulated PKB activity seemed to be more clarify the functional role of either isoform. tightly coupled with the overexpression of IR-B (Fig- Little is known about selective insulin signaling via A- ure 6E). and B-type IR. Besides the affinity for insulin, differences in their kinase activity (Kellerer et al., 1992) as well as Conclusion internalization and recycling (Vogt et al., 1991; Yama- Selectivity in insulin signaling is currently discussed as guchi et al., 1991) have been described. These data have the result of the activation of specific signal transduction implied differences in the function of either IR isoform, pathways. This selectivity may be gained by activating but no isoform-specific insulin-induced effect has been specific adaptor proteins (i.e., IRS and Shc proteins) reported so far. In the present paper, we provide a “read- that “channel” the insulin signal in a more defined way out” system for discriminating selective signaling via by specifically interacting with downstream located ef- the two IR isoforms. We have demonstrated that the fector proteins (Myers and White, 1996; Virkama¨ ki et molecular basis for this selectivity could be provided by al., 1999). Whereas the importance of IRS proteins in the different localization of the two IR isoforms in the achieving insulin effects in different tissues is currently plasma membrane and their different sensitivity for insu- under extensive investigation, the possibility of selective lin. Mechanistically, this enables preferential activation insulin signaling via the two isoforms of the IR has been of IR-A/PI3K Ia/p70s6k in pancreatic ␤ cell glucose/ neglected. Studies on general and tissue-specific IR insulin–stimulated insulin gene transcription and IR-B/ knockout models have demonstrated that a defect IR- PI3K class II–like/PKB in glucose/insulin–stimulated mediated insulin signaling leads to a type 2 diabetes–like ␤GK transcription (Figure 7). That this specificity in sig- phenotype (reviewed in Taylor, 1999). However, these naling has a wider implication than to the pancreatic ␤ knockouts do not discriminate between the two IR iso- cell is demonstrated by the data obtained in non-insulin- forms. This is of importance, since earlier studies clearly producing HEK293 cells. established differences in tissue-specific IR isoform ex- Thus, our data clearly demonstrate that selectivity of pression as well as in their activation profile. Whereas insulin signaling can be gained by signaling through IR-B, which shows a 2-fold lesser affinity for insulin in the two IR isoforms and reinforce the concept of the comparison to IR-A (Mosthaf et al., 1990; Yamaguchi et pancreatic ␤ cell as a target for positive insulin action. al., 1991; McClain, 1991), is predominantly expressed in liver and muscle, IR-A is mainly expressed in brain Experimental Procedures (Moller et al., 1989; Seino and Bell, 1989; Mosthaf et al., Materials 1990). Although it is tempting to link the phenotypes of Bisindolylmalemide I, PD98059, wortmannin, LY294002, rapamycin, the liver- and brain-specific IR knockouts (Bru¨ ning et HNMPA-(AM)3, autocamtide-2 related inhibitory peptide, and nifedi- Molecular Cell 568

pine were purchased from Calbiochem. Actinomycin D was from RNA Analysis Sigma. Microcystin-LR and PD169316 were purchased from Alexis Levels of ␤GK mRNA were analyzed by comparative RT–PCR as Biochemicals. Rabbit anti-Insulin Receptor ␣ and Rabbit anti-Insulin described in Leibiger et al. (1998b) using primers 5Ј-GTTCCTACTG Receptor B antibodies were from Biodesign. Mouse monoclonal GAGTATGACC-3Ј and 5Ј-CCTCCTCTGATTCGATGAAG-3Ј for char- IGF-IR␣ was from Pharmingen. Oligonucleotides were synthesized acterizing ␤GK mRNA in HIT cells, and primers 5Ј-TGGATGACA at Genset (France). GAGCCAGGATGG-3Ј and 5Ј-ACTTCTGAGCCTTCTGGGGTG-3Ј for ␤GK mRNA in rat and mouse pancreatic islets and islet cells. Levels Expression Constructs of ␤ actin mRNA were analyzed by RT–PCR using primers 5Ј-AACTGG The construction of prIns1.GFP has been described earlier (Leibiger AACGGTGAAGGCGA-3Ј and 5Ј-AACGGTCTCACGTCAGTGTA-3Ј. et al., 1998a). prIns1.DsRed was generated by exchanging the GFP PCR conditions were chosen that guaranteed the amplification of expression cassette versus the DsRed-expression cassette from ␤GK and actin fragments within the linear range, as verified by pDsRed1–1 (Clontech). pr␤GK.GFP was generated by exchanging testing various numbers of amplification cycles (10–35). PCR prod- the CAT expression cassette of pr␤GK-278.CAT (Leibiger et al., 1994a) ucts were separated on a 6% polyacrylamide sequencing gel and versus the GFP-expression cassette obtained from pRcCMVi.GFP analyzed by phosphorimaging. Quantification was performed with (Moede et al., 1999). Constructs for expression of the human IR-A TINA software 2.07d (Raytest). Values of ␤GK mRNA were normal- and -B types, pRcCMVi.hIR(A) and pRcCMVi.hIR(B), were generated ized by ␤ actin values. by subcloning the respective IR-A and IR-B sequences from pCMVHIR(A) and pCMVHIR(B) (kindly provided by A. Ullrich, MPI Analysis of PKB Activity for Biochemistry, Martinsried, Germany) into pRcCMVi. GFP- and Analysis of PKB activity was performed employing the Akt1/PKB␣ DsRed-tagged variants were generated by introducing a ClaI-site Immunoprecipitation Kinase Assay Kit (Upstate Biotech.) according ␤ into the IR 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- tion into the IR cDNA was performed by employing the Quik- Analysis of p70s6k Activity Change Mutagenesis Kit (Stratagene). Adenovirus-based vector p70 s6 kinase from cell lysates was immunoprecipitated using a Ad.r␤GK.GFP was constructed by subcloning the r␤GK.GFP cas- p70s6k antibody (Upstate Biotech.). Analysis of p70s6k activity was sette into pAC.CMV.pLpA and performing homologous recom- performed employing the S6 Kinase Assay Kit (Upstate Biotech.) bination as described in Moitoso de Vargas et al. (1997). All according to the manufacturer’s instructions. vector constructions were verified by DNA sequence analysis. Plas- ␣ ␣⌬ mids pCMV5.PKB , pCMV5.PKB 308/437, pCMV5.PDK1, and Analysis of PI3K Activity GFP orف(pCMV5.PDK1 antisense were kindly provided by D.R. Alessi (MRC HIT cells were transfected with either pRcCMVi.hIR(A GFP. Cell lysates containing 2 mg of protein wereف(Phosphorylation Unit, University of Dundee, UK), and pcDNA3- pRcCMVi.hIR(B ⌬ Zeo. p85 was a gift from C.P. Downes (Department of Biochemistry, subjected to immunoprecipitation using anti-GFP antibody A-11122 University of Dundee, UK). (Molecular Probes). PI3K activity was analyzed in the GFP immunoprecipitates as described in Krook et al. (1997) using L-␣-phosphati- Cell Culture and Transfection dylinositol from Avanti Polar-Lipids, Inc. Isolation of pancreatic islets, the culture of islets, islet cells, and HIT-T15 cells, and their transfection have been described in Leibiger et al. (1998a; 1998b). Following transfection, HIT cells were cultured On-Line Monitoring of GFP and DsRed Expression for 12–18 hr in RPMI 1640 medium containing 0.1 mM glucose, 10% and Detection of Fluorescence fetal calf serum and supplemented with 100 U/ml penicillin, 100 Detection of fluorescence by digital imaging fluorescence microscopy was performed as described previously (Leibiger et al., 1998a; ␮g/ml streptomycin, 2 mM glutamine at 5% CO2 and 37ЊC. Before stimulation, islets and islet cells were incubated for 2 hr in RPMI 1998b). The following filter settings were used: for GFPS65T: excitation 1640 medium supplemented as above but containing 3 mM glucose. at 485 nm, a 505 nm dicroic mirror, and an emission band-pass filter HEK293 cells were grown in DMEM containing 5.5 mM glucose, of 500–530 nm; for DsRed: excitation at 558 nm, a 565 nm dicroic 10% fetal calf serum, 100 U/ml penicillin, 100 ␮g/ml streptomycin, mirror, and a 580 nm long-pass filter for emission. On-line monitoring was initiated 60 min following the start of stimulation and the 60 2 mM glutamine at 5% CO2 and 37ЊC, and were transfected by the calcium phosphate/coprecipitation technique as described earlier min value of GFP or DsRed fluorescence was set to 1.0. Cells to be (Leibiger et al., 1994a). Islets were transduced with Ad.r␤GK.GFP monitored were chosen randomly at minute 60 from six fields of as described in Moitoso de Vargas et al. (1997) and analyzed by vision, and fluorescence was monitored up to minute 240. By over- laser scanning confocal microscopy. laying the fluorescence and phase-contrast images, the positions Cultured islets, islet cells, and HIT cells were stimulated with either of cells on the coverslip could be checked. Fluorescence intensity 16.7 mM glucose for 15 min or 50 mM KCl, 1 ␮M glibenclamide or was calculated by using the Isee software for UNIX (Inovision Corpo- with various concentrations of insulin for 5 min at substimulatory ration). glucose concentrations (3 mM for islets/islet cells and 0.1 mM for Laser scanning confocal microscopy was performed using a HIT cells). All experiments were performed in RPMI 1640 medium, LEICA TCS SP2 (Leica Lasertechnik GmbH) with the following set- ϫ supplemented as above. Protein kinase inhibitors, antibodies, or tings: Leica HCX PL APO 63 /1.20/0.17 UV objective lens, excitation nifedipine were added to the culture medium 30 min prior to stimula- wavelength 488 nm (Ar Laser) and 543 nm (HeNe laser), a 488/543 tion and were kept in the medium throughout stimulation. After double dichroic mirror, and detection of GFP at 505–525 nm and stimulation, islets, islet cells, and HIT cells were cultured further at DsRed at 605–670 nm. Laser scanning confocal microscopy of trans- substimulatory glucose concentrations. For RNA analysis, islets and duced islets was performed as described in Leibiger et al. (1998b). cells were harvested 60 min after start of stimulation, if not indicated otherwise. For analysis of protein kinase activities or tyrosine phos- Acknowledgments phorylation, cells were harvested immediately following stimulation. The authors wish to thank Drs. A.M. Bertorello, K. Michelsen, T. Nuclear Run-Off Analysis Schwarz-Romond, X. Wang, and N. Welsh for sharing material and Nuclear run-off analysis was performed as described previously unpublished data. This work was supported by funds from Karolin- (Leibiger et al., 1998b), except that labeled RNA was hybridized to ska Institutet; by grants from Juvenile Diabetes Foundation Interna- 2.5 ␮g of cDNA of glucokinase, insulin, ␤ actin, and control pBlue- tional (JDFI), the Novo Nordisk Foundation, the Nordic Insulin Foun- script DNA, which were immobilized on nitrocellulose filters. For dation Committee, EC Biotechnology Project (BIO4-CT98-0286), the nuclear run-off analysis on islets, nuclei from 2000 islets per experi- Swedish Diabetes Association, and the Swedish Medical Research ment were used. Data were analyzed by phosphorimaging. Values Council (72X-12594, 03X-13394, 72X-00034, 72XS-12708, 72X- obtained for ␤GK mRNA were normalized by ␤ actin mRNA values. 09890); and in part by NIH GRASP Center grant DK34928 and NIH Selective Signaling via A and B Insulin Receptors 569

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