Selective insulin signaling in the pancreatic beta-cell via the two insulin receptor isoforms

Insulin exhibits pleiotropic effects that are tissueas well as development-dependent. However, the mechanisms by which insulin gains selective effects are poorly understood. Selectivity in insulin signaling is currently discussed as the result of the activation of specific signal transduction pathways. This may be gained by activating specific adapter proteins, such as IRS proteins and Shc, that ‘channel’ the insulin signal in a more defined way by specifically interacting with downstream located effector proteins. The insulin receptor (IR), the first step in these cascades, exists in two isoforms as a result of alternative mRNA splicing of the 11th exon of the pro-receptor transcript. IR-A lacks whereas IR-B contains the respective sequence coding for 12 amino acids in the C-terminus of the α-chain of the receptor. Studies on general and tissue-specific IR knockout models have demonstrated that a defect IR-mediated insulin signaling leads to a type 2 diabetes-like phenotype. However, these knockouts do not discriminate between the two IR isoforms. Besides their different affinity for insulin, differences in kinase activity as well as internalization and recycling for IR-A and IR-B have been described. These data implied differences in the function of either IR isoform. Although all cell types express both isoforms to a various degree, little is known about the mechanisms that underlie IR isoform-specific signaling and their biological importance remains obscure. Besides the classical insulin target tissues liver, muscle and fat, recent research disclosed the pancreatic β-cell as an important target for pleiotropic insulin action, here involving signal transduction through IR and IGF-I receptors. The overall objective of the present thesis work was to test the hypothesis that the two IR isoforms contribute to selective insulin signaling. Specifically, we aimed to investigate the molecular mechanisms that allow simultaneous and selective transcriptional activation of three model genes encoding insulin, β-cell glucokinase (βGK) and c-fos by insulin signal transduction via the two IR isoforms in the pancreatic β-cell. We show here that insulin activates the transcription of these three genes by different mechanisms. Insulin activates transcription of its own gene by signaling via IR-A and IRS/PI3K Ia/mTOR/p70s6k. In contrast, βGK and c-fos genes are activated by insulin signaling via IR-B but employing different signaling cascades. While insulin-stimulated βGK promoter up-regulation requires the integrity of the IR-B NPEY-motif and signaling via PI3K-C2α/PDK1/PKB, c-fos gene activation needs the intact YTHM-motif and signaling via PI3K Ia/p52-Shc/MEK1/ERK1/2. Studying the molecular mechanisms that underlie the selective signaling via IR-A versus IR-B, we found that both IR-A-mediated insulin and IR-B-mediated βGK promoter activation are not dependent on IR isoform-specific differences in internalization but on their spatial segregation in the plasma membrane. Our data demonstrate that localization and function of the two receptor types depend on the 12 amino acids encoded by exon 11. Moreover, our data suggest that selective activation of the insulin and βGK promoters occurs by signaling from non-caveolae plasma membrane micro-domains that are differently sensitive towards cholesterol depletion. Analyzing the mechanisms that allow activation of selective signaling cascades downstream of IR-B, we found that insulin activates the βGK promoter from membrane-standing IR-B, while c-fos promoter activation is dependent on clathrin-mediated IR-B endocytosis. In conclusion, the results of the present thesis work clearly demonstrate that spatial segregation of selective signaling pathways originating from IR-A and IR-B allows the simultaneous activation of discrete signaling cascades that lead to specific insulin effects. LIST OF PUBLICATIONS I. Leibiger, B. , Leibiger, I.B. , Moede, T. , Kemper, S. *, Kulkarni, R.N. , Kahn, C.N. , Moitoso de Vargas, L. , and Berggren, P.-O. (2001). Selective insulin signaling through A and B insulin receptors regulates transcription of insulin and glucokinase genes in pancreatic β cells. Mol. Cell 7, 559-570. II. Uhles, S. , Moede, T. , Leibiger. B. , Leibiger. I.B. , and Berggren, P.-O. PI3K-C2α is involved in insulin receptor B-type mediated activation of glucokinase gene transcription in insulin-producing HIT-T15 cells. Manuscript. III. Uhles, S. , Moede, T. , Leibiger, B. , Berggren, P.-O. , and Leibiger, I.B. (2003). Isoform-specific insulin receptor signaling involves different plasma membrane domains. J. Cell. Biol. 163, 1327-1337. IV. Uhles, S. , Moede, T. , Leibiger, B. , Berggren, P.-O. , and Leibiger, I.B. Spatial segregation of insulin receptor B-type signaling allows selective and simultaneous activation of glucokinase and c-fos gene transcription. Manuscript submitted. * Uhles, S. – former Kemper, S. All previously published papers were reproduced with permission from Elsevier (Paper I) and the Rockefeller University Press (Paper III).