Exocytosis of Insulin Promotes Insulin Gene Transcription via the Insulin Receptor/PI-3 Kinase/p70 s6 Kinase and CaM Kinase Pathways  Ingo B Leibiger, Barbara Leibiger, Tilo Moede, Per-Olof Berggren  Molecular Cell  Volume 1, Issue 6, Pages 933-938 (May 1998) DOI: 10.1016/S1097-2765(00)80093-3

Figure 1 Effect of Secretagogues and Voltage-Dependent L-Type Ca2+ Channel Blockers on Endogenous PPI mRNA Levels and Insulin Promoter-Driven GFP Expression (A) Endogenous PPI mRNA levels in cultured rat pancreatic islets and HIT-T15 cells following stimulation with either glucose or KCl. Treatment with 10 μM nimodipine (nim) lasted from 2 min prior to stimulation up to 2 min after stimulation. Amounts of PPI mRNA are presented as percentage of mRNA levels of nonstimulated control (given as 100%). PPI mRNA was quantified by comparative RT–PCR. The increase in PPI mRNA following glucose stimulation was verified by RNase-protection analysis and showed similarly elevated values. Data shown are from a representative experiment. This experiment has been done a minimum of three times in duplicate with similar results. Elevation of PPI mRNA levels in response to glucose varied in islets from 3.5- to 5-fold and in HIT cells from 2- to 3-fold. (B) On-line monitoring of insulin promoter-driven GFP expression in transfected islet cells and HIT-T15 cells. Islet cells and HIT cells were transfected with prIns1GFP (R1GFP) or with pRcCMVGFP (CMVGFP). Mean values for 7 islet cells and for 10 HIT cells are presented. SD values never exceeded 10% of the shown mean values. Molecular Cell 1998 1, 933-938DOI: (10.1016/S1097-2765(00)80093-3)

Figure 2 Stimulus-Dependent Insulin Gene Transcription (A) Effect of various protein kinase inhibitors on insulin promoter-driven GFP expression. On-line monitoring of GFP expression in transfected islet cells (n = 7) and HIT-T15 cells (n = 10) following stimulation with either glucose or KCl. SD values never exceeded 10% of the shown mean values. (B) Effect of various protein kinase inhibitors on endogenous PPI mRNA levels. Amounts of PPI mRNA from islet cells and HIT cells were quantified by comparative RT–PCR and are presented as percentage of mRNA levels of nonstimulated control (given as 100%). Data are from a representative experiment. This experiment has been done a minimum of three times in duplicate with similar results. (A and B) (LY), 25 μM LY294002; (PD), 20 μM PD98059; (SB), 20 μM SB203580; (BIM), 150 nM bisindolylmaleimide I; (RP), 100 μM Rp isomer of cAMP; (K6), 10 μM KN-62; (rap), 10 nM rapamycin. (C) Effects of exogenous insulin on endogenous PPI mRNA levels. Amounts of PPI mRNA were quantified by RNase-protection analysis and are presented as percentage of mRNA levels of nonstimulated control (given as 100%). Data are from a representative experiment. This experiment has been done a minimum of three times in duplicate with similar results. Elevation of PPI mRNA levels in response to 5 mU/ml insulin varied in islet cells from 4- to 5-fold and in HIT cells from 3- to 4-fold. (D) Effects of exogenous insulin on insulin promoter-driven GFP expression. On-line monitoring of GFP expression in transfected islet cells (n = 7). SD values never exceeded 10% of the shown mean values. Molecular Cell 1998 1, 933-938DOI: (10.1016/S1097-2765(00)80093-3)

Figure 3 Role of Insulin Receptors and p70 s6k in Signal Transduction (A) Effects of inhibition of insulin receptor activity by HNMPA and overexpression of human insulin receptors A and B [HIR(A) and HIR(B)] on insulin promoter-driven GFP expression in stimulated HIT cells. Cells were cotransfected with prIns1GFP and with either pCMVHIR(A), pCMVHIR(B), or pCMVStop (mock). Tyrosine kinase activity of endogenous insulin receptors was blocked by pretreatment of transfected HIT cells with 100 μM HNMPA-(AM)3 (HNMPA) for 30 min prior to and throughout stimulation. Mean values for 10 HIT cells are presented. SD values never exceeded 10% of the shown mean values. (B) Role of p70 s6k in glucose/insulin-stimulated insulin promoter-driven GFP expression in HIT cells. Cells were cotransfected with prIns1GFP and with either wild-type p70 s6k (p70), the rapamycin-resistant form of p70 s6k, i.e., p70Δ2−46/ΔCT104 (p70Δ), or pCMVstop (mock). Data are presented as the ratio of GFP-fluorescence values obtained at minutes 240 and 60 and represent mean values ± SD for at least 7 monitored HIT cells. Molecular Cell 1998 1, 933-938DOI: (10.1016/S1097-2765(00)80093-3)

Figure 4 Role of Insulin Promoter cis Elements in the Glucose/Insulin-Stimulated rInsGFP Expression Islet cells and HIT-T15 cells were transfected with either the wild-type prIns1GFP or with constructs carrying mutations of one of the following cis elements: A1, E1, CAAT, CRE, E2, or A3/4. Data are presented as the ratio of GFP-fluorescence values obtained at minutes 240 and 60 and represent mean values ± SD for at least 9 monitored HIT cells and 5 monitored islet cells. Nucleotide sequences of the respective cis elements are given. Upper sequences show the wild type. Mutations in the lower sequences are shown as lowercase letters. Molecular Cell 1998 1, 933-938DOI: (10.1016/S1097-2765(00)80093-3)

Figure 5 Insulin-Secretion/Insulin-Gene-Transcription Coupling The scheme illustrates the coupling between insulin exocytosis (briefly outlined under the Results and Discussion) and insulin gene transcription. (KATP channel), ATP-sensitive K+ channel; (L-type VDCC), L-type voltage-dependent Ca2+ channel; (Glc), glucose. Molecular Cell 1998 1, 933-938DOI: (10.1016/S1097-2765(00)80093-3)