Glucagon regulates its own synthesis by autocrine signaling

Abstract

Peptide hormones are powerful regulators of various biological processes. To guarantee continuous availability and function, peptide hormone secretion must be tightly coupled to its biosynthesis. A simple but efficient way to provide such regulation is through an autocrine feedback mechanism in which the secreted hormone is "sensed" by its respective receptor and initiates synthesis at the level of transcription and/or translation. Such a secretion-biosynthesis coupling has been demonstrated for insulin; however, because of insulin's unique role as the sole blood glucose-decreasing peptide hormone, this coupling is considered an exception rather than a more generally used mechanism. Here we provide evidence of a secretion-biosynthesis coupling for glucagon, one of several peptide hormones that increase blood glucose levels. We show that glucagon, secreted by the pancreatic α cell, up-regulates the expression of its own gene by signaling through the glucagon receptor, PKC, and PKA, supporting the more general applicability of an autocrine feedback mechanism in regulation of peptide hormone synthesis.

Fig. 1.

Secreted or exogenously added glucagon stimulates (prepro)glucagon gene transcription and glucagon biosynthesis. (A) (Prepro)glucagon mRNA levels in cultured human and mouse islets at 60 min after the start of stimulation with exogenously added glucagon (200 nM for 15 min). (Prepro)glucagon mRNA levels represent the percentage of mRNA levels of nonstimulated control (given as 100%); n = 5. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01. (B and C) (Prepro)glucagon mRNA levels in cultured mouse islets (B) and clonal αTC1-9 cells (C) at indicated time points after the start of stimulation with exogenously added glucagon (200 nM for 15 min). (Prepro)glucagon mRNA levels represent the percentage of mRNA levels of nonstimulated control (given as 100%); n = 3. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (D and E) Online monitoring of glucagon promoter-driven DsRed2 expression in transfected αTC1-9 cells. (E) Effects of different amounts of exogenously added glucagon on glucagon promoter-driven and CMV promoter-driven DsRed2 expression. Data represent the ratio of DsRed2 fluorescence values obtained at 240 min and 60 min, expressed as mean ± SEM for at least 15 monitored cells. *P < 0.05; **P < 0.01. (F) Effect of immunoabsorption of secreted glucagon on glucagon promoter-driven DsRed2 expression in transfected αTC1-9 cells. Shown are the effects of glucose at 16.7 mM (1) and 1 mM (2) without immunoabsorption; effects of immunoabsortion with anti-glucagon antibodies at 1 mM glucose (3) and 16.7 mM glucose (4); and effect of control IgG at 1 mM glucose (5). Data represent the ratio of DsRed2 fluorescence values obtained at 240 min and 60 min and are expressed as mean ± SEM for at least 15 monitored cells. (–5) Significance vs. (1): **P < 0.01. ns: not significant. (–5) Significance vs. (2): ^#P < 0.01. ns: not significant. (G) Effects of exogenously added or secreted glucagon on glucagon biosynthesis. αTC1-9 cells were incubated at 16.7 mM glucose without stimulation (1) or stimulated with either 200 nM exogenous glucagon at 16.7 mM glucose (2) or 1 mM glucose (3) for 15 min, and glucagon biosynthesis was analyzed by incorporation of ³H-labeled leucine for 60 min. Data are expressed as mean ± SEM for five independent experiments. **P < 0.01.

Fig. 2.

Glucagon stimulates its own expression by signaling via the glucagon receptor. (A) Identification of the glucagon receptor in lysates of αTC1-9, mouse islets, and control tissues (insulin-producing cell lines HIT-T15, MIN6, and INS1 and mouse muscle) by Western blot analysis. (B) Identification of the glucagon receptor by immunohistochemistry in αTC1-9, mouse islets, and human islets (green). In islets, α cells were identified by costaining with an anti-glucagon antibody (red). The images are single confocal frames. (Scale bar: 10 µm.) (C and D) Effects of GRA II on (prepro)glucagon mRNA levels in mouse and human islets (C) and clonal αTC1-9 cells (D). Islets and αTC1-9 cells were cultured at 16.7 mM glucose and stimulated for 15 min with 200 nM glucagon or left unstimulated. GRA II (400 nM) was added 30 min before the start of stimulation and maintained throughout the stimulation. (Prepro)glucagon mRNA levels represent the percentage of mRNA levels of nonstimulated control (given as 100%); n = 3. Data are expressed as mean ± SEM. Significance vs. stimulated expression without GRA II, *P < 0.05. (E and F) Effect of GRA II on glucagon promoter-driven DsRed2 expression in transfected αTC1-9 cells stimulated with either exogenously added glucagon (200 nM at 16.7 mM glucose for 15 min) (E) or 1 mM glucose for 15 min (F) to trigger glucagon secretion. Significance vs. stimulated expression without GRA II, *P < 0.05; ^#P < 0.01. GRA II (400 nM) was added 30 min before the start of stimulation and maintained throughout the stimulation. (G) Effect of [des-His1, Glu9]glucagon ([ΔH1,E9]glucagon) on glucagon-stimulated glucagon promoter-driven DsRed2 expression. Transfected αTC1-9 cells were preincubated for 30 min with 1 µM [ΔH1,E9]glucagon and then stimulated with 200 nM glucagon for 15 min at 16.7 mM glucose or left unstimulated. Significance vs. stimulated expression without [ΔH1,E9]glucagon, *P < 0.05; ^#P < 0.05. Data in E–G represent the ratio of DsRed2 fluorescence values obtained at 240 min and 60 min and are expressed as mean ± SEM for at least 15 monitored cells.

Fig. 3.

Glucagon stimulates its own expression by signaling via PKA and PKC. (A and B) Effect of exogenously added (200 nM for up to 15 min) or secreted glucagon (in response to 1 mM glucose, for up to 15 min) on the activity for PKA (A) or PKC (B). Protein kinase activities are presented as percentage of activity in nonstimulated control (given as 100%); n = 6. Data are expressed as mean ± SEM. *P < 0.05 in A; *P < 0.01 in B. (C–E) Effect of inhibitors of PKA (Rp-cAMP) and PKC (BIM) on glucagon-stimulated (prepro)glucagon mRNA levels (C and E) and glucagon promoter-driven DsRed2 expression (D). Transfected αTC1-9 cells or islets were preincubated with either 100 µM Rp-cAMP or 150 nM BIM or vehicle (control) for 30 min and then stimulated with exogenous glucagon (200 nM at 16.7 mM glucose) or left unstimulated. (Prepro)glucagon mRNA levels at 60 min after the start of stimulation are presented as percentage of mRNA levels of nonstimulated control (given as 100%); n = 3. Data are expressed as mean ± SEM. Significance vs. stimulated expression without inhibitor, *P < 0.05. Data in D represent the ratio of DsRed2 fluorescence values obtained at 240 min and 60 min and are expressed as mean ± SEM for at least 15 monitored cells.

Fig. 4.

Glucagon-stimulated (prepro)glucagon gene transcription is mediated via CREB and the CRE motif in the (prepro)glucagon promoter. (A and B) CREB binds in electrophoretic mobility shift assays to ds oligonucleotides containing an intact CRE motif (CRE), but not to ds oligonucleotides containing a mutated CRE (CRE-mut). The normal ds oligonucleotide/CREB complex 1 (B, lanes 2, 4, and 6) is “supershifted” and forms complex 2 after incubation with an anti-CREB antibody (B, lane 5). (C–E) Effect of CRE-mut on basal (C) or stimulated (prepro)glucagon promoter activity by secreted (D) or exogenous (E) glucagon. In C, αTC1-9 cells were cotransfected with pGL4.CMV.hRlucCP and either WT pGlcg1.luc2neo (CRE) or mutant pGlcg1.CRE-mut.luc2neo (CRE-mut). Basal promoter activity was analyzed as described in Materials and Methods and calculated by dividing Glcg1.CRE or Glcg1.CRE-mut promoter-controlled firefly luciferase luminescence by CMV promoter-controlled Renilla luciferase luminescence. Glucagon promoter activity is presented as percentage of WT promoter activity (given as 100%); n = 3. Data are expressed as mean ± SEM; *P < 0.05. In D and E, αTC1-9 cells were transfected with either WT pGlcg1.DsRed2 (CRE) or mutant pGlcg1.mut.DsRed2 (CRE-mut). Cells were left unstimulated or were stimulated with either 1 mM glucose (D) or 200 nM glucagon (at 16.7 mM glucose) (E) for 15 min. Data represent the ratio of DsRed2 fluorescence values obtained at 240 min and 60 min and are expressed as mean ± SEM for at least 15 monitored cells. Significance vs. stimulated expression of CRE, *P < 0.05.