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. 2008 Sep;132(3):329-38.
doi: 10.1085/jgp.200810044. Epub 2008 Aug 11.

Glucose and GLP-1 stimulate cAMP production via distinct adenylyl cyclases in INS-1E insulinoma cells

Lavoisier S Ramos  1 , Jonathan Hale Zippin, Margarita Kamenetsky, Jochen Buck, Lonny R Levin
Affiliations

Glucose and GLP-1 stimulate cAMP production via distinct adenylyl cyclases in INS-1E insulinoma cells

Lavoisier S Ramos et al. J Gen Physiol. 2008 Sep.

Abstract

In beta cells, both glucose and hormones, such as GLP-1, stimulate production of the second messenger cAMP, but glucose and GLP-1 elicit distinct cellular responses. We now show in INS-1E insulinoma cells that glucose and GLP-1 produce cAMP with distinct kinetics via different adenylyl cyclases. GLP-1 induces a rapid cAMP signal mediated by G protein-responsive transmembrane adenylyl cyclases (tmAC). In contrast, glucose elicits a delayed cAMP rise mediated by bicarbonate, calcium, and ATP-sensitive soluble adenylyl cyclase (sAC). This glucose-induced, sAC-dependent cAMP rise is dependent upon calcium influx and is responsible for the glucose-induced activation of the mitogen-activated protein kinase (ERK1/2) pathway. These results demonstrate that sAC-generated and tmAC-generated cAMP define distinct signaling cascades.

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Figures

Figure 1.
Figure 1.
Effects of GLP-1 and glucose on cAMP levels in INS-1E cells. Total cellular cAMP was measured in INS-1E cells at the indicated times after incubation in Krebs Ringer buffer with 2.5 mM (▪) glucose, 16 mM (▴) glucose, or 2.5 mM glucose with 30 nM GLP-1 (▾) in the presence of 0.5 mM IBMX. Values represent means ± SEM (n = 4) of total cAMP content per well, with each well containing equivalent number of cells (2.5 × 105 cells were plated into each well two days before the assay).
Figure 2.
Figure 2.
GLP-1-induced cAMP production is mediated via tmACs. Cyclic AMP was measured in INS-1E cells after incubation for 5 min in 2.5 mM glucose in the presence of 0.5 mM IBMX, with either vehicle control, 30 μM KH7, 50 μM 2′5′ ddAdo, 30 nM GLP-1, GLP-1 and KH7, or GLP-1 and 2′5′ ddAdo. 2 d before the assay, 2.5 × 105 cells were plated into each well. Values represent means ± SEM (n = 8). ANOVA statistical analyses were performed with the Newman-Keuls post-hoc test. ***, indicates P < 0.001; *, P < 0.05.
Figure 3.
Figure 3.
Individual contribution of tmACs and sAC to glucose-induced cAMP. Total cellular cAMP was measured in INS-1E cells at the indicated times in Krebs Ringer buffer with 2.5 mM glucose (triangle symbols) or 16 mM glucose (square symbols) in the presence of IBMX, with either vehicle control (A, B, and C, closed symbols and solid lines: ▴ and ▪), or in the presence of the inhibitors (open symbols with dotted lines: ▵ and □); 50 μM 2′5′ ddAdo (A), 30 μM KH7 (B), or both ddAdo and KH7 (C). Values represent means ± SEM (n = 4) of total cAMP content per well, with each well containing equivalent number of cells. ANOVA statistical analyses were performed with the Bonferroni comparison test and provided in Table S1 (available at http://www.jgp.org/cgi/content/full/jgp.200810044/DC1).
Figure 4.
Figure 4.
sAC is present in pancreatic β cells. (A) Western blot (WB) using biotinylated anti-sAC mAb R21 of an immunoprecipitation using anti-sAC mAb R37 or nonspecific mouse IgG from INS-1E cells. Specificity of the Western blot was confirmed using streptavidin alone (not depicted). (B) Immunocytochemistry of INS-1E cells using anti-sAC mAb R21 (green) alone (left top) or in combination with the nuclear stain, DAPI (blue) (right top). All specific staining was lost when the sAC mAb was preincubated with excess peptide antigen (bottom; left panel is fluorescence; right panel is bright field). Immunostains with secondary antibodies alone were also negative (not depicted).
Figure 5.
Figure 5.
RNAi confirms the role of sAC in glucose-induced cAMP production. (A) Western blot, along with quantitation, using sAC-specific mAb R21 (top) or anti–β-actin mAb (middle) of INS-1E cells transfected with no RNA (mock; white bar), sAC-specific RNAi oligonucleotide (sAC-siRNA; black bar), or nonspecific oligonucleotide (Neg C; gray bar). sAC bands from two independent transfections were quantified by densitometry and normalized to β-actin (bottom). (B) Total cellular cAMP in untransfected INS-1E cells (Un) or INS-1E cells transfected with no RNA (mock), sAC-specific RNAi (sAC siRNA), or nonspecific oligonucleotide (neg C) after incubation for 15 min in Krebs Ringer buffer with low (2.5 mM; white bars) or high (16 mM; black bars) glucose. Values represent means ± SEM (n = 6) of total cAMP content per well, where each well contained equivalent number of cells upon RNAi transfection. High glucose stimulated cAMP production 1.78-fold in controls (untransfected, mock-transfected, and negative control–transfected cells) and 1.34-fold in sAC-siRNA-transfected INS-1E cells. ANOVA statistical analysis was performed with the Newman-Keuls post-hoc test. ns, not significant; **, P < 0.01; ***, P < 0.001.
Figure 6.
Figure 6.
Glucose leads to ERK (p42/44) activation in a sAC-dependent manner. Western blots using anti-phosphoERK (pERK) or total ERK (ERK) of INS-1 cells incubated in Krebs Ringer buffer with 2.5 mM glucose (LG) or 16 mM glucose (HG) (in the absence of IBMX) (A) for 5, 10, 15, 20, and 30 min or (B) for 15 min in the presence or absence of 30 μM KH7 or 50 μM 2′5′ ddAdo or KH7 and 0.5 mM Sp-8Br-cAMP. Western blot shows phosphorylation and total protein for both ERK1 and ERK2, p44 and p42. Shown are representative experiments repeated multiple times (for A, n = 4; for B, n = 7).
Figure 7.
Figure 7.
Glucose-induced calcium entry is necessary for sAC activation. Cyclic AMP was measured in INS-1E cells after incubation for 15 min in 2.5 or 16 mM glucose in the presence of 0.5 mM IBMX, with either vehicle control (white bars) or (A) 100 μM EGTA-AM (gray bars), 300 μM diazoxide (black bars) or (B) 60 μM verapamil (black bars). Values represent means ± SEM (n ≥ 4). ANOVA statistical analyses were performed with the Newman-Keuls post-hoc test. ***, P < 0.001.
Figure 8.
Figure 8.
Depolarization-induced calcium entry is sufficient to induce functional sAC-generated cAMP. (A) Cyclic AMP was measured in INS-1E cells after incubation for 15 min in 2.5 mM glucose (white bars) or 2.5 mM glucose plus 60 mM KCl (gray bars) in the presence of 0.5 mM IBMX, with either vehicle control, 50 μM 2′5′ ddAdo, and/or 30 μM KH7. Values represent means ± SEM (n ≥ 4). ANOVA statistical analyses were performed with the Bonferroni comparison test. ***, P < 0.001. (B) Western blots using anti-phosphoERK (pERK) or total ERK (ERK) of INS-1E cells incubated in Krebs-Ringer buffer with 2.5 mM glucose (LG) or 2.5 mM glucose with 60 mM KCl (LG+KCl), in the absence of IBMX, for 15 min in the presence or absence of 50 μM 2′5′ ddAdo or 30 μM KH7. Shown are representative experiments repeated four times.
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References

    1. Arnette, D., T.B. Gibson, M.C. Lawrence, B. January, S. Khoo, K. McGlynn, C.A. Vanderbilt, and M.H. Cobb. 2003. Regulation of ERK1 and ERK2 by glucose and peptide hormones in pancreatic β cells. J. Biol. Chem. 278:32517–32525. - PubMed
    1. Asfari, M., D. Janjic, P. Meda, G. Li, P.A. Halban, and C.B. Wollheim. 1992. Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology. 130:167–178. - PubMed
    1. Ashcroft, F.M., and P. Rorsman. 1989. Electrophysiology of the pancreatic β-cell. Prog. Biophys. Mol. Biol. 54:87–143. - PubMed
    1. Baillie, G.S., J.D. Scott, and M.D. Houslay. 2005. Compartmentalisation of phosphodiesterases and protein kinase A: opposites attract. FEBS Lett. 579:3264–3270. - PubMed
    1. Benes, C., M.P. Roisin, H.V. Tan, C. Creuzet, J. Miyazaki, and R. Fagard. 1998. Rapid activation and nuclear translocation of mitogen-activated protein kinases in response to physiological concentration of glucose in the MIN6 pancreatic β cell line. J. Biol. Chem. 273:15507–15513. - PubMed

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