Differential Regulation of ERK1/2 and mTORC1 Through T1R1/T1R3 in MIN6 Cells

Abstract

The MAPKs ERK1/2 respond to nutrients and other insulin secretagogues in pancreatic β-cells and mediate nutrient-dependent insulin gene transcription. Nutrients also stimulate the mechanistic target of rapamycin complex 1 (mTORC1) to regulate protein synthesis. We showed previously that activation of both ERK1/2 and mTORC1 in the MIN6 pancreatic β-cell-derived line by extracellular amino acids (AAs) is at least in part mediated by the heterodimeric T1R1/T1R3, a G protein-coupled receptor. We show here that AAs differentially activate these two signaling pathways in MIN6 cells. Pretreatment with pertussis toxin did not prevent the activation of either ERK1/2 or mTORC1 by AAs, indicating that G(I) is not central to either pathway. Although glucagon-like peptide 1, an agonist for a G(s-)coupled receptor, activated ERK1/2 well and mTORC1 to a small extent, AAs had no effect on cytosolic cAMP accumulation. Ca(2+) entry is required for ERK1/2 activation by AAs but is dispensable for AA activation of mTORC1. Pretreatment with UBO-QIC, a selective G(q) inhibitor, reduced the activation of ERK1/2 but had little effect on the activation of mTORC1 by AAs, suggesting a differential requirement for G(q). Inhibition of G(12/13) by the overexpression of the regulator of G protein signaling domain of p115 ρ-guanine nucleotide exchange factor had no effect on mTORC1 activation by AAs, suggesting that these G proteins are also not involved. We conclude that AAs regulate ERK1/2 and mTORC1 through distinct signaling pathways.

Figure 1.

AAs activate ERK1/2 and mTORC1. MIN6 cells (A, C, and D) were placed in KRBH without AAs (see Materials and Methods) and 4.5 mM glucose for 2–3 hours prior to stimulation with AAs for the times indicated on each panel. Human islets (B) were treated similarly except that 2 mM glucose was used. After treatment, cell lysates were resolved on gels and transferred to nitrocellulose for immunoblotting of pERK1/2, pS6K, pS6 (240/244), or p4EBP1 as indicated. ERK1/2, S6K, and S6 were loading controls used to normalize the p protein signals to total protein amount (eg, pS6K/S6K). The value for the untreated control signal is set to 1. A, Stimulation with AAs for the indicated times and is representative of 10 experiments. Quantification of 4 is shown in Supplemental Figure 1A. B, Stimulation of islets with AAs for 2 minutes. One of two experiments with islets from different donors is shown. C, Stimulation with individual AAs at 0.25, 0.5, or 5 mM for 2 minutes. One of six experiments is shown. D, Stimulation with individual AAs at 1 mM for 30 minutes. Immunoblots show one of three experiments is shown. Bar graphs are means of quantification ± SEM (n = 3). *, P < .05; **, P < .01, using one-way ANOVA.

Figure 2.

Comparison of AA activation ERK1/2 and mTORC1 in MIN6 cells. A, Stimulation with Ala (lanes 1–5) or Ser (lanes 7–11) at 0.05, 0.16, 0.5, 1, or 5 mM for 2 minutes or unstimulated (lane 6). One of four (for Ser) or three (for Ala) experiments are shown. B, Stimulation with 0.25, 0.5, 1, or 5 mM Ala or Ser for 30 minutes. Top panel, Immunoblots for one of three experiments. Bottom panel, Quantification (n = 3). *, P < .05, **, P < .01, using one-way ANOVA with Fisher's posttest. Stimulation of cells with 0.5 mM Gln, EAA, or NEAA, with or without Gln for 2 minutes (C) and 30 minutes (D), which is representative of four (C) and six (D) experiments. Cells were preincubated for 1 hour without or with 1 mM Gln (Gln loaded) with the indicated dilutions of the AA mixture for 2 minutes (E) or 30 minutes (F), which is representative of three (E) and six (F) independent experiments. Numbers under blots are normalized to total ERK1/2 and fold change relative to the control as in Figure 1 (C and E) or S6 (D and F). Gln (1- to 7-fold), EAA (4- to 25-fold), NEAA (13- to 147-fold), Gln/EAA (6- to 31-fold), and Gln/NEAA (3- to 188-fold).

Figure 3.

AAs activate ERK1/2 and mTORC1 through T1R1/T1R3. MIN6 cells were preincubated in KRBH and ERK1/2, pERK1/2, and S6 and pS6 (S235–236) and were immunoblotted in lysate proteins. MIN6 cells stably expressing either control nontarget shRNA or T1R3 shRNA were stimulated with the indicated concentrations of Ala for 2 minutes (A) and the AA mixture for 30 minutes (B). Numbers under the blot are the fold change in pS6/S6 as in Figure 1. One of two (A) or three (B) independent experiments is shown. C, Immunoblots of T1R3 in triplicate lysates of MIN6 cells expressing control nontarget shRNA or T1R3 shRNA as in panels A and B. Stimulation with the indicated dilutions of AAs with or without 2.5 mM IMP for 2 minutes (D) or 30 minutes (E), which is representative of three (D) or two (E) independent experiments (also see Supplemental Figure 2, A and B). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 4.

Effects of Ca²⁺ on activation of ERK1/2 and mTORC1. A, Cells were preincubated in KRBH with 4.5 mM Glc for 2 hours, treated with 100 μM diazoxide (DZ) or vehicle (DMSO) for 15 minutes, and then stimulated with 100 μM carbachol (Carb), AAs, or 100 nM EXD-4 for 2 minutes prior to harvest. One of four experiments is shown. B, Cells treated as in panel A were stimulated with AAs, 100 nM GLP-1, or 100 ng/mL EGF as indicated for either 2 or 30 minutes. One of two experiments is shown. Numbers under blots are fold change as in Figure 1. C, Cells were placed in fresh KRBH with or without Ca²⁺ plus 2 mM EGTA for 5 minutes before stimulation with AAs for 30 minutes. One of five experiments is shown. D, Cells preincubated in KRBH were stimulated with AAs or 100 μM Carb for the indicated times. pS6K and pS6 (S235/236) were immunoblotted. One of two experiments is shown.

Figure 5.

G proteins and activation of ERK1/2 and mTORC1 by AAs. A, Intracellular cAMP was measured in MIN6 cells stably expressing the CAMYEL BRET sensor. Cells in KRBH were stimulated with AAs (n = 4) or GLP-1 (n = 5). Data were analyzed as in Materials and Methods. B and C, MIN6 cells were treated with or without 50 ng/mL of pertussis toxin (PTX) for 18 hours in normal growth medium before incubation in KRBH with 4.5 mM Glc ± 50 ng/mL pertussis toxin (PTX) for 2 hours. After a 15-minute treatment with 10 μM UK14304 or vehicle (DMSO), cells were stimulated with AAs for 2 minutes (B) or 15 minutes (C). pERK1/2, pS6K, or pS6 (240/244) was immunoblotted. D and E, Cells treated as in panels B and C were loaded with fura 2, pretreated with 10 μM UK14304, and stimulated with AAs, as indicated to measure effects on intracellular Ca²⁺. One of three (panels B, D, and E) or four (panel C) experiments is shown. F and G, MIN6 cells in KRBH with 4.5 mM Glc for 2 hours were treated with or without 300 nM of the Gq inhibitor UBO-GIC for 15 minutes prior to the addition of AAs for 2 minutes (F) or 30 minutes (G). Lysate proteins were immunoblotted for pERK1/2 and ERK1/2 (F) or pS6K and S6K (G) (n = 3 for panels F and G). G, Bar graph, means ± inhibitor; AA stimulation relative to control + or − inhibitor. Numbers obtained as described in Figure 1. **, P < .01. H, HeLa cells were cotransfected with pGL4 vectors encoding SRE.L-dependent firefly luciferase and pCMV5 vector, either empty or encoding the p115 ρGEF RGS domain. Amounts of p115 ρGEF RGS domain plasmid DNA were as indicated. Cells were placed in serum-free medium for 16 hours prior to preincubation in KRBH for 30 minutes and then stimulated with AAs for 30 minutes. **, P < .01, student's t test. Top panel, Means of pS6K/S6K were expressed as fold increase compared with starved cells without p115 ρGEF RGS DNA. Middle panel, Immunoblots showing pS6K, S6K, and actin. Bottom panel, As indicated, cells were stimulated with 10 μM S1P for 5 hours and lysed, and firefly luciferase activities were measured. The means of activities are presented as fold stimulation relative to firefly luciferase activity from cells transfected with control (empty) pCMV5 vector in the absence of drug. This panel demonstrates the inhibitory effect of the RGS domain in a system that is dependent on G_12/13.