Glucagon-like peptide-1 receptor activation stimulates PKA-mediated phosphorylation of Raptor and this contributes to the weight loss effect of liraglutide
- Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, United States
- Department of Medicine, Vanderbilt University Medical Center, United States
Figures
Figure 1 with 2 supplements
The GLP-1R agonist liraglutide increases mTOR activity in a PKA- dependent manner (Resubmission Rev 2 Figure 1—source data 1).
(A) Immunoblot of pS6, S6, pCREB, and CREB and quantification of pS6 expression (pS6 /S6) in CHO-K1 cells transfected with the a vector expressing the hGLP1R and treated with DMSO or the mTORC1 inhibitor rapamycin (100 nM) for 30 min followed by treatment with 1 X PBS (Veh) or liraglutide (Lira, 10 nM) for 1 hr (Interaction: F (1, 8) = 6.926, p=0.0301; Inhibitor: F (1, 8) = 6.926, p=0.0301; Treatment: F (1, 8) = 22.50, p=0.0015). (B–C) Immunoblot of pS6, S6, pCREB, and CREB and quantification of pS6 expression (pS6/S6) in CHO-K1 cells stably expressing the hGLP1R treated with DMSO (Veh) or either of the PKA inhibitors, (B) H89 (10 µM) or (C) KT5720 (10 µM or 50 μM), for 30 min followed by treatment with vehicle (PBS), Lira (10 nM), forskolin (Fsk, 10 µM), or insulin (Ins, 10 mg/mL) for 1 hr (H89: Interaction: F (1, 12) = 4.782, p=0.0493; Inhibitor: F (1, 12) = 4.782, p=0.0493; Treatment: F (1, 12) = 12.50, p=0.0041). KT5720: (Interaction: F (2, 18) = 15.32, p=0.0001; Inhibitor: F (2, 18) = 15.32, p=0.0001; Treatment: F (1, 18) = 36.14, p<0.0001). Quantification of pCREB expression (pCREB/CREB) is shown in Figure 1—figure supplement 1. (D) Immunoblot of pS6, S6, pAkt, and Akt and quantification of pS6 expression (pS6/S6) in CHO-K1 cells stably expressing the hGLP1R treated with DMSO or the pan-Akt inhibitor MK-2206 (5 μM or 20 µM) for 30 min followed by treatment with vehicle (PBS), Lira (10 nM), Fsk (10 µM), or Ins (10 mg/mL) for 1 hr (Interaction: F (1, 12) = 0.02486, p=0.8773; Inhibitor: F (1, 12) = 0.02486, p=0.8773; Treatment: F (1, 12) = 110.2, p<0.0001). Quantification of pAkt expression (pAkt/Akt) is shown in Figure 1—figure supplement 1. Analysis was done using two-way ANOVA followed by Tukey’s multiple comparisons. Data are normalized to the respective control (Veh or Veh and inhibitor). Absolute data are shown in Figure 1—figure supplement 1. All data are shown as mean ± SEM, ns not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, n=3–4 biological replicates.
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Figure 1—source data 1
Graphs of absolute quantification of data from Figure 1 and raw data in Excel spreadsheet.
- https://cdn.elifesciences.org/articles/80944/elife-80944-fig1-data1-v2.zip
Figure 1—figure supplement 1
Absolute pS6, pCREB, and pAkt expression in CHO-K1 cells stably expressing the hGLP1R and treated with liraglutide.
(Resubmission Rev 2 Figure 1—figure supplement 1—source data 1). (A) Quantification of pS6 expression (pS6 /S6) in CHO-K1 cells transfected with the a vector expressing the hGLP1R and treated with DMSO or the mTORC1 inhibitor rapamycin (100 nM) for 30 min followed by treatment with 1 X PBS (Veh) or liraglutide (Lira, 10 nM) for 1 hr (Interaction: F (1, 8) = 31.10, p=0.0005; Inhibitor: F (1, 8) = 50.10, p=0.0001; Treatment: F (1, 8) = 39.17, p=0.0002). (B–C) Quantification of pS6 (pS6/S6) and pCREB (pCREB/CREB) expression in CHO-K1 cells stably expressing the hGLP1R treated with DMSO (Veh) or either of the PKA inhibitors, (B) H89 (10 µM) or (C) KT5720 (10 µM or 50 μM), for 30 min followed by treatment with vehicle (PBS) or Lira (10 nM) for 1 hr (H89 pS6/S6: Interaction: F (1, 12) = 13.40, p=0.0033; Inhibitor: F (1, 12) = 21.00, p=0.0006; Treatment: F (1, 12) = 18.40, p=0.0011. H89 pCREB/CREB: Interaction: F (1, 8) = 356.7, p<0.0001; Inhibitor: F (1, 8) = 578.5, p<0.0001; Treatment: F (1, 8) = 361.3, p<0.0001. KT5720 pS6/S6: Interaction: F (2, 18) = 15.32, p=0.0001; Inhibitor: F (2, 18) = 15.32, p=0.0001; Treatment: F (1, 18) = 36.14, p<0.0001. KT5720 pCREB/CREB: Interaction: F (2, 12) = 30.76, p<0.0001; Inhibitor: F (2, 12) = 43.93, p<0.0001; Treatment: F (1, 12) = 63.66, p<0.0001). Quantification of pS6 and pAkt (pAkt/Akt) in CHO-K1 cells stably expressing the hGLP1R treated with DMSO or the pan-Akt inhibitor MK-2206 (5 μM or 20 µM) for 30 min followed by treatment with vehicle (PBS), Lira (10 nM) or Ins (10 mg/mL) for 1 hr (pS6/S6: Interaction: F (1, 12) = 17.67, p=0.0012; Inhibitor: F (1, 12) = 31.37, p=0.0001; Treatment: F (1, 12) = 181.6, p<0.0001. pAkt/Akt: Interaction: F (2, 12) = 60.57, p<0.0001; Inhibitor: F (1, 12) = 62.79, p<0.0001; Treatment: F (2, 12) = 61.73, p<0.0001). Analysis was done using two-way ANOVA followed by Tukey’s multiple comparisons. Data are presented as absolute densitometry values and are shown as mean ± SEM, ns not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, n=3–4 biological replicates.
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Figure 1—figure supplement 1—source data 1
Quantification of absolute values from data in Figure 1—figure supplement 1 and raw data.
- https://cdn.elifesciences.org/articles/80944/elife-80944-fig1-figsupp1-data1-v2.zip
Figure 1—figure supplement 2
Dose response and time course of absolute pS6 and pCREB expression in CHO-K1 cells stably expressing the hGLP1R and treated with liraglutide.
(Resubmission Rev 2 Figure 1—figure supplement 2—source data 1). (A) Quantification of pS6 expression (pS6/S6) and pCREB (pCREB/CREB) in CHO-K1 cells stably expressing the hGLP1R and treated with DMSO (Veh) or increasing doses of Lira for 1 h (pS6/S6: F (5, 12) = 70.69, p<0.0001; pCREB/CREB: F (5, 12) = 52.89, p<0.0001). (B) Quantification of pS6 expression (pS6/S6) and pCREB (pCREB/CREB) in CHO-K1 cells stably expressing the hGLP1R and treated with DMSO (Veh) or Lira (10 nM) for different time points (pS6/S6: F (4, 15) = 73.02, p<0.0001; pCREB/CREB: F (4, 15) = 178.2, p<0.0001). Analysis was done using one-way ANOVA followed by Brown-Forsythe test. Data are presented as absolute densitometry values and are shown as mean ± SEM, ** p<0.01, **** p<0.0001, n=3–4 biological replicates.
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Figure 1—figure supplement 2—source data 1
Figure 1—figure supplement 2 and raw blots and raw data for Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/80944/elife-80944-fig1-figsupp2-data1-v2.zip
Figure 2
PKA phosphorylates Raptor at S791 upon GLP-1R activation (Resubmission Rev 2 Figure 2—source data 1).
(A) Immunoblot and quantification of phospho-PKA substrate (pRRXS*/T*) and Myc in CHO-K1 cells stably expressing the hGLP1R transfected with Myc-wild-type (WT) Raptor or Myc-Ser791Ala Raptor and treated with vehicle (DMSO) or H89 (10 µM) for 30 min followed by treatment with Lira (10 nM), forskolin (Fsk, 10 μM), or insulin (Ins, 10 mg/mL) for 1 hr (Interaction: F (4, 21) = 3.695, p=0.0071; Genotype: F (2, 21) = 10.51, p=0.0007; Treatment: F (2, 21) = 6.328, p=0.0071). (B) Immunoblot and quantification of phospho-PKA substrate and Myc in CHO-K1 cells stably expressing the hGLP1R transfected with Myc-wild-type (WT) Raptor and treated with vehicle (DMSO) or MK-2206 (20 μM) for 30 min followed by treatment with Lira (10 nM), Fsk (10 μM), or Ins (10 mg/mL) for 1 hr (Interaction: F (3, 16) = 0.4599, p=0.7141; Inhibitor: F (1, 16) = 0.04511, p=0.8345; Treatment: F (3, 16) = 44.26, p<0.0001). (C) Immunoblot and quantification of phospho-PKA substrate and Myc in β-TC3 cells transfected with Myc-wild-type (WT) Raptor or Myc-Ser791Ala Raptor and treated with vehicle (DMSO) or MK-2206 (10 µM) for 30 min followed by treatment with Lira (10 nM), forskolin (Fsk, 10 μM), or insulin (Ins, 10 mg/mL) for 1 hr (Interaction: F (3, 16) = 7.345, p=0.0026; Genotype: F (1, 16) = 22.18, p=0.0002; Treatment: F (3, 16) = 10.93, p=0.0004). Analysis was done using two-way ANOVA followed by Tukey’s multiple comparisons. Data are shown as mean ± SEM, no symbol = not significant, * p<0.05, ** p<0.01, *** p<0.001, n=3–4 biological replicates.
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Figure 2—source data 1
Figure 2, raw blots from Figure 2, and raw data for Figure 2.
- https://cdn.elifesciences.org/articles/80944/elife-80944-fig2-data1-v2.zip
Figure 3 with 2 supplements
PKA phosphorylation of Raptor at S791 contributes to the weight-lowering effects of liraglutide in lean mice (Resubmission Rev 2 Figure 3—source data 1).
Body weight, body composition, and energy balance parameters in male wild-type (WT) and CMV-Ser791Ala Raptor knock-in (S791A) mice given vehicle (0.9% saline) or liraglutide (200 μg/kg) subcutaneously twice a day for 14 days. Absolute body weight (A), total mass (C), fat mass (E), and lean mass (G). Change in body weight (B), total mass (D), fat mass (F), and lean mass (H) from baseline (Day 0). Absolute daily food intake (I) and EE (J) and change in daily food intake (K) and EE (L) relative to baseline (Day 0) during a 10-day treatment period in metabolic cages. ((A), Interaction: F (42, 700)=14.10, p<0.0001. (B), Interaction: F (42, 700)=14.10, p<0.0001. (C), Interaction: F (3, 50)=42.94, p<0.0001. (D), F (3, 50)=42.94, p<0.0001. (E), Interaction: F (3, 36)=30.94, p<0.0001. (F), F (3, 36)=30.94, p<0.0001. (G), Interaction: F (3, 36)=12.19, p<0.0001. (H), F (3, 36)=12.19, p<0.0001. (I), Interaction: F (30, 112)=1.370, p<0.0213. (J), Interaction: F (30, 112)=1.706, p<0.0240. (K), Interaction: F (30, 120)=3.827, p<0.0001. (L), Interaction: F (30, 112)=3.827, p<0.0001) Analysis was done using mixed-effects analysis followed by Holm-Sidak’s multiple comparisons between liraglutide-treated WT and CMV-Ser791Ala Raptor mice. Data are shown as mean ± SEM, * p<0.05, ** p<0.01, **** p<0.0001, n=13–14 (A–D), 9–12 (E–H), or 4 (I–L) mice per group.
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Figure 3—source data 1
- https://cdn.elifesciences.org/articles/80944/elife-80944-fig3-data1-v2.zip
Figure 3—figure supplement 1
Blood glucose prior to drug treatment and blood glucose and insulin post-treatment in male wild-type (WT) and Ser791Ala Raptor knock-in mice (Resubmission Rev 2 Figure 3—figure supplement 1—source data 1).
(A–D) Total (A), fat (B), lean (C), and free body fluid (D) measurements in WT and CMV-Ser791Ala Raptor mice prior to vehicle or liraglutide administration. (E– F) 5 hr fasting blood glucose in male WT and CMV-Ser791Ala Raptor mice before drug treatment (E) and on treatment day 14 (F). (G) 5 hr fasting insulin in male WT and CMV-Ser791Ala Raptor mice before on treatment day 14. Analysis was done using one-way ANOVA followed by Holm-Sidak’s multiple comparisons. Data are shown as mean ± SEM, ns not significant, n=4–8 mice per group.
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Figure 3—figure supplement 1—source data 1
Figure 3—figure supplement 1 and raw data for Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/80944/elife-80944-fig3-figsupp1-data1-v2.zip
Figure 3—figure supplement 2
Generation of CMV-Ser791Ala Raptor knock-in mice.
(A) Targeting vector used by Ingenious Targeting Laboratory, Inc to introduce a Cre-inducible two base pair point mutation in an inverted exon 20 of the Raptor coding sequence. (B) Verification of correct target sequence expression by genomic DNA digestion. Wild-type (Control) DNA contains a SacI sequence that is absent in the Ser791Ala point mutant (red). SacI digestion of a subcloned 911 base pair (bp) genomic DNA fragment obtained from liver tissue of control mice produces two products at 371 bp and 540 bp. The corresponding 989 bp genomic DNA sequence obtained from CMV-Ser791Ala Raptor knock-in mice cannot be digested by SacI.
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Figure 3—figure supplement 2—source data 1
Powerpoint slide of Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/80944/elife-80944-fig3-figsupp2-data1-v2.zip
Figure 4 with 1 supplement
PKA phosphorylation of Raptor at S791 does not significantly contribute to the weight-lowering effects of liraglutide in obese mice (Resubmission Rev 2 Figure 4—source data 1).
Body weight in male wild-type (WT) and CMV-Ser791Ala Raptor knock-in (S791A) mice fed a 60% HFD for 8–10 weeks and then given vehicle (0.9% saline) or liraglutide (200 μg/kg) subcutaneously twice a day for 9 days. Absolute body weight (A) and change in body weight (B) from baseline (Day 0). Absolute total mass (C), fat mass (E), and lean mass (G) on Days 0 and 9. Change in total mass (D), fat mass (F), and lean mass (H) from baseline (Day 0). Change in body weight in mice with a baseline body weight >43.4 g (I, J) and mice with a baseline body weight <43.4 g (K, L). ((A), Interaction: F (27, 297)=22.22, p<0.0001. (B), Interaction: F (27, 297)=22.22, p<0.0001. (C), Interaction: F (3, 33) = 39.82, p<0.0001. (D), F (3, 33) = 39.82, p<0.0001. (E), Interaction: F (3, 33) = 30.17, p<0.0001. (F), F (3, 33) = 30.17, p<0.0001. (G), Interaction: F (3, 33) = 25.69, p<0.0001. (H), F (3, 33) = 25.69, p<0.0001. (I), Interaction: F (27, 126)=40.94, p<0.0001. (J), F (3, 14) = 131.2, p<0.0001. (K), Interaction: F (27, 135)=17.98, p<0.0001. (L), F (3, 15) = 29.48, p<0.0001). Analysis was done using mixed-effects analysis followed by Holm-Sidak’s multiple comparisons between liraglutide-treated WT and CMV-Ser791Ala Raptor mice. Data are shown as mean ± SEM, * p<0.05, ** p<0.01, n=7–12 mice per group (A – H), 3–5 (I, J), and 2–7 (K, L).
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Figure 4—source data 1
- https://cdn.elifesciences.org/articles/80944/elife-80944-fig4-data1-v2.zip
Figure 4—figure supplement 1
Relationship between baseline body weight and liraglutide-induced weight loss in male wild-type (WT) and Ser791Ala Raptor knock-in mice (Resubmission Rev 2 Figure 4—figure supplement 1—source data 1).
(A) Relationship between baseline body weight and liraglutide-induced weight loss in HFD-fed wild-type (WT) and CMV-Ser791Ala Raptor knock-in (S791A) mice (Test if slopes are non-zero: WT, F (1,10) = 9.666, p<0.01; S791A Raptor: F (1, 17) = 36.714, p<0.001. Test if slopes are different: WT vs. S791A: F (1, 17) = 1.266, p=0.28). Absolute body weight in mice with >43.3 g baseline body weight (B) and <43.3 g baseline body weight (C). ((A), F (3, 14) = 9.005, p=0.0014. (B), F (3, 15) = 0.3796, p=0.7691). Analysis was done using linear regression or one-way ANOVA followed by Holm-Sidak’s multiple comparisons. Data are shown as mean ± SEM, ** p<0.01, n=3–5 (B) and 2–7 (C) mice per group.
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Figure 4—figure supplement 1—source data 1
Figure 4—figure supplement 1 and its raw data for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/80944/elife-80944-fig4-figsupp1-data1-v2.zip
Figure 5
PKA phosphorylation of Raptor at S791 is not required for the weight-lowering effect of setmelanotide (Resubmission Rev 2 Figure 5—source data 1).
Body weight and body composition in male wild-type (WT) and CMV-Ser791Ala Raptor knock-in (S791A) mice given vehicle (0.9% saline) or setmelanotide (4 mg/kg) subcutaneously once a day for 9 days. Absolute body weight (A), total mass (C), fat mass (E), and lean mass (G). Change in body weight (B), total mass (D), fat mass (F), and lean mass (H) from baseline (Day 0). (A), Interaction: F (27, 171)=4.955, p<0.0001. (B), Interaction: F (27, 171)=4.600, p<0.0001. (C), Interaction: F (3, 19) = 12.93, p<0.0001. (D, F) (3, 19) = 11.28, p=0.0002. (E), Interaction: F (3, 19) = 6.087, p=0.0044. (F), F (3, 19) = 6.087, p=0.0044. (G), Interaction: F (3, 19) = 1.311, p=0.2999. (H, F) (3, 1) = 1.311, p=0.2999. Analysis was done using mixed-effects analysis followed by Holm-Sidak’s multiple comparisons between liraglutide-treated WT and CMV-Ser791Ala Raptor mice. Data are shown as mean ± SEM, * p<0.05, ** p<0.01, n=5–7 mice per group.
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Figure 5—source data 1
- https://cdn.elifesciences.org/articles/80944/elife-80944-fig5-data1-v2.zip
Author response image 1
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Glucagon-like peptide-1 receptor activation stimulates PKA-mediated phosphorylation of Raptor and this contributes to the weight loss effect of liraglutide
eLife 12:e80944.
https://doi.org/10.7554/eLife.80944
