. 2023 Jun:72:101718.

doi: 10.1016/j.molmet.2023.101718. Epub 2023 Apr 7.

Fibroblast growth factor-21 is required for weight loss induced by the glucagon-like peptide-1 receptor agonist liraglutide in male mice fed high carbohydrate diets

Thao D V Le¹, Payam Fathi², Amanda B Watters³, Blair J Ellis⁴, Gai-Linn K Besing⁵, Nadejda Bozadjieva-Kramer⁶, Misty B Perez⁷, Andrew I Sullivan⁸, Jesse P Rose⁹, Laurie L Baggio¹⁰, Jacqueline Koehler¹¹, Jennifer L Brown¹², Michelle B Bales¹³, Kaitlyn G Nwaba⁴, Jonathan E Campbell¹⁴, Daniel J Drucker¹⁵, Matthew J Potthoff¹⁶, Randy J Seeley¹⁷, Julio E Ayala¹⁸

Affiliations

¹ Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA. Electronic address: thao.d.le@vanderbilt.edu.
² Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA. Electronic address: payam.fathi@vanderbilt.edu.
³ Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA. Electronic address: awatters@tulane.edu.
⁴ Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA.
⁵ Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA. Electronic address: gai-linn.besing.1@vanderbilt.edu.
⁶ Department of Surgery, University of Michigan, 1500 E. Medical Center Drive, Ann Arbor, MI 48109, USA; Veterans Affairs Ann Arbor Healthcare System, Research Service, 2215 Fuller Road, Ann Arbor, MI 48105, USA. Electronic address: nibozad@med.umich.edu.
⁷ Department of Neuroscience and Pharmacology, University of Iowa Carver College of Medicine, 375 Newton Road, Iowa City, IA 52242, USA. Electronic address: misty-perez@uiowa.edu.
⁸ Department of Neuroscience and Pharmacology, University of Iowa Carver College of Medicine, 375 Newton Road, Iowa City, IA 52242, USA. Electronic address: andrew-sullivan-1@uiowa.edu.
⁹ Department of Neuroscience and Pharmacology, University of Iowa Carver College of Medicine, 375 Newton Road, Iowa City, IA 52242, USA. Electronic address: jesse-rose@uiowa.edu.
¹⁰ Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Department of Medicine, University of Toronto, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada. Electronic address: baggio@lunenfeld.ca.
¹¹ Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Department of Medicine, University of Toronto, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.
¹² Duke Molecular Physiology Institute, Duke University, 300 N. Duke Street, Durham, NC 27701, USA.
¹³ Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA. Electronic address: bales@psy.fsu.edu.
¹⁴ Duke Molecular Physiology Institute, Duke University, 300 N. Duke Street, Durham, NC 27701, USA. Electronic address: jonathan.campbell@duke.edu.
¹⁵ Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Department of Medicine, University of Toronto, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada. Electronic address: drucker@lunenfeld.ca.
¹⁶ Department of Neuroscience and Pharmacology, University of Iowa Carver College of Medicine, 375 Newton Road, Iowa City, IA 52242, USA. Electronic address: matthew-potthoff@uiowa.edu.
¹⁷ Department of Surgery, University of Michigan, 1500 E. Medical Center Drive, Ann Arbor, MI 48109, USA. Electronic address: seeleyrj@med.umich.edu.
¹⁸ Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA; Vanderbilt Mouse Metabolic Phenotyping Center, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA; Vanderbilt Center for Addiction Research, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA. Electronic address: julio.e.ayala@vanderbilt.edu.

PMID: 37030441
PMCID: PMC10131131
DOI: 10.1016/j.molmet.2023.101718

Fibroblast growth factor-21 is required for weight loss induced by the glucagon-like peptide-1 receptor agonist liraglutide in male mice fed high carbohydrate diets

Thao D V Le et al. Mol Metab. 2023 Jun.

. 2023 Jun:72:101718.

doi: 10.1016/j.molmet.2023.101718. Epub 2023 Apr 7.

Authors

Affiliations

¹ Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA. Electronic address: thao.d.le@vanderbilt.edu.
² Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA. Electronic address: payam.fathi@vanderbilt.edu.
³ Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA. Electronic address: awatters@tulane.edu.
⁴ Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA.
⁵ Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA. Electronic address: gai-linn.besing.1@vanderbilt.edu.
⁶ Department of Surgery, University of Michigan, 1500 E. Medical Center Drive, Ann Arbor, MI 48109, USA; Veterans Affairs Ann Arbor Healthcare System, Research Service, 2215 Fuller Road, Ann Arbor, MI 48105, USA. Electronic address: nibozad@med.umich.edu.
⁷ Department of Neuroscience and Pharmacology, University of Iowa Carver College of Medicine, 375 Newton Road, Iowa City, IA 52242, USA. Electronic address: misty-perez@uiowa.edu.
⁸ Department of Neuroscience and Pharmacology, University of Iowa Carver College of Medicine, 375 Newton Road, Iowa City, IA 52242, USA. Electronic address: andrew-sullivan-1@uiowa.edu.
⁹ Department of Neuroscience and Pharmacology, University of Iowa Carver College of Medicine, 375 Newton Road, Iowa City, IA 52242, USA. Electronic address: jesse-rose@uiowa.edu.
¹⁰ Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Department of Medicine, University of Toronto, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada. Electronic address: baggio@lunenfeld.ca.
¹¹ Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Department of Medicine, University of Toronto, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.
¹² Duke Molecular Physiology Institute, Duke University, 300 N. Duke Street, Durham, NC 27701, USA.
¹³ Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA. Electronic address: bales@psy.fsu.edu.
¹⁴ Duke Molecular Physiology Institute, Duke University, 300 N. Duke Street, Durham, NC 27701, USA. Electronic address: jonathan.campbell@duke.edu.
¹⁵ Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Department of Medicine, University of Toronto, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada. Electronic address: drucker@lunenfeld.ca.
¹⁶ Department of Neuroscience and Pharmacology, University of Iowa Carver College of Medicine, 375 Newton Road, Iowa City, IA 52242, USA. Electronic address: matthew-potthoff@uiowa.edu.
¹⁷ Department of Surgery, University of Michigan, 1500 E. Medical Center Drive, Ann Arbor, MI 48109, USA. Electronic address: seeleyrj@med.umich.edu.
¹⁸ Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA; Vanderbilt Mouse Metabolic Phenotyping Center, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA; Vanderbilt Center for Addiction Research, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232, USA. Electronic address: julio.e.ayala@vanderbilt.edu.

PMID: 37030441
PMCID: PMC10131131
DOI: 10.1016/j.molmet.2023.101718

Abstract

Objective: Glucagon-like peptide-1 receptor (GLP-1R) agonists (GLP-1RA) and fibroblast growth factor-21 (FGF21) confer similar metabolic benefits. GLP-1RA induce FGF21, leading us to investigate mechanisms engaged by the GLP-1RA liraglutide to increase FGF21 levels and the metabolic relevance of liraglutide-induced FGF21.

Methods: Circulating FGF21 levels were measured in fasted male C57BL/6J, neuronal GLP-1R knockout, β-cell GLP-1R knockout, and liver peroxisome proliferator-activated receptor alpha knockout mice treated acutely with liraglutide. To test the metabolic relevance of liver FGF21 in response to liraglutide, chow-fed control and liver Fgf21 knockout (Liv^Fgf21-/-) mice were treated with vehicle or liraglutide in metabolic chambers. Body weight and composition, food intake, and energy expenditure were measured. Since FGF21 reduces carbohydrate intake, we measured body weight in mice fed matched diets with low- (LC) or high-carbohydrate (HC) content and in mice fed a high-fat, high-sugar (HFHS) diet. This was done in control and Liv^Fgf21-/- mice and in mice lacking neuronal β-klotho (Klb) expression to disrupt brain FGF21 signaling.

Results: Liraglutide increases FGF21 levels independently of decreased food intake via neuronal GLP-1R activation. Lack of liver Fgf21 expression confers resistance to liraglutide-induced weight loss due to attenuated reduction of food intake in chow-fed mice. Liraglutide-induced weight loss was impaired in Liv^Fgf21-/- mice when fed HC and HFHS diets but not when fed a LC diet. Loss of neuronal Klb also attenuated liraglutide-induced weight loss in mice fed HC or HFHS diets.

Conclusions: Our findings support a novel role for a GLP-1R-FGF21 axis in regulating body weight in a dietary carbohydrate-dependent manner.

Keywords: Carbohydrate; Fibroblast growth factor-21; Food intake; Glucagon-like peptide-1 receptor agonists; Liraglutide; Weight loss.

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Figures

**Figure 1**
**GLP-1RA stimulate FGF21 independent of their effect on food intake.** (A) Study outline for B–C. (B) Plasma FGF21 levels at 0 and 7 h following treatment in fasted C57BL6/J male mice treated with vehicle or liraglutide (400 μg/kg) (Mixed-effects analysis: Interaction, F (1, 12) = 6.62, P = 0.0244; N = 6–8). (C) Relative change in liver *Fgf21* gene expression in fasted C57BL6/J male mice 7 h following treatment with vehicle or liraglutide (400 μg/kg) (Unpaired t-test: P = 0.0152; N = 6–8). (D) Study outline for E–F. (**E–F**) Changes in body weight (E; One-way ANOVA: Interaction, F (2, 13) = 12.02, P = 0.0011; N = 5–6)) and plasma FGF21 levels (F; One-way ANOVA: Interaction, F (2, 13) = 4.111, P = 0.0414; N = 5–6) in C57BL6/J male mice *ad libitum*-fed and treated with vehicle or liraglutide (200 μg/kg, *b.i.d*), or pair-fed to weight match the liraglutide-treated group for 48 h. (**G–H**) Plasma FGF21 levels in C57BL/6J mice following 7 day treatment with (G) vehicle or exendin-4 (10 μg/kg, *b.i.d*) (Unpaired t-test: P = 0.0425; N = 4–5) and (H) vehicle or liraglutide (200 μg/kg, *b.i.d*) (Unpaired t-test: P = 0.0053, N = 8). Data are shown as mean ± SEM, ns not significant, ∗P < 0.05, ∗∗ <0.01.

**Figure 2**
**Central GLP-1R and liver PPARα are required for liraglutide to stimulate plasma FGF21.** Absolute (A, C, E) and relative change (B, D, F) in FGF21 levels at 0 and 7 h post treatment with vehicle or liraglutide (400 μg/kg) in fasted control and respective knockout mice. (**A–B**) Control and vGLUT2+ neuron-*Glp1r* knockout (Vglut2^Glp1r−/−) mice (A, Mixed-effects analysis: Interaction, F (1, 23) = 2.021, P = 0.1686; Genotype Effect, F (1, 23) = 5.965, P = 0.0227; B, Mixed-effects analysis: Interaction, F (1, 23) = 4.571, P = 0.0434; N = 5–6). (**C–D**) Control and β cell-*Glp1r* knockout (β cell^Glp1r−/−) mice (C, Mixed-effects analysis: Interaction, F (1, 56) = 0.05070, P = 0.8227; Genotype Effect, F (1, 56) = 11.28, P = 0.0014; D, Mixed-effects analysis: Interaction, F (1, 28) = 0.01663, P = 0.8983; Genotype Effect, F (1, 28) = 10.41, P = 0.0032; N = 6–10). (**E–F**) Control and liver *Ppara* knockout (Liv^Ppara−/−) mice (E, Mixed-effects analysis: Interaction, F (1, 66) = 9.627, P = 0.0039; liraglutide-treated control vs. Liv^Ppara−/− at 7 h, P < 0.0001; F, Mixed-effects analysis: Interaction, F (1, 33) = 8.775, P = 0.0056; N = 6–13). Data are shown as mean ± SEM, ns not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

**Figure 3**
**Liver FGF21 mediates the appetite- and weight-lowering actions of liraglutide.** Chow-fed control and liver *Fgf21* knockout (Liv^Fgf21−/−) mice housed in metabolic cages and treated with vehicle or liraglutide (200 μg/kg, *b.i.d*) for 11 days (N = 7–9). (**A–B**) Relative weight loss over time (A; Mixed-effects analysis: Interaction, F (11, 156) = 1.920, P = 0.0405) and relative weight loss on the last day of treatment (B; Mixed-effects analysis: Interaction, F (1, 27) = 13.11 P = 0.0012). (C) Time course of food intake (Mixed-effects analysis: Interaction, F (36, 324) = 2.154, P = 0.0003; Genotype Effect, F (3, 27) = 1.271, P = 0.3042). (D) Total food intake during the treatment period (One-way ANOVA: F (3,27) = 4.728, P = 0.0089). (E) Time course of energy expenditure (EE) (Mixed-effects analysis: Interaction, F (36, 324) = 1.151, P = 0.2606; Genotype Effect, F (3, 27) = 5.035, P = 0.0067). (F) Total EE during the treatment period (One-way ANOVA: F (3, 27) = 2.946, P = 0.0507). (**G–I**) Total body mass (G; Mixed-effects analysis: Interaction, F (1, 27) = 10.60, P = 0.0030), fat-free (or lean) mass (H; Mixed-effects analysis: Interaction, F (1, 27) = 1.383, P = 0.2499; Genotype Effect, F (1, 27) = 1.983, P = 0.1705), and fat mass (I; Mixed-effects analysis: Interaction, F (1, 27) = 0.2012, P = 0.6573; Genotype Effect, F (1, 27) = 0.4077, P = 0.5285) at the end of treatment. Data are shown as mean ± SEM, ns not significant, ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001 for comparisons between liraglutide-treated control vs. Liv^Fgf21−/− mice (A, C, E) and those delineated by lines (B, F, G–I).

**Figure 4**
**Liver FGF21 contributes to liraglutide-induced weight loss in the context of high-carbohydrate diets.** Control and liver *Fgf21* knockout (Liv^Fgf21−/−) mice fed a low fat, low or high carbohydrate diet for 4 weeks (A–D), a high fat, high sugar diet for 4 weeks of 1 week (E–H), or a high fat, low carbohydrate diet for 4 weeks (I–J) followed by treatment with vehicle or liraglutide (200 μg/kg, *b.i.d*) for 14 days. (**A–B**) Relative weight loss over time (A; Mixed-effects analysis: Interactions, F (14, 168) = 0.5293, P = 0.9134; Genotype Effect, F (1, 12) = 0.02792, P = 0.8701; N = 7–8) and on the last day of treatment (B; Mixed-effects analysis: Interactions, F (1, 25) = 0.5317, P = 0.4727; N = 7–8) of mice fed a low fat, low carbohydrate diet for 4 weeks. (**C–D**) Relative weight loss over time (C; Mixed-effects analysis: Interactions, F (14, 266) = 1.865, P = 0.0303; N = 7–14) and on the last day of treatment (D; Mixed-effects analysis: Interactions, F (1, 37) = 10.35, P = 0.0027; N = 7–14) of mice fed a low fat, high carbohydrate diet for 4 weeks. (**E–F**) Relative weight loss over time (E; Mixed-effects analysis: Interactions, F (14, 434) = 7.323, P < 0.0001; N = 14–19) and on the last day of treatment (F; Mixed-effects analysis: Interactions, F (1, 62) = 13.12, P = 0.0006; N = 14–19) of mice fed a high fat, high sugar diet for 4 weeks. (**G–H**) Relative weight loss over time (G; Mixed-effects analysis: Interactions, F (14, 210) = 4.004, P < 0.0001; N = 7–9) and on the last day of treatment (H; Mixed-effects analysis: Interactions, F (1, 29) = 2.291, P = 0.1410; Genotype Effect, F (1, 29) = 2.303, P = 0.1400; N = 7–9) of mice fed a high fat, high sugar diet for 1 week. (**I–J**) Relative weight loss over time (I; Mixed-effects analysis: Interactions, F (42, 322) = 44.58, P < 0.0001; N = 7) and on the last day of treatment (H; Mixed-effects analysis: Interactions, F (3, 23) = 65.53, P < 0.0001; N = 7) of mice fed a high fat, low carbohydrate diet for 4 weeks. Data are shown as mean ± SEM, ns not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001 for comparisons between liraglutide-treated control vs. Liv^Fgf21−/− mice (A, C, E, G, I) and those delineated by lines (B, D, F, H, J).

**Figure 5**
**Liraglutide promotes weight loss in the context of high-carbohydrate diets via brain FGF21 action.** Control and Camk2a *Klb* knockout (Camk2a^Klb−/−) mice fed a low fat, high carbohydrate diet for 4 weeks (A–B) and control and Vglut2 *Klb* knockout (Vglut2^Klb−/−) mice fed a high fat, high sugar diet for 1 week (C–D) followed by treatment with vehicle or liraglutide (200 μg/kg, *b.i.d*) for 14 days. (**A–B**) Relative weight loss over time (A; Mixed-effects analysis: Interactions, F (14, 294) = 3.552, P < 0.0001; N = 9–15) and on the last day of treatment (B; Mixed-effects analysis: Interactions, F (1, 38) = 7.686, P = 0.009; N = 9–15). (**C–D**) Relative weight loss over time (C; Mixed-effects analysis: Interactions, F (14, 252) = 5.031, P < 0.0001; N = 9–11) and on the last day of treatment (D; Mixed-effects analysis: Interactions, F (1, 35) = 7.296, P = 0.0106; N = 9–11). Data are shown as mean ± SEM, ns not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001 for comparisons between liraglutide-treated control vs. Camk2a^Klb−/− mice (A), liraglutide-treated control vs. Vglut2^Klb−/− mice (C) and those delineated by lines (B, D).

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Update of

Liver Fibroblast Growth Factor 21 (FGF21) is Required for the Full Anorectic Effect of the Glucagon-Like Peptide-1 Receptor Agonist Liraglutide in Male Mice fed High Carbohydrate Diets.
Le TDV, Fathi P, Watters AB, Ellis BJ, Bozadjieva-Kramer N, Perez MB, Sullivan AI, Rose JP, Baggio LL, Koehler J, Brown JL, Bales MB, Nwaba KG, Campbell JE, Drucker DJ, Potthoff MJ, Seeley RJ, Ayala JE. Le TDV, et al. bioRxiv [Preprint]. 2023 Jan 3:2023.01.03.522509. doi: 10.1101/2023.01.03.522509. bioRxiv. 2023. Update in: Mol Metab. 2023 Jun;72:101718. doi: 10.1016/j.molmet.2023.101718. PMID: 36711605 Free PMC article. Updated. Preprint.

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Fibroblast growth factor-21 is required for weight loss induced by the glucagon-like peptide-1 receptor agonist liraglutide in male mice fed high carbohydrate diets

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Fibroblast growth factor-21 is required for weight loss induced by the glucagon-like peptide-1 receptor agonist liraglutide in male mice fed high carbohydrate diets

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