Research ArticleHEALTH AND MEDICINE

Sustained release of a GLP-1 and FGF21 dual agonist from an injectable depot protects mice from obesity and hyperglycemia

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Science Advances  26 Aug 2020:
Vol. 6, no. 35, eaaz9890
DOI: 10.1126/sciadv.aaz9890
  • Fig. 1 GLP-1 and FGF21 cotreatment augments the blood glucose–lowering and weight gain–inhibiting effects of single-drug treatment.

    Six-week-old db/db mice (n = 6 to 7) were subcutaneously injected with ELP-FGF21 (1000 nmol/kg), GLP1-ELP (1000 nmol/kg), or GLP1-ELP and ELP-FGF21 (1000 nmol/kg each). Ambient blood glucose levels (A) and body weights (B) were measured 48 hours after injection and reported as a magnitude change from pretreatment baseline and a percentage change from preinjection weight. Data are presented as means ± SEM and were analyzed by one-way ANOVA, followed by Tukey’s tests. *, treatment compared to vehicle; ^, comparisons between treatments; ^P < 005, **P < 0.01, ***P < 0.001, and ****/^^^^P < 0.0001. NS, not significant (P > 0.05).

  • Fig. 2 A recombinant GLP1-ELP-FGF21 fusion protein has dual agonism and LCST phase behavior.

    (A) SDS-PAGE analysis of the 72-kDa fusion protein following recombinant expression in E. coli and ITC-based purification. 1, molecular weight ladder (kilodaltons); 2, cell lysate; 3, insoluble lysate fraction; 4, soluble lysate fraction; 5, hot spin supernatant; 6 to 8, ITC rounds 1 to 3. (B and C) In vitro GLP-1 and FGF21 activity assays for GLP1-ELP-FGF21. GLP-1R agonism (B) was measured by quantifying adenosine 3′,5′-monophosphate (cAMP) production in human embryonic kidney (HEK) 293 cells stably expressing the GLP-1R and the cAMP-inducible luciferase reporter. Cells were stimulated for 5 hours with GLP1-ELP-FGF21, the GLP1-ELP single-agonist control, or native GLP-1. FGF21 receptor agonism (C) was measured by quantifying extracellular signal–regulated kinase 1/2 (ERK1/2) phosphorylation in HEK293 cells stably expressing FGF receptor 1 (FGFR1) and β-klotho and by normalizing phospho-ERK1/2 to total ERK1/2. Cells were stimulated for 5 min with GLP1-ELP-FGF21, the ELP-FGF21 single-agonist control, or native FGF21. Data are presented as means ± SEM, n = 3. (D to F) LCST phase transition behavior of GLP1-ELP-FGF21. (D) The optical density (OD) at 350 nm of GLP1-ELP-FGF21 at the indicated concentration in phosphate-buffered saline (PBS) was measured as a function of temperature, with temperature ramped at a rate of 1°C/min. (E) Turbidity versus temperature scans were repeated as in (D) for the indicated concentrations (n = 3). Tt values were defined as the temperature corresponding to the 50% maximum OD and are plotted as a function of fusion protein concentration. The horizontal dashed line indicates the approximate temperature of the subcutaneous space in a mouse (27). (F) A turbidity scan was repeated for GLP1-ELP-FGF21 at an injection-relevant concentration (150 μM), with the temperature ramped up to 37°C and then down to 20°C.

  • Fig. 3 The GLP1-ELP-FGF21 dual-agonist fusion protein has potent and sustained effects on glycemia and body weight.

    (A to D) Six-week-old db/db mice (n = 3 to 4) received a single subcutaneous injection of GLP1-ELP-FGF21 at the indicated dose or vehicle. Ambient blood glucose levels were measured every 24 hours until animals returned to baseline levels and are reported as blood glucose versus time AUC or raw values (A and B). Body weights were recorded daily and are reported as a percentage change from preinjection weight over time (C) or on day 7 after injection (D). (E) GLP1-ELP-FGF21 (1000 nmol/kg) was subcutaneously administered to 6-week-old db/db mice (n = 4) as a radiolabeled protein, blood samples were collected at indicated time points following injection, and plasma gamma counts were correlated to fusion protein concentration. Regression curves were fit to the terminal portion of the dataset, and data could be described by either a first-order (dotted) or a zero-order (solid) elimination model. Data are presented as means ± SEM and were analyzed by one-way ANOVA, followed by Dunnett’s tests. *P < 0.05 and **P < 0.01.

  • Fig. 4 A GLP1-ELP-FGF21 dual agonist confers greater glycemic control and protection from weight gain compared to a long-acting GLP-1RA.

    (A) A mixed ELP depot releases GLP-1 and FGF21 at different rates. Six-week-old db/db mice (n = 4 to 5) received a single subcutaneous injection consisting of a 1:1 mixture of the synthesized GLP1-ELP and previously reported ELP-FGF21 (18). The mixture was tested once when GLP1-ELP was radiolabeled and once when ELP-FGF21 was radiolabeled. Fusions were injected at 200 μM and dosed at 1000 nmol/kg each of GLP1-ELP and ELP-FGF21. Blood samples were collected at indicated time points following injection, and plasma gamma counts were measured and correlated to fusion protein concentration. Lines represent regression curves fit to the terminal portion of each dataset. Data can be described by both the first-order (dotted) and zero-order (solid) elimination models. (B to G) Six-week-old db/db mice (n = 6 to 7) were treated weekly for 4 weeks with GLP1-ELP-FGF21, GLP1-ELP, ELP-FGF21, or PBS vehicle. Fusion proteins were administered subcutaneously at 1000 nmol/kg. (B to D) Glucose challenge. Seventy-two hours after the first treatment cycle, mice were fasted 5 hours, baseline blood glucose levels were measured (B), and mice were injected intraperitoneally with glucose (0.75 g/kg). Blood glucose levels were measured at indicated time points (C), and blood glucose versus time AUC values were calculated (D). (E and F) Body weights and food consumption were measured every 1 to 2 days and are reported as percentage change from preinjection weights (E) and cumulative food intake per mouse (F). (G) Before the first treatment (day 0) and 6 days following the final treatment (day 27), percent glycated hemoglobin A1c (%HbA1c) was measured in all cohorts and reported as a magnitude change from prestudy values. Data are presented as means ± SEM and were analyzed by one-way ANOVA or two-way repeated-measures ANOVA, followed by Dunnett’s tests. *P < 0.05, ***P < 0.001, and ****P < 0.0001.

  • Fig. 5 A GLP1-ELP-FGF21 dual-agonist format is therapeutically preferable to a GLP-1/FGF21 single-agonist mixture.

    (A to F) Six-week-old db/db mice (n = 7) received a single subcutaneous injection of GLP1-ELP-FGF21 (1000 nmol/kg), a 1:1 equimolar mixture of GLP1-ELP and ELP-FGF21, or an ELP-only control. Ambient blood glucose levels were measured every 24 hours until animals returned to baseline levels and are reported as raw values (A) or blood glucose versus time AUC (B). (C to E) Glucose challenge. Six days after treatment, mice were fasted 5 hours, baseline blood glucose levels were measured (C), and mice were injected intraperitoneally with glucose (0.75 g/kg). Blood glucose levels were measured at indicated time points (D), and blood glucose versus time incremental AUC (iAUC) values were calculated (E). Body weights were recorded daily and are reported as a percentage change from preinjection weight over time (F). (G) Six-week-old db/db mice (n = 6 to 7) received a single subcutaneous injection of GLP1-ELP-FGF21 (1000 nmol/kg), a 1:1 equimolar mixture of GLP1-ELP and ELP-FGF21, or vehicle control. Food consumption was measured every 1 to 2 days and is reported as cumulative food intake per mouse. Data are presented as means ± SEM and were analyzed by one-way ANOVA or two-way repeated-measures ANOVA, followed by Dunnett’s tests. *P < 0.05 and ****P < 0.0001.

Supplementary Materials

  • Supplementary Materials

    Sustained release of a GLP-1 and FGF21 dual agonist from an injectable depot protects mice from obesity and hyperglycemia

    C. A. Gilroy, M. E. Capozzi, A. K. Varanko, J. Tong, D. A. D'Alessio, J. E. Campbell, A. Chilkoti

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