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. 2025 Apr;27(4):2294-2298.
doi: 10.1111/dom.16216. Epub 2025 Jan 30.

The physiological impact of an N-terminal Halo-tag on glucose-dependent insulinotropic polypeptide receptor function in mice

Iona Davies  1 , Yusman Manchanda  2 , Kyle W Sloop  3 , Stephen R Bloom  1 , Tricia M-M Tan  1 , Alejandra Tomas  2 , Ben Jones  1
Affiliations

The physiological impact of an N-terminal Halo-tag on glucose-dependent insulinotropic polypeptide receptor function in mice

Iona Davies et al. Diabetes Obes Metab. 2025 Apr.
No abstract available

Keywords: GIP; incretin physiology; mouse model; type 2 diabetes.

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Conflict of interest statement

KWS is an employee of Eli Lilly and Company and may own company stock. SRB is an employee of Zihipp Ltd. TM‐MT is a former consultant for and shareholder in Zihipp Ltd. AT has received funding from Eli Lilly and Company and Sun Pharma. BJ has received funding from Eli Lilly and Company, Metsera Inc and Sun Pharma, and acts as a consultant for Metsera Inc.

Figures

FIGURE 1
FIGURE 1
N‐terminally Halo‐tagged glucose dependent insulinotropic polypeptide receptor (GIPR) displays a partially impaired receptor function in vivo. (A) Schematic of GIPR tagging with Halo‐tag protein and subsequent receptor labelling. (B) Wild‐type Gipr and Halo‐Gipr alleles. (C, D) Body mass (g) of Gipr Wt/Wt (male: N = 11, female: N = 13), Gipr Halo/Wt (male: N = 17, female: N = 17) and Gipr Halo/Halo (male: N = 8, female: N = 8), measured at 6 weeks of age. Males are displayed in (C) and females in (D). (E, F) Oral glucose tolerance test (OGTT) conducted in Gipr Wt/Wt (n = 20), Gipr Halo/Wt (n = 21) and Gipr Halo/Halo (n = 8) mice. (G–R) Crossover Intraperitoneal glucose tolerance tests (IPGTTs) conducted in Gipr Wt/Wt mice (G: N = 24, K: N = 11, O: N = 11), Gipr Halo/Wt mice (H: N = 33, L: N = 20, P: N = 20) and Gipr Halo/Halo mice (I: N = 12, M: N = 14, Q: N = 13–14), in response to human GIP (hGIP, 50 nmol/kg) (G–J), hGIP (200 nmol/kg) (K–N) and hGIP 500 nmol/kg (O–R). (E, G–I, K–M, O–Q) Plasma glucose time‐course. (F, J, N, R) Glucose area under the curve (AUC) derived from corresponding glucose curves. Blood glucose at specific time‐points were analysed using a two‐way analysis of variance (ANOVA) with time and subgroup as co‐variables. Šídák test used to correct for multiple comparisons. Body mass at 6 weeks (C, D) and glucose AUC in (F) were analysed with a one‐way ANOVA. Dunnett's test was used to correct for multiple comparisons. Glucose AUCs in (J), (N) and (R) were analysed using a two‐way ANOVA with genotype and subgroup as co‐variables. Šídák test was used to correct for multiple comparisons. All values are presented as a mean ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001.
FIGURE 2
FIGURE 2
Investigating the causes of reduced functionality of the Halo‐tagged glucose dependent insulinotropic polypeptide receptor (GIPR) and visualising endogenous GIPR via anti‐Halo antibody staining. (A–F) cAMP signal as a percentage of FSK/IBMX responses in cADDis‐transduced dispersed islet cells from Gipr Wt/Wt mice (n = 3–4) (A, D) or Gipr Halo/Halo mice (n = 4) (B, E), in response to stepwise addition of human GIP (hGIP; A, B) and mouse GIP (mGIP; D, E). (C, F) cAMP dose–response curves derived from area under the curve in each time interval in (A) and (B) (for C) and (D) and (E) (for F) minus vehicle control, with three parameter fits shown. (G) Representative images of experiment displayed in (H) (scale bars = 20 μm). (H) Fluorescence intensity of AD293 cells transiently transfected with mouse and human untagged and Halo‐tagged GIPR following 1 h of labelling with GIP‐TMR (100 nM) (n = 6), expressed as a percentage of the experimental average. Data were analysed using a one‐way analysis of variance. The Šídák test was used to correct for multiple comparisons. (I, J) cAMP dose responses in AD293 cells transiently transfected with mouse (I) and human (J) untagged and Halo‐tagged GIPR, stimulated for 30 min with increasing doses of hGIP (n = 4–5). Three parameter fits are shown, with values normalised to the % max of the mouse or human untagged receptor. (K, L) Representative images of a pancreatic islet from Gipr Wt/Wt (K) and Gipr Halo/Wt (L) mice labelled with an anti‐Halo antibody. Scale bar = 20 μm. (M) Mean intensity of cell surface anti‐Halo staining normalised to wild‐type from 9 to 21 islets from Gipr Wt/Wt (n = 4) versus littermate Gipr Halo/Wt (n = 4) mice. Changes in fluorescence intensity were analysed using Student's t test. Values are presented as a mean ± SEM; *p < 0.05. hGIP, human GIP; mGIP, mouse GIP.
See this image and copyright information in PMC

References

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