In vivo studies of glucagon secretion by human islets transplanted in mice

Abstract

Little is known about regulated glucagon secretion by human islet α-cells compared to insulin secretion from β-cells, despite conclusive evidence of dysfunction in both cell types in diabetes mellitus. Distinct insulins in humans and mice permit in vivo studies of human β-cell regulation after human islet transplantation in immunocompromised mice, whereas identical glucagon sequences prevent analogous in vivo measures of glucagon output from human α-cells. Here, we use CRISPR–Cas9 editing to remove glucagon codons 2–29 in immunocompromised NSG mice, preserving the production of other proglucagon-derived hormones. Glucagon knockout NSG (GKO-NSG) mice have metabolic, liver and pancreatic phenotypes associated with glucagon-signalling deficits that revert after transplantation of human islets from non-diabetic donors. Glucagon hypersecretion by transplanted islets from donors with type 2 diabetes revealed islet-intrinsic defects. We suggest that GKO-NSG mice provide an unprecedented resource to investigate human α-cell regulation in vivo.

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Fig. 1: Generation of GKO-NSG mice.
Fig. 2: Transplanted human islets retain regulated glucagon secretion in GKO-NSG mice.
Fig. 3: Human islet transplantation establishes a glucagon-signalling axis that corrects liver phenotypes in GKO-NSG mice.
Fig. 4: Human islet-derived glucagon corrects GKO-NSG α-cell hyperplasia.
Fig. 5: Improved glucose and insulin regulation in transplanted GKO-NSG mice.
Fig. 6: Excessive glucagon secretion by transplanted T2D islets in GKO-NSG mice.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data for Fig.1 are presented with the paper.

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Acknowledgements

We thank past and current members of the Kim group for advice and encouragement, S. Park for assistance with gene targeting, K. Abraham (NIDDK/NIH) for guidance in the initial stages of this work, O. McGuinness and the Vanderbilt University Medical Center Hormone Core (DK059637 and DK020593) for amino acid measurements and advice, E. Walker for advice on glycogen quantification, the Stanford University Veterinary Service Center for animal care and advice, C. Sabatti and the Stanford Department of Biomedical Data Science Data Studio for advice on statistical analyses, the Stanford Cell Sciences Imaging Facility for microscope usage, and D. Serreze (The Jackson Laboratory) for generation of mouse lines. We also thank the Integrated Islet Distribution Program (UC4 DK098085-02), Alberta Diabetes Institute IsletCore, and International Institute for the Advancement of Medicine for processing and coordinating human islet distribution. This work was supported by the Type 1 Diabetes Mouse Resource (1UC4DK097610 to D. Serreze), a graduate research fellowship award from the National Science Foundation (DGF-114747 to K. Tellez), RO1 awards (DK107507; DK108817; CA21192701 to S. K. Kim) and a U01 award (DK120447 to P. MacDonald, University of Alberta). Work in the Stein lab was supported by NIH grants (DK106755, DK050203, and DK090570 to R. W. Stein). Work in the Kim lab was also supported by NIH grant P30 DK116074, the HL Snyder Foundation, the Mulberry Foundation, a gift from S. and M. Kirsch, and by the Stanford Islet Research Core, and the Diabetes Genomics and Analysis Core of the Stanford Diabetes Research Center.

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S.K.K., K.T. and Y.H. conceptualized the study; K.T., Y.H. and S.K.K. designed the methodology; K.T., Y.H. and X.G validated the results; K.T. and X.G. performed the formal analysis; K.T., Y.H., X.G. and C.A.C. carried out the investigation; K.T. curated the data; K.T. and S.K.K. wrote the original draft; K.T., Y.H., R.W.S., and S.K.K. contributed to writing, reviewing and editing the manuscript; K.T. and Y.H. carried out visualization studies; R.W.S. and S.K.K supervised the study; S.K.K. administered the project and S.K.K acquired funding for the study.

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Correspondence to Seung K. Kim.

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Extended data

Extended Data Fig. 1 Design and characterization of GKO-NSG mice.

Related to Fig. 1. (a) Sequence from GKO-NSG founder (18-1) showing an in-frame deletion of 93 base pairs within exon 3 of the Gcg gene compared to the wild type NSG sequence (WT). Pink bar on top depicts exon 3 of Gcg. Blue bar represents nucleotide sequences encoding mature glucagon peptide. Red-highlighted dashes indicate deleted nucleotides in founder 18-1. (b) Representative immunostaining of GKO-NSG pancreatic islets with antibodies raised against mature glucagon (GCG, green) and proglucagon (Pro-GCG, red) - peptide sequences of GLP-1 (7-17). Similar results were seen across n = 3 NSG littermate control, n = 3 GKO-NSG, and n = 2 GKO-NSG Tx mice. (c) Body weight of male and female GKO-NSG and NSG control littermates at 3 and 8-weeks of age (3-week old female mice P = 0.025667 and 3-week old male mice P = 0.000454 by Repeated Measures ANOVA, with Tukey’s multiple comparisons test) (NSG mice, n = 7 males and 5 females; GKO-NSG mice, n = 11 males and 6 females). (d) Blood glucose measures of 2-3 month old GKO-NSG and NSG control mice during ad libitum feeding or after a 3-hour fast (fed: P = 0.000223; fasted: P = 0.003003 by Repeated Measures ANOVA, with Bonferroni’s multiple comparisons test) (NSG mice, n = 6 males and 4 females; GKO-NSG mice, n = 4 males and 8 females). (e) GKO-NSG and NSG control blood glucose measures over 180 minutes post oral glucose gavage (60 min: P = 0.001383; 90 min: P = 0.002618; 120 min: P = 0.040657 by Repeated Measures ANOVA, with Bonferroni’s multiple comparisons test) (6g/kg body weight) (NSG mice, n = 5 males and 3 females; GKO-NSG mice, n = 8 males and 2 females) and (f) plasma total GLP-1 levels from 2.5-3 month old GKO-NSG and NSG controls following oral glucose challenge (15 min: P = 0.000194, and 30 min: P = 0.000034 by Repeated Measures ANOVA, with Bonferroni’s multiple comparisons test) (NSG mice, n = 4 males; GKO-NSG mice, n = 5 males). (g) Quantification of active GLP-1 present in islet lysates from 5-7 month old NSG (n = 3 males) and GKO-NSG (n = 3 males) mice (P = 0.035323 by two-tailed Student’s t-test). Dashed lines indicate limit of detection. Scale bars, 50 μm. Data are represented as mean of biological replicates with individual data points overlaid and error bars indicate ± SEM. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Extended Data Fig. 2 Blood glucose reduction following insulin challenge.

Related to Fig. 2. Percent of basal blood glucose 30-minutes post insulin injection (1U/kg body weight) from 4.5-6.5 month old NSG, GKO-NSG, and GKO-NSG Tx mice (P = 0.017712 by one-way ANOVA, with Tukey’s multiple comparison test) (NSG mice, n = 5 males; GKO-NSG mice, n = 2 males and 1 female; GKO-NSG Tx mice, n = 5 males). Data are represented as mean of biological replicates with individual data points overlaid and error bars indicate ± SEM. * P ≤ 0.05.

Extended Data Fig. 3 Concentrations of individual plasma amino acids showing no change in GKO-NSG mice.

Related to Fig. 3. Concentration of individual plasma amino acids that showed no significant changes in 6-7 month old GKO-NSG mice (NSG mice, n = 10 males and 2 females; GKO-NSG mice, n = 5 males and 3 females; GKO-NSG Tx mice, n = 6 males). Data are represented as mean of biological replicates with individual data points overlaid and error bars indicate ± SEM.

Extended Data Fig. 4 Further assessment of blood glucose, plasma insulin, and glucagon phenotypes in GKO-NSG mice after human islet transplantation.

Related to Fig. 5. Data are from 4-6 month old NSG control, GKO-NSG, and GKO-NSG mice post-transplantation (GKO-NSG Tx). (a) Plasma glucagon levels in ad libitum-fed mice (NSG vs. GKO-NSG Tx: P = 0.005112 by two-tailed Student’s t-test). Due to the distribution of data from GKO-NSG mice, these data points were omitted from statistical analysis. (NSG mice, n = 10 males and 3 females; GKO-NSG mice, n = 12 males and 1 female; GKO-NSG Tx mice, n = 6 males). Blood glucose (b) (P = 0.013846 by one-way ANOVA, with Tukey’s multiple comparison test) (NSG mice, n = 10 males and 3 females; GKO-NSG mice, n = 8 males and 2 females; GKO-NSG Tx mice, n = 6 males) and plasma insulin levels (c) (NSG mice, n = 10 males and 3 females; GKO-NSG mice, n = 7 males and 2 females; GKO-NSG Tx mice, n = 6 males) in fasted mice. (d) Mouse and human plasma insulin levels in ad libitum-fed GKO-NSG Tx mice (n =6 males). Dashed lines indicate limit of detection (d: black dashed line indicates limit of detection of mouse insulin and red dashed line indicates limit of detection of human insulin). Data are represented as mean of biological replicates with individual data points overlaid and error bars indicate ± SEM. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Extended Data Fig. 5 In vitro characterization of donor human islets and more physiological assessment of GKO-NSG mice transplanted with islets either non-diabetic or T2D diabetic donors.

Related to Fig. 6. (a) In vitro glucagon secretion assay on islets from non-diabetic (n = 4 donors) and type 2 diabetic donors (n = 3 donors), shown as technical replicates from individual donors. (b) Glucagon content of donor islets transplanted into GKO-NSG mice (P =0.558605 by two-tailed Student’s t-test; non-diabetic donor n = 5, type 2 diabetic donor n = 3). Data in (c-e) are from 4-6 month old GKO-NSG mice post-transplantation with islets from non-diabetic (GKO-NSG Tx) or type 2 diabetic donors (GKO-NSG Tx T2D). For data presented in (c-e): GKO-NSG Tx mice, n = 6 males; GKO-NSG Tx T2D mice n = 1 male and 2 females. (c) Plasma glucagon levels in ad libitum-fed mice (P = 0.034687 by two-tailed Student’s t-test). Blood glucose (d) (P = 0.042886 by two-tailed Student’s t-test) and plasma insulin levels (e) in 6-hour fasted mice. (f) Percent of basal blood glucose 30-minutes post insulin injection (1U/kg body weight) from 4.5-6.5 month old GKO-NSG Tx and GKO-NSG Tx T2D mice (GKO-NSG Tx mice, n = 5 males; GKO-NSG Tx T2D n = 1 male and 1 female). Dashed lines indicate limit of detection. Data are represented as mean of biological replicates with individual data points overlaid, except in a, where individual data points represent technical replicates from single donors. Error bars indicate ± SEM. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

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Unprocessed genotyping gel

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Tellez, K., Hang, Y., Gu, X. et al. In vivo studies of glucagon secretion by human islets transplanted in mice. Nat Metab 2, 547–557 (2020). https://doi.org/10.1038/s42255-020-0213-x

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