The Role of Pancreatic Preproglucagon in Glucose Homeostasis in Mice

Abstract

Glucagon-like peptide 1 (GLP-1) is necessary for normal gluco-regulation, and it has been widely presumed that this function reflects the actions of GLP-1 released from enteroendocrine L cells. To test the relative importance of intestinal versus pancreatic sources of GLP-1 for physiological regulation of glucose, we administered a GLP-1R antagonist, exendin-[9-39] (Ex9), to mice with tissue-specific reactivation of the preproglucagon gene (Gcg). Ex9 impaired glucose tolerance in wild-type mice but had no impact on Gcg-null or GLP-1R KO mice, suggesting that Ex9 is a true and specific GLP-1R antagonist. Unexpectedly, Ex-9 had no effect on blood glucose in mice with restoration of intestinal Gcg. In contrast, pancreatic reactivation of Gcg fully restored the effect of Ex9 to impair both oral and i.p. glucose tolerance. These findings suggest an alternative model whereby islet GLP-1 also plays an important role in regulating glucose homeostasis.

Keywords: GLP-1; glucose homeostasis; incretin.

Figure 1. Targeting construct and glucose phenotype of GcgRA^ΔNull mice

A&B. A Lox P flanked transcriptional blocking cassette consisting of a splice acceptor (SA), an internal ribosomal entry site (IRES), and a green fluorescent protein (GFP) with a poly-A signal (pA) and was inserted into the intron between exons 2 and 3 of the Gcg gene. In the absence of Cre recombinase, expression of the targeted allele is suppressed generating a null allele. C. Gcg gene expression relative to GAPDH was nearly undetectable in GcgRA^ΔNull vs. WT mice (n=4M-WT and 6M-GcgRA^ΔNull). D. Glucose response to Ensure® (200μl) was significantly lower at 15 and 30min after the gavage in GcgRA^ΔNull vs. WT mice (n=4F;6M/genotype). E. Endogenous glucose production (EGP) during baseline and during the final 30min of a hyperinsulinemic euglycemic clamp (n=6M/genotype) was similar in WT and GcgRA^ΔNull (main effect of time; p<0.05). F. Glucose clearance was similar in WT and GcgRA^ΔNull at baseline and during the final 30 minutes of the hyperinsulinemic euglycemic clamp (main effect of time; p<0.05). G. Glucose and H. insulin response to an IV glucose load (0.5 g/kg) were similar between WT and GcgRA^ΔNull (n=8M-WT; 7M- GcgRA^ΔNull). *p<0.01 vs. GcgRA^ΔNull. Significance in C was determined using a One-way ANOVA. Signifcance in D–H was determine using a Two-way Anova for genotype and time. Data is represented as mean±SEM.

Figure 2. Validation and glucose phenotype of mice with intestinal reactivation of endogenous Gcg

A. Gcg expression was significantly greater in Vil Cre vs. GcgRA^Δvilcre in the duodenum but similar in the jejunum, ileum, and colon. Gcg expression was undetectable in the GcgRA^Δvilcre in the pancreas and hindbrain and in all tissues in the GcgRA^ΔNull mice (n=7F;5M/genotype). B. Tissue GLP-1 levels were not significantly different in the duodenum and ileum in Vil Cre vs. GcgRA^Δvilcre and were undetectable in the pancreas of GcgRA^Δvilcre and in all tissues of the GcgRA^ΔNull mice (n=9M/genotype). C. Circulating levels of active GLP-1 15 min after a glucose (3 g/kg) gavage was similar between Vil Cre and GcgRA^Δvilcre mice and undetectable in GcgRA^ΔNull (n=7F;5M/genotype). D. Pancreatic protein levels of glucagon were undetectable in GcgRA^ΔNull and GcgRA^ΔVilCre mice (n=10M; Vil Cre & GcgRA^ΔNull; 9M GcgRA^ΔVilCre). E. Plasma glucagon levels 15min after 1U/kg of insulin were undetectable in GcgRA^Δvilcre (n=8 GcgRA^ΔVilCre and n=10 Vil Cre mice). F. Oral glucose tolerance (2g/kg) was significantly lower at 15, 30 and 45 min in both GcgRA^ΔNull and GcgRA^ΔVilCre mice vs. WT mice (n=10M; Vil Cre & GcgRA^ΔNull; 9M GcgRA^ΔVilCre). G. IP glucose tolerance (2g/kg) was not significantly different between GcgRA^ΔNull, GcgRA^ΔVilCre, and WT mice (n=10M; Vil Cre & GcgRA^ΔNull; 9M GcgRA^ΔVilCre). *p<0.01 GcgRA^ΔNull vs. Vil Cre and GcgRA^ΔVilCre; **p<0.05 Vil Cre vs. GcgRA^ΔVilCre; ₊ p<0.01 Vil Cre vs. GcgRA^ΔNull and GcgRA^ΔVilCre. H–K. Ex9 (50ug) impaired oral (H–I) and IP (J–K) glucose tolerance over saline (100μl) in WT and Vil Cre vs. GcgRA^ΔNull and GcgRA^ΔVilCre, respectively (n=15M; WT and GcgRA^ΔNull/drug treatment; 15M; Vil Cre and GcgRA^ΔVilCre/drug treatment). *p<0.05 in WT or Vil Cre vs. all other groups; +p<0.05 Vil Cre Ex9 vs. saline. Significance in A–E was determined using a One-way ANOVA for genotype. Significance in F–G was determine using a Two-way ANOVA for genotype and time. Significance in H–K was determined using a three-way ANOVA for genotype, drug and time. Data is represented as mean±SEM.

Figure 3. Validation and glucose phenotype of mice with pancreatic reactivation of endogenous Gcg

A. Gcg expression was significantly greater in PDXcre control vs. GcgRA^ΔPDX1Cre mice in the ileum and hindbrain, was not different in the duodenum, and was significantly lower in the pancreas compared to the GcgRA^ΔPDX1Cre mice. GcgRA^ΔNull had undetectable Gcg expression across all tissues. n=10M/genotype. B. Plasma glucagon levels 15 min after IP insulin (1U/kg) were similar between control and GcgRA^ΔPDX1Cre mice and were below detection limits in all GcgRA^ΔNull mice (GcgRA^ΔNull and PDX1Cre, n=6M; GcgRA^ΔPDX1Cre, n=8M). C. Plasma GLP-1 levels 15min after a glucose (3g/kg) gavage (GcgRA^ΔNull and PDX1Cre, n=10M; GcgRA^ΔPDX1Cre, n=7M). D. Oral (2g/kg) glucose tolerance in GcgRA^ΔNull, PDX1Cre, and GcgRA^ΔPDX1cre mice (n=13M/genotype). E. IP (2g/kg) glucose tolerance in GcgRA^ΔNull vs. PDX1Cre and GcgRA^ΔPDX1cre mice (n=10M/genotype). Oral (F.) and IP (G.) glucose tolerance in response to saline or Ex9 (50ug; n=7M/genotype). H. IP glucose tolerance in response to chronic (21d) infusion of saline or Ex9 (50 μg/day; n=11M/genotype). *p<0.05 PDX1Cre vs. GcgRA^ΔNull and GcgRA^ΔPDX1Cre mice; +p<0.05 PDX1Cre vs. GcgRA^ΔNull; **p<0.05 GcgRA^ΔPDX1Cre vs. GcgRA^ΔNull; #p<0.05 GcgRA^ΔPDX1Cre Ex9 vs. saline; @p<0.05 Ex9 vs. Sal in PDX1Cre and GcgRA^ΔPDX1Cre groups. Significance in A–C was determined using a One-way ANOVA for genotype. Significance in D–E was determine using a Two-way ANOVA for genotype and time. Significance in F-H was determined using a three-way ANOVA for genotype, drug and time. Data is represented as mean±SEM.

Figure 4. Role of extra-pancreatic GLP-1R

A–D. Ex9 (50ug) impaired oral (A&C) and IP (B&D) glucose tolerance over saline (100μl) in GLP-1R f/f vs. GLP-1RKD^ΔNestin and GLP-1RKD^ΔNkx2.1, respectively (n=7M; GLP-1R f/f and GLP-1RKD^ΔNestin/drug treatment; n=10M; GLP-1R f/f and GLP-1RKD^ΔNkx2.1/drug treatment). *p<0.05 in Ex9 vs. Saline within a both genotypes; +p<0.05 Ex9 vs. saline within GLP1R f/f only. Significance in A–D was determined using a three-way ANOVA for genotype, drug and time. Data is represented as mean±SEM.