Keywords: GLP-1, incretin, glucose homeostasis

Gcg loss-of-function

We inserted a constitutive stop signal flanked by loxP sites in the proximal portion ofGcg to prevent gene transcription (GcgRA^ΔNull;Figure 1A–B). Intestinal, pancreatic, and brainstemGcg expression was nearly undetectable in GcgRA^ΔNull vs. WT littermates (Figure 1C). GcgRA^ΔNull mice had similar body mass, but lower proportions of fat and higher proportions of lean mass compared to WT mice (Supplemental Figure 1A–B). Consistent with other models in which glucagon signaling is abolished (Gelling et al., 2009;Hayashi et al., 2009), the glucose response to a mixed meal gavage (200 μl of Ensure® nutrient shake) was lower in chow-fed GcgRA^ΔNull vs. WT mice even when animals were weight-matched between genotypes (Figure 1D). However, the improved glucose response was not due to differences in insulin sensitivity as clamped glycemia, glucose infusion rates (Supplementary Figure 1C–D), basal and insulin regulated hepatic glucose production and glucose clearance were all similar between chow-fed GcgRA^ΔNull and WT mice during a hyperinsulinemic-euglycemic clamp (Figure 1E–F). In addition, plasma glucose and insulin increased to a similar extent in response to an IV glucose load in GcgRA^ΔNull vs. WT mice (Fig 1G–H). GcgRA^ΔNull mice also had increased pancreatic mass, and α-cell hyperplasia similar to what has been previously reported in glucagon receptor KO mice (Gelling et al., 2003) (Supplemental Figure 1D–E). Together these data demonstrate that our newly generated GcgRA^ΔNull exhibits features previously reported in models that lack glucagon signaling with lower than normal glucose responses to a mixed nutrient load and increases in pancreatic mass, but differs in not having enhanced insulin secretion.

Gut-specific reactivation of the Gcg gene

We next assessed the effect of intestinalGcg reactivation, and thus gut production of GLP-1, on glucose tolerance. GcgRA^ΔNull mice were crossed with mice that express Cre recombinase in villus and crypt epithelial cells of the small and large intestine (Villin-1 Cre; elMarjou et al., 2004).Figure 2A demonstrates thatGcg expression was not significantly different from controls in the jejunum, ileum, or colon in male and female offspring, hereafter termed GcgRA^Δvilcre mice. In addition, these mice did not expressGcg in the pancreas or brainstem confirming gut specific-reactivation. Although levels ofGcg expression in the duodenum were only 50% of control values, tissue (duodenum and ileum) protein and glucose-stimulated plasma levels of active GLP-1 were not significantly different between Vil Cre and GcgRA^Δvilcre mice (Figure 2B–C). Consistent with our initial observations,Gcg gene expression and tissue GLP-1 levels were nearly undetectable in GcgRA^ΔNull mice (Figure 2A–C) and pancreatic glucagon was undetectable in both GcgRA^ΔNull mice and GcgRA^Δvilcre mice (Figure 2D). Because intestinal contribution to plasma glucagon has been reported in pancreatectomy patients, we also measured plasma glucagon responses to hypoglycaemia in WT vs. GcgRA^Δvilcre mice. We found plasma glucagon in response to insulin-induced hypoglycaemia remained undetectable in GcgRA^Δvilcre mice (Figure 2E).

Glucagon receptor KO mice have lower glycemic excursions in response to both oral and IP glucose, but this occurs in the setting of elevated islet and plasma GLP-1(Gelling et al., 2003). To determine if reactivation of intestinalGcg, which normalized meal-induced increases in plasma GLP-1, would also improve IP glucose tolerance, we performed an oral and IPGTT with identical doses of glucose over 1 week in a cross-over design. In contrast to the results in glucagon receptor KOs, the blood glucose profiles were only lower during the oral, but not during the IPGTT in GcgRA^ΔNull and GcgRA^Δvilcre compared to Vil Cre mice (Figure 2F–G). These data suggest that lack of glucagon has a potent influence on postprandial glucose regulation, and restoration of intestinal GLP-1 production does not affect glucose homeostasis beyond this.

To directly test the physiologic importance of intestinal GLP-1 on glucose tolerance, we administered the GLP-1R antagonist Ex9, during oral and IPGTTs. Importantly, while Ex9 caused glucose intolerance in control mice, there was no effect of Ex9 on blood glucose in the GcgRA^Δnull mice, regardless of route of glucose administration (Figure 2H&J). These data demonstrate thatin vivo, Ex9 lacks inverse agonist properties and instead acts as a pure GLP-1R antagonist. As a demonstration of an absolute requirement for the canonical GLP-1R, Ex9 had no impact on oral or IP glucose tolerance in GLP-1R KO mice (Supplementary Figure 2A–B). Given the specificity and pure GLP-1R antagonism of Ex9, it was very surprising that Ex9 failed to impair either oral or IP glucose tolerance (Figure 2I&K) even with normalized postprandial GLP-1 levels in the GcgRA^{ΔVil Cre} mice.

Pancreas-specific reactivation of the Gcg gene

α-cell production of GLP-1 is thought to play a role in pancreatic function only under conditions of high metabolic demand and/or inflammation (Donath and Burcelin, 2013). To determine whether α-cell production of GLP-1 plays a physiological role in glucose regulation, and given the fact that Ex9 had no effect on glucose tolerance in mice with restoration ofGcg in the intestine, we next restored pancreatic expression ofGcg (GcgRA^ΔPDX1Cre) by crossing our GcgRA^ΔNull mice with mice that express Cre recombinase under the control of the mouse pancreatic duodenal homebox promoter (PDX1).

Pancreatic, but not hindbrain or ilealGcg expression was restored in the Gcg RA^ΔPDX1Cre mice (Figure 3A). As PDX1 is also expressed in the duodenum, we also observed some restoration ofGcg expression in the duodenum (Figure 3A). Confirming pancreatic reactivation, plasma glucagon responses to insulin-induced hypoglycemia were restored and, in fact, were significantly greater than in control mice (Figure 3B). In addition, glucose-induced increases in circulating total GLP-1 were approximately 50% of control values in Gcg RA^ΔPDX1Cre (Figure 3C).

The glycemic responses to oral and IP glucose were similar to controls in the Gcg RA^ΔPDX1Cre mice and both of these lines had significantly higher oral glucose excursions than GcgRA^ΔNull mice (Figure 3D–E). In contrast to the GcgRA^ΔVilCre mice, treatment with Ex9 impaired both oral and IP glucose tolerance in control and GcgRA^ΔPDX1Cre groups relative to saline-treatment (Figure 3F–G). These data suggest thatGcg expression and GLP-1 synthesis within the pancreas is necessary for the normal regulation of glucose. To further confirm these results, we implanted a mini-osmotic pump into these mice and found that 30d treatment of Ex9, at a dose that had no significant effect on body or fat mass (Supplementary Figure 3), impaired glucose tolerance in control and GcgRA^ΔPDX1Cre mice but had no effect in GcgRA^ΔNull mice (Figure 3H).

Role of extra-pancreatic GLP-1R

Our data suggest that the pancreatic source of GLP-1 is necessary for glucose regulation. Previous work, including our own, has suggested that brain GLP-1R are important for glucoregulation. Because plasma GLP-1 levels were also significantly increased, we tested the hypothesis that this source of GLP-1 could activate CNS GLP-1R and regulate glucose levels. We found that Ex9 impaired both oral and IP glucose tolerance equally in GLP-1R f/f controls and both whole brain (GLP-1RKD^ΔNestin) and hypothalamic (GLP-1RKD^ΔNkx2.1) GLP-1R KO animals (Figure 4A–D). These findings indicate that the effect of Ex9 to cause glucose intolerance is not mediated in the CNS.

Animals

Male and female mice on a primarily C57BL6/J background were singly housed and maintained on a 12-hour light/dark cycle with ad libitum access to chow diet (Harlan Teklad no. 7012). Studies were conducted in animals 8–20 weeks of age and included age- and sex-matched littermate control mice. All procedures were approved by either the University of Cincinnati or University of Michigan Institutional Animal Care and Use Committee. All animals included in the data analysis did not display any health impairments. The numbers of animals studied per genotype are indicated within each experiment, however the minimal number of animals was 6/genotype in the IVGTT and hyperglycemic-euglycemic clamp studies.

Genetic mouse generation

A conditional GcgRA^ΔNull mouse was generated by introducing a transcriptional blocking cassette into the intron between exons 2 and 3 of theGcg gene (Figure 1A). Flanking this blocking cassette wereloxP sites recognized by cre-recombinase. To generate these mice, theGcg targeting vector (University of Cincinnati Gene Targeting and Transgenic Mouse Models Core) which consisted of a splice acceptor (SA), an internal ribosomal entry site (IRES) with green fluorescent protein (GFP), a poly-A signal (pA), the selection marker, neo, and loxP sites flanking this cassette, was inserted into the intron between exons 2 and 3 (Figure 1A). This vector was then electroporated into 129 ES and positiveGcg ES clones, identified by Southern blotting, were injected into blastocysts to generate chimera by the ES core at the University of Cincinnati. Germline transmission was verified in agouti mice, and the neomycin resistance cassette was removed by breeding then these mice Flp-recombinase mice (Jackson Laboratory, stock number 003800; C57BL/6). Offspring were then bred to C57BL/6 mice to remove theFlp gene and generate breeding pairs for studies. The GLP-1R floxed animals were generated similarly except thatloxP sites flanking exons 6 and 7 or theGLP1R gene were inserted (Smith et al., 2014; Wilson-Pérez et al., 2013). The whole body GLP-1R KO mice were provided by Dr. Dan Drucker and were generated by isolating the mouseGLP1R gene and using homologous recombination in mouse embryonic stem cells to generate a null mutation in both alleles (Scrocchi et al., 1996).

Mice with intestinal or pancreatic reactivation of the endogenousGcg gene were generated by crossing GcgRA^ΔNull mice with villin 1-Cre (Jax Laboratories, stock number 004586) and PDX1-Cre (Jax Laboratories, stock number 014647) mice, respectively, to produce mice with Cre-specific reactivation of theGcg gene, GcgRA^ΔNull, and Cre littermate controls. The GLP-1R f/f mice were crossed to nestin Cre (Jax Laboratories, stock number 003771) and Nkx2.1 Cre (Jax Laboratories, stock number 0086611) promoter mice to delete the GLP-1R from the CNS and hypothalamus, respectively. See the Key Resources Table for all mouse lines utilized for this manuscript.

Mouse validation

To validate proper gene targeting, mice were sacrificed by CO₂ asphyxiation and tissues (hindbrain, duodenum, jejunum, ileum, colon, and pancreas) were rapidly removed and frozen. Tissue was homogenized in Trizol reagent and tissue RNA was extracted using a Pure Link RNA mini kit (Invitrogen, Carlsbad, CA). cDNA was isolated (iScript cDNA synthesis kit, BioRad, Hercules, CA), and real-time quantitative PCR (qPCR) was performed using a TaqMan 7900 Sequence Detection System with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays (all from Applied Biosystems, Foster City, CA) or with the BioRad CFX 96 Touch RT PCR detection system with Sso Advanced Universal Probes supermix and iTaq Universal probes supermix (BioRad, Hercules, CA). Relative mRNA expression for theGcg (Mm01269055_m1, Therma Scientific, Waltham, MA) was calculated relative to L32 using the ΔΔCT method.

Tissue and protein levels of GLP-1 and glucagon were also measured to validate appropriate ablation or reactivation of gene function. Ileal and pancreatic tissue was homogenized in T-Per Protein Extraction Reagent (Thermo Scientific, Waltham, MA) with protease/phosphatase inhibitors and a DPP4 inhibitor. Total protein was quantified using a BCA protein assay kit (Thermo Scientific, Waltham, MA).

Plasma GLP-1 was measured 10 minutes after a gavage of 25% dextrose (3g/kg), CO₂ asphyxiation, and cardiac puncture. Plasma glucagon response was measured 15 minutes after an IP injection of insulin (1U/kg), CO₂ asphyxiation, and cardiac puncture. In both cases, blood was collected in heparinized syringes and placed into a tube with a mixture of heparin, EDTA, and aprotonin. Plasma levels of total and active GLP-1 and glucagon in protein-extracted tissue were measured using sandwich ELISA kits (Mesoscale Discovery, Rockville, MD, USA).

Glucose tolerance tests

For all glucose tolerance tests, mice were fasted for 4h after the onset of the light phase. For the IP and oral glucose tolerance tests, blood was taken via tail nick. Basal blood glucose was sampled at -15min, and glucose administered as an oral gavage or by IP injection at a dose of 2mg/kg of 25% dextrose or Ensure® (200μl) was administered via oral gavage. Blood samples were taken at 15, 30, 45, 60, and 120 min after glucose administration.

On another occasion, oral or IP glucose tolerance tests (100μl of 20% dextrose) were performed 15-min after an IP injection of Ex9 (50μg/100μl) or vehicle control and blood was sampled as described above.

IVGTT

Catheters were placed in the left common carotid artery for sampling and right jugular vein for infusions under isoflurane anesthesia in GcgRA^ΔNull and WT littermate controls (n=6M/genotype). The catheters were tunneled under the skin to the back of the neck and attached via stainless steel connectors to tubing made of Micro-Renathane (0.033 OD); the tubing was externalized and sealed with stainless steel plugs. Lines were filled with saline containing 200 units/ml heparin and 5 mg/ml ampicillin. Body mass was recorded daily and animals were studied once body mass was within 10% of their presurgery weight (~5d). Animals not reaching this weight were excluded from the study.

One the day of the study, mice were fasted and moved to the procedure room 4h prior to the IVGTT. Approximately 30-min after lines were lengthened for ease of access and patency was verified, all animals received an intravenous bolus of 20% dextrose (0.5 mg/kg). Blood samples for glucose and insulin were taken at baseline, at 1, 2, 5, 10, 12, 16, 20, and 30 min after glucose administration.

Hyperinsulinemic euglycemic clamp

For this experiment, 8-week old GcgRA^ΔNull and WT (n=6M/genotype) littermate controls had catheters placed as described for the IVGTT. A hyperglycemic euglycemic clamp with HPLC purified [3–3H]glucose and a continuous infusion of human insulin (2.5 mU/kg/min; Humulin R; Eli Lilly, Indianapolis, IN) was performed as described previously (Ayala et al., 2006). Specific activity for individual time points did not vary by >15% from the average specific activity during the last 40 min of the clamp, and the slope of specific activity over time was not significantly different from zero.

Mini-Osmotic Pump Study

Body weight-matched mice within a genotype (PDX1 Cre, GcgRA^ΔNull, and GcgRA^ΔPDX1Cre) had a 30d subcutaneous mini-osmotic pump (Alzet Osmotic Pumps, Cupertino, CA) filled with saline or Ex9 implanted under isoflurane anesthesia. An IPGTT was performed on treatment day 21 as described above.

For all studies above, glucose tolerance tests with and without Ex9 were replicated for data that will be used in publication elsewhere. Animals were placed in groups based on genotype and within each genotype were weight matched between drug and vehicle administrations. Due to the necessity of ensuring the proper drug and glucose dosage administration, no formal blinding was performed in any of these studies. Animals were only excluded from a study if there were apparent and noted experimental mistakes made with drug dosage, injections, with the assay procedures or if the data point in question was greater than 3 standard deviations from the mean of the group.

Statistical analysis

The statistical procedures used are indicated in the figure legends. Briefly, normally distributed data were analysed utilizing standard parametric statistics including ANOVA’s and t-tests where applicable. Both male (M) and female (F) mice were used based on availability from the breeding cohorts. For experiments requiring surgery, males were used to avoid the potential surgical complications of doing surgeries in the smaller age-matched females. Statistical analysis were performed using either GraphPad Prism v.6.02 or Statistica v.13 for Windows. Data are expressed as mean ± SEM, and statistical significance was accepted when p<0.05.

Highlights.

• Intestinally-secreted GLP-1 is presumed to regulate glucose via incretin action.

• Exendin-9 does not alter glucose in mice that only produce GLP-1 in the intestine.

• Exendin-9 does impair glucose in mice that only produce GLP-1 in the pancreas.

• Alternative to the incretin model, islet GLP-1 is crucial for gluco-regulation.

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Supplementary Materials