Neuronal GLP1R mediates liraglutide’s anorectic but not glucose-lowering effect (original) (raw)
To discriminate between the effects of GLP1R expression on peripheral versus central nerves, we developed mouse models with selective reduction of key GLP1R–expressing components of the central or peripheral nervous system. This was accomplished through the insertion of loxP sites surrounding exons 6 and 7 of the Glp1r gene (Figure 1A) and subsequent breeding to either nestin-Cre (7) or Phox2b-Cre (8) mice, respectively. Nestin is an intermediate filament protein expressed in neuroepithelial cells that, when used as a promoter for Cre expression, specifically disrupts the gene of interest in the brain (7, 9, 10). Mice with site-specific knockdown (KD) of the GLP1R in the CNS (nestin-Cre Glp1rflox/flox, herein referred to as GLP1R KDΔNestin) showed markedly decreased Glp1r mRNA in the hypothalamus and brainstem compared with control mice, but no reduction in the nodose ganglion or lung (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI72434DS1). We also observed a decrease in pancreatic Glp1r expression (Supplemental Figure 1A). However, while some data show expression of nestin in pancreatic progenitor cells (11), it has not been shown to be present in endocrine cells (12). The gene for Glp1r is located in the nodose ganglia, a cell body of the vagus nerve, and the protein is found within neurons innervating the hepatic portal vein (13). Phox2b is a homeobox gene expressed in the autonomic nervous system, including the nucleus of the solitary tract and dorsomotor nucleus of the vagus (14). Thus, using a Cre-recombinase driven by a Phox2b promotor, we generated mice with site-specific KD of the GLP1R in visceral afferent and efferent nerves (Phox2b-Cre Glp1rflox/flox, herein referred to as GLP1R KDΔPhox2b). Accordingly, GLP1R KDΔPhox2b had decreased expression of the Glp1r in the nodose ganglion, but no change in the hypothalamus or hindbrain (Supplemental Figure 1B).
Characterization of GLP1R KDΔNestin and GLP1R KDΔPhox2b mice. (A) Glp1r floxed construct. (B) Body weight analysis of chow-fed Glp1r mutant mice. (C) Body composition analysis on chow diet. (D) Body weight analysis of high-fat–fed Glp1r mutant mice. (E) Body composition analysis after 7 weeks of high-fat diet. (F) Seven-day high-fat diet intake. Statistical analysis was with 2-way ANOVA (B–E) or 1-way ANOVA (F) with Tukey’s post-hoc. Black, controls; red, GLP1R KDΔNestin; gray, GLP1R KDΔPhox2b.
Since endogenous GLP1 has been hypothesized to regulate food intake and body weight (6), we tested the role of CNS and vagal GLP1Rs in the regulation of food intake and body weight. In chow and high-fat diet conditions, body weight did not differ among GLP1R KDΔNestin, GLP1R KDΔPhox2b, or controls (Figure 1, B and D). Consistent with this, no differences existed in body composition (Figure 1, C and E). Although no differences existed in food intake among the groups on high-fat diet (Figure 1F), we have observed conflicting data regarding the food intake of chow-fed GLP1R KDΔNestin mice. In separate cohorts of GLP1R KDΔNestin mice, we have seen either similar or increased food intakes when compared with controls (Supplemental Figure 2, A and B, respectively). These discrepancies will be the subject of future investigations. However, given the high degree of GLP1R KD in both strains of mice, these data suggest that CNS and vagal GLP1Rs are not necessary for the normal regulation of food intake or body weight.
Despite these data, long-acting GLP1R agonists are well documented as having potent food intake–reducing effects. Prior research has pointed to a role for central GLP1 signaling in the anorectic effects of long-acting GLP1R agonists, but the respective contributions of central versus vagal GLP1R is unclear. Thus, a crucial question is whether such agonists act directly in the CNS or on vagal afferents to mediate these effects. In response to an acute s.c. injection of liraglutide, control and GLP1R KDΔPhox2b chow-fed mice had significantly decreased food intake at 4 and 24 hours (Figure 2, A and B). However, acute liraglutide administration caused a modest reduction in food intake at 4 hours but not at 24 hours in GLP1R KDΔNestin mice (Figure 2, A and B). In a new cohort of high-fat–fed GLP1RNestin mice, we repeated this study and found that liraglutide was able to decrease food intake compared with saline, but this response was attenuated compared with that of controls (Figure 2, C and D). Thus, CNS but not vagal GLP1Rs are necessary for the full acute anorectic response to a long-acting GLP1R agonist.
Acute effects of liraglutide in GLP1R KDΔNestin and GLP1R KDΔPhox2b mice. (A and B) Anorectic effects of liraglutide at 4 (A) and 24 (B) hours in chow-fed mice. (C and D) Anorectic effects of liraglutide in GLP1R KDΔNestin high-fat–fed mice at 4 (C) and 24 (D) hours. (E) CTA test after acute liraglutide administration in chow-fed mice. Statistical analysis was with 2-way ANOVA with repeated measures (A and B) or without repeated measures (C–E) with Tukey’s post-hoc. *P < 0.05 vs. same genotype; §P < 0.05 vs. same drug, different genotype
Acute administration of either GLP1 or long-acting GLP1R agonists causes visceral illness, which is reflected in rodent models by a pronounced conditioned taste aversion (CTA) (15, 16). Whether this effect is the result of activation of GLP1Rs in the CNS or a secondary result of reduced gastric emptying is controversial. Thus, we next tested whether liraglutide caused a CTA in our mouse models. Both chow-fed control and GLP1R KDΔPhox2b mice developed a CTA to a novel flavor paired with liraglutide. However, the pairing had no effect in GLP1R KDΔNestin mice, despite the ability to develop a CTA to lithium chloride (Figure 2E). Combined with our previous data, these results highlight the importance of CNS GLP1R in the acute anorectic effects of a long-acting GLP1R agonist.
Chronic administration of liraglutide continues to cause weight loss long after attenuation of its aversive effects (15). Thus, to understand the mechanism underlying weight loss associated with chronic administration of a long-acting GLP1R agonist (17), we administered liraglutide or saline daily for 15 days in these mouse models after 5 weeks of high-fat feeding. GLP1R KDΔNestin mice treated with liraglutide lost more weight than saline controls, but significantly less weight than liraglutide-treated GLP1R KDΔPhox2b or control mice (Figure 3, A and B). The body weight loss was proportional to the food intake changes observed with chronic liraglutide treatment (Figure 3C). Over the last 7 days of treatment, liraglutide decreased food intake in GLP1R KDΔPhox2b and control mice, but not in GLP1R KDΔNestin mice (Figure 3D). Liraglutide treatment also significantly decreased fat mass in both control and GLP1R KDΔPhox2b, mice but not in GLP1R KDΔNestin mice (Figure 3E). There were no significant differences in lean mass across groups or treatments (Figure 3F). Together, these data show that CNS but not vagal GLP1R are necessary for the chronic anorectic and weight-lowering effects of liraglutide.
Liraglutide requires CNS GLP1R for anorectic and weight-lowering effects in high-fat–fed mice. (A) Longitudinal body weights of Glp1r mutant mice treated with a long-acting GLP1R agonist or saline. (B) Cumulative weight loss of 14 days. (C) Longitudinal 14-day cumulative food intake of mice. (D) Total food intake during last 7 days of treatment. (E) Fourteen-day fat-mass change of mice. (F) Fourteen-day lean-mass change in mice. Black, controls; red, GLP1R KDΔNestin; gray, GLP1R KDΔPhox2b; solid, saline treatment; patterned, liraglutide treatment. Statistical analysis was with 2-way ANOVA with Tukey’s post-hoc with repeated measures (A and C) or without repeated measures (B and D–F). *P < 0.05 compared with saline treatment, same genotype unless otherwise indicated.
Although GLP1 has been widely hypothesized to lower glucose by increasing β cell insulin secretion, other data implicate vagal and CNS GLP1R contributions (13, 18). We performed both oral and i.p. glucose tolerance tests (GTTs) to assess the role of the central and vagal CNS GLP1R in endogenous GLP1 signaling. GLP1R KDΔNestin mice had no differences in oral or i.p. glucose tolerance compared with controls whether studied under chow- or high-fat–fed conditions (Figure 4, A–I). The chow-fed oral GTT was run in a separate cohort of mice without the availability of concurrent analysis of GLP1R KDΔPhox2b mice. GLP1R KDΔPhox2b did not show any differences in glucose tolerance in the chow-fed i.p. GTT or high-fat diet fed i.p. and oral GTTs (Figure 4, A, B, and E–I). During the i.p. GTT on high-fat–fed mice, there was no interaction of genotype on insulin secretion at baseline or at 15 minutes (Figure 4F). Homeostasis model assessment estimated insulin resistance (HOMA-IR) calculations, a measure of insulin resistance, also found no differences at baseline, nor were there differences in the insulin times glucose product at the 15-minute time point (data not shown). Thus, these data imply that neither CNS nor vagal GLP1R are necessary for the physiological regulation of glucose.
Glucose tolerance phenotypes of Glp1r mutant mice. (A) i.p. GTT on chow diet. (B) AUC for A. (C) Oral GTT on chow diet. Main effect of genotype. (D) AUC for C. (E) i.p. GTT on high-fat diet. (F) AUC for E. (G) Insulin response to i.p. glucose. (H) Oral GTT on high-fat diet. (I) AUC for H. Statistical analysis was with 2-way ANOVA with repeated measures (A, C, E, G, and H) or 1-way ANOVA (B, D, F, and I) with Tukey’s post-hoc.
Given the previously published studies on the effects of GLP1 in the brain on glucose homeostasis (1, 13), it is reasonable to hypothesize that the actions of long-acting GLP1 agonists to improve glucose regulation are not limited to their direct actions on β cells. Thus, we next tested the necessity of CNS or vagal GLP1R to meditate the glucose-lowering effects of liraglutide. Liraglutide caused significant decreases in glucose excursions in all genotypes during i.p. GTTs after both acute and chronic administration (Figure 5, A and B). There was no effect of genotype on insulin levels during an i.p. GTT after 14 days of liraglutide (measured at 0 and 15 minutes after i.p. glucose) (Figure 5C). These data demonstrate that neither vagal nor CNS GLP1R are necessary for the glucose-lowering effects of liraglutide and suggest that these effects are also independent of insulin concentrations.
Liraglutide lowers glucose tolerance despite KD of central GLP1 receptors. (A) i.p. GTT on chow diet. (B) i.p. GTT on high-fat diet after 14 days of chronic liraglutide (from Figure 3A). (C) Insulin level during the GTT in B. Statistical analysis was with 2-way ANOVA with repeated measures with Tukey post-hoc.