A second-generation glucagon-like peptide-1 receptor agonist mitigates vomiting and anorexia while retaining glucoregulatory potency in lean diabetic and emetic mammalian models

Abstract

Aim: To develop a conjugate of vitamin B12 bound to the glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (Ex4) that shows reduced penetrance into the central nervous system while maintaining peripheral glucoregulatory function.

Methods: We evaluated whether a vitamin B12 conjugate of Ex4 (B12-Ex4) improves glucose tolerance without inducing anorexia in Goto-Kakizaki (GK) rats, a lean type 2 diabetes model of an understudied but medically compromised population of patients requiring the glucoregulatory effects of GLP-1R agonists without anorexia. We also utilized the musk shrew (Suncus murinus), a mammalian model capable of emesis, to test B12-Ex4 on glycaemic profile, feeding and emesis.

Results: In both models, native Ex4 and B12-Ex4 equivalently blunted the rise in blood glucose levels during a glucose tolerance test. In both GK rats and shrews, acute Ex4 administration decreased food intake, leading to weight loss; by contrast, equimolar administration of B12-Ex4 had no effect on feeding and body weight. There was a near absence of emesis in shrews given systemic B12-Ex4, in contrast to reliable emesis produced by Ex4. When administered centrally, both B12-Ex4 and Ex4 induced similar potency of emesis, suggesting that brain penetrance of B12-Ex4 is required for induction of emesis.

Conclusions: These findings highlight the potential therapeutic value of B12-Ex4 as a novel treatment for type 2 diabetes devoid of weight loss and with reduced adverse effects and better tolerance, but similar glucoregulation to current GLP-1R agonists.

Keywords: animal pharmacology; antidiabetic drug.

FIGURE 1

Systemically delivered B12-Ex4 enhances glucose clearance during an intraperitoneal glucose tolerance test (IPGTT) and co-localizes with insulin in pancreatic β-cells in shrews. (A) In an IPGTT, Ex4 (5 nmol/kg, i.e. ~20 μg/kg) and B12-Ex4 (5 nmol/kg) showed a similar potency in suppressing blood glucose (BG) levels after intraperitoneal (IP) glucose administration (2 g/kg, IP) compared with saline; saline versus B12-Ex4: *** P < .001; saline versus Ex4: ### P < .001. (B) Area under the curve (AUC) analysis from 0 (i.e. postglucose bolus) to 120 minutes; B12-Ex4 and Ex4 similarly reduced AUCs compared with saline. (C, D) To investigate the effects of B12-Ex4 and Ex4 on baseline glucose homeostasis, the same doses of Ex4, B12-Ex4 or saline were administered without subsequent glucose administration. Ex4 and B12-Ex4 treatments were no longer effective in reducing BG levels and had no effect on AUC. (E) Systemically injected fluorophore-labelled B12-Ex4 (Cy5-B12-Ex4, 5 nmol/kg) co-localized with insulin in shrew pancreatic tissue supporting the hypothesis that B12-Ex4 acts at the pancreas to improve glycaemic control. All data are expressed as mean ± SEM. Data in (A) and (C) were analysed with repeated measurements two-way ANOVA followed by Tukey’s post hoc test. Data in (B) and (D) were analysed with repeated measurements one-way ANOVA followed by Tukey’s post hoc test. Means with different letters are significantly different (P < .05). In (A, B), n = 13, within subject; in (C-D), n = 5, within subject

FIGURE 2

Pharmacological and supra-pharmacological doses of B12-Ex4 do not affect feeding and body weight in shrews. (A, B) Ex4 (5 nmol/kg) suppressed feeding at 3, 6, 12 and 24 hours, whereas equimolar doses of B12-Ex4 had no effect on food intake. (C) Ex4-induced anorexia was accompanied by body weight loss. No significant changes in body weight were observed after B12-Ex4 administration compared with controls. (D, E) Ex4 (50 nmol/kg, i.e. ~200 μg/kg) suppressed eating at all measured time points. By strict contrast, supra-pharmacological equimolar doses of B12-Ex4 did not show any effects. (F) While severe body weight loss occurred following supra-pharmacological doses of Ex4, no significant changes occurred in the B12-Ex4–treated animals. All data are expressed as mean ± SEM. Data were analysed with repeated measurements one-way ANOVA followed by Tukey’s post hoc test. Means with different letters are significantly different (P < .05). In (A-C), n = 8, within subject; in (D-F), n = 6, within subject

FIGURE 3

Emesis in shrews is significantly reduced following B12-Ex4 treatment compared with native Ex4, denoting improved tolerance across different doses. (A) The percentage of shrews experiencing emesis was significantly different between Ex4 (5 nmol/kg) and equimolar B12-Ex4 (*** P < .001). None of the animals experienced emesis following saline administration (data not shown). (B) The number of single emetic episodes following Ex4 (5 nmol/kg), equimolar B12-Ex4 or saline systemic administration was recorded for 90 minutes. Ex4 induced robust emetic responses that were not observed after B12-Ex4 or saline injections. (C) The number of emetic bouts was also lower after B12-Ex4 treatment compared with Ex4 and it did not differ from saline controls. (D) Latency to the first emetic episode in Ex4 animals that exhibited emesis. (E) Heatmap showing the emetic latency and intensity, as well as the number of emetic episodes induced by Ex4 or B12-Ex4 for each animal across time. A similar experiment was conducted with supra-pharmacological doses of Ex4 and B12-Ex4. Astonishingly, no emesis occurred after B12-Ex4 administration. (F) The percentage of shrews experiencing emesis was completely reversed between Ex4 (50 nmol/kg, i.e. ~200 μg/kg) and equimolar B12-Ex4 (*** P < .001). No animal experienced emesis following saline injection (data not shown). (G, H) The number of emetic episodes and bouts following Ex4 (50 nmol/kg), equimolar B12-Ex4 or saline administration was analysed over 90 minutes. Also, at this dosage Ex4 induced robust emetic responses. (I) Latency to the first emetic episode in Ex4 shrews that exhibited emesis. (J) Heatmap showing the emetic latency and intensity, as well as the number of emetic episodes for each animal across time. All data are expressed as mean ± SEM. Data in (A, F) were analysed with Fisher’s exact test. Data in (B, C, G, H) were analysed with repeated measurements one-way ANOVA followed by Tukey’s post hoc test. In (A-C), n = 16, within-subject; in (F-H), n = 8, within subject. Means with different letters are significantly different (P < .05)

FIGURE 4

Systemic administered B12-Ex4 does not activate the area postrema (AP) or the nucleus tractus solitarius (NTS) in shrews but it causes emesis when administered centrally. (A) Representative immunostainings of the AP/NTS region showing the c-Fos response following saline (n = 4), Ex4 (5 nmol/kg, n = 4) and equimolar B12-Ex4 (n = 5) systemic treatment. (B) Peripheral Ex4 administration significantly increased the number of c-Fos immunoreactive (IR) cells in the AP/NTS of shrews 3 hours after injection. The number of c-Fos–positive cells in the AP/NTS was significantly lower in B12-Ex4–treated animals and it did not differ from saline-treated shrews. (C) Ex4 (0.24 nmol, i.e. 1 μg), equimolar B12-Ex4 or vehicle was infused into the lateral ventricle. The percentage of shrews showing emesis was similar between Ex4 and B12-Ex.4. (D, E) The number of single emetic episodes or bouts following Ex4, B12-Ex4 or saline was recorded for 120 minutes. Both Ex4 and B12-Ex4 induced comparable emetic responses, while none of the animals experienced emesis following vehicle delivery (data not shown). (F) Latency to the first emetic episode in Ex4- and B12-Ex4–treated animals that exhibited emesis did not differ. Data in (B) were analysed with one-way ANOVA followed by Tukey post hoc test. Means with different letters are significantly different from each other (P < .05). In (C) the analysis was performed with Fisher’s exact test. Data in (D-F) were analysed with Student t-test (n = 8, within subject). Values are expressed as mean ± SEM. Scale bar 100 μm

FIGURE 5

B12-Ex4 enhances glucose clearance during an oral glucose tolerance test (OGTT) without inducing anorexia and body weight loss in the Goto-Kakizaki lean diabetic rat model. (A) In an OGTT, liraglutide (100 μg/kg, i.e. 26.6 nmol/kg) and 5 nmol/kg B12-Ex4 suppressed blood glucose levels after intraperitoneal (IP) glucose administration (1 g/kg, PO) compared with 5 nmol/kg Ex4 (i.e. ~20 μg/kg) and saline. * saline versus others; #: Ex4 versus others; $: liraglutide versus others. *, #, $: P < .05; **, ##, $$: P < .01; ***, ###, $$$: P < .001. (B) Liraglutide and Ex4 induced a strong anorectic effect, which was not observed after B12-Ex4 treatment. (C) Hypophagia was accompanied by body weight loss in liraglutide- and Ex4-treated animals, while no significant body weight change was observed following B12-Ex4. Data were analysed with repeated measure two-way ANOVA (A) or one-way ANOVA (B, C) followed by Tukey post hoc test; n = 21, within subject. All data are expressed as mean ± SEM. Means with different letters are significantly different from each other (P < .05)