Pharmacological targeting of α3β4 nicotinic receptors improves peripheral insulin sensitivity in mice with diet-induced obesity

Abstract

Aims/hypothesis: Treatment with the α3β4 nicotinic acetylcholine receptor (nAChR) agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), improves glucose tolerance in diet-induced obese (DIO) mice, but the physiological and molecular mechanisms are unknown.

Methods: DMPP (10 mg/kg body weight, s.c.) was administered either in a single injection (acute) or daily for up to 14 days (chronic) in DIO wild-type (WT) and Chrnb4 knockout (KO) mice and glucose tolerance, tissue-specific tracer-based glucose metabolism, and insulin signalling were assessed.

Results: In WT mice, but not in Chrnb4 KO mice, single acute treatment with DMPP induced transient hyperglycaemia, which was accompanied by high plasma adrenaline (epinephrine) levels, upregulated hepatic gluconeogenic genes, and decreased hepatic glycogen content. In contrast to these acute effects, chronic DMPP treatment in WT mice elicited improvements in glucose tolerance already evident after three consecutive days of DMPP treatment. After seven days of DMPP treatment, glucose tolerance was markedly improved, also in comparison with mice that were pair-fed to DMPP-treated mice. The glycaemic benefit of chronic DMPP was absent in Chrnb4 KO mice. Chronic DMPP increased insulin-stimulated glucose clearance into brown adipose tissue (+69%), heart (+93%), gastrocnemius muscle (+74%) and quadriceps muscle (+59%), with no effect in white adipose tissues. After chronic DMPP treatment, plasma adrenaline levels did not increase following an injection with DMPP. In glucose-stimulated skeletal muscle, we detected a decreased phosphorylation of the inhibitory Ser640 phosphorylation site on glycogen synthase and a congruent increase in glycogen accumulation following chronic DMPP treatment.

Conclusions/interpretation: Our data suggest that DMPP acutely induces adrenaline release and hepatic glycogenolysis, while chronic DMPP-mediated activation of β4-containing nAChRs improves peripheral insulin sensitivity independently of changes in body weight via mechanisms that could involve increased non-oxidative glucose disposal into skeletal muscle.

Keywords: Catecholamine; Glucose metabolism; Glucose tolerance; Hyperglycaemia; Insulin sensitivity; Nicotinic acetylcholine receptor; Pharmacology.

Fig. 1

DMPP acutely elicits hyperglycaemia, while chronically it improves glucose tolerance. (a) Effect of first injection (i.e. day 0) of DMPP (10 mg/kg) or vehicle injected at time point 0 min on blood glucose excursion with AUC in DIO WT mice. (b) Glucose tolerance with incremental AUC (iAUC) determined 24 h after the first injection (i.e. day 1) of DMPP or vehicle. (c) Effect of the third injection (i.e. day 2) of DMPP or vehicle on blood glucose excursion and AUC. (d) Glucose tolerance with iAUC determined 18 h after the third daily injection (i.e. day 3) of DMPP or vehicle. All data are presented as mean ± SEM (n = 7–8). Data in line graphs were assessed by two-way repeated measures ANOVA (time × drug) with a subsequent Bonferroni post hoc test. Data in bar graphs were probed with two-tailed Student’s t tests, comparing the means of vehicle and DMPP; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared with vehicle

Fig. 2

Acute DMPP increases circulating adrenaline and induces hepatic gluconeogenesis and glycogenolysis. (a–d) Effect of first injection of DMPP (10 mg/kg) or vehicle in DIO WT mice on (a) plasma noradrenaline (NAd) and adrenaline (Ad) concentrations determined in blood collected 80 min after vehicle or DMPP was injected (n = 8); on (b) plasma insulin concentrations in blood collected 80 min after vehicle or DMPP was injected (n = 8); on (c) liver glycogen at 150 min after vehicle or DMPP injection (n = 4–5); and on (d) expression of indicated genes in the liver at 150 min after vehicle or DMPP injection (n = 4–5). All data are presented as mean ± SEM. Data were probed with two-tailed Student’s t tests, comparing the means of vehicle and DMPP; *p ≤ 0.05, **p ≤ 0.01 compared with vehicle. ww, wet weight

Fig. 3

Chronic DMPP improves glucose tolerance independent of body weight loss and specifically via CHRNB4. (a, b) Body weight loss (in %) and cumulative food intake (in g) of DMPP-treated and vehicle-treated DIO WT mice with either ad libitum (Ad lib) food access or pair-fed to mice receiving 10 mg/kg DMPP for 10 days. (c) Glucose tolerance with incremental AUC (iAUC) conducted 18 h after the seventh injection of daily vehicle or DMPP (i.e. day 7). (d) Plasma glucose (at 0 and 30 min) and (e) plasma insulin at 30 min after i.p. injection of glucose (injected at time point 0 min), 18 h after the tenth injection of daily vehicle or DMPP (i.e. day 10). (f, g) Body weight loss (in %) and cumulative food intake (in g) in DIO WT or Chrnb4 KO mice receiving vehicle or DMPP. (h) Glucose tolerance with respective iAUC 18 h after seventh injection of daily vehicle or DMPP. (i) Plasma glucose (0 and 30 min) and (j) plasma insulin at 30 min after i.p. injection of glucose (injected at time point 0 min) 18 h after tenth injection of daily vehicle or DMPP. All data are presented as mean ± SEM; (n = 7–8). Data in line graphs were assessed by two-way repeated measures ANOVA (time × drug) within genotypes with a subsequent Bonferroni post hoc test. Data in (e) and bar graph in (c) were assessed by one-way ANOVA, and with a subsequent Bonferroni post hoc test (for c). Data in (j) and in the bar graph in (h) were assessed with two-tailed Student’s t tests within the genotypes. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 for vehicle compared with DMPP; ^†p ≤ 0.05, ^††p ≤ 0.01, ^†††p ≤ 0.001 for vehicle compared with pair-fed; ^‡p ≤ 0.05, ^‡‡‡p ≤ 0.001 for pair-fed compared with DMPP; ^§p ≤ 0.05, ^§§p ≤ 0.01, ^§§§p ≤ 0.001 for vehicle compared with DMPP within WT

Fig. 4

Chronic DMPP selectively increases glucose clearance in the BAT and the muscles. (a) Glucose excursion after injection with glucose 18 h after the eighth injection of daily DMPP (10 mg/kg) or vehicle in DIO WT mice. Glucose clearance into the (b) BAT, (c) gastrocnemius (Gastroc) muscle, (d) quadriceps (Quad) muscle, (e) heart, (f) iWAT, and (g) eWAT. All data are presented as mean ± SEM (n = 7). Data in line graph in (a) were assessed by two-way repeated measures ANOVA (time × drug) with a subsequent Bonferroni post hoc test. All other data (b–g) were probed with two-tailed Student’s t tests, comparing the means of vehicle and DMPP. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 for the effect of drug at the indicated time point or, in bar graphs, compared with vehicle

Fig. 5

DMPP increases non-oxidative glucose disposal in skeletal muscle. (a–c) Representative western blots and quantification of indicated protein phosphorylation residues or total proteins, relative to total protein, in quadriceps muscle from DIO WT mice treated as described in Fig. 4. (d, e) [¹⁴C]Glucose incorporation into glycogen and glycogen content in quadriceps (Quad) and gastrocnemius (Gastroc) muscles from DIO WT mice treated as described in Fig. 4. (f) Quadriceps muscle glycogen from vehicle-treated pair-fed (PF) and DMPP-treated mice (10 mg/kg); and (g) from vehicle- or DMPP-treated DIO WT and Chrnb4 KO mice (10 mg/kg). The key next to (e) applies to (b–e). All data are presented as mean ± SEM. For (a–e) n = 7, except for DMPP-treated quadriceps muscle in (e) for which n = 6, because of insufficient material for one of the samples; (f) n = 8; (g) n = 7–8. Differences were probed with two-tailed Student’s t tests for (b, c, d, f, g) comparing the means of vehicle and DMPP. Data in (e) were analysed with two-way repeated measures ANOVA (muscle × drug). *p ≤ 0.05, **p ≤ 0.01 for DMPP compared with vehicle; ^§p = 0.064 for DMPP compared with vehicle; ^¶p ≤ 0.05 main effect of DMPP; ^‡p ≤ 0.05 vehicle-treated pair-fed compared with DMPP. DPM, disintegrations/min; GS, glycogen synthase; HKII, hexokinase II; RU, relative units; ww, wet weight