Coordinated targeting of cold and nicotinic receptors synergistically improves obesity and type 2 diabetes

Abstract

Pharmacological stimulation of brown adipose tissue (BAT) thermogenesis to increase energy expenditure is progressively being pursued as a viable anti-obesity strategy. Here, we report that pharmacological activation of the cold receptor transient receptor potential cation channel subfamily M member 8 (TRPM8) with agonist icilin mimics the metabolic benefits of cold exposure. In diet-induced obese (DIO) mice, treatment with icilin enhances energy expenditure, and decreases body weight, without affecting food intake. To further potentiate the thermogenic action profile of icilin and add complementary anorexigenic mechanisms, we set out to identify pharmacological partners next to icilin. To that end, we specifically targeted nicotinic acetylcholine receptor (nAChR) subtype alpha3beta4 (α3β4), which we had recognized as a potential regulator of energy homeostasis and glucose metabolism. Combinatorial targeting of TRPM8 and nAChR α3β4 by icilin and dimethylphenylpiperazinium (DMPP) orchestrates synergistic anorexic and thermogenic pathways to reverse diet-induced obesity, dyslipidemia, and glucose intolerance in DIO mice.

Fig. 1

TRPM8 agonist icilin increases energy expenditure and lowers body weight in DIO mice. a Effects on body weight, b energy expenditure, c food intake, d locomotor activity following daily s.c. injections of vehicle (black, n = 8 for a, c; n = 5 for b, d), icilin 0.6 mg/kg (light blue, n = 8), and icilin 6 mg/kg (dark blue, n = 6) in DIO male C57Bl6j mice for 14 days. e, f Oxygen consumption following a single injection of vehicle (black/gray, n = 8) or icilin (blue/green, n = 8) to DIO male WT mice or DIO TRPM8 KO mice. *p < 0.05, ***p < 0.001 by two-way ANOVA (a, c) with Tukey post-hoc test and two-tailed Student’s t-test (d). All data are presented as mean ± SEM

Fig. 2

The nicotinic receptor agonist DMPP lowers body weight and improves glucose tolerance in DIO mice. a Effects on body weight, b food intake, and c, d glucose tolerance following daily s.c. injections to DIO male C57Bl6j mice of vehicle (black, n = 8 for a, b; n = 7 for c, d), DMPP 1 mg/kg (light red, n = 8), DMPP 5 mg/kg (pink, n = 7 for a, b; n = 8 for c, d), and DMPP 10 mg/kg (dark red, n = 7) for 14 days. *p < 0.05, ***p < 0.001 by two-way ANOVA (a, b, c) and one-way ANOVA with Tukey post-hoc test (d). All data are presented as mean ± SEM

Fig. 3

Icilin and DMPP synergistically lower body weight in DIO mice via central mechanisms. a Effects on body weight, b cumulative food intake, and c cFOS positive neurons in the hypothalamus following daily s.c. injections to DIO male C57Bl6j mice of vehicle (black, n = 8 for a, b; n = 6 for c), DMPP 10 mg/kg (red, n = 8 for a, b; n = 6 for c), icilin 5 mg/kg (blue, n = 8 for a, b; n = 6 for c), or the combination of DMPP 10 mg/kg and icilin 5 mg/kg (purple, n = 7 for a, b; n = 8 for c) for 4 days (a, b) or 3 days (c), respectively. d cFOS positive neurons in the hypothalamus following daily s.c. injections to DIO male WT C57Bl6j mice or to DIO male CHRNB4 KO mice of vehicle (black, n = 5/gray, n = 4) or the combination of DMPP 10 mg/kg and icilin 5 mg/kg (purple, n = 5/light purple, n = 5) for 3 days. e Effects on body weight and f cumulative food intake following daily s.c. injections to HFD-fed male MC4R KO mice of vehicle (black, n = 8), DMPP 5 mg/kg (red, n = 8), icilin 5 mg/kg (blue, n = 8), or the combination of DMPP 5 mg/kg and icilin 5 mg/kg (purple, n = 7) for 7 days. All scale bars are 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA (a, e) and one-way ANOVA with Tukey post-hoc test (b, c, d, f). All data are presented as mean ± SEM

Fig. 4

Icilin and DMPP combination therapy increases sympathetic nervous system-linked BAT thermogenesis. a Effects on body weight and b cumulative food intake following daily s.c. injections to DIO male betaless mice or DIO male WT C57Bl6j mice of vehicle (black, n = 8/gray, n = 8) or the combination of DMPP 5 mg/kg and icilin 5 mg/kg (purple, n = 8/bordeaux, n = 7) for 7 days. c Effects on body weight and d cumulative food intake following daily s.c. injections to global UCP1 KO DIO male mice or DIO male C57Bl6j control mice of vehicle (black, n = 7/gray, n = 7) or the combination of DMPP 5 mg/kg and icilin 5 mg/kg (purple, n = 7/light blue, n = 7) for 7 days. e Mean optoacoustic signal intensity and f blood oxygen saturation (SO₂) measured by Multi-Spectral Optoacoustic Tomography (MSOT) in selected BAT regions of male C57Bl6j control mice of vehicle (black, n = 6), DMPP 10 mg/kg (red, n = 6), icilin 5 mg/kg (blue, n = 6), or the combination of DMPP 10 mg/kg and icilin 5 mg/kg (purple, n = 6). (g–a to g–d) MSOT image of BAT at 800 nm (gray scale) with overlaid of SO₂ (green-red scale) in: a control- (g–a), DMPP- (g–b), icilin- (g–c), and combination-injected mouse (g-d). All scale bars are 5 mm.*p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA (a, c), one-way ANOVA (e, f) with Tukey post-hoc test and two-tailed Student’s t-test (within genotype) (b, d). All data are presented as mean ± SEM

Fig. 5

Icilin and DMPP reverse obesity and increase BAT UCP1 at thermoneutral conditions. a Effects on body weight, b cumulative food intake, changes in c fat mass and d lean mass, e BAT H & E staining, and f UCP1 immunoreactivity staining in BAT with g quantification of UCP1 immunoreactivity staining (% area). BAT expression of genes involved in h thermogenesis, i mitochondrial transport, and j creatine signaling pathway following daily s.c. injections of vehicle (black or white, n = 8), DMPP 5 mg/kg (red, n = 7), icilin 5 mg/kg (blue, n = 8), or the combination of DMPP 5 mg/kg and icilin 5 mg/kg (purple, n = 7) to DIO male C57Bl6j chronically housed at 30 °C for 14 days. All scale bars are 50 µm. *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA (a) and one-way ANOVA (b, c, d, g, h, i, j) with Tukey post-hoc test. All data are presented as mean ± SEM

Fig. 6

Icilin and DMPP reverse diet-induced glucose intolerance, insulin resistance, and hepatic steatosis in DIO mice. a, b Effects on glucose tolerance and c, d glucose-stimulated insulin secretion following daily s.c. injections to DIO mice of vehicle (black, n = 8), DMPP 10 mg/kg (red, n = 8), icilin 5 mg/kg (blue, n = 8), or DMPP 10 mg/kg and icilin 5 mg/kg (purple, n = 7) for 7 days. e Effects on 6-h fasted insulin levels, f HOMA-IR score, and g insulin tolerance following daily s.c. injections to DIO mice of vehicle (black, n = 8), DMPP 10 mg/kg (red, n = 8), icilin 5 mg/kg (blue, n = 8), or DMPP 10 mg/kg and icilin 5 mg/kg (purple, n = 7) for 7 days at 23 °C. h Effect on insulin tolerance following 14 days of daily s.c. injections of vehicle (black, n = 8), DMPP 5 mg/kg (red, n = 7), icilin 5 mg/kg (blue, n = 8), or the combination of DMPP 5 mg/kg and icilin 5 mg/kg (purple, n = 7) in DIO mice housed at 30 °C. i and j Effect on pyruvate tolerance following daily s.c. injections to DIO mice of vehicle (black, n = 7), DMPP 5 mg/kg (red, n = 7), icilin 5 mg/kg (blue, n = 7), or DMPP 5 mg/kg and icilin 5 mg/kg (purple, n = 7) for 20 days. k Plasma lipoprotein fractions following daily s.c. injections to DIO mice of vehicle (black, n = 7), DMPP 5 mg/kg (red, n = 7), icilin 5 mg/kg (blue, n = 7), or DMPP 5 mg/kg and icilin 5 mg/kg (purple, n = 7) for 21 days. l Effect on hepatic steatosis score following daily s.c. injections to DIO mice housed at 23 °C of vehicle (n = 8), DMPP 10 mg/kg (n = 8), icilin 5 mg/kg (n = 8), or DMPP 10 mg/kg and icilin 5 mg/kg (n = 7) for 14 days. m Liver H & E stainings and n NASH severity (%) following daily s.c. injections to DIO mice housed at 30 °C of vehicle (black, n = 8), DMPP 5 mg/kg (red, n = 7), icilin 5 mg/kg (blue, n = 8), or DMPP 5 mg/kg and icilin 5 mg/kg (purple, n = 7) for 14 days. All scale bars are 200 µm. *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA (a, c, g, h, i) and one-way ANOVA (b, d, e, f, j) with Tukey post-hoc test. ^#p < 0.05 vehicle vs. icilin and ⁺p < 0.05 vehicle vs. DMPP (g). All data are presented as mean ± SEM

Fig. 7

The melanocortin system is indispensable for the glycemic benefits of icilin and DMPP combination therapy. a and b Effects on glucose tolerance following daily s.c. injections to DIO male C57Bl6j mice or c, d MC4R KO mice of vehicle (black, n = 8/striped white, n = 8), DMPP 5 mg/kg (red, n = 8/striped red, n = 8), icilin 5 mg/kg (blue, n = 8/striped blue, n = 8), or the combination of DMPP 5 mg/kg and icilin 5 mg/kg (purple, n = 8/striped purple, n = 7) for 7 days. e and f Effects on glucose tolerance following daily s.c. injections to DIO male C57Bl6j mice or g, h UCP1 KO mice of vehicle (black, n = 7/gray, n = 7) or the combination of DMPP 5 mg/kg and icilin 5 mg/kg (purple, n = 7/light blue, n = 7) for 7 days. *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA (a, c, e, g), one-way ANOVA wit Tukey post-hoc test (b, d), or two-tailed Student’s t-test (f, h). All data are presented as mean ± SEM