The cannabinoid Δ(9)-tetrahydrocannabivarin (THCV) ameliorates insulin sensitivity in two mouse models of obesity

Abstract

Background: Cannabinoid type-1 (CB1) receptor inverse agonists improve type 2 diabetes and dyslipidaemia but were discontinued due to adverse psychiatric effects. Δ(9)-Tetrahydrocannabivarin (THCV) is a neutral CB1 antagonist producing hypophagia and body weight reduction in lean mice. We investigated its effects in dietary-induced (DIO) and genetically (ob/ob) obese mice.

Methods: We performed two dose-ranging studies in DIO mice; study 1: 0.3, 1, 2.5, 5 and 12.5 mg kg(-1), oral twice daily for 30 days and study 2: 0.1, 0.5, 2.5 and 12.5 mg kg(-1), oral, once daily for 45 days. One pilot (study 3: 0.3 and 3 mg kg(-1), oral, once daily) and one full dose-ranging (study 4: 0.1, 0.5, 2.5 and 12.5 mg kg(-1), oral, once daily) studies in ob/ob mice for 30 days. The CB1 inverse agonist, AM251, oral, 10 mg kg(-1) once daily or 5 mg kg(-1) twice daily was used as the positive control. Cumulative food and water intake, body weight gain, energy expenditure, glucose and insulin levels (fasting or during oral glucose tolerance tests), plasma high-density lipoprotein and total cholesterol, and liver triglycerides were measured. HL-5 hepatocytes or C2C12 myotubes made insulin-resistant with chronic insulin or palmitic acid were treated with 0, 1, 3 and 10 μM THCV or AM251.

Results: THCV did not significantly affect food intake or body weight gain in any of the studies, but produced an early and transient increase in energy expenditure. It dose-dependently reduced glucose intolerance in ob/ob mice and improved glucose tolerance and increased insulin sensitivity in DIO mice, without consistently affecting plasma lipids. THCV also restored insulin signalling in insulin-resistant hepatocytes and myotubes.

Conclusions: THCV is a new potential treatment against obesity-associated glucose intolerance with pharmacology different from that of CB1 inverse agonists/antagonists.

Figure 1

Effect of THCV on body weight gain, cumulative food intake and energy expenditure in DIO mice (study 1). (a) Body weight gain in the dose–response study, n=9 mice per treatment. (b) Cumulative food intake in mice given AM251 or THCV (12.5 mg kg⁻¹ p.o.), n=3 groups of three mice per treatment (other dose levels of THCV excluded from graph for sake of clarity since identical to controls). However, all treatments were included in the one-way ANOVA statistical analysis. (c) Twenty-four-hour energy expenditure after 9 days treatment in DIO mice given AM251, (d) THCV (5 mg kg⁻¹) or (e) THCV (12.5 mg kg⁻¹ p.o.) in study 1, n=3 groups of three mice per treatment. ****P<0.0001 as compared to vehicle treated animals.

Figure 2

Effect of THCV on glucose tolerance and indexes of insulin sensitivity in DIO mice (study 1). (a) Glucose tolerance after 7 days treatment with AM251 or THCV, n=9 mice per treatment. (b) Blood glucose concentration in 5-h fasted mice after 7 days treatment with AM251 or THCV, n=9 mice per treatment. (c) Plasma insulin in 5-h fasted mice and at 30 min post glucose load after 7 days treatment with AM251 or THCV, n=9 mice per treatment. (d) Homeostatic model assessment (HOMA) index (glucose × insulin) in 5-h fasted mice after 7 days treatment with AM251 or THCV. (e) Glucose tolerance after 3 weeks treatment with AM251 or THCV, n=9 mice per treatment. (f) Plasma insulin in 5-h fasted mice at 30 min post glucose load after 3 weeks treatment with AM251 or THCV, n=9 mice per treatment. (g) HOMA index (glucose × insulin) in 5-h fasted mice after 3 weeks treatment with AM251 or THCV. (h) HOMA index (glucose × insulin) at 30 min post glucose load after 3 weeks treatment with AM251 or THCV. *P<0.05; **P<0.01 as compared to vehicle treated animals.

Figure 3

Effect of THCV on body weight gain, cumulative food intake and energy expenditure in ob/ob mice (study 3). (a) Body weight gain in mice given AM251 or THCV, n=8 mice. (b) Cumulative food intake in mice given AM251 or THCV, n=2 groups of four mice per treatment. (c) Twenty-four-hour energy expenditure in mice given AM251 or THCV, n=2 groups of four mice per treatment. *P<0.05; **P<0.01 as compared to vehicle treated animals.

Figure 4

Effect of THCV on glucose tolerance and liver triglycerides in ob/ob mice (study 4). (a) Glucose tolerance after 3 weeks treatment with AM251 or THCV, n=8. (b) Area under the glucose tolerance curve, n=8. (c) Liver triglycerides content after 4 weeks treatment with AM251 or THCV, n=8. *P<0.05 as compared to vehicle treated animals.

Figure 5

Effect of THCV on insulin-induced phosphorylation of Akt in insulin-resistant human HHL-5 hepatocytes. (b, c) HHL-5 cells were rendered insulin-resistant following 72 h incubation with 100 nℳ insulin (Ins.) (b) or 48 h incubation with 0.25 mℳ palmitic acid (PA) (c). A representative western blot for insulin stimulation of Akt phosphorylation in insulin-sensitive cells is shown in (a), the right panel indicating the fold-stimulation by insulin of basal phosphoAkt (pAKT)/total Akt (AKT), quantified by densitometry in absence (DMSO) or presence of THCV 1–10 μℳ. (b, c) Representative western blots for insulin stimulation of Akt phosphorylation in insulin-resistant hepatocytes incubated for the final 24 h of chronic insulin or PA incubation with the indicated concentrations of THCV. The middle panels, obtained by densitometry quantification of n=3 separate western blots, indicate the lower stimulation by acute insulin of pAKT/AKT levels in desensitized cells as compared to insulin-sensitive cells (naïve), considered as 1. The right panels indicate the effect of THCV on insulin-induced stimulation of pAKT/AKT levels in insulin-resistant cells as compared with insulin-resistant cells only treated with acute insulin and THCV vehicle (DMSO), considered as 1. (d, e) Representative western blots for insulin stimulation of Akt phosphorylation in hepatocytes made insulin-resistant with a 24-h treatment with insulin or PA and co-incubated with the indicated concentrations of THCV, AM251 or vehicle. The middle panels, obtained by densitometry quantification of n=3 separate western blots, indicate the lower stimulation by acute insulin of pAKT/AKT levels in desensitized cells as compared with insulin-sensitive cells (naïve), considered as 1. The right panels indicate the effect of THCV or AM251 on insulin-induced stimulation of pAKT/AKT levels in insulin-resistant cells as compared with insulin-resistant cells only treated with acute insulin and vehicle (DMSO), considered as 1. ^#P<0.05 or ^##P<0.01 vs naïve. *P<0.05 or **P<0.01 vs DMSO, as assessed by ANOVA followed by the Bonferroni's test.

Figure 5

Effect of THCV on insulin-induced phosphorylation of Akt in insulin-resistant human HHL-5 hepatocytes. (b, c) HHL-5 cells were rendered insulin-resistant following 72 h incubation with 100 nℳ insulin (Ins.) (b) or 48 h incubation with 0.25 mℳ palmitic acid (PA) (c). A representative western blot for insulin stimulation of Akt phosphorylation in insulin-sensitive cells is shown in (a), the right panel indicating the fold-stimulation by insulin of basal phosphoAkt (pAKT)/total Akt (AKT), quantified by densitometry in absence (DMSO) or presence of THCV 1–10 μℳ. (b, c) Representative western blots for insulin stimulation of Akt phosphorylation in insulin-resistant hepatocytes incubated for the final 24 h of chronic insulin or PA incubation with the indicated concentrations of THCV. The middle panels, obtained by densitometry quantification of n=3 separate western blots, indicate the lower stimulation by acute insulin of pAKT/AKT levels in desensitized cells as compared to insulin-sensitive cells (naïve), considered as 1. The right panels indicate the effect of THCV on insulin-induced stimulation of pAKT/AKT levels in insulin-resistant cells as compared with insulin-resistant cells only treated with acute insulin and THCV vehicle (DMSO), considered as 1. (d, e) Representative western blots for insulin stimulation of Akt phosphorylation in hepatocytes made insulin-resistant with a 24-h treatment with insulin or PA and co-incubated with the indicated concentrations of THCV, AM251 or vehicle. The middle panels, obtained by densitometry quantification of n=3 separate western blots, indicate the lower stimulation by acute insulin of pAKT/AKT levels in desensitized cells as compared with insulin-sensitive cells (naïve), considered as 1. The right panels indicate the effect of THCV or AM251 on insulin-induced stimulation of pAKT/AKT levels in insulin-resistant cells as compared with insulin-resistant cells only treated with acute insulin and vehicle (DMSO), considered as 1. ^#P<0.05 or ^##P<0.01 vs naïve. *P<0.05 or **P<0.01 vs DMSO, as assessed by ANOVA followed by the Bonferroni's test.

Figure 6

Effect of THCV on insulin-induced phosphorylation of Akt in insulin-resistant rat C₂C₁₂ myotubes. Differentiated C₂C₁₂ cells were rendered insulin-resistant following 24 h incubation with 0.25 mℳ palmitic acid (PA) and co-treated with either THCV, AM251 or vehicle, at the concentrations shown. (a) Representative western blot for insulin stimulation of Akt phosphorylation in insulin-resistant cells. (b) Densitometric quantification of n=2 separate western blots, indicating the lower stimulation by acute insulin of pAKT/AKT levels in desensitized cells as compared to insulin-sensitive cells (naïve), considered as 1. (c) Effect of THCV or AM251 on insulin-induced stimulation of pAKT/AKT levels in insulin-resistant cells as compared to insulin-resistant cells only treated with acute insulin and vehicle (DMSO), considered as 1. ^#P<0.05 vs naïve. *P<0.05 vs DMSO, as assessed by ANOVA followed by the Bonferroni's test.