Hierarchical glucocorticoid-endocannabinoid interplay regulates the activation of the nucleus accumbens by insulin

Abstract

Here we asked if insulin activation of the nucleus accumbens in vitro is reflected by an increase in (3)H-deoxyglucose ([(3)H]DG) uptake, thus subserving a new model to study molecular mechanisms of central insulin actions. Additionally, we investigated the dependence of this insulin effect on endocannabinoids and corticosteroids, two major culprits in insulin resistance. We found that in acute accumbal slices, insulin (3 and 300nM but not at 0.3nM) produced an increase in [(3)H]DG uptake. The synthetic cannabinoid agonist, WIN55212-2 (500nM) and the glucocorticoid dexamethasone (10μM), impaired insulin (300nM) action on [(3)H]DG uptake. The glucocorticoid receptor (GcR) antagonist, mifepristone (10μM) prevented dexamethasone from inhibiting insulin's action. Strikingly, this anti-insulin action of dexamethasone was also blocked by two CB1 cannabinoid receptor (CB1R) antagonists, O-2050 (500nM) and SR141716A (500nM), as well as by tetrahydrolipstatin (10μM), an inhibitor of diacylglycerol lipases-the enzymes responsible for the synthesis of the endocannabinoid, 2-arachidonoyl-glycerol (2-AG). On the other hand, the blockade of the post-synaptic 2-AG metabolizing enzymes, α,β-serine hydrolase domain 6/12 by WWL70 (1μM) also prevented the action of insulin, probably via increasing endogenous 2-AG tone. Additionally, an anti-insulin receptor (InsR) antibody immunoprecipitated CB1Rs from accumbal homogenates, indicating a physical complexing of CB1Rs with InsRs that supports their functional interaction. Altogether, insulin stimulates glucose uptake in the nucleus accumbens. Accumbal GcR activation triggers the synthesis of 2-AG that in turn binds to the known CB1R-InsR heteromer, thus impeding insulin signaling.

Keywords: 2-Arachidonoylglycerol; CB(1)R cannabinoid receptor; Glucocorticoid receptor; Glucose uptake; Insulin; Nucleus accumbens.

Fig. 1

Insulin receptor activation stimulates glucose uptake in acute accumbal slices from the rat. (A) Flow-chart depicting the experimental steps in the principal assay and their timing. Abbreviations: dexa, dexamethasone; DMAQB1, demethylasterriquinone B1 THL, tetrahydrolipstatin; WIN, WIN55212-2. DMSO (0.1%) was given as vehicle control in place of the lipophilic compounds in the same time. (B) Insulin, rather than IGF-1 receptors mediate the action of insulin on glucose uptake. Bars represent the mean ± S.E.M. of n ≥ 7 independent observations (rats). The control values from each experiment are averaged and taken as 100%, while effect amplitudes are normalized to the appropriate controls. Due to the relative comparison, the ordinate is preferably labeled as [³H]DG ([³H]deoxyglucose) uptake which is more exact than glucose uptake, although the two are used interchangeably in this study. *P < 0.05, ***P < 0.001, n.s., not significant.

Fig. 2

Release diagrams and relative values of the release of [³H]GABA and [¹⁴C]glutamate in rat accumbal slices. Insulin was administered in the superfusion medium according to the horizontal bars. FR%, fractional release %, i.e. the release value relative to the total radioactivity content in the slice at the given time-point. All data are mean ± S.E.M. of n = 4, in duplicate.

Fig. 3

Glucocorticoid receptor activation prevents insulin from stimulating glucose uptake. Bars represent the mean ± S.E.M. of n ≥ 6 independent observations (rats). Abbreviations; dexa, dexamethasone; spirono, spironolactone (10 μM). *P < 0.05, ***P < 0.001, n.s., not significant.

Fig. 4

CB₁ cannabinoid receptors mediate the anti-insulin action of dexamethasone. While the synthetic cannabinoid agonist, WIN55212-2 (500 nM) and the CB₁R antagonists, O-2050 (500 nM) and SR141716A (500 nM) had no effect on glucose uptake per se (Table 1), WIN55212-2 prevented insulin from stimulating glucose uptake. Both CB₁R antagonists opposed the anti-insulin action of dexamethasone (10 μM). The diacylglycerol lipase inhibitor, tetrahydrolipstatin (THL, 10 μM) mimicked the effect of the CB₁R antagonists. Finally, the α,βHD6/12 inhibitor, WWL70 (1 μM) but not that the monoacylglycerol lipase inhibitor, JZL184 (1 μM) mimicked the anti-insulin action of dexamethasone, thus supporting the involvement of 2-AG in the glucocorticoid-induced insulin resistance. Bars represent the mean ± S.E.M. of n ≥ 6 independent observations (rats). *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.

Fig. 5

The CB₁R was readily detected at around 53 kDa molecular weight, and enriched in complexes immunoprecipitated with the anti-insulin receptor β-chain antibody (anti-InsRβ; Sigma-Aldrich), but not with rabbit polyclonal IgG (44 kDa). InsRβ was detected at 95 kDa (with a different anti-InsRβ antibody from Santa-Cruz Biotechnology).