Insulin in the ventral tegmental area reduces hedonic feeding and suppresses dopamine concentration via increased reuptake

Abstract

Mesolimbic dopamine (DA) signaling has been implicated in the incentive, reinforcing and motivational aspects of food intake. Insulin receptors are expressed on dopaminergic neurons of the ventral tegmental area (VTA), and insulin may act in the VTA to suppress feeding. However, the neural mechanisms underlying insulin effects in the VTA are poorly understood. Here, we measured the effects of insulin on evoked DA concentration in the VTA using fast-scan cyclic voltammetry. Insulin concentration-dependently reduced evoked somatodendritic DA in the VTA, requiring activation of phosphoinositol 3-kinase and mTOR signaling. Insulin depression of somatodendritic DA was abolished in the presence of a selective DA transporter (DAT) inhibitor, GBR 12909, as well as in VTA slices of DAT knockout mice, suggesting that insulin upregulated the number or function of DAT to reduce DA concentration. Finally, insulin administered to the VTA depressed sated feeding of sweetened high-fat food. Taken together, these results indicate that insulin depresses DA concentration in the VTA via increased reuptake of DA through DAT. Insulin-mediated decrease of DA in the VTA may suppress salience of food once satiety is reached.

Figure 1. Insulin attenuates evoked somatodendritic DA

A. An example voltammogram from a single experiment for electrically evoked [DA]_o before (black line) and 40 min after insulin (500 nM, grey line) application. B. Bath application of insulin (500 nM, 10 min, filled circles) inhibited evoked [DA]_o in VTA slices (n=6) compared to control slices (n=6, open circles) (P < 0.05). C. A representative current-time plot from a single experiment showing DA evoked before (open circles) and 40 min after (filled circles) application of insulin (500 nM). The signals decay was fit with a one phase exponential curve to determine the rate of decay 5 min before insulin application (r²=0.99) and 40 min after insulin application (r²=0.98). D. A triangular waveform (−0.4 to 1.0 V vs Ag/AgCl at 400 V/s) was used to measure catecholamine concentrations with FSCV. An example experiment showing carbon fiber electrodes were 10 fold more sensitive to exogenously applied DA (1 μM) than norepinephrine (NE, 1, 10 μM). Symbols represent mean and bars represent SEM.

Figure 2. Insulin-mediated suppression of [DA]_o is concentration-dependent

A. Insulin produced a concentration-dependent decrease of the evoked somatodendritic [DA]_o (n=5 for 10nM, 100nM, and 1000nM; n=6 for 500nM). The maximal effect was measured 40 min after insulin application. B. Slices were pre-incubated with HNMPA[AM]₃ (300 μM), an inhibitor of insulin receptor tyrosine kinase, for 1 hour. HNMPA[AM]₃ blocked insulin-mediated suppression of somatodendritic [DA]_o (n=7). Symbols represent mean and bars represent SEM.

Figure 3. Insulin decreases evoked somatodendritic [DA]_o via PI3K and mTOR

A. The PI3K inhibitor, wortmannin (100 nM) applied for 40 min to the VTA slices did alter evoked [DA]_o (n=4). B. Slices were pre-incubated with wortmannin (100 nM) 30 min prior to and during recording. Insulin (500 nM, 10 min) did not inhibit evoked [DA]_o in the presence of wortmannin (n=6). C. The mTOR blocker, rapamycin (50 nM) applied for 60 min to VTA slices did not produce significant changes in evoked [DA]_o (n=5). D. Slices were incubated with rapamycin (50 nM) 20 min prior to and during recording. Insulin (500 nM, 10 min) did not inhibit evoked [DA]_o in the presence of rapamycin (n=7). Symbols represent mean and bars represent SEM.

Figure 4. Insulin suppression of [DA]_o is Ca²⁺-independent and suppression is greater at higher stimulation frequencies

A. Current-time plot indicating that somatodendritic [DA]_o was evoked in 0.5 mM external Ca²⁺ (open circles, n=4) or 2.4 mM external Ca²⁺ (filled circles, n=4). B. In the presence of 0.5 mM external Ca²⁺, insulin (500 nM, 10 min) suppressed [DA]_o (n=6). C. Somatodendritic [DA]_o increased with increasing pulse frequency (filled triangles, n=4). The maximal effect of insulin (500 nM, 10 min) measured 40 min after application was greater at higher frequencies (open triangles; n=4). D. The difference between maximal-evoked [DA]_o before and 40 min after insulin (500 nM, 10 min) observed at each frequency calculated based on data from C (*P < 0.05, one way ANOVA). Bars represent means and SEM from 4 slices.

Figure 5. Insulin-suppression of somatodendritic [DA]_o is absent in the present of a DA transporter (DAT) inhibitor

A. GBR 12909 (500 nM), a selective DAT blocker, did not alter the peak concentration of evoked DA (n=6). B. Representative current time plots of evoked [DA]_o from a single slice show GBR 12909 (500 nM, filled circles) 50 min after application prolonged DA reuptake compared to [DA]_o evoked prior to GBR 12909 application (open circles). C. VTA slices were preincubated with GBR 12909 (500 nM) 1 hr prior to and during recording. In the presence of GBR 12909, insulin-mediated suppression of [DA]_o was abolished (n=5). D. An example current time plot demonstrating the reuptake of DA was not significantly different before (open circles) or after insulin (500 nM, filled circles) application in the presence of GBR 12909 (500 nM). Symbols represent mean, bars represent SEM.

Figure 6. Insulin increases the reuptake of DA via DAT

A. Insulin (500 nM, 10 min, n=5) did not significantly alter evoked [DA]_o compared to slices treated with vehicle (control; open circles, n=4) in VTA slices of DAT −/− mice (P > 0.05). B. A representative current-time plot of evoked [DA]_o before (open circles) and 40 min after insulin (filled circles) application in VTA slices of DAT −/− mice. C. Insulin significantly inhibited evoked [DA]_o (filled circles, n=5) compared to controls (open circles, n=4) in VTA slices of NET −/− KO mice (P < 0.05). D. A representative current-time plot before (open circles) and 40 min after insulin application (filled circles) to VTA slices of NET −/− mice. E. Insulin significantly decreased evoked [DA]_o (filled circles, n=6) compared to controls (open circles, n=3) in the wild-type mice (P < 0.05). F. A representative current-time plot showing DA release and reuptake before (open circles) and 40 min after insulin (500 nM) application to VTA slices of wild-type mice. Symbols represent mean, bars represent SEM.

Figure 7. Insulin-mediated reduction of [DA]_o is independent of protein synthesis

A. Protein synthesis inhibitor, cycloheximide (100 nM), applied for one hour to the VTA slices did not produce any significant changes in the evoked [DA]_o (n=8). B. VTA slices were incubated with cycloheximide (100 nM) 20 min prior to and during recording. Insulin (500 nM, 10 min) suppressed evoked [DA]_o in the presence of cycloheximide (n=8).

Figure 8. Intra-VTA microinfusion of insulin reduces hedonic feeding

Mice were entrained to consume their daily caloric needs within 4 hours of food access. A. Reconstructed injection sites in the VTA are shown in coronal sections. Distance from bregma is shown to the right of each section (in millimeters). B. Animals microinjected with vehicle (filled bars, n = 6) or insulin (open bars, n = 6) in VTA 10 min prior to food access did not consume significantly different amounts of regular chow during either the first hour (left) or total period of food access (right) compared to naive mice (shaded bars, n=6, P < 0.05). C. Mice consumed at least 50 % of their daily food within the first hour of food access and ate progressively less towards the end of the 4 hour food-access (left). Subsequent intra-VTA administration of insulin (open bars, n = 9) significantly decreased the 1 hour sated consumption of sweetened high-fat food compared to vehicle-treated mice (right; filled bars, n = 8, P < 0.001). Bars represent mean ± s.e.m. A one-way ANOVA followed by a Bonfferoni’s multiple comparisons test was used.