Using optogenetic stimulation, we show that GLP-1 excites medial habenular (MHb) projections to the interpeduncular nucleus (IPN). Here we show that nicotine activates glucagon-like peptide-1 (GLP-1) neurons in the nucleus tractus solitarius (NTS). A growing body of preclinical evidence indicates that glucagon-like peptide-1 (GLP-1) receptor agonists reduce drug reinforcement. In mice lacking the MCH gene the prominent phenotype is hypophagia (reduced desire for food intake) and lean body mass. The antidiabetic drugs sitagliptin and ColonBroom fiber blend exenatide, which inhibit GLP-1 breakdown and stimulate GLP-1 receptors, respectively, decreased nicotine intake in mice. Conversely, Glp1r knockout mice consumed greater quantities of nicotine than wild-type mice. Tobacco smokers titrate their nicotine intake to avoid its noxious effects, sensitivity to which may influence vulnerability to tobacco dependence, yet mechanisms of nicotine avoidance are poorly understood. In addition to NPY this family is composed of two gut hormones, pancreatic polypeptide (PP) and peptide tyrosine-tyrosine (PYY) both of which are discussed below. Peptide identification was based on a search with an initial mass deviation of the precursor ion of up to 6 ppm, and the allowed fragment mass deviation was set to 20 ppm for HCD and 0.5 Da for CID fragmentation. 60,000 at m/z 400. The six most intense ions were sequentially isolated with a window of 2 Th for MS/MS sequencing, the multistage activation algorithm was enabled using the neutral loss values of 97.97, 48.99, 32.66 and 24.49 m/z units to improve the fragmentation of phosphopeptides.

Top twelve ions were selected at an isolation window of 2.0 m/z units and accumulated to a target value of 2e5 for MS/MS sequencing. The "match between runs" option was enabled with a time window of 1 min to transfer identifications in the same fraction, only for samples analyzed using the same chromatography and nanospray conditions. Phosphorylation sites satisfied with one of the following two criterions were determined as medium-confidence regulated sites: (i), if phosphorylation sites were quantified in only one of the forward and reverse labeling replicates (sites quantified with different Nmods phosphopeptides in forward and reverse replicates also belonged to this situation), the ratio condition B/A should reach the 1.75-fold change threshold in the adopted quantified experiment; (ii), if phosphorylation sites were quantified in both forward and reverse labeling replicates with the same Nmods phosphopeptides, the ratio condition B/A should reach the 1.75-fold change threshold in one replicate and 1.5-fold in the other. For each experiment (Exp1, Exp2 and Exp3 in Fig. 1A), phosphorylation sites were quantified using the ratio from forward and ColonBroom fiber blend reverse labeling replicates which used the least Nmods phosphopeptides to quantify the corresponding sites. High-confidence regulated sites were defined if the ratio condition B/A reached the 1.75-fold change threshold (determined by boxplot algorithm) in both forward and reverse labeling replicates.

After quenching by adding ammonia solution and formic acid, the "light" and "heavy" dimethyl-labeled samples were mixed in 1:1 ratio based on total peptide amount determined by measuring fluorescence as described above. The peptide sequences were recovered directly from sorted cells by PCR and cloned into lentiviral vectors to construct libraries for next round selection. RNA levels in INS-1 cells and isolated rodent and human islets treated with GLP-1 in vitro and in vivo (with osmotic pumps) were measured by real-time quantitative PCR.