We have begun to explore the specific CNS location(s) whereby insulin and leptin may decrease food reward, based on our studies of ICV administration. A minimum set of criteria for identifying target sites would have to include the expression of receptors for insulin or leptin, and functional activity of these receptors demonstrated at both the cellular and behavioral level. We have obtained evidence which suggests that the VTA may serve as a direct target for insulin and leptin action. Using fluorescence immunocytochemistry we identified neurons positive for insulin receptors or leptin receptors in the VTA, and some in the adjacent substantia nigra (34
). In the medial hypothalamus, a well-characterized target for insulin and leptin effects in the CNS (35
), insulin and leptin activate the intracellular PI3 kinase cascade (as initially identified in classic peripheral target tissues), which results in the generation of the signaling molecule PIP3 (36
). As shown in , acute administration of insulin or leptin (approximately fifteen min prior to brain excision) directly into the VTA likewise results in enhanced PIP3 immunoexpression relative to artificial cerebrospinal fluid (CSF)-injected controls. DiLeone and colleagues have subsequently demonstrated mRNA expression of the leptin receptor, and have demonstrated phosphorylation of STAT3, another downstream marker of leptin action, within the VTA following IP leptin administration (40
Figure 1 PIP3-positive neurons were identified by fluorescence immunocytochemistry following acute injection of CSF (upper),5 mU insulin (middle), or 2.5 μg leptin (lower) directly into the VTA (2 μl/10 min). PIP3 immunofluorescence was assayed (more ...)
In addition to the demonstration of activation of a cell-signalling pathway, we have identified a critical regulatory protein in dopaminergic neurons which is a target for insulin action, the dopamine re-uptake transporter (DAT). Synaptically released dopamine is retro-transported into dopaminergic nerve terminals where it is re-packaged or degraded and this function of the DAT represents the major mechanism whereby dopamine signaling is terminated within the CNS (41
). We have demonstrated that ICV insulin increases DAT mRNA (42
), and that in vitro
insulin reverses the effect of acute, in vivo
, food restriction to decrease DA re-uptake (i.e., insulin stimulates DAT function) (10
). Subsequent studies in cellular systems have demonstrated that this action of insulin is caused by enhanced recruitment of DATs to the cell membrane and is dependent on insulin-stimulation of the PI3 kinase pathway (43
). This collective set of evidence strongly suggests that the DAT is likely a physiological target of insulin action. To date, comparable studies have not been carried out for leptin. The functional significance of increased DAT numbers or activity is that released dopamine should be cleared from the synapse more effectively, hence dopamine signaling will be decreased.
Direct acute injection of insulin or leptin unilaterally into the VTA blocks VTA-initiated feeding (). Feeding can be stimulated by the administration of a mu-opioid agonist into the VTA such as DAMGO, and this feeding is dopamine-dependent (44
). We used a two-day paradigm where rats had ad libitum
access to sucrose pellets and were tested in the sated condition. On the first day all rats received a CSF injection and baseline sucrose pellet intake was recorded. On the second day, rats received either a second CSF injection (vehicle control); an injection of DAMGO; an injection of insulin or leptin alone; or a combined injection of DAMGO with either leptin or insulin. The data shown in represent the within-subjects' response of each rat relative to its baseline intake, which was approximately fifty sucrose pellets. Injection of DAMGO stimulated pellet intake and this was reversed when insulin or leptin were co-administered. Insulin and leptin alone did not change baseline sucrose intake, and a speculative interpretation of this is that insulin and leptin effects on food reward may only be relevant when there is adequate stimulation or drive within the VTA.
Unilateral DAMGO (3 nmol) administration stimulates 60-min sucrose intake in sated rats, which is reversed by co-administration of 5 mU insulin or 0.2 μg leptin.
Other candidate CNS sites are beginning to be evaluated as mediators of the ICV actions of insulin and leptin to blunt food reward. Because insulin or leptin signals at the medial hypothalamus are relayed to the lateral hypothalamus (45
), which may serve as an integrating site for signals from reward/motivational circuitry (46
), we are beginning to test the ability of insulin or leptin administered directly into the arcuate nucleus to decrease performance in food reward paradigms such as those described above. Additionally, we are beginning to explore the possibility that the amygdala (either central or basolateral nucleus) may be an important anatomical mediator of some aspects of food reward. This somewhat speculative hypothesis is based upon the observations that the amygdala is implicated in the expression (but not the learning) of place preference conditioning (47
); its important role in cue-conditioned feeding (48
) and its important role in the circuitry of nucleus accumbens-stimulated feeding of palatable food (49
). This is a limited body of evidence gathered from laboratories that are asking different types of questions; however it seems clear that the amygdala is a good candidate for the systematic evaluation of its role in food reward/motivation paradigms. We are beginning such studies and have preliminary evidence of the expression of insulin receptors in both central and basolateral regions of the amygdala.