FROM HISTORICAL PERSPECTIVE TO MODERN PERSPECTIVE

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In 1979, Porte, Woods, and colleagues (130) made the observation that putting a small amount of the metabolic hormone insulin directly into the cerebrospinal fluid (CSF) of nonhuman primates resulted in a significant decline in the animals' food intake and body weight. This observation was made in the context of ongoing studies in the field that suggested that a circulating humoral factor or factors could regulate both size of individual meals, as well as food intake and body weight, over a longer time course (31, 83, 90, 117). In a subsequent review, Porte and Woods (94) proposed that insulin served as an "adiposity signal" whose levels in the brain reflected the size of adipose stores integrated over time and which served to complete a negative feedback loop that links the behavior of feeding with size of adipose stores, such that adipose mass remains fairly constant in adults over a relatively long time. Many studies over the intervening decades have essentially validated this basic concept (e.g., 3, 4, 15, 25, 84). In the mid-90s, a second candidate adiposity signal, Ob-protein or leptin, was identified (133), and the function of these two signals appears to be similar. A major research focus has been on the actions of these two hormones at the medial hypothalamus, which historically has been identified as playing a major role in the CNS regulation of metabolism, energy balance, and caloric intake in terms of physiological need. Because these studies are reviewed frequently, this work will not be described here but the reader is directed to several recent reviews (1, 10-12).

A great deal has been learned about the effects of insulin on CNS regulation of body weight, but several questions remain unanswered, for example, how environmental factors such as diet composition can interact with this feedback loop to impair the effectiveness of adiposity signals such as insulin. Data collected by the Centers for Disease Control document a pervasive increase of moderate obesity in adults across the United States in the 1990s (86, 87). This finding has been interpreted as indicative of a significant environmental influence over the neural circuitry associated with the physiological maintenance of energy homeostasis. The epidemiologic finding also emphasizes that attention must now be focused, in terms of both basic research and future drug development, on additional CNS circuitry, which is either directly or indirectly connected with the more extensively characterized hypothalamic circuitry to modulate feeding behavior. One obvious target for study is the CNS circuitry, which mediates motivation and reward. Components of this circuitry are activated with, and contribute to, complex behaviors, such as food seeking and food intake (13, 14, 116, 129; and see below). In recent years, new evidence is forthcoming in support of a role for insulin and leptin in the modulation of reward and motivated behaviors (see below). The hypothesis that we have been evaluating in our recent studies is that adiposity signals can modulate food reward. In this review, I will first summarize briefly the anatomical, biochemical, and behavioral evidence garnered in support of the role of insulin and leptin as CNS adiposity signals (i.e., an evaluation of the original hypothetical construct), then I will summarize the newer evidence for the involvement of insulin and leptin in motivational components of behavior, particularly in terms of food as the rewarding stimulus.

	INSULIN AND LEPTIN: ADIPOSITY SIGNALING IN THE CNS

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As reviewed previously (41), two critical issues needed to be addressed to argue for a role for peripherally derived adiposity signals in modulating any aspect of CNS function. First, the signals must be present in the CNS; second, their receptors must be localized in the relevant areas of the CNS. With regard to the first issue, it is now generally appreciated that neither insulin nor leptin are synthesized to any significant degree in the adult brain. Although early studies using an immunoassay approach suggested that insulin might be synthesized in the developed (adult) brain (58), subsequent studies (30, 52) suggest that, in fact, most if not all insulin that is found in the mature brain is transported there from the circulation after its release from the beta cells of the endocrine pancreas. A limitation to studies that have reported insulin synthesis within primary or transformed mammalian neuronal cell lines (36, 112) based on the use of fetal or embryonic neurons, is that proteins are expressed in developing neurons with specific and differential time courses. It is possible that insulin synthesis is "turned on" during a specific phase of brain development, or as a result of the culture conditions used to maintain neurons viable, and thus insulin may have a role in CNS development. This concept is supported by the recent report of a critical developmental role for insulin-like peptides produced by neurosecretory cells in larval Drososphila (99). As summarized in Refs. 11 and 104, however, there is good correspondence between plasma and cerebrospinal fluid (CSF) insulin levels when measured in a variety of animal models under different metabolic conditions. Schwartz and colleagues (106) developed a model for the transport of insulin from plasma to CSF across the blood-brain barrier. Kinetic analysis following prolonged intravenous insulin infusions fit data to a model whereby insulin distributes itself into three compartments: blood, CSF, and a compartment intermediate between blood and CSF, which probably represents brain interstitial fluid. Such a model is consistent with both the proposed transit mechanism of insulin from the blood to peripheral target tissues such as muscle and a role for the unique insulin receptor found in capillary endothelial cells, which serves to carry insulin across the endothelial cell in a transcytotic process (34, 35, 77). The relationship between CSF and plasma insulin concentrations is not linear but is saturable, also consistent with a receptor-mediated transport process. This nonlinear relationship has been observed in moderate dietary-obese rats (69). High-fat diet-induced moderate obesity is associated with reduced brain insulin transport in dogs (71) and genetically obese Zucker fa/fa rats have impaired brain insulin transport (119), which can be ascribed to a decrease in the number of brain capillary insulin receptors (102). Studies comparable to those described for insulin have been carried out to evaluate the CNS transport of leptin, an adipose secretory product (72). Similar to insulin, leptin can be transported via a saturable process across capillary endothelial cells (82) and a specific non-signaling isoform of the leptin receptor appears to be responsible for this (64). There is regional variation of uptake within the CNS (6). The transport rate of leptin is reduced in some models of obesity (7, 8); however, leptin transport into the CNS persists in both ob/ob and db/db mice (81) perhaps via a second, not yet defined transporter mechanism (9). Similar to what we observed for insulin, relative levels of leptin in the CSF are decreased in association with obesity (18, 103). The functional implication of this is that, in circumstances of chronic hyperinsulinemia and hyperleptinemia, such as obesity, relatively less adiposity signaling would be available to the CNS.

The second basic issue relates to the presence of insulin and leptin receptors in the CNS. Receptors for both insulin and leptin are expressed ubiquitously throughout the CNS. Insulin receptors in the CNS were first identified by the lab of Jesse Roth in 1978 (57). This was confirmed by a number of subsequent studies (32, 122, 124, 131), along with the observation that they are present in relatively higher concentrations in specific brain regions (e.g., hypothalamus, hippocampus, olfactory bulb). Both neurons and glia express insulin receptors as the identical gene product (76), but with modification of the posttranslationally added carbohydrate moiety on the neuronal subtype (62). Numerous studies have been carried out to determine whether plasma membrane concentrations of insulin receptors are regulated by mechanisms similar to their regulation in peripheral target tissues: the overall finding seems to be that they are not, although membrane concentrations have been shown to be decreased in rat models of genetic obesity (43, 46). The relevance of the brain insulin receptor to body weight regulation has been recently confirmed by the observation of obesity in a neuron-specific knockout of the insulin receptor in mice (16). The leptin receptor is likewise ubiquitously expressed and is present as different splice-variant isoforms in the CNS, most notably "the short form" alluded to above, which is thought to play a role in leptin transport, and the "signaling" form OBRb, which is thought to play a major role in leptin signaling (37). The obese db/db mouse and Zucker fa/fa rat represent naturally occurring "knockouts" (27) of the leptin receptor.

Intracellular signaling by the brain insulin receptor has been presumed to be similar if not identical to that of the receptor expressed in peripheral target tissues such as muscle or adipose. Thus the receptor functions as an auto-tyrosine kinase (75, 110). Although some putative substrate proteins have been reported as downstream targets for insulin-dependent phosphorylation (59-61, 63), and we reported in vitro effects of insulin to stimulate myo-inositol incorporation into inositol phosphates and membrane inositol lipids in brain slices from lean Zucker fa/fa rats [an effect that was absent in obese fa/fa Zucker rats (42)], the role of these intracellular events in the energy balance-regulatory action(s) of insulin has not been substantiated. Activation of the intracellular PI3 kinase pathway has been shown to play an important role in postreceptor signaling in peripheral insulin target tissues (55). PI3 kinase signaling is implicated in the food intake suppressive actions of leptin (91). Carvalheira and colleagues, in a preliminary report (23), have demonstrated that intraventricular insulin stimulates hypothalamic PI3 kinase activity. Both leptin- and insulin-induced decreases of food intake can be blocked by administration of a PI3 kinase inhibitor (Carvalheira et al., unpublished observations; Ref. 91), and insulin can act at the hypothalamus to enhance leptin-induced phosphorylation of the transcription factor STAT3 (24). This finding fits with the recent report of additive behavioral effects of insulin and leptin to decrease body weight (2), suggesting that insulin and leptin may act on common target neurons in the hypothalamus. These studies represent an important advance in our understanding of the cellular effects of insulin and leptin at the hypothalamus.

As peptides transported into the CNS, leptin and insulin must function in a neuromodulatory role, as opposed to functioning as synaptically released neurotransmitters. Thus studies have focused on other identified neural systems or pathways that might mediate the effects of insulin and leptin on food intake and energy balance. We hypothesized that one mechanism could be through the enhancement of a peptide signal that plays a role in meal termination, CCK. A dose of exogenous CCK that is subthreshold for decreasing meal size becomes effective when insulin is coadministered into the CSF, also at a concentration that is subthreshold for decreasing body weight (47, 49). Similar observations have now been made for leptin-CCK interactions (e.g., Ref. 85), and leptin receptors (54) and leptin enhancement of neural activation (39) have been observed in the brain stem. After the initial report in 1984 that the recently discovered brain peptide neuropeptide Y (NPY) robustly stimulated food intake when administered into the CNS (28, 29), there have been literally hundreds of studies evaluating the role of this and other peptides to act in the hypothalamus to modulate food intake, body weight, and energy balance. Insulin and leptin decrease the expression of hypothalamic NPY (11, 107, 113, 125), and these observations have led to an extensive investigation of the medial hypothalamus as a target region for insulin and leptin effects on body weight regulation. Over the past decade, additional peptides within the hypothalamus [e.g., melanocortins, MCH, CRH, orexins, agouti-related peptide (56)] have been implicated in hypothalamic-mediated energy balance, and the interaction of adiposity signals with these neuropeptides has served as a focus for many investigations; see Refs. 68, 88, 89, 95, and 105 for present status of this work.

	INSULIN, LEPTIN, REWARD, AND PALATABILITY

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The model of an adipose-brain feedback loop has thus received significant substantiation since originally proposed. In metabolic circumstances in which plasma insulin or leptin levels are low (starvation and reduced adiposity), negative feedback signaling would be decreased and drive for food intake would be increased. Obesity (excessive adiposity) would represent a pathophysiological state in which either adipose signals are present in relatively decreased amounts in the CNS (discussed above) or there is direct CNS resistance to their action [e.g., obese Zucker rats (67)]. Finally, and perhaps not surprisingly, the hypothalamus has proven to be a critical site for this physiological regulation. However, the need for additional inputs into the conceptual model of a hypothalamus-adiposity signal feedback loop is clear. As mentioned above, afferent and efferent connections with brain stem nuclei implicated in taste and feeding and the role of adiposity signals in this connectivity are ongoing areas of research (66). However, this circuitry in and of itself does not account for experimental or public health data that implicate a role for environmental factors such as availability and composition of food in the energy balance equation. In 1988, Arase et al. (5) made the observation that putting rats on a high-fat diet resulted in an impairment in the action of insulin, given directly into the CNS, to decrease body weight. This finding was subsequently replicated and extended in a study by Chavez and colleagues (26), in which rats were fed diets with four different concentrations of fat. At a concentration of 39% fat, similar to the concentration of fat in a typical American diet, intraventricular insulin was no longer effective at decreasing food intake and body weight. A similar observation has been made for peripheral leptin, although studies with intraventricular leptin have yielded conflicting results (see Ref. 80). These observations hold great significance for Westernized populations consuming relatively high-fat meals, because they imply that endogenous adiposity signals, once in the CNS, may also become ineffective at providing feedback signaling. The mechanism(s) underlying this phenomenon still remain to be elucidated. CNS circuitry that participates in food intake activity can be functionally grouped: the brain stem circuitry, implicated in oral sensory and motor events in relation to the act of eating; the hypothalamus, as described above, important in the physiological regulation of energy balance; and limbic circuitry including portions of the cerebral cortex, hippocampus, amygdala, and the striatonigral pathways, which is implicated in transposing motivational aspects of stimuli into motor responses, as well as hedonic evaluation of the stimulus and associative learning (40, 97). In terms of neural connectivity, the hypothalamus is essentially embedded in this motivational circuitry of the CNS, and numerous mono- and multisynaptic pathways between different components of the limbic circuitry and the hypothalamus have been identified (e.g., Ref. 98). The anatomical and functional relevance of motivational circuitry to food intake is discussed in several recent reviews (13, 73, 74). Understanding the role of CNS circuitry that is involved in hedonic and reward valuation of food and food choices would seem to be a priority in terms of human nutrition research, given the ready economic availability of high caloric density, palatable foods, a circumstance that is not modeled by studies in laboratory rodents maintained on a constant diet of chow. Insulin and leptin receptors are expressed throughout the limbic forebrain, including the striatum, the hippocampus, the amygdala, and the lateral hypothalamic/zona incerta area (Fig. 1). This provides one rationale for exploring the possibility that the limbic forebrain may itself be a direct target for insulin or leptin. Whereas these types of studies of the lateral hypothalamus (LH) and striatum are currently being done and are summarized below, studies of the hippocampus and amygdala remain to be initiated and offer an opportunity for new experimentation. Evaluation of the potential relevance or importance of insulin and leptin interaction with reward circuitry as part of their action to decrease food intake probably cannot be done by using an experimental model of ad libitum chow feeding, with a measure of chow intake as the endpoint. However, behavioral paradigms evaluating reward and food reward provide evidence for significant effects of metabolic status on performance in these paradigms and support the idea that both insulin and leptin may play a role in modulating reward function in the CNS (50). For example, it is long established that food restriction or fasting paradigms (in which endogenous insulin and leptin are decreased) enhance the addictive or reinforcing properties of drugs of abuse, as found in both drug self-administration and relapse to drug-taking paradigms; intraventricular leptin can reverse food deprivation-induced relapse to heroin self-administration (22, 108, 109).

View larger version (48K): [in this window] [in a new window]   Fig. 1.   Immunofluorescent labeling of leptin receptors (A) and insulin receptors (B) in the lateral hypothalamic area. Unpublished results, Figlewicz Lattemann, Evans, Murphy, Hoen, and Baskin. See Ref. 44 for methods.

One critical synaptic relay area between medial hypothalamus and other limbic circuitry is the LH. A current research emphasis on identifying afferent and efferent projections of the LH and their role in feeding behavior per se (e.g., Ref. 120) should provide new insights into the way that forebrain and limbic inputs can modify the function of the medial hypothalamic circuitry. Data from the experimental approach of brain self-stimulation, in which electrodes are placed in the lateral hypothalamus close to the fornix (perifornical), have shown that chronic food restriction shifts the dose response curve for self-stimulation in some perifornical hypothalamus sites to the left, such that weaker electrical current that normally would not support sustained self-stimulation activity at these sites becomes efficacious when animals are maintained on a food-restriction paradigm (19, 20). As summarized in a recent review from Shizgal et al. (111), this effect appears to be linked to long-term energy stores, rather than acute caloric need. Given that insulin and leptin provide information to the CNS regarding energy stores in the form of adipose, studies evaluating their effect(s) on this phenomenon would seem to be a logical follow-up, and in fact such studies have been carried out. Carr and colleagues (21) demonstrated that intraventricular administration of insulin into both food-restricted and ad libitum-feeding rats is effective in increasing the threshold current needed to sustain lateral hypothalamic self-stimulation, both reversing the decreased threshold observed with fasting and elevating the threshold above its "free-feeding" level. A similar observation by Fulton and colleagues (51, 111) was made for leptin: intraventricular leptin administration shifts the dose-response curve for self-stimulation in food restriction-sensitive perifornical hypothalamic sites to the right (i.e., reverses the effect of food restriction). Whether the effects of centrally administered insulin and leptin are direct within the LH has not been determined. However, we have identified leptin receptors and insulin receptors in the LH (Fig. 1), thus the LH may serve as one anatomical substrate for these effects.

As the medial hypothalamus has been identified as a central anatomical substrate for energy balance, the striatum represents a major anatomical component of CNS reward circuitry, and of critical importance is the extensive projection of dopamine (DA) neurons from the midbrain ventral tegmental area (VTA) and associated DA cell groups (substantia nigra) to the striatum and its functionally specialized subregion, the nucleus accumbens (40). Activation of these midbrain DA neurons has been implicated in the motivating, rewarding, reinforcing, and incentive salience properties of natural stimuli such as food and water as well as pharmacotherapeutic and addictive drugs (13, 14, 65, 78, 96, 116, 128, 129). The functions mediated by mesocorticolimbic DA remain a topic of very active debate as patterns of DA release change in association with familiarity or repeated exposure (e.g., training) to a stimulus: activity of the midbrain DA neurons may change depending on the time course (e.g., tonic vs. phasic) of DA release and the experience of the animal. The reader is referred to recent reviews (101, 118, 127) that address the complexity of this function (associative learning, hedonic valuation, incentive salience) as discussion of these issues is outside the scope of this review. Appreciation of these different aspects of DA function would seem to be an important consideration in the design of studies of food choice, eating habits, and even therapeutic weight loss regimens that include diets.

Measurement of DA levels in nucleus accumbens interstitial fluid has shown that there is a greater DA release in the nucleus accumbens in response to a food reward in rats that are food deprived vs. ad libitum-fed rats (126). Mechanism(s) responsible for this altered DA release have not been determined. Studies from our laboratory have focused on putative insulin (and more recently, leptin) effects on the midbrain DA neurons at both the cellular and the behavioral level. Insulin receptors have been identified in the VTA and the striatum in previous anatomical studies using receptor autoradiography and receptor immunocytochemistry approaches (122, 124), and insulin and leptin receptor mRNA is expressed in the substantia nigra (38). Recently, we used double-label fluorescence immunocytochemistry and localized both insulin receptors and leptin receptors on midbrain DA neurons, including those of the VTA, as well as medial and lateral substantia nigra (44). Thus this critical motivational circuitry has the potential to serve as a direct target for adiposity signals. In addition to identification of insulin receptors on these neurons, we identified one potential cellular target for insulin action: the dopamine reuptake transporter (DAT). This synaptic protein functions to inactivate DA signaling by transporting DA back into the DA nerve terminal from the synapse (70). Both the synthesis and the activity or synaptic concentrations of the DAT can be regulated by intracellular signaling systems such as protein kinase C (123). We observed both in vivo and in vitro effects of insulin on expression and activity of the DAT (48, 93). Insulin can increase mRNA levels of, and synaptic activity of, the DAT. The functional implication of this would be that increased DAT activity could result in increased clearance of DA from the synapse and, hence, decreased DA signaling. Given the role of striatal DA in motivation and reward, we hypothesized that one of insulin's actions in the CNS is to decrease the rewarding aspect of food. Comparable cellular studies to identify action(s) of leptin on DA neurons, either at the cell body or terminal projection field, have not been done. Presumably, the specific anatomic localization of leptin receptors on DA neurons now provides the rationale for such studies.

Pursuing the behavioral significance of insulin/DA interactions for ingestive behavior, we tested the ability of insulin to synergize with a DA receptor antagonist to decrease performance of rats in a 5-min sucrose lick-rate task. This task was established by Davis and Smith (33) to evaluate the rewarding properties of food solutions and to investigate the neuropharmacologic basis for intake of these hedonic solutions. The short time frame of this task (seconds to minutes) prevents or minimizes the influence of endogenous gastrointestinal or endocrine factors, and thus the licking activity reflects a "pure" hedonic response to the offered solution. Schneider et al. (100) demonstrated that administration of DA receptor antagonists could decrease the lick rate for a series of sucrose solutions in a manner consistent with dilution of the solutions, hence, a decrease of the rewarding value of these solutions. We reasoned that if a DA antagonist were acting postsynaptically and insulin were acting presynaptically to enhance DA clearance, then a combination of subthreshold doses of the two should be effective in decreasing performance in the lick-rate task. We tested this with a dose of insulin (5 mU) that was ineffective in suppressing 24-h food intake or decreasing body weight, even when administered repeatedly over a time course of weeks. Additionally, this dose of insulin by itself did not alter performance in the 5-min lick-rate task. However, when given a few hours before the 5-min lick-rate test, insulin could facilitate the action of (a subthreshold dose of) the D2 receptor antagonist raclopride to decrease 5-min lick rate of sucrose solutions (115). The efficacy of an insulin dose that is subthreshold for decreasing body weight suggests that insulin may have effects on reward circuitry that are independent of its effects to regulate caloric homeostasis.

Although the lick-rate data are consistent with an acute role for insulin in decreasing performance of a DA-dependent ingestive task, they do not address the possibility that insulin (or leptin) may have effects to decrease the rewarding properties of food independent of ingestive effects. Thus we evaluated the ability of leptin and insulin to decrease performance in a second reward task, the conditioned place preference (CPP) paradigm. In this task, the ability of a palatable food, available during training sessions, to condition a subsequent preference for rats to be in the "place" that they associate with having the food (on a test day when no food is available) is assessed (79). The conditioning and expression of this preference is dependent on intact DA circuitry (92). It has been demonstrated that food is more effective at conditioning a place preference in rats that are food restricted (and presumably hypoinsulinemic and hypoleptinemic) vs. ad libitum fed (121). Because those studies did not control the amount of food that was eaten by the rats during their training sessions (and because the food-restricted rats would eat more than ad libitum-fed rats), we repeated this study and demonstrated that the food restriction effect occurs even when the amount of food available during training sessions is restricted to a small, and identical, amount (10 45-mg sucrose pellets) for either food-restricted or free-feeding rats (45). This effect of food restriction is dependent on intact DA circuitry, as administration of a DA antagonist during training blocked the formation of a CPP. This finding is also consistent with the observation by Wilson et al. (126) that an identical food reward stimulates greater nucleus accumbens DA release in food-restricted vs. fed rats. Furthermore, the formation and expression of a CPP was blocked if food-restricted rats received peripheral minipump infusions of leptin at a dose that was chosen to replace baseline free-feeding levels but was subthreshold for decreasing body weight. To evaluate the potential role of insulin and leptin within the CNS in decreasing the CPP to a food reward in ad libitum-fed rats, we carried out a further study. Rats were trained with a high-fat diet (132) reward over a 6-day training period (alternate days of 5 g diet or no treatment). Whereas rats receiving control infusions of artificial CSF expressed a significant place preference (CPP score, 133 ± 34), chronic third ventricular infusion of doses of insulin (5 mU/day) or leptin (0.2 µg/day) that are subthreshold for decreasing food intake and body weight completely blocked the ability of the high-fat diet to condition a place preference (insulin CPP score, 29 ± 46; leptin CPP score, 22 ± 32). These findings are consistent with those of the hypothalamic self-stimulation studies in that insulin and leptin appear to modify performance in paradigms where food intake per se is not the endpoint. However, whereas self-stimulation is an instrumental task, the CPP reflects associative learning. In our paradigm, rats received infusions during both the training period and on the test day, thus we cannot differentiate between whether rats did not learn the association, whether expression of the preference was blocked on the test day by insulin or leptin, or both. The doses of insulin and leptin were chosen so that the three groups of rats consumed identical amounts of the diet during training (CSF, 62 ± 5 kcal; insulin, 70 ± 4 kcal; leptin, 65 ± 3 kcal). Thus diet experience was identical and the absence of CPP in the insulin- and leptin-treated rats cannot be due to a lack of experience with the stimulus.

Taken together, the results of these three very different types of behavioral paradigm, self-stimulation, lick-rate task, and CPP, demonstrate that insulin and leptin, across a concentration range from fasting to free feeding to elevated (as would occur postprandially) levels, are able to modify behaviors that reflect learning and reward evaluation, independent of their action(s) to regulate body adiposity. There appear to be both rapid and chronic effects for both insulin and leptin, and whether these are mediated via the same circuitry and the same neural mechanisms remains to be elucidated. An additional issue is whether these effects hold uniquely for food reward or generalize to other rewarding stimuli. As mentioned above, intraventricular leptin can blunt the food restriction-induced enhancement of relapse to heroin self-administration (109), which suggests that leptin-sensitive CNS circuitry includes sites that can be substrates for drugs of abuse, although this experimental approach does not allow conclusions to be drawn regarding different classes of motivating stimuli. The possibility of stimulus specificity is shown by studies from Carelli (17) who showed that in the nucleus accumbens there is response specificity for natural stimuli (food, water) vs. psychostimulants (cocaine) at the level of individual neurons (17), which are evaluated electrophysiologically in rats trained to self-administer cocaine, water, or sucrose.

To date, all of these studies have been carried out with insulin and leptin being administered into the cerebral ventricles or peripherally. While the results argue compellingly for a CNS locus of action for leptin and insulin, they do not help delineate specific anatomical site(s) mediating these effects. Because we have observed insulin (and leptin) receptors in the VTA, we are presently testing the ability of insulin to act directly in the VTA to block short-term feeding of highly palatable sucrose pellets in nondeprived rats. Mu-opioid agonists administered into the VTA can enhance feeding (97). In this study, we examined the effect of 5 mU insulin on mu-agonist-stimulated sucrose intake. All rats receive an injection of artificial CSF on day 1 and then one of four injection conditions on day 2 [artificial CSF; 5 mU insulin; 1 nmol of the mu opioid agonist [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO); or a combination of insulin and DAMGO]. On both days, rats have 60 min access to sucrose pellets. As shown in Fig. 2, CSF or 5 mU insulin had no effect on baseline sucrose pellet intake, whereas DAMGO stimulated intake. This was completely reversed when insulin was given concurrently with DAMGO into the VTA. Thus insulin may act directly at the VTA to blunt intake of a palatable food, and we would hypothesize that leptin, either alone or perhaps acting in concert with insulin, has similar acute effects.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effect of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and insulin in the ventral tegmental area (VTA) on 60-min sucrose intake in nondeprived rats. Baseline (day 1) pellet intakes did not differ among the 4 treatment groups [cerebrospinal fluid (CSF), 57 ± 13; insulin (INS), 64 ± 13; DAMGO, 64 ± 5; DAMGO/insulin, 66 ± 8]. Data are expressed as the within-subjects difference in sucrose consumption (sucrose pellet counts) between day 2 and day 1, and are shown as means ± SE. Evans, Pu, and Figlewicz Lattemann, unpublished results.

The feeding that occurs with VTA stimulation is not based on caloric need as it was elicited in fed rats, and in our studies the food offered is a highly palatable one. Work from Glass et al. (53) and other labs has documented the role for opioid peptides to preferentially enhance intake of palatable food over less palatable food (rat chow). The question of whether adiposity signals can blunt palatability-induced feeding has not been addressed, and we have begun to evaluate this. In an experimental design similar to the one described above for the sucrose lick-rate task, we tested whether intraventricular insulin (5 mU icv, time 3 h) can acutely blunt the ability of the kappa opioid agonist U50,488 (26 µg icv, time 15 min) to stimulate intake of sucrose pellets in nondeprived rats. We observed that insulin could both blunt the ability of the exogenous agonist to stimulate sucrose pellet intake and also synergize with a subthreshold dose of the dynorphin antagonist nor-binaltorphimine (5 µg, time 15 min) to decrease baseline intake of sucrose pellets (114). This is the first report of an interaction between an adiposity signal and an endogenous opioid-mediated feeding system, and we are now working to identify the specific population(s) of dynorphin-receptive (or downstream) neurons that may be targets for insulin action.

	INSULIN, LEPTIN, CALORIC NEED, AND FOOD REWARD

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Teleologically, it would appear advantageous for an animal that is food restricted or starved to have enhanced motivation to seek food and to experience that food as more rewarding. Although to date only a limited number of studies have investigated the possible role of adiposity signals in modulating reward behavior, the findings collectively are consistent and compelling for such an action for these signals. The possibility for multiple sites of action for these signals within the CNS should be considered: insulin and leptin may act directly at the VTA and also may act indirectly via signaling at the medial hypothalamus, with subsequent activation of pathways that project to the limbic circuitry. We speculate that the efficacy of insulin and leptin at low concentrations (levels that reflect the switch from fasting to fed status) is predominantly at the VTA, because administration of insulin or leptin at doses or in a paradigm that does not result in decreased long-term food intake or body weight (presumptively via the medial hypothalamus) are nonetheless effective at altering the reward threshold. With postprandial elevations of insulin and leptin, recruitment of inputs via the medial hypothalamus may occur, such that CNS circuits mediating reward/motivation and those mediating energy balance regulation are now inhibited in a synergistic manner to result in decreased appetitive or ingestive behavior. A further level of synergism may occur in reward circuitry itself, with insulin and leptin acting together either on identical neurons or (and perhaps more effectively) on a particular pathway, but via activation of separate neurons. Such synergism is being identified for energy balance circuitry, described above, and should be considered for reward circuitry as well, given that in general insulin and leptin levels change in tandem with altered metabolic status. The possibility that palatability and hedonic attributes of food may lead to enhanced activation of motivation circuitry, temporarily overriding the effectiveness of adiposity signals such as insulin and leptin is a hypothetical construct that we are currently investigating. Studies examining the anatomical, neurochemical, and behavioral relationships between circuitry for "reward" and circuitry for "caloric regulation" represent an obvious and important area for new studies in ingestive behavior, because, as is clear in this review, the field currently has many more questions than it has answers.

In conclusion, the role of regulation of metabolism and energy balance has been ascribed to insulin and leptin in the CNS. More recent anatomic and behavioral studies suggest that this role must be expanded for the consideration of both physiological and pathological effects of these two adiposity signals on limbic circuitry. From a broader perspective, potential interactions between insulin, leptin, metabolic status, and brain reward pathways may prove significant in the expression and treatment of several major classes of mental illness (eating disorders, depression, schizophrenia, substance abuse) and should provide the impetus for several new directions in neuroscience research in the immediate future.