Obesity is caused by an imbalance between energy intake and expenditure and has become a major health-care problem in western society. The central melanocortin system plays a crucial role in the regulation of feeding and energy expenditure, and functional loss of melanocortin receptor 4 (MC4R) is the most common genetic cause of human obesity. In this study, we present the first functional Mc4r knockout model in the rat, resulting from an N-ethyl-N-nitrosourea mutagenesis–induced point mutation. In vitro observations revealed impaired membrane-binding and subsequent nonfunctionality of the receptor, whereas in vivo observations showed that functional loss of MC4R increased body weight, food intake, white adipose mass, and changed substrate preference. In addition, intracerebroventricular (ICV) administration of Agouti-Related Protein79–129 (AgRP79–129), an MC4R inverse agonist, or Melanotan-II (MTII), an MC4R agonist, did affect feeding behavior in wild-type rats but not in homozygous mutant rats, confirming complete loss of MC4R function in vivo. Finally, ICV administration of MTII induced excessive grooming behavior in wild-type rats, whereas this effect was absent in homozygous mutant rats, indicating that MTII-induced grooming behavior is exclusively regulated via MC4R pathways. Taken together, we expect that the MC4R rat model described here will be a valuable tool for studying monogenic obesity in humans. More specifically, the relative big size and increased cognitive capacity of rats as compared to mice will facilitate complex behavioral studies and detailed mechanistic studies regarding central function of MC4R, both of which ultimately may help to further understand the specific mechanisms that induce obesity during loss of MC4R function.

The melanocortin-4 receptor (MC4R) plays an important role in the regulation of body weight in rodents. Mutations in the coding region of the MC4R are found more frequently in obese individuals, supporting the hypothesis that also in humans deficient melanocortin signaling may lead to obesity. Family studies that were carried out to demonstrate the relevance of single mutations for obesity were mostly inconclusive, most likely due to small sample size and complexity of the trait. In addition, the existing pharmacological data of the mutant receptors are limited in that for most mutations the effect on receptor expression level and Agouti-related protein (AgRP) pharmacology have not been studied. The aim of the present study was to gain further insight into the impact of the MC4R mutations on receptor function. Eleven missense mutations were tested for cell surface expression, affinity for ␣-melanocyte-stimulating hormone (␣-MSH) and AgRP-(83-132), and the biological response to ␣-MSH.

Presentation of antigen to T cells is generally restricted by MHC type but the mixed leukocyte reaction (MLR) was thought to involve direct stimulation by dendritic cells (DC) of allogeneic T cells. However, here we showed that DC bearing allogeneic MHC class II acted synergistically with responder-type DC. Removal of residual DC from 'purified' responder T cell populations was achieved using treatment with DC-specific antibody and complement. These DC-depleted cells showed a significantly reduced response to allogeneic DC which was restored by addition of DC syngeneic with responder T cells. The studies support the concept that a major component of the MLR is the secondary presentation of alloantigens acquired from stimulator DC by DC of responder type. To investigate the reasons why DC and not other cells stimulate an MLR, synergy between DC and other cell types was investigated. Synergy was found exclusively between DC; macrophages, B cells or L cells transfected with MHC class II molecules did not contribute. When allogeneic DC were mixed in culture, transfer of MHC molecules between DC was observed as assessed by flow cytometry. Freshly obtained cell-free supernatants from cultured DC contained MHC class II and stimulated primary allogeneic MLR. DC of responder type acquired allogeneic MHC molecules from the supernatants and stimulated proliferation in syngeneic T cells. The capacity of DC both to shed and to acquire MHC molecules may contribute to their potency in stimulating primary responses, and could explain why passenger DC within allografts provide a potent stimulus for graft rejection.

Altered mesolimbic dopamine signaling has been widely implicated in addictive behavior. For the most part, this work has focused on dopamine within the striatum, but there is emerging evidence for a role of the auto-inhibitory, somatodendritic dopamine D2 receptor (D2R) in the ventral tegmental area (VTA) in addiction. Thus, decreased midbrain D2R expression has been implicated in addiction in humans. Moreover, knockout of the gene encoding the D2R receptor (Drd2) in dopamine neurons has been shown to enhance the locomotor response to cocaine in mice. Therefore, we here tested the hypothesis that decreasing D2R expression in the VTA of adult rats, using shRNA knockdown, promotes addiction-like behavior in rats responding for cocaine or palatable food. Rats with decreased VTA D2R expression showed markedly increased motivation for both sucrose and cocaine under a progressive ratio schedule of reinforcement, but the acquisition or maintenance of cocaine self-administration were not affected. They also displayed enhanced cocaine-induced locomotor activity, but no change in basal locomotion. This robust increase in incentive motivation was behaviorally specific, as we did not observe any differences in fixed ratio responding, extinction responding, reinstatement or conditioned suppression of cocaine, and sucrose seeking. We conclude that VTA D2R knockdown results in increased incentive motivation, but does not directly promote other aspects of addiction-like behavior.

Neural growth regulator 1 (Negr1) is among the first common variants that have been associated with the regulation of body mass index. Using AAV technology directed to manipulate Negr1 expression in vivo, we find that decreased expression of Negr1 in periventricular hypothalamic areas leads to increases in body weight, presumably via increased food intake. Moreover, we observed that both increased and decreased levels of Negr1 lead to reduced locomotor activity and body temperature. In sum, our results provide further support for a role of hypothalamic expressed Negr1 in the regulation of energy balance.