Mammalian reproductive function depends upon a neuroendocrine circuit that evokes the pulsatile release of gonadotropin hormones (luteinizing hormone and follicle-stimulating hormone) from the pituitary. This reproductive circuit is sensitive to metabolic perturbations. When challenged with starvation, insufficient energy reserves attenuate gonadotropin release, leading to infertility. The reproductive neuroendocrine circuit is well established, composed of two populations of kisspeptin-expressing neurons (located in the anteroventral periventricular hypothalamus, Kiss1AVPV, and arcuate hypothalamus, Kiss1ARH), which drive the pulsatile activity of gonadotropin-releasing hormone (GnRH) neurons. The reproductive axis is primarily regulated by gonadal steroid and circadian cues, but the starvation-sensitive input that inhibits this circuit during negative energy balance remains controversial. Agouti-related peptide (AgRP)-expressing neurons are activated during starvation and have been implicated in leptin-associated infertility. To test whether these neurons relay information to the reproductive circuit, we used AgRP-neuron ablation and optogenetics to explore connectivity in acute slice preparations. Stimulation of AgRP fibers revealed direct, inhibitory synaptic connections with Kiss1ARH and Kiss1AVPV neurons. In agreement with this finding, Kiss1ARH neurons received less presynaptic inhibition in the absence of AgRP neurons (neonatal toxin-induced ablation). To determine whether enhancing the activity of AgRP neurons is sufficient to attenuate fertility in vivo, we artificially activated them over a sustained period and monitored fertility. Chemogenetic activation with clozapine N-oxide resulted in delayed estrous cycles and decreased fertility. These findings are consistent with the idea that, during metabolic deficiency, AgRP signaling contributes to infertility by inhibiting Kiss1 neurons.

Leptin-deficient (Lep ob/ob ) mice are obese, diabetic, and infertile. Ablation of neurons that make agouti-related protein (AgRP) in moderately obese adult Lep ob/ob mice caused severe anorexia. The mice stopped eating for 2 wk and then gradually recovered. Their body weight fell to within a normal range for WT mice, at which point food intake and glucose tolerance were restored to that of WT mice. Remarkably, both male and female Lep ob/ob mice became fertile. Ablation of neurons that express melanin-concentrating hormone (MCH) in adult Lep ob/ob mice had no effect on food intake, body weight, or fertility, but resulted in improved glucose tolerance. We conclude that AgRP-expressing neurons play a critical role in mediating the metabolic syndrome and infertility of Lep ob/ob mice, whereas MCHexpressing neurons have only a minor role.he spontaneous nonsense mutation in the leptin gene that generated Lep ob/ob mice results in obesity, hyperphagia, diabetes, and infertility (1, 2). These phenotypes can be reversed by chronic delivery of recombinant leptin (3, 4). The neural circuitry underlying these phenotypes has been the subject of intense investigation (5-7). Mice lacking leptin have elevated levels of neuropeptide Y (NPY) and melanin-concentrating hormone (MCH) in the hypothalamus (8, 9), and genetic experiments have demonstrated that Lep ob/ob mice lacking either of these peptides have an improved metabolic phenotype (10, 11).Because hypothalamic NPY-expressing neurons send projections to the vicinity of MCH-expressing neurons (12), which have electrophysiological responses to NPY (13), the two neuronal populations may be part of the same circuitry mediating the phenotype of Lep ob/ob mice. NPY-expressing neurons in the arcuate nucleus also express agouti-related protein (AgRP), which exerts the same orexigenic effects as NPY but by a different mechanism (14).We take advantage of the restricted expression of AgRP and MCH to hypothalamic neurons by targeting the human diphtheria toxin (DT) receptor to the genes encoding those neuropeptides, thereby allowing their selective ablation by administration of DT (15). Ablation of AgRP-expressing neurons (AgRP neurons) in adult mice results in severe anorexia and death in ∼6 d (15,16). In this study, we applied this same methodology to MCH-expressing neurons (MCH neurons) and asked whether ablation of either of these neuronal populations in adult Lep ob/ob mice is beneficial (as predicted from the constitutive loss of the neuropeptides) or lethal (as predicted from the loss of AgRP neurons in a WT background). ) gradually stopped feeding and lost 20% of their body weight during the next 6 d and would have succumbed, whereas mice without the Agrp DTR allele maintained their feeding and body weight (Fig. 1). Statistical analyses of our results are reported in the figure legends. Analysis of the brains of DT-treated mice at the end of the experiment revealed a virtual absence of AgRP neurons in Agrp DTR/+ mice, as measured by immunocytochemistry or quantitative PCR for...

The thermodynamic and structural effects of macrocyclization as a tactic for stabilizing the biologically-active conformation of Grb2 SH2 binding peptides were investigated using isothermal titration calorimetry and x-ray crystallography. 23-Membered macrocycles containing the sequence pYVN were slightly more potent than their linear controls; however, preorganization did not necessarily eventuate in a more favorable binding entropy. Structures of complexes of macrocycle 7 and its acyclic control 8 are similar except for differences in relative orientations of corresponding atoms in the linking moieties of 7 and 8. There are no differences in the number of direct or water-mediated protein-ligand contacts that might account for the less favorable binding enthalpy of 7; however, an intramolecular hydrogen bond between the pY and pY+3 residues in 8 that is absent in 7 may be a factor. These studies highlight the difficulties associated with correlating energetics and structure in protein-ligand interactions.