Julie E. McMinn

Leptin receptor signaling in POMC neurons is required for normal body weight homeostasis

Neuroanatomical and electrophysiological studies have shown that hypothalamic POMC neurons are targets of the adipostatic hormone leptin. However, the physiological relevance of leptin signaling in these neurons has not yet been directly tested. Here, using the Cre/loxP system, we critically test the functional importance of leptin action on POMC neurons by deleting leptin receptors specifically from these cells in mice. Mice lacking leptin signaling in POMC neurons are mildly obese, hyperleptinemic, and have altered expression of hypothalamic neuropeptides. In summary, leptin receptors on POMC neurons are required but not solely responsible for leptins regulation of body weight homeostasis.

An allelic series for the leptin receptor gene generated by CRE and FLP recombinase.

Body weight regulation is mediated through several major signaling pathways, some of which have been delineated by positional cloning of spontaneous genetic mutations in mice. Leprdb/db mice are obese due to a defect in the signaling portion of the leptin receptor, which has led to extensive study of this highly conserved system over the past several years. We have created an allelic series at Lepr for the further examination of LEPR signaling phenotypes using both the FLP/frt and CRE/loxP systems. By inserting a frt-PGK-neo-frt sequence in Lepr intron 16, we have generated a conditional gene repair Lepr allele (Lepr-neo) that elicits morbid obesity, diabetes, and infertility in homozygous mice, recapitulating the obesity syndrome of Leprdb/db mice. Thus, in vivo excision of the PGK-neo cassette with a FLP recombinase transgene restores the lean and fertile phenotype to Leprflox/flox mice. In the same construct, we have also inserted loxP sites that flank Lepr coding exon 17, a region that encodes a JAK docking site required for STAT3 signaling. CRE-mediated excision of Lepr coding exon 17 from Lepr with a frameshift in subsequent exons results in a syndrome of obesity, diabetes, and infertility in LeprΔ17/Δ17 mice, which is indistinguishable from Leprneo/neo and Leprdb/db mice. We conclude that suppression of Lepr gene expression by PGK-neo is phenotypically equivalent to deletion of the Lepr signaling motifs, and therefore the Leprneo/neo mouse may be used to investigate conditional gene repair of Lepr signaling deficiency.

Increased expression of hypothalamic leptin receptor and adiponectin accompany resistance to dietary- induced obesity and infertility in female C57BL/6J mice

Background:Obesity is strongly associated with female infertility, but the mechanisms underlying this relationship are largely unknown.Methods:We investigated the effect of increasing dietary fat percentage upon body mass, hypothalamic neuropeptide gene expression, adipose hormone secretion and fertility in females of the inbred mouse strains C57BL/6J and DBA/2J. To assess the effect of obesity independent of dietary influence, we also compared these parameters in wild-type female C57BL/6J mice to those congenic for the obesogenic mutations ob/ob and Ay/a.Results:After 24 weeks, rather than exhibiting an obese, leptin-resistant phenotype like their female DBA/2J counterparts, wild-type female C57BL/6J mice remained lean, fertile and manifested increased hypothalamic LEPR-B expression. Although both mutant genotypes were associated with obesity and subfertility, ob/ob mice demonstrated significantly increased hypothalamic LEPR-B expression, whereas Ay/a mice had a significant reduction. Interestingly, wild-type female C57BL/6J mice were noted to manifest significantly higher and lower levels of adiponectin and tissue plasminogen activator inhibitor-1 (tPAI-1), respectively, than weight-matched wild-type female DBA/2J mice.Conclusions:We conclude that (1) resistance to the obese-infertile phenotype in female C57BL/6J mice is associated with increased hypothalamic leptin receptor expression and alterations in adipokine levels consistent with decreased adipose tissue inflammation and (2) that long-standing hyperleptinemic obesity in mice is associated with a downregulation of the hypothalamic leptin receptor.

NPY-induced overfeeding suppresses hypothalamic NPY mRNA expression : potential roles of plasma insulin and leptin

To test the hypothesis that NPY-induced overfeeding activates compensatory responses that inhibit hypothalamic NPY gene expression, we investigated the effect of chronically administered neuropeptide Y (NPY) on plasma hormones involved in energy balance and on the level of mRNA for hypothalamic neuropeptides. After cannulation of the third cerebral ventricle, male Long-Evans rats received a 4.5-day intracerebroventricular (i.c.v.) infusion of either human NPY (12 microg per day), or synthetic cerebrospinal fluid (CSF). NPY-treated animals were either allowed ad libitum access to food or were pairfed to the intake of CSF-treated controls. In rats fed ad libitum, i.c.v. NPY induced significant increases in food intake (75%), body weight (9%), plasma insulin (150%) and plasma leptin levels (300%) as compared to the i.c.v. CSF group. Levels of plasma leptin, but not insulin, remained elevated in NPY-treated rats that were pairfed to the intake of the CSF group. NPY mRNA levels in the midregion of the arcuate nucleus (ARC) were reduced by 50% in NPY-treated rats that were allowed to overeat, but not in the pairfed group, as determined by in situ hybridization. In contrast, mRNA for corticotropin-releasing hormone (CRH) in the paraventricular nucleus (PVN) and proopiomelanocortin (POMC) in the rostral ARC were not significantly different among groups. These findings indicate that NPY-induced overfeeding suppresses ARC NPY mRNA expression, and that this effect unlikely to be mediated by a direct action of NPY, since it was abolished by limiting food intake in NPY-treated animals to that observed in controls. NPY-induced overfeeding was also associated with elevated plasma levels of leptin and insulin. The effect of these hormones to inhibit NPY gene expression may therefore have contributed to the decrease of NPY mRNA.