Carol F. Elias

Leptin targets in the mouse brain.

The central actions of leptin are essential for homeostatic control of adipose tissue mass, glucose metabolism, and many autonomic and neuroendocrine systems. In the brain, leptin acts on numerous different cell types via the long‐form leptin receptor (LepRb) to elicit its effects. The precise identification of leptins cellular targets is fundamental to understanding the mechanism of its pleiotropic central actions. We have systematically characterized LepRb distribution in the mouse brain using in situ hybridization in wildtype mice as well as by EYFP immunoreactivity in a novel LepRb‐IRES‐Cre EYFP reporter mouse line showing high levels of LepRb mRNA/EYFP coexpression. We found substantial LepRb mRNA and EYFP expression in hypothalamic and extrahypothalamic sites described before, including the dorsomedial nucleus of the hypothalamus, ventral premammillary nucleus, ventral tegmental area, parabrachial nucleus, and the dorsal vagal complex. Expression in insular cortex, lateral septal nucleus, medial preoptic area, rostral linear nucleus, and in the Edinger‐Westphal nucleus was also observed and had been previously unreported. The LepRb‐IRES‐Cre reporter line was used to chemically characterize a population of leptin receptor‐expressing neurons in the midbrain. Tyrosine hydroxylase and Cre reporter were found to be coexpressed in the ventral tegmental area and in other midbrain dopaminergic neurons. Lastly, the LepRb‐IRES‐Cre reporter line was used to map the extent of peripheral leptin sensing by central nervous system (CNS) LepRb neurons. Thus, we provide data supporting the use of the LepRb‐IRES‐Cre line for the assessment of the anatomic and functional characteristics of neurons expressing leptin receptor. J. Comp. Neurol. 514:518–532, 2009.

Distinct hypothalamic neurons mediate estrogenic effects on energy homeostasis and reproduction.

Estrogens regulate body weight and reproduction primarily through actions on estrogen receptor-α (ERα). However, ERα-expressing cells mediating these effects are not identified. We demonstrate that brain-specific deletion of ERα in female mice causes abdominal obesity stemming from both hyperphagia and hypometabolism. Hypometabolism and abdominal obesity, but not hyperphagia, are recapitulated in female mice lacking ERα in hypothalamic steroidogenic factor-1 (SF1) neurons. In contrast, deletion of ERα in hypothalamic pro-opiomelanocortin (POMC) neurons leads to hyperphagia, without directly influencing energy expenditure or fat distribution. Further, simultaneous deletion of ERα from both SF1 and POMC neurons causes hypometabolism, hyperphagia, and increased visceral adiposity. Additionally, female mice lacking ERα in SF1 neurons develop anovulation and infertility, while POMC-specific deletion of ERα inhibits negative feedback regulation of estrogens and impairs fertility in females. These results indicate that estrogens act on distinct hypothalamic ERα neurons to regulate different aspects of energy homeostasis and reproduction.

Hypothalamic Pathways Linking Energy Balance and Reproduction

During periods of metabolic stress, animals must channel energy toward survival and away from processes such as reproduction. The reproductive axis, therefore, has the capacity to respond to changing levels of metabolic cues. The cellular and molecular mechanisms that link energy balance and reproduction, as well as the brain sites mediating this function, are still not well understood. This review focuses on the best characterized of the adiposity signals: leptin and insulin. We examine their reproductive role acting on the classic metabolic pathways of the arcuate nucleus, NPY/AgRP and POMC/CART neurons, and the newly identified kisspeptin network. In addition, other hypothalamic nuclei that may play a role in linking metabolic state and reproductive function are discussed. The nature of the interplay between these elements of the metabolic and reproductive systems presents a fascinating puzzle, whose pieces are just beginning to fall into place.

Direct insulin and leptin action on pro-opiomelanocortin neurons is required for normal glucose homeostasis and fertility.

Circulating leptin and insulin convey information regarding energy stores to the central nervous system, particularly the hypothalamus. Hypothalamic pro-opiomelanocortin (POMC) neurons regulate energy balance and glucose homeostasis and express leptin and insulin receptors. However, the physiological significance of concomitant leptin and insulin action on POMC neurons remains to be established. Here, we show that mice lacking both leptin and insulin receptors in POMC neurons (Pomc-Cre, Lepr(flox/flox) IR(flox/flox) mice) display systemic insulin resistance, which is distinct from the single deletion of either receptor. In addition, Pomc-Cre, Lepr(flox/flox) IR(flox/flox) female mice display elevated serum testosterone levels and ovarian abnormalities, resulting in reduced fertility. We conclude that direct action of insulin and leptin on POMC neurons is required to maintain normal glucose homeostasis and reproductive function.

Palmitic acid mediates hypothalamic insulin resistance by altering PKC-θ subcellular localization in rodents

Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-theta, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-theta was expressed in discrete neuronal populations of the arcuate nucleus, specifically the neuropeptide Y/agouti-related protein neurons and the dorsal medial nucleus in the hypothalamus. CNS exposure to palmitic acid via direct infusion or by oral gavage increased the localization of PKC-theta to cell membranes in the hypothalamus, which was associated with impaired hypothalamic insulin and leptin signaling. This finding was specific for palmitic acid, as the monounsaturated fatty acid, oleic acid, neither increased membrane localization of PKC-theta nor induced insulin resistance. Finally, arcuate-specific knockdown of PKC-theta attenuated diet-induced obesity and improved insulin signaling. These results suggest that many of the deleterious effects of high-fat diets, specifically those enriched with palmitic acid, are CNS mediated via PKC-theta activation, resulting in reduced insulin activity.

Segregation of Acute Leptin and Insulin Effects in Distinct Populations of Arcuate Proopiomelanocortin Neurons

Acute leptin administration results in a depolarization and concomitant increase in the firing rate of a subpopulation of arcuate proopiomelanocortin (POMC) cells. This rapid activation of POMC cells has been implicated as a cellular correlate of leptin effects on energy balance. In contrast to leptin, insulin inhibits the activity of some POMC neurons. Several studies have described a “cross talk” between leptin and insulin within the mediobasal hypothalamus via the intracellular enzyme, phosphoinositol-3-kinase (PI3K). Interestingly, both insulin and leptin regulate POMC cellular activity by activation of PI3K; however, it is unclear whether leptin and insulin effects are observed in similar or distinct populations of POMC cells. We therefore used dual label immunohistochemistry/in situ hybridization and whole-cell patch-clamp electrophysiology to map insulin and leptin responsive arcuate POMC neurons. Leptin-induced Fos activity within arcuate POMC neurons was localized separate from POMC neurons that express insulin receptor. Moreover, acute responses to leptin and insulin were largely segregated in distinct subpopulations of POMC cells. Collectively, these data suggest that cross talk between leptin and insulin occurs within a network of cells rather than within individual POMC neurons.

Leptin’s effect on puberty in mice is relayed by the ventral premammillary nucleus and does not require signaling in Kiss1 neurons

Studies in humans and rodents indicate that a minimum amount of stored energy is required for normal pubertal development. The adipocyte-derived hormone leptin is a key metabolic signal to the neuroendocrine reproductive axis. Humans and mice lacking leptin or the leptin receptor (LepR) (ob/ob and db/db mice, respectively) are infertile and fail to enter puberty. Leptin administration to leptin-deficient subjects and ob/ob mice induces puberty and restores fertility, but the exact site or sites of leptin action are unclear. Here, we found that genetic deletion of LepR selectively from hypothalamic Kiss1 neurons in mice had no effect on puberty or fertility, indicating that direct leptin signaling in Kiss1 neurons is not required for these processes. However, bilateral lesions of the ventral premammillary nucleus (PMV) of ob/ob mice blunted the ability of exogenous leptin to induce sexual maturation. Moreover, unilateral reexpression of endogenous LepR in PMV neurons was sufficient to induce puberty and improve fertility in female LepR-null mice. This LepR reexpression also normalized the increased hypothalamic GnRH content characteristic of leptin-signaling deficiency. These data suggest that the PMV is a key site for leptins permissive action at the onset of puberty and support the hypothesis that the multiple actions of leptin to control metabolism and reproduction are anatomically dissociated.

Disruption of leptin receptor expression in the pancreas directly affects β cell growth and function in mice

Obesity is characterized by hyperinsulinemia, hyperleptinemia, and an increase in islet volume. While the mechanisms that hasten the onset of diabetes in obese individuals are not known, it is possible that the adipose-derived hormone leptin plays a role. In addition to its central actions, leptin exerts biological effects by acting in peripheral tissues including the endocrine pancreas. To explore the impact of disrupting leptin signaling in the pancreas on beta cell growth and/or function, we created pancreas-specific leptin receptor (ObR) KOs using mice expressing Cre recombinase under the control of the pancreatic and duodenal homeobox 1 (Pdx1) promoter. The KOs exhibited improved glucose tolerance due to enhanced early-phase insulin secretion, and a greater beta cell mass secondary to increased beta cell size and enhanced expression and phosphorylation of p70S6K. Similar effects on p70S6K were observed in MIN6 beta cells with knockdown of the ObR gene, suggesting crosstalk between leptin and insulin signaling pathways. Surprisingly, challenging the KOs with a high-fat diet led to attenuated acute insulin secretory response to glucose, poor compensatory islet growth, and glucose intolerance. Together, these data provide direct genetic evidence, from a unique mouse model lacking ObRs only in the pancreas, for a critical role for leptin signaling in islet biology and suggest that altered leptin action in islets is one factor that contributes to obesity-associated diabetes.

Characterization of Kiss1 neurons using transgenic mouse models

Humans and mice with loss-of-function mutations of the genes encoding kisspeptins (Kiss1) or kisspeptin receptor (Kiss1r) are infertile due to hypogonadotropic hypogonadism. Within the hypothalamus, Kiss1 mRNA is expressed in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (Arc). In order to better study the different populations of kisspeptin cells we generated Kiss1-Cre transgenic mice. We obtained one line with Cre activity specifically within Kiss1 neurons (line J2-4), as assessed by generating mice with Cre-dependent expression of green fluorescent protein or β-galactosidase. Also, we demonstrated Kiss1 expression in the cerebral cortex and confirmed previous data showing Kiss1 mRNA in the medial nucleus of amygdala and anterodorsal preoptic nucleus. Kiss1 neurons were more concentrated towards the caudal levels of the Arc and higher leptin-responsivity was observed in the most caudal population of Arc Kiss1 neurons. No evidence for direct action of leptin in AVPV Kiss1 neurons was observed. Melanocortin fibers innervated subsets of Kiss1 neurons of the preoptic area and Arc, and both populations expressed melanocortin receptors type 4 (MC4R). Specifically in the preoptic area, 18-28% of Kiss1 neurons expressed MC4R. In the Arc, 90% of Kiss1 neurons were glutamatergic, 50% of which also were GABAergic. In the AVPV, 20% of Kiss1 neurons were glutamatergic whereas 75% were GABAergic. The differences observed between the Kiss1 neurons in the preoptic area and the Arc likely represent neuronal evidence for their differential roles in metabolism and reproduction.

Hypothalamic cocaine- and amphetamine-regulated transcript neurons project to areas expressing gonadotropin releasing hormone immunoreactivity and to the anteroventral periventricular nucleus in male and female rats.

Cocaine- and amphetamine-regulated transcript (CART) and CART-derived peptides are widely expressed in the hypothalamus. CART is involved in food intake control and is regulated by circulating leptin, a hormone implicated in a variety of endocrine functions. Lack of leptin (ob/ob mice) is associated with obesity, hypogonadism and infertility. In the arcuate nucleus, dorsomedial nucleus of the hypothalamus, and ventral premammillary nucleus, CART neurons also express leptin receptor long-form splice-variant. Recent studies have suggested that the facilitatory effect of leptin on gonadotropin-releasing hormone (GnRH) secretion is mediated by CART. In the present study, using dual- and triple-label immunohistochemistry, we identified CART fibers in close apposition with GnRH neurons expressing Fos in the afternoon of the proestrous day, as well as with GnRH neurons in male rats. In order to investigate the origin of these fibers, we injected the retrograde tracer Fluorogold into areas containing GnRH cell bodies. In male and female rats, the tracer was injected around the vascular organ of lamina terminalis, median preoptic nucleus and medial preoptic nucleus, as well as in the anteroventral periventricular nucleus. We observed retrogradely labeled neurons in various hypothalamic nuclei, including the arcuate, dorsomedial and ventral premammillary. In these areas, dual-label immunohistochemistry/in situ hybridization revealed that part of the retrogradely labeled neurons also express CART mRNA. As a control, we injected the anterograde tracer biotinylated dextran amine into the ventral premammillary nucleus of both males and females. Most projections targeted brain areas related to reproductive behavior and few fibers were closely associated with GnRH neurons. Our findings indicate that ventral premammillary nucleus CART neurons intermingle with brain circuitry involved in reproduction. Therefore, these neurons are well positioned to mediate leptin effect on reproductive control.