Jennifer W. Hill

Acute effects of leptin require PI3K signaling in hypothalamic proopiomelanocortin neurons in mice

Normal food intake and body weight homeostasis require the direct action of leptin on hypothalamic proopiomelanocortin (POMC) neurons. It has been proposed that leptin action requires PI3K activity. We therefore assessed the contribution of PI3K signaling to leptins effects on POMC neurons and organismal energy balance. Leptin caused a rapid depolarization of POMC neurons and an increase in action potential frequency in patch-clamp recordings of hypothalamic slices. Pharmacologic inhibition of PI3K prevented this depolarization and increased POMC firing rate, indicating a PI3K-dependent mechanism of leptin action. Mice with genetically disrupted PI3K signaling in POMC cells failed to undergo POMC depolarization or increased firing frequency in response to leptin. Insulins ability to hyperpolarize POMC neurons was also abolished in these mice. Moreover, targeted disruption of PI3K blunted the suppression of feeding elicited by central leptin administration. Despite these differences, mice with impaired PI3K signaling in POMC neurons exhibited normal long-term body weight regulation. Collectively, these results suggest that PI3K signaling in POMC neurons is essential for leptin-induced activation and insulin-induced inhibition of POMC cells and for the acute suppression of food intake elicited by leptin, but is not a major contributor to the regulation of long-term organismal energy homeostasis.

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.

Central insulin and leptin-mediated autonomic control of glucose homeostasis

Largely as a result of rising obesity rates, the incidence of type 2 diabetes is escalating rapidly. Type 2 diabetes results from multi-organ dysfunctional glucose metabolism. Recent publications have highlighted hypothalamic insulin- and adipokine-sensing as a major determinant of peripheral glucose and insulin responsiveness. The preponderance of evidence indicates that the brain is the master regulator of glucose homeostasis, and that hypothalamic insulin and leptin signaling in particular play a crucial role in the development of insulin resistance. This review discusses the neuronal crosstalk between the hypothalamus, autonomic nervous system, and tissues associated with the pathogenesis of type 2 diabetes, and how hypothalamic insulin and leptin signaling are integral to maintaining normal glucose homeostasis.

PI3K Signaling in the Ventromedial Hypothalamic Nucleus Is Required for Normal Energy Homeostasis

Phosphatidyl inositol 3-kinase (PI3K) signaling in the hypothalamus has been implicated in the regulation of energy homeostasis, but the critical brain sites where this intracellular signal integrates various metabolic cues to regulate food intake and energy expenditure are unknown. Here, we show that mice with reduced PI3K activity in the ventromedial hypothalamic nucleus (VMH) are more sensitive to high-fat diet-induced obesity due to reduced energy expenditure. In addition, inhibition of PI3K in the VMH impaired the ability to alter energy expenditure in response to acute high-fat diet feeding and food deprivation. Furthermore, the acute anorexigenic effects induced by exogenous leptin were blunted in the mutant mice. Collectively, our results indicate that PI3K activity in VMH neurons plays a physiologically relevant role in the regulation of energy expenditure.

Attenuation of Luteinizing Hormone Surges in Neuropeptide Y Knockout Mice

To clarify the role of neuropeptide Y (NPY) in the regulation of the reproductive axis, these experiments evaluated the extent to which reproductive hormone secretions may be compromised in the absence of NPY expression. In NPY knockout (NPY-KO) and wild-type (WT) mice, hormone secretions were analyzed under conditions of basal release, following ovariectomy (OVX), in proestrus, after estrogen treatments which induce gonadotropin surges and after injection of gonadotropin-releasing hormone (GnRH). Radioimmunoassays of serum from metestrous females revealed that basal luteinizing hormone (LH), follicular-stimulating hormone (FSH), estrogen and progesterone levels, as well as hypothalamic GnRH tissue concentrations, were not different between the two genotypes. The LH and FSH levels and GnRH tissue concentrations were likewise similar in WT and NPY-KO mice 5 and 10 days following OVX. Significant differences in LH levels were observed however when animals were exposed to pheromone stimulation (male mouse urine) to induce preovulatory LH surges. In proestrous animals, mean LH levels at 18.30–19.00 h were reduced by about 66% in NPY-KO versus WT mice (4.33 ± 1.12 ng/ml in the WT mice vs. 1.47 ± 0.42 ng/ml in the NPY-KO mice, p = 0.028). Despite diminishment of LH surges in NPY-KO mice, corpora lutea were equally abundant in the ovaries of NPY-KO and WT mice. In an additional experiment, a surge-inducing regimen of estradiol-17-β (E2) and estradiol benzoate (E2B) was administered to OVX animals. The LH surges in the NPY-KO animals treated in this manner were again diminished by approximately 50% compared to corresponding values in WT animals (WT mice 7.33 ± 0.97 ng/ml, NPY-KO mice 3.58 ± 0.74 ng/ml; p = 0.0063). To assess the contribution of altered pituitary responsiveness to the diminishment of LH surges, LH responses to a GnRH challenge (200 ng/kg subcutaneously) were determined; NPY-KO animals exhibited LH responses that were significantly reduced compared to values in WT mice (WT mice 4.88 ± 0.56 ng/ml, NPY-KO mice 3.00 ± 0.41 ng/ml; p = 0.013). Taken together, these observations do not support the idea that NPY plays a major role in the regulation of basal gonadotropin secretion or in mediating negative feedback actions of gonadal hormones. They demonstrate however that preovulatory NPY release is required for normal amplification of the LH surge that occurs on proestrus. Involvement of NPY in the generation of normal LH surges is partially mediated by the ability of the peptide to prime the anterior pituitary gland to GnRH stimulation.

Phosphatidyl Inositol 3-Kinase Signaling in Hypothalamic Proopiomelanocortin Neurons Contributes to the Regulation of Glucose Homeostasis

Recent studies demonstrated a role for hypothalamic insulin and leptin action in the regulation of glucose homeostasis. This regulation involves proopiomelanocortin (POMC) neurons because suppression of phosphatidyl inositol 3-kinase (PI3K) signaling in these neurons blunts the acute effects of insulin and leptin on POMC neuronal activity. In the current study, we investigated whether disruption of PI3K signaling in POMC neurons alters normal glucose homeostasis using mouse models designed to both increase and decrease PI3K-mediated signaling in these neurons. We found that deleting p85alpha alone induced resistance to diet-induced obesity. In contrast, deletion of the p110alpha catalytic subunit of PI3K led to increased weight gain and adipose tissue along with reduced energy expenditure. Independent of these effects, increased PI3K activity in POMC neurons improved insulin sensitivity, whereas decreased PI3K signaling resulted in impaired glucose regulation. These studies show that activity of the PI3K pathway in POMC neurons is involved in not only normal energy regulation but also glucose homeostasis.

Delayed puberty but normal fertility in mice with selective deletion of insulin receptors from Kiss1 cells.

Pubertal onset only occurs in a favorable, anabolic hormonal environment. The neuropeptide kisspeptin, encoded by the Kiss1 gene, modifies GnRH neuronal activity to initiate puberty and maintain fertility, but the factors that regulate Kiss1 neurons and permit pubertal maturation remain to be clarified. The anabolic factor insulin may signal nutritional status to these neurons. To determine whether insulin sensing plays an important role in Kiss1 neuron function, we generated mice lacking insulin receptors in Kiss1 neurons (IR(ΔKiss) mice). IR(ΔKiss) females showed a delay in vaginal opening and in first estrus, whereas IR(ΔKiss) males also exhibited late sexual maturation. Correspondingly, LH levels in IR(ΔKiss) mice were reduced in early puberty in both sexes. Adult reproductive capacity, body weight, fat composition, food intake, and glucose regulation were comparable between the 2 groups. These data suggest that impaired insulin sensing by Kiss1 neurons delays the initiation of puberty but does not affect adult fertility. These studies provide insight into the mechanisms regulating pubertal timing in anabolic states.

Monitoring FoxO1 Localization in Chemically Identified Neurons

The PI3K-Akt-FoxO1 pathway contributes to the actions of insulin and leptin in several cell types, including neurons in the CNS. However, identifying these actions in chemically identified neurons has proven difficult. To address this problem, we have developed a reporter mouse for monitoring PI3K-Akt signaling in specific populations of neurons, based on FoxO1 nucleocytoplasmic shuttling. The reporter, FoxO1 fused to green fluorescent protein (FoxO1GFP), is expressed under the control of a ubiquitous promoter that is silenced by a loxP flanked transcriptional blocker. Thus, the expression of the reporter in selected cells is dependent on the action of Cre recombinase. Using this model, we found that insulin treatment resulted in the nuclear exclusion of FoxO1GFP within POMC and AgRP neurons in a dose- and time-dependent manner. FoxO1GFP nuclear exclusion was also observed in POMC neurons following in vivo administration of insulin. In addition, leptin induced transient nuclear export of FoxO1GFP in POMC neurons in a dose dependent manner. Finally, insulin-induced nuclear export was impaired in POMC neurons by pretreatment with free fatty acids, a paradigm known to induce insulin resistance in peripheral insulin target tissues. Thus, our FoxO1GFP mouse provides a tool for monitoring the status of PI3K-Akt signaling in a cell-specific manner under physiological and pathophysiological conditions.

Testosterone decreases the potential for song plasticity in adult male zebra finches.

Zebra finches are age-limited learners; males crystallize their songs at 90 days and do not subsequently alter those songs. However, a variety of interventions, including deafening and syringeal denervation, result in long-term changes to the crystallized song. These changes can be prevented by lesioning nucleus LMAN. As different social contexts for song production result in differential activation of LMAN, we asked whether the social context experienced by adult males would affect their ability to alter their songs in response to syringeal denervation. Males able to see and direct their songs to females made fewer changes to their songs than did males that could hear but not see females, but this trend was not significant. The volume of a males HVc, a forebrain song control nucleus, also failed to predict the degree to which a male would change his song. However, testis mass was significantly correlated with the number of changes made to the song, indicating that variations in testosterone modulate adult song plasticity. We directly tested the effect of circulating testosterone on adult song plasticity by implanting adult males with either testosterone or flutamide, a testosterone receptor blocker, and tracking song changes triggered by ts nerve injury. As predicted, males implanted with testosterone changed their songs less than did males that received flutamide implants. These results suggest that the high testosterone concentrations associated with sexual maturity and song crystallization in zebra finches continue to act in adult males to reduce the potential for vocal plasticity.