James L. Smart

Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus

The administration of leptin to leptin-deficient humans, and the analogous Lepob/Lepob mice, effectively reduces hyperphagia and obesity. But common obesity is associated with elevated leptin, which suggests that obese humans are resistant to this adipocyte hormone. In addition to regulating long-term energy balance, leptin also rapidly affects neuronal activity. Proopiomelanocortin (POMC) and neuropeptide-Y types of neurons in the arcuate nucleus of the hypothalamus are both principal sites of leptin receptor expression and the source of potent neuropeptide modulators, melanocortins and neuropeptide Y, which exert opposing effects on feeding and metabolism. These neurons are therefore ideal for characterizing leptin action and the mechanism of leptin resistance; however, their diffuse distribution makes them difficult to study. Here we report electrophysiological recordings on POMC neurons, which we identified by targeted expression of green fluorescent protein in transgenic mice. Leptin increases the frequency of action potentials in the anorexigenic POMC neurons by two mechanisms: depolarization through a nonspecific cation channel; and reduced inhibition by local orexigenic neuropeptide-Y/GABA (γ-aminobutyric acid) neurons. Furthermore, we show that melanocortin peptides have an autoinhibitory effect on this circuit. On the basis of our results, we propose an integrated model of leptin action and neuronal architecture in the arcuate nucleus of the hypothalamus.

A Transgenic Marker for Newly Born Granule Cells in Dentate Gyrus

Neurogenesis in the dentate gyrus continues into adulthood, yet little is known about the function of newly born neurons or how they integrate into an existing network of mature neurons. We made transgenic mice that selectively and transiently express enhanced green fluorescent protein (EGFP) in newly born granule cells of the dentate gyrus under the transcriptional control of proopiomelanocortin (POMC) genomic sequences. Analysis of transgenic pedigrees with truncation or deletion mutations indicated that EGFP expression in the dentate gyrus required cryptic POMC promoter regions dispensable for arcuate hypothalamic or pituitary expression. Unlike arcuate neurons, dentate granule cells did not express the endogenous POMC gene. EGFP-positive neurons had immature properties, including short spineless dendrites and small action potentials. Colocalization with bromodeoxyuridine indicated that EGFP-labeled granule cells were ∼2 weeks postmitotic. EGFP-labeled cells expressed markers for immature granule cells but not the glial marker GFAP. The number of EGFP-labeled neurons declined with age and increased with exercise, paralleling neurogenesis. Our results indicate that POMC-EGFP marks immature granule cells and that adult-generated granule cells integrate quite slowly into the hippocampal circuitry.

Proopiomelanocortin Neurons in Nucleus Tractus Solitarius Are Activated by Visceral Afferents: Regulation by Cholecystokinin and Opioids

The nucleus tractus solitarius (NTS) receives dense terminations from cranial visceral afferents, including those from the gastrointestinal (GI) system. Although the NTS integrates peripheral satiety signals and relays this signal to central feeding centers, little is known about which NTS neurons are involved or what mechanisms are responsible. Proopiomelanocortin (POMC) neurons are good candidates for GI integration, because disruption of the POMC gene leads to severe obesity and hyperphagia. Here, we used POMC-enhanced green fluorescent protein (EGFP) transgenic mice to identify NTS POMC neurons. Intraperitoneal administration of cholecystokinin (CCK) induced c-fos gene expression in NTS POMC-EGFP neurons, suggesting that they are activated by afferents stimulated by the satiety hormone. We tested the synaptic relationship of these neurons to visceral afferents and their modulation by CCK and opioids using patch recordings in horizontal brain slices. Electrical activation of the solitary tract (ST) evoked EPSCs in NTS POMC-EGFP neurons. The invariant latencies, low failure rates, and substantial paired-pulse depression of the ST-evoked EPSCs indicate that NTS POMC-EGFP neurons are second-order neurons directly contacted by afferent terminals. The EPSCs were blocked by the glutamate antagonist 2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline. CCK increased the amplitude of the ST-stimulated EPSCs and the frequency of miniature EPSCs, effects attenuated by the CCK1 receptor antagonist lorglumide. In contrast, the orexigenic opioid agonists [d-Ala(2), N-Me-Phe(4), Gly-ol(5)]-enkephalin and met-enkephalin inhibited both ST-stimulated EPSCs and the frequency of miniature EPSCs. These findings identify a potential satiety pathway in which visceral afferents directly activate NTS POMC-EGFP neurons with excitatory inputs that are appropriately modulated by appetite regulators.

Glucocorticoids exacerbate obesity and insulin resistance in neuron-specific proopiomelanocortin-deficient mice

Null mutations of the proopiomelanocortin gene (Pomc) cause obesity in humans and rodents, but the contributions of central versus pituitary POMC deficiency are not fully established. To elucidate these roles, we introduced a POMC transgene (Tg) that selectively restored peripheral melanocortin and corticosterone secretion in Pomc mice. Rather than improving energy balance, the genetic replacement of pituitary POMC in PomcTg mice aggravated their metabolic syndrome with increased caloric intake and feed efficiency, reduced oxygen consumption, increased subcutaneous, visceral, and hepatic fat, and severe insulin resistance. Pair-feeding of PomcTg mice to the daily intake of lean controls normalized their rate of weight gain but did not abolish obesity, indicating that hyperphagia is a major but not sole determinant of the phenotype. Replacement of corticosterone in the drinking water of Pomc mice recapitulated the hyperphagia, excess weight gain and fat accumulation, and hyperleptinemia characteristic of genetically rescued PomcTg mice. These data demonstrate that CNS POMC peptides play a critical role in energy homeostasis that is not substituted by peripheral POMC. Restoration of pituitary POMC expression to create a de facto neuronal POMC deficiency exacerbated the development of obesity, largely via glucocorticoid modulation of appetite, metabolism, and energy partitioning.

Identification of neuronal enhancers of the proopiomelanocortin gene by transgenic mouse analysis and phylogenetic footprinting

ABSTRACT The proopiomelanocortin (POMC) gene is expressed in the pituitary and arcuate neurons of the hypothalamus. POMC arcuate neurons play a central role in the control of energy homeostasis, and rare loss-of-function mutations in POMC cause obesity. Moreover, POMC is the prime candidate gene within a highly significant quantitative trait locus on chromosome 2 associated with obesity traits in several human populations. Here, we identify two phylogenetically conserved neuronal POMC enhancers designated nPE1 (600 bp) and nPE2 (150 bp) located approximately 10 to 12 kb upstream of mammalian POMC transcriptional units. We show that mouse or human genomic regions containing these enhancers are able to direct reporter gene expression to POMC hypothalamic neurons, but not the pituitary of transgenic mice. Conversely, deletion of nPE1 and nPE2 in the context of the entire transcriptional unit of POMC abolishes transgene expression in the hypothalamus without affecting pituitary expression. Our results indicate that the nPEs are necessary and sufficient for hypothalamic POMC expression and that POMC expression in the brain and pituitary is controlled by independent sets of enhancers. Our study advances the understanding of the molecular nature of hypothalamic POMC neurons and will be useful to determine whether polymorphisms in POMC regulatory regions play a role in the predisposition to obesity.

Lack of Proopiomelanocortin Peptides Results in Obesity and Defective Adrenal Function but Normal Melanocyte Pigmentation in the Murine C57BL/6 Genetic Background

Abstract: Mice deficient in proopiomelanocortin peptides (Pomc−/−) generated on a 129 (Aw/ Aw) genetic background were back‐crossed onto the C57BL/6 (a/a) genetic background. These mice exhibited most of the phenotypic characteristics previously reported on the 129 genetic background (Yaswen et al. 1999. Nat. Med. 5: 1066–1070). Adult mice became obese, their adrenals were atrophied, and they had undetectable plasma corticosterone in basal and stressed states. The partial perinatal lethality previously reported was also present on the C57BL/6 background. In addition, we found that both male and female homozygote (−/−) adults were fertile, but when homozygous males were intercrossed with homozygous females, all the pups died in the perinatal period. Attempts to rescue the perinatal lethality of pups from homozygous breeder pairs by supplementing the mothers drinking water with glucocorticoids were unsuccessful. Furthermore, failure to stimulate adrenal development and corticosterone production/release with daily exogenous adreno‐corticotropin‐stimulating hormone (ACTH) injections indicates an adrenal dependence on POMC peptides for normal development and function. While the original Pomc−/− mice, bred on a mixed white‐bellied agouti (Aw/ Aw) 129 genetic background, had patchy alternations in their coat color, they clearly were not a uniform yellow like the lethal yellow (Ay/a) mice. Our Pomc−/− mice bred onto the C57BL/6 (a/a) genetic background had a black coat color indistinguishable from that of the wild‐type C57BL/6 mice, further suggesting that the POMC peptide melanocyte‐stimulating hormone (α‐MSH) is not essential for the production of eumelanin (black/brown) pigmentation.

Spontaneous and Induced Genetic Mutations of the POMC System

Two decades ago a series of biochemical experiments conclusively demonstrated that adrenocorticotropin (ACTH) and P-lipotropin (PLPH) peptides were stoichiometrically generated from a common precursor (1). This result was heralded by one of the landmark studies in the nascent field of eukaryotic cloning, the sequencing of a pituitary cDNA whose deduced amino acids encoded both peptides separated by a pair of basic amino acids (Lys-Arg) (2). Subsequent work demonstrated that the proopiomelanocortin (POMC) prohormone is posttranslationally processed into several biologically active peptides in addition to ACTH and PLPH. Among these are the melanocortin melanocyte-stimulating hormone (aMSH) and the potent opioid s—endorphin (PEND). The mechanism of POMC processing became a paradigm in cellular biology for the elucidation of vesicular trafficking and enzymatic cleavage of secreted peptides from prohormone precursors. A renaissance of scientific interest in POMC has occurred in more recent years with the cloning of a gene family of melanocortin receptors and the demonstration of central melanocortin effects on energy homeostasis. This chapter will review the ongoing role that spontaneous and induced (transgenic and “knockout”) mutant mouse models have played in the analysis of POMC gene regulation and biological function.