RHEBI Expression in Embryonic and Postnatal Mouse

Ras homolog enriched in brain (RHEB1) is a member within the superfamily of GTP-binding proteins encoded by the RAS oncogenes. RHEB1 is located at the crossroad of several important pathways including the insulin-signaling pathways and thus plays an important role in different physiological processes. To understand better the physiological relevance of RHEB1 protein, the expres- sion pattern of RHEB1 was analyzed in both embryonic (at E3.5–E16.5) and adult (1-month old) mice. RHEB1 immu- nostaining and X-gal staining were used for wild-type and Rheb1 gene trap mutant mice, respectively. These inde- pendent methods revealed similar RHEB1 expression pat- terns during both embryonic and postnatal developments. Ubiquitous uniform RHEB1/β-gal and/or RHEB1 expres- sion was seen in preimplantation embryos at E3.5 and post- implantation embryos up to E12.5. Between stages E13.5 and E16.5, RHEB1 expression levels became complex: In particular, strong expression was identified in neural tis- sues, including the neuroepithelial layer of the mesenceph- alon, telencephalon, and neural tube of CNS and dorsal root ganglia. In addition, strong expression was seen in certain peripheral tissues including heart, intestine, muscle, and urinary bladder. Postnatal mice have broad spatial RHEB1 expression in different regions of the cerebral cortex, sub- cortical regions (including hippocampus), olfactory bulb, medulla oblongata, and cerebellum (particularly in Purkinje cells). Significant RHEB1 expression was also viewed in internal organs including the heart, intestine, urinary blad- der, and muscle. Moreover, adult animals have complex tis- sue- and organ-specific RHEB1 expression patterns with different intensities observed throughout postnatal develop- ment. Its expression level is in general comparable in CNS and other organs of mouse. Thus, the expression pattern of RHEB1 suggests that it likely plays a ubiquitous role in the development of the early embryo with more tissue-specific roles in later development

The Anorexigenic Fatty Acid Synthase Inhibitor, C75, Is a Nonspecific Neuronal Activator

C75, a recently derived compound that potently suppresses feeding and induces weight loss, has been proposed to act mainly by inhibiting fatty acid synthase (FAS) in central neurons that control feeding. For example, normal, fasting- associated, hypothalamic increases in neuropeptide Y (NPY)/Agouti-related protein (AGRP) expression and decreases in proopiomelanocortin (POMC)/cocaine and amphetamine regulated transcript (CART) expression were reported to be blocked by C75. Using loose-patch extracellular recording in acute slices, we tested the effect of C75 on anorexigenic POMC neurons and orexigenic NPY neurons of the hypothalamic arcuate nucleus, which were identified by promoter-driven GFP expression, as well as on feeding-unrelated cerebellar Purkinje neurons. We expected C75 to activate POMC neurons, inhibit NPY neurons, and have no effect on Purkinje neurons. Instead, C75 activated all cell types, suggesting that it lacks target specificity. This activation was probably not caused by FAS inhibition, because the classical FAS inhibitor, cerulenin, did not have this effect when tested on POMC and NPY neurons. Nonspecific neuronal activation and resulting neurological effects might contribute to the decreased feeding reported to follow centrally administered C75. Injection, ip, of C75 induced severe loosening or liquefaction of stools, weight loss, and decreased food intake in both wild-type and melanocortin-4 receptor knockout mice. In contrast, cerulenin failed to loosen stools, even at a molar dose over 9-fold greater than C75, and had a much smaller effect on body weight. FAS inhibitory activity, by itself, seems to be insufficient to reproduce all of the effects of ip-injected C75

RHEB1 Expression in Embryonic and Postnatal Mouse

Ras homolog enriched in brain (RHEB1) is a member within the superfamily of GTP-binding proteins encoded by the RAS oncogenes. RHEB1 is located at the crossroad of several important pathways including the insulin-signaling pathways and thus plays an important role in different physiological processes. To understand better the physiological relevance of RHEB1 protein, the expres-sion pattern of RHEB1 was analyzed in both embryonic (at E3.5–E16.5) and adult (1-month old) mice. RHEB1 immu-nostaining and X-gal staining were used for wild-type and Rheb1 gene trap mutant mice, respectively. These inde-pendent methods revealed similar RHEB1 expression pat-terns during both embryonic and postnatal developments. Ubiquitous uniform RHEB1/β-gal and/or RHEB1 expres-sion was seen in preimplantation embryos at E3.5 and post-implantation embryos up to E12.5. Between stages E13.5 and E16.5, RHEB1 expression levels became complex: In particular, strong expression was identified in neural tis-sues, including the neuroepithelial layer of the mesenceph-alon, telencephalon, and neural tube of CNS and dorsal root ganglia. In addition, strong expression was seen in certain peripheral tissues including heart, intestine, muscle, and urinary bladder. Postnatal mice have broad spatial RHEB1 expression in different regions of the cerebral cortex, sub-cortical regions (including hippocampus), olfactory bulb, medulla oblongata, and cerebellum (particularly in Purkinje cells). Significant RHEB1 expression was also viewed in internal organs including the heart, intestine, urinary blad-der, and muscle. Moreover, adult animals have complex tis-sue- and organ-specific RHEB1 expression patterns with different intensities observed throughout postnatal develop-ment. Its expression level is in general comparable in CNS and other organs of mouse. Thus, the expression pattern of RHEB1 suggests that it likely plays a ubiquitous role in the development of the early embryo with more tissue-specific roles in later development

Brainstem Raphe Pallidus and the Adjacent Area Contain a Novel Action Site in the Melanocortin Circuitry Regulating Energy Balance

The central melanocortin system plays a critical role in the regulation of energy balance in rodents and humans. The melanocortin signals in both the hypothalamus and brainstem contribute to this regulation. However, how the melanocortin signals of the hypothalamus interact with those intrinsic to the brainstem in the regulation of energy balance is poorly understood. The brainstem raphe pallidus (RPa) and adjacent areas contain melanocortin 4 receptor (MC4-R)-bearing neurons and sympathetic premotor neurons regulating thermogenesis. Here we report that α-melanocyte-stimulating hormone (α-MSH)-immunoreactive (IR) fibers are in close apposition to MC4-R neurons in the RPa. Retrograde tracing studies revealed a unique direct projection from hypothalamic proopiomelanocortin (POMC) neurons to the RPa and adjacent areas of the brainstem in mice and rats. Furthermore, microinjection of the MC3/4-R agonist MTII into the RPa area dose-dependently stimulated oxygen consumption and inhibited feeding, whereas microinjection of the antagonist, SHU9119, enhanced feeding. These data suggest a novel pathway of hypothalamic POMC neuronal efferents to brainstem RPa area MC4-R neurons in the melanocortin circuitry that contribute to coordinate regulation of energy balance

Proopiomelanocortin Physiological Roles: Pituitary Versus Hypathalamic Functions

The fact that the proopiomelanocortin (POMC) gene is a critical component of energy homeostasis and the stress response, two distinct yet not exclusively separate biological functions, distinguishes this gene as very intriguing and unique for st lllldy. The POMC gene encodes a preprohormone that is post-translationally processed into multiple bioactive peptides. The tissue specific regulation and tissue specific post-translational modifications provide a means for the broad spectrum of the gene\u27s biological activities. Understanding the POMC gene\u27s cell-specific regulation and the physiological functions of its encoded peptides has been an ongoing project of multiple labs spanning the last two decades. Initial studies predominately revolved around the role of the POMC peptide adrenocorticotropin-stimulating hormone\u27s (ACTH) function as a key descending-component of the hypothalamicpituitary adrenal axis. Recently, the majority of studies have transitioned to a focus on the hypothalamic POMC peptide, a.-MSH, and its anorexigenic effects. This focus is with good reason. Within affluent societies, the rampant increase in obesity and overweight prevalence and the associated risk for several chronic diseases has accentuated the need for understanding the biological mechanisms of energy homeostasis. Using the murine rodent for its genetic advantages, this thesis attempts to further advance our understanding of the POMC system regarding its gene expression, neuron physiology, and biological functions. The fact that the proopiomelanocortin (POMC) gene is a critical component of energy homeostasis and the stress response, two distinct yet not exclusively separate biological functions, distinguishes this gene as very intriguing and unique for st lllldy. The POMC gene encodes a preprohormone that is post-translationally processed into multiple bioactive peptides. The tissue specific regulation and tissue specific post-translational modifications provide a means for the broad spectrum of the gene\u27s biological activities. Understanding the POMC gene\u27s cell-specific regulation and the physiological functions of its encoded peptides has been an ongoing project of multiple labs spanning the last two decades. Initial studies predominately revolved around the role of the POMC peptide adrenocorticotropin-stimulating hormone\u27s (ACTH) function as a key descending-component of the hypothalamicpituitary adrenal axis. Recently, the majority of studies have transitioned to a focus on the hypothalamic POMC peptide, a.-MSH, and its anorexigenic effects. This focus is with good reason. Within affluent societies, the rampant increase in obesity and overweight prevalence and the associated risk for several chronic diseases has accentuated the need for understanding the biological mechanisms of energy homeostasis. Using the murine rodent for its genetic advantages, this thesis attempts to further advance our understanding of the POMC system regarding its gene expression, neuron physiology, and biological functions. Whether the hypothalamic POMC peptides have functions that are exclusive to hypothalamic neurons or whether pituitary POMC cells provide a redundancy for these hypothalamic neurons was not known. Utilizing promoter-mapping data, we generated a transgene that would selectively rescue pituitary POMC while retaining the neuronal POMC deficiency when crossed onto a POMC null mutant that we obtained from the Hochgeschwender laboratory. Our studies revealed that replacing pituitary POMC (Pomc_1 _; Tg/+) was not sufficient to rescue the phenotypes caused by the ubiquitous absence of POMC peptides in the Pomc_,_ mice. In fact some of the phenotypes present in the Pomc_,_ mouse, such as obesity and diabetes, were accentuated in the mice lacking only CNS POMC. Obesity in the Pomc_,_ mice resulted from a decrease in their basal metabolic rate (BMR). Replacing pituitary POMC and thus glucocorticoids resulted in a further depression in the mouse\u27s BMR. Replacing pituitary POMC did not normalize the HPA axis but instead further suggested a regulation of the HP A axis, independent of glucocorticoids, by hypothalamic POMC neurons. The lack of CRH suppression in the presence of elevated circulating corticosterone in the Pomc-1 -; Tgl+ mice lead us to the previous conclusion. CRH is a known anorexigenic peptide and evidence supporting the opposing regulation of these neurons by POMC peptides, a-MSH and P-endorphin, makes this hypothalamic circuitry very intriguing for not only future stress axis studies but energy homeostasis studies as well

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

The nucleustractus 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

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

Hypothalamic Proopiomelanocortin Neurons Are Glucose Responsive and Express KATP Channels

Hypothalamic proopiomelanocortin (POMC) neurons are critical for controlling homeostatic functions in the mammal. We used a transgenic mouse model in which the POMC neurons were labeled with enhanced green fluorescent protein to perform visualized, whole-cell patch recordings from prepubertal female hypothalamic slices. The mouse POMC-enhanced green fluorescent protein neurons expressed the same endogenous conductances (a transient outward K current and a hyperpolarization-activated, cation current) that have been described for guinea pig POMC neurons. In addition, the selective -opioid receptor agonist DAMGO induced an outward current (maximum of 12.8 1.2 pA), which reversed at K equilibrium potential (EK), in the majority (85%) of POMC neurons with an EC50 of 102 nM. This response was blocked by the opioid receptor antagonist naloxone with an inhibition constant of 3.1 nM. In addition, the -aminobutyric acidB receptor agonist baclofen (40 M) caused an outward current (21.6 4.0 pA) that reversed at EK in these same neurons. The ATP-sensitive potassium channel opener diazoxide also induced an outward K current (maximum of 18.7 2.2 pA) in the majority (92%) of POMC neurons with an EC50 of 61 M. The response to diazoxide was blocked by the sulfonylurea tolbutamide, indicating that the POMC neurons express both Kir6.2 and sulfonylurea receptor 1 channel subunits, which was verified using single cell RT-PCR. This pharmacological and molecular profile suggested that POMC neurons might be sensitive to metabolic inhibition, and indeed, we found that their firing rate varied with changes in glucose concentrations. Therefore, it appears that POMC neurons may function as an integrator of metabolic cues and synaptic input for controlling homeostasis in the mammal