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

Capacity and kinetics of light-induced cytochrome oxidation in intact cells of photosynthetic bacteria

Light-induced oxidation of the reaction center dimer and periplasmic cytochromes was detected by fast kinetic difference absorption changes in intact cells of wild type and cytochrome mutants ( cycA , cytC4 and pufC ) of Rubrivivax gelatinosus and Rhodobacter sphaeroides . Constant illumination from a laser diode or trains of saturating flashes enabled the kinetic separation of acceptor and donor redox processes, and the electron contribution from the cyt bc 1 complex via periplasmic cytochromes. Under continuous excitation, concentrations of oxidized cytochromes increased in three phases where light intensity, electron transfer rate and the number of reduced cytochromes were the rate liming steps, respectively. By choosing suitable flash timing, gradual steps of cytochrome oxidation in whole cells were observed; each successive flash resulted in a smaller, damped oxidation. We attribute this damping to lowered availability of reduced cytochromes resulting from both exchange (unbinding/binding) of the cytochromes and electron transfer at the reaction center interface since a similar effect is observed upon deletion of genes encoding periplasmic cytochromes. In addition, we present a simple model to calculate the damping effect; application of this method may contribute to understanding the function of the diverse range of c-type cytochromes in the electron transport chains of anaerobic phototrophic bacteria

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

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

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

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