Charles Rosenblum

A Receptor in Pituitary and Hypothalamus That Functions in Growth Hormone Release

Small synthetic molecules termed growth hormone secretagogues (GHSs) act on the pituitary gland and the hypothalamus to stimulate and amplify pulsatile growth hormone (GH) release. A heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs. On the basis of its pharmacological and molecular characterization, this GPC-R defines a neuroendocrine pathway for the control of pulsatile GH release and supports the notion that the GHSs mimic an undiscovered hormone.

Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass.

Genetic and pharmacological studies have defined a role for the melanocortin-4 receptor (Mc4r) in the regulation of energy homeostasis. The physiological function of Mc3r, a melanocortin receptor expressed at high levels in the hypothalamus, has remained unknown. We evaluated the potential role of Mc3r in energy homeostasis by studying Mc3r-deficient (Mc3r−/−) mice and compared the functions of Mc3r and Mc4r in mice deficient for both genes. The 4–6-month Mc3r−/− mice have increased fat mass, reduced lean mass and higher feed efficiency than wild-type littermates, despite being hypophagic and maintaining normal metabolic rates. (Feed efficiency is the ratio of weight gain to food intake.) Consistent with increased fat mass, Mc3r−/− mice are hyperleptinaemic and male Mc3r−/− mice develop mild hyperinsulinaemia. Mc3r−/− mice did not have significantly altered corticosterone or total thyroxine (T4) levels. Mice lacking both Mc3r and Mc4r become significantly heavier than Mc4r−/− mice. We conclude that Mc3r and Mc4r serve non-redundant roles in the regulation of energy homeostasis.

A role for the melanocortin 4 receptor in sexual function

By using a combination of genetic, pharmacological, and anatomical approaches, we show that the melanocortin 4 receptor (MC4R), implicated in the control of food intake and energy expenditure, also modulates erectile function and sexual behavior. Evidence supporting this notion is based on several findings: (i) a highly selective non-peptide MC4R agonist augments erectile activity initiated by electrical stimulation of the cavernous nerve in wild-type but not Mc4r-null mice; (ii) copulatory behavior is enhanced by administration of a selective MC4R agonist and is diminished in mice lacking Mc4r; (iii) reverse transcription (RT)-PCR and non-PCR based methods demonstrate MC4R expression in rat and human penis, and rat spinal cord, hypothalamus, brainstem, pelvic ganglion (major autonomic relay center to the penis), but not in rat primary corpus smooth muscle cavernosum cells; and (iv) in situ hybridization of glans tissue from the human and rat penis reveal MC4R expression in nerve fibers and mechanoreceptors in the glans of the penis. Collectively, these data implicate the MC4R in the modulation of penile erectile function and provide evidence that MC4R-mediated proerectile responses may be activated through neuronal circuitry in spinal cord erectile centers and somatosensory afferent nerve terminals of the penis. Our results provide a basis for the existence of MC4R-controlled neuronal pathways that control sexual function.

The role of melanocortins in body weight regulation: opportunities for the treatment of obesity

Five G-protein-coupled melanocortin receptors (MC(1)-MC(5)) are expressed in mammalian tissues. The melanocortin receptors support diverse physiological functions, including the regulation of hair color, adrenal function, energy homeostasis, feed efficiency, sebaceous gland lipid production and immune and sexual function. The melanocortins (adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), beta-MSH and gamma-MSH) are agonist peptide ligands for the melanocortin receptors and these peptides are processed from the pre-prohormone proopiomelanocortin (POMC). Peptide antagonists for the melanocortin MC(1), MC(3) and MC(4) receptors include agouti-related protein (AgRP) and agouti. Diverse lines of evidence, including genetic and pharmacological data obtained in rodents and humans, support a role for the melanocortin MC(3) and MC(4) receptors in the regulation of energy homeostasis. Recent advances in the development of potent and selective peptide and non-peptide melanocortin receptor ligands are anticipated to help unravel the roles for the melanocortin receptors in humans and to accelerate the clinical use of small molecule melanocortin mimetics.

Apolipoprotein E4 Domain Interaction Mediates Detrimental Effects on Mitochondria and Is a Potential Therapeutic Target for Alzheimer Disease

Apolipoprotein (apo) E4 is the major genetic risk factor for late-onset Alzheimer disease (AD). ApoE4 assumes a pathological conformation through an intramolecular interaction mediated by Arg-61 in the amino-terminal domain and Glu-255 in the carboxyl-terminal domain, referred to as apoE4 domain interaction. Because AD is associated with mitochondrial dysfunction, we examined the effect of apoE4 domain interaction on mitochondrial respiratory function. Steady-state amounts of mitochondrial respiratory complexes were examined in neurons cultured from brain cortices of neuron-specific enolase promoter-driven apoE3 (NSE-apoE3) or apoE4 (NSE-apoE4) transgenic mice. All subunits of mitochondrial respiratory complexes assessed were significantly lower in NSE-apoE4 neurons compared with NSE-apoE3 neurons. However, no significant differences in levels of mitochondrial complexes were detected between astrocytes expressing different apoE isoforms driven by the glial fibrillary acidic protein promoter, leading to our conclusion that the effect of apoE4 is neuron specific. In neuroblastoma Neuro-2A (N2A) cells, apoE4 expression reduced the levels of mitochondrial respiratory complexes I, IV, and V. Complex IV enzymatic activity was also decreased, lowering mitochondrial respiratory capacity. Mutant apoE4 (apoE4-Thr-61) lacking domain interaction did not induce mitochondrial dysfunction in N2A cells, indicating that the effect is specific to apoE4-expressing cells and dependent on domain interaction. Consistent with this finding, treatment of apoE4-expressing N2A cells with a small molecule that disrupts apoE4 domain interaction restored mitochondrial respiratory complex IV levels. These results suggest that pharmacological intervention with small molecules that disrupt apoE4 domain interaction is a potential therapeutic approach for apoE4-carrying AD subjects.

Activation of MAP kinase by MC4-R through PI3 kinase.

The melanocortin 4 receptor (MC4-R) is a Galpha s-coupled receptor known to increase cAMP production following agonist stimulation. We demonstrate that the mitogen-activated protein kinases p42 (ERK2) and p44 (ERK1) are also activated by MC4-R following treatment with the MC4-R agonist NDP-alpha-MSH in stably transfected CHO-K1 cells. This time- and dose-dependent response is abolished by the MC4-R antagonist SHU-9119. p42/p44 MAPK activation is blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 but not by the protein kinase A (PKA) inhibitor Rp-cAMPS, indicating that that signal activating the p42/p44 MAPK pathway is conveyed through inositol triphosphate.

Diminished Hepatic Growth Hormone Receptor Binding in Sex-Linked Dwarf Broiler and Leghorn Chickens:

Abstract Hepatic growth hormone (GH) receptor binding was compared in normal and sex-linked dwarfs (SLD) from both Hubbard and Cornell strain chickens. At 6, 8, and 20 weeks of age, hepatic GH receptor binding in the Hubbard SLD chickens was significantly lower than that of normal fast-growing birds. At 20 weeks of age, only 2 of 22 SLD chickens in the Hubbard broiler strain showed positive binding at a high enough level to allow for Scatchard analysis. The affinity constants and binding capacities of these two SLD chickens were numerically (but not significantly) lower than those of the normal fast-growing birds. We further examined hepatic GH receptor binding in two closely related White Leghorn strains of chickens that have been maintained as closed breeding populations for many years. We observed no detectable hepatic GH binding in the Cornell SLD chickens (N = 20), as compared to the normal-growing control strain (K strain). In both SLD strains, pretreatment with 4 M MgCl2 did not enhance GH binding, suggesting that there was no endogenous GH binding to the receptor. Based on these data, we suggest that the lack, or greatly reduced number, of GH receptors may be a major contributing factor to the dwarfism observed in these strains.

Monitoring leptin activity using the chicken leptin receptor

We report on the construction of a leptin bioassay based on the activation of chicken leptin receptor in cultured cells. A human embryonic kidney (HEK)-293 cell line, stably transfected with the full-length cDNA of chicken leptin receptor together with a STAT3-responsive reporter gene specifically responded to recombinant human and Xenopus leptins. The observed higher sensitivity of chicken leptin receptor to the former is in agreement with the degree of sequence similarity among these species (about 60 and 38% identical amino acids between humans and chickens, and between humans and Xenopus respectively). The specific activation of signal transduction through the chicken leptin receptor, shown here for the first time, suggests that the transition of Gln269 (implicated in the Gln-to-Pro Zucker fatty mutation in rats) to Glu in chickens does not impair its activity. Analysis of leptin-like activity in human serum samples of obese and lean subjects coincided well with leptin levels determined by RIA. Serum samples of pre- and post partum cows showed a tight correlation with the degree of adiposity. However, specific activation of the chicken leptin receptor in this assay was not observed with serum samples from broiler or layer chickens (representing fat and lean phenotypes respectively) or with those from turkey. Similar leptin receptor activation profiles were observed with cells transfected with human leptin receptor. Further work is needed to determine whether the lack of leptin-like activity in the chicken serum samples is due to a lack of leptin in this species or simply to a serum level of leptin that is below the detection threshold.

In ovo transfection of chicken embryos using cationic liposomes

It is reported that cationic liposomes are capable of transfecting embryos in unincubated fertile chicken eggs and that the cationic liposome, TransfectAceTM, has superior properties to LipofectinTM. In order to determine the duration of expression of genes introduced in this way, embryos were transfected with an expression vector encoding the firefly luciferase cDNA under the control of the Rous sarcoma virus long terminal repeat (LTR). Luciferase activity could be observed consistently in day 3 embryos and activity was detectable up to day 8 of incubation. The relative expression of luciferase under the control of different viral promoters was compared in transfected chicken embryo fibroblasts and day 3 embryos. The cytomegalovirus immediate early promoter and the SV40 early promoter directed the highest amount of expression in fibroblasts while the Rous sarcoma virus LTR caused the highest amount of expression in embryos. Chicken embryo fibroblasts were transfected with the luciferase vector in order to examine duration of reporter gene expressionin vitro. Luciferase expression was decreased exponentially over a 24-day period after which point luciferase activity could no longer be detected. These data suggest that stable integration of transfected DNA using liposomes is a rare event. Nevertheless, liposome-mediated transfection of embryos is suitable for the examination of promoter activityin vivo and may be a useful method to transfect genes to study embryonic development.

Lack of leptin activity in blood samples of Adélie penguin and Bar-tailed godwit

Unsuccessful attempts to identify the leptin gene in birds are well documented, despite the characterization of its receptor (LEPR). Since leptin and LEPR have poor sequence conservation among vertebrates, we speculated that a functional assay should represent the best way to detect leptin in birds. Using a leptin bioassay that is based on activation of the chicken LEPR in cultured cells, blood samples from wild birds with extreme seasonal variation in voluntary food intake and fat deposition (Adélie penguins and bar-tailed godwits) were tested for leptin activity. In these experiments, blood samples collected during the pre-incubation and the chick-rearing periods of Adélie penguins, and during the migratory flight and refueling stages of bar-tailed godwits, were found to contain no detectable leptin activity, while the sensitivity of the assay to activation by human blood samples from donor subjects representing a variety of body mass indices and fat contents was clearly demonstrated. These results suggest that in birds, an alternative control mechanism to that of mammals operates in the communication between the body fat tissues and the central control on energy homeostasis.