Aurawan Vongs

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.

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.

A rapid, quantitative functional assay for measuring leptin

At present, leptin is quantitated using immuno-assays that measure leptin mass. Leptin biological activity is determined using protocols that measure feed consumption and weight reduction. These in vivo protocols are semi-quantitative and require large quantities of leptin. We describe a rapid, sensitive and quantitative in vitro assay for leptin using HEK-293 cells stably co-transfected with the leptin receptor Ob-Rb isoform and a STAT-inducible promoter regulating the firefly luciferase cDNA. The assay, performed in a 96-well format, has an EC50 of 150 pM and is linear from 3 to 700 pM of leptin. We demonstrate that the assay is capable of measuring leptin in plasma samples. We demonstrate that bacterially-expressed, recombinant leptin and in vivo expressed leptin are equipotent. Furthermore, we demonstrate that a leptin-derived peptide, leptin fragment 22-56, previously shown to be capable of reducing feed intake following ICV injection does not act directly through the leptin receptor.

Optimization of privileged structures for selective and potent melanocortin subtype-4 receptor ligands.

Design, syntheses and structure-activity relationships of N-acetylated piperazine privileged structures containing MC4R agonist compounds were described. The most potent derivatives were low nM MC4R selective full agonists. Several compounds from the series had modest pharmacokinetic properties.

A Miniaturized Homogenous Assay of Mitochondrial Membrane Potential

Mitochondrial dysfunction is increasingly associated with disease states. These organelles, responsible for adenosine triphosphate production, have been targeted for improved function in such diseases as Parkinsons, Alzheimers, type 2 diabetes, and sarcopenia. In addition, the importance of determining if a clinical drug candidate adversely effects mitochondria function, which could lead to overt toxicity, has been recognized. Hence, assays that measure mitochondria activity have become essential in early stage drug development. Limitations of current assays that measure mitochondria membrane potential have prohibited the high-throughput performance necessary to screen current chemical space. Here, we describe a homogeneous assay to measure mitochondria membrane potential that can utilize either adherent or suspension cell types. The assay has been miniaturized to 1,536-well plate format, and was used to perform a fully automated robotic high-throughput screen of a small molecule chemical library.

Apolipoprotein E4-induced mitochondrial dysfunction depends on its structure and can be rescued by a structure corrector

in cholesterol-rich lipid rafts of cell membrane, their activities are affected by the levels of cholesterol in cell membrane. We have shown previously that there was significant cholesterol retention in AD brain as compared to agematched non-demented controls (ND) and cholesterol is capable of stimulating b-and g-secretase activities (Xiong et al. Neurobiol. Dis. 29:422-437, 2008). The objective of this study is to investigate apoE alleles and the levels of cholesterol in Alzheimer’s and ND brains. Methods: Genotyping and cholesterol assays were used to determine apoE alleles and the levels of cholesterol in ND and AD brains, respectively. Results: Nine ND and 19 AD brain samples were used in the study. All NDs carried two apoE3 alleles. One AD patient carried one apoE2 and one apoE3 allele. Eight AD patients carried two apoE3 alleles. Nine AD patients carried one apoE4 and one apoE3 allele. One AD patient carried two apoE4 alleles. Cholesterol assay showed that the level of cholesterol is significantly higher in 19 AD brains (9.7962.24mg/mg protein) than that of 9 ND brains (7.961.11) (t test, p1⁄40.0243). The level of cholesterol in 10 apoE4 carrier AD patients is significantly higher (10.4562.21) than that of 9 NDs (7.961.11) (t test, p1⁄40.0041) but not than that of 9 apoE3 carrier AD patients (9.0661.99) (t test, p1⁄40.0909). The level of cholesterol in 9 apoE3 carrier AD patients is higher but not statistically significant than that of 9 NDs (t test, p1⁄4 0.0728). Conclusions: The results suggest that apoE4 allele is associated with higher brain cholesterol and may contribute to altered cholesterol metabolism in AD brain.