J Korean Soc Endocrinol.  1999 Mar;14(1):14-17.

Leptin and Diabetes Mellitus


BACKGROUND: Leptin secreted from adipose tissue activates not only its receptors expressed abundantly in the brain, including arcuate nucleus but the POMC neurons resulting in secretion of a-MSH. Activation of MC4R by a-MSH increases the energy expenditure and decreases the food intake, and promotes the sympathetic activity.
To analyze the biologic activity of a-MSH analogues, the rat MC3R DNA and human MC4R DNA were stably and independently expressed in CHO cell lines, and we performed the competitive receptor binding assay with [125I]NDP-MSH as a radioligand and cyclic AMP accumulation assay following treatment with different ligands. a-MSH (Ac-Ser-Tyr-Ser- Met-Glue-His-Phe-Arg-Trp-Gly-Lys-Val-NH2) and its analogue a-MSH-ND (Ac-Nle-Asp-His-Dphe- Arg-Trp-Lys-NH2) were synthesized by Genosys Biotechnologies, Inc. The HPLC purified samples for NMR experiments were dissolved in 90% HO/10% DO or 99.9% DO solutions at pH 7.0 with concentrations of 2 mM in 50 mM sodium phosphate buffer. The NMR spectra were acquired on a Bruker AMX-500 or DMX-600 spectrometer in quadrature detection mode equipped with a triple-resonance probe with an actively shielded pulsed field gradient coil.
The preferential order of binding activity for MC3R was NDP-MSH > a-MSH-ND > [Nle4]-MSH with IC50 values (nM) being 0.367 +/- 0.074, 47.20 +/- 9.08 and 106.70 +/- 32.25, respectively. For MC4R, the same preferential order, NDP-MSH (0.566 +/- 0.194) > a-MSH-ND (6.70 +/- 1.07) > [Nle4]a-MSH (238.30 +/- 30.47), were obtained. In c-AMP generation assay, the potency order of peptides was NDP-MSH > a-MSH-ND > a-MSH at both MC3R and MCAR. Accumulated food intake of Sprague Dawley rats was low significantly for six hours after injection of a-MSH-ND into peritoneum. a-MSH forms a hair pin loop conformation, whereas a-MSH-ND, a linear form of MTII, prefers a type I B-turn comprising residues Asp5-His6-DPhe7-Arg8 in NMR study.
we developed in vitro and in vivo bioassay system to evaluate the biologic activity of a-MSH analogues and we also showed that the type I B-turn structure of a-MSH-ND might enhance the binding activity to MC4R.

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