To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

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The synthesis of (4-bromomethyl-4'-methylbipyridine) [bis(bipyridine)]ruthenium(II) hexafluorophosphate is described. This new reagent was found to selectively label the single sulfhydryl group at Cys-102 on yeast iso-1-cytochrome c to form the (dimethylbipyridine-Cys-102-cytochrome c)[bis(bipyridine)]ruthenium derivative (Ru-102-cyt c). Excitation of Ru-102-cyt c with a short light flash resulted in formation of excited-state Ru(II*), which rapidly transferred an electron to the ferric heme group to form Fe(II). When the cytochrome c peroxidase compound I (CMPI) was present in the solution, electron transfer from photoreduced Fe(II) in Ru-102-cyt c to the radical site in CMPI was observed. At high ionic strength (100 mM sodium phosphate and 25 mM EDTA, pH 7), second-order kinetics were observed with a rate constant of (7.5 +/- 0.7) x 10(7) M-1 s-1. The second-order rate constant for native iso-1-cytochrome c was (6.7 +/- 0.7) x 10(7) M-1 s-1 under these conditions. The second-order rate constant for electron transfer from Ru-102-cyt c to the radical site in CMPI increased as the ionic strength was decreased, reaching a value of (4.8 +/- 0.5) x 10(8) M-1 s-1 in 40 mM EDTA, pH 7. At lower ionic strength, a complex was formed between Ru-102-cyt c and CMPI, and the rate for intracomplex electron transfer to the radical site was found to be greater than 50,000 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Targeted deletion of the gene encoding the neuronal and neuroendocrine secreted polypeptide VGF (nonacronymic) produces a lean, hypermetabolic mouse. Consistent with this phenotype, VGF mRNA levels are regulated in the hypothalamic arcuate nucleus in response to fasting. To gain insight into the site(s) and mechanism(s) of action of VGF, we further characterized VGF expression in the hypothalamus. Double-label studies indicated that VGF and pro-opiomelanocortin were coexpressed in lateral arcuate neurons in the fed state, and that VGF expression was induced after fasting in medial arcuate neurons that synthesize neuropeptide Y (NPY). Like NPY, VGF mRNA induction in this region of the hypothalamus in fasted mice was inhibited by exogenous leptin. In leptin-deficient ob/ob and receptor-mutant db/db mice, VGF mRNA levels in the medial arcuate were elevated. To identify neural pathways that are functionally compromised by Vgf ablation, VGF mutant mice were crossed with obese A(y)/a (agouti) and ob/ob mice. VGF deficiency completely blocked the development of obesity in A(y)/a mice, whereas deletion of Vgf in ob/ob mice attenuated weight gain but had no impact on adiposity. Hypothalamic levels of NPY and agouti-related polypeptide mRNAs in both double-mutant lines were dramatically elevated 10- to 15-fold above those of wild-type mice. VGF-deficient mice were also found to resist diet- and gold thioglucose-induced obesity. These data and the susceptibility of VGF mutant mice to monosodium glutamate-induced obesity are consistent with a role for VGF in outflow pathways, downstream of hypothalamic and/or brainstem melanocortin 4 receptors, that project via the autonomic nervous system to peripheral metabolic tissues and regulate energy homeostasis.

Background: DGAT1 is a triglyceride biosynthetic enzyme with a possible role in metabolic disorders. Results: T-863, a potent DGAT1 inhibitor acting on the acyl-CoA binding site of DGAT1, decreased body weight, improved insulin sensitivity, and alleviated hepatic steatosis in diet-induced obese mice. Conclusion: These data support further exploration of DGAT1 inhibitors for metabolic disorders. Significance: Our study reveals mechanisms of action for DGAT1 inhibitors.