Recently Zhang et al. cloned a gene that is expressed only in adipose tissue of the mouse. The obese phenotype of the ob/ob mouse is linked to a mutation in the obese gene that results in expression of a truncated inactive protein. Human and rat homologues for this gene are known. Previous experiments predict such a hormone to have a hypothalamic target. Hypothalamic neuropeptide Y stimulates food intake, decreases thermogenesis, and increases plasma insulin and corticosterone levels making it a potential target. Here we express the obese protein in Escherichia coli and find that it suppresses food intake and decreases body weight dramatically when administered to normal and ob/ob mice but not db/db (diabetic) mice, which are thought to lack the appropriate receptor. High-affinity binding was detected in the rat hypothalamus. One mechanism by which this protein regulated food intake and metabolism was inhibition of neuropeptide-Y synthesis and release.

Mutations in the obese gene (OB) or in the gene encoding the OB receptor(OB-R) result in obesity, infertility and diabetes in a variety of mouse phenotypes. The demonstration that OB protein (also known as leptin) can normalize body weight in ob/ob mice has generated enormous interest. Most human obesity does not appear to result from a mutant form of leptin: rather, serum leptin concentrations are increased and there is an apparent inability to transport it to the central nervous system (CNS). Injection of leptin into the CNS of overfed rodents resistant to peripheral administration was found to induce biological activity. Consequently, for the leptin to act as a weight-lowering hormone in human obesity, it appears that appropriate concentrations must be present in the CNS. This places a premium on understanding the structure of the hormone in order to design more potent and selective agonists. Here we report the crystal structure at 2.4A resolution of a human mutant OB protein (leptin-E100) that has comparable biological activity to wild type but which crystallizes more readily. The structure reveals a four-helix bundle similar to that of the long-chain helical cytokine family.

The DNA fragment coding for the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, has been inserted into the high-level expression vectors, pIN-HI. A foreign DNA fragment can be cloned in any one of the three reading frames at the unique EcoRI, HindlU or BamHI sites immediately after the ompA signal peptide coding sequence. The cloned foreign gene is under the control of both the lpp promoter and the lac promoteroperator. The expression of the gene is regulated by the lac repressor produced by the same vectors. Using the pIN-III-ompA vector, the DNA fragment coding for only the mature portion of ,B-lactamase was inserted into the EcoRI site. Upon induction of gene expression, ,B-lactamase was secreted into the periplasmic space. The ompA signal peptide was correctly removed resulting in the production of fl-lactamase with four extra amino acid residues (Gly-Ile-Pro-Gly) at its amino terminus due to the linker sequence in the vector. After a 3-h induction, ,B-lactamase was accumulated to 2007o of total ceflular protein without any detectable accumulation of pro-,B-lactamase. Using oligonucleotide-directed site-specific mutagenesis, we have also removed the linker sequence and upon induction of gene expression, 3-lactamase with the authentic NHrterminal sequence was produced, in even larger amounts than the ,3-lactamase with the linker sequence.

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are two closely related peptides that bind two homologous G protein-coupled receptors, VIP/PACAP receptor 1 (VPAC1R) and VIP/PACAP receptor II (VPAC2R), with equally high affinity. Recent reports suggest that VPAC2R plays a role in circadian rhythm and T cell functions. To further elucidate the functional activities of VPAC2R, we generated VPAC2R-deficient mice by deleting exons VIII-X of the VPAC2R gene. The VPAC2R-deficient mice showed retarded growth and had reduced serum IGF-I levels compared with gender-matched, wild-type siblings. The mutant mice appeared healthy and fertile at a young adult age. However, older male mutant mice exhibited diffuse seminiferous tubular degeneration with hypospermia and reduced fertility rate. The mutant mice appeared to have an increase in insulin sensitivity. VPAC2R-deficient mice had increased lean mass and decreased fat mass with reduced serum leptin levels. Indirect calorimetry experiments showed that the respiratory quotient values immediately following the transition into the dark cycle were significantly higher in male knockout mice for about 4 h. Additionally, male and female VPAC2R-deficient mice presented an increased basal metabolic rate (23% and 10%, respectively) compared with their wild-type siblings. Our results suggest that VPAC2R plays an important role in growth, basal energy expenditure, and male reproductive functions.