Mice harboring mutations in the obese (ob) and diabetes (db) genes display similar phenotypes, and it has been proposed that these genes encode the ligand and receptor, respectively, for a physiologic pathway that regulates body weight. The cloning of ob, and the demonstration that it encodes a secreted protein (leptin) that binds specifically to a receptor (OB-R) in the brain, have validated critical aspects of this hypothesis. Here it is shown by genetic mapping and genomic analysis that mouse db, rat fatty (a homolog of db), and the gene encoding the OB-R are the same gene.

The rat fatty (fa) mutation produces profound obesity of early onset caused by hyperphagia, defective nonshivering thermogenesis, and preferential deposition of energy into adipose tissue. Genetic mapping studies indicate that fa and diabetes (db) are homologous loci in the rat and mouse genomes, respectively. It has been shown that db alleles carry mutations in the Lepr (leptin receptor) gene. This paper describes a point mutation in the fatty allele of Lepr. A nucleotide substitution at position 880 (A-->C) causes an amino acid substitution at position 269 (Gln-->Pro). The mutation generates a novel Msp I site that cosegregates with fa in 1,028 meioses examined in obese F2 progeny from two crosses (Bnx13M and WKYx13M) and is still segregating in three rat colonies. PCR-based mutagenesis was used to introduce the fa mutation into the mouse Lepr cDNA. Transient transfection studies indicate that the mutant Lepr cDNA has greatly reduced binding of leptin (Lep) at the cell surface. These data are strong evidence that the single nucleotide substitution in the fa allele of Lepr (Leprfa) is responsible for the obese phenotype.

Progesterone receptors play a central role in neuroendocrine and behavioural regulation. To gain insight into the sex- and tissue-specific regulation of progesterone receptors, protein binding on a progesterone receptor-oestrogen response element and mRNA levels for progesterone receptor (PR)-A and PR-B were compared between female and male rats following oestradiol benzoate replacement treatment in hypothalamic and pituitary tissue. Both male and female pituitary protein extracts demonstrated an increase in nuclear protein binding activity to a progesterone receptor-oestrogen response element following oestradiol benzoate treatment. However, there was a greater difference in total binding activity seen in the female pituitary extracts compared to male pituitary protein extracts. In both cases, reflecting the binding data, oestradiol benzoate pretreatment led to an increase in pituitary PR-B messenger RNA, although this increase was significantly larger in females than in males. Oestradiol benzoate treatment also led to a significant increase in specific binding of hypothalamic nuclear proteins to the progesterone receptor oestrogen response element from both females and male hypothalamic extracts. In addition, PR-B messenger RNA was induced by oestradiol benzoate treatment in the female rat hypothalamus, under circumstances where no PR-A could be detected. The male also demonstrated an increase in PR-B messenger RNA following oestradiol benzoate treatment, with undetectable levels of PR-A, although to a lesser degree than that seen in the female. The predominance of PR-B over PR-A messenger RNA in rat hypothalamus and pituitary, and the quantitative differences between female and male rats, could both contribute to the greater responsiveness of female rats to progesterone with respect to control over luteinizing hormone release from the pituitary, and lordosis behaviour regulated by hypothalamic neurones.

We have previously confirmed the estrogen-induced protein of rat uterus to be creatine kinase B (CKB), and demonstrated a 1.7-kilobase pair fragment encompassing the promoter and adjoining 5'-flank to be capable of conferring estrogen responsiveness in HeLa cells. In this study we find an element at -550, aGGTCAgaaCACCCt, with limited similarity to the estrogen response element consensus, to be involved in conferring estrogen responsiveness on the CKB promoter. This element can bind estrogen receptor (ER) and is flanked by two GC boxes, which we find capable of binding bacterially expressed Sp1. Additional responsiveness is found closely associated with the CKB promoter at high levels of cotransfected ER construct. No potential response element was identified in this region, but we find the ER DNA-binding domain to be required.

The obese phenotypes of the diabetes (db) mouse and fatty (fa) rat are due to functional null mutations of the leptin receptor (Lepr). The recessive mutation in the Koletsky (f) obese rat maps to the same genetic intervals as db and fa and fails to complement the fa mutation. Comparison of the sequence of brain Lepr cDNA from +/+ and f/f animals reveals a T2349A transversion resulting in a Tyr763Stop nonsense mutation in the gene just before the transmembrane domain. Virtual absence of Lepr mRNA in whole brain from /^animals is consistent with the presence of a null mutation. The predicted reduced cerebrospinal fluid (CSF) transport of leptin in both f/f and fa/fa mutants is reflected in thẽ 10-fold lower ratio of CSF/plasma leptin concentration in the obese versus lean animals. However, equivalent CSF leptin concentration between lean and obese rats (fa/fa, f/f) indicates that leptin can enter the CSF through a non-Lepr-mediated mechanism, which may be saturated at normal physiological plasma leptin concentration. Diabetes 46:513-518, 1997 T he obese (ob) and diabetes (db) mutations have recently been shown to be in the Lep (leptin) and Lepr (leptin receptor) genes, respectively (1,2). Leptin is secreted from adipose tissue and is a ligand for the receptor whose five or more splice variants are expressed in a tissue-specific fashion (3,4). Because leptin (16 kDa) is produced only in adipose tissue and is too large to From the Laboratory of Human Behavior and Metabolism (X.S.W.-R, S.C.C., cross the blood-brain barrier, it is thought to enter the brain parenchyma via a saturable transport system, possibly constituted by one or more of the various membrane bound splice variants of Lepr.The identical phenotypes of ob and db mutant micewhich include early onset, severe obesity, insulin resistance, and strain-dependent diabetes-and the results of cross circulation experiments between ob and db animals provided the initial evidence that these were mutations in a signaling pathway for the size of body fat stores (5). Rats with a similar autosomal recessive phenotype were described first by Zucker et al. (fatty =/a) (6) and then by Koletsky (Koletsky =f) (7) in two different rodent colonies and rat strains. Based on genetic mapping experiments (8,9), it was suspected (and subsequently proven) that the Zucker fatty mutation was in the same gene as the db mutation (10). The high likelihood that fa and/are mutations in the same gene was demonstrated by the obese phenotype offa/f compound mutants (11-13) and the recent molecular mapping of/to the same genomic interval as/a (13).The recently cloned Lepr gene encodes a single transmembrane-spanning protein that resembles gpl30, a member of the class I cytokine receptor superfamily (1,14). The db and db Pas mutations have been shown to be, respectively, novel splice donor (3) and gene duplication (9) mutations in Lepr. The fa mutation is a Gln269Pro transversion in Lepr that impairs intracytoplasmic transport of the protein (10,15).In this report, we show that / is a nonsense ...