Plasma agouti-related protein level: a possible correlation with fasted and fed states in humans and rats.

We measured plasma concentrations of agouti-related protein (AGRP) in humans and rats and determined whether these were affected by ingestion of a meal after fasting. In 17 healthy human subjects, the mean plasma concentration of AGRP was lower in the fed state than in the fasted state. Two hours after a breakfast meal, AGRP levels dropped by 39%. By contrast, a continued fast for 2 h increased the average AGRP concentration by 73%. In rats with diet-induced obesity, refeeding resulted in a 50% decrease in plasma AGRP concentrations following a fasting-refeeding protocol. Our results support the notion that plasma AGRP may serve as a biomarker for the transition from a fasted to the satiated state.

Molecular determinants of ligand binding to the human melanocortin-4 receptor.

To elucidate the molecular basis for the interaction of ligands with the human melanocortin-4 receptor (hMC4R), agonist structure-activity studies and receptor point mutagenesis were performed. Structure-activity studies of [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) identified D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full agonist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R transmembrane (TM) residues were mutated (the rationale for choosing specific receptor residues for mutation is outlined in the Results section). Mutation of TM3 residues D122 and D126 and TM6 residues F261 and H264 decreased the binding affinity of NDP-MSH 5-fold or greater, thereby identifying these receptor residues as sites potentially involved in the sought after ligand-receptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found that core melanocortin peptide residue Arg8 interacts at a molecular level with hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to alanine decreased the binding affinity of AGRP (87-132), a C-terminal derivative of the endogenous melanocortin antagonist, 8-fold, and simultaneous mutations D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4R antagonist SHU 9119. These results provide further insight into the molecular determinants of hMC4R ligand binding.

Molecular interaction of Agouti protein and Agouti-related protein with human melanocortin receptors.

Agouti protein and the Agouti-related protein (AGRP) are antagonists of the melanocortin-3 receptor and melanocortin-4 receptor. Both proteins contain 10 cysteines in the C-terminal domain arranged in five disulfide bonds. One possible arrangement of the disulfide bonds predicts an octapeptide loop, and the chemical properties of four residues within this loop (residues 111-114 in human AGRP) bear striking resemblance to those of several melanocortin peptides, including alpha-MSH, MT-II, and SHU-9119. We showed that cyclic synthetic octapeptides based on the sequence of this loop from Agouti protein or human AGRP are functional antagonists of the human melanocortin-4 receptor. All peptides had a lower affinity for the melanocortin-3 receptor than for the melanocortin-4 receptor. Substitution of serines for cysteines resulted in linear peptides which had reduced binding affinities for both receptors. Mutational analysis of human AGRP indicated that its C-terminal domain is functionally equivalent to the intact human AGRP. The RFF111-113 triplet appears to be the most critical portion of AGRP in determining the binding affinity for both melanocortin-3 and melanocortin-4 receptors. These data strongly suggest that the loop defined by Cys-110 and Cys-117 is critical in determining the antagonist activity of human AGRP. Our data provide indirect evidence for the suggestion that the Cys-110 to Cys-117 octapeptide loop of human AGRP mimics the conformation of alpha-MSH, MT-II, and SHU-9119.

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.

13C NMR study of the effects of leptin treatment on kinetics of hepatic intermediary metabolism.

The recent discovery of leptin receptors in peripheral tissue raises questions about which of leptin's biological actions arise from direct effects of the hormone on extraneural tissues and what intracellular mechanisms are responsible for leptin's effects on carbohydrate and lipid metabolism. The present study is focused on the action of leptin on hepatic metabolism. Nondestructive 13C NMR methodology was used to follow the kinetics of intermediary metabolism by monitoring flux of 13C-labeled substrate through several multistep pathways. In perfused liver from either ob/ob or lean mice, we found that acute treatment with leptin in vitro modulates pathways controlling carbohydrate flux into 13C-labeled glycogen, thereby rapidly enhancing synthesis by an insulin-independent mechanism. Acute treatment of ob/ob liver also caused a rapid stimulation of long-chain fatty acid synthesis from 13C-labeled acetyl-CoA by the de novo synthesis route. Chronic leptin treatment in vivo induced homeostatic changes that resulted in a tripling of the rate of glycogen synthesis via the gluconeogenic pathway from [2-13C]pyruvate in ob/ob mouse liver perfused in the absence of the hormone. Consistent with the 13C NMR results, leptin treatment of the ob/ob mouse in vivo resulted in significantly increased hepatic glycogen synthase activity. Chronic treatment with leptin in vivo exerted the opposite effect of acute treatment in vitro and markedly decreased hepatic de novo synthesis of fatty acids in ob/ob mouse liver. In agreement with the 13C NMR findings, activities of hepatic acetyl-CoA carboxylase and fatty acid synthase were significantly reduced by chronic treatment of the ob/ob mouse with leptin. Our data represent a demonstration of direct effects of leptin in the regulation of metabolism in the intact functioning liver.

Leptin gene therapy and daily protein administration: a comparative study in the ob/ob mouse.

We have compared the efficacy of daily injection of recombinant leptin protein (rh-leptin) with adenovirus-mediated delivery of the murine or human leptin gene (Ad-leptin) for treatment of obesity in the obese (ob/ob) mouse model. We demonstrate an improved correction profile for obesity and associated surrogate markers using the adenovirus delivery method. Rate of weight loss and percentage satiety were significantly greater in the mice treated with Adleptin. These findings were associated with lower peak serum leptin levels with Ad-leptin (22.9 +/- 2.6 ng/ml for the human gene, and 48.9 +/- 11.5 ng/ml for the murine gene) compared to rh-leptin (385.2 +/- 36.0 ng/ml). (Values are given as mean +/- standard error of the mean.) Importantly rh-leptin and ex vivo-expressed Ad-leptin were equivalently active in a functional cell-based assay. The primary difference in the two therapeutic approaches is the continuous chronic secretion of leptin mediated by gene delivery, versus the intermittent bolus delivery and rapid clearance of the daily injection of rh-leptin protein. Thus, in vivo findings suggest that leptin effects are better achieved at lower steady-state levels, a pharmacological feature attained here by gene therapy. These findings may have implications for the potential use of leptin in the treatment of obesity.

Localization of leptin binding domain in the leptin receptor.

The leptin receptor is a member of the class I cytokine receptor family and is involved in the control of appetite and body weight. The predicted amino acid sequence of the extracellular region of the cloned leptin receptor differs from that of many other cytokine receptors in that it contains two homologous segments representing potential ligand binding sites. After the analysis of various deletion and substitution mutants of the leptin receptor, we found that the first potential binding motif is not required for leptin binding and receptor activation, whereas modification of the second potential binding motif can lead to inactive receptor mutants. Further deletion analysis generated a minimal binding domain that retains high affinity leptin binding. The leptin binding domain thus has been localized to residues 323-640, which contain the second segment of cytokine receptor domain/fibronectin type 3 domain (residues 428-635). Coexpression of the active isoform of leptin receptor (OB-Rb) with an inactive mutant lacking high affinity leptin binding site led to suppression of the activity mediated by OB-Rb, suggesting that the leptin receptor may exist as a multimeric complex in the absence of leptin.

Production of leptin in Escherichia coli: a comparison of methods.

A procedure is described for gram-scale refolding of Escherichia coli-derived human leptin inclusion bodies. Refolding was achieved by gradually reducing denaturant using a diafiltration method. Refolded leptin is characterized by in vivo modulation of food intake, reduction in body weight, and lowering of insulin and glucose levels in ob/ob mice. In addition, refolded leptin is characterized by radioimmunoassay (RIA) and activation of the leptin receptor in a cell-based assay. For comparison we also refolded leptin by a simple dilution method and produced periplasmic derived leptin, which did not require ex vivo folding. Leptin produced by these three methods and leptin obtained from commercial sources were compared using the RIA and the cell-based assay and appeared to be of comparable quality and potency.

ART (protein product of agouti-related transcript) as an antagonist of MC-3 and MC-4 receptors.

The mRNA encoding an agouti related protein (ART) of unknown biochemical function was previously reported to be up-regulated in the hypothalamus of two genetically obese mouse strains. We have expressed human ART as a secreted protein in COS-7 cells, and show that recombinant ART is functionally active in inhibiting the binding of a radiolabeled alpha-melanocyte stimulating hormone (alpha-MSH) analog to the human melanocortin-3 (MC-3) and melanocortin-4 (MC-4) receptors, while it is not a potent inhibitor of the human melanocortin-5 (MC-5) receptor. ART is an antagonist of the human MC-3 and MC-4 receptors as determined in functional assay. ART appears to be approximately 100-fold more potent than agouti with reference to the MC-3R and MC-4R binding affinity. These data suggest that ART may be a physiological regulator of feeding behavior.

Characterization of the binding and activity of a high affinity, pseudoirreversible morpholino tachykinin NK1 receptor antagonist.

2(S)-((3,5-Bis(trifluoromethyl)benzyl)-oxy)-3(S)-phenyl-4-((3-oxo-1,2,4- triazol-5-yl)methyl)morpholine (L-742,694) is a selective morpholino tachykinin NK1 receptor antagonist that inhibits the binding of 125I-substance P to the human tachykinin NK1 receptor with a Kd = 37 pM. Increasing concentrations of L-742,694 added to cells 15 min prior to agonist progressively increase the apparent EC50 of substance P for inducing the synthesis of inositol phosphate in Chinese hamster ovary (CHO) cells expressing human tachykinin NK1 receptor and decrease the maximal level of stimulation observed. In contrast, addition of substance P and L-742,694 to the cells at the same time results in an increase in the EC50 for substance P with no decrease in the maximal level of stimulation. The compound also decreases the apparent number of binding sites for 125I-substance P observed by Scatchard analysis. Analysis of the binding of [3H]L-742,694 to the tachykinin NK1 receptor shows that it associates with the receptor with k(a) = 3.98 x 10(8) M(-1) min(-1), and dissociates with k(d) = 0.026 min(-1) and t1/2 = 27 min at 22 degrees C. The slow rate of dissociation of L-742,694 from the tachykinin NK1 receptor and the observation that altering the order of addition of antagonist and substance P attenuates the effect of the antagonist on the maximal activation suggest that L-742,694 is a competitive antagonist that can behave as a pseudoirreversible antagonist under some experimental conditions. L-742,694 has reduced affinity for tachykinin NK1 receptors in which alanine has been substituted for Gln165, His197 or His265 in transmembrane helices 4, 5 and 6, respectively. These three residues have previously been shown to be present in the binding site of tachykinin NK1 receptor antagonists of several structural classes. In addition, L-742,694 inhibits binding of the quinuclidine antagonist (2S,3S)-cis-2-(diphenyl methyl)-N-[(2-iodophenyl)-methyl]-1-azabicyclo[2.2.2]octane 3-amine ([125I]L-703,606) with the same affinity as it inhibits binding of 125I-substance P. These data indicate that L-742,694 binds to the same site within the transmembrane domain of the receptor as previously described competitive antagonists.

Fluorescein-Trp25-exendin-4, a biologically active fluorescent probe for the human GLP-1 receptor.

Exendin-4, a reptilian GLP-1 analogue, has been fluorescently labeled by covalently linking a fluorescein moiety onto the Trp residue yielding fluorescein-Trp25-exendin-4 (FLEX). FLEX is equipotent to GLP-1(7-36)-amide and exendin-4 as an inhibitor of [125I] GLP-1 binding to the human GLP-1 receptor stably expressed in CHO cells, and maintains full biological potency and efficacy as measured by the stimulation of cAMP accumulation in these cells. FLEX binding to CHO/hGLP-1R membranes results in an increase in fluorescence anisotropy. The binding is specific and saturable (Kd = 2.0 +/- 0.4 nM), and GLP-1(7-36)-amide and exendin-4 are equipotent inhibitors of FLEX binding to the human GLP-1 receptor. Thus, FLEX is a potent, biologically active ligand that is useful for the study of the binding and functional characteristics of the human GLP-1 receptor.

Purification and reconstitution of a recombinant human neurokinin-1 receptor.

Recombinant human neurokinin-1 receptors expressed in insect cells have been purified to near homogeneity by sequential metal-chelating chromatography and gel filtration chromatography. The purified receptor consists of a single polypeptide with an apparent molecular weight of 50 kD as revealed by SDS gel electrophoresis, and exhibits a specific activity of 19 nmol of L-703,606 bound per mg of protein. Immunoblot experiments further confirm the identity of the stained protein band. The purified receptor binds the antagonist L-703,606 with an affinity similar to that of native human neurokinin-1 receptor, and binds the agonist substance P with an affinity similar to that of the low affinity state of uncoupled native receptor. The purified receptor can be reconstituted with membranes from uninfected insect cells, and the reconstitution results in an increased affinity for substance P, consistent with the reappearance of the high affinity state of the receptor for agonist in the presence of endogenous G proteins. These data indicate that the purified neurokinin-1 receptor is functional with respect to agonist and antagonist binding and G protein coupling.

Interaction of [fluorescein-Trp25]glucagon with the human glucagon receptor expressed in Drosophila Schneider 2 cells.

The human glucagon receptor was expressed at high density in Drosophila Schneider 2 (S2) cells. Following selection with G418 and induction with CuSO4, the cells expressed the receptor at a level of 250 pmol/mg of membrane protein. The glucagon receptor was functionally coupled to increases in cyclic AMP in S2 cells. Protein immunoblotting with anti-peptide antibodies revealed the expressed receptor to have an apparent molecular mass of 48 kDa, consistent with low levels of glycosylation in this insect cell system. Binding of [fluorescein-Trp25]glucagon to S2 cells expressing the glucagon receptor was monitored as an increase in fluorescence anisotropy along with an increase in fluorescence intensity. Anisotropy data suggest that the mobility of the fluorescein is restricted when the ligand is bound to the receptor. Kinetic analysis indicates that the binding of glucagon to its receptor proceeds via a bimolecular interaction, with a forward rate constant that is several orders of magnitude slower than diffusion-controlled. These data would be consistent with a conformational change upon the binding of agonist to the receptor. The combination of [fluorescein-Trp25]glucagon with the S2 cell expression system should be useful for analyzing glucagon receptor structure and function.

The family of G-protein-coupled receptors.

The family of G-protein-coupled receptors can be defined by their similar structural and functional characteristics. Although their primary sequences are quite diverse, these proteins share several common structural features that reflect their common mechanism of action. Mutagenesis and biophysical analysis of several of these receptors indicate that small molecule agonists and antagonists bind to a hydrophobic pocket buried in the transmembrane core of the receptor. In contrast, peptide ligands bind to both the extracellular and transmembrane domains. The mechanisms by which these peptide and small molecule agonists cause receptor activation are being explored by various approaches, but are not yet well defined. A deeper understanding of structural basis for the functional activity of this large family of receptors will have important implications for drug design in a variety of therapeutic areas.

Characterization of a fluorescent substance P analog.

We describe the development and characterization of substance P labeled at Lys3 with fluorescein ([fluorescein Lys3]SP) as a fluorescent probe for the neurokinin 1 (NK1) receptor. [fluorescein Lys3]SP is an agonist at the human NK1 receptor, with an affinity for both the high-affinity and low-affinity binding states of the receptor approximately 6-fold lower than that of substance P. Binding of the probe to the human NK1 receptor expressed in Sf9 insect cells was observed directly by monitoring either a decrease in fluorescence intensity or an increase in anisotropy of the [fluorescein Lys3]SP. Detection by anisotropy gave the larger signal and thus was used to characterize the interaction of [fluorescein Lys3]SP with the receptor. The anisotropy of the bound ligand was 0.17, compared to 0.04 for the free ligand. The fluorescence was quenched by about 15% upon binding to the receptor. Bound [fluorescein Lys3]SP was displaced by unlabeled SP and by the quinuclidine antagonist L-703,606. As expected for an agonist, binding was also reduced by the addition of the nonhydrolyzable guanine nucleotide analog GppNHp. [fluorescein Lys3]SP should provide a useful structural and kinetic probe for the NK1 receptor.

Characterization of the binding domain of the beta-adrenergic receptor with the fluorescent antagonist carazolol. Evidence for a buried ligand binding site.

The antagonist carazolol has been used as a fluorescent probe for the binding site of the beta-adrenergic receptor (beta AR). The fluorescence properties of carazolol are dominated by the emission of the carbazole group, with the fine structure of the spectrum, but not the quantum yield, sensitive to the environment of the probe. The fluorescence emission spectrum of the bound probe is consistent with an extremely hydrophobic environment in the binding site of the receptor. Binding of carazolol to the purified beta AR increases the polarization of the fluorophore. Exposure to collisional quenchers has demonstrated the bound carazolol to be completely inaccessible to the solvent. Furthermore, the fluorescence of bound carazolol is not quenched by exposure to sodium nitrite, a Förster energy acceptor which has an R0 value of 11.7 A with carazolol. Thus, physical analysis of the binding site of the beta AR by carazolol fluorescence indicates that the antagonist binds to the beta AR in a rigid hydrophobic environment which is buried deep within the core of the protein.

Partial agonist effects on the interaction between the atrial muscarinic receptor and the inhibitory guanine nucleotide-binding protein in a reconstituted system.

The mechanism of action of the partial muscarinic agonist pilocarpine was analyzed in a reconstituted system consisting of the purified porcine atrial muscarinic receptor and the purified porcine atrial inhibitory guanine nucleotide-binding protein, Gi. When GTPase activity was measured as a function of receptor.agonist complex concentration at saturating concentrations of either the full agonist carbachol or pilocarpine, both ligands gave similar values of kcat (4.3 +/- 0.2 min-1 for carbachol and 5.4 +/- 0.7 min-1 for pilocarpine); however, the observed dissociation constant for the ligand.receptor complex binding to Gi was about 4-fold lower for carbachol (0.81 +/- 0.19 nM) than for pilocarpine (3.02 +/- 0.83 nM). These results suggested that, in this system, the reduced activity of the partial agonist compared with the full agonist was the result of a decrease in affinity of the receptor.ligand complex for Gi, as opposed to differences in their relative abilities to activate the guanine nucleotide-binding protein. Several analogues of oxotremorine were also tested to determine their effects on the GTPase activity of Gi. Results from these studies indicate that the reconstituted system may be useful in determining structure-function relationships for muscarinic agonists with regard to receptor.Gi interactions.

Reconstitution of muscarinic receptor-mediated inhibition of adenylyl cyclase.

Inhibition of bovine brain calmodulin-sensitive adenylyl cyclase was examined in a system consisting of the reconstituted purified porcine atrial muscarinic acetylcholine receptor, the purified inhibitory guanine nucleotide-binding protein (Gi), and the partially purified stimulatory guanine nucleotide-binding protein.adenylyl cyclase complex. Under conditions where Gi existed mainly as the Gi.GDP complex, adenylyl cyclase was selectively preactivated with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Addition of carbachol formed the receptor.carbachol complex, which catalyzed the exchange of GDP bound to Gi for GTP gamma S, initiating Gi-mediated inhibition of adenylyl cyclase. Adenylyl cyclase activated by calcium plus calmodulin was more sensitive to inhibition by carbachol than either unstimulated adenylyl cyclase or adenylyl cyclase activated by GTP gamma S or forskolin. Studies using the resolved subunits of Gi showed that the beta gamma subunit could inhibit adenylyl cyclase activated by GTP gamma S or calcium plus calmodulin, as well as the unactivated enzyme. The alpha subunit of Gi inhibited adenylyl cyclase only when adenylyl cyclase was activated by calcium plus calmodulin. Possible explanations for these results are discussed.