The imaging of insulinomas using a radionuclide-labelled molecule of the GLP-1 analogue liraglutide: a new application of liraglutide.

OBJECTIVE: This study explores a new, non-invasive imaging method for the specific diagnosis of insulinoma by providing an initial investigation of the use of 125I-labelled molecules of the glucagon-like peptide-1 (GLP-1) analogue liraglutide for in vivo and in vitro small-animal SPECT/CT (single-photon emission computed tomography/computed tomography) imaging of insulinomas. METHODS: Liraglutide was labelled with 125I by the Iodogen method. The labelled 125I-liraglutide compound and insulinoma cells from the INS-1 cell line were then used for in vitro saturation and competitive binding experiments. In addition, in a nude mouse model, the use of 125I-liraglutide for the in vivo small-animal SPECT/CT imaging of insulinomas and the resulting distribution of radioactivity across various organs were examined. RESULTS: The labelling of liraglutide with 125I was successful, yielding a labelling rate of approximately 95% and a radiochemical purity of greater than 95%. For the binding between 125I-liraglutide and the GLP-1 receptor on the surface of INS-1 cells, the equilibrium dissociation constant (Kd) was 128.8 ± 30.4 nmol/L(N = 3), and the half-inhibition concentration (IC50) was 542.4 ± 187.5 nmol/L(N = 3). Small-animal SPECT/CT imaging with 125I-liraglutide indicated that the tumour imaging was clearest at 90 min after the 125I-liraglutide treatment. An examination of the in vivo distribution of radioactivity revealed that at 90 min after the 125I-liraglutide treatment, the target/non-target (T/NT) ratio for tumour and muscle tissue was 4.83 ± 1.30(N = 3). Our study suggested that 125I-liraglutide was predominantly metabolised and cleared by the liver and kidneys. CONCLUSION: The radionuclide 125I-liraglutide can be utilised for the specific imaging of insulinomas, representing a new non-invasive approach for the in vivo diagnosis of insulinomas.

Expression, purification and preliminary characterization of glucagon receptor extracellular domain.

Glucagon is a pancreatic hormone that plays pivotal roles in regulating glucose homeostasis and metabolism. Glucagon exerts its action by binding to its receptor, glucagon receptor (GCGR), one of class B G-protein coupled receptors (GPCRs). Diabetes is a bihormonal disease in which excessive glucagon secretion is a major contributor in the pathogenesis of this disease; elucidation of how glucagon binds to GCGR will facilitate the rational design of the GCGR antagonist for treating diabetic hyperglycemia. Here we report the successful expression and purification of the GCGR extracellular domain (GCGR-ECD) and its fusion protein with the glucagon peptide at its C-terminus (GCGR-ECD-Gc). We utilized the maltose binding protein (MBP) fusion method and disulfide bond isomerase DsbC co-expression approach for the success of the soluble expression of both GCGR-ECD and GCGR-ECD-Gc in Escherichia coli. We also obtained a high yield production of secreted GCGR-ECD with the baculovirus expression system by optimizing its N-terminal secreting signal. We first utilized isothermal titration calorimetry approach to determine the in vitro binding affinities of glucagon to the GCGR-ECD. No significant differences were found between the prokaryotic expressed GCGR-ECD (7.6µM) and the eukaryotic glycosylated one (6.6µM). The observation of the intra ligand-receptor binding within the fusion protein GCGR-ECD-Gc suggests it as a good candidate for further structural study.

Expression of glucagon receptors in tetracycline-inducible HEK293S GnT1- stable cell lines: an approach toward purification of receptor protein for structural studies.

Glucagon is a 29-amino acid polypeptide hormone secreted by pancreatic A cells. Together with insulin, it is an important regulator of glucose metabolism. Type 2 diabetes is characterized by reduced insulin secretion from pancreatic B cells and increased glucose output by the liver which has been attributed to abnormally elevated levels of glucagon. The glucagon receptor (GR) is a member of family B G protein-coupled receptors, ligands for which are peptides composed of 30-40 amino acids. The impetus for studying how glucagon interacts with its membrane receptor is to gain insight into the mechanism of glucagon action in normal physiology as well as in diabetes mellitus. The principal approach toward this goal is to design and synthesize antagonists of glucagon that will bind with high affinity to the GR but will not activate it. Site-directed mutagenesis of the GR has provided some insight into the interactions between glucagon and GR. The rational design of potent antagonists has been hampered by the lack of structural information on receptor-bound glucagon. To obtain adequate amounts of receptor protein for structural studies, a tetracycline-inducible HEK293S GnT1(-) cell line that stably expresses human GR at high-levels was developed. The recombinant receptor protein was characterized, solubilized, and isolated by one-step affinity chromatography. This report describes a feasible approach for the preparation of human GR and other family B GPCRs in the quantities required for structural studies.

In vitro folding, functional characterization, and disulfide pattern of the extracellular domain of human GLP-1 receptor.

The N-terminal, extracellular domain of the receptor for glucagon-like peptide 1 (GLP-1 receptor) was expressed at a high level in E. coli and isolated as inclusion bodies. Renaturation with concomitant disulfide bond formation was achieved from guanidinium-solubilized material. A soluble and active fraction of the protein was isolated by ion exchange chromatography and gel filtration. Complex formation with GLP-1 was shown by cross-linking experiments, surface plasmon resonance measurements, and isothermal titration calorimetry. The existence of disulfide bridges in the N-terminal receptor fragment was proven after digestion of the protein with pepsin. Further analysis revealed a disulfide-binding pattern with links between cysteines 46 and 71, 62 and 104, and between 85 and 126.

Characterization of glucagon-like peptide-1 receptor-binding determinants.

Glucagon-like peptide 1 (GLP-1) is a potent insulinotropic hormone currently under study as a therapeutic agent for type 2 diabetes. Since an understanding of the molecular mechanisms leading to high-affinity receptor (R) binding and activation may facilitate the development of more potent GLP-1R agonists, we have localized specific regions of GLP-1R required for binding. The purified N-terminal fragment (hereafter referred to as NT) of the GLP-1R produced in either insect (Sf9) or mammalian (COS-7) cells was shown to bind GLP-1. The physical interaction of NT with GLP-1 was first demonstrated by cross-linking ((125)I-GLP-1/NT complex band at approximately 28 kDa) and secondly by attachment to Ni(2+)-NTA beads. The GLP-1R NT protein attached to beads bound GLP-1, but with lower affinity (inhibitory concentration (IC(50)): 4.5 x 10(-7) M) than wild-type (WT) GLP-1R (IC(50): 5.2 x 10(-9)M). The low affinity of GLP-1R NT suggested that other receptor domains may contribute to GLP-1 binding. This was supported by studies using chimeric glucose-dependent insulinotropic polypeptide (GIP)/GLP-1 receptors. GIP(1-151)/GLP-1R, but not GIP(1-222)/GLP-1R, exhibited specific GLP-1 binding and GLP-1-induced cAMP production, suggesting that the region encompassing transmembrane (TM) domain 1 through to TM3 was required for binding. Since it was hypothesized that certain charged or polar amino acids in this region might be involved in binding, these residues (TM2-TM3) were analyzed by substitution mutagenesis. Five mutants (K197A, D198A, K202A, D215A, R227A) displayed remarkably reduced binding affinity. These studies indicate that the NT domain of the GLP-1R is able to bind GLP-1, but charged residues concentrated at the distal TM2/extracellular loop-1 (EC1) interface (K197, D198, K202) and in EC1 (D215 and R227) probably contribute to the binding determinants of the GLP-1R.

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.

Glucagon-like peptide-1 binding to rat skeletal muscle.

We have found [125I]glucagon-like peptide-1(7-36)-amide-specific binding activity in rat skeletal muscle plasma membranes, with an estimated M(r) of 63,000 by cross-linking and SDS-PAGE. The specific binding was time and membrane protein concentration dependent, and displaceable by unlabeled GLP-1(7-36)-amide with an ID50 of 3 x 10(-9) M of the peptide; GLP-1(1-36)-amide also competed, whereas glucagon and insulin did not. GLP-1(7-36)-amide did not modify the basal adenylate cyclase activity in skeletal muscle plasma membranes. These data, together with our previous finding of a potent glycogenic effect of GLP-1(7-36)-amide in rat soleus muscle, and also in isolated hepatocytes, which was not accompanied by a rise in the cell cyclic AMP content, lead use to believe that the insulin-like effects of this peptide on glucose metabolism in the muscle could be mediated by a type of receptor somehow different to that described for GLP-1 in pancreatic B cells, where GLP-1 action is mediated by the cyclic AMP-adenylate cyclase system.

Localization and diffusion of glucagon receptor in rat hepatocytes.

The lateral diffusion rate of glucagon receptor in rat hepatocyte plasma membrane in the absence and presence of glucagon was measured to be approximately 7.0 x 10(-10) cm2/s. The percentage of glucagon receptor molecules remaining on the cell surface after the activation of signal transduction process by 100 nM glucagon was approximately 74% of its original number. Although the number of glucagon receptors on the plasma membrane capable of interacting with its signal transduction partners decreases on addition of glucagon, the lateral diffusion rate and the percentage of mobile receptors remain essentially unchanged. A hypothesis has been developed that for signal transduction to occur, the random diffusion-dependent collision of one, two, or all three components is an essential part, and it may be the rate-limiting step. An approximate calculation has been made of random diffusion-dependent theoretical and experimental collision frequencies using experimentally measured concentrations and reasonable value for diffusion rate of G protein to investigate the role of diffusion in signal transduction. These calculations indicate that the diffusion of individual components is important and may be the rate-limiting step in the signal transduction process. The diffusion rate and percent mobile fraction of glucagon receptor data presented in this article are the first step toward elucidating the validity of the diffusion-dependent signal transduction hypothesis.

[Molecular cloning and functional expression of glucagon receptors in the liver, the heart and various other tissues]. / Le clonage moléculaire et l'expression fonctionnelle du récepteur à glucagon dans le foie, le coeur et quelques autres tissus.

The mRNA for a glucagon receptor is present in five glucagon-responsive tissues of rat, including the liver, heart, pancreatic islets, kidneys and adipose tissue. The mature mRNA of this receptor is likely to derive from one gene only in all tissues investigated. The maturation of the pre-mRNA requires the splicing of 11 introns. This proceeds stepwise at the 5' end but a single intronless ORF is finally operative. The receptor is a glycoprotein endowed with 485 amino acids (including the signal peptide).