C-terminal Monoclonal Antibody Research Articles

62-OR: Evidence of Gut-Derived Glucagon in Man

We have previously shown that totally pancreatectomized patients (PX) and patients with type 2 diabetes (T2D) exhibit hypersecretion of glucagon in response to oral glucose stimulation whereas intravenous glucose suppresses plasma concentrations of glucagon. Here, we evaluated the expression of the gene encoding glucagon (GCG), glucagon peptide content and density of glucagon-positive cells in mucosal biopsies from the upper gastrointestinal tract of PX patients, patients with T2D and nondiabetic controls. Eight PX patients, eight patients with T2D and eight sex, age and BMI-matched healthy controls underwent upper enteroscopy with intestinal biopsy sampling followed by an intraluminal infusion of 100 ml 50% (w/v) glucose solution with frequent blood sampling. The mucosal biopsies were subjected to mRNA sequencing, immunohistochemistry using a new glucagon-directed C-terminal monoclonal antibody, peptide extraction and subsequent measurements of tissue concentrations of glucagon and other proglucagon-derived peptides. GCG expression in the small intestine was 3 to 4-fold higher in PX and T2D patients compared to controls (P<0.01). Extractable glucagon in small intestinal biopsies was 12-fold greater in PX and 5-fold greater in T2D compared to controls (P<0.05). The density of glucagon-positive cells was more than 3-fold higher in PX compared to controls (P<0.05). Plasma glucagon responses to intraluminal administration of glucose was significantly higher in PX and T2D (649±206 vs. 557±142 pmol/l × min [mean±SEM], P<NS) compared to controls (142±39 pmol/l × min, P<0.005). We conclude that glucagon was identifiable in the small intestine of PX and T2D patients, respectively. This may explain the hyperglucagonemia observed after oral glucose in these groups of patients. The role of gut-derived glucagon in diabetes warrants further investigation. Disclosure M. Baekdal: None. A.B. Lund: None. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S. Speaker's Bureau; Self; Merck Sharp & Dohme Corp. J.I. Egholk: None. T. Jorsal: None. C.T. Juel: None. K.D. Galsgaard: None. J. Hunt: None. J.F. Rehfeld: None. K. Rigbolt: Employee; Self; Gubra. Stock/Shareholder; Self; Gubra. S.U. Friis: Stock/Shareholder; Self; Alk-Abello B, Bavarian Nordic, H. Lundbeck A/S, Lundbeck, Novo Nordisk A/S. C. Orskov: None. S.S. Poulsen: None. J.J. Holst: Advisory Panel; Self; Novo Nordisk A/S. T. Vilsbøll: None. F.K. Knop: Advisory Panel; Self; AstraZeneca, MedImmune, Merck Sharp & Dohme Corp., Mundipharma, Novo Nordisk A/S, Sanofi. Consultant; Self; Amgen Inc., Carmot Therapeutics, Novo Nordisk A/S. Research Support; Self; AstraZeneca, Novo Nordisk A/S. Speaker's Bureau; Self; AstraZeneca, MedImmune, Merck Sharp & Dohme Corp., Mundipharma, Norgine, Novo Nordisk A/S. Funding European Foundation for the Study of Diabetes; Novo Nordisk Foundation; Aase og Ejnar Danielsens Foundation; A.P. Møller Foundation

Specific Sorting and Post-Golgi Trafficking of Dendritic Potassium Channels in Living Neurons

Proper membrane localization of ion channels is essential for the function of neuronal cells. Particularly, the computational ability of dendrites depends on the localization of different ion channels in specific subcompartments. However, the molecular mechanisms that control ion channel localization in distinct dendritic subcompartments are largely unknown. Here, we developed a quantitative live cell imaging method to analyze protein sorting and post-Golgi vesicular trafficking. We focused on two dendritic voltage-gated potassium channels that exhibit distinct localizations: Kv2.1 in proximal dendrites and Kv4.2 in distal dendrites. Our results show that Kv2.1 and Kv4.2 channels are sorted into two distinct populations of vesicles at the Golgi apparatus. The targeting of Kv2.1 and Kv4.2 vesicles occurred by distinct mechanisms as evidenced by their requirement for specific peptide motifs, cytoskeletal elements, and motor proteins. By live cell and super-resolution imaging, we identified a novel trafficking machinery important for the localization of Kv2.1 channels. Particularly, we identified non-muscle myosin II as an important factor in Kv2.1 trafficking. These findings reveal that the sorting of ion channels at the Golgi apparatus and their subsequent trafficking by unique molecular mechanisms are crucial for their specific localizations within dendrites.

Loss of estrogen receptor beta expression in malignant human prostate cells in primary cultures and in prostate cancer tissues.

The aim of this study was to investigate the expression of estrogen receptor (ER) beta and alpha genes in normal (N) and malignant (C) primary cultures of human prostate epithelial cells (PEC) and fibroblasts (PFC) and in the prostate tissue donors. Both ERbeta and ERalpha messenger ribonucleic acids were found by RT-PCR analysis in six NPECs and normal prostate tissues and in only one of six CPECs and in the respective cancer tissue donor. The other five CPECs and related cancer tissue donors and all normal and cancer PFCs expressed ERalpha messenger ribonucleic acid alone. Immunoblot analysis, using a polyclonal anti-ERbeta (C-terminal) antibody, demonstrated ERbeta protein in all NPEC lysates and in one of the six CPECS: ERalpha protein was expressed in both NPECs and CPECs when a polyclonal antibody directed against the ERalpha N-terminal domain was used. In contrast, ERalpha protein was not detected in two of the six CPEC lysates when ERalpha C-terminal monoclonal antibodies were used. Using a set of primers designed to amplify the region from exons 6-8, RT-PCR analysis demonstrated the absence of the expected transcript in these cells. The present study shows that the ERbeta gene is expressed together with ERalpha in normal prostates and NPECs, whereas it is barely detectable in prostate cancer and CPECS: Moreover, in some CPECs, the ERalpha gene may be transcribed in a changed protein, resulting from the expression of a deletion variant. Together, these data suggest that prostate malignancy is associated with a potential disorder of ER-mediated pathways.

Phenotypic Duchenne muscular dystrophy with C-terminal domain

We report a patient with X-linked muscular dystrophy who had rapidly progressive muscle weakness and became wheelchair-bound at age 10 years. Clinically, he was diagnosed as having Duchenne muscular dystrophy; however, he was diagnosed as having Becker muscular dystrophy by dystrophin tests using a C-terminal monoclonal antibody. No immunolabelling was observed with a monoclonal antibody against the N-terminal domain. Multiplex polymerase chain reaction analysis revealed the deletion of exons 3–19. The data suggest that the deletion of the N-terminal domain of dystrophin can cause a severe phenotype even when the C-terminus of the protein is well preserved.

Production and characterization of N-terminally and C-terminally directed monoclonal antibodies against pancreatic glucagon

Hybridoma technology has been successfully applied to the production of monoclonal antibodies against a variety of small soluble peptides. We report herein for the first time on the development of monoclonal antibodies to pancreatic glucagon. Twenty-three stable positive hybridomas were detected by radioimmunoassay from five separate fusions and cloned by the limiting dilution method. Four selected monoclonal antibodies were all of the IgG 2a subclass type κ and bound to protein A. One monoclonal antibody (23.8B6) was shown to be directed toward the C-terminal region and another (23.6B4) toward the N-terminal to central region of the glucagon molecule. These antibodies did not cross-react with any of the other peptides tested. Two further monoclonal antibodies (23.4A1, 22.3A6) reacted with the C-terminal third of the glucagon molecule and showed a cross-reaction with the structurally related gastric inhibitory polypeptide of 0.7% and 9.1%, respectively. All but the C-terminal monoclonal antibody 23.8B6 showed a marked cross-reaction with ileal extracts. The N-terminally directed monoclonal antibody 23.6B4 was of sufficient avidity for use in the radio-immunoassay of pancreatic glucagon and gut-glucagonlike immunoreactivity in tissue extracts, being sensitive to changes of pancreatic glucagon of 2.0 fmol/tube at a final titer of culture supernatant of 1:1.4 × 105. In gel permeation chromatography of intestinal extracts, two major peaks were detectable (Kav 0.27 and 0.54). The present findings show that monoclonal antibodies provide sensitive tools for detecting pancreatic glucagon and gut-glucagonlike immunoreactivity. They will be valuable immunoreactants for the development of immunoradiometric assays as well as for large-scale immunoaffinity purification of gut-glucagonlike immunoreactivity.