We report on the culture of human insulinoma cells derived from a 32-year-old male patient with hyperinsulinism due to an insulinoma of the pancreas. A single-cell suspension was made by passing insulinoma fragments through a fine-gauge stainless-steel mesh. Cluster-forming insulinoma cells resembling pancreatic islets grew in the presence of fibroblasts. The insulinoma cell clusters could be differentiated from fibroblasts by using in situ pan optic staining and specific immunocytochemical staining (anti-human insulin and anti-human insulinoma monoclonal antibody (mAb) D24). mAb D24 was generated using insulinoma cells as antigen for immunization of a Balb/C mouse and cell fusion by the hybridoma cell technique. The anti-insulinoma cell mAb recognized a 32 kDa protein on immunoblot analysis of neuroendocrine tumor cells. D24 mAb also reacted immunohistochemically with normal pancreatic beta-cells and tumors such as vipoma, gastrinoma and carcinoid. Insulinoma cell clusters separated from fibroblasts by micromanipulation and plated into multiwell culture dishes exhibited an insulin-secretion rate of approximately 30 U/100 cells per 24 h with no insulin-secretory response to elevated glucose concentration. Purified insulinoma cells incubated with 1 ng/ml human nerve growth factor expressed neurofilament and neurite extension. These findings together with earlier observations in animal models suggest that human pancreatic beta-cells share some properties with neurons and are related to other neuroendocrine cells in the gastrointestinal tract.
L Wagner, E Templ, G Reining, W Base, M Weissel, P Nowotny, K Kaserer and W Waldhausl
W Karges, K Jostarndt, S Maier, A Flemming, M Weitz, A Wissmann, B Feldmann, H Dralle, P Wagner and BO Boehm
Germ line mutations of the multiple endocrine neoplasia type 1 (MEN1) tumour suppressor gene cause MEN1, a rare familial tumour syndrome associated with parathyroid hyperplasia, adenoma and hyperparathyroidism (HP). Here we investigated the role of the MEN1 gene in isolated sporadic and familial HP. Using RT-PCR single-strand conformational polymorphism screening, somatic (but not germ line) mutations of the MEN1 coding sequence were identified in 6 of 31 (19.3%) adenomas from patients with sporadic primary HP, but none in patients (n=16) with secondary HP due to chronic renal failure. MEN1 mutations were accompanied by a loss of heterozygosity (LOH) for the MEN1 locus on chromosome 11q13 in the adenomas as detected by microsatellite analysis. No DNA sequence divergence within the 5' region of the MEN1 gene, containing the putative MEN1 promoter, was detectable in HP adenomas. Clinical characteristics were not different in HP patients with or without MEN1 mutation. Heterozygous MEN1 gene polymorphisms were identified in 9.6% and 25% of patients with primary and secondary HP respectively. In a large kindred with familial isolated familial HP, MEN1 germ line mutation 249 del4 and LOH was associated with the HP phenotype and a predisposition to non-endocrine malignancies. We suggest that the bi-allelic somatic loss of MEN1 wild-type gene expression is involved in the pathogenesis of a clinically yet undefined subset of sporadic primary HP adenomas. MEN1 genotyping may further help define the familial hyperparathyroidism-MEN1 disease complex, but it seems dispensable in sporadic primary HP.
S M Baumgartner-Parzer, L Wagner, G Reining, V Sexl, P Nowotny, M Müller, M Brunner and W Waldhäusl
Hyperthyroidism is associated with elevated plasma levels of endothelium-derived proteins such as von Willebrand factor (vWF), fibronectin (FN) and endothelin-1 (ET-1). This study was designed to characterize the mechanisms involved in this phenomenon at the cellular level. vWF, FN and ET-1 secretion and mRNA expression were measured in human umbilical vein endothelial cells (HUVECs) exposed to tri-iodothyronine (T3) for 13 ± 1 days, using ELISA, Western blot, RIA and Northern blot analysis respectively. Exposure of HUVECs to T3 significantly increased vWF secretion (50 ng T3/ml: 117 ± 5%, P<0·01; 100 ng T3/ml: 127 ± 26%, P<0·01) as well as vWF mRNA expression (50 ng/ml: 116 ± 13%, P<0·001; 100 ng/ml: 136 ± 30%, P<0·002) (results are means ± s.d. analysed by the Wilcoxon signed rank test). FN secretion was significantly affected by 50 (145 ± 42% of control, P<0·05) and 100 (116·8 ± 16% of control, P<0·05) ng T3/ml, and FN mRNA expression by 50 ng T3/ml (123 ± 20%, P<0·05). Long-term incubation with T3 increased both ET-1 secretion (25 ng/ml: 124 ± 25%, P<0·001; 50 ng/ml: 165 ± 53%, P<0·05; 100 ng/ml: 116 ± 17%, P<0·05) and prepro-ET-1 mRNA expression (25 ng/ml: 112 ± 16%, P<0·05; 50 ng/ml: 134 ± 43%, P<0·02; 100 ng/ml: 120 ± 20%, P<0·02). Protein kinase C (PKC) isoforms ε and βII were not significantly affected by T3, whereas PKC α was increased in whole cell lysates and in membrane fractions of cells incubated with 100 but not 50 ng T3/ml. Prepro-ET-1 mRNA stability, cell numbers and proliferation, measured by [3H]thymidine assays, remained unaffected in HUVECs after exposure to T3. These data indicate thyroid hormone-induced upregulation of mRNA expression and protein synthesis of vWF, FN and ET-1, by PKC α-, βII- and ε-independent pathways, explaining, at least in part, increased plasma concentrations of endothelial proteins and peptides in the hyperthyroid state.
Journal of Endocrinology (1997) 154, 231–239