Transgenic mice expressing either human proinsulin cDNA or mutated proinsulin cDNA in the liver were created. The human proinsulin cDNA was mutated to generate a protein cleavable by the ubiquitous prohormone convertase furin, thus leading to mature insulin peptide. All transgenic lines expressed human C-peptide in the blood, whose level varied according to nutritional conditions. High performance liquid chromatography fractionation of mouse serum revealed that mutant proinsulin was effectively processed into mature insulin in vivo. This transgenic mouse model provides a useful tool for further prospects of gene therapy of insulin-dependent diabetes mellitus.z 1998 Federation of European Biochemical Societies.

It is generally accepted that human 13-melanocyte-stimulating hormone (h.BMSH) does not normally exist in humans but was merely an artifactually generated 22-amino acid peptide corresponding to a lipotropin (LPH) fragment (residues 35-56). We examined whether the shorter 18-amino acid peptide hBMSH-(5-22) could be detected in some human tissues. MATERIALS AND METHODSTissue Collection. The following tumors were surgically obtained from five patients with ectopic ACTH syndrome: tumors E1-E4 were bronchial carcinoid tumors; tumor E5 was a thymic carcinoid tumor. Fragments of pituitary corticotropic adenomas from two patients with Nelson syndrome (N1, N2) were surgically obtained by the transsphenoidal approach. In all cases, macroscopically homogeneous tumor fragments with no evidence of local necrosis were quickly selected by the surgeon, immediately frozen in liquid nitrogen, and subsequently stored at -85°C. Two normal human pituitaries (P1, P2) and hypothalami (HT1, HT2) were obtained at autopsy 2-and 6-hr post-mortem from patients with no evidence of endocrine disease and no history of prior hormonal treatment. They were frozen and stored as indicated. Patients with ACTH-producing tumors had not been subjected to either radiotherapy or chemotherapy.Tissue Extraction. Tissue fragments were homogenized in ice-cold 5 M acetic acid (10 ml/g of tissue) containing 1 mM phenylmethylsulfonyl fluoride, by three to five 15-sec bursts in a PCU2 Polytron (Polytron, Elkhart, IN). The homogenates were centrifuged at 5000 x g for 30 min at 4°C, and the supernatants were lyophilized in multiple aliquots that were subsequently used for RIA and/or chromatographic studies.

A specific propiomelanocortin (POMC) immunoradiometric assay was developed using antibodies directed against ACTH and beta-endorphin (beta end). Partially purified standard POMC was prepared from the human small cell lung carcinoma cell line DMS-79 culture medium. Ten units (U) POMC had the same displacement ability as one pg beta end in a C-terminal beta end radioimmunoassay and thus were close if not equal to 10 pg POMC. This POMC assay was used to investigate patients with ACTH-dependent Cushing's syndrome. Plasma POMC was undetectable (< 60 U/mL) in 17 normal controls and in 4 patients with Addison's disease (concomitant ACTH plasma levels between 362 and 1058 pg/mL). Forty-two patients with Cushing's disease were studied, either before (n = 25) or after (n = 17) bilateral adrenalectomy: 7 patients with highly invasive macroadenomas had high POMC plasma levels, between 240 and 4200 U/ml (concomitant ACTH plasma levels between 77 and 5730 pg/mL); 35 patients, including one with an invasive macroadenoma, had undetectable POMC plasma levels (concomitant ACTH plasma levels between 31 and 2820 pg/mL). Among 20 patients with histologically proven ectopic ACTH syndrome, 16 had high POMC plasma levels, between 80 and 8000 U/mL (concomitant ACTH plasma levels between 45 and 9265 pg/mL); all those tumors were malignant, and the highest POMC/ACTH plasma levels ratios (taken as an index of altered POMC processing) were observed in the 3 patients with small cell carcinomas of the lung; in one of these patients, ACTH and POMC plasma levels both decreased during the course of chemotherapy, in parallel with the reduction of the tumoral mass. Four patients with ectopic ACTH syndrome had undetectable POMC plasma levels (concomitant ACTH plasma levels between 78 and 335 pg/mL): they were all typical bronchial carcinoids. These data show that high POMC plasma level is neither specific for nor constant in ectopic ACTH syndrome. Rather it should be considered as a marker of tumor aggressivity, in pituitary- and non-pituitary tumors. Its diagnostic help appears limited for the most frequent cause of occult ectopic ACTH syndrome, the typical bronchial carcinoids.

Pregnancy is the only condition in which POMC is produced and released physiologically, similar in some respects to the ectopic ACTH syndrome. POMC is derived solely from the placenta, with no interference from maternal pituitary secretion, and is thus a new and specific placental marker.