Glucagon is a 29-amino acid polypeptide hormone synthesized by the A cells of the endocrine pancreas. Its primary site of action is the liver where it stimulates glycogenolysis, gluconeogenesis and ketogenesis. In mammals, biosynthetic studies have shown that glucagon is derived from a precursor of molecular weight (Mr) approximately 18,000 which is five to six times larger than glucagon. Glucagon-containing polypeptides and immunoreactants of various sizes have also been described from stomach, intestine, brain and salivary gland. Here, we have determined the structure of hamster pancreatic preproglucagon from the sequence of its cDNA. This 180-amino acid precursor contains the sequence of glucagon and two glucagon-like polypeptides arranged in tandem. The precursor also contains the sequences of several non-pancreatic glucagon-containing polypeptides which suggests that, in mammals, both pancreatic and non-pancreatic glucagon and glucagon-containing polypeptides may be derived from a common precursor by tissue-specific processing. We have tentatively identified each of the glucagon-like immunoreactants which have been described with respect to the sequence of proglucagon and have proposed a scheme for the processing of pancreatic proglucagon.

A clonal hamster beta cell line (HIT) was established by simian virus 40 transformation of Syrian hamster pancreatic islet cells. Cytoplasmic insulin was detected in all cells by indirect fluorescent antibody staining, and membrane-bound secretory granules were observed ultrastructurally. Acidified-ethanol extracts of HIT cell cultures contained hamster insulin as determined by radioimmunoassay, radioreceptor assay, and bioassay. One subclone at passage 39 contained 2.6 ,ug of insulin per mg of cell protein.[3H]Leucine-labeled HIT insulin and proinsulin were identical to islet-derived proteins when compared by NaDodSO4 polyacrylamide gel electrophoresis of immunoprecipitates. HIT cell insulin secretion was stimulated by glucose, glucagon, and 3-isobutyl-l-methylxanthine. Insulin secretion at ogtimal glucose concentration (7.5 mM) was 2.4 milliunits per 10 cells per hr. Somatostatin and dexamethasone markedly inhibited HIT insulin secretion. The HIT cell line represents a unique in vitro system for studying beta cell metabolism and insulin biosynthesis.Much of the current understanding of insulin biosynthesis and beta cell metabolism has been derived from in vitro studies utilizing isolated islets, either intact or dissociated in monolayer culture. Such studies have been limited by (i) the difficulty of preparing even small quantities of islets, (ii) cellular and hormonal heterogeneity within islets, and (iii) rapid loss of insulin production in vitro. The recent availability of a highly differentiated rat insulinoma tumor line (1) has provided experimental material for biochemical studies including purification and characterization ofpreproinsulin mRNA (2) and cloning and sequence analysis ofcDNA (3). Further in vitro studies would be facilitated by the availability of permanent cell lines possessing functions characteristic of differentiated beta cells. Attempts to establish stable insulin-producing cell lines from primary islet monolayer cultures (4-6), insulinomas (7,8), and hybrids ofislet cells and continuous cell lines (9) have met with only limited success. Simian virus 40 (SV40) transformation of rat islets has yielded continuous cell lines that produced a 30,000-dalton protein antigenically related to insulin (10). Rae et al. (11) have recently derived continuous cell lines from the poorly differentiated Kirkman hamster insulinoma which did not make insulin in vitro but resumed insulin synthesis in vivo.In this communication we describe a continuous beta cell line, established from SV40-transformed Syrian hamster islet cells, that produces substantial quantities of insulin and proinsulin in vitro. Preliminary studies characterizing the insulin product and comparing stimulus-secretion coupling between this transformed hamster beta cell line (HIT) and primary islet cultures are also described.MATERIALS AND METHODS Isolation of Pancreatic Islets. Pancreata were dissociated by the collagenase-digestion technique of Lacy and Kostianovsky (12). DNase I (Worthington; code DP, 23 units/ml) was added ...

Human liver cDNA coding for protein C has been synthesized, cloned and sequenced. The abundance of protein C message is approximately 0.02% of total mRNA. Three overlapping clones contain 1,798 nucleotides of contiguous sequence, which approximates the size of the protein's mRNA, based upon Northern hybridization. The cDNA sequence consists of 73 5'-noncoding bases, coding sequence for a 461 amino acid nascent polypeptide precursor, a TAA termination codon, 296 3'-noncoding bases, and a 38 base polyadenylation segment. The nascent protein consists of a 33 amino acid "signal", a 9 amino acid propeptide, a 155 amino acid "light" chain, a Lys-Arg connecting dipeptide, and a 262 amino acid "heavy" chain. Human protein C and Factor IX and X precursors possess about one third identical amino acids (59% in the gamma-carboxyglutamate domain), including two forty-six amino acid segments homologous to epidermal growth factor. Human protein C also has similar homology with prothrombin in the "leader", gamma-carboxyglutamate and serine protease domains, but lacks the two "kringle" domains found in prothrombin.

We describe a new, highly sensitive semiquantitative method for rapid measurement of in vitro mineralization using calcein. Fluorescence analysis of the calcein bound to the calcium phosphate (hydroxyapatite) allows direct quantitation of extracellular matrix mineral content in monolayer cultures of bone-forming cells such as primary osteoblasts or osteosarcoma cells. Osteosarcoma cell lines UMR 106 and SaOS-2 were used to demonstrate that qualitatively, calcein was bound to the same regions of the mineralized cell monolayer as seen by conventional histological staining with von Kossa or Alizarin Red S. Moreover, total bound calcein could be quantitated by direct fluorescence analysis using a Cytofluor II plate reader. Changes in cell monolayer calcein fluorescence were shown to correlate well with direct colorimetric measurement of acid-solubilized Ca+2 from parallel cultures. Relative mineral quantitation by calcein fluorescence is rapid and more sensitive than colorimetric Ca+2 assays, can be performed directly on unfixed or fixed cell monolayers, and does not require the use of radioisotopes. The cell monolayer remains intact and potentially available for further analysis.

Osteocalcin is a major noncollagenous protein component of bone extracellular matrix, synthesized and secreted exclusively by osteoblastic cells in the late stage of maturation, and is considered indicator of osteoblast differentiation. Osteocalcin expression is modulated by parathyroid hormone (PTH) and a variety of other factors. The cAMP-dependent protein kinase pathway has been shown previously to have an essential role in PTH signaling and regulation of osteocalcin expression. To determine the extent to which other pathways may also participate in osteocalcin expression, we used rat and human osteoblast-like cell lines to generate stably transfected clones in which the osteocalcin promoter was fused to a luciferase reporter gene. These clones were examined for their responsiveness to agents known to activate or interfere with protein kinase A (PKA)-and protein kinase C (PKC)-dependent pathways. We have found that forskolin, cAMP, and PTH, as well as insulinlike growth factor I (IGF-I) and basic fibroblast growth factor, all were effective in activating the osteocalcin promoter. Phorbol 12-myristate 13-acetate (PMA) was also a strong inducer of the promoter, indicating that PKC plays a role in expression of osteocalcin. In combination with PTH or forskolin, the effect of PMA was additive to synergistic. Calphostin C, a selective inhibitor of PKC, decreased the PMA-, PTH-, and IGF-I-induced luciferase activity in a dose-dependent manner; a PKA inhibitor, H-89, also blocked the induction by PTH and IGF-I but not by PMA. We conclude that regulation of osteocalcin transcription is mediated by both PKAdependent and PKC-dependent mechanisms and that the respective kinases reside on a linear or convergent pathway.