Pancreatic Explants Research Articles

In vivo inhibition of cysteine proteinases delays the onset of growth of human pancreatic cancer explants.

An animal model was used to study the effects of oral treatment with a small molecular selective inhibitor of cysteine proteinases, Z-Phe-Arg-fluoromethylketone (Z-Phe-Arg-FMK) on primary tumour development. Poorly differentiated rapidly growing and moderately differentiated slowly growing human pancreatic tumours were implanted in the neck of nude mice that were orally treated or not with the inhibitor. Growth rates of the tumours were determined during 38 days after implantation. The poorly differentiated tumours were not affected by treatment with the inhibitor. Development of the moderately differentiated tumours was inhibited significantly by Z-Phe-Arg-FMK treatment. Moreover, the amount of stroma was increased and the volume of cancer cells was reduced in the moderately differentiated tumours that had grown in the treated animals. Reduction in size of the tumours was not achieved by reduction in growth rate but in a delay of the onset of growth. It is concluded that cysteine proteinases play a transient role at the start of tumour development only when cancer cells are surrounded by stroma as was the case in the moderately differentiated but not in the poorly differentiated pancreatic tumours. However, this role of cysteine proteinases can easily be taken over by other proteinases. © 2000 Cancer Research Campaign

Interferon regulatory factor-2 point mutations in human pancreatic tumors

Interferon regulatory factor (IRF)-2, a member of the IRF family, is a transcription factor involved in the regulation of various interferon and virus-stimulated genes and other genes. For example, IRF-2 is an activator of the interferon (IFN)-gamma-inducible MHC class II transactivator (CIITA) type IV promoter. It cooperates with IRF-1 in the activation of the CIITA type IV promoter and can co-occupy the IRF-E of the promoter with IRF-1. In a previous study, we identified an inactivating point mutation in the DNA binding domain of IRF-2 expressed in a human pancreatic tumor cell line that does not express CIITA or MHC class II in response to IFN-gamma. To further assess the potential impact of IRF-2 mutations in tumorigenesis, we screened fresh pancreatic tumor explants and identified 2 IRF-2 point mutations in the 2 alleles of IRF-2 from a single tumor specimen. Both mutations occurred in the DNA binding domain of IRF-2. DNA binding assays demonstrated that the IRF-2 point mutations impaired IRF-2 DNA binding. The transactivation function of the mutant IRF-2s was similarly impaired. This is the first report of IRF-2 mutations in human tumor explants.

Signaling through fibroblast growth factor receptor 2b plays a key role in the development of the exocrine pancreas.

The development of the pancreas depends on epithelial-mesenchymal interactions. Fibroblast growth factors (FGFs) and their receptors (FGFRs 1-4) have been identified as mediators of epithelial-mesenchymal interactions in different organs. We show here that FGFR-2 IIIb and its ligands FGF-1, FGF-7, and FGF-10 are expressed throughout pancreatic development. We also show that in mesenchyme-free cultures of embryonic pancreatic epithelium FGF-1, FGF-7, and FGF-10 stimulate the growth, morphogenesis, and cytodifferentiation of the exocrine cells of the pancreas. The role of FGFs signaling through FGFR-2 IIIb was further investigated by inhibiting FGFR-2 IIIb signaling in organocultures of pancreatic explants (epithelium + mesenchyme) by using either antisense FGFR-2 IIIb oligonucleotides or a soluble recombinant FGFR-2 IIIb protein. Abrogation of FGFR-2 IIIb signaling resulted in a considerable reduction in the size of the explants and in a 2-fold reduction of the development of the exocrine cells. These results demonstrate that FGFs signaling through FGFR-2 IIIb play an important role in the development of the exocrine pancreas.

Sonic hedgehog directs specialised mesoderm differentiation in the intestine and pancreas

The generation of the pancreas and small intestine from the embryonic gut depends on intercellular signalling between the endodermal and mesodermal cells of the gut [1–5]. In particular, the differentiation of intestinal mesoderm into smooth muscle has been suggested to depend on signals from adjacent endodermal cells [1–3]. One candidate mediator of endodermally derived signals in the embryonic hindgut is the secreted protein Sonic hedgehog (Shh) [6]. The Shh gene is expressed throughout the embryonic gut endoderm [7,8] with the exception of the pancreatic bud endoderm, which instead expresses high levels of the homeodomain protein Ipf1/Pdx1 (insulin promoter factor 1/pancreatic and duodenal homeobox 1), an essential regulator of early pancreatic development [9–12]. Here, we have examined whether the differential expression of Shh in the embryonic gut tube controls the differentiation of the surrounding mesoderm into specialised mesoderm derivatives of the small intestine and pancreas. To test this, we used the promoter of the Ipf1/Pdx1 gene to selectively express Shh in the developing pancreatic epithelium. In Ipf1/Pdx1-Shh transgenic mice, the pancreatic mesoderm developed into smooth muscle and interstitial cells of Cajal, characteristic of the intestine, rather than into pancreatic mesenchyme and spleen. Also, pancreatic explants exposed to Shh underwent a similar program of intestinal differentiation. These results provide evidence that the differential expression of endodermally derived Shh controls the fate of adjacent mesoderm at different regions of the gut tube.

Development of fetal sheep pancreas after transplantation into athymic mice

The capacity of the fetal sheep pancreas to grow and function when transplanted into athymic mice was examined to determine whether this source of tissue might be of potential use in reversing diabetes. For this purpose fetal sheep pancreases were obtained in the period between 50 days of gestation and fullterm (148 days). Explants (1 mm 3) in organ culture secreted insulin for at least 7 days, but in steadily diminishing amounts. Acute exposure to arginine (10 mM) and theophylline (10 mM), but not glucose (20 mM), calcium chloride (10 mM), and sodium butyrate (10 mM), caused acute secretion of insulin. Explants survived for many months when grafted beneath the renal capsule of athymic mice, but their growth was less, the epithelia-like component smaller, and the percentage of endocrine cells (31 ±5%) fewer than the case of transplanted fetal human pancreas. The β cell was the predominant endocrine cell in the ungrafted fetal sheep pancreas. In the transplanted fetal sheep pancreas this was not so, the ot and PP cells being dominant—β:α:δPP = 3:14:3:11. This pattern was unchanged when the recipient mice were hyperglycaemic—α:δ:PP = 4:13:4:28, with no reduction of blood glucose levels being observed for up to 4 mo after transplantation. Altering the site of transplantation to the spleen or liver did not improve survival of the endocrine cells. Fetal sheep pancreatic explants when transplanted into athymic rats failed to survive. Thus, although the unusual pattern of endocrine differentiation in fetal sheep pancreas transplanted into athymic mice makes it an interesting model for further studies of fetal development, it is not of benefit in normalizing the blood glucose levels of the recipients.

Development of fetal sheep pancreas after transplantation into athymic mice.

The capacity of the fetal sheep pancreas to grow and function when transplanted into athymic mice was examined to determine whether this source of tissue might be of potential use in reversing diabetes. For this purpose fetal sheep pancreases were obtained in the period between 50 days of gestation and fullterm (148 days). Explants (1 mm3) in organ culture secreted insulin for at least 7 days, but in steadily diminishing amounts. Acute exposure to arginine (10 mM) and theophylline (10 mM), but not glucose (20 mM), calcium chloride (10 mM), and sodium butyrate (10 mM), caused acute secretion of insulin. Explants survived for many months when grafted beneath the renal capsule of athymic mice, but their growth was less, the epithelial-like component smaller, and the percentage of endocrine cells (31 +/- 5%) fewer than the case of transplanted fetal human pancreas. The beta cell was the predominant endocrine cell in the ungrafted fetal sheep pancreas. In the transplanted fetal sheep pancreas this was not so, the alpha and PP cells being dominant--beta:alpha:delta:PP = 3:14:3:11. This pattern was unchanged when the recipient mice were hyperglycaemic--beta:alpha:delta:PP = 4:13:4:28, with no reduction of blood glucose levels being observed for up to 4 mo after transplantation. Altering the site of transplantation to the spleen or liver did not improve survival of the endocrine cells. Fetal sheep pancreatic explants when transplanted into athymic rats failed to survive. Thus, although the unusual pattern of endocrine differentiation in fetal sheep pancreas transplanted into athymic mice makes it an interesting model for further studies of fetal development, it is not of benefit in normalizing the blood glucose levels of the recipients.

Insulin, transforming growth factors, and substrates modulate growth of guinea pig pancreatic duct cells in vitro

Background & Aims : Little is known of the physiological mechanisms that control cellular renewal in the pancreatic excretory duct system. This study investigated the effects of potential regulatory substances on the growth of cultured guinea pig pancreatic duct epithelial monolayers. Methods : Pancreatic duct explants were cultured for 3 days on plastic and on permeable filters in the presence and absence of different substances. Growth of epithelial monolayers from these explants was measured by 5-bromo-2′-deoxyuridine incorporation and morphometric procedures. Results : Epidermal growth factor and insulin both enhanced monolayer growth and together had an additive effect. Transforming growth factor α enhanced and transforming growth factor β inhibited growth, whereas glucagon, somatostatin, pancreatic polypeptide, secretin, cerulein, bombesin, and dexamethasone had no significant effects. Monolayer growth on type I collagen-coated filters was enhanced when compared with that of monolayers grown on tissue culture plastic. Cell growth from explants on filters coated with type IV collagen and fibronectin was comparable with that on plastic, whereas growth on Matrigel- or laminin-coated filters was reduced. Conclusions : Insulin, transforming growth factors, and substrate components modulate growth of pancreatic duct epithelial cells in vitro, suggesting that they are important regulators of cell division in the excretory duct system of the intact pancreas.

Ectopic mineralized cartilage formation in human undifferentiated pancreatic adenocarcinoma explants grown in nude mice.

Mineralized as well as nonmineralized cartilage-like structures enclosing cells resembling chondrocytes were found in human-derived undifferentiated but not in poorly differentiated pancreatic adenocarcinoma explants grown in nude mice. The structures reacted with anti-mouse IgG but not with antibodies against human cytokeratin 19, indicating that the newly formed tissue was of mouse origin. High activity of alkaline phosphatase was found in cell layers surrounding the structures and in cells embedded in the matrix. The extracellular matrix was strongly positive after Sirius red staining, reacted with anti-collagen type II antibodies, and the presence of proteoglycans was demonstrated with Alcian blue staining and by metachromasia after Giemsa staining. Electron microscopic inspection revealed the presence of bundles of both thick collagenous fibrils with low levels of fine filamentous material and thin collagenous fibrils with high concentrations of filamentous components. The majority of both types of matrices was found to be partially or completely calcified. The mean area density of the cartilage-like structures in the undifferentiated tumors was 0.31%. The frequent formation of the cartilage-like structures in the rapidly growing undifferentiated explants and its absence in the slowly growing, more differentiated explants suggest that low oxygen tensions in combination with altered levels of growth factors, such as members of the transforming growth factor beta superfamily, create conditions that induce differentiation of fibroblasts to chondrocytes. It is concluded that these human tumors grown in nude mice can be used as an in vivo model to study ectopic formation of mineralized cartilage.

THE LACK OF INTERACTION BETWEEN TRANSPLANTED HUMAN FETAL PANCREAS AND LIVER

Trophism between transplanted hepatocytes and pancreatic endocrine tissue has been demonstrated with both adult and late gestational fetal tissue. Since this effect has not been looked for with fetal tissue obtained early in pregnancy, we conducted a series of experiments transplanting human liver and pancreas, which was obtained early in the second trimester (15-20 weeks gestation), beneath the renal capsule of athymic mice. Fetal pancreatic explants increased in size after transplantation into nondiabetic mice, but their insulin content 11 weeks later was not different from that of grafts that included liver explants. Reversal of diabetes was achieved in 2 of 5 diabetic mice transplanted with pancreas alone, but none of the mice that received pancreas and liver became normoglycemic. Histological examination of grafted liver explants, which consist of hepatocytes and hematopoietic cells, showed that hepatocytes survived for only two weeks regardless of the presence of pancreatic explants. Bile ducts differentiated by this time in both groups and were still present at 7 weeks. In conclusion, there was no trophic effect observed between transplanted fetal human liver and pancreatic endocrine tissue obtained early in pregnancy; bile duct differentiation is a feature of fetal human liver xenografted into the athymic mouse.

Chronic Hyperglycemia and the Human Fetal β Cell

It is well known that the ability of the immature rodent fetal beta cell to release insulin in response to a glucose challenge can be enhanced by chronic exposure to a high concentration of glucose in vitro. It might be thought that the human fetal beta cell would mature similarly in vitro, because neonates born of diabetic mothers release insulin in a more mature manner than normal infants. Using an organ culture of human fetal pancreatic explants, we have examined this possibility by exposing the tissue to 0-30 mM glucose. Six weeks of exposure of pancreatic explants to as high a concentration of glucose as 30 mM did not cause significant enhancement of the insulinogenic response to an acute challenge with 20 mM glucose. In contrast, chronic insulin release was enhanced, although culture medium containing 2.8 mM glucose was equally as efficacious as that with 30 mM glucose. Just as with insulin, proinsulin levels in the culture media containing no glucose also were suppressed. Degranulation of the beta cell exposed to high concentrations of glucose did not occur, the insulin content of the explants at the end of culture being enhanced in those maintained in 5.6-30 mM but not 2.8 mM glucose. Desensitization to the acute stimulatory effect of 10 mM theophylline did not eventuate, even in explants exposed to 30 mM glucose. In contrast to the human explants, rat fetal pancreatic explants did mature when exposed to 11.2 mM glucose for 1 week.(ABSTRACT TRUNCATED AT 250 WORDS)

Resistance of the Human Fetal P-Cell to the Toxic Effect of Multiple Low-Dose Streptozotocin

The human fetal beta-cell is different from the adult beta-cell not only in that it is unable to release significant amounts of insulin during an acute glucose challenge, but also in that it is resistant to the toxic effects of a bolus of streptozotocin (STZ) and chronic exposure to cytokines tumour necrosis factor-alpha and interferon-gamma. To examine further the differences between the immature and mature beta-cell, these cells were exposed to multiple low doses of STZ (MLS) both in vivo and in vitro, the regimen used being classically toxic to rodent adult beta-cells. The in vivo experiments were conducted after the tissue had been grafted into the athymic mouse. There was no adverse effect of MLS on the insulin content of engrafted human fetal pancreatic explants, either 1, 4, or 15 weeks after the injection of 70 mg/kg/i.p. STZ daily for 5 days. Diabetes, induced in the mice by MLS or a bolus of STZ, was reversed in five of seven MLS-treated mice at 59 +/- 8 days and four of seven STZ-treated animals at 75 +/- 10 days (no significant difference). The growth rate of the implanted tissues also was not adversely affected by the MLS, nor was the percentage of beta-cells 2 weeks after completion of the MLS. In contrast, mouse fetal beta-cells transplanted into the athymic mouse were adversely affected, their insulin content 2 weeks after MLS being only 0.7% that of controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Colonization of the developing pancreas by neural precursors from the bowel.

Neurons in ganglia of the myenteric plexus of the duodenum and stomach have recently been demonstrated to innervate pancreatic ganglia and transsynaptically to excite acinar and islet cells. The hypothesis that crest-derived cells first colonize the foregut and secondarily enter the pancreas by way of the pancreatic buds was tested. Studies were done with fetal rats (days E11-E15). Pancreatic rudiments and foregut were explanted separately and in co-culture. The development of neurons in the explants, identified by demonstrating the immunoreactivities of neurofilaments and growth-associated protein-43 (GAP-43), provided an indirect assay for the presence of neural precursors in the tissue at the time of explantation. Cells of putative neural crest origin were visualized immunocytochemically using the monoclonal antibody, NC-1. Additional markers included the immunoreactivities of dopamine-beta-hydroxylase (DBH), which is expressed by vagal crest-derived cells that colonize the bowel, neuropeptides (substance P and neuropeptide Y [NPY]) found in mature pancreatic neurons, and serotonin (5-HT), which is located in the cell bodies of enteric but not pancreatic neurons. Neurons were detected in cultures of foregut, but not pancreas, when these tissues were explanted by themselves at days E11 and E12. At E11 neural precursors did not leave explants of bowel or migrate into co-cultured pancreatic rudiments. When the foregut was explanted at E12, however, neural precursors migrated away from the bowel, giving rise both to distant ganglia and to neurons within co-cultured pancreatic rudiments. Intrapancreatic ganglia developed in the co-cultures even when the pancreatic attachment to the bowel was severed. Neurons appeared in pancreatic rudiments explanted by themselves on day E13. Neurons developing in pancreatic explants expressed the immunoreactivities of DBH, substance P, and NPY, but not 5-HT. These observations support the idea that pancreatic ganglia develop from crest-derived cells that first colonize the fetal rat foregut and there acquire the ability to colonize the pancreas. A later migration into the pancreatic rudiments of a subset of the original émigrés or their progeny between days E12 and E13 gives rise to a network of pancreatic ganglia that can be regarded as an extension of the enteric nervous system.

Vitellogenin induction by estradiol in channel catfish, Ictalurus punctatus

In oviparous vertebrates estrogens induce hepatic synthesis of vitellogenin (VG), a blood protein sequestered in vitellogenic oocytes and from which lipovitellin (LV) and phosvitin are derived. Our objective was to identify VG in the channel catfish, Ictalurus punctatus. An intraperitoneal injection of estradiol-17β into adult male fish induced a dose-dependent accumulation of a 150 kDa protein (EP) in the plasma. EP was detectable in Coomassie blue-stained polyacrylamide gels within 24 hr after injection of 2 mg hormone/100 g body weight. During the next 4 days, EP increased from 5 to about 25% of the total plasma protein. Electrophoretic mobility, peptide mapping, and immunological crossreactivity showed EP to be indistinguishable from a plasma protein in adult females with vitellogenic ovaries. Two major yolk polypeptides, YP1 (120 kDa) and YP2 (29.6 kDa), were precipitated by (NH 4) 2SO 4 from a yolk protein extract. YP1 but not YP2 reacted with an anti-EP polyclonal antiserum in Western blots. Peptide mapping after proteolysis with trypsin showed YPs 1 and 2 to be unique and revealed structural homologies between YP1 and EP. Liver but not pancreatic explants from an estradiol-treated male synthesized and secreted a [ 35S]methionine-labeled, 150 kDa protein beginning about 2 hr after initial exposure to the label. We tentatively conclude that EP and YP1 represent VG and LV, respectively. YP2 remains unidentified.

30 ORGAN CULTURE OF FETAL RAT PANCREAS: EFFECT OF CCK AND INSULIN ON AMYLASE ACTIVITY

In fetal rats, just before birth, there is a dramatic increase in pancreatic amylase activity. Besides the well demonstrated role of corticosteroids in this process, in vivo studies have suggested that CCK, a trophic hormone for the pancreas in adult rats, might also be involved (Werlin, Biol Neonate 1983; 44: 287-94). Since in vivo studies cannot rule out an indirect maternal effect through corticosteroid secretion, we studied the direct effect of CCK on fetal rat pancreas in organ culture. Furthermore, the role of insulin, another important hormone for the exocrine pancreas in adult rats, was checked by using streptozotocin (STZ). Pancreata from 20 day-old rat fetuses were cultured in a serum-free medium (SFM) for 6 days, with or without dexamethasone 3.10−6M (DXM) and/ or CCK8 2.10−6M. In the presence of these two hormones, and with or without insulin (0.1 U/ml), cultures were exposed to STZ 10−7M for the first day of culture. Pancreatic explants were assayed for proteins and amylase on days 0, 2, 4, 6. Amylase specific activity (ASA) was expressed as percentage of day 0. With SFM alone, almost all the ASA disappeared by day 2 (17.7 % ± 12.5), but was partially but significantly maintained when CCK (48.6% ± 8.8) or DXM (47.9% ±14.4) was added. The DXM effect was significantly maintained for 6 days but not the CCK effect which lasted only 2 days. On day 2 of culture the latter was dose-dependent with a maximum occuring between 10 md 10−10M and was inhibited by asperlicin 10 μM. A combination of CCK and DXM gave the best results in maintenance of ASA (87.4 % ± 21.8 on day 2; 81.9% ± 15.9 on day 4; 62.2% ± 10.3 on day 6). When cultures in this optimal medium (CCK + DXM) were exposed to streptozotozin for the first day of culture, a significant decrease of the ASA was found on days 4 (63.2% ± 21)and 6 {47.6% ± 12.8). This difference was corrected when, in the same protocol, insulin was added for the complete time of culture. Conclusions: CCK, like DXM, is important in the prenatal development of the pancreas in the rat. Its action on ASA is different from and additive, but not synergistic, to that of DXM. The effect of STZ, corrected by insulin, suggests also a role for this hormone. The paracrine effect of insulin on exocrine pancreas, well demonstrated in the adult rat, might be already efficient in the fetus.

Immunomodulation of human fetal cells by the fungal metabolite gliotoxin

Gliotoxin (GT) is a fungal metabolite that reduces the ability of murine macrophages to react immunologically in vitro. It is also capable of modulating the immunogenicity of murine bone marrow cells, so that the onset of graft-versus-host disease in fully allogeneic bone marrow chimeras is delayed. The present study examines the effect of GT on human fetal cells, both in terms of reduction of immunogenicity and toxicity. GT (10 micrograms/ml) significantly decreased the responsiveness in mixed lymphocyte cultures of cells derived from human fetal pancreas, spleen, liver and bone marrow. This concentration of GT was, however, mildly toxic to explants of the pancreas, with a significant reduction in insulin secretion from this tissue during the first day of its organ culture, but not thereafter. GT-treated pancreatic explants were lighter and contained less insulin than the untreated controls 3 months after the tissue had been implanted beneath the renal capsule of nude mice. This difference was not apparent 3 weeks after transplantation into these animals. It is hypothesized that the immunomodulating effect of GT (at a concentration less than 10 micrograms/ml) may be of benefit in treating allografted human fetal pancreas before it is transplanted, as it has for murine adult bone marrow cells.

Tissue culture of human fetal pancreas. Effects of human serum on development and endocrine function of isletlike cell clusters.

The human fetal pancreas represents a source of insulin-producing beta-cells with a potential for transplantation to diabetic patients. It has previously been shown that such cells can be viably maintained in tissue culture media containing fetal calf serum (FCS) and that these explants continue to synthesize and release insulin. In this study the effects of human serum (HS) on the growth and function of human fetal pancreatic explants have been compared with those of FCS. For this purpose, pancreatic glands, obtained after prostaglandin-induced abortions, were briefly exposed to collagenase, and the digest was cultured in RPMI-1640 medium plus 10% pooled HS or FCS. The outgrowth of isletlike cell clusters (ICCs) was monitored. In 31 of 58 consecutively explanted glands, development of ICCs was observed. In the presence of FCS the outgrowth of ICC took place on top of a fibroblast monocellular cell layer; HS effected less growth of fibroblasts and increased the formation of ICCs about sevenfold compared with explants from the same glands maintained in FCS. However, in the explant cultures with HS, the cell number per ICC, expressed as DNA content, was reduced by 50%. In both FCS and HS the insulin content of the medium showed great variability and progressively declined from day 2 to day 5. The medium glucagon concentration also decreased but not to the same extent as that of insulin. Immunocytochemical-stained ICCs showed insulin- and glucagon-positive cells scattered among most nonstained, presumably nonendocrine cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of human placental lactogen and growth hormone on the production of insulin and somatomedin C/insulin-like growth factor I by human fetal pancreas in tissue culture.

We have investigated the ability of glucose, human GH and human placental lactogen (hPL) to alter the content and release of somatomedin C/insulin-like growth factor I (SM-C/IGF-I), and the biosynthesis, content and release of insulin from cultured human fetal pancreas. Fetal pancreatic explants obtained from glands following prostaglandin-induced abortion between 12 and 21 weeks of gestation were maintained in free-floating culture for 3-5 days before the experiments. The explants were then cultured for 3 days in medium containing either 2.7 or 16.7 mmol glucose/l with or without GH (4.5 or 45.5 nmol/l) or hPL (4.6 or 46.5 nmol/l). Serum-free medium from the final 24 h of culture was collected and SM-C/IGF-I and insulin were measured radioimmunologically in both conditioned medium and tissue explants extracted with acid ethanol. Insulin biosynthesis, determined by immunoprecipitation of [3H]leucine incorporated into insulin, was not significantly altered by any experimental variable. Incubation in the presence of 16.7 mmol glucose/l caused an increase of insulin release from explants, but had no consistent action on insulin content, compared with medium containing 2.7 mmol glucose/l. The pancreatic content and release of SM-C/IGF-I were independent of these glucose concentrations. Neither GH nor hPL altered insulin or SM-C/IGF-I content or release in the presence of the lower glucose concentration. At the higher glucose concentration, 45.5 nmol GH/l did not alter insulin release but caused a significant increase in SM-C/IGF-I content.(ABSTRACT TRUNCATED AT 250 WORDS)

Somatomedin-C in human fetal pancreas. Cellular localization and release during organ culture.

The presence of somatomedin-C/insulin-like growth factor I (SM-C/IGF-I) was investigated in human fetal pancreatic glands obtained after prostaglandin-induced abortion at 14-17 wk of gestation. Pancreatic explants were cultured in medium containing 11.1 or 22.2 mM glucose and 20% fetal calf serum for 8-10 days. During this period they were exposed, on two separate occasions, to serum-free culture medium for 24 h. SM-C/IGF-I and insulin were measured radioimmunologically in the serum-free media and in acid-ethanol-extracted homogenates of the cultured explants. SM-C/IGF-I was measurable in the conditioned media only after extraction by reverse-phase chromatography to remove somatomedin-binding proteins. On gel filtration at neutral pH of extracted medium samples, the immunoreactive SM-C/IGF-I was recovered in the region of the homogeneous peptide with an apparent molecular weight of 7000-8000. Explants cultured in 22.2 mM glucose contained more SM-C/IGF-I and had a tendency to release more of the peptide into the culture medium than explants cultured in 11.1 mM. There was no difference in insulin content or release between the two groups. By immunocytochemistry, SM-C/IGF-I was localized to the beta-cells of the endocrine pancreas in both freshly fixed tissue and cultured explants. We conclude that the human fetal pancreas contains SM-C/IGF-I and secretes the peptide during tissue culture. The presence of SM-C/IGF-I in the islets of Langerhans may contribute to the growth and development of the insulin-producing beta-cell.

Effect of phorbol and glucose on insulin secretion from the human fetal pancreas

It has been reported previously that 12-0-tetradecanoylphorbol-13-acetate is capable of stimulating the release of insulin from adult and neonatal pancreatic tissue. The data from this study show that this agent at a conentration of 1.3 uM, in the presence of 2.8 mM glucose, was unable to cause significant secreation of insulin from cultured human fetal pancreatic explants. By contrast 20 mM glucose was able to cause a small but significant immediate increase in secretion of insulin, but was unable to maintain this response beyond ten minutes. When the two agents were combined, a synergistic effect was seen throughout the entire 50 minute period of stimulation. The reason for this synergism is unclear since, whilst both secretagogues were able to cause a rise in the levels of diacylglycerol, together no extra effect was observed.

DNA adduct formation in rat, human and hamster pancreas treated with methylnitrosourea.

The methylation of cellular macromolecules with dimethylnitrosamine (DMN) and methylnitrosourea (MNU) was studied in organ cultured rat, hamster and human pancreatic explants. At concentrations of DMN and MNU that caused similar methylation of protein in human explants DMN caused only 2.6% and 0.3% of the methylation of DNA and RNA that was produced by MNU. The DNA of explants treated with MNU was analyzed. The O6-methylguanine (O6-MeG)/7-methylguanine (7-MeG) ratio was greater in the hamster DNA than in DNA isolated from either rat or human. The time course of removal of methyl adducts from DNA was followed for 6 h after treatment with MNU. No decline in O6-MeG occurred during this period in hamster explants, although there was a decline in the content of 7-MeA and 3-MeA, whereas there was removal of O6-MeG in the DNA from human pancreas explants.