Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01275 (glucagon)
 26,492 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Primary liver cells, isolated from 16- 17-day-old chick embryos, were incubated in a serum-free chemically defined medium (Ham's F12) supplemented with hormones for up to 6 days. The culture method also includes the complete removal of contaminating red cells before the initiation of culture. On the 2nd day in cluture, the level of amino-levulinate (ALA) synthase activity in response to allylisopropylacetamide (AIA) was increased 6-fold in cells grown in F12. Insulin, hydrocortisone, and triiodothyronine alone had no appreciable effects on ALA synthase levels. On the other hand, when added with AIA, insulin, insulin plus hydrocortisone, insulin plus hydrocortisone triiodothyronine increased ALA synthase levels 17-, 50-, 110-fold, respectively. The maximally induced levels of ALA synthase activity by AIA in the presence of insulin, hydrocortisone, and triiodothyronine were approximately 15 nmol of ALA/mg of protein/h, 37 degrees or 3 micronmol of ALA/g of tissue/h, 37 degrees, a value similar to that found in ovo or at least 5 times greater than that found in rat liver. The morphology of hepatocytes was maintained for at least 6 days in culture, although the induction of ALA synthase was reduced after the 4th day unless triiodothyronine was present. Dibutyryl adenosine 3':5'-monophosphate (10(8) M) or glucagon (5x10(8) M) had little effect on the induced as well as noninduced levels of ALA synthase or porphyrins. These data demonstrate a "permissive" effect of insulin, hydrocortisone, and triiodothyronine on the induction of ALA synthase and porphyrins by AIA in cultured chick embryo liver cells. In the absence of insulin hydrocortisone, or triiodothyronine, AIA produces only a slight increase in ALA synthase activity or porphyrins (or both); on the other hand, it produces a marked increase in the enzyme activity and porphyrins when these hormones are added to the culture medium. The term "permissive" is applied to these hormone-dependent effects. A sensitive spectrofluorometric method for heme quantitation allowed us to follow changes in the cellular heme content in hemoglobin-free cultured liver cells. Heme content in the cultured liver cells was approximately 250 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein during 48 h of incubation. The apparent decrease in heme content may be accounted for by the concomitant increase in protein content in these cells.
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PMID:Induction of aminolevulinate synthase and porphyrins in cultured liver cells maintained in chemically defined medium. Permissive effects of hormones on induction process. 32 58

High yields of human hepatocytes (up to 23 X 10(6) viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham's F12 containing 0.2% bovine serum albumin, 10(-8) M insulin, and 10(-8) M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250 +/- 177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50 +/- 0.17 nmol glucose.mg-1.min-1) similar to that reported for human liver. Insulin at 10(-8) M activated glycolysis (X1.40) and glycogenesis (X1.34), and glucagon at 10(-9) M stimulated gluconeogenesis (X1.35) and glycogenolysis (X2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein, haptoglobin, alpha 2-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10(-9) M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol.mg-1 cell protein.min-1, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol.mg-1). The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.
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PMID:Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo. 215 94

We have developed a method to circumvent the use of exogenous proteolytic enzymes in the isolation of islets of Langerhans from the perinatal rodent pancreas. Advantage is taken of the propensity of fibroblastlike cells to attach and migrate on polystyrene at low-serum concentrations (5%). In contrast, at this serum level, rat islet epithelial cells tend not to adhere to the substrate. At 3 d of culture, islets are visible at the edges of the explants. With further fibroblast outgrowth the majority of islets are freefloating by 7 d. Simple agitation of the medium and centrifugation yields approximately 50 micrograms of islet tissue per perinatal pancreas. Further purification of the islets can be obtained by subculture. Rat islets can be maintained in this manner for several months in Medium F12 supplemented with 25% horse serum in an atmosphere of 5% CO2 and air at 37 degrees C. Hormone content of the islet tissue remains constant during prolonged subculture and such islets continue to exhibit appropriate insulin and glucagon responses to glucose and theophylline. The morphological integrity of the endocrine cells within the cultured islets was confirmed by immunocytochemistry and ultrastructural study. Nonendocrine cells are not identifiable within the long-term cultured islets.
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PMID:Nonenzymic in vitro isolation of perinatal islets of Langerhans. 613 59

In this study, in vitro responsiveness to glucose of fresh and cultured islets from adult pigs was tested under both static (incubation) and dynamic (perifusion) conditions. Islets were isolated by an automated method from pancreases of 24-month-old animals and cultured overnight in CMRL 1066 and 10% FCS plus antibiotics; islets, perifused immediately after the overnight culture, showed a paradoxical decrease in insulin release when exposed to an acute glucose stimulus (16.7 mmol/L), and a normal response to acute glucose when isobutylmethylxanthine (IBMX) was added to the perifusing buffer. In addition, an acute reduction of glucose concentration in the perifusate elicited a paradoxical insulin release. At the microscope, islets appeared loose and irregularly shaped after the overnight culture; immunohistochemistry showed loss of peripheral A and other mantle cells. After the overnight culture, islets were divided into 5 groups and were cultured for a further 48 hr in different tissue culture media: CMRL 1066; RPMI 1640 (without glucose); RPMI 1640 (plus 11.1 mmol/L glucose); Ham's F12; and medium 199 (all media were supplemented with 10% FCS and antibiotics). During this period, insulin release was 11.4 +/- 1.1 pg/islet/min in islets cultured in CMRL 1066, 16.2 +/- 2.4 in islets cultured in RPMI 1640 (11.1 mmol/L glucose), 1.8 +/- 0.2 (P < 0.001 vs. all the other groups), and 9.0 +/- 0.6 and 8.4 +/- 0.9 pg/islet/min in islets cultured in RPMI 1640 (without glucose), Ham's F12, and medium 199, respectively. After the 48-hr culture in different media, the islets' responsiveness to an acute glucose stimulus (16.7 mmol/L; static incubation) was evaluated: islets cultured in CMRL 1066 and in RPMI 1640 (with and without glucose) showed no insulin response to the acute glucose stimulus; in contrast, insulin release rose from 0.42 +/- 0.06 to 0.60 +/- 0.12 pg/islet/min (NS) in islets cultured in Ham's F12, and from 0.24 +/- 0.06 to 0.48 +/- 0.06 pg/islet/min (P < 0.001) in islets cultured in medium 199. During perifusions, the paradoxical insulin release in response to an acute fall in glucose concentration disappeared, but a significant increase in response to high (16.7 mmol/L) glucose was observed only in islets previously cultured in medium 199. To assess the possible role of glucagon and of cAMP, additional perifusions were done in islets cultured for 48 hr in CMRL 1066 in the presence of glucagon (10 mumol/L) and IBMX (10 mumol/L); glucagon and IBMX were unable to modify the insulin response to 16.7 mmol/L glucose.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Paradoxical release of insulin by adult pig islets in vitro. Recovery after culture in a defined tissue culture medium. 768 94

The objective of these studies was to develop serum-free culture conditions for dissociated acini from rat submandibular glands. Acini were isolated from the submandibular glands of 42-46 d old rats and cultured on reconstituted rat tail collagen containing laminin in 1:1 Ham's F12 and Dulbecco's media, supplemented with BSA, transferrin, insulin, T3, EGF, dexamethasone, retinoic acid, carbamylcholine, and trace elements, and gassed with 50% O2. The acini became partly embedded in the collagen gel and rapidly enlarged throughout the first 22 d of culture, maintaining modest seromucous acinar differentiation, as judged morphologically and by mucin secretion. Parallel cultures then were grown under 20, 35, 50, and 65% O2, and evaluated morphologically and by DNA content. Growth and retention of seromucous acinar characteristics were best with 35% O2, but lipid accumulation and cell death were unacceptably high. A spectrum of concentrations of insulin and glucagon then were tried. With 0.05 micrograms/ml insulin, cellular growth and organization were orderly, lipid accumulations were not excessive, and moderate differentiation was retained through 15 d of culture. With more than 0.1 microgram/ml insulin added to or subtracted from the optimum, the detrimental effects recurred. Addition of sufficient glucagon counteracted the effects of both optimum and excessive concentrations of insulin. We now have achieved an orderly growth of moderately differentiated rat submandibular acini for 15 d in serum-free primary culture.
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PMID:Effects of oxygen, insulin, and glucagon concentrations on rat submandibular acini in serum-free primary culture. 789 74

The model of rat primary hepatocytes incubated in DMEM/F12 (Ham) medium was used for studying the influence of the cAMP-effectors epinephrine (100 microM), norepinephrine (100 microM), glucagon (1 microM) and isoproterenol (1-1000 microM) as well as the synthetic cAMP-analogon dibutyryl-cAMP on the metabolism of metallothionein. Liver parenchymal cells isolated by a two-step collagenase perfusion were incubated with DMEM/F12 containing 5% (v/v) fetal calf serum (FCS) and 20 microM zinc in Petri dishes. Experiments were initiated after a 24 h equilibration period by adding the agonists for 18 h. MT in hepatocyte homogenates was quantified by the 109Cd-hemoglobin-binding assay. Cell viability was assessed by the activity of the cytosolic enzyme lactate dehydrogenase (LDH) liberated into the culture medium and by trypan blue exclusion. Isoproterenol and glucagon produced a significant increase of cytosolic MT about 50%. In contrast, incubation with epinephrine and norepinephrine did not lead to any significant effects in the amount of hepatic metallothionein. Simulating the influence of cAMP by dibutyryl-cAMP (500 microM) did not affect the content of hepatic metallothionein. To examine transcriptional and translational regulatory effects supplementation of cycloheximide (0.1-500 microM) and actinomycin D (0.1-100 microM) showed a total inhibition of the agonist induced amounts. Particularly in combination with isoproterenol low LDH activities reflected a high viability of hepatocytes. In conclusion, in primary hepatocyte cultures cAMP-mobilizing-agonists like isoproterenol and glucagon indicate an independent effect on the MT-metabolism. This is possibly due to the de novo synthesis of the protein because suppression by actinomycin D can be observed. However, cAMP-effectors do not seem to be involved in the induction of metallothionein because theophylline and dibutyryl-cAMP did not affect MT-metabolism by suppressing the phosphodiesterase or by stimulating the cAMP-cascade.
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PMID:Influence of cAMP-effector-agonists on the synthesis of metallothionein in rat primary hepatocytes. 858 45

The parenchymal cell fraction was isolated from abattoir adult porcine livers and cultured in Dulbecco's Modified Eagles' medium/Ham's F12 medium (DMEM/F12; 1:1) medium supplemented with 5% foetal calf serum, 10 ng/mL glucagon, 10 microg/mL insulin, 60 ng/mL hydrocortisone and eight other factors (NAIR-1 medium). The fraction contained a number of epithelial cells other than hepatocytes, some of which attached to the culture plates as cell clusters and began to grow after 3 days in culture. These epithelial cells growing as colonies were found to express cytokeratin 18 by immunocytochemistry. After 7-8 days, duct-like structures emerged in the central parts of the colonies. The cells constituting the duct-like structures and some cells located outside the structures were positive for cytokeratin 19 and gamma-glutamyltransferase (GGT). The albumin-positive cells were located in the outer parts of the colonies rather than their central parts. Albumin was also detectable in the cells surrounded by the duct-like structures. Moreover, cytochrome P450 IA1 was induced by 3-methylcholanthrene (3-MC) on day 16. These results suggest that porcine liver epithelial cell clusters may contain stem-like cells which can differentiate into mature hepatocytes or bile duct epithelial cells.
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PMID:Colonial growth and differentiation of epithelial cells derived from abattoir adult porcine livers. 979 36

We assessed the potential role of all-trans-retinoic acid on the developing chick pancreas, specifically with regard to the proportions of insulin cells. The endodermal component of the dorsal pancreatic bud of 5-d-old chick embryos was cultured on Matrigel. Retinoic acid (10(-6) or 10(-5) M) was added to a standard serum-free medium, Ham's F12 containing insulin, transferrin and selenium (F12.ITS). Control grafts were cultured in F12.ITS alone or in F12.ITS with DMSO (the diluent for retinoic acid). After 7 d the explants were retrieved, freeze-dried, vapor-fixed, and embedded in resin. Endocrine cell types were identified by immunocytochemistry. The numbers of insulin cells were expressed as a proportion of the sum of insulin plus glucagon cells. Retinoic acid had a dose-related effect; the proportion of insulin cells in explants treated with the lower dose of retinoic acid (10(-6) M) was more than twice the proportion of insulin cells in explants treated with the higher dose (10(-5) M) of retinoic acid and more than three times that of the control grafts.
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PMID:The effect of retinoic acid on the proportion of insulin cells in the developing chick pancreas. 1069 Oct 36

Previous studies have suggested that nicotinamide increases the number of insulin cells both in vivo and in vitro. However, the question remains as to whether there is in fact an increase and whether this increase is caused by the proliferation of progenitor cells, or by replication of existing insulin cells. In order to investigate this, the endodermal component of dorsal pancreatic buds of 5-day-old chick embryos was cultured on Matrigel in a serum-free medium (Ham's F12-ITS) to which nicotinamide, at a concentration of 5 and 10 mM, respectively, was added. Control explants were cultured in Ham's F12-ITS medium without nicotinamide. After 7 days in culture the buds were incubated with bromodeoxyuridine (BrdU) and then processed for immunocytochemistry. Localization of insulin, BrdU and glucagon was carried out on adjacent serial sections. The proportion of insulin cells was 6.76, 11.32 and 16.86% in control, 5 and 10 mM nicotinamide-treated explants, respectively. Hence adding nicotinamide to the culture medium induced a 1.7- and 2.5-fold increase in the proportion of insulin cells when compared to the controls. These proportions were significantly different from that of control explants (P < 0.05). However, a very small number of insulin cells were found to be proliferating, suggesting that the increase in the proportion of insulin cells had resulted from stimulation of progenitor cells and not proliferation of existing insulin cells.
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PMID:Beneficial effect of nicotinamide on the proportion of insulin cells in developing chick pancreas. 1083 Apr 42

We are interested in the regulation of early pancreatic differentiation, particularly with regard to factors that enhance insulin cell proliferation. Both retinoic acid and insulin-like growth factor 1 (IGF-1) are known to be important in the proliferation and differentiation of insulin cells. Individually, they have the ability to increase the proportion of insulin cells when added to cultures of chick dorsal pancreatic buds. The aim of this study was to define the action of retinoic acid (RA) in combination with IGF-1 on the proportion of insulin cells. The dorsal pancreatic bud of 5-day-old chick embryos was excised and the endodermal component, with minimal adherent mesenchyme, was explanted onto Matrigel. RA (10(-6) M) and IGF-1 (50 ng/ml) were added to Ham's F12 culture medium containing transferrin (5 microg/ml) and selenium (10(-10) M) (F12.TS). Control explants were cultured in F12.TS alone or in F12.TS containing dimethyl sulphoxide (DMSO) [F12.TS (DMSO)]. After 7 days in culture, insulin and glucagon cells were localized immunocytochemically; numbers of insulin cells were expressed as a percentage of insulin plus glucagon cell counts. Addition of RA plus IGF-1 to the medium increased the proportion of insulin cells markedly (23.43%) compared with the proportions in control explants (11.3% with F12.TS (DMSO), 13.2% with F12.TS). This increase represents a more than twofold increase in the proportion of insulin cells over that of control explants.
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PMID:Regulation of embryonic chick insulin cells: effect of retinoic acid and insulin-like growth factor 1. 1134 Feb 60


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