Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The objective of the present study was to determine the effects of insulin on amphibian hepatocytes in primary culture. Hepatocytes were isolated from adult bullfrogs by collagenase perfusion and maintained as monolayers in serum-free medium. Cells cultured in the continuous presence of insulin exhibited a relatively constant rate of protein secretion over the first four to five days, whereas controls showed an almost three-fold decrease over the same time period. The decline in secreted proteins was equally represented in most exported proteins, except that serum albumin secretion showed twice as much of a decrease relative to the other proteins. The maintenance of protein secretion by insulin was the result of its effect on protein synthesis. The rate of protein synthesis was measured by the incorporation of (3H)-leucine into protein using culture medium containing 0.5 mM leucine, a condition where the specific radioactivity of leucyl-tRNA was shown to be equal to that of (3H)-leucine in the medium. Cultures maintained with insulin for 60 hours synthesized protein at two to three times the rate found in non-insulin treated controls whose rate of protein synthesis was first detectably decreased after nine hours of culture in the insulin-free medium. Sedimentation profiles of polyribosomes from hepatocytes maintained for 60 hours without insulin showed proportionately fewer ribosomes in large polysomes and more in monosomes and free ribosomal subunits than ribosomes from cells cultured with insulin. This result suggests that the decrease in protein synthesis found in the absence of insulin is due to a defect in initiation. Insulin does not exert its effect by regulating cellular levels of ATP; no change in ATP content was found in cells maintained with or without insulin. The results show that insulin maintains high levels of protein synthesis and secretion in amphibian hepatocytes. The hepatocytes in monlayer culture provide a system to study the molecular mechanisms involved in the translational control of protein synthesis by insulin.
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PMID:Insulin effects on protein synthesis and secretion in primary cultures of amphibian hepatocytes. 31 46

Six specimens of human cadaveric pancreas were collagenase-dispersed and placed in tissue culture. Media were changed at 2 day intervals and assayed for insulin and amylase. Experiments were terminated after 8 days, and phase-contrast and light microscopy were performed on cultured tissue. Insulin content in media remained high for 6 days in five cases and for 8 days in four experiments; this correlated directly with morphologic viability of cultured tissue. Media amylase fell to zero after 4 days in four cases and after 6 days in two cases. These data support the concept that tissue culture may be an efficient method for (1) islet cell purification, since acinar tissue and amylase activity disappear, and (2) islet preservation.
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PMID:Tissue culture isolation and preservation of human cadaveric pancreatic islets. 32 Jun 96

There are conflicting data in the literature regarding the role of monamines in the secretion of insulin. In order to clarify the contribution that species variation may make to these divergent results, the uptake of serotinin (5-HT), dopamine (DA), and their precursor amino acids, 5-hydroxytryptophan (5-HTP) and L-dopa, into islets was studied. Islets from golden hamsters, rabbits, guinea pigs, and obese, hyperglycemic mice were isolated by the collagenase technique. The islets were incubated in Krebs-Ringer buffer in the presence of 14C-labeled monamines or their precursors. At 30-minute intervals after initiating the study, the incubation mixture was passed through a Millipore filter. The retained islets were disrupted by sonication and the radioactivity counted. The ratio of the uptake of 5-HTP to 5-HT was at least 3:1 in the hamster, guinea pig, and mouse. In the rabbit the ratio was 1:1. A similar relationship was noted for the uptake of L-dopa and DA. The in-vitro results were confirmed by the in-vivo studies, in which hamsters were injected with 14C5-HT or 5-HTP, followed by isolation of the islets. We conclude that there is significant species variation inthe uptake of these monoamines and their precursors.
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PMID:Species variation in pancreatic islet monoamine uptake and action. 32 Dec 87

The effect of various inhibitors of insulin secretion such as mannoheptulose (20 mM), atropine (1 mM), diphenylhydantoin (20 microng/ml), high concentration of Mg++ (5.3 mM) in the presence of 20 mM glucose (control) on insulin content and secretion from collagenase-isolated rat pancreatic islets was studied in vitro by cultivation of islets up to 5 or 9 days in glass Petri dishes without attachment. In a following short-term incubation for 60 min the glucose-induced insulin release without and with theophylline (5 mM) was investigated. Islets cultivated at 5 mM glucose and at 20 mM glucose with the inhibitors mannoheptulose or atropine lost the responsiveness to glucose and theophylline whereas such islets cultivated at 20 mM glucose alone or with diphenylhydantoin (DPH) or 5.3 mg Mg++ showed a stimulation of insulin secretion by glucose and theophylline. Compared, however, with freshly isolated islets all cultivated islets were restricted in their maximal glucose response and this defect was not evoked alone by quantitative changes in islet insulin content. Nevertheless, culture conditions which facilitate a net increase of insulin (content and release) during cultivation influenced also positively the glucose-induced insulin release without and with 5 mM theophylline in the following short-term experiments.
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PMID:Successful cultivation of isolated islets of Langerhans without attachment: relationship between Glucose- and theophylline-induced insulin release and insulin content in rats islets after cultivation. 32

The effect of glucose on acute 45Ca uptake and efflux in collagenase-isolated islets was studied using a double-isotope incubation technique with [3H]sucrose as an extracellular marker. Both 45Ca uptake (0-70 min) and efflux (0-80 min) were measured in either 3 or 20 mM glucose. Calcium-45 uptake and efflux were biphasic demonstrating a rapid phase (0-1 min) followed by a slow phase. Glucose (20 mM) increased the rate constant for slow-phase 45Ca uptake 7-fold and had no effect on the rapid phase. Suppression of insulin release by D2O (100%) did not affect glucose-induced 45Ca uptake indicating that this increased uptake occurred independent of insulin release. Rapid-phase and slow-phase 45Ca efflux rate constants were unaltered by 20 mM glucose and inhibition of insulin release by D2O did not influence 45Ca efflux. The rapid-phase movement of 45Ca may represent a rapidly exchangeable calcium pool at the cell membrane whereas the slow phase may be transmembranous calcium movement, as has been reported for calcium transport in HeLa and kidney cells.
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PMID:The effect of glucose on the acute uptake and efflux of calcium-45 in isolated rat islets. 33 Jan 51

To study the influence of insulin on its own secretion, collagenase-isolated islets of rat pancreas were prelabelled with [3H]leucine for 2 h. After washing the islets, (pro-)insulin release was stimulated by glucose in the presence or absence of exogenous insulin (up to 2-5 mu./ml). Hormone release was unchanged by the presence of exogenous insulin as judged by determination of both immunoreactive insulin and radioactivity incorporated into the proinsulin and insulin fractions of the medium. No direct feedback mechanism for insulin secretion was apparent from this study.
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PMID:Release of immunoreactive and radioactively prelabelled endogenous (pro)-insulin from isolated islets of rat pancreas in the presence of exogenous insulin. 33 Jul 86

A procedure was developed for the isolation of intact islets of Langerhans from sheep pancreas. The pancreas was disrupted by syringe injection of Hanks solution followed by collagenase incubation and islet separation by sedimentation. The islets were incubated in varying concentrations of glucose and butyrate. The rate of insulin release was approximately linear while the glucose and butyrate concentrations were increased. In additional studies at 2.5 and 5.0 mM levels of substrate concentration, the stimulation of insulin had the following pattern: octanoate greater than hexanoate greater than butyrate, whereas beta-hydroxybutyrate, lactate, acetate, and propionate had only slight stimulatory effects that were not statistically significant. Decanoate did not alter insulin release from isolated islets. These data confirm earlier in vivo reports that fatty acids stimulate pancreatic hormone release in sheep and that the stimulus is related to chain length of the fatty acid through C-8 but that C-10 has no effect. A hypothesis was suggested to explain these results based on chain length, solubility, and plasma membrane alterations.
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PMID:Effect of fatty acids on isolated ovine pancreatic islets. 34 24

3[H]-Leucine incorporation into (pro)insulin and insulin secretion was investigated using islets prepared by collagenase digestion from pancreata of rats pretreated with the cholimimetic agent pilocarpine or with saline (controls). Under the influence of pilocarpine pretreatment the [3H]-leucine incorporation into islet proteins with insulin immunoreactivity is enhanced at 6 mM glucose in the incubation medium of the islets but the incorporated radioactivity at 18 mM glucose is independent of the pretreatment of the animals. Only small or no changes were found regarding insulin secretion. It is concluded that an influence of pilocarpine pretreatment should be taken into consideration using such islets for studies on the regulation of (pro)insulin biosynthesis.
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PMID:Influence of pilocarpine pretreatment on (pro)insulin biosynthesis of isolated islets of rats. 35 84

Kinetics of (45)Ca efflux and insulin release were studied in collagenase-isolated rat islets during 2-h perifusions with calcium-depleted (0.05 mM) bicarbonate-phosphate buffer containing 2.2 mM glucose. Addition of glucose (16.7 mM) suppressed (45)Ca efflux by 30%. Removal of glucose caused an "off response" of insulin release. The perifusion of a normal concentration of Ca (2.3 mM) greatly stimulated (45)Ca efflux, indicating Ca <--> (45)Ca exchange. When Ca and glucose were superimposed, the effects on (45)Ca efflux and insulin release depended upon the order of presentation of the stimuli: when Ca was added to an ongoing 16.7-mM glucose perifusion, biphasic patterns of (45)Ca and insulin release were seen; when glucose was superimposed on a Ca perifusion, an inhibition of the Ca-stimulated (45)Ca efflux occurred, and a reduced but clearly biphasic insulin response was seen. The subsequent insulin off response after with-drawal of the glucose was also reduced. Mathematical "peeling" of (45)Ca efflux curves from unstimulated islets suggests that there are at least two, and probably three, different intracellular Ca compartments (not including the extracellular sucrose space). At the beginning of perifusion, these three compartments (I, II, III) contain 25, 56, and 19% of the intracellular (45)Ca, and their rates of efflux are 6.7, 1.2, and 0.1%/min, respectively. Glucose appears to suppress efflux from the largest compartment (II); Ca appears to exchange with (45)Ca from a more inert compartment (III). The relationship between insulin and (45)Ca release is not stoichiometric.
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PMID:Glucose-stimulated 45Calcium efflux from isolated rat pancreatic islets. 35 48

Six normal weight subjects without any heredity of diabetes (group 1), 3 obese subjects with normal (group 2) and 9 with pathological carbohydrate tolerance (group 3) were characterized by a 2-h glucose infusion test. Adipose tissue fragments were obtained from the abdominal wall by surgical biopsy under intracutaneous anesthesia. Adipocytes were isolated by collagenase digestion and incubated in buffer containing [1-14C] glucose and different concentrations of insulin. The metabolic effect of insulin was expressed as percent increase above control 14CO2 production. Maximal CO2 raised to 207 +/- 25% and 154 +/- 9% in groups 1 and 2, respectively. These values were significantly higher than in obese subjects displaying a pathological carbohydrate tolerance (group 3; 119 +/- 6%). A negative correlation was found between blood glucose levels and biological activity of insulin on adipocytes. The results suggest that insulin sensitivity of target tissue seems to play an important role in development of carbohydrate intolerance.
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PMID:Relationship between carbohydrate tolerance, insulin secretion, and insulin sensitivity of isolated fat cells from obese protodiabetics. 36 Jul 50


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