Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 sensitivity to lipolytic agents is altered in diabetic vs. control animals. Because of its role as a diabetogenic hormone and its ability to elicit lipolysis, GH was studied in isolated fat cells (IFC) from control and streptozotocin-diabetic (STZ-DM) rats. IFCs from the epididymal fat of 150 to 200-g normal and STZ-DM Holtzman rats were prepared by collagenase digestion. Lipolysis was measured by glycerol release after either incubation or perifusion with the following concentrations: epinephrine (EPI), 0.01-0.1 microM; theophylline, 0.01-1.0 mg/ml; adenosine deaminase (ADA), and bovine GH (bGH), 0.01-1.0 microgram/ml. Rats, rendered diabetic by STZ (65 mg/kg), were used on day 3. In a dose-response study comparing glycerol release from control and STZ-DM IFC, IFC were preincubated with 1.0 microgram/ml bGH and then incubated with varying concentrations of EPI or bGH. In STZ-DM, we noted increased lipolytic sensitivity to low concentrations of EPI or bGH. Furthermore, in perifusion, STZ-DM IFC did not require obligatory preincubation with bGH for optimal glycerol release. The addition of ADA increased glycerol release from incubated IFC (STZ-DM and controls). In both systems an enhanced lipolytic response to theophylline was seen in the presence of bGH in control and STZ-DM. It was thus concluded that IFC from normal animals do not respond to GH without preincubation. IFC from STZ-DM rats show a lipolytic response to GH without preincubation. Preincubation with GH increases the lipolytic response of IFC from STZ-DM to all lipolytic agents compared to control responses. In addition, ADA greatly enhanced lipolysis in IFC from STZ-DM compared to that in controls. Together these data demonstrate enhanced sensitivity to both lipolytic stimuli and adenosine suppression of lipolysis in IFC from STZ-DM.
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PMID:Lipolysis in diabetic adipocytes: differences in response to growth hormone and adenosine. 362 74

1. Incubation of intact epididymal adipose tissue from fed rats at 37 degrees in an albumin solution at pH7.4 in vitro results in rapid loss of clearing-factor lipase activity until a low activity, stable to prolonged incubation, is attained. The clearing-factor lipase activity of intact tissue from starved rats, which is initially much less than that of tissue from fed rats, is mainly stable to incubation at 37 degrees . 2. Much of the clearing-factor lipase activity of intact epididymal adipose tissue from fed rats is inactivated by collagenase. The enzyme activity of intact tissue from starved rats is not inactivated by collagenase. 3. The clearing-factor lipase activity of fat cells isolated from the epididymal adipose tissue of fed rats is stable to prolonged incubation at 37 degrees . It represents only a small proportion of the total activity of the intact tissue. In starved rats, the isolated fat cells contain a much higher proportion of the activity of the intact tissue. Their activity is also stable at 37 degrees . 4. Incubation of isolated fat cells in a serum-based medium leads to a progressive rise in clearing-factor lipase activity. Actinomycin increases the extent of this rise in activity. No rise in clearing-factor lipase activity occurs when stromal-vascular cells isolated from epididymal adipose tissue are incubated in the medium. 5. The findings indicate that less than 20% of the activity of intact adipose tissue from fed rats is retained when fat cells are isolated from the tissue by collagenase treatment. The activity that is lost could be that which normally functions in the uptake of triglyceride fatty acids by the tissue.
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PMID:Clearing-factor lipase in adipose tissue. Distinction of different states of the enzyme and the possible role of the fat cell in the maintenance of tissue activity. 430 92

Sucrose gradient centrifugation has been used to examine the triglyceride lipases present in extracts of rat epididymal adipose tissue. The aqueous infranatant recovered between the pellet and fat cake of tissue homogenates which had been centrifuged at 40,000 g was shown to contain two types of triglyceride lipase activity. One of these appears in the 15s region and has been identified as the active form of the "hormone-sensitive lipase" believed to be responsible for initiating the hydrolysis of tissue triglyceride stores in response to lipolytic stimuli. The activity of this enzyme was selectively increased in extracts prepared from tissue exposed to epinephrine and decreased in extracts of insulin-treated tissue. The increased lipolytic activity of extracts of tissue from fasted or fasted-refed rats was also found largely in this region. When the tissue was incubated with orthophosphate-(32)P, radioactivity was incorporated into a protein migrating at 15s. A second peak of triglyceride lipase activity appeared in the 6s region coincident with the location of the monoglyceride and diglyceride lipase activities. The amount of 6s triglyceride lipase activity did not correlate with changes in the lipolytic activity of the tissue from which the extracts were prepared, and its physiological function remains to be elucidated. The lipoprotein lipase and the short-chain triglyceride lipase ("tributyrinase") each moved more slowly in the gradient than the 6s triglyceride lipase. Both the 6s and 15s enzymes were shown to be present in washed adipocytes isolated from the tissue by collagenase digestion.
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PMID:Studies on the hormone-sensitive lipase of adipose tissue. 432 8

1. A method for measuring the lipogenesis from [(14)C]glucose by single fat cells is described: (i) after incubation with ;carrier-free' [U-(14)C]glucose (0.55 mu-mole/ml.), collagenase-isolated fat cells were fixed with osmium tetroxide; (ii) similarly incubated pieces of epididymal fat pads were treated with osmium tetroxide for 90 sec, whereby only the superficial cells are fixed, and the tissue was then disintegrated by shaking with collagenase. The osmium-fixed free cells were washed, sucked into a micropipette, measured under a microscope and assayed individually for (14)C-activity.2. There was a quantitative recovery of (14)C-lipid activity from osmium-fixed single cells.3. Both collagenase-isolated cells and in situ fixed surface cells were normally distributed with respect to diameters (for both cell groups from ad lib. fed rats of ca. 110 g; mean diameter, about 55 mum; S.D. about 7 mum).4. Frequency distribution curves (number of fat cells versus (14)C-lipogenesis per cell) were asymmetric and very broad (coefficient of variation about 50%) for collagenase-isolated cells incubated with insulin (10(3) mu-u./ml.). Frequency distribution curves for surface cells obtained from similarly incubated pieces of epididymal fat pads showed a coefficient of variation of the same magnitude, whereas the mean lipogenesis of these cells was only about one third of that of the isolated cells.5. Collagenase-isolated cells incubated in the presence of insulin (10(3) mu-u./ml.) showed a weak but highly significant positive correlation between fat cell diameter and (14)C-lipogenesis (eight rats, r about 0.5 and P < 0.001 for each rat). Analysis of the relationship: lipogenesis = k x diameter to the exponent of beta showed that the estimates of beta varied significantly from rat to rat (range: 1.3-2.9). Similar correlations between cell size and lipogenesis were found both for cells incubated with insulin in various submaximal concentrations and for cells incubated without insulin.6. Small and large cells from the same rat were equally sensitive to insulin.7. Statistical analysis of frequency distribution curves (number of cells versus (14)C-lipogenesis per unit surface area) representing cells from the same rat incubated with insulin 0, 2.5, 5, 10, and 10(3) mu-u./ml., respectively, suggests that insulin exerts a graded influence on the lipogenesis of each fat cell.
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PMID:Lipogenesis and insulin sensitivity of single fat cells. 436 2

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.
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PMID:The intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm. 439 82

Capillaries isolated by collagenase digestion of hamster epididymal fat pads were used to examine the properties of endothelial adenylate cyclase and cyclic nucleotide phosphodiesterase. Adenylate cyclase activity in capillary homogenates was increased by 10 microM GTP or 100 microM isoproterenol. Lower concentrations of the catecholamine and 5.7 microM prostaglandin E1 did not stimulate endothelial adenylate cyclase activity unless GTP was included in the assay system. The effects of isoproterenol on capillary adenylate cyclase activity were blocked by propranolol, but were not affected by phentolamine. Phosphodiesterase activity in endothelial homogenates showed anomalous kinetic behavior with either cyclic AMP or cyclic GMP as the enzyme substrate. At substrate concentrations below 1 microM, capillary phosphodiesterase activity hydrolyzed cyclic GMP 2-6 times faster than cyclic AMP. However, at high substrate levels, e.g., 100 microM, cyclic AMP and cyclic GMP were degraded at similar rates. Hydrolysis of 1 microM cyclic AMP by capillary homogenates was stimulated by 0.1 and 1 microM cyclic GMP. Caffeine, 1-methyl-3-isobutylxanthine, papaverine and dipyridamole SQ 20009 were effective inhibitors of capillary phosphodiesterase activity. In contrast, imidazole enhanced the activity of the enzyme. The presence of adenylate cyclase and phosphodiesterase activities in hamster isolated capillaries is consistent with a role for cyclic AMP in the regulation of endothelial function. Moreover, the experiments described here indicate that hamster isolated capillaries are useful model systems for studying the metabolism of vascular endothelium.
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PMID:Properties of adenylate cyclase and cyclic nucleotide phosphodiesterase in hamster isolated capillary preparations. 624 1

Adipocytes isolated from the epididymal fat of hypophysectomized rats by digestion with collagenase failed to respond to insulin with an increase in glucose utilization. These cells also exhibited an anomalous response to insulin when added in the presence of epinephrine. While insulin antagonized the lipolytic actions of epinephrine in normal adipocytes or in segments of epididymal fat from normal or hypophysectomized rats, it potentiated lipolysis in adipocytes isolated from hypophysectomized rats. This anomalous effect was also evident when isoproterenol, ACTH, or glucagon was used as the lipolytic agent, but the expected antilipolytic response was obtained when theophylline served as the lipolytic agonist. The lipolytic effects of insulin were not seen until 4-7 days after hypophysectomy. Treatment of hypophysectomized rats with a combination of GH (100 micrograms/rat . day), cortisone acetate (1 mg/rat . day), and T3 (1 micrograms/rat . day) for 5 days restored the antilipolytic response to insulin in cells of hypophysectomized rats, but no one hormone alone was effective. The data indicate that adipocytes of hypophysectomized rats retain their ability to recognize and response to insulin, but the ability of insulin to stimulate glucose oxidation or antagonize epinephrine-induced lipolysis is abolished by the cell isolation procedure. The findings underscore the need to consider the impact of hormonal status on the ability of cells to retain normal responsiveness during the rigors of the cell isolation procedure and suggest that failure to do so might lead to erroneous interpretations of the physiological actions of hormones.
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PMID:Lipolytic effects of insulin in adipocytes isolated from hypophysectomized rats. 627 27

Most effects of thyroid hormones appear to be mediated by the binding of triiodothyronine (T3) to nuclear triiodothyronine binding sites (NT3BS). Although thyroid hormones influence adipocyte metabolism, NT3BS have not been described in mature adipocytes yet. This report describes T3 nuclear binding in isolated nuclei from rat epididymal fat pad adipocytes. Nuclei were isolated by exposing collagenase-dispersed adipocytes to STM (sucrose, 0.25 mol/L; TRIS, 20 mmol/L; MgCl2, 1.1 mmol/L, pH 7.85) containing 0.5% (vol/vol) Triton X-100. Incubation of nuclei suspended in STM/EDTA (2 mmol/L)/DTT (5 mmol/L) with 125I-T3 and varying concentrations of unlabeled T3 at 37 degrees C for one hour revealed the presence of high-affinity, low-capacity NT3BS. Their MBC was 0.39 +/- 0.04 (SD) ng of T3 per milligram of DNA and their Kd was 1.4 +/- 0.5 (SD) X 10(-10) mol/L T3. Specific binding reached a plateau between 30 minutes and two hours of incubation. The addition of 5 X 10(-7) mol/L T3 to nuclei incubated for one hour with 2 X 10(-11) mol/L T3 completely displaced the specifically bound 125I-T3 within 30 minutes. Thyroxine (T4) and 3, 3', 5'-triiodothyronine (rT3) could displace 125I-T3 from the NT3BS but were less than 10% and 1% as effective, respectively, as T3. Rat epididymal fat pad adipocytes contain NT3BS, the binding characteristics of which are similar to those of rat hepatic NT3BS.
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PMID:Nuclear triiodothyronine binding sites in rat adipocytes. 632 15

In order to attempt to characterize the adipocyte precursor cell in situ in developing adipose tissues from young rats, the light and electron microscopic characteristics were determined of cells in situ in these tissues, as well as in suspensions from the same tissues of cells liberated with collagenase subsequently developing to adipocytes in culture. The functional characteristics of adipose precursor cells at different stages of development in culture is known from previous work, and could therefore be added in parallel to the morphological characteristics. In order to allow comparison with fibroblasts from a site where adipocyte formation does not occur, lung-fibroblasts were also studied in parallel cultures. In culture, a rapidly dividing cell confluenced and reached full expression to adipocytes. This cell had morphological characteristics developing at the same time as early lipid accumulation making it easily distinguishable from a lung fibroblast in culture. A similar cell was found in abundance in the peripheral, non-developed part of the intact epididymal fat pad, here often surrounded by collagen. However, the early preadipocyte (without lipid droplets) and the adipoblast cannot be morphologically distinguished in situ from the fibroblast; therefore it cannot be excluded that fibroblasts and adipoblasts are the same cells in adipose tissue, possibly deriving from the pericyte.
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PMID:A morphological study of the adipocyte precursor. 632 21

Endothelial cells from rat epididymal fat pad capillaries were isolated from rats immediately after weaning. The cells were obtained after an initial brief incubation with collagenase under conditions of minimal breakage of cells. Adipocytes were removed by flotation and endothelial cells were then obtained as cell aggregates by fractional filtration procedures whereby intact tissue as well as free cells were removed. These aggregates were then dispersed and cultured in supplemented medium 199 whereby a monolayer of cells with a growth pattern, numerous pinocytotic vesicles, and intercellular junctions typical of endothelial cells were obtained. Minor contaminations of precursor cells to adipocytes were absent after one subculture. Here greater than 95% of the cells showed the presence of Factor VIII. Further subcultures produced nonhomogenous cells and decreasing rates of replication. The endothelial cells showed a very low rate of triglyceride synthesis and release, and collected no visible lipid upon prolonged cultures in the presence of an abundance of triglyceride substrate. They bound lipoprotein lipase from rat adipocytes, whereby the lipase was stabilized. This binding was released by heparin, and the cells did not synthesize the enzyme.
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PMID:Isolation and characterization of endothelial cells from the epididymal fat pad of the rat. 683 87


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