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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)

Adipocytes were prepared by collagenase digestion of rat epididymal adipose tissue and incubated for 5, 15 or 30 minutes in Krebs-Ringer bicarbonate buffer containing albumin (40 mg/ml), glucose (1 mg/ml) and epinephrine. Calcium ion was present in some incubations at concentration of 2.5 mM and omitted from others; media with no added calcium contained 1.0 mM EGTA thereby producing a final calcium concentration of less than 10(-7) M. Glycerol release and accumulation of cyclic AMP were measured. Basal lipolysis and cell cyclic AMP levels were increased slightly but not significantly when adipocytes were incubated in calcium free media. Lipolysis could be activated with epinephrine in the absence of calcium but the sensitivity of the lipolytic response was greatly reduced; however, the maximum lipolytic response to epinephrine was not decreased in calcium free media. Similarly, incubation of adipocytes in calcium free media resulted in decreased accumulation of cyclic AMP in response to epinephrine but only when sub-maximum concentrations of the catecholamine were present. Varying the extracellular calcium concentration showed that a concentration of at least 10(-5) M was optimal for epinephrine activation of lipolysis. These observations are considered in accord with the view that activation of adenylate cyclase is facilitated by calcium ion.
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PMID:The role of calcium ion in epinephrine activation of lipolysis. 18 5

To identify cells developing into adipocytes by accumulation of triglyceride, rat epididymal fat pad cells from small rats were exposed to (3)H-labeled chylomicron fatty acids in vivo and then liberated with collagenase. Tissue remnants were removed by filtration and mature fat cells by flotation. Aggregating cells were then removed by filtration through a 25- micro m nylon screen. Further purification of cells labeled in vivo was obtained by removing floating cells from those adhering to the bottom of a culture dish. The adhering cells multiplied to a confluent monolayer when cultured in Medium 199 containing serum, glucose, insulin, and a triglyceride emulsion. The cells then gradually enlarged due to granulation of the cytoplasm by a lipid-staining material. After about 2 weeks these granules had coalesced forming mature adipocytes of typical signet-ring appearance. Free adipocytes could then be recovered from the cultures by collagenase treatment. After about 2 weeks of culture these cells had the same size (about 30 micro m) as adipocytes recovered in the original collagenase preparation of the rat epididymal fat pad. They contained triglyceride lipase activity and incorporated glucose into triglycerides to the same extent as cells developed in vivo but had higher lipoprotein lipase activity. In vitro, heparin in a low concentration, prostaglandin E(1), isobutylmethylxanthine, and cholera toxin markedly promoted the development of these cells into adipocytes. This could be shown to occur almost completely indicating that this fraction of cells was homogeneous and consisted of cells with the capacity to form adipocytes. The duplication time was about 2 days and did not change with subculturing. Preadipocytes could be obtained by density gradient centrifugation, isolating triglyceride-containing cells either directly from the pad or after 3 days in culture. All of these cells developed into adipocytes as described above but did not multiply as readily. It was concluded that cells from the epididymal fat pad from small rats can be isolated in a homogenous fraction that develops in culture into cells of identical morphology and function as adipocytes formed in vivo. The differentiation of these cells into adipocytes may be manipulated in vitro.
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PMID:Isolation and characterization of cells from rat adipose tissue developing into adipocytes. 20 38

This study supports the possibility for multiple subcellular forms of lipoprotein lipase. 1. The total activity of lipoprotein lipase per g of intact epididymal adipose tissue from fed rats is much higher than that from starved rats. 2. The isolated fat-cells of fed and of starved rats have lipoprotein lipase of almost the same activity per g of fat-pads. The isolated fat-cells of starved rats have a much higher proportion of total activity per g of the intact tissue than do those of fed rats. 3. Under the conditions of homogenization used, only a small proportion of the total activity per g of intact tissue from fed rats was associated with the fat layer which floated to the top of the homogenate during low-speed centrifugation. The different proportions of the specific enzyme activity found in each subcellular fraction are described. 4. Lipoprotein lipase from plasma membranes and microsomal fractions from starved and fed rats was purified by affinity chromatography. 5. The total activity of microsomal lipoprotein lipase per g of intact adipose tissue is enhanced by a normal diet. 6. In intact epididymal adipose tissue from fed rats, the activity per g of tissue of lipoprotein lipase of plasma membranes is much higher than that in the same fraction from starved rats. By contrast, the activities per g of tissue in plasma membranes obtained from starved or from fed rats by collagenase treatment were similar.
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PMID:Effect of nutrition on subcellular localization of rat fat-cell lipoprotein lipase. 66 42

1. Rats of four different age groups were injected intraperitoneally with labelled thymidine and killed 1, 7 or 12 days later. 2. The epididymal fat-pads were separated into fat-cells and stromal elements by collagenase digestion. 3. The incorporation of labelled thymidine into the deoxyribonucleic acid (DNA) of both fractions was greatest in the 6-week-old animals. Uptake was significantly decreased in 12- and 15-week-old animals and was lowest in 22-week-old rats.
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PMID:The effect of age on deoxyribonucleic acid synthesis in rat adipose tissue. 112 23

To detect the presence in adipose tissue of peptides known to affect tissue growth and to investigate potential regional differences, epididymal and perirenal adipose tissue depots from male Sprague-Dawley rats were separated into adipocyte and stroma-vascular fractions by collagenase digestion, sequential centrifugation and filtration. Identity and integrity of the fractions were demonstrated by light and electron microscopy, while dose-response curves for angiotensin-converting enzyme (ACE) were performed, revealing maintained functional capacity of the stroma-vascular fraction. ACE, atrial natriuretic peptide (ANP), and transforming growth factor-alpha (TGF-alpha) concentrations were significantly greater in epididymal than perirenal stroma-vascular tissue. Adipocyte fractions from both depots contained significant concentrations of ANP and TGF-alpha. There was no detectable ACE in the adipocyte fractions, indicating that no contaminating stromal-vascular cells were present in these fractions. These data show significant concentrations of peptides with effects on growth in subfractions of adipose tissue and demonstrate regional differences in concentrations between fat depots.
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PMID:Transforming growth factor alpha and atrial natriuretic peptide in white adipose tissue depots in rats. 133 61

Recent studies have found that angiotensinogen is expressed in white and brown fat pads, and adipocytes have been implicated as a primary source of angiotensinogen in several other tissues. The functional significance of this unexpected expression is unknown. To address this, we studied angiotensinogen messenger RNA (mRNA) expression and angiotensinogen secretion in adipose tissue and isolated adipocytes comparing fasted and refed rodents and those with genetic obesity with normal controls. Control 2-month-old Sprague-Dawley rats, those fasted for 3 days, or those fasted for 2 days and refed for 6 days were killed, and adipocytes were isolated from epididymal fat pads using collagenase digestion. Angiotensinogen mRNA was reduced to 14.6 +/- 2.3% of control levels under fasted conditions and increased to 228 +/- 53% of control levels after refeeding. Angiotensinogen release from adipocytes was reduced to 33% of control levels by fasting and increased to 183% by refeeding. These effects of fasting and refeeding on angiotensinogen regulation were tissue specific since liver angiotensinogen mRNA and serum angiotensinogen concentrations were unaffected. Systolic blood pressure, however, was modulated by fasting and refeeding in a manner parallel to adipocyte angiotensinogen expression. In related experiments, angiotensinogen secretion per epididymal fat pad of the ob/ob mouse model of obesity was increased an average of 3.4-fold compared with control. We conclude angiotensinogen expression in white adipocytes is regulated nutritionally in a tissue-specific manner. We propose that adipocyte angiotensinogen could play a previously unrecognized role in regulating adipose tissue blood supply and thereby fatty acid efflux from fat.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tissue-specific nutritional regulation of angiotensinogen in adipose tissue. 155 65

Exposure of rat epididymal fat pad to phorbol 12-myristate 13-acetate (TPA), an activator of protein kinase C, results in an 85% increase in isoproterenol-stimulated cyclic AMP (cAMP) accumulation, an effect which was antagonized by H7, a protein kinase C inhibitor. This promoting action of TPA appears to be related to (i) an increase in the catalytic activity of adenylate cyclase, (ii) an increase in the maximal response of adenylate cyclase to fluoride and guanylimidodiphosphate (GppNHp) with no change in the EC50 value for GppNHp, and (iii) a reduction of the isoproterenol-stimulated low-Km cAMP phosphodiesterase activity present in the 30,000 g pellet of fat pad homogenates. In contrast with fat pads, exposure of isolated rat fat cells to TPA failed to influence their adenylate cyclase response to GppNHp and their cAMP accumulation and lipolysis. However, the other alterations caused by TPA in fat pads were still observed in fat cells. These results suggest that (i) the major alteration responsible for the promoted isoproterenol-stimulated cAMP response observed in fat pads after exposure to TPA is an increased interaction between the alpha s subunit of Gs and the catalytic site of adenylate cyclase and (ii) this increased interaction is dependent on protein kinase C activation and is abolished by collagenase digestion.
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PMID:Differential modulation of the adenylate cyclase/cyclic AMP stimulatory pathway by protein kinase C activation in rat adipose tissue and isolated fat cells. Influence of collagenase digestion. 165 98

The antibodies of keratin and vimentin were used as the histochemical probes determined by the immuno-fluorescent technique to recognize the rat epididymal epithelial cells in the different ages from the connective tissue both in intact epididymides and in isolated cultured cells. It also showed that an enriched suspension of epididymal epithelial cells could be obtained by sequential digestion with 0.05% trypsin and 0.1% collagenase. The morphological characteristics were appeared during the cells in culture. Therefore the epididymal epithelial cells isolated and cultured by present methods could be used as a research model to study their functions.
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PMID:[The recognition of rat epididymal epithelial cells]. 170 22

Responsiveness to lipolytic agents and glycerol output from rat adipocytes is altered by the diabetic process. We have confirmed reports that preincubation is required for growth hormone-induced lipolysis in isolated fat cells. Isolated fat cells were prepared from the epididymal fat pads of normal and spontaneously diabetic BB Wistar rats (weight, 250-400 gm) and their nondiabetic littermates by collagenase digestion. Lipolysis was measured by glycerol release after sequential perifusion with buffer alone, bovine growth hormone 1 microgram/ml, buffer alone, and epinephrine, 0.5 mumol/L. In each case isolated fat cells from control, nondiabetic, and spontaneously diabetic rats were perifused under two conditions, with and without preincubation with bovine growth hormone. Isolated fat cells from control and nondiabetic rats did not respond to bovine growth hormone without preincubation. When preincubation with bovine growth hormone, response in control rats increased from nonpreincubated glycerol values of 4.9 to 13.5 nmol glycerol released/10(6) cells/min. In contrast to controls, non-preincubated isolated fat cells from spontaneously diabetic rats that were stimulated with 1 microgram/ml bovine growth hormone went from 18.0 to 42.6 nmol of glycerol released/10(6) cells/min. No preincubation was necessary in spontaneously diabetic rats. In addition, in all situations in which preincubation or the diabetic state enhanced lipolysis with growth hormone, similar enhancement was seen with epinephrine. For nondiabetic rats both preincubated and nonpreincubated isolated fat cells respond minimally to bovine growth hormone. In conclusion, preincubation with bovine growth hormone is not required to elicit lipolysis in perifused isolated fat cells from spontaneously diabetic BB rats.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Growth hormone-enhanced lipolysis in the spontaneously diabetic BB rat. 206 50

Epididymal epithelial fragments, free of stromal elements were isolated from mature rats using two sequential collagenase digestions. Within 24 h these attached efficiently to a variety of substrates including glass, plastic, placental collagen, type IV collagen and epididymal extracellular matrix material. Cells spreading away from the fragments rapidly assumed a flattened, overlapping, monolayer appearance typical of epithelial cells in culture. Cells still associated with the fragments or adjacent to them remained more polarized and more closely resembled epididymal principal cells in vivo than did cells that had migrated to the periphery of the monolayer. Apical microvilli characteristic of these cells in vivo were common during the first 4 days in culture but diminished in number and size thereafter. Cultured cells maintained many of the structural features characteristic of principal cells in vivo, including a well developed Golgi apparatus, coated pits and vesicles, and many multivesicular bodies. An extensive filamentous network, shown immunocytochemically to consist of keratin, was present in the cytoplasm of all cells but was more obvious in flattened cells at the periphery of the monolayer. Rhodamine phalloidin labelling of filamentous actin showed that concentrations of actin occurred corresponding to microvilli on the apical surface, in a continuous ring just below the apical surface, and also in stress fibres at the base of the cells. Cells isolated and cultured from the distal caput epididymidis possessed lobulated nuclei, in contrast to the round or oval nuclei found in cells cultured from the proximal caput epididymidis. Cells from the distal caput epididymidis were also characterized by the presence of many lipid droplets in their cytoplasm. Autofluorescent granules were observed in epithelial cells from both regions but were larger and more numerous in cells isolated from the distal caput epididymidis. Tritiated thymidine incorporation by the cells after 4 days in culture showed that cells adjacent to the parent epithelial fragment were dividing at a greater rate than cells that had migrated to the periphery of the monolayer.
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PMID:Structural features of rat epididymal epithelial cells in vitro. 241 3


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