394
Biochimica ei Biophysics &la, @ Elsevier Scientific Pubjishin~
398 (1975)
Company,
394-400
Amsterdam
- Printed in The Netherlands
BBA 56656
EFFECTS OF COLCHICINE AND ~YCLOHEXIM~DE ON THE FUN~IONAL AND NON-FUNCTIONAL LrPOPROTErN LIPASE FRACTIONS OF RAT HEART
J. BORENSZTAJN,
M.S. RONE and T. SANDROS
De~~r~rne~~ of Pathology, Ill. 60637 (U.S.A.J
(Received
The Pritzker
School
of medicine,
~tli~er~~ty of Chicago,
Chicago,
March 17th, 1975)
Summary
In response to food deprivation, total myocardial lipoprotein lipase activity increased gradually over a period of 9 h. Although lipoprotein lipase exists in a functional and non-functional form in the myocardium, most of the increase in activity occurred in the functional (heparin-releasable) lipoprotein lipase fraction. The administration of colchicine, while having no effect on the increase seen in total lipoprotein lipase activity, did inhibit the increase in the functional fraction, while at the same time, caused a marked rise in the activity of the non-functional (non-releasable) fraction. In rats injected with colchicine after a 24-h fast, total lipoprotein lipase activity was not affected, but activity levels in the functional fraction declined while that in the non-functional fraction increased. These results suggest that the functional lipoprotein lipase is constantly being formed in sites not readily accessible to heparin (presumably the myocardial cells) and transported to its site of action, the surface of the endothelial cells of the capillaries. Cycloheximide administration to rats starved for 24 h caused a decline in activity in both the functional (half-life of about 2 h) and the non-functional (half-life of about 4 h) lipoprotein lipase fractions. These results suggest that the functional and non-functional lipoprotein lipase fractions may correspond to two distinct enzyme species. -_ Introduction
Considerable evidence is presently available demonstrating that in heart and adipose tissue, as well as other extra-hepatic tissues, lipoprotein lipase exists in a functional and a non-functional form [l-3] . The functional lipoprotein lipase, that which is directly involved in the hydrolysis of plasma lipoprotein triacylglycerol, is believed to be localized on the surface of the endo-
395
thelial cells of capillaries [P-3]. This functional enzyme can be distinguished from the non-functional lipoprotein lipase by its property of being readily released from the tissues by heparin [1,3]. The non-functions lipopro~in lipase, that which does not participate in the hydrolysis of plasma triacylglycer01, is not readily accessible to heparin and is believed to be localized mainly in the parenchymal cells of the tissues [1,3] . Recent studies by Davies et al. [4] on adipose tissue and by Schotz and Garfinkel [5,6] on heart and adipose tissue have suggested that the parenchymal, non-functional lipoprotein lipase may represent the precursor of the functions hep~in-releasable lipopro~in lipase fraction. Based on these, as well as in previous studies [l--3], it has been postulated that the functional heparin-releasable lipoprotein lipase fraction is transported from its site of formation, the parenchymal cells, to its site of action, the surface of the endothelial cells of capillaries. The postulated existence of such a lipoprotein lipase transport system has recently received support from the work of Chajek et al. [7], These authors provided indirect evidence, based on the measurement of plasma post-heparin lipoprotein lipase activities, which suggested that the transport of lipoprotein lipase to the endothelial surface is inhibited by colchicine and vinblastine, compounds which are known to affect the intracellular transport of various hormones, glycoproteins and lipoproteins [S-15]. In the present study we examined the effects of colchicine and cycloheximide on the heart lipoprotein lipase activity and we provide further evidence that the functional lipoprotein lipase is formed in sites which are not readily accessible to heparin (presumably the myocardial cells), and is then transported to the luminal surface of the endothelial cells of the capillaries. Methods Male Sprague-Dawley rats (200-240 g) were housed in rooms in which the lights were switched on at 6 a.m. and switched off at 6 p.m. Fed animals are defined as rats which had free access to water and food (rat laboratory chow) and which were killed between 8 and 9 a.m. Starved animals are defined as rats which having been maintained on their normal diets, were deprived of food starting between 8 and 9 a.m. Colchicine (5.5 mg/kg body weight) and cycloheximide (38 mg/kg body weight) were dissolved in 0.9% NaCl and injected intraperitoneally. Control animals were injected intraperitoneally with 0.5 ml of 0.9% NaCl. All animals were sacrificed under ether anesthesia. For the measurement of the hepa~n-rele~able (functional) and non-releasable (non-functional) lipoprotein lipase fractions, the hearts were quickly dissected out, and perfused for 30 s in a non-recirculatory system with Krebs-Ringer bicarbonate buffer containing 1% rat serum and 5 units of heparin/ml, as previously described [ 1]. Lipoprotein lipase activities were measured in the perfusates (hep~in-rele~able fraction) and in the hearts (non-releasable fraction) after the 30-s perfusion as previously described [16]. The total lipoprotein lipase activity was obtained by adding the activities of the heparin-releasable and non-releasable fractions. The enzyme activities are expressed as units + standard error of the mean, 1 unit representing 1 pmol of free fatty acids released into the assay medium per hour incubation per gram wet weight of
396
tissue. The significance of the difference between means was analyzed using the student’s t-test. Colchicine and cycloheximide were purchased from Sigma Chemical Co., St. Louis, Missouri and sodium heparin from Upjohn Co., Kalamazoo, Michigan. Results and Discussion Effect 9h
of colchicine
on heart lipoprotein
lipase activity of rats starved for up to
When fed rats are deprived of food the total lipoprotein lipase activity in the heart gradually increases to reach maximal levels within 10 h [ 171. Most of this increase in activity occurs in the heparin-releasable fraction [ 171. These findings have been interpreted to suggest that most of the lipoprotein lipase formed in the myocardial cells during the first 10 h of starvation is transported to the surface of the endothelial cells of the capillaries. If this hypothesis is correct, blocking the lipoprotein lipase transport in these hearts would result in a similar increase in total cardiac lipoprotein lipase activity but with the increment concentrated mainly in the non-releasable fraction. To test this possibility fed rats were injected with colchicine and, at the same time, deprived of food. The heparin-releasable and non-releasable lipoprotein lipase fractions were measured at 3-h intervals over a period of 9 h. The results in Fig. 1 show that colchicine administration had no effect on the increase in the total lipoprotein lipase activity of the hearts. Significant differences were observed, however, in the activities of the heparin-releasable and non-releasable fractions (Fig. 1). In control animals the activity of the heparin-releasable fraction was significantly increased after 3 h of starvation (P < 0.001) and continued to increase for the remaining 6 h. In the colchicine-injected animals the activity of the heparinreleasable lipoprotein lipase fraction was also significantly increased after 3 h of starvation (P < O.OOl), but no further increases were observed in the remaining
25
d-.
f -’
6
-+
TIME IHOURS)
TIME LHOURS)
Fig. 1. Effect of colchicine on the (A) total, (B) heparin-releasable tein lipase (LPL) fractions. Rats fed ad libitum were deprived of neally with colchine () (5.5 mg/kg body weight) or saline of rats were sacrificed and their isolated hearts perfused for 30 containing 1% rat serum and 5 units of heparinlml. The procedures activities in the different fractions are as described in Methods. point is shown in parentheses.
TIME (HOURS)
and (C) non-releasable heart lipoprofood at 8 a.m. and injected intraperito(- - - - - -). At the times indicated groups s with Krebs-Ringer bicarbonate buffer used to measure the lipoprotein lipase The number of hearts perfused at each
397
6 h. Thus, after 9 h the activity of the hep~n-rele~able lipoprotein lipase fraction of the control animals had increased from 7 + 1 to 75 * 4 units, while in the colchicine-injected animals the increase was only from 7 + 1 to 17 rt 3 units. The activity of the non-releasable lipoprotein lipase fraction (Fig. 1) of control animals increased from 45 f 2 to 99 k 6 units after 9 h of starvation (P < 0.001). In the colchicine-injected animals, however, the increase in activity of the non-releasable fraction at 6 and 9 h of starvation was about 70% greater (P < 0.001) than that of control animals, and accounted for most of the increase in total lipoprotein lipase activity observed in these hearts. These results show that in colchicine-treated rats the increase in heparinreleasable lipoprotein lipase activity was inhibited by about 80%. In addition, they strongly suggest that in these animals the activity of the non-releasable lipoprotein lipase which is greater than that of the control animals represents the lipoprotein lipase fraction whose transport to the surface of the endothelial cells is inhibited. Effect of colchicine on heart lipoprotein lipase activities of rats starved for 24 h Shown in Fig. 2 are the results obtained when rats starved for 24 h were injected with colchicine and the heart heparin-releasable and non-releasable lipoprotein lipase fractions measured. Confirming previous observations [ 1’71 the total heart lipoprotein lipase activity in the control animals remained constant for a period of 6 h. This was also observed in the colchicine-treated animals (Fig. 2). The activity of the hep~in-reIeasable and non-releasable lipoprotein lipase fractions of control animals also remained constant during the 6 h. In the colchicine-treated animals, however, the activity of the non-releasable fraction increased from 107 + 7 to 137 + 5 units within 270 min (P < O.OOl), after which no further increases were observed. The activity of the heparin-releasable lipoprotein lipase fraction, in contrast, decreased by about 50% in the first 3 h following the colchicine injection, and by a further 15% in the following 3 h. However, the minimal lipoprotein lipase activities of the heparin-releasable fraction reached between 270 and 360 min (about 25 units),
rw4E
(MINUTES)
TIME (MINUTES)
Fig. 2. Effect Of cokhieine on the (A) total, (B) heparin-releasable and (C} non-releasable lipoprotein KPase (LPL)fractions from hearts of rats starved for 24 h. The procedures used were as described in the legend to Fig. 1. , colchicine; - - - - - -. saline.
were significantly greater (P < 0.001) than the minimal activity observed in fed animals (7 rt 1 units) (Fig. 1). These results indicate that in starved animals the heparin-releasable lipoprotein lipase fraction is being constantly degraded and that the stable activity observed in control animals over the period of 6 h most probably derives from the constant transport of newly formed lipoprotein lipase to the endothelial surface. Confirming the results obtained with fed rats (Fig. 1) at the dose of colchicine used lipoprotein lipase transport was inhibited by about 70% resulting in a correponding increase in activity of the non-releasable lipoprotein lipase fraction. It is noteworthy, however, that 270 min after the colchicine injection the non-releasable lipoprotein lipase activity reached a plateau indicating that, at that time, the rate of formation of lipoprotein lipase for transport was decreased. The reason for this phenomenon is not presently known. Effect 24 h
of c~clo~le~i~~i~e on ~~a~~ lipoprotein
lipase activities of rats starved for
The finding that the stable heparin-releasable lipoprotein lipase activity in the heart of starved control rats was being degraded and replaced at a rapid rate (Fig. 2) raised the possibility that in these animals the stability of the nonreleasable lipoprotein hpase fraction might also be the result of a constant turnover process. To examine this possibility rats starved for 24 h were injected with cycloheximide and the heparin-releasable and non-releasable lipoprotein lipase activities measured at different time intervals (Fig. 3). The heparin-releasable lipoprotein lipase activity decreased very rapidly with a half-life of about 2 h. The lowest activity (7 + 1 units) reached 4 h after the injection of cycloheximide was similar to that found in fed rats (Fig. l), suggesting that, at that time, the formation of lipoprotein lipase for transport to the endothelial surface was inhibited by about 85%. Cycloheximide also caused a decline in the activity of the non-releasable lipoprotein lipase fraction, albeit at a much lower rate (half-life of about 4 h). This finding can be interpreted as indicating that in
heparin-releasable (B, - - - - -) and non-releasable Fig. 3. Effect of cycloheximide on the total (A. -), (C, - -_) heart lipoprotein lipase (LPL) fractions. Rats were starved for 24 h and injected intraperitoneally with cycloheximide (38 m&/kg body weight). The procedures for measurement of the different lipoprotein lipase fractions are described in Methods. The number of hearts perfused at each point is shown in parentheses.
399
starved animals the stability of the non-releasable ‘lipoprotein lipase activity (Fig. 2) depends on the constant synthesis of lipoprotein lipase. Wing et al. [18] have reported that in cycloheximide-treated rats the half-life of the total lipoprotein lipase activity is of about 2 h. In the present study the half-life of the total lipoprotein lipase activity was found to be about 4 h. The reasons for these marked differences are not presently known. The findings that the turnover rate of the non-releasable lipoprotein lipase fraction is about half of the heparin-releasable fraction could be interpreted as indicating that the rate of degradation of the enzyme is dependent on its localization in the heart. Another possibility is that the two enzyme fractions have similar rates of de~dation but the hep~in-relea~ble fraction, in addition, being present on the surface of the endothelial cells of the capillaries, could be released into the circulation associated with plasma lipoproteins [19]. A third possibility is that the heparin-releasable and non-releasable fractions correspond to two different lipoprotein lipase species with distinct turnover rates. Schotz and Garfinkel [6] using gel filtration chromatography have recently been able to separate two distinct lipoprotein lipase fractions (lipoprotein lipases a and b) from heart as well as adipose tissue. In adipose tissue lipoprotein lipase b is apparently present only in the fat cell, while lipoprotein lipase a seems to be present only in the endothelial cell [4-63. It is possible, therefore, that, as it has been proposed to be the case in adipose tissue [ 4-61, the heart hep~in-rele~able fraction corresponds to lipoprotein lipase a and the non-releasable fraction corresponds to lipoprotein lipase b. The results which demonstm~d that the hepa~n-rele~able and non-releasable lipoprotein lipase have different turnover rates (Figs 2 and 3) could also be interpreted to indicate that in hearts of starved rats these two distinct lipoprotein lipase fractions are synthesized at different rates. Another possibility is that only the non-functional, non-releasable lipoprotein lipase is synthesized in the myocardial cell, transformed by activation into the functional lipoprotein lipase, and then transported to the surface of the endothelium. While no evidence is presently available supporting either of these possibilities, the activation of a precursor form of lipoprotein lipase and its subsequent transport to the endothelial cells have been proposed to occur in the adipose tissue [4-61. If this hypothesis is correct, the results of the present study indicate that colchicine, at the dose administered, inhibited the transport of the functions lipoprotein lipase, but had no effect on the postulated activation process (Figs 1 and 2). The mechanism whereby colchicine affects the transport of lipoprotein lipase remains to be elucidated.
Acknowledgements This work was supported by Public I-Iealth Research Grants AM-16831 and A~-17046 and by the Chicago Heart Association. The authors are grateful to Drs G.S. Getz and LB Oscai for helpful discussion during the preparation of the manuscript.
400
References 1 Borensztajn, J. and Robinson, D.S. (1970) J. Lipid Res. ll, lll-117 2 Cunningham, VI. and Robinson, D.S. (1969) Biochem. J. 106. 667676 3 Robinson, D.S. (1970) Comprehensive Biochemistry (Florkin, M. and St&z, E.H.. eds), pp. 51~~116, Elsevier, Amsterdam 4 Davies, P., Cryer, A. and Robinson, D.S. (1974) PEBS Lett. 45, 271-275 5 Garfinkel, AS. and Schotz, M.C. (1973) Biochim. Biophys. Acta 306, 128-133 6 Schotz, M.C. and Garfinkel, AS. (1972) Biochim. Biophys. Acta 270, 472-478 7 Chajek, T., Stein, 0. and Stein, Y. (1975) Biochim. Biophys. Acta 380, 127-131 8 Lacy, P.E.. Howell, S.L., Young, D.A. and Fink. C..J. (1968) Nature 219, 1177-1179 9 Williams, J.A. and Wolff, J. (1972) J. Cell. Biol. 54, 157-165 10 Marchand, Y., Singh, A., AssimacopoulosJeannet, F.. Orci, L., Roviller, C. and JeanRenaud. B. (1973) J. Biol. Chem. 248, 6862-6870 11 Stein. 0. and Stein, Y. (1973) Biochim. Biophys. Acta 306, 142-147 12 Stein, 0.. Sanger, L. and Stein, Y. (1974) J. Cell Biol. 62, 90-103 13 Pelletier, G. and Bronstein, M.B. (1972) Exp. Cell Res. 70, 221-223 14 Douglas, W.W. and Sorimachi, M. (1962) Br. J. Pharmacol. 45, 129-132 15 Rossignoi. B., Herman, G. and Keryer, G. (1972) FEBS L&t. 21, 189-194 16 Borensztajn. J., Samols, D.R. and Rubenstein. A.H. (1972) Am. J. Physiol. 223, 1271-1275 17 Borensztajn, J.. Otway, S. and Robinson, D.S. (1970) J. Lipid Res. 11, 102-110 18 Wing, D.R., Fielding, C.J. and Robinson, D.S. (1967) Biochem. J. 104, 45c--46~ 19 Shafrir, E. and Biale, Y. (1970) Eur. J. Clin. Invest. 1, 19-24
Biochimica ei Biophysics &la, @ Elsevier Scientific Pubjishin~
398 (1975)
Company,
394-400
Amsterdam
- Printed in The Netherlands
BBA 56656
EFFECTS OF COLCHICINE AND ~YCLOHEXIM~DE ON THE FUN~IONAL AND NON-FUNCTIONAL LrPOPROTErN LIPASE FRACTIONS OF RAT HEART
J. BORENSZTAJN,
M.S. RONE and T. SANDROS
De~~r~rne~~ of Pathology, Ill. 60637 (U.S.A.J
(Received
The Pritzker
School
of medicine,
~tli~er~~ty of Chicago,
Chicago,
March 17th, 1975)
Summary
In response to food deprivation, total myocardial lipoprotein lipase activity increased gradually over a period of 9 h. Although lipoprotein lipase exists in a functional and non-functional form in the myocardium, most of the increase in activity occurred in the functional (heparin-releasable) lipoprotein lipase fraction. The administration of colchicine, while having no effect on the increase seen in total lipoprotein lipase activity, did inhibit the increase in the functional fraction, while at the same time, caused a marked rise in the activity of the non-functional (non-releasable) fraction. In rats injected with colchicine after a 24-h fast, total lipoprotein lipase activity was not affected, but activity levels in the functional fraction declined while that in the non-functional fraction increased. These results suggest that the functional lipoprotein lipase is constantly being formed in sites not readily accessible to heparin (presumably the myocardial cells) and transported to its site of action, the surface of the endothelial cells of the capillaries. Cycloheximide administration to rats starved for 24 h caused a decline in activity in both the functional (half-life of about 2 h) and the non-functional (half-life of about 4 h) lipoprotein lipase fractions. These results suggest that the functional and non-functional lipoprotein lipase fractions may correspond to two distinct enzyme species. -_ Introduction
Considerable evidence is presently available demonstrating that in heart and adipose tissue, as well as other extra-hepatic tissues, lipoprotein lipase exists in a functional and a non-functional form [l-3] . The functional lipoprotein lipase, that which is directly involved in the hydrolysis of plasma lipoprotein triacylglycerol, is believed to be localized on the surface of the endo-
395
thelial cells of capillaries [P-3]. This functional enzyme can be distinguished from the non-functional lipoprotein lipase by its property of being readily released from the tissues by heparin [1,3]. The non-functions lipopro~in lipase, that which does not participate in the hydrolysis of plasma triacylglycer01, is not readily accessible to heparin and is believed to be localized mainly in the parenchymal cells of the tissues [1,3] . Recent studies by Davies et al. [4] on adipose tissue and by Schotz and Garfinkel [5,6] on heart and adipose tissue have suggested that the parenchymal, non-functional lipoprotein lipase may represent the precursor of the functions hep~in-releasable lipopro~in lipase fraction. Based on these, as well as in previous studies [l--3], it has been postulated that the functional heparin-releasable lipoprotein lipase fraction is transported from its site of formation, the parenchymal cells, to its site of action, the surface of the endothelial cells of capillaries. The postulated existence of such a lipoprotein lipase transport system has recently received support from the work of Chajek et al. [7], These authors provided indirect evidence, based on the measurement of plasma post-heparin lipoprotein lipase activities, which suggested that the transport of lipoprotein lipase to the endothelial surface is inhibited by colchicine and vinblastine, compounds which are known to affect the intracellular transport of various hormones, glycoproteins and lipoproteins [S-15]. In the present study we examined the effects of colchicine and cycloheximide on the heart lipoprotein lipase activity and we provide further evidence that the functional lipoprotein lipase is formed in sites which are not readily accessible to heparin (presumably the myocardial cells), and is then transported to the luminal surface of the endothelial cells of the capillaries. Methods Male Sprague-Dawley rats (200-240 g) were housed in rooms in which the lights were switched on at 6 a.m. and switched off at 6 p.m. Fed animals are defined as rats which had free access to water and food (rat laboratory chow) and which were killed between 8 and 9 a.m. Starved animals are defined as rats which having been maintained on their normal diets, were deprived of food starting between 8 and 9 a.m. Colchicine (5.5 mg/kg body weight) and cycloheximide (38 mg/kg body weight) were dissolved in 0.9% NaCl and injected intraperitoneally. Control animals were injected intraperitoneally with 0.5 ml of 0.9% NaCl. All animals were sacrificed under ether anesthesia. For the measurement of the hepa~n-rele~able (functional) and non-releasable (non-functional) lipoprotein lipase fractions, the hearts were quickly dissected out, and perfused for 30 s in a non-recirculatory system with Krebs-Ringer bicarbonate buffer containing 1% rat serum and 5 units of heparin/ml, as previously described [ 1]. Lipoprotein lipase activities were measured in the perfusates (hep~in-rele~able fraction) and in the hearts (non-releasable fraction) after the 30-s perfusion as previously described [16]. The total lipoprotein lipase activity was obtained by adding the activities of the heparin-releasable and non-releasable fractions. The enzyme activities are expressed as units + standard error of the mean, 1 unit representing 1 pmol of free fatty acids released into the assay medium per hour incubation per gram wet weight of
396
tissue. The significance of the difference between means was analyzed using the student’s t-test. Colchicine and cycloheximide were purchased from Sigma Chemical Co., St. Louis, Missouri and sodium heparin from Upjohn Co., Kalamazoo, Michigan. Results and Discussion Effect 9h
of colchicine
on heart lipoprotein
lipase activity of rats starved for up to
When fed rats are deprived of food the total lipoprotein lipase activity in the heart gradually increases to reach maximal levels within 10 h [ 171. Most of this increase in activity occurs in the heparin-releasable fraction [ 171. These findings have been interpreted to suggest that most of the lipoprotein lipase formed in the myocardial cells during the first 10 h of starvation is transported to the surface of the endothelial cells of the capillaries. If this hypothesis is correct, blocking the lipoprotein lipase transport in these hearts would result in a similar increase in total cardiac lipoprotein lipase activity but with the increment concentrated mainly in the non-releasable fraction. To test this possibility fed rats were injected with colchicine and, at the same time, deprived of food. The heparin-releasable and non-releasable lipoprotein lipase fractions were measured at 3-h intervals over a period of 9 h. The results in Fig. 1 show that colchicine administration had no effect on the increase in the total lipoprotein lipase activity of the hearts. Significant differences were observed, however, in the activities of the heparin-releasable and non-releasable fractions (Fig. 1). In control animals the activity of the heparin-releasable fraction was significantly increased after 3 h of starvation (P < 0.001) and continued to increase for the remaining 6 h. In the colchicine-injected animals the activity of the heparinreleasable lipoprotein lipase fraction was also significantly increased after 3 h of starvation (P < O.OOl), but no further increases were observed in the remaining
25
d-.
f -’
6
-+
TIME IHOURS)
TIME LHOURS)
Fig. 1. Effect of colchicine on the (A) total, (B) heparin-releasable tein lipase (LPL) fractions. Rats fed ad libitum were deprived of neally with colchine () (5.5 mg/kg body weight) or saline of rats were sacrificed and their isolated hearts perfused for 30 containing 1% rat serum and 5 units of heparinlml. The procedures activities in the different fractions are as described in Methods. point is shown in parentheses.
TIME (HOURS)
and (C) non-releasable heart lipoprofood at 8 a.m. and injected intraperito(- - - - - -). At the times indicated groups s with Krebs-Ringer bicarbonate buffer used to measure the lipoprotein lipase The number of hearts perfused at each
397
6 h. Thus, after 9 h the activity of the hep~n-rele~able lipoprotein lipase fraction of the control animals had increased from 7 + 1 to 75 * 4 units, while in the colchicine-injected animals the increase was only from 7 + 1 to 17 rt 3 units. The activity of the non-releasable lipoprotein lipase fraction (Fig. 1) of control animals increased from 45 f 2 to 99 k 6 units after 9 h of starvation (P < 0.001). In the colchicine-injected animals, however, the increase in activity of the non-releasable fraction at 6 and 9 h of starvation was about 70% greater (P < 0.001) than that of control animals, and accounted for most of the increase in total lipoprotein lipase activity observed in these hearts. These results show that in colchicine-treated rats the increase in heparinreleasable lipoprotein lipase activity was inhibited by about 80%. In addition, they strongly suggest that in these animals the activity of the non-releasable lipoprotein lipase which is greater than that of the control animals represents the lipoprotein lipase fraction whose transport to the surface of the endothelial cells is inhibited. Effect of colchicine on heart lipoprotein lipase activities of rats starved for 24 h Shown in Fig. 2 are the results obtained when rats starved for 24 h were injected with colchicine and the heart heparin-releasable and non-releasable lipoprotein lipase fractions measured. Confirming previous observations [ 1’71 the total heart lipoprotein lipase activity in the control animals remained constant for a period of 6 h. This was also observed in the colchicine-treated animals (Fig. 2). The activity of the hep~in-reIeasable and non-releasable lipoprotein lipase fractions of control animals also remained constant during the 6 h. In the colchicine-treated animals, however, the activity of the non-releasable fraction increased from 107 + 7 to 137 + 5 units within 270 min (P < O.OOl), after which no further increases were observed. The activity of the heparin-releasable lipoprotein lipase fraction, in contrast, decreased by about 50% in the first 3 h following the colchicine injection, and by a further 15% in the following 3 h. However, the minimal lipoprotein lipase activities of the heparin-releasable fraction reached between 270 and 360 min (about 25 units),
rw4E
(MINUTES)
TIME (MINUTES)
Fig. 2. Effect Of cokhieine on the (A) total, (B) heparin-releasable and (C} non-releasable lipoprotein KPase (LPL)fractions from hearts of rats starved for 24 h. The procedures used were as described in the legend to Fig. 1. , colchicine; - - - - - -. saline.
were significantly greater (P < 0.001) than the minimal activity observed in fed animals (7 rt 1 units) (Fig. 1). These results indicate that in starved animals the heparin-releasable lipoprotein lipase fraction is being constantly degraded and that the stable activity observed in control animals over the period of 6 h most probably derives from the constant transport of newly formed lipoprotein lipase to the endothelial surface. Confirming the results obtained with fed rats (Fig. 1) at the dose of colchicine used lipoprotein lipase transport was inhibited by about 70% resulting in a correponding increase in activity of the non-releasable lipoprotein lipase fraction. It is noteworthy, however, that 270 min after the colchicine injection the non-releasable lipoprotein lipase activity reached a plateau indicating that, at that time, the rate of formation of lipoprotein lipase for transport was decreased. The reason for this phenomenon is not presently known. Effect 24 h
of c~clo~le~i~~i~e on ~~a~~ lipoprotein
lipase activities of rats starved for
The finding that the stable heparin-releasable lipoprotein lipase activity in the heart of starved control rats was being degraded and replaced at a rapid rate (Fig. 2) raised the possibility that in these animals the stability of the nonreleasable lipoprotein hpase fraction might also be the result of a constant turnover process. To examine this possibility rats starved for 24 h were injected with cycloheximide and the heparin-releasable and non-releasable lipoprotein lipase activities measured at different time intervals (Fig. 3). The heparin-releasable lipoprotein lipase activity decreased very rapidly with a half-life of about 2 h. The lowest activity (7 + 1 units) reached 4 h after the injection of cycloheximide was similar to that found in fed rats (Fig. l), suggesting that, at that time, the formation of lipoprotein lipase for transport to the endothelial surface was inhibited by about 85%. Cycloheximide also caused a decline in the activity of the non-releasable lipoprotein lipase fraction, albeit at a much lower rate (half-life of about 4 h). This finding can be interpreted as indicating that in
heparin-releasable (B, - - - - -) and non-releasable Fig. 3. Effect of cycloheximide on the total (A. -), (C, - -_) heart lipoprotein lipase (LPL) fractions. Rats were starved for 24 h and injected intraperitoneally with cycloheximide (38 m&/kg body weight). The procedures for measurement of the different lipoprotein lipase fractions are described in Methods. The number of hearts perfused at each point is shown in parentheses.
399
starved animals the stability of the non-releasable ‘lipoprotein lipase activity (Fig. 2) depends on the constant synthesis of lipoprotein lipase. Wing et al. [18] have reported that in cycloheximide-treated rats the half-life of the total lipoprotein lipase activity is of about 2 h. In the present study the half-life of the total lipoprotein lipase activity was found to be about 4 h. The reasons for these marked differences are not presently known. The findings that the turnover rate of the non-releasable lipoprotein lipase fraction is about half of the heparin-releasable fraction could be interpreted as indicating that the rate of degradation of the enzyme is dependent on its localization in the heart. Another possibility is that the two enzyme fractions have similar rates of de~dation but the hep~in-relea~ble fraction, in addition, being present on the surface of the endothelial cells of the capillaries, could be released into the circulation associated with plasma lipoproteins [19]. A third possibility is that the heparin-releasable and non-releasable fractions correspond to two different lipoprotein lipase species with distinct turnover rates. Schotz and Garfinkel [6] using gel filtration chromatography have recently been able to separate two distinct lipoprotein lipase fractions (lipoprotein lipases a and b) from heart as well as adipose tissue. In adipose tissue lipoprotein lipase b is apparently present only in the fat cell, while lipoprotein lipase a seems to be present only in the endothelial cell [4-63. It is possible, therefore, that, as it has been proposed to be the case in adipose tissue [ 4-61, the heart hep~in-rele~able fraction corresponds to lipoprotein lipase a and the non-releasable fraction corresponds to lipoprotein lipase b. The results which demonstm~d that the hepa~n-rele~able and non-releasable lipoprotein lipase have different turnover rates (Figs 2 and 3) could also be interpreted to indicate that in hearts of starved rats these two distinct lipoprotein lipase fractions are synthesized at different rates. Another possibility is that only the non-functional, non-releasable lipoprotein lipase is synthesized in the myocardial cell, transformed by activation into the functional lipoprotein lipase, and then transported to the surface of the endothelium. While no evidence is presently available supporting either of these possibilities, the activation of a precursor form of lipoprotein lipase and its subsequent transport to the endothelial cells have been proposed to occur in the adipose tissue [4-61. If this hypothesis is correct, the results of the present study indicate that colchicine, at the dose administered, inhibited the transport of the functions lipoprotein lipase, but had no effect on the postulated activation process (Figs 1 and 2). The mechanism whereby colchicine affects the transport of lipoprotein lipase remains to be elucidated.
Acknowledgements This work was supported by Public I-Iealth Research Grants AM-16831 and A~-17046 and by the Chicago Heart Association. The authors are grateful to Drs G.S. Getz and LB Oscai for helpful discussion during the preparation of the manuscript.
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