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Lepid transport across the intestinal epithelial cell
291
Biochimica et Biophysics Acta, 431 (1976) 297-302 @ Elsevier Scientific Publishing Company, Amsterdam
- Printed
in The Netherlands
BBA 56718
LIPID TRANSPORT EFFECT
ACROSS THE INTESTINAL
EPITHELIAL
CELL
OF COLCHICINE
C.A. ARREAZA-PLAZA,
V. BOSCH and M.A. OTAYEK
Chtedra de Patologh General y Fisiopatologio, Facultad de Medicinn, Apdo. 50587, Grande, Universidad Central de Venezuela, Caracas (Venezuela) (Received
November
24th,
Sabana
1975)
summary Rats injected with colchicine (0.5 mg/lOO g of body weight) 1 h before ingestion of a margarine emulsion (1 g in 2 ml of saline) do not show the rise in plasma triacylglycerol concentration found in controls during the subsequent hours. The effect of colchicine is more dramatic when the experiment is performed after prior administration of Triton WR-1339, a substance known to inhibit the catabolism of lipoproteins. Colchicine-treated rats also showed a fivefold increase in the content of triacylglycerol in proximal jejunum, when compared to controls. These results are consistent with the idea that colchicine interferes with the intracellular phase of fat absorption, suggesting that the microtubular-microfilamentous system could be involved in the release of chylomicrons from the intestinal cell into the circulation.
Introduction It has been demonstrated that the secretion of substances stored in granules or vesicles is affected by mitotic poisons such as colchicine, and the Vinca alkaloids (vincristine and vinblastine) [l-7]. These drugs affect the microtubularmicrofilamentous system, which participates in the intracellular transfer of molecules from the sites of synthesis to the extracellular space. In the case of colchicine a specific binding to the microtubule protein tubulin has been demonstrated, resulting in the inhibition of microtubule activity and impairment of the organell function [8]. Microtubules have been observed in liver parenchymal cells [9], and it has been reported that the secretion of lipoproteins by the perfused mouse liver [lo] and in the whole rat [ll] is significantly diminished by colchicine and the Vinca alkaloids. In addition, we have already shown that these drugs have an inhibitory effect on the secretion of lipoproteins and proteins by incubated
298
rat liver slices. 2Hz0 or low calcium concentrations had the same effect [12]. However, despite the similarities of lipoprotein synthesis by the liver and the intestine, we do not know about any work done concerning the effect of colchicine on chylomicron synthesis and release by the intestine. In this communication we present our results on the influence of colchicine on the process of lipid transport across the intestinal epithelial cell. Materials and Methods
Animals and experiment procedure Female Sprague-Dawley rats weighing between 230 and 250 g, reared in our own animal facilities, were fasted overnight. An initial blood sample was taken from the cut end of the tail vein of every animal. Colchicine (Sigma-Chemical Co., St. Louis, MO.) freshly dissolved in 0.15 M NaCl was injected intraperitoneally in a single dose of 0.5 mg/lOO g of body weight. Control rats were injected with saline. 1 h after the administration of colchicine or saline, 1 g of emulsified margarine * was given to all animals by gastric intubation in 2 ml of saline. 1 h later another blood sample from the tail vein was obtained. 2 h after the administration of margarine, the rats were decapitated with a guillotine and blood was collected from the cervical wound. All blood samples were anticogulated with EDTA and centrifuged at 2000 rev./min for 20 min in an Intemational Refrigerated centrifuge. Chylomicrons were floated from approximately 4 ml of plasma by ultracentrifugation at 105 000 X g for 1 h in a Spine0 L5-65 ultracentrifuge with a 50 Ti Rotor at 16°C. Then they were recentrifuged in saline solution at density 1.006 g/ml, using the same procedure as above. Triacylglycerol concentrations were determined in aliquots of plasma and of chylomicron suspensions [ 131.
Measurement of rat intestinal triacylglycerol con tent A segment of proximal jejunum was obtained immediately after decapitation and thoroughly washed with saline, From this, 1 g of wet tissue was homogenized in a Polytron type PT-10-20-3500 (Kinematica, Switzerl~d), the lipids were extracted [14] and triacylglycerols analyzed quantitatively as before [ 131. Results are expressed as mg/g wet tissue.
Experiments with Triton WR-1339 (‘Superinone’),
colchicine and margarine
In order to study the effect of Triton WR-1339 on plasma t~acylglycerol and chylomicron accumulation in colchicine-treated and control rats, we maintained the same experimental protocol described above except that, before the margarine was given, rats were anesthetized with ether, a femoral vein was exposed and 0.5 ml of 20% Triton WR-1339 solution in saline was injected. 2 h later the animals were decapitated and the blood was collected. Control rats were submitted to the same operation, but injected with saline.
* Prepared with partially hydrogenated cottonseed, coconut, seeame oils and powdered milk. Fatty acid composition was determined by gas-liquid chromatography: lam-k. 11%. myristic 5%. Palmitic 21%. stearie 6%. oleic 51% and linoleic 6%.
299
Absorption of D-(+)-Xyhe by the intestine in colchicine-treated and control rats 50 mg of xylose dissolved in 2 ml of distilled water were administered by gastric intubation to control rats subjected 1 h earlier to ligation of the most proximal urethra. 3 h later, the rats were decapitated and the clean urine collected in the bladder, approximately 1.5 ml per rat, was withdrawn with a syringe. Another group of rats was similarly treated, except that the same dose of xylose was given 1 h after the administration of colchicine (0.5 mg/ 100 g body weight). All urine samples were analyzed for D-(+)-xylose [ 291 .
Analysis of pancreatic lipase activity in the intestinal juice Non-fasted rats, treated 1 h before with colchicine or saline, as described above, were anesthetized with ether, the abdominal cavity was opened and two catgut ligatures were placed in order to obtain a closed duodenum loop which was replaced into the abdomen. 1 h later, the animals were decapitated, the abdominal cavity was reopened and the entire content of the duodenal juice (about 1.5 ml) was collected and frozen at below 65°C until analyzed [25]. Results The ingestion of margarine by the rat is followed by a rising curve of triacylglycerol concentration in plasma. This postprandial hypertriglyceridemia is inhibited by the action of colchicine when the drug is given shortly before the absorptive process (Fig. 1). Triton WR-1339 interferes with the hydrolysis of chylomicrons and VLDL * by preventing the action of the lipoprotein lipase complex [ 151. Therefore, its administration blocks the exit of triacylglycerol moiety of VLDL and chylomicrons from the plasma compartment. The results of the experiments performed with Triton WR-1339 alone, Triton WR-1339 plus margarine and Triton WR-1339 plus colchicine plus margarine are given in Table I. As can be seen, the treatment with Triton WR-1339 alone produces a marked increase in plasma triacylglycerol concentration in the fasting rat. This effect is mainly due to the plasma accumulation of VLDL secreted by the liver. If Triton WR-1339 is given in conjuction with margarine, the elevation of triacylglycerol concentration in plasma reaches higher values than when Triton WR-1339 is given alone (P < 0.001). However, pretreatment with colchicine blocks the margarine induced rise in triacylglycerol concentration in the presence of Triton WR1339. In Table II we show the results obtained when triacylglycerol quantification was performed on the chylomicrons alone, to avoid interference from other triacylglycerol-containing lipoproteins. Triton WR-1339 produced an increased concentration of chylomicron triacylglycerol in fasted rats. Furthermore, margarine, when given in addition to Triton WR-1339, produces an even higher rise in chylomicron triacylglycerol concentration in plasma (P < 0.001). However, the margarine-induced rise in triacylglycerol concentration in Triton WR-1339* Very low density intestinal origin.
lipoproteins:
lipoprotein
particles
with a density
< 1.006
g/ml.
from
hepatic
or
-‘-d-
CONTROLS
-4~
TREATED
0
; TIME
; ( hours)
Fig. 1. Curve of cumulative plasma triacylglycerol concentration in rats after margarine administration in control and colchicine-treated rats. Results are expressed as means of results from 8 rats k S.E.M. (P < 0.001).
treated rats is completely blocked by the previous adm~is~ation of colchicine, as found for whole plasma triacylglycerol. In addition, colchicine also blocks to some extent (59%) the increase in chylomicron triacylclyerol seen in the Triton WR-13394reated animals. This would suggest that the observed rise in chylomicron triacylglycerol seen after Triton WR-1339 administration could be due to coprophagia, which was unavoidable in the conditions of our experiments, or to phospholipids secreted to the bile. Colchicine could also interfere with the release into the circulation of chylomicrons whose triacylglycerol moiety is derived from the bile phospholipids. To prove that colchicine-mediated inhibition of triacylglycerol concentration rise in blood was due to interference with chylomicron released by the intestine, we measure the content of triacylglycerol in a jejunal segment after margarine and colchicine treatment as compared to margarine treatment alone. The amount of triacylglycerol in six control animals was 3.7 rt 1 mg/g wet TABLE I TRIACYLGLYCEROL CONCENTRATION IN PLASMA OF FASTED RATS AFTER THE ADMINISTRATION OF TRITON WR-1339 ALONE, TRITON PLUS MARGARINE. AND COLCHICINE PLUS TRITON PLUS MARGARINE Results are expressed as mg/lOO ml of plasma, mean ?; S.E.M.. Number of animals in each experiment is given in parenthesis. Degree of significance: * P < 0.001 when compared with Triton + margarine-treated rats. Results of 0 and 120 min were analyzed in each experimental group and all were found to differ significalltlY (P < 0.001). Time (mm)
Triton (61
0 120
45.2 + 4 920 f 31*
Triton + margarine (61 46.7 + 4 1588 * 36
._
Colchicine + Trlton + margarine (8) 48 f 4.5 978 f 80 *
301 TABLE II PLASMA AFTER TREATED
CHYLOMICRONS ADMINISTERING
TRIACYLGLYCEROL CONCENTRATIONS IN FASTED RATS 2 h SALINE, MARGARINE AND COLCHICINE PLUS MARGARINE,
OR NOT WITH TRITON WR-1339
Results are mean f S.E.M., mg/lOO ml of plasma. Number of animals is given in parenthesis. Saline (6) Without Triton With Triton
27f
1
289 + 12
Margarine (8) 755
9
461 t 14
Colchicine + margarine (10) 44*
8
171-?: 14
weight. A 5-fold increase occurred in the six rats treated with colchicine (20.2 f 1 mg/g wet weight, P < 0.001). In order to explore the possibility that colchicine could have a general toxic effect on the intestine, we performed a xylose absorption test. The result of this experiment showed that the 3 h urine excretion of xylose in control (6.9 + 0.5 mg/3 h) and treated rats (7.9 f 1.6 mg/ 3 h) were not statistically different. Pancreatic lipase concentrations in intestinal juice gave similar results in control and treated rats. Discussion There is an obvious resemblance between VLDL and chylomicrons, not only in their chemical and physicochemical properties but in the complex process leading to their progress along the endoplasmic reticulum, Golgi apparatus and secretory vesicles, on their way to the extracellular space [16-231. Therefore, it was natural to postulate that colchicine, which has been shown to inhibit VLDL secretion by the liver, could interfere with the intracellular phase of fat absorption. The results presented here support this hypothesis, since we have produced an almost complete inhibition on the release of the absorbed fat to the chyle after administration of colchicine to rats. This transport inhibition produced a remarkable accumulation of triacylglycerol in the intestine. Moreover, colchicine treatment did not seem to induce any abnormality in the digestion of the fat load as judged by the normal amount of pancreatic lipase found in the intestinal juice of the treated animals. The fact that controls and colchicine-treated rats showed the same capacity to absorb xylose indicates that the dose of colchicine used in present experiments did not produce a general toxic effect upon the intestinal mucosa. Our results do not give information about the intimate mechanism by which colchicine interferes with the intracellular phase of fat absorption. A microtubular-microfilamentous system has been described in the intestinal epithelial cells [24]. The inactivation of this system by colchicine might be the main factor responsible for its interference with fat release from the intestine but, of course, many other possibilities should be investigated before a final explanation about this subject could be given. The dose of colchicine used in these experiments is rather high when compared to that employed in the treatment of symptomatic gout in man. It would
302
be interesting to know if orally administered colchicine could produce, acting directly in the intestinal mucosa, the same inhibitory effect of fat transport in to the circulation. Colchicine has been recently shown to reduce lipoprotein lipase activity [ 261, to block lipoprotein secretion by the liver [lo--121 , to inhibit free fatty acid release by the adipose tissue [27] and to suppress reversibly lactation in the goat [28]. All these effects, plus the block on fat transport presented in this communication, indicates that the drug will find a more extensive use as a probe for study of many facets of lipid metabolism. Acknowledgements This work was supported by grants from Consejo National de Investigaciones Cientificas y Tecnologicas (CONICIT) No. 31-26-Sl-0696, and Consejo de Desarrollo Cientifico y Humanistic0 de la Universidad Central de Venezuela (1974). In addition, we acknowledge the technical assistance of Mrs. Marta de Rodtiguez, the care and handling of the experimental animals to Miss Emilia Perez Ayuzo and the secretarial work of Mrs. Luisa de Ferrari. Winthrop de Venezuela kindly provided us with Triton WR-1339 (“Superinone”). References 1 Lacy, P.E.. Howell. S.L., Young. D.A. and Fink, C.J. (1968) Nature 219. 1177-1179 2 MaIaisse. W.J.. MaIaisse-Lagae, F., Walker, M.O. and Lacy, P.E. (1971) Diabetes 20.257-265 3 Van Obberghen, E., Somers, G., Devis. G.. Ravazzola, M.. Malaisse-Lagae. F.. Orci. L. and Malaisse. W. 4 5 6 7 8
J. (1974) Endocrinology 95.1518-1528 Williams, J.A. and Wolff, J. (1970) Proc. Natl. Acad. Sci. U.S. 67.1901-1908 Poisner, A.M. and Berstain, J. (1971) J. Pharmacol. EXP. Ther. 177. 102-108 Gillespie. E. and Lichtenstein, L.M. (1972) J. CIin. Invest. 51, 2941-2947 Gillespie. E.. Levine, R.S. and Malawista. S.E. (1968) J. Pharmacol. EXP. Ther. 164,158-165 OwelIen, R.J., Owens, Jr., A.H. and Donigian. C. (1972) Biochem. Biophys. Res. Commtm. 47. 685-
691 9 Bruni. C. and Porter, K.R. (1965) Am. J. Pathol. XLVI. 691-755 10 Le Marchand. Y., Singh, A., AssimacopouIus-Jeannet. F.. Orci, L., RouilIer, C. and Jeanrenaud. B. (1973) J. Biol. Chem. 248.6862-6870 11 Stein, O., Sanger. L. and Stein, Y. (1974) J. Cell. Biol. 62.90-103 I2 Arreaza-Plaza. C.. Bosch, V., Otayek. M. and Rodriguez. M. (1975) Acta Cient. Venez. 26. (SUPPI. 1). 13 14 15 16 17 18 19 20 21 22 23
125-126 Van Handel, E. and Zilversmith. D.V. (1957) J. Lab. Clin. Med. 50, 152-157 Folch. J., Lees, M. and Sloan-Stanley. G.H. (1957) J. Biol. Chem. 226.497-509 Otway. S. and Robinson, D.S. (1967) J. Physiol. 190.321-332 Johnston, J.M. and BorgstrGm. B. (1964) Biochim. Biophys. Acta 84,412--423 Senior, J.R. (1964) J. Lipid Res. 5,495-521 Friedman, H.J. and CardeB. R.R. (1972) J. Cell. Biol. 52.1540 Jersild. Jr., R.A. (1966) Am. J. Anat. 118.136-141 Lacy, D. and Taylor, A.B. (1962) Am. J. Anat. 110.155-163 Sage, J.A. and Jersild, Jr., R.A. (1971) J. Cell. Biol. 51.333-338 Trier, J.S. and Rubin. C.E. (1965) Gastroenterology 49, 574-603 Mahley, R.W., Bennett, B.D.. Morr6, D.J., Gray. M.E.. Thistlethwait, W. and Le
24 25 26 27 28 29
Lab. Invest. 25, 435444 Tilney, L.G. and Mooseker, M. (1971) Proc. Natl. Acad. Sci. U.S. 68.2611-2615 Shihabi. Z.K. and Bishop, C. (1971) Clin. Chem. 17.1150-1153 Chajeck. T., Stein, 0. and Stein, Y. (1975) Biochim. Biophys. Acta 380. 127-131 Schimmel. R.J. (1974) J. Lipid Res. 15, 206-210 Patton, S. (1974) FEBS Lett. 48. 85-87 Roe, J.H. and Rice, E.W. (1948) J. Biol. Chem. 173. 507-512
Quire. V.S.(1971)
Biochimica et Biophysics Acta, 431 (1976) 297-302 @ Elsevier Scientific Publishing Company, Amsterdam
- Printed
in The Netherlands
BBA 56718
LIPID TRANSPORT EFFECT
ACROSS THE INTESTINAL
EPITHELIAL
CELL
OF COLCHICINE
C.A. ARREAZA-PLAZA,
V. BOSCH and M.A. OTAYEK
Chtedra de Patologh General y Fisiopatologio, Facultad de Medicinn, Apdo. 50587, Grande, Universidad Central de Venezuela, Caracas (Venezuela) (Received
November
24th,
Sabana
1975)
summary Rats injected with colchicine (0.5 mg/lOO g of body weight) 1 h before ingestion of a margarine emulsion (1 g in 2 ml of saline) do not show the rise in plasma triacylglycerol concentration found in controls during the subsequent hours. The effect of colchicine is more dramatic when the experiment is performed after prior administration of Triton WR-1339, a substance known to inhibit the catabolism of lipoproteins. Colchicine-treated rats also showed a fivefold increase in the content of triacylglycerol in proximal jejunum, when compared to controls. These results are consistent with the idea that colchicine interferes with the intracellular phase of fat absorption, suggesting that the microtubular-microfilamentous system could be involved in the release of chylomicrons from the intestinal cell into the circulation.
Introduction It has been demonstrated that the secretion of substances stored in granules or vesicles is affected by mitotic poisons such as colchicine, and the Vinca alkaloids (vincristine and vinblastine) [l-7]. These drugs affect the microtubularmicrofilamentous system, which participates in the intracellular transfer of molecules from the sites of synthesis to the extracellular space. In the case of colchicine a specific binding to the microtubule protein tubulin has been demonstrated, resulting in the inhibition of microtubule activity and impairment of the organell function [8]. Microtubules have been observed in liver parenchymal cells [9], and it has been reported that the secretion of lipoproteins by the perfused mouse liver [lo] and in the whole rat [ll] is significantly diminished by colchicine and the Vinca alkaloids. In addition, we have already shown that these drugs have an inhibitory effect on the secretion of lipoproteins and proteins by incubated
298
rat liver slices. 2Hz0 or low calcium concentrations had the same effect [12]. However, despite the similarities of lipoprotein synthesis by the liver and the intestine, we do not know about any work done concerning the effect of colchicine on chylomicron synthesis and release by the intestine. In this communication we present our results on the influence of colchicine on the process of lipid transport across the intestinal epithelial cell. Materials and Methods
Animals and experiment procedure Female Sprague-Dawley rats weighing between 230 and 250 g, reared in our own animal facilities, were fasted overnight. An initial blood sample was taken from the cut end of the tail vein of every animal. Colchicine (Sigma-Chemical Co., St. Louis, MO.) freshly dissolved in 0.15 M NaCl was injected intraperitoneally in a single dose of 0.5 mg/lOO g of body weight. Control rats were injected with saline. 1 h after the administration of colchicine or saline, 1 g of emulsified margarine * was given to all animals by gastric intubation in 2 ml of saline. 1 h later another blood sample from the tail vein was obtained. 2 h after the administration of margarine, the rats were decapitated with a guillotine and blood was collected from the cervical wound. All blood samples were anticogulated with EDTA and centrifuged at 2000 rev./min for 20 min in an Intemational Refrigerated centrifuge. Chylomicrons were floated from approximately 4 ml of plasma by ultracentrifugation at 105 000 X g for 1 h in a Spine0 L5-65 ultracentrifuge with a 50 Ti Rotor at 16°C. Then they were recentrifuged in saline solution at density 1.006 g/ml, using the same procedure as above. Triacylglycerol concentrations were determined in aliquots of plasma and of chylomicron suspensions [ 131.
Measurement of rat intestinal triacylglycerol con tent A segment of proximal jejunum was obtained immediately after decapitation and thoroughly washed with saline, From this, 1 g of wet tissue was homogenized in a Polytron type PT-10-20-3500 (Kinematica, Switzerl~d), the lipids were extracted [14] and triacylglycerols analyzed quantitatively as before [ 131. Results are expressed as mg/g wet tissue.
Experiments with Triton WR-1339 (‘Superinone’),
colchicine and margarine
In order to study the effect of Triton WR-1339 on plasma t~acylglycerol and chylomicron accumulation in colchicine-treated and control rats, we maintained the same experimental protocol described above except that, before the margarine was given, rats were anesthetized with ether, a femoral vein was exposed and 0.5 ml of 20% Triton WR-1339 solution in saline was injected. 2 h later the animals were decapitated and the blood was collected. Control rats were submitted to the same operation, but injected with saline.
* Prepared with partially hydrogenated cottonseed, coconut, seeame oils and powdered milk. Fatty acid composition was determined by gas-liquid chromatography: lam-k. 11%. myristic 5%. Palmitic 21%. stearie 6%. oleic 51% and linoleic 6%.
299
Absorption of D-(+)-Xyhe by the intestine in colchicine-treated and control rats 50 mg of xylose dissolved in 2 ml of distilled water were administered by gastric intubation to control rats subjected 1 h earlier to ligation of the most proximal urethra. 3 h later, the rats were decapitated and the clean urine collected in the bladder, approximately 1.5 ml per rat, was withdrawn with a syringe. Another group of rats was similarly treated, except that the same dose of xylose was given 1 h after the administration of colchicine (0.5 mg/ 100 g body weight). All urine samples were analyzed for D-(+)-xylose [ 291 .
Analysis of pancreatic lipase activity in the intestinal juice Non-fasted rats, treated 1 h before with colchicine or saline, as described above, were anesthetized with ether, the abdominal cavity was opened and two catgut ligatures were placed in order to obtain a closed duodenum loop which was replaced into the abdomen. 1 h later, the animals were decapitated, the abdominal cavity was reopened and the entire content of the duodenal juice (about 1.5 ml) was collected and frozen at below 65°C until analyzed [25]. Results The ingestion of margarine by the rat is followed by a rising curve of triacylglycerol concentration in plasma. This postprandial hypertriglyceridemia is inhibited by the action of colchicine when the drug is given shortly before the absorptive process (Fig. 1). Triton WR-1339 interferes with the hydrolysis of chylomicrons and VLDL * by preventing the action of the lipoprotein lipase complex [ 151. Therefore, its administration blocks the exit of triacylglycerol moiety of VLDL and chylomicrons from the plasma compartment. The results of the experiments performed with Triton WR-1339 alone, Triton WR-1339 plus margarine and Triton WR-1339 plus colchicine plus margarine are given in Table I. As can be seen, the treatment with Triton WR-1339 alone produces a marked increase in plasma triacylglycerol concentration in the fasting rat. This effect is mainly due to the plasma accumulation of VLDL secreted by the liver. If Triton WR-1339 is given in conjuction with margarine, the elevation of triacylglycerol concentration in plasma reaches higher values than when Triton WR-1339 is given alone (P < 0.001). However, pretreatment with colchicine blocks the margarine induced rise in triacylglycerol concentration in the presence of Triton WR1339. In Table II we show the results obtained when triacylglycerol quantification was performed on the chylomicrons alone, to avoid interference from other triacylglycerol-containing lipoproteins. Triton WR-1339 produced an increased concentration of chylomicron triacylglycerol in fasted rats. Furthermore, margarine, when given in addition to Triton WR-1339, produces an even higher rise in chylomicron triacylglycerol concentration in plasma (P < 0.001). However, the margarine-induced rise in triacylglycerol concentration in Triton WR-1339* Very low density intestinal origin.
lipoproteins:
lipoprotein
particles
with a density
< 1.006
g/ml.
from
hepatic
or
-‘-d-
CONTROLS
-4~
TREATED
0
; TIME
; ( hours)
Fig. 1. Curve of cumulative plasma triacylglycerol concentration in rats after margarine administration in control and colchicine-treated rats. Results are expressed as means of results from 8 rats k S.E.M. (P < 0.001).
treated rats is completely blocked by the previous adm~is~ation of colchicine, as found for whole plasma triacylglycerol. In addition, colchicine also blocks to some extent (59%) the increase in chylomicron triacylclyerol seen in the Triton WR-13394reated animals. This would suggest that the observed rise in chylomicron triacylglycerol seen after Triton WR-1339 administration could be due to coprophagia, which was unavoidable in the conditions of our experiments, or to phospholipids secreted to the bile. Colchicine could also interfere with the release into the circulation of chylomicrons whose triacylglycerol moiety is derived from the bile phospholipids. To prove that colchicine-mediated inhibition of triacylglycerol concentration rise in blood was due to interference with chylomicron released by the intestine, we measure the content of triacylglycerol in a jejunal segment after margarine and colchicine treatment as compared to margarine treatment alone. The amount of triacylglycerol in six control animals was 3.7 rt 1 mg/g wet TABLE I TRIACYLGLYCEROL CONCENTRATION IN PLASMA OF FASTED RATS AFTER THE ADMINISTRATION OF TRITON WR-1339 ALONE, TRITON PLUS MARGARINE. AND COLCHICINE PLUS TRITON PLUS MARGARINE Results are expressed as mg/lOO ml of plasma, mean ?; S.E.M.. Number of animals in each experiment is given in parenthesis. Degree of significance: * P < 0.001 when compared with Triton + margarine-treated rats. Results of 0 and 120 min were analyzed in each experimental group and all were found to differ significalltlY (P < 0.001). Time (mm)
Triton (61
0 120
45.2 + 4 920 f 31*
Triton + margarine (61 46.7 + 4 1588 * 36
._
Colchicine + Trlton + margarine (8) 48 f 4.5 978 f 80 *
301 TABLE II PLASMA AFTER TREATED
CHYLOMICRONS ADMINISTERING
TRIACYLGLYCEROL CONCENTRATIONS IN FASTED RATS 2 h SALINE, MARGARINE AND COLCHICINE PLUS MARGARINE,
OR NOT WITH TRITON WR-1339
Results are mean f S.E.M., mg/lOO ml of plasma. Number of animals is given in parenthesis. Saline (6) Without Triton With Triton
27f
1
289 + 12
Margarine (8) 755
9
461 t 14
Colchicine + margarine (10) 44*
8
171-?: 14
weight. A 5-fold increase occurred in the six rats treated with colchicine (20.2 f 1 mg/g wet weight, P < 0.001). In order to explore the possibility that colchicine could have a general toxic effect on the intestine, we performed a xylose absorption test. The result of this experiment showed that the 3 h urine excretion of xylose in control (6.9 + 0.5 mg/3 h) and treated rats (7.9 f 1.6 mg/ 3 h) were not statistically different. Pancreatic lipase concentrations in intestinal juice gave similar results in control and treated rats. Discussion There is an obvious resemblance between VLDL and chylomicrons, not only in their chemical and physicochemical properties but in the complex process leading to their progress along the endoplasmic reticulum, Golgi apparatus and secretory vesicles, on their way to the extracellular space [16-231. Therefore, it was natural to postulate that colchicine, which has been shown to inhibit VLDL secretion by the liver, could interfere with the intracellular phase of fat absorption. The results presented here support this hypothesis, since we have produced an almost complete inhibition on the release of the absorbed fat to the chyle after administration of colchicine to rats. This transport inhibition produced a remarkable accumulation of triacylglycerol in the intestine. Moreover, colchicine treatment did not seem to induce any abnormality in the digestion of the fat load as judged by the normal amount of pancreatic lipase found in the intestinal juice of the treated animals. The fact that controls and colchicine-treated rats showed the same capacity to absorb xylose indicates that the dose of colchicine used in present experiments did not produce a general toxic effect upon the intestinal mucosa. Our results do not give information about the intimate mechanism by which colchicine interferes with the intracellular phase of fat absorption. A microtubular-microfilamentous system has been described in the intestinal epithelial cells [24]. The inactivation of this system by colchicine might be the main factor responsible for its interference with fat release from the intestine but, of course, many other possibilities should be investigated before a final explanation about this subject could be given. The dose of colchicine used in these experiments is rather high when compared to that employed in the treatment of symptomatic gout in man. It would
302
be interesting to know if orally administered colchicine could produce, acting directly in the intestinal mucosa, the same inhibitory effect of fat transport in to the circulation. Colchicine has been recently shown to reduce lipoprotein lipase activity [ 261, to block lipoprotein secretion by the liver [lo--121 , to inhibit free fatty acid release by the adipose tissue [27] and to suppress reversibly lactation in the goat [28]. All these effects, plus the block on fat transport presented in this communication, indicates that the drug will find a more extensive use as a probe for study of many facets of lipid metabolism. Acknowledgements This work was supported by grants from Consejo National de Investigaciones Cientificas y Tecnologicas (CONICIT) No. 31-26-Sl-0696, and Consejo de Desarrollo Cientifico y Humanistic0 de la Universidad Central de Venezuela (1974). In addition, we acknowledge the technical assistance of Mrs. Marta de Rodtiguez, the care and handling of the experimental animals to Miss Emilia Perez Ayuzo and the secretarial work of Mrs. Luisa de Ferrari. Winthrop de Venezuela kindly provided us with Triton WR-1339 (“Superinone”). References 1 Lacy, P.E.. Howell. S.L., Young. D.A. and Fink, C.J. (1968) Nature 219. 1177-1179 2 MaIaisse. W.J.. MaIaisse-Lagae, F., Walker, M.O. and Lacy, P.E. (1971) Diabetes 20.257-265 3 Van Obberghen, E., Somers, G., Devis. G.. Ravazzola, M.. Malaisse-Lagae. F.. Orci. L. and Malaisse. W. 4 5 6 7 8
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