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Lymphatic lipid transport is not impaired in ageing rat intestine
Mechanisms of Ageing and Development 113 (2000) 219 – 225 www.elsevier.com/locate/mechagedev
Lymphatic lipid transport is not impaired in ageing rat intestine Hiroshi Hayashi a,b,*, Yuko Sato b, Setsuko Kanai b, Masao Masuda b, Minoru Ohta b, Akihiro Funakoshi c, Koji Nagao d, Katsumi Imaizumi d, Kyoko Miyasaka b a
Department of Internal Medicine, Yokohama Red Cross Hospital, 2 -85 Negishi-cho, Naka-ku, Yokohama 231 -0836, Japan b Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan c Department of Gastroenterology, National Kyushu Cancer Center, Fukuoka, Japan d Laboratory of Nutrition Chemistry, Kyushu Uni6ersity School of Agriculture, Fukuoka, Japan Received 21 September 1999; received in revised form 15 November 1999; accepted 19 November 1999
Abstract Lymphatic lipid transport in the intestine of adult and ageing rats was compared. Adult (8–10 months old) and old (24–26 months old) male Wistar rats were cannulated into the mesenteric lymph under ethrane anesthesia. On the following day, lipid emulsion containing 35.4 mg/h of olive oil was infused intraduodenally for 7 h and lymph collected hourly was assayed for triglyceride and apolipoprotein A-IV (apo A-IV). The results showed there was no difference in lymphatic lipid and apo A-IV transport between adult and old rats. Since apo A-IV synthesis in the enterocytes is linked to the intracellular assembly of lipoprotein, it is likely that in addition to lymphatic transport, production of chylomicrons is not impaired in ageing rats. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ageing rat; Lipid; Lymph transport; Intestine; Apolipoprotein A-IV
* Corresponding author. Tel.: +81-45-6220101; fax: +81-45-6220106. E-mail address: [email protected] (H. Hayashi) 0047-6374/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 9 9 ) 0 0 1 1 0 - 4
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1. Introduction One of the major causes of death in western countries is arteriosclerosis and related organ failure. In the treatment of arteriosclerosis, recent attention has focused on lipid disorders that resulted in hyperlipidemia. Several interventions which improve hyperlipidemia were shown to reduce arteriosclerosis, although the effects were sometimes limited (Scandinavian Simvastatin Survival Study Group, 1994; Shepherd et al., 1995). To devise a more effective strategy for normalizing lipid metabolism to control arteriosclerosis, the mechanism of lipid metabolism especially in the aged must be better clarified. Intestinal lipid metabolism is an extremely important determinant of plasma lipid levels, because the intestine serves as a major extrahepatic source of lipids. Intestinal lipid metabolism consists of several steps including intraluminal digestion, mucosal uptake, intracellular reesterification and lipoprotein assembly, and lymphatic transport (Tso and Balint, 1986). Lipid digestion and uptake seem to be generally well preserved in both ageing humans and animals (Holt and Balint, 1993). However, little remains known about the later intracellular events in intestinal lipid metabolism and final lymphatic transport. Holt and Dominguez suggested that there was delayed removal of reesterified lipid from the mucosa in the older rats, although they did not measure the lymphatic lipid transport directly (Holt and Dominguez, 1981). Most of lipids derived from normal foods are largely composed of long fatty acids and they enter the body via the mesenteric lymph. Since arteriosclerosis is generally a disease of the elderly, a comparison of events such as lymphatic lipid transport in younger and older subjects may identify age-related determinants of lipid metabolism. The aim of this study was to observe the lymphatic lipid transport directly in ageing animals. We prepared a mesenteric lymph fistula in ageing rats and measure the lymphatic lipid and apolipopotein A-IV (apo A-IV) levels. Apo A-IV was measured because in addition to being produced in the intestine (Imaizumi et al., 1978), it is also directly involved in intracellular lipoprotein assembly (Hayashi et al., 1990b), which means we can measure intracellular lipid metabolism.
2. Materials and methods
2.1. Animals Adult (8 – 10-months-old; 350 –410 g body weight) and old (24–26-months-old; 425 – 460 g body weight) male Wistar rats were used. They were purchased from Charles River Japan, Atsugi, at 4 weeks of age and maintained in a specific pathogen-free (SPF) room at the Tokyo Metropolitan Institute of Gerontology at a controlled temperature (24 – 25°C) with a 12-h dark/light cycle (17:00–05:00 h dark cycle), and given a standard chow (CRF-1, Oriental Yeast Co., Tokyo) as previously reported (Miyasaka et al., 1997, 1998). After overnight fasting, the main mesenteric lymph duct was cannulated with clear vinyl tubing (0.8 mm OD) under ethrane anesthesia according to the method of Bollman et al. (Bollman et al., 1948).
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Silicone tubing (2.2 mm OD) was introduced 2 cm down the duodenum through the fundus of the stomach. The fundal incision was closed by a purse-string suture. Postoperatively, the rats were infused intraduodenally at 3 ml/h with a glucose – saline solution (145 mM NaCl, 4 mM KCl, 0.28 M glucose) and allowed to recover in restraint cages for at least 24 h before lipid infusion.
2.2. Experimental protocol On the day following surgery, the saline-glucose infusion was replaced by a lipid infusion containing 35.4 mg/h of olive oil, which corresponds to about 40 mmol/h of triglyceride (TG), 7.8 mmol/h of egg lecithin, and 57 mmol/h of taulocholate emulsified in phosphate buffer saline (pH 6.4) as previously described (Tso et al., 1980). The lipid emulsion was infused at 3 ml/h for 7 h. Lymph was collected before and hourly during the lipid infusion.
2.3. Determination of lipid and apo A-IV outputs Lymph TG mass corrected for free glycerol was determined by an enzymatic procedure (Boehringer Mannheim kit 450 032) (Ly et al., 1992). Apo A-IV in lymph was measured by rocket immunoelectrophoresis as described by Imaizumi et al. (1987). The concentration of apo A-IV in lymph was determined by comparing rocket heights with those in a series of dilutions of pooled serum obtained from 7-week-old male Sprague-Dawley rats and expressed using arbitrary units.
2.4. Statistics All values are expressed as mean 9 SD. A repeated measures analysis of variance was used to determine whether differeces existed in and between groups for each dependent variable. Differences were considered significant at PB 0.05.
3. Results
3.1. Lymph flow The fasting lymph flow in the adult (n= 5) and old (n= 4) rats were 2.579 1.28 and 2.18 9 1.10 ml/h, respectively. In both groups, lymph flow decreased at 1 h, and was further decreased in the adult group at 2 h. Thereafter lymph flow began to increase in both groups and there was no significant difference in lymph flow between the two groups over the 7-h period (Table 1). The initial decrease of lymph flow after the change of infusate from saline to lipid emulsion seen in this study was consistent with previous studies (Hayashi et al., 1990a,b).
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3.2. Lymphatic TG transport There was no significant difference in the fasting TG output in lymph between the adult (3.491.0 mg/h) and old (4.79 4.2 mg/h) rats. The lymphatic TG output in adult rats began to increase sharply after the change of infusate to lipid emulsion and by 3 – 4 h it reached an almost stable phase of transport (Table 1). A similar trend in TG output was observed in the old rats although the stable phase was reached a little earlier at 2 – 3 h. The lymphatic TG outputs in the adult and old rats at 7 h were 21.29 3.6 and 19.79 3.7 mg/h, respectively, and were not different.
3.3. Lymphatic apo A-IV outputs The lymphatic apo A-IV outputs in the fasting state were equivalent in both groups; 156.7958.8 a.u./h in adult rats and 163.09 105.8 a.u./h in old rats. The change in apo A-IV levels in lymph after the beginning of lipid infusion in each group was similar to the trend of lymphatic TG output, but was more gradual (Table 1). Apo A-IV outputs at 7 h were 255.0 9 40.8 a.u./h in the adult rats and 281.19 61.0 a.u./h in the old rats. While lymphatic apo A-IV outputs in both groups increased significantly, there was no significant difference between the two groups.
4. Discussion To our knowledge, this is the first report of the direct measurement of the intestinal lipid transport in ageing animals. The mesenteric lymph-fistula rat adopted in this experiment is a very suitable model for observing intestinal lipid metabolism in the conscious animal because most lipids absorbed in the intestine are transported in mesenteric lymph as chylomicron and very low density lipopotein (VLDL) components, except for medium- or short-chain fatty acids (Kiyasu et al., Table 1 Lymph flow and lymphatic triglyceride and apo A-IV outputsa Hour
0 1 2 3 4 5 6 7
Lymph flow (ml/h)
TG (mg/h)
Adultb
Oldc
Adult
Old
2.57 1.87 1.78 2.05 2.24 2.35 2.39 2.42
2.18 1.93 2.37 2.44 2.51 2.83 2.92 2.75
3.4 9.1 15.1 18.0 19.3 20.4 20.5 21.2
4.7 9.6 20.1 21.4 19.1 20.5 20.1 19.7
(1.28) (1.05) (0.87) (0.67) (0.60) (0.72) (0.77) (0.72)
(1.10) (0.14) (0.57) (0.28) (0.35) (0.41) (0.51) (0.35)
(1.0) (3.8) (3.2) (3.8) (3.4) (3.1) (3.3) (3.6)
Apo A-IV (a.u./h) Adult (4.2) (2.2) (6.9) (5.6) (5.4) (5.3) (4.6) (3.7)
156.7 174.8 203.2 211.6 199.9 236.7 229.6 255.0
Old (58.8) (25.5) (50.9) (44.0) (34.1) (37.8) (39.2) (40.8)
163.0 199.5 295.5 273.6 264.5 288.7 285.2 281.1
(105.8) (44.9) (82.7) (61.5) (79.4) (66.1) (61.2) (61.0)
a All data are mean 9SD in parentheses. There were no significant differences between adult and old rats in lymph flow, TG transport, and apo A-IV transport. b n =5. c n =4.
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1952) and high loads of long-chain fatty acids (Mansbach et al., 1991), which are transported to the liver via the portal vein. The infused lipid in this study was olive oil, which is mostly composed of long-chain fatty acids, especially oleic acids. In a previous study, 40 mmol/h of triolein, an equivalent dose of triglyceride to that used in this study, was infused into the rat duodenum and most of the absorbed lipid was confirmed to be transported in lymph (Hayashi et al., 1990a,b). In the younger rats, about 60% of the infused lipid was transported in lymph in a stable phase (Table 1). The net transfer of infused lipid, that is total TG mass in lymph minus intrinsic TG secreted into the intestinal lumen and absorbed into lymph, must be lower than this figure. In previous studies (Hayashi et al., 1990a,b), about 75% of infused TG mass was transported in lymph. The discrepancy between these previous studies and the present study may be due to different experimental conditions, such as the species of rats or the infused lipids. Our results clearly showed that ageing has no effect on intestinal lipid transport under these experimental conditions. Rather, it may even enhance the lymphatic lipid transport because both the lymphatic outputs of TG and apo A-IV began to increase sooner after the infusion of lipid in the old rats than in the adult rats, although there was no significant difference between the two groups (Table 1). Our data is in direct contrast to the report of Holt and Dominguez which found that removal of reesterified lipid from the mucosa was delayed in older rats (Holt and Dominguez, 1981). They infused radiolabeled triolein into Sprague-Dawley rats at a rate of 93, 183, or 358 mmol/h and calculated the transintestinal transport rate as the difference between the lipid infused intraduodenally and the lipid recovered from the intestinal lumen plus the small intestinal mucosa. They concluded the transport rate of lipid from the mucosa to lymph was impaired in the ageing rats and the lipoprotein assembly or discharge from the enterocytes was defective because they could find no evidence for impaired mucosal triolein reesterification. In addition to the differences in measurement of lymphatic lipid transport, the infusion rate of lipid into the duodenum adopted by Holt and Dominguez was far higher than physiological conditions. The maximal transport capacity of the proximal half of the rat intestine, where most triglycerides are absorbed into the mucosa, was measured to be 100 –130 mmol/h of fatty acids (Tso et al., 1982), which corresponds to up to about 80 mmol/h of intraluminally infused TG. We infused lipid at a rate of about 40 mmol/h, which should be close to physiological conditions. Therefore we believe it is reasonable to assume that the lymphatic lipid transport in old rats does not differ from that in adult rats. We could not draw any definite conclusion from this experiment on the intracellular process of intestinal lipid metabolism, especially the assembly of lipoproteins. A previous study showed that lymphatic apo A-IV secretion is closely related to lymphatic TG transport, and that synthesis of apo A-IV in the enterocytes is tightly linked to the assembly of lipoproteins in lymph-fistula rats treated with Pluronic L-81 (L-81) (Hayashi et al., 1990b). L-81, a hydrophobic surfactant, can reversibly inhibit formation of chylomicron, but not VLDL, in the enterocytes (Tso et al.,
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1981). When L-81 is infused intraduodenally with lipid emulsion, lipid is absorbed and reesterified normally in the enterocytes, but is not assembled into chylomicron resulting in the intracellular storage of lipid droplets. The intracellular synthesis of apo A-IV is not enhanced in this situation, but once the infusion of L-81 ceases, the formation and lymphatic secretion of chylomicron begin with the enhanced synthesis and secretion of apo A-IV. In this experiment, lymphatic apo A-IV increased along with TG transport both in the adult and old rats (Table 1). Therefore, based on our results, we conclude that the intracellular processes of lipid metabolism, especially the assembly of chylomicron, are not affected in the old rats. In clinical practice, hyperlipidemic patients may therefore be eligible for the same diet therapy regardless of age, but further studies in humans are warranted.
References Bollman, J.L., Cain, J.C., Grindlay, J.H., 1948. Techniques for the collection of lymph from the liver, small intestine or thoracic duct of the rat. J. Lab. Clin. Med. 33, 1349 – 1352. Hayashi, H., Fujimoto, K., Cardelli, J.A., Nutting, D.F., Bergstedt, S., Tso, P., 1990a. Fat feeding increases size, but not number, of chylomicrons produced by small intestine. Am. J. Physiol. 259, G709–G719 (Gastrointest. Liver Physiol. 22). Hayashi, H., Nutting, D.F., Fujimoto, K., Cardelli, J.A., Black, D., Tso, P., 1990b. Transport of lipid and apolipoproteins A-I and A-IV in intestinal lymph of the rat. J. Lipid Res. 31, 1613 – 1625. Holt, P.R., Dominguez, A.A., 1981. Intestinal absorption of triglyceride and vitamin D3 in aged and young rats. Dig. Dis. Sci. 26, 1109–1115. Holt, P.R., Balint, J.A., 1993. Effects of aging on intestinal lipid absorption. Am. J. Physiol. 264, G1–G6 (Gastrointest. Liver Physiol. 27). Imaizumi, K., Fainaru, M., Havel, R.J., 1978. Composition of proteins of mesenteric lymph chylomicrons in the rat and alterations produced upon exposure of chylomicrons to blood serum and serum proteins. J. Lipid Res. 19, 712–722. Imaizumi, K., Lu, Y.-F., Sugano, M., 1987. Characterization of serum apolipoprotein patterns in rats during suckling and post-weaning periods. Biochim. Biophys. Acta 917, 269 – 278. Kiyasu, J.Y., Bloom, B., Chaikoff, I.L., 1952. The portal transport of absorbed fatty acids. J. Biol. Chem. 199, 415–419. Ly, H.L., Mortimer, B-C., Baker, E., Redgrave, T.G., 1992. Clearance from plasma of lymph chylomicrons and chylomicron remnants labelled with 125I-tyramine-cellobiose. Biochem. J. 286, 937–943. Mansbach II, C.M., Dowell, R.F., Pritchett, D., 1991. Portal transport of absorbed lipids in rats. Am. J. Physiol. 261, G530–G538 (Gastrointest. Liver Physiol. 24). Miyasaka, K., Kanai, S., Ohta, M., Funakoshi, A., 1997. Aging impairs release of central and peripheral cholecystokinin (CCK) in male but not in female rats. J. Gerontol. Med. Sci. 52, M14 – M18. Miyasaka, K., Jimi, A., Kanai, S., Sato, Y., Masuda, M., Ohta, M., et al., 1998. Incidence of apoptosis in the pancreas of young and old rats. Arch. Gerontol. Geriatr. 26, 235 – 245. Scandinavian Simvastatin Survival Study Group, 1994. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 344, 1383–1389. Shepherd, J., Cobbe, S.M., Ford, I., Isles, C.G., Lorimer, A.R., MacFarlane, P.W., et al., 1995. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. N. Engl. J. Med. 333, 1301–1307.
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Tso, P., Balint, J.A., Rodgers, J.B., 1980. Effect of hydrophobic surfactant (Pluronic L-81) on lymphatic lipid transport in the rat. Am. J. Physiol. 239, G348 – G353 (Gastrointest. Liver Physiol. 2). Tso, P., Balint, J.A., Bishop, M.B., Rodgers, J.B., 1981. Acute inhibition of intestinal lipid transport by Pluronic L-81 in the rat. Am. J. Physiol. 241, G487 – G497 (Gastrointest. Liver Physiol. 4). Tso, P., Bush, K.L., Balint, J.A., Rodgers, J.B., 1982. Maximal lymphatic triglyceride transport rate from the rat small intestine. Am. J. Physiol. 242, G408 – G415 (Gastrointest. Liver Physiol. 2). Tso, P., Balint, J.A., 1986. Formation and transport of chylomicrons by enterocytes to the lymphatics. Am. J. Physiol. 250, G715–G726 (Gastrointest. Liver Physiol. 13).
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Lymphatic lipid transport is not impaired in ageing rat intestine Hiroshi Hayashi a,b,*, Yuko Sato b, Setsuko Kanai b, Masao Masuda b, Minoru Ohta b, Akihiro Funakoshi c, Koji Nagao d, Katsumi Imaizumi d, Kyoko Miyasaka b a
Department of Internal Medicine, Yokohama Red Cross Hospital, 2 -85 Negishi-cho, Naka-ku, Yokohama 231 -0836, Japan b Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan c Department of Gastroenterology, National Kyushu Cancer Center, Fukuoka, Japan d Laboratory of Nutrition Chemistry, Kyushu Uni6ersity School of Agriculture, Fukuoka, Japan Received 21 September 1999; received in revised form 15 November 1999; accepted 19 November 1999
Abstract Lymphatic lipid transport in the intestine of adult and ageing rats was compared. Adult (8–10 months old) and old (24–26 months old) male Wistar rats were cannulated into the mesenteric lymph under ethrane anesthesia. On the following day, lipid emulsion containing 35.4 mg/h of olive oil was infused intraduodenally for 7 h and lymph collected hourly was assayed for triglyceride and apolipoprotein A-IV (apo A-IV). The results showed there was no difference in lymphatic lipid and apo A-IV transport between adult and old rats. Since apo A-IV synthesis in the enterocytes is linked to the intracellular assembly of lipoprotein, it is likely that in addition to lymphatic transport, production of chylomicrons is not impaired in ageing rats. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ageing rat; Lipid; Lymph transport; Intestine; Apolipoprotein A-IV
* Corresponding author. Tel.: +81-45-6220101; fax: +81-45-6220106. E-mail address: [email protected] (H. Hayashi) 0047-6374/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 9 9 ) 0 0 1 1 0 - 4
220
H. Hayashi et al. / Mechanisms of Ageing and De6elopment 113 (2000) 219–225
1. Introduction One of the major causes of death in western countries is arteriosclerosis and related organ failure. In the treatment of arteriosclerosis, recent attention has focused on lipid disorders that resulted in hyperlipidemia. Several interventions which improve hyperlipidemia were shown to reduce arteriosclerosis, although the effects were sometimes limited (Scandinavian Simvastatin Survival Study Group, 1994; Shepherd et al., 1995). To devise a more effective strategy for normalizing lipid metabolism to control arteriosclerosis, the mechanism of lipid metabolism especially in the aged must be better clarified. Intestinal lipid metabolism is an extremely important determinant of plasma lipid levels, because the intestine serves as a major extrahepatic source of lipids. Intestinal lipid metabolism consists of several steps including intraluminal digestion, mucosal uptake, intracellular reesterification and lipoprotein assembly, and lymphatic transport (Tso and Balint, 1986). Lipid digestion and uptake seem to be generally well preserved in both ageing humans and animals (Holt and Balint, 1993). However, little remains known about the later intracellular events in intestinal lipid metabolism and final lymphatic transport. Holt and Dominguez suggested that there was delayed removal of reesterified lipid from the mucosa in the older rats, although they did not measure the lymphatic lipid transport directly (Holt and Dominguez, 1981). Most of lipids derived from normal foods are largely composed of long fatty acids and they enter the body via the mesenteric lymph. Since arteriosclerosis is generally a disease of the elderly, a comparison of events such as lymphatic lipid transport in younger and older subjects may identify age-related determinants of lipid metabolism. The aim of this study was to observe the lymphatic lipid transport directly in ageing animals. We prepared a mesenteric lymph fistula in ageing rats and measure the lymphatic lipid and apolipopotein A-IV (apo A-IV) levels. Apo A-IV was measured because in addition to being produced in the intestine (Imaizumi et al., 1978), it is also directly involved in intracellular lipoprotein assembly (Hayashi et al., 1990b), which means we can measure intracellular lipid metabolism.
2. Materials and methods
2.1. Animals Adult (8 – 10-months-old; 350 –410 g body weight) and old (24–26-months-old; 425 – 460 g body weight) male Wistar rats were used. They were purchased from Charles River Japan, Atsugi, at 4 weeks of age and maintained in a specific pathogen-free (SPF) room at the Tokyo Metropolitan Institute of Gerontology at a controlled temperature (24 – 25°C) with a 12-h dark/light cycle (17:00–05:00 h dark cycle), and given a standard chow (CRF-1, Oriental Yeast Co., Tokyo) as previously reported (Miyasaka et al., 1997, 1998). After overnight fasting, the main mesenteric lymph duct was cannulated with clear vinyl tubing (0.8 mm OD) under ethrane anesthesia according to the method of Bollman et al. (Bollman et al., 1948).
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Silicone tubing (2.2 mm OD) was introduced 2 cm down the duodenum through the fundus of the stomach. The fundal incision was closed by a purse-string suture. Postoperatively, the rats were infused intraduodenally at 3 ml/h with a glucose – saline solution (145 mM NaCl, 4 mM KCl, 0.28 M glucose) and allowed to recover in restraint cages for at least 24 h before lipid infusion.
2.2. Experimental protocol On the day following surgery, the saline-glucose infusion was replaced by a lipid infusion containing 35.4 mg/h of olive oil, which corresponds to about 40 mmol/h of triglyceride (TG), 7.8 mmol/h of egg lecithin, and 57 mmol/h of taulocholate emulsified in phosphate buffer saline (pH 6.4) as previously described (Tso et al., 1980). The lipid emulsion was infused at 3 ml/h for 7 h. Lymph was collected before and hourly during the lipid infusion.
2.3. Determination of lipid and apo A-IV outputs Lymph TG mass corrected for free glycerol was determined by an enzymatic procedure (Boehringer Mannheim kit 450 032) (Ly et al., 1992). Apo A-IV in lymph was measured by rocket immunoelectrophoresis as described by Imaizumi et al. (1987). The concentration of apo A-IV in lymph was determined by comparing rocket heights with those in a series of dilutions of pooled serum obtained from 7-week-old male Sprague-Dawley rats and expressed using arbitrary units.
2.4. Statistics All values are expressed as mean 9 SD. A repeated measures analysis of variance was used to determine whether differeces existed in and between groups for each dependent variable. Differences were considered significant at PB 0.05.
3. Results
3.1. Lymph flow The fasting lymph flow in the adult (n= 5) and old (n= 4) rats were 2.579 1.28 and 2.18 9 1.10 ml/h, respectively. In both groups, lymph flow decreased at 1 h, and was further decreased in the adult group at 2 h. Thereafter lymph flow began to increase in both groups and there was no significant difference in lymph flow between the two groups over the 7-h period (Table 1). The initial decrease of lymph flow after the change of infusate from saline to lipid emulsion seen in this study was consistent with previous studies (Hayashi et al., 1990a,b).
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3.2. Lymphatic TG transport There was no significant difference in the fasting TG output in lymph between the adult (3.491.0 mg/h) and old (4.79 4.2 mg/h) rats. The lymphatic TG output in adult rats began to increase sharply after the change of infusate to lipid emulsion and by 3 – 4 h it reached an almost stable phase of transport (Table 1). A similar trend in TG output was observed in the old rats although the stable phase was reached a little earlier at 2 – 3 h. The lymphatic TG outputs in the adult and old rats at 7 h were 21.29 3.6 and 19.79 3.7 mg/h, respectively, and were not different.
3.3. Lymphatic apo A-IV outputs The lymphatic apo A-IV outputs in the fasting state were equivalent in both groups; 156.7958.8 a.u./h in adult rats and 163.09 105.8 a.u./h in old rats. The change in apo A-IV levels in lymph after the beginning of lipid infusion in each group was similar to the trend of lymphatic TG output, but was more gradual (Table 1). Apo A-IV outputs at 7 h were 255.0 9 40.8 a.u./h in the adult rats and 281.19 61.0 a.u./h in the old rats. While lymphatic apo A-IV outputs in both groups increased significantly, there was no significant difference between the two groups.
4. Discussion To our knowledge, this is the first report of the direct measurement of the intestinal lipid transport in ageing animals. The mesenteric lymph-fistula rat adopted in this experiment is a very suitable model for observing intestinal lipid metabolism in the conscious animal because most lipids absorbed in the intestine are transported in mesenteric lymph as chylomicron and very low density lipopotein (VLDL) components, except for medium- or short-chain fatty acids (Kiyasu et al., Table 1 Lymph flow and lymphatic triglyceride and apo A-IV outputsa Hour
0 1 2 3 4 5 6 7
Lymph flow (ml/h)
TG (mg/h)
Adultb
Oldc
Adult
Old
2.57 1.87 1.78 2.05 2.24 2.35 2.39 2.42
2.18 1.93 2.37 2.44 2.51 2.83 2.92 2.75
3.4 9.1 15.1 18.0 19.3 20.4 20.5 21.2
4.7 9.6 20.1 21.4 19.1 20.5 20.1 19.7
(1.28) (1.05) (0.87) (0.67) (0.60) (0.72) (0.77) (0.72)
(1.10) (0.14) (0.57) (0.28) (0.35) (0.41) (0.51) (0.35)
(1.0) (3.8) (3.2) (3.8) (3.4) (3.1) (3.3) (3.6)
Apo A-IV (a.u./h) Adult (4.2) (2.2) (6.9) (5.6) (5.4) (5.3) (4.6) (3.7)
156.7 174.8 203.2 211.6 199.9 236.7 229.6 255.0
Old (58.8) (25.5) (50.9) (44.0) (34.1) (37.8) (39.2) (40.8)
163.0 199.5 295.5 273.6 264.5 288.7 285.2 281.1
(105.8) (44.9) (82.7) (61.5) (79.4) (66.1) (61.2) (61.0)
a All data are mean 9SD in parentheses. There were no significant differences between adult and old rats in lymph flow, TG transport, and apo A-IV transport. b n =5. c n =4.
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1952) and high loads of long-chain fatty acids (Mansbach et al., 1991), which are transported to the liver via the portal vein. The infused lipid in this study was olive oil, which is mostly composed of long-chain fatty acids, especially oleic acids. In a previous study, 40 mmol/h of triolein, an equivalent dose of triglyceride to that used in this study, was infused into the rat duodenum and most of the absorbed lipid was confirmed to be transported in lymph (Hayashi et al., 1990a,b). In the younger rats, about 60% of the infused lipid was transported in lymph in a stable phase (Table 1). The net transfer of infused lipid, that is total TG mass in lymph minus intrinsic TG secreted into the intestinal lumen and absorbed into lymph, must be lower than this figure. In previous studies (Hayashi et al., 1990a,b), about 75% of infused TG mass was transported in lymph. The discrepancy between these previous studies and the present study may be due to different experimental conditions, such as the species of rats or the infused lipids. Our results clearly showed that ageing has no effect on intestinal lipid transport under these experimental conditions. Rather, it may even enhance the lymphatic lipid transport because both the lymphatic outputs of TG and apo A-IV began to increase sooner after the infusion of lipid in the old rats than in the adult rats, although there was no significant difference between the two groups (Table 1). Our data is in direct contrast to the report of Holt and Dominguez which found that removal of reesterified lipid from the mucosa was delayed in older rats (Holt and Dominguez, 1981). They infused radiolabeled triolein into Sprague-Dawley rats at a rate of 93, 183, or 358 mmol/h and calculated the transintestinal transport rate as the difference between the lipid infused intraduodenally and the lipid recovered from the intestinal lumen plus the small intestinal mucosa. They concluded the transport rate of lipid from the mucosa to lymph was impaired in the ageing rats and the lipoprotein assembly or discharge from the enterocytes was defective because they could find no evidence for impaired mucosal triolein reesterification. In addition to the differences in measurement of lymphatic lipid transport, the infusion rate of lipid into the duodenum adopted by Holt and Dominguez was far higher than physiological conditions. The maximal transport capacity of the proximal half of the rat intestine, where most triglycerides are absorbed into the mucosa, was measured to be 100 –130 mmol/h of fatty acids (Tso et al., 1982), which corresponds to up to about 80 mmol/h of intraluminally infused TG. We infused lipid at a rate of about 40 mmol/h, which should be close to physiological conditions. Therefore we believe it is reasonable to assume that the lymphatic lipid transport in old rats does not differ from that in adult rats. We could not draw any definite conclusion from this experiment on the intracellular process of intestinal lipid metabolism, especially the assembly of lipoproteins. A previous study showed that lymphatic apo A-IV secretion is closely related to lymphatic TG transport, and that synthesis of apo A-IV in the enterocytes is tightly linked to the assembly of lipoproteins in lymph-fistula rats treated with Pluronic L-81 (L-81) (Hayashi et al., 1990b). L-81, a hydrophobic surfactant, can reversibly inhibit formation of chylomicron, but not VLDL, in the enterocytes (Tso et al.,
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1981). When L-81 is infused intraduodenally with lipid emulsion, lipid is absorbed and reesterified normally in the enterocytes, but is not assembled into chylomicron resulting in the intracellular storage of lipid droplets. The intracellular synthesis of apo A-IV is not enhanced in this situation, but once the infusion of L-81 ceases, the formation and lymphatic secretion of chylomicron begin with the enhanced synthesis and secretion of apo A-IV. In this experiment, lymphatic apo A-IV increased along with TG transport both in the adult and old rats (Table 1). Therefore, based on our results, we conclude that the intracellular processes of lipid metabolism, especially the assembly of chylomicron, are not affected in the old rats. In clinical practice, hyperlipidemic patients may therefore be eligible for the same diet therapy regardless of age, but further studies in humans are warranted.
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