丁香实验_LOGO
登录
提问
我要登录
|免费注册
点赞
收藏
wx-share
分享

Methods for Studying Rodent Intestinal Lipoprotein Production and Metabolism

互联网

827
  • Abstract
  • Table of Contents
  • Materials
  • Literature Cited

Abstract

 

Lipid absorption begins with the digestion of dietary triacylglycerol and ultimately results in the secretion of triacylglycerol in chylomicrons into the lymphatics. Additionally, the intestine also secretes numerous proteins and peptides involved in lipid and lipoprotein metabolism in response to food. Ultimately, chylomicrons and these proteins, peptides, and hormones are found in lymph. The lymph fistula rat model has traditionally been used to study this intestinal absorption of nutrients, especially lipids, but recently, this model has also been used for studying the secretion of hormones by the small intestine. The protocols described in this unit include the lymph fistula rat and mouse model, as well as in vivo chylomicron metabolism studies. These experimental models are helpful for the study of metabolic phenotypes, the characterization of intestinal lipid absorption and transport, and determining peripheral metabolism of intestinally derived lipoproteins. Curr. Protoc. Mouse Biol. 2:219?230 © 2012 by John Wiley & Sons, Inc.

Keywords: lipids; lipoproteins; chylomicrons; lymph; mouse; lymph fistula model

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Lymph Fistula in the Rat
  • Basic Protocol 2: Lymph Fistula in the Mouse
  • Basic Protocol 3: In Vivo Chylomicron Metabolism Assay
  • Commentary
  • Literature Cited
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Lymph Fistula in the Rat

  Materials
  • Adult male rats, 280 to 250 g
  • Inhaled anesthetic (recommended: isoflurane, e.g., from Butler Schein) and necessary equipment to administer anesthetic
  • Liposyn III (Hospira) or olive oil (food grade), optional
  • Cyanoacrylate adhesive (Krazy Glue)
  • Sterile normal saline with 5% glucose
  • Ice
  • Mixed meal bolus (e.g., Ensure, Abbott Nutrition; 3.125 kcal/animal – 0.075 g fat (21.6%), 0.5 g carbohydrate (64.0%), 0.1125 g protein (14.4%)
  • 0.9% sterile saline
  • Sterile surgical equipment including:
  • Fine forceps
  • Iris scissors
  • Drapes
  • Lighting
  • Heating pad
  • PVC cannula (highly recommended): o.d 0.80 × i.d. 0.5 mm (Critchley Electrical Products Pty)
  • Duodenal infusion tube (e.g., 0.040‐in. i.d. × 0.085‐in. o.d; VWR, cat. no. 60985‐708)
  • Temperature‐controlled recovery housing
  • Peristaltic pump; low flow (1 to 10 ml)
  • 1.5‐ml microcentrifuge tubes
  • Conical vials suitable for lymph collection

Basic Protocol 2: Lymph Fistula in the Mouse

  Materials
  • Adult C57BL/6 mice (30 g or larger)
  • Anesthetic (Ketamine/Xylazine)
  • Cyanoacrylate adhesive (e.g., Krazy Glue)
  • Sterile 0.9% saline w/0.5% glucose
  • Sterile 0.9% saline
  • Liposyn III (Hospira)
  • 1‐ml syringe equipped with a 25‐G needle
  • PVC cannula: i.d. 0.20 × o.d. 0.50 mm (Critchley Electrical Products Pty)
  • Silicone feeding tube for duodenal infusion (0.5‐mm i.d, 0.8‐mm o.d.; Tyco Electronics)
  • Temperature‐controlled restraint cages for overnight recovery
  • Peristaltic pump; very low flow
  • Conical vials suitable for lymph collection

Basic Protocol 3: In Vivo Chylomicron Metabolism Assay

  Materials
  • Lymph fistula animals (either rats or mice; protocol 1 , steps 1 to 9, or protocol 2 , steps 1 to 8, respectively)
  • 5.0 mCi 3 H‐labeled glycerol Trioleate (PerkinElmer)
  • 1.0 mCi 14 C‐cholesterol (PerkinElmer)
  • Liposyn III (Hospira)
  • Triglyceride assay kit (recommended: Randox TG kits, Randox Laboratories)
  • Cholesterol assay kit (recommended: Infinity cholesterol kits, Thermo Electron)
  • Recipient rodents of the same breed, age, and sex as selected lymph fistula animals
  • Conical tubes suitable for lymph collection
  • Refrigerated centrifuge with Beckman 50.3 Ti rotor (Beckman Instruments)
  • Liquid scintillation counter with dual window (for 3 H and 14 C) capability
  • 1‐ml insulin syringes
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Abrams, C.K., Hamosh, M., Lee, T.C., Ansher, A.F., Collen, M.J., Lewis, J.H., Benjamin, S.B., and Hamosh, P. 1988. Gastric lipase: Localization in the human stomach. Gastroenterology 95:1460‐1464.
   Austin, M.A. 1991. Plasma triglyceride and coronary heart disease. Arterioscler. Thromb. 11:2‐14.
   Barrowman, J.A. 1978. Physiology of the gastro‐intestinal lymphatic system. Monogr. Physiol. Soc. 33:ix‐xii, 1‐312
   Bearnot, H.R., Glickman, R.M., Weinberg, L., Green, P.H., and Tall, A.R. 1982. Effect of biliary diversion on rat mesenteric lymph apolipoprotein‐I and high density lipoprotein. J. Clin. Invest. 69:210‐217.
   Bennett, S. and Simmonds, W.J. 1962. Absorptive capacity and intestinal motility in unanaesthetized rats during intraduodenal infusion of fat. Q. J. Exp. Physiol. Cogn. Med. Sci. 47:32‐38.
   Bollman, J.L., Cain, J.C., and 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.
   Borgström, B. 1964. Influence of Bile Salt, Ph, and Time on the Action of Pancreatic Lipase; Physiological Implications. J. Lipid. Res. 5:522‐531.
   Borgström, B. 1985. The micellar hypothesis of fat absorption: Must it be revisited? Scand. J. Gastroenterol. 20:389‐394.
   Gantz, D., Bennett Clark, S., Derksen, A., and Small, D.M. 1990. Size and shape determination of fixed chylomicrons and emulsions with fluid or solid surfaces by three‐dimensional analysis of shadows. J. Lipid. Res. 31:163‐171.
   Grand, R.J., Watkins, J.B., and Torti, F.M. 1976. Development of the human gastrointestinal tract. A review. Gastroenterology 70:790‐810.
   Hofmann, A.F. and Borgström, B. 1962. Physico‐chemical state of lipids in intestinal content during their digestion and absorption. Fed. Proc. 21:43‐50.
   Hofmann, A.F. and Borgström, B. 1964. The intraluminal phase of fat digestion in man: The lipid content of the micellar and oil phases of intestinal content obtained during fat digestion and absorption. J. Clin. Invest. 43:247‐257.
   Jandacek, R.J., Rider, T., Yang, Q., Woollett, L.A., and Tso, P. 2009. Lymphatic and portal vein absorption of organochlorine compounds in rats. Am. J. Physiol. Gastrointest. Liver Physiol. 296:G226‐G234.
   Jandacek, R.J., Rider, T., Keller, E.R., and Tso, P. 2010. The effect of olestra on the absorption, excretion and storage of 2,2′,5,5′ tetrachlorobiphenyl; 3,3′,4,4′ tetrachlorobiphenyl; and perfluorooctanoic acid. Environ. Int. 36:880‐883.
   Ji, Y., Sakata, Y., and Tso, P. 2011. Nutrient‐induced inflammation in the intestine. Curr. Opin. Clin. Nutr. Metab. Care 14:315‐321.
   Johnston, J.M. 1976. Triglyceride biosynthesis in the intestinal mucosa. In Lipid Absorption: Biochemical and Clinical Aspects (K. Rommel, Goebell, H., and Bohmer, R., eds.) pp. 85‐94. MTP Press, Lancaster, U.K.
   Kannel, W.B., Dawber, T.R., Friedman, G.D., Glennon, W.E., and McNamara, P.M. 1964. Risk factors in coronary heart disease. An evaluation of several serum lipids as predictors of coronary heart disease; the framingham study. Ann. Intern. Med. 61:888‐899.
   Kohan, A., Yoder, S., and Tso, P. 2010. Lymphatics in intestinal transport of nutrients and gastrointestinal hormones. Ann. N.Y. Acad. Sci. 1207:E44‐E51.
   Kohan, A.B., Qing, Y., Cyphert, H.A., Tso, P., and Salati, L.M. 2011a. Chylomicron remnants and nonesterified fatty acids differ in their ability to inhibit genes involved in lipogenesis in rats. J. Nutr. 141:171‐176.
   Kohan, A.B., Yoder, S.M., and Tso, P. 2011b. Using the lymphatics to study nutrient absorption and the secretion of gastrointestinal hormones. Physiol. Behav. 105:82‐88.
   Kohan, A.B., Wang, F., Li, X., Bradshaw, S., Yang, Q., Caldwell, J.L., Bullock, T.M., and Tso, P. 2012. Apolipoprotein A‐IV regulates chylomicron metabolism—mechanism and function. Am. J. Physiol. Gastrointest. Liver Physiol. 302:G628‐G636.
   Kuksis, A. and Manganaro, F. 1986. Biochemical characterization and purification of intestinal acylglycerol acyltransferases. In Fat Absorption (A. Kuksis, ed.) pp. 233‐259. CRC Press, Boca Raton, Florida.
   Lehner, R. and Kuksis, A. 1995. Triacylglycerol synthesis by purified triacylglycerol synthetase of rat intestinal mucosa. Role of acyl‐CoA acyltransferase. J. Biol. Chem. 270:13630‐13636.
   Levy, D. and Kannel, W.B. 1988. Cardiovascular risks: New insights from Framingham. Am. Heart J. 116:266‐272.
   Liao, T.H., Hamosh, P., and Hamosh, M. 1984. Fat digestion by lingual lipase: Mechanism of lipolysis in the stomach and upper small intestine. Pediatr. Res. 18:402‐409.
   Lindgren, F.T., Jensen, L.C., and Hatch, F.T. 1972. The isolation and quantitative analysis of serum lipoproteins. In Blood Lipids and Lipoproteins: Quantitation, Composition, and Metabolism. (G.J. Nelson, ed.) pp. 181‐274. John Wiley & Sons, New York.
   Lo, C.M., Nordskog, B.K., Nauli, A.M., Zheng, S., Vonlehmden, S.B., Yang, Q., Lee, D., Swift, L.L., Davidson, N.O., and Tso, P. 2008. Why does the gut choose apolipoprotein B48 but not B100 for chylomicron formation? Am. J. Physiol. Gastrointest. Liver Physiol. 294:G344‐G352.
   Luchoomun, J. and Hussain, M.M. 1999. Assembly and secretion of chylomicrons by differentiated Caco‐2 cells. Nascent triglycerides and preformed phospholipids are preferentially used for lipoprotein assembly. J. Biol. Chem. 274:19565‐19572.
   Mahley, R.W., Bennett, B.D., Morre, D.J., Gray, M.E., Thistlethwaite, W., and LeQuire, V.S. 1971. Lipoproteins associated with the Golgi apparatus isolated from epithelial cells of rat small intestine. Lab. Invest. 25:435‐444.
   Mansbach, C.M. and Siddiqi, S.A. 2010. The biogenesis of chylomicrons. Annu. Rev. Physiol. 72:315‐333.
   Martins, I.J., Mortimer, B.C., Miller, J., and Redgrave, T.G. 1996. Effects of particle size and number on the plasma clearance of chylomicrons and remnants. J. Lipid. Res. 37:2696‐2705.
   Mattson, F.H. and Volpenhein, R.A. 1964. The digestion and absorption of triglycerides. J. Biol. Chem. 239:2772‐2777.
   Miura, S., Tsuzuki, Y., Hokari, R., and Ishii, H. 1998. Modulation of intestinal immune system by dietary fat intake: Relevance to Crohn's disease. J. Gastroenterol. Hepatol. 13:1183‐1190.
   Mortimer, B.C., Simmonds, W.J., Joll, C.A., Stick, R.V., and Redgrave, T.G. 1988. Regulation of the metabolism of lipid emulsion model lipoproteins by a saturated acyl chain at the 2‐position of triacylglycerol. J. Lipid Res. 29:713‐720.
   Mortimer, B.C., Kenrick, M.A., Holthouse, D.J., Stick, R.V., and Redgrave, T.G. 1992. Plasma clearance of model lipoproteins containing saturated and polyunsaturated monoacylglycerols injected intravenously in the rat. Biochim Biophys Acta 1127:67‐73.
   Mortimer, B.C., Tso, P., Phan, C.T., Beveridge, D.J., Wen, J., and Redgrave, T.G. 1995. Features of cholesterol structure that regulate the clearance of chylomicron‐like lipid emulsions. J. Lipid Res. 36:2038‐2053.
   Nauli, A.M., Nassir, F., Zheng, S., Yang, Q., Lo, C.M., Vonlehmden, S.B., Lee, D., Jandacek, R.J., Abumrad, N.A., and Tso, P. 2006. CD36 is important for chylomicron formation and secretion and may mediate cholesterol uptake in the proximal intestine. Gastroenterology 131:1197‐1207.
   Ockner, R.K., Hughes, F.B., and Isselbacher, K.J. 1969. Very low density lipoproteins in intestinal lymph: Role in triglyceride and cholesterol transport during fat absorption. J. Clin. Invest. 48:2367‐2373.
   Rao, G.A. and Johnston, J.M. 1966. Purification and properties of triglyceride synthetase from the intestinal mucosa. Biochim Biophys Acta 125:465‐473.
   Redgrave, T.G., Roberts, D.C., and West, C.E. 1975. Separation of plasma lipoproteins by density‐gradient ultracentrifugation. Anal. Biochem. 65:42‐49.
   Redgrave, T.G., Ly, H.L., Quintao, E.C., Ramberg, C.F., and Boston, R.C. 1993. Clearance from plasma of triacylglycerol and cholesteryl ester after intravenous injection of chylomicron‐like lipid emulsions in rats and man. Biochem. J. 290:843‐847.
   Redgrave, T.G., Mortimer, B.C., and Martins, I.J. 1996. Receptor‐ligand interactions in the hepatic uptake of chylomicron remnants studied with genetically manipulated mice. Z. Gastroenterol. 34:105‐107.
   Sabesin, S.M. and Frase, S. 1977. Electron microscopic studies of the assembly, intracellular transport, and secretion of chylomicrons by rat intestine. J. Lipid Res. 18:496‐511.
   Siddiqi, S., Saleem, U., Abumrad, N.A., Davidson, N.O., Storch, J., Siddiqi, S.A., and Mansbach, C.M. 2nd. 2010. A novel multiprotein complex is required to generate the prechylomicron transport vesicle from intestinal ER. J. Lipid Res. 51:1918‐1928.
   Simmonds, W.J., Redgrave, T.G., and Willix, R.L. 1968. Absorption of oleic and palmitic acids from emulsions and micellar solutions. J. Clin. Invest. 47:1015‐1025.
   Tso, P., Drake, D.S., Black, D.D., and Sabesin, S.M. 1984. Evidence for separate pathways of chylomicron and very low‐density lipoprotein assembly and transport by rat small intestine. Am. J. Physiol. 247:G599‐G610.
   Vigne, J.L. and Havel, R.J. 1981. Metabolism of apolipoprotein A‐I of chylomicrons in rats and humans. Can. J. Biochem. 59:613‐618.
   Woollett, L.A., Wang, Y., Buckley, D.D., Yao, L., Chin, S., Granholm, N., Jones, P.J., Setchell, K.D., Tso, P., and Heubi, J.E. 2006. Micellar solubilisation of cholesterol is essential for absorption in humans. Gut 55:197‐204.
   Wu, A.L. and Windmueller, H.G. 1978. Identification of circulating apolipoproteins synthesized by rat small intestine in vivo. J. Biol. Chem 253:2525‐2528.
   Wu, A.L. and Windmueller, H.G. 1979. Relative contributions by liver and intestine to individual plasma apolipoproteins in the rat. J Biol Chem. 254:7316‐7322.
   Yoder, S.M., Kindel, T.L., and Tso, P. 2010. Using the lymph fistula rat model to study incretin secretion. Vitam. Horm. 84:221‐249.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
 
提问
扫一扫
丁香实验小程序二维码
实验小助手
丁香实验公众号二维码
扫码领资料
反馈
TOP
打开小程序