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Medicine

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

Published: February 26th, 2019

DOI:

10.3791/58533

1Department of Biomedical Sciences and NNF Centre for Basic Metabolic Research, The Panum Institute, University of Copenhagen

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption — the isolated perfused rat small intestine.

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate nutrient absorption and nutrient disposition, intestinal growth and modulate appetite1. Gut hormones are, therefore, involved in many fundamental physiological processes, and understanding these patterns of secretion and the molecular details that control secretion of the respective hormones is thus important for our basic physiological understanding and for addressing translational aspects of gut hormone actions; but how can one study the molecular sensing mechanisms underlying gut hormone secretion? In general, hormone secretion....

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All studies were conducted with permission from the Danish Animal Experiments Inspectorate (2013-15-2934-00833) and the local ethical committee, in accordance with the guidelines of Danish legislation governing animal experimentation (1987) and the National Institutes of Health (publication number 85-23).

1. Experimental Animals

  1. Obtain male Wistar rats (250 g) and house 2 per cage, with ad libitum access to standard chow and water, and maintain in­­­ a 12:12 h light-.......

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The ability to determine whether a given stimulus causes secretion of the gut hormone of interest relies on a steady baseline secretion. Furthermore, if no response to the stimulus is observed, a robust secretory response to the positive control must be evident to exclude that the lack of response to the test stimulus does not reflect a general lack of responsiveness. Figure 2A and 2B shows an example of good quality data; GLP-1 secretion fro.......

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The isolated perfused rat small intestine is a powerful research tool that allows the dynamics and molecular mechanisms underlying gut hormone secretion to be studied in detail. The most critical step for the successful production of data with this model is the surgical operation. Handling of the gut will inevitably cause some damage to the intestine and should therefore be kept to an absolute minimum. Even more importantly, the speed of operation is key, particularly with regard to the time for the catheter placement in.......

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This work was supported by an unrestricted grant to Prof. Jens Juul Holst from the Novo Nordisk Center for Basic Metabolic Research (Novo Nordisk Foundation, Denmark), a separate grant from the Novo Nordisk Foundation for doing rodent perfusion studies (grant no. NNF15OC0016574), a grant to Prof. Holst from the European Research Council (Grant no.695069) and theEuropean Union's Seventh Framework Programme for Research, TechnologicalDevelopment, and Demonstration Activities (Grant No. 266408) as well as a postdoc grant to Rune E. Kuhre from the Lundbeck Foundation (R264-2017-3492). We thank Jenna E. Hunt and Carolyn F. Deacon for careful proofreading.

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Name Company Catalog Number Comments
Chemicals for perfusion buffer
Bovine serum albumin (BSA) Merck 1.12018.0500
Calcium chloride dihydrate (CaCl2 x 2 H2O) Merck 102382
Dextran 70 Pharmacosmos 40014
Fumaric acid disodium salt (C4H2Na2O4) Sigma Aldrich F9642
Glucose (C6H12O6) Merck 108342
Magnesium sulfate hepatahydrate (MgSO4) Merck 105886
Potasium chloride (KCl) Merck 104936
Potassium dihydrogen phosphate (KH2PO4) Merck 104873
Pyruvic acid sodium salt (C3H3NaO4) Merck 106619
Sodium bicarbonate (NaHCO3) Merck
Sodium chloride (NaCl) Merck 106404
Sodium L-glutamate monohydrate (C5H8NNaO4 x H2O) Merck 106445
Name Company Catalog Number Comments
Perfusion equitment
Universial perfusion system Harvard Bioscience, Inc. 732316
BASIC UNIT UNIPER UP-100, TYPE 834 Harvard Bioscience, Inc.
Roller Pump, with four channels Harvard Bioscience, Inc. 730100
Windkessel Harvard Bioscience, Inc. 732068
Thermostatic Circulator,Bath Volume 3L, 230V/50Hz Harvard Bioscience, Inc. 730125
Operating table, heated on tripod stand, type 873 Harvard Bioscience, Inc. 733776
Cannula with basked, OD= 2.0mm, ID= 1.0mm Harvard Bioscience, Inc. 733313

  1. Rindi, G., Leiter, A. B., Kopin, A. S., Bordi, C., Solcia, E. The "normal" endocrine cell of the gut: changing concepts and new evidences. Annals of the New York Academy of Sciences. 1014, 1-12 (2004).
  2. Brighton, C. A., et al. Bile Acids Trigger GLP-1 Release Predominantly by Accessing Basolaterally Located G Protein-Coupled Bile Acid Receptors. Endocrinology. 156, 3961-3970 (2015).
  3. Kuhre, R. E., et al. Bile acids are important direct and indirect regulators of the secretion of appetite- and metabolism-regulating hormones from the gut and pancreas. Molecular Metabolism. , (2018).
  4. Roberge, J. N., Brubacker, P. L. Secretion of Proglucagon-Derived Peptides in Response to Intestinal Luminal Nutrients. Endocrinology. 128, 3169-3174 (1991).
  5. Brubaker, P. L., Schloos, J., Drucker, D. J. Regulation of glucagon-like peptide-1 synthesis and secretion in the GLUTag enteroendocrine cell line. Endocrinology. 139, 4108-4114 (1998).
  6. Jacobsen, S., et al. Changes in Gastrointestinal Hormone Responses, Insulin Sensitivity, and Beta-Cell Function Within 2 Weeks After Gastric Bypass in Non-diabetic Subjects. Obesity Surgery. 22, 1084-1096 (2012).
  7. Kuhre, R. E., Frost, C. R., Svendsen, B., Holst, J. J. Molecular mechanisms of glucose-stimulated GLP-1 secretion from perfused rat small intestine. Diabetes. 64, 370 (2014).
  8. Svendsen, B., Holst, J. J. Regulation of gut hormone secretion. Studies using isolated perfused intestines. Peptides. 77, 47-53 (2016).
  9. Ratner, C., et al. Effects of Peripheral Neurotensin on Appetite Regulation and Its Role in Gastric Bypass Surgery. Endocrinology. 157, 3482-3492 (2016).
  10. Wewer Albrechtsen, N. J., et al. Dynamics of glucagon secretion in mice and rats revealed using a validated sandwich ELISA for small sample volumes. American Journal of Physiology Endocrinology and Metabolism. 311, E302-E309 (2016).
  11. Vilsboll, T., Agerso, H., Krarup, T., Holst, J. J. Similar elimination rates of glucagon-like peptide-1 in obese type 2 diabetic patients and healthy subjects. The Journal of Clinical Endocrinology and Metabolism. 88, 220-224 (2003).
  12. Windelov, J. A., et al. Why is it so difficult to measure glucagon-like peptide-1 in a mouse?. Diabetologia. 60, 2066-2075 (2017).
  13. Gorboulev, V., et al. Na+-d-glucose Cotransporter SGLT1 is Pivotal for Intestinal Glucose Absorption and Glucose-Dependent Incretin Secretion. Diabetes. 61, 187-196 (2012).
  14. Kuhre, R. E., Bechmann, L. E., Wewer Albrechtsen, N. J., Hartmann, B., Holst, J. J. Glucose stimulates neurotensin secretion from the rat small intestine by mechanisms involving SGLT1 and GLUT2, leading to cell depolarization and calcium influx. American Journal of Physiology Endocrinology and Metabolism. 308, E1123-E1130 (2015).
  15. Kuhre, R. E., Christiansen, C. B., Saltiel, M. Y., Wewer Albrechtsen, N. J., Holst, J. J. On the relationship between glucose absorption and glucose-stimulated secretion of GLP-1, neurotensin, and PYY from different intestinal segments in the rat. Physiological Reports. 5, (2017).
  16. Svendsen, B., et al. An analysis of cosecretion and coexpression of gut hormones from male rat proximal and distal small intestine. Endocrinology. 156, 847-857 (2015).
  17. Goldspink, D. A., et al. Mechanistic insights into the detection of free fatty and bile acids by ileal glucagon-like peptide-1 secreting cells. Molecular Metabolism. 7, 90-101 (2018).
  18. Sun, E. W., et al. Mechanisms Controlling Glucose-Induced Glp-1 Secretion in Human Small Intestine. Diabetes. , (2017).
  19. Kuhre, R. E., et al. Peptide production and secretion in GLUTag, NCI-H716 and STC-1 cells: a comparison to native L-cells. Journal of Molecular Endocrinology. 56, 11 (2016).
  20. Reimann, F., et al. Glucose sensing in L cells: a primary cell study. Cell Metabolism. 8, 532-539 (2008).
  21. Reimann, F., et al. Characterization and functional role of voltage gated cation conductances in the glucagon-like peptide-1 secreting GLUTag cell line. The Journal of Physiology. 563, 161-175 (2005).
  22. Kuhre, R. E., et al. Fructose stimulates GLP-1 but not GIP secretion in mice, rats and humans. American journal of physiology. Gastrointestinal and Liver Physiology. 306, G622-G630 (2014).
  23. Reimann, F., Tolhurst, G., Gribble, F. M. G-Protein-Coupled Receptors in Intestinal Chemosensation. Cell Metabolism. 15, 421-431 (2012).
  24. Parker, H. E., et al. Molecular mechanisms underlying bile acid-stimulated glucagon-like peptide-1 secretion. British Journal of Pharmacology. 165, 414-423 (2012).
  25. Petersen, N., et al. Generation of L cells in mouse and human small intestine organoids. Diabetes. 63, 410-420 (2014).
  26. Parker, H., et al. Predominant role of active versus facilitative glucose transport for glucagon-like peptide-1 secretion. Diabetologia. 55, 2445-2455 (2012).
  27. Roberts, G. P., et al. Comparison of Human and Murine Enteroendocrine Cells by Transcriptomic and Peptidomic Profiling. Diabetes. , (2019).
  28. Reimann, F., Gribble, F. M. Glucose-Sensing in Glucagon-Like Peptide-1-Secreting Cells. Diabetes. 51, 2757-2763 (2002).
  29. Drucker, D. J., Jin, T., Asa, S. L., Young, T. A., Brubaker, P. L. Activation of proglucagon gene transcription by protein kinase-A in a novel mouse enteroendocrine cell line. Molecular Endocrinology. 8, 1646-1655 (1994).
  30. Svendsen, B., et al. GLP1- and GIP-producing cells rarely overlap and differ by bombesin receptor-2 expression and responsiveness. The Journal of Endocrinology. 228, 39-48 (2016).
  31. Bak, M. J., et al. Specificity and sensitivity of commercially available assays for glucagon-like peptide-1 (GLP-1): implications for GLP-1 measurements in clinical studies. Diabetes, Obesity & Metabolism. 16, 1155-1164 (2014).
  32. Jacobsen, S. H., et al. Effects of gastric bypass surgery on glucose absorption and metabolism during a mixed meal in glucose-tolerant individuals. Diabetologia. 56, 2250-2254 (2013).

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