Name: mechanisms underlying gut hormone secretion using the isolated perfused rat small intestine
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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&#39;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.

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
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	<li>Obtain male Wistar rats (250 g) and house 2 per cage, with ad libitum access to standard chow and water, and maintain in&#173;&#173;&#173; a 12:12 h light-...

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+insights+into+the+detection+of+free+fatty+and+bile+acids+by+ileal+glucagon-like+peptide-1+secreting+cells.">Mechanistic insights into the detection of free fatty and bile acids by ileal glucagon-like peptide-1 secreting cells.</a> Molecular Metabolism. 7, 90-101 (2018).</li><li>Sun, E. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+Controlling+Glucose-Induced+Glp-1+Secretion+in+Human+Small+Intestine.">Mechanisms Controlling Glucose-Induced Glp-1 Secretion in Human Small Intestine.</a> Diabetes. , (2017).</li><li>Kuhre, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+production+and+secretion+in+GLUTag,+NCI-H716+and+STC-1+cells:+a+comparison+to+native+L-cells.">Peptide production and secretion in GLUTag, NCI-H716 and STC-1 cells: a comparison to native L-cells.</a> Journal of Molecular Endocrinology. 56, 11 (2016).</li><li>Reimann, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+sensing+in+L+cells:+a+primary+cell+study.">Glucose sensing in L cells: a primary cell study.</a> Cell Metabolism. 8, 532-539 (2008).</li><li>Reimann, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+functional+role+of+voltage+gated+cation+conductances+in+the+glucagon-like+peptide-1+secreting+GLUTag+cell+line.">Characterization and functional role of voltage gated cation conductances in the glucagon-like peptide-1 secreting GLUTag cell line.</a> The Journal of Physiology. 563, 161-175 (2005).</li><li>Kuhre, R. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Human+and+Murine+Enteroendocrine+Cells+by+Transcriptomic+and+Peptidomic+Profiling.">Comparison of Human and Murine Enteroendocrine Cells by Transcriptomic and Peptidomic Profiling.</a> Diabetes. , (2019).</li><li>Reimann, F., Gribble, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose-Sensing+in+Glucagon-Like+Peptide-1-Secreting+Cells.">Glucose-Sensing in Glucagon-Like Peptide-1-Secreting Cells.</a> Diabetes. 51, 2757-2763 (2002).</li><li>Drucker, D. J., Jin, T., Asa, S. L., Young, T. A., Brubaker, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+proglucagon+gene+transcription+by+protein+kinase-A+in+a+novel+mouse+enteroendocrine+cell+line.">Activation of proglucagon gene transcription by protein kinase-A in a novel mouse enteroendocrine cell line.</a> Molecular Endocrinology. 8, 1646-1655 (1994).</li><li>Svendsen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GLP1-+and+GIP-producing+cells+rarely+overlap+and+differ+by+bombesin+receptor-2+expression+and+responsiveness.">GLP1- and GIP-producing cells rarely overlap and differ by bombesin receptor-2 expression and responsiveness.</a> The Journal of Endocrinology. 228, 39-48 (2016).</li><li>Bak, M. 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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...

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 can be studied in intact organisms (humans or experimental animals), from isolated gut preparations or from gut hormone-secreting primary cell cultures or immortalized cell cultures2,3,4,5,6. Our preferred model is the isolated perfused rat small intestine, which is a physiologically relevant model that allows the secretion of gut hormones to be studied in detail with optimal time resolution (secretion rates can be determined with any time base down to the second), and the results are likely transferrable to an in vivo situation7. Here, we provide a detailed protocol on how to perform this procedure, but first we will discuss other methods for studying gut hormone secretion, including the benefits and limitations of these models compared to the isolated perfused rat small intestine.
If one wishes to establish whether a specific compound regulates the secretion of certain gut hormones, studies in humans are the ultimate goal. Thus, if a compound shows great effects on the secretion of one or several gut hormones in rodents (in vivo or perfusions) or from hormone secreting cells (cell lines or primary cells), this effect is only relevant to medicine and human physiology if it can be confirmed in humans. However, there are clear limits for the type of studies that can be performed in humans, and in vivo studies on experimental animals are, therefore, often the second-best option for such studies. Mice and rats are the most frequently used experimental animals presumably due to their convenient size, low cost and the option to genetically alter the genes suspected of being involved in the specific study questions (e.g., knock out of a certain transporter or receptor). In general, in vivo models benefit from being physiologically intact, but also have several limitations. Most importantly, the small size of rodents, particularly mice, is a limiting factor, as most assays for gut hormone quantification require at least 20 &#181;L of plasma (and often much more), meaning that at least 100 &#181;L of blood has to be withdrawn to make a duplicate quantification. Therefore, it is only possible to obtain very few samples corresponding to baseline samples and one or two post-stimulation samples (the total blood volume in a mouse of 20 g is ~1.4 mL). Consequently, potential secretory responses (e.g., rapidly or late-occurring responses) may therefore be missed.
In the perfusion model, this issue is overcome, as large sample volumes are obtained (flow rate: 7.5 mL/min) and the collection intervals can be adjusted as needed to ensure that the rapid and short-lasting responses are not missed (we collect samples every min)7. Another issue with in vivo studies in rodents is that most gut hormones are even more rapidly eliminated or metabolized than in humans8,9,10, which may complicate the subsequent biochemical analysis. For instance, we showed that GLP-1 is metabolized in mice at an even faster rate than in humans (where T1/2 is 1-2 min11) and, more importantly, that the cleavage of GLP-1 in mice involves, in addition to N-terminal cleavage by dipeptidyl-peptidase-4 (DPP4) (which is the major GLP-1 degrading enzyme in humans), further cleavage by the enzyme neutral endopeptidase 24.1112. Consequently, current commercial assays for the quantification of GLP-1, which are either based on the intact isoform of GLP-1 (7-36amide) or the DPP-4 cleaved isoform (9-39amide), vastly underestimate GLP-1 secretion in mice and result in misleading results12. In the isolated perfused rat small intestine, most of the metabolism of secreted hormones is eliminated or markedly reduced, since plasma-mediated degradation is avoided, and liver/kidney/lung extraction/degradation is prevented (because perfusate is collected as it leaves the gut).
Of course, important insight can be generated by the use of genetically modified animals, e.g., sodium-glucose transporter-1 knockout mice13, but a detailed assessment of the molecular sensors involved in secretion often requires consideration of multiple molecular sites, ranging from molecular transporters to ion channels and from different G-protein-coupled receptors to intracellular proteins. For instance, we targeted the activity of nine different molecular sites when unraveling the molecular sensors responsible for glucose-stimulated GLP-1 secretion7. A similar investigation would not be possible in vivo as some of the compounds used have unspecific or harmful/lethal effects. For instance, when using the perfused gut, it was possible to assess the role of intra-cellular glucose metabolism for the secretion of GLP-1 and neurotensin by blocking ATP formation with 2-4-dinitrophenol7,14 as well as the role of voltage-gated calcium channels for bile acid stimulated GLP-1, NT and PYY secretion3. Indeed, the highly toxic sodium channel blocker tetrodotoxin can be successfully applied in the perfusion studies. Finally, in the perfusion model it can be directly assessed where in the gut a certain compound stimulates secretion of a certain hormone, as the investigator can simply choose and prepare the desired region to perfuse, and at the same time it can be investigated whether a stimulus causes secretion by activation of molecular sensors from the luminal or vascular side of the intestine3,15,16.
The secretory mechanisms underlying gut hormone secretion may also be studied by use of gut tissue pieces (including human tissue), primary intestinal cultures (usually from mice), immortalized hormone secreting cell lines (of mouse or human origin), by gut tissue mounted in Ussing chambers or by organoids (both most often from mice)2,3,4,5,6,17,18. Compared to intestinal perfusions, studies on human gut pieces, primary cell cultures and cell lines are technically easier to perform and are a faster and cheaper way of generating data, but of course the study of gut pieces requires access to fresh human gut specimens. However, in these models the normal cell polarization of the gut is inherently lost, meaning that these models cannot be used to assess normal activation of molecular sensors, and absorption processes also cannot be studied. Moreover, such studies usually employ static incubations (for up to several h) which is highly non-physiological and has nothing to do with the cells&#39; normal secretory dynamics, because the secreted product is not removed and thus may feedback influence the secretion of hormones. In contrast, in the perfused intestine, secreted and absorbed molecules are efficiently removed by the mucosal microcirculation as they are in vivo, ensuring that the transmucosal gradients are maintained so absorption and secretion can occur at a normal rate. Furthermore, cell cultures may have dedifferentiated from their native enteroendocrine cell origin, meaning that they are no longer representative of the native cells in terms of peptide content and expression of molecular sensors, although they may still secrete the hormone in question. This is, for instance, the case for GLP-1 secreting cell lines19.
It is, therefore, our opinion that primary cell cultures or cell line studies are most suited for screening purposes and for performing types experiments that cannot be performed in vivo or in the isolated perfused gut. For instance, a true strength of the primary cell cultures and cell line cultures is that intra-cellular secondary messengers (e.g. Ca2+, cAMP, NAD(P)H) can be monitored in real time, and electrical signaling of the hormone secreting cells can be investigated20,21,22. In addition, siRNA knockdown can be done, which is particularly useful if specific inhibitors are not available20,21,22,23,24. Gut tissue from mice mounted in Ussing chambers has recently been used for studying the molecular mechanisms underlying bile-acid stimulated GLP-1 secretion, while intestinal organoids (from mice) and human gut pieces have also been used for studying the molecular details of gut hormone secretion17,25. Whereas the former benefits from being polarized2 all of these models involve static incubations. Studies on human gut pieces, however, benefit from using human, rather than rodent, tissue which is important since species difference in tissue expression of 7TM receptors and molecular transporters may result in different molecular sensing pathways between species. In fact, most data in this field has been generated by studies on either pigs, mice or rats, and it remains elusive whether these findings can be transferred to humans. It is, however, reassuring that the molecular sensing mechanisms that underlie glucose-stimulated GLP-1 secretion appear to be similar between mouse, rat,&#160;man,&#160;and transcriptomic and peptidomic profiling of mouse and human L-cells&#160; revealed strong global similarities between the two species7,18,26,27.
The isolated perfused rat small intestine, however, also has some limitations that should be considered. Most importantly, it is impossible to determine whether a given secretory response results from direct activation by the test substance of the targeted hormone-producing cells or rather is caused by an indirect mechanism. For instance, KCl instantly increases GLP-1 secretion from the perfused intestine7, but it remains unknown whether this is a consequence of direct depolarization of the L-cell or results from depolarization of neurons close to the L-cells or effects of simultaneously released paracrine stimulators/inhibitors. Data arising from studies using the perfused intestine which aim to elucidate the molecular mechanisms underlying secretion should, therefore, always be put into context with data obtained from other more specific models to increase the ability to establish causality. For instance, glucose-stimulated GLP-1 secretion from the GLP-1 secreting cell line GLUTag28,29&#160;and from primary mouse L-cells depend on the activity of the glucose transporters (SGLT1 and GLUT2). Blocking these transporters in the perfused rat small intestine also attenuates secretion20, meaning that it is likely that glucose-stimulated GLP-1 secretion is largely mediated by direct actions of glucose on the L-cell. Another important limitation of the isolated perfused intestine is that some of the lipids are difficult to study due to their hydrophobicity. Although it is possible to investigate the final products of lipid digestion (fatty acids, diacyl glycerols, lysophosphatidylglycerols, etc.) and although the preparation may be able to re-esterify the lipids intra-cellularly and perhaps pack them into chylomicrons, the transport of the chylomicrons out of the cells and their subsequent uptake by the lacteals of the villi is disrupted, since the lymph flow in the isolated gut cannot be secured. Most likely, therefore, lipid absorption is halted once the absorbed products start to accumulate in the cells. The in vitro cell systems are even less suitable for lipid studies because of their lack of polarization. Obviously, this limitation is only relevant for lipids that are absorbed and transported via the lacteals, whereas those absorbed via the intestinal blood vessels are likely to be handled normally.

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

mechanisms underlying gut hormone secretion using 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 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...

Watch this Scientific Journal Video about Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine at JoVE.com

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

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; 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, ...

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