Name: oral glucose tolerance test (ogtt): measuring glucose metabolism in mice
Uploaded: 2023-04-30T12:50:12.000+00:00
Duration: 1 min 3 s
Description: Source: Ajoy, R. and Chou, S. Y. Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into ...

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Oral Glucose Tolerance Test (OGTT)

<ol>
	<li>Glucose solution preparation: Before conducting the experiment, prepare the glucose solution by dissolving 3.75 g glucose in 15 mL distilled H2O.</li>
	<li>Set up a timetable to record the readings during the experimental procedure (Table 1). 
	NOTE: It is important to set up a timetable with proper intervals between each blood examination to allow accurate recording during the experiment.</li>
	<li>Weigh each mouse after fasting and calculate the appropriate amount of glucose to be injected. For example, if the mouse weighs 30 g, the amount of glucose solution to be administered should be 300 &#181;L.</li>
	<li>Prepare the following instruments at the workbench:
	<ol>
		<li>Glucometer (Press the Start button to check the battery status, make sure it is functioning before the test.)</li>
		<li>Glucose chip</li>
		<li>Insulin syringe (0.3 mL Insulin syringe)</li>
		<li>Razor blades</li>
		<li>Timer</li>
	</ol>
	</li>
	<li>Once the bench is set up, measure and record the blood glucose level as follows: Put a clean and new glucose chip into the glucometer and press the Start button to zero it.</li>
	<li>Pick up the mouse by the back of the neck and stroke the tail a few times to ensure sufficient blood flow to the tail region.</li>
	<li>Using a new razor blade, cut off a small piece of the tail and squeeze out one small drop of blood (about 10-20 &#181;L) into the glucose chip. The blood should fill the chip to allow accurate measurement. The glucometer will display the glucose level immediately. If the machine shows &#34;error&#34;, repeat the procedure with a new glucose chip. 
	NOTE: The glucose chip requires only one drop of blood. When the blood sample needs to be collected more than once, simply apply pressure by running your fingers along the mouse&#39;s tail several times while holding the end of the tail directly on top of the chip to collect blood. It is not necessary to cut the tail end each time while collecting blood samples.</li>
	<li>Next, feed the mice glucose (0.25 g/mL) orally by using the intragastric gavage technique. The amount of glucose to be administered should be calculated by using the formula: 10X body weight (BW) &#181;L glucose solution (for example, if the mouse weighs 30 g, the amount of glucose solution to be administered will be 300 &#181;L). Start the timer immediately after administering oral glucose.</li>
	<li>Repeat the glucose measurement procedure at 15, 30, 60, 90, and 120 min.</li>
	<li>After all the glucose level readings have been recorded, discard the razor blades and glucose chips in a biohazard container. Put the food back into the mice cages and return them to the animal room.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#</td>
			<td>Mouse ID</td>
			<td>Body</td>
			<td>Glucose</td>
			<td>Start</td>
			<td>0</td>
			<td>15</td>
			<td>30</td>
			<td>60</td>
			<td>90</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>weight</td>
			<td>&#956;L =10xBW</td>
			<td>time</td>
			<td>mins</td>
			<td>mins</td>
			<td>mins</td>
			<td>mins</td>
			<td>mins</td>
		</tr>
		<tr>
			<td>1</td>
			<td>501</td>
			<td>25.8g</td>
			<td>258</td>
			<td>9:00</td>
			<td>9:00</td>
			<td>9:00</td>
			<td>9:15</td>
			<td>9:30</td>
			<td>10:00</td>
		</tr>
		<tr>
			<td>2</td>
			<td>502</td>
			<td>25.3g</td>
			<td>253</td>
			<td>9:07</td>
			<td>9:07</td>
			<td>9:07</td>
			<td>9:22</td>
			<td>9:37</td>
			<td>10:07</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Timetable for Oral Glucose Tolerance Test (OGTT) recording

<table><tbody><tr><td>Glucometer</td><td>Roche</td><td>#06870244001</td><td>Used to measure the blood glucose level</td></tr><tr><td>Glucose chip</td><td>Roche</td><td>#06454011020</td><td>Used to load the blood sample</td></tr><tr><td>10 mM glucose</td><td></td><td>Filter sterilize with a 0.22 &amp;micro;m filter apparatus and store at 4&amp;deg;C.</td><td></td></tr><tr><td>Animals: C57BL/6</td><td>NAR Labs</td><td></td><td>Wild type mice strain used in the study</td></tr></tbody></table>

oral glucose tolerance test (ogtt): measuring glucose metabolism in mice

Source: Ajoy, R. and Chou, S. Y. <a href="https://www.jove.com/video/56410/studying-hypothalamic-insulin-signal-to-peripheral-glucose">Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain</a>.&#160;J. Vis. Exp. (2018).
This video introduces how to perform an oral glucose tolerance test on a laboratory mouse.

Table 1.&#160;Timetable for Oral Glucose Tolerance Test (OGTT) recording
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody style="font-size:14px; text-align:center">
		<tr>
			<td>#</td>
			<td>Mouse ID</td>
			<td>Body</td>
			<td>Glucose</td>
			<td>Start</td>
			<td>0</td>
			<td>15</td>
			<td>30</td>
			<td>60</td>
			<td>90</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>weight</td>
			<td>&#181;L =10xBW</td>
			<td>time</td>
			<td>mins</td...

Oral Glucose Tolerance Test (OGTT): Measuring Glucose Metabolism in Mice

Source: Ajoy, R. and Chou, S. Y. Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into ...

Prepare for the test by fasting the mice for 6 hours. Then, weigh each mouse to determine its body mass, which will be used to calculate the dose of glucose to be used. To measure the baseline blood glucose for each mouse, we&#39;ll use a glucometer and glucose chip, commonly known as a test strip.
Collect blood by cutting off 1 to 2 millimeters of the tail tip. Place a drop of this blood on the glucose chip and record the measurement. Next, feed glucose to the mouse by gavage and immediately record the time. Repeat the blood glucose measurements over 2 hours.
This test will monitor how much the blood glucose increases as oral glucose is taken up by the body and how quickly it returns to normal as peripheral tissues take up the glucose to be used or stored.

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5.4K visualizações. Fonte: Ajoy, R. e Chou, S. Y. Estudando o sinal de insulina hipotalâmica para intolerância periférica à glicose com um sistema contínuo de infusão de drogas no cérebro de camundongos. J. Vis. Exp. (2018). Este vídeo apresenta como realizar um teste oral de tolerância à glicose em um camundongo de laboratório. 

Vídeo: Teste oral de tolerância à glicose (OGTT): medindo o metabolismo da glicose em camundongos - Conceito

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

Combinado de microscopia Intravital e ultra-sonografia contraste aprimorado do membro posterior Mouse para estudar a vasodilatação induzida por insulina e perfusão muscular

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

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Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

Estudo do metabolismo in vivo da glicose em camundongos alimentados com dieta de alto teor em gordura usando o teste oral de tolerância à glicose (OGTT) e o teste de tolerância à insulina (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Medição da captação de glicose e resposta à estimulação de insulina em miotubos primários humanos cultivados in vitro

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Oral Glucose Tolerance Test (OGTT): Measuring Glucose Metabolism in Mice

Transcrição

Oral Glucose Tolerance Test (OGTT): Measuring Glucose Metabolism in Mice