Name: hyperinsulinemic-euglycemic clamp in the conscious rat
Uploaded: 2011-02-07T17:00:00
Description: Watch this Scientific Journal Video about Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat at JoVE.com

This study was supported by the Canadian Institutes of Health Research and Genome Canada.JS holds salary support awards from the Alberta Heritage Foundationfor Medical Research, Heart and Stroke Foundation of Canadaand the Canadian Diabetes Association. Special thanks to Dr. David Wasserman and Bingle Bracy for teaching this procedure to the Shearer laboratory.

A: Surgical Catheterization of Arterial and Venous Circulation
Part 1: Arterial and Venous Catheter Preparation
<ol>
	<li>Cut 15 cm of PE-50 (internal diameter of .58mm (.023") x an outer diameter of .965mm (.038"). Cut a 1mm section of silastic tubing (0.76mm (0.030") internal diameter x 1.65mm (0.065") outer diameter) for use as restraining bead. The restraining bead prevents the rat from pulling out the catheter once it is in place.</li>
	<li>Insert the tips of micro dissecting forceps i...

<ol>
	<li>Halseth, A. E., Bracy, D. P., Wasserman, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+to+basal+and+insulin-stimulated+skeletal+muscle+glucose+uptake+in+the+high-fat-fed+rat.">Limitations to basal and insulin-stimulated skeletal muscle glucose uptake in the high-fat-fed rat.</a> Am J Physiol Endocrinol Metab. 279, E1064-E1071 (2000).</li><li>Halseth, A. E., Bracy, D. P., Wasserman, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+to+muscle+glucose+uptake+due+to+high+fat+feeding.">Limitations to muscle glucose uptake due to high fat feeding.</a> Am. J. Physiol. 279, E1064-E1071 (2000).</li><li>Muniyappa, R., Lee, S., Chen, H., Quon, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+for+assessing+insulin+sensitivity+and+resistance+in+vivo:+advantages,+limitations,+and+appropriate+usage.">Current approaches for assessing insulin sensitivity and resistance in vivo: advantages, limitations, and appropriate usage.</a> Am J Physiol Endocrinol Metab. 294, 15-26 (2008).</li><li>Cho, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+resistance+and+a+diabetes+mellitus-like+syndrome+in+mice+lacking+the+protein+kinase+Akt2+(PKB+beta).">Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta).</a> Science. 292, 1728-1731 (2001).</li><li>Dubois, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SHP-1+protein+tyrosine+phosphatase+negatively+modulates+glucose+homeostasis.">The SHP-1 protein tyrosine phosphatase negatively modulates glucose homeostasis.</a> Nat Med. 12, 549-556 (2006).</li><li>Pacini, G., Thomaseth, K., Ahren, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+to+glucose+tolerance+of+insulin-independent+vs.+insulin-dependent+mechanisms+in+mice.">Contribution to glucose tolerance of insulin-independent vs. insulin-dependent mechanisms in mice.</a> Am J Physiol Endocrinol Metab. 281, 693-703 (2001).</li><li>Herbach, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant-negative+effects+of+a+novel+mutated+Ins2+allele+causes+early-onset+diabetes+and+severe+beta-cell+loss+in+Munich+Ins2C95S+mutant+mice.">Dominant-negative effects of a novel mutated Ins2 allele causes early-onset diabetes and severe beta-cell loss in Munich Ins2C95S mutant mice.</a> Diabetes. 56, 1268-1276 (2007).</li><li>Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet-induced+insulin+resistance+in+mice+lacking+adiponectin/ACRP30.">Diet-induced insulin resistance in mice lacking adiponectin/ACRP30.</a> Nat Med. 8, 731-737 (2002).</li><li>Potenza, M. A., Marasciulo, F. L., Tarquinio, M., Quon, M. J., Montagnani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+spontaneously+hypertensive+rats+with+rosiglitazone+and/or+enalapril+restores+balance+between+vasodilator+and+vasoconstrictor+actions+of+insulin+with+simultaneous+improvement+in+hypertension+and+insulin+resistance.">Treatment of spontaneously hypertensive rats with rosiglitazone and/or enalapril restores balance between vasodilator and vasoconstrictor actions of insulin with simultaneous improvement in hypertension and insulin resistance.</a> Diabetes. 55, 3594-3603 (2006).</li><li>Ayala, J. E., Bracy, D. P., McGuinness, O. P., Wasserman, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+in+the+design+of+hyperinsulinemic-euglycemic+clamps+in+the+conscious+mouse.">Considerations in the design of hyperinsulinemic-euglycemic clamps in the conscious mouse.</a> Diabetes. 55, 390-397 (2006).</li><li>Wasserman, D. H., Ayala, J. E., McGuinness, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lost+in+translation.">Lost in translation.</a> Diabetes. 58, 1947-1950 (2009).</li></ol>

Initially developed for investigation of insulin sensitivity in humans, the clamp procedure has now been adapted to other species including laboratory rats and mice. Investigating animal models of insulin resistance provides a significant aid in understanding the pathophysiology of insulin sensitivity and associated pathologies as well as identifying therapeutic interventions that have clinical value1,2. Several methods to evaluate insulin sensitivity in animals have been employed3. Such techniques ...

<table border="1">
	<tbody>
		<tr valign="top">
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr valign="top">
			<td>Intramedic Polyethylene Tubing (PE-50)</td>
			<td>Fisher Scientific</td>
			<td>14-170-12B</td>
			<td>Internal diameter of .58mm (.023") x Outer diameter of .965mm (.038")</td>
		</tr>
		<tr valign="top">
			<td>Dow Corning Silastic Laboratory Tubing</td>
			<td>Fisher Scientific</td>
			<td>11-189-15C</td>
			<td>Internal diameter of .76mm (0.030") x Outer diameter of 1.65mm (0.065") </td>
		</tr>
		<tr valign="top">
			<td>Tygon S-50-HL Medical Tubing</td>
			<td>Harvard Apparatus</td>
			<td>PY2 72-1251</td>
			<td>Internal diameter of 3.2mm (0.125") x Outer diameter of 4.7mm (0.1875")</td>
		</tr>
		<tr valign="top">
			<td>Loctite Super Glue</td>
			<td>Grand &amp; Toy</td>
			<td>32237</td>
			<td>Gel Control</td>
		</tr>
		<tr valign="top">
			<td>Sterile Surgical Blade</td>
			<td>VWR</td>
			<td>BD371610</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Curved Micro Dissecting Forceps</td>
			<td>George Tiemann &amp; Co.</td>
			<td>160-20</td>
			<td>x 2</td>
		</tr>
		<tr valign="top">
			<td>Straight Micro Dissecting Forceps</td>
			<td>George Tiemann &amp; Co.</td>
			<td>160-15</td>
			<td>x 2</td>
		</tr>
		<tr valign="top">
			<td>Curved Hemostat</td>
			<td>George Tiemann &amp; Co.</td>
			<td>105-1135</td>
			<td>x 2</td>
		</tr>
		<tr valign="top">
			<td>Straight Hemostat</td>
			<td>George Tiemann &amp; Co.</td>
			<td>105-1130</td>
			<td>x 2</td>
		</tr>
		<tr valign="top">
			<td>Hemostat Tip Guards</td>
			<td>Robbins Instruments, Inc.</td>
			<td>15.09-2-004</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Straight Surgery Scissors</td>
			<td>George Tiemann &amp; Co.</td>
			<td>105-402</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>VENOJECT Multi-Sample Luer Adapter</td>
			<td>Terumo Medical Products</td>
			<td>810127A</td>
			<td>21 guage, 1 in.</td>
		</tr>
		<tr valign="top">
			<td>Sterile Catheter Introducer</td>
			<td>Becton Dickinson</td>
			<td>406999</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>14-gauge Blunt Needle</td>
			<td>Becton Dickinson</td>
			<td>511310</td>
			<td>14 guage, 2 in.</td>
		</tr>
		<tr valign="top">
			<td>Sterile Surgical Suture</td>
			<td>Johnson &amp; Johnson Medical Products</td>
			<td>1679H</td>
			<td>Silk, size 3-0</td>
		</tr>
		<tr valign="top">
			<td>Non-Sterile Surgical Suture </td>
			<td>Angiotech Pharmaceuticals, Inc.</td>
			<td>SP116</td>
			<td>Silk, size 4-0</td>
		</tr>
		<tr valign="top">
			<td>Cotton Swabs</td>
			<td>VWR</td>
			<td>10806-005</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>4ply Gauze Pads</td>
			<td>VWR</td>
			<td>CA43845-062</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Small Animal Cordless Clippers</td>
			<td>Harvard Apparatus</td>
			<td>729063</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Isoflurane</td>
			<td>Halocarbon Products Corp.</td>
			<td>IPN-45</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Anesthetic Cart</td>
			<td>Benson Medical Industries, Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>70 % Ethanol</td>
			<td>Fisher Scientific</td>
			<td>HC-1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Betadine Antiseptic Solution</td>
			<td>Western Drug Distribution Centre Ltd.</td>
			<td>105267</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Model 11 Plus Syringe Pump</td>
			<td>Harvard Apparatus</td>
			<td>702208</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Stainless Steel Tubing Couplers</td>
			<td>Harvard Apparatus</td>
			<td>72-4434</td>
			<td>23 gauge, 0.3 in.</td>
		</tr>
		<tr valign="top">
			<td>Stainless Steel Tubing Plugs</td>
			<td>Harvard Apparatus</td>
			<td>72-4436</td>
			<td>23 gauge, 0.5 in.</td>
		</tr>
		<tr valign="top">
			<td>Stainless Steel Blunt Needles</td>
			<td>Instech Laboratories, Inc.</td>
			<td>LS22</td>
			<td>22 gauge</td>
		</tr>
		<tr valign="top">
			<td>60 Degree Y-Connectors</td>
			<td>Small Parts</td>
			<td>STCY-22-05</td>
			<td>22 gauge</td>
		</tr>
		<tr valign="top">
			<td>CritSpin Micro-hematocrit Centrifuge</td>
			<td>Iris Sample Processing</td>
			<td>CS12</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Mini Centrifuge</td>
			<td>Fisher Scientific</td>
			<td>05-090-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Micro Centrifuge Tubes</td>
			<td>VWR</td>
			<td>53550-778</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>50ml polypropylene centrifuge tubes</td>
			<td>VWR</td>
			<td>89004-364</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>1ml Plastic Slip Tip Syringes</td>
			<td>Becton Dickinson</td>
			<td>309602</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>3ml Plastic Luerlok Tip Syringes</td>
			<td>Becton Dickinson</td>
			<td>309585</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Heparin Anticoagulant Injection</td>
			<td>Western Drug Distribution Centre Ltd.</td>
			<td>102824</td>
			<td>Manufacturer: LEO Pharma Inc.
				Conc. 1000 IU</td>
		</tr>
		<tr valign="top">
			<td>EDTA Solution</td>
			<td>Promega Corp.</td>
			<td>V4231</td>
			<td>0.5 M, pH 8.0</td>
		</tr>
		<tr valign="top">
			<td>Saline</td>
			<td>Western Drug Distribution Centre Ltd.</td>
			<td>ABB7983154</td>
			<td>Manufacturer: Hospira
				0.9% Sodium Chloride</td>
		</tr>
		<tr valign="top">
			<td>50% Dextrose</td>
			<td>Vetoquinol</td>
			<td>8DEX012D</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Humulin-R </td>
			<td>Eli Lilly</td>
			<td>HI-210</td>
			<td>100U/ml</td>
		</tr>
		<tr valign="top">
			<td>1ml Insulin Syringes </td>
			<td>Becton Dickinson</td>
			<td>309311</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Fisherbrand* Hemato-Seal Sealant</td>
			<td>Fisher Scientific</td>
			<td>02-678</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Fisherbrand* Microhematocrit Capillary Tubes</td>
			<td>Fisher Scientific</td>
			<td>22-362-574</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>One Touch Ultra Test Strips</td>
			<td>LifeScan, Inc.</td>
			<td>AW 085-314H</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>One Touch Ultra Blood Glucose Meter</td>
			<td>LifeScan, Inc.</td>
			<td>AW 085-314B</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Sodium Pentobarbitol</td>
			<td>Ceva Sante Animale</td>
			<td>1715 138</td>
			<td>Conc. 54.7mg/ml</td>
		</tr>
		<tr valign="top">
			<td>Red Laboratory Labeling Tape</td>
			<td>VWR</td>
			<td>89097-932</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Blue Laboratory Labeling Tape</td>
			<td>VWR</td>
			<td>89097-936</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Weigh Scale</td>
			<td>Fisher Scientific</td>
			<td>01-913-88</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Vortex</td>
			<td>VWR</td>
			<td>58815-234</td>
			<td>&nbsp;</td>
		</tr>
		<tr valign="top">
			<td>Timer</td>
			<td>VWR</td>
			<td>62344-641</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

hyperinsulinemic-euglycemic clamp in the conscious rat

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet specific experimental requirements. It is further enhanced by the use of radioactive tracers that can determine tissue specific insulin-stimulated glucose uptake as well as whole body glucose turnover.

Watch this Scientific Journal Video about Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat at JoVE.com

Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

The hyperinsulinemic-euglycemic clamp is the "gold standard" for the assessment of insulin action. Insulin is infused at a constant rate stimulating glucose uptake. The amount of exogenous glucose infused to counter this drop is indicative of insulin sensitivity. Here the procedure is performed on a conscious, unrestrained rat.

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, ...

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