Name: insulin injection and hemolymph extraction to measure insulin sensitivity in adult drosophila melanogaster
Uploaded: 2011-06-30T13:00:00
Description: Watch this Scientific Journal Video about Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster at JoVE.com

This work was supported by grants from the NIA to Y-W.C.F (AG21068, AG31086).

1. Insulin Solution Preparation
<ol>
<li>Prepare fresh bovine insulin solution by dissolving insulin in PBS to achieve the concentration of 0.01 mg/ml. Both insulin/PBS and control PBS solutions must be kept on ice throughout the injection procedure. These solutions should be prepared with 0.5% (v/v) FD&amp;C Blue no. 1 food coloring.</li>
</ol>
2. Needle Preparation and Injection Set-up
<ol>
<li>Prepare capillary glass needles using a micropipette puller.The following puller settings produc...

<ol>
	<li>Rulifson, E. J., Kim, S. K., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ablation+of+insulin-producing+neurons+in+flies:+growth+and+diabetic+phenotypes.">Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes.</a> Science. 296, 1118-1118 (2002).</li><li>Kim, S. K., Rulifson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+mechanisms+of+glucose+sensing+and+regulation+by+Drosophila+corpora+cardiaca+cells.">Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells.</a> Nature. 431, 316-316 (2004).</li><li>Broughton, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longer+lifespan,+altered+metabolism,+and+stress+resistance+in+Drosophila+from+ablation+of+cells+making+insulin-like+ligands.">Longer lifespan, altered metabolism, and stress resistance in Drosophila from ablation of cells making insulin-like ligands.</a> Proc Natl Acad Sci U S A. 102, 3105-3105 (2005).</li><li>Haselton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+ablation+of+adult+Drosophila+insulin-producing+neurons+modulates+glucose+homeostasis+and+extends+life+span+without+insulin+resistance.">Partial ablation of adult Drosophila insulin-producing neurons modulates glucose homeostasis and extends life span without insulin resistance.</a> Cell Cycle. 9, 3063-3063 (2010).</li><li>Haselton, A. T., Fridell, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+Drosophila+melanogaster+as+a+model+for+the+study+of+glucose+homeostasis.">Adult Drosophila melanogaster as a model for the study of glucose homeostasis.</a> Aging (Albany NY). 2, 523-523 (2010).</li><li>Belgacem, Y. H., Martin, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+insulin+pathways+alters+trehalose+level+and+abolishes+sexual+dimorphism+in+locomotor+activity+in+Drosophila.">Disruption of insulin pathways alters trehalose level and abolishes sexual dimorphism in locomotor activity in Drosophila.</a> J Neurobiol. 66, 19-19 (2006).</li><li>Demerec, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of Drosophila. , (1950).</li></ol>

The technique described in this report is potentially useful in any study that investigates physiological processes resulting in detectable alterations in Drosophila hemolymph composition. By combining injection and hemolymph collection in this manner, it is possible to ascertain the immediate physiologically relevant effects of a particular experimental treatment or manipulation. The primary advantage of this "bloodletting" technique in hemolymph collection over previous techniques involving decapitation <sup...

<table border="1">
<tbody>
	<tr>
 	<th>Name of the reagent/equipment</th>
 <th>Company</th>
 <th>Catalogue number</th>
 
 </tr>
 <tr>
 	<td>Bovine insulin</td>
 <td>Sigma</td>
 <td>I5500</td>
 </tr>
 <tr>
 	<td>Infinity Glucose Reagent </td>
 <td>Thermo Electron Corporation</td>
 <td>TR1541</td>
 </tr>
 <tr>
 	<td>Manual microinjector </td>
 <td>Sutter Instrument </td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>P-87 Flamming/Brown micropipette puller</td>
 <td>Sutter Instrument </td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>Single barrel borosilicate capillary glass</td>
 <td>A-M Systems</td>
 <td>626000</td>
 </tr>

 <tr>
 	<td>FD&amp;C Blue No. 1</td>
 <td>McCormick &amp; Company</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>1 &mu;l microcapillary tubes</td>
 <td>Drummond</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>Three-axis manual micromanipulator and base</td>
 <td>World Precision Instruments</td>
 <td>&nbsp;</td>
 </tr>
</tbody>
</table>

insulin injection and hemolymph extraction to measure insulin sensitivity in adult drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Watch this Scientific Journal Video about Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster at JoVE.com

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

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