1Department of Biology, State University of New York, 2Allied Health Sciences, University of Connecticut
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Haselton, A. T., Fridell, Y. C. Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster. J. Vis. Exp. (52), e2722, doi:10.3791/2722 (2011).
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.
1. Insulin Solution Preparation
2. Needle Preparation and Injection Set-up
3. Fly Preparation
4. Injection Procedure
5. Hemolymph Collection
6. Hemolymph Glucose Determination
7. Representative Results
A typical insulin tolerance response is detected in insulin-injected flies where a drop in circulating glucose levels is detected 15 minutes post-injection. In contrast, such response is not seen in PBS injected flies (Fig. 3). This response in peripheral glucose disposal continues in insulin-injected flies up to 30 minutes post-injection. We routinely extract 0.2-0.5 μl of hemolymph per 4-5 flies in each injection group. Three injection groups are included in each experiment.
Figure 1. Left side of Drosophila thorax showing needle insertion site (Modified from Demerec, 1950) 7. Insert the needle through the center of the prescutum on the anterior, dorsal region of the left side of the thorax.
Figure 2. Frontal view of Drosophila head showing puncture location for hemolymph extraction (Modified from Demerec, 1950) 7. Puncture the head capsule with a finely sharpened tungsten probe in the center of the head capsule just above the ptilinal suture.
Figure 3. A typical insulin tolerance response detected in control adult flies. Control w1118 flies were injected with bovine insulin (1 ng in PBS) or PBS only. Flies in replicate groups were then allowed to recover for 0, 15 or 30 minutes and circulating glucose levels were measured.
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 2 is that this technique minimizes contamination of hemolymph samples with gut contents.If care is taken not to collect any pericerebral fat body tissue, which may sometimes appear in the exuded hemolymph droplet, then samples should accurately reflect the state of in vivo circulatory fluids.
A potentially confounding factor inherent in any injection experiment performed at this scale is the initial dilution of total circulatory fluids following injection. This can easily be controlled for, however, by injecting the same volumes of PBS in experimental flies or insulin into age-matched controls and normalizing results. To ensure reproducible circulating glucose readings, the quality of hemolymph droplets is of most importance. Only clear droplets of hemolymph devoid of pericerebral fat body or other tissue debris should be collected for glucose determination. It is also noteworthy that only up to 8 samples should be collected before determining relative glucose concentrations. We noticed a "drift" in OD340 readings when the hemolymph samples were left on ice for an extended period of time.
Our protocol leaves much room for modification based on available equipment. Pipet puller settings may need to be modified based on the model and condition of the puller. We have found that settings adequate for producing intracellular recording electrode needles are ideal for injection needles. Additionally, while we employed a three-axis micromanipulator for maintaining needle position under the stereoscope, this may also be achieved with a sturdy ringstand and clamp system as the needle remains in a fixed position throughout the injection procedure. The high resistance of the needles used necessitated the development of an alternative method for determining the volume of injected fluid as a 1:1 syringe plunger movement to volume displacement was not achievable. We developed a graduated scale that could be printed and attached to the side of the needle shaft. Depending on the system used, it may be necessary to displace some fluid from the needle beyond the needle holder of the microinjector so that the fluid meniscus may be aligned with graduations the calibration card affixed to the side of the needle shaft.
Finally, two limitations of this technique are noted in our protocol. First, two persons are usually required to coordinate both injection and hemolymph extraction steps in order to maximize the number of samples processed. Second, variations in insulin tolerance response are sometimes observed within the same genotype. We believe this is due to variable responses individual flies may have following ice immobilization and recovery on agar. Therefore, a sufficient number of samples should be examined before reliable conclusions can be drawn.
No conflicts of interest declared.
This work was supported by grants from the NIA to Y-W.C.F (AG21068, AG31086).
|Infinity Glucose Reagent||Thermo Fisher Scientific, Inc.||TR1541|
|Manual microinjector||Sutter Instrument Co.|
|P-87 Flamming/Brown micropipette puller||Sutter Instrument Co.|
|Single barrel borosilicate capillary glass||A-M Systems||626000|
|FD&C Blue No. 1||McCormick & Co.|
|1 Ál microcapillary tubes||Drummond Scientific|
|Three-axis manual micromanipulator and base||World Precision Instruments, Inc.|