This work was funded by the Austrian Research Promotion Agency (FFG; project number 850681), the University of Applied Sciences Upper Austria Basic Funding initiative (project GlucoSTAR) and the Center for Technological Innovation in Medicine (TIMed Center) that receives basic funding by the province of Upper Austria.

According to the directive 2010/63/EU, experiments with non-hatched avian embryos during the first two-thirds of embryonic development do not need permission by an ethics committee.
1. Storage and Breeding of the Eggs
<ol>
	<li>Obtain fertilized hen eggs (Table of Materials) from a local breeder. 
	NOTE: Eggs may be stored at 14 &#176;C in a humidified atmosphere for up to 10 days after laying before use for experiments.</li>
	<li>Incubate the eggs at 38 &#176;C with an average humidity of 40 - 60% for 10 or 11 days in an incubator that constantly turns the eggs.</li>
</ol>
2. Selection of the Eggs
<ol>
	<li>Check the eggs for fertilization after incubation of up to 11 days. Use a candling light for this step. Dip the fringe of the candling lamp into an inkpad and candle the pointed side of the egg.</li>
	<li>To identify the air bladder, check for differences in brightness on the top of the egg. 
	NOTE: The air bladder appears as a round, more transparent region, while the albumen is darker when the egg is candled.</li>
	<li>After detection of the air bladder, mark the location by pressing the lamp slightly against the egg with the previously applied ink.</li>
	<li>Exclude non-fertilized eggs at this step, which do not show a difference in brightness between the albumen and air bladder.</li>
</ol>
3. Injection of Substances
<ol>
	<li>Prepare the substance of interest (e.g., the herbal extract or insulin) by diluting the substance in the desired concentration in Hank&#39;s balanced salt solution (HBSS) buffer. For example, 3.3 U/mL of a commercially available insulin analog in HBSS.</li>
	<li>For the application of the selected compound, carefully peck the eggshell with a pointed pair of tweezers in the marked area of the air bladder. Do not form a hole larger than the diameter of the needle of the syringe. Inject the buffer solution (300 &#181;L) containing the substance of interest into the pecked area via a syringe. 
	NOTE: Use sterile, 1-ml single-use syringes, pipette tips, and vessels. Wear gloves and a lab coat for the experiments.</li>
	<li>To determine the blood glucose-reducing effect of a substance, place the eggs back into the incubator for 60, 120, and 180 min, respectively. To determine basal blood glucose levels, include at least 10 - 15 non-treated control eggs.</li>
</ol>
4. Toxicity Tests
<ol>
	<li>After application of the selected compound for 24 h, equilibrate the eggshell membrane with phosphate buffered saline (PBS) buffer (enough to cover the whole eggshell membrane) for at least 30 s.</li>
	<li>Remove excess PBS buffer by pouring it away.</li>
	<li>Pull off the eggshell membrane carefully with a pointed pair of tweezers.</li>
	<li>Check blood vessels for lesions and confirm vitality (e.g., movement) of the chick embryo. 
	NOTE: See Figure 1 for comparison of vital versus non-vital embryos after application of a toxic herbal extract.</li>
</ol>
5. Measurement of the Blood Glucose Value
<ol>
	<li>At the respective time point, carefully remove the eggshell above the air bladder and equilibrate the eggshell membrane with PBS buffer (enough to cover the whole eggshell membrane).</li>
	<li>Remove excess PBS buffer by pouring it away.</li>
	<li>Carefully pull off the eggshell membrane with a pointed pair of tweezers.</li>
	<li>Cut and remove the chorioallantoic membrane with a micro-scissor, to enable good access to suitable blood vessels. Cut the membrane at least 2 - 3 cm. Do not cut any large vessels in the membrane itself to avoid unwanted blood loss of the embryo.</li>
	<li>Locate the large vessel originating from the abdomen of the embryo. Lift this vessel out of the albumen with a closed micro-scissor and place it on a plastic pH-strip. Move the pH strip under the vessel, which is held with the micro-scissor, and pull back the micro-scissor away carefully.</li>
	<li>Remove 1 - 2 mL of the liquid beneath the chorioallantoic membrane with a pipette to avoid dilution of the blood in the next step. Dry the vessel and pH-strip with filter paper, before the vessel is cut.</li>
	<li>Cut the blood vessel carefully with a micro-scissor on one side. Do not cut through the vessel completely. 
	NOTE: The vessel is approximately 1-mm thick. Do not cut more than half of the vessel to avoid cutting through the vessel entirely, as it may slip off the strip and no blood collection is possible.</li>
	<li>Collect the leaking blood on the pH-strip using a pipette. Ten microliters of blood are required to determine glucose levels using the blood glucose meter.</li>
</ol>
6. Statistical Evaluation
<ol>
	<li>Calculate the mean value of at least 10 individual eggs per time point and normalize these values to one of the control eggs (non- or buffer-treated eggs).</li>
	<li>Subsequently, determine the percentage of decrease. In addition, use insulin as a positive control. 
	NOTE: Repeat each experiment at least three times.</li>
</ol>

<ol>
	<li>Defronzo, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Banting+Lecture.+From+the+triumvirate+to+the+ominous+octet:+a+new+paradigm+for+the+treatment+of+type+2+diabetes+mellitus.">Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus.</a> Diabetes. 58 (4), 773-795 (2009).</li><li>American_Diabetes_Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+classification+of+diabetes+mellitus.">Diagnosis and classification of diabetes mellitus.</a> Diabetes Care. 37, S81-S90 (2014).</li><li>Han Cho, N., Cavan, D., 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=.">.</a> IDF Diabetes Atlas. 7, (2015).</li><li>Bailey, C. J., 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=Individualized+glycaemic+targets+and+pharmacotherapy+in+type+2+diabetes.">Individualized glycaemic targets and pharmacotherapy in type 2 diabetes.</a> Diabetes &amp; vascular disease research. 10 (5), 397-409 (2013).</li><li>Maruthur, N. M., 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=Diabetes+Medications+as+Monotherapy+or+Metformin-Based+Combination+Therapy+for+Type+2+Diabetes:+A+Systematic+Review+and+Meta-analysis.">Diabetes Medications as Monotherapy or Metformin-Based Combination Therapy for Type 2 Diabetes: A Systematic Review and Meta-analysis.</a> Annals of internal medicine. 164 (11), 740-751 (2016).</li><li>Aleman-Gonzalez-Duhart, D., Tamay-Cach, F., Alvarez-Almazan, S., Mendieta-Wejebe, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Advances+in+the+Biochemical+and+Physiological+Aspects+of+the+Treatment+of+Type+2+Diabetes+Mellitus+with+Thiazolidinediones.">Current Advances in the Biochemical and Physiological Aspects of the Treatment of Type 2 Diabetes Mellitus with Thiazolidinediones.</a> PPAR research. , 7614270 (2016).</li><li>Cohen, F. J., Neslusan, C. A., Conklin, J. E., Song, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+antihyperglycemic+prescribing+trends+for+US+privately+insured+patients+with+type+2+diabetes.">Recent antihyperglycemic prescribing trends for US privately insured patients with type 2 diabetes.</a> Diabetes care. 26 (6), 1847-1851 (2003).</li><li>Desai, N. R., 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=Patterns+of+medication+initiation+in+newly+diagnosed+diabetes+mellitus:+quality+and+cost+implications.">Patterns of medication initiation in newly diagnosed diabetes mellitus: quality and cost implications.</a> The American journal of medicine. 125 (3), e301-e307 (2012).</li><li>Martel, J., 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=Anti-obesogenic+and+antidiabetic+effects+of+plants+and+mushrooms.">Anti-obesogenic and antidiabetic effects of plants and mushrooms.</a> Nature reviews. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+new+methodological+aspects+of+the+hen's+egg+test+for+micronucleus+induction+(HET-MN).">Some new methodological aspects of the hen's egg test for micronucleus induction (HET-MN).</a> Mutation research. 514 (1-2), 59-76 (2002).</li></ol>

The authors have nothing to disclose.

The HET-CAM assay is a widely used alternative test system to animal testing in the industry12. The in-ovo system described here represents a modified version of the HET-CAM assay. While in its original form, HET-CAM experiments are performed to study the irritating properties of compounds and formulation or the analysis of angiogenesis14,15,16, we have adapted the approach to test compounds with putative insulin-mimetic characteristics12. According to the directive 2010/63/EU, experiments with non-hatched avian embryos during the first two-thirds of embryonic development do not need permission by an ethics committee since these experiments are not considered animal experiments. Recent studies have proven the suitability of the in-ovo system to characterize the efficacy of selected herbal extracts, which have been identified in a GLUT4-translocation-based primary screen10, to reduce blood glucose levels in the absence of insulin12,13. Critical points for a good performance of the in-ovo system include: First, a reliable breeder delivering the fertilized eggs. The Lohmann classic brown chicken is a good choice of breed. Second, the implementation of toxicity tests to exclude toxic effects of the investigated compound needs to be done. In addition, the preparation of a suitable blood vessel for blood collection is important. It is important to avoid the cutting of large vessels in the chorioallantoic membrane itself, as this can lead to a non-preferable loss of blood. Furthermore, before the vessel is cut onto the pH strip, it has to be patted dry using filter paper to prevent dilution of the collected blood. Finally, a sufficient number of experiments appears to be important. We recommend using at least 10 eggs for each time point, and a three-fold repetition of the respective experiment.
Naturally, there are some limitations of this in-ovo approach. Since it takes up to 30 minutes until the substances are absorbed through the eggshell membrane and come into close contact with the chorioallantoic membrane, it is not possible to quantify a rapid response of the embryo to the applied compound. Furthermore, some compounds may be toxic in the applied concentration, which frequently results in lesions of the blood vessels in the chorioallantoic membrane. Thus, cytotoxicity testing based on long-term incubation of the compounds for about 24 hours appears reasonable. Finally, we found that the buffer system used for application of the compounds has a significant effect on the performance of the assay.12 Currently, HBSS buffer is used because it results in the smallest effect on the blood glucose levels of the embryo.
For future applications, additional buffer systems as an alternative to HBSS might be tested. Furthermore, the influence of herbal extracts on additional blood parameters such as cholesterol, triglycerides or lipoproteins could be an attractive question.
Our new in-ovo system is a promising and important tool to test substances with insulin-mimetic characteristics in a living organism without the need of an animal experiment. Therefore, it fills the gap between in-vitro and in-vivo approaches. In comparison to existing in-ovo model systems17 there is no need to induce diabetes by streptozotocin (STZ) treatment, as we use embryos at day 10 or 11 of incubation. At this stage, insulin production has not started, but the embryos are already insulin sensitive. Additionally, permission by an ethics committee might be required if embryos aged 14 - 17 days are used17. Furthermore, compared to alternative strategies18, the compound application as described here is less harmful and laborious, increasing experimental through-put rates.
Taken together, this in-ovo approach is an attractive system if a blood-decreasing effect of an insulin-mimetic substance needs to be tested in a living organism.

Diabetes mellitus is a metabolic disease characterized by hyperglycemia and is caused by defects in insulin secretion or insulin action1. Ninety percent of all diabetes cases are category type 2 diabetes mellitus (T2DM), where individuals demonstrate insulin resistance and mostly insulin deficiency2. Several factors are known to increase the global incidence of T2DM, including changes in lifestyle, particularly those related to overnutrition, aging, and physical inactivity. Approximately 400 million people have diabetes mellitus worldwide according to the International Diabetes Federation (IDF). This number is expected to reach 600 million within the next 20 years3.
Diabetic complications caused by hyperglycemia and insulin-resistance, such as hypertension, dyslipidemia, glucose intolerance, coronary heart disease, and cerebral vascular disease, lead to a significantly reduced life expectancy and quality4. Affected individuals require glucose-lowering pharmacotherapy; however, the usage of drugs, such as metformin5 is often associated with dramatic side-effects6,7,8. Therefore, further antidiabetic approaches that are safe, widely available and inexpensive are necessary.
Different herbal compounds, extracts, plants, and nutraceuticals have been ascribed to feature insulin-mimetic properties by modulating various cellular functions. An important feature is stimulation of the translocation of glucose transporter 4 (GLUT4) to the plasma membrane from intracellular storage compartments, resulting in an increased uptake of glucose in muscle and adipose tissue in the absence of insulin, known as insulin-mimetic properties9. Previously, we implemented a fluorescence microscopy-based approach to quantify the translocation process of GLUT410. Analysis of numerous plant extracts from an extract library11 using this assay resulted in the identification of promising candidates. However, further tests in living organisms are required. The approach described in detail in this paper, a modified chick embryo model, has proven to be a promising model for identifying substances with insulin-mimetic properties. It represents an attractive tool filling the wide gap between in-vitro and in-vivo studies and offers several advantages in comparison to existing systems such as acceptable costs, simple handling procedures, and adequate throughput rates. In addition, permission by an ethics committee is not required.

<table><tbody><tr><td>Phosphate buffered saline (PBS)</td><td>Biochrom</td><td>L1825</td><td></td></tr><tr><td>Hank&amp;rsquo;s balanced salt solution (HBSS)</td><td>Thermo Scientific</td><td>SH30268.02</td><td></td></tr><tr><td>NovoRapid</td><td>Novo Nordisk</td><td>8-0905-82-304-6</td><td></td></tr><tr><td>Hens eggs (Lohmann classic brown chicken)</td><td>Local Breeder</td><td></td><td></td></tr><tr><td>Accu-check performa</td><td>Roche</td><td>6454011</td><td></td></tr><tr><td>Accu-check Inform II Teststreifen</td><td>Roche&amp;nbsp;</td><td>5942861</td><td></td></tr><tr><td>Incubator&amp;nbsp; HEKA- Turbo 288</td><td>HEKA Brutger&amp;auml;te</td><td></td><td></td></tr><tr><td>Syringe Omnican F 1 mL</td><td>Braun</td><td>09161502S&amp;nbsp;</td><td></td></tr><tr><td>Extracts</td><td>PEKISH extract library</td><td></td><td></td></tr><tr><td>Lamp Tempo Nr. 119</td><td>ORBAN</td><td></td><td></td></tr></tbody></table>

an in ovo model for testing insulin-mimetic compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

Description of the Gluc-HET approach:
After incubation with the substances of interest, the fertilized eggs are opened and prepared for blood collection by removal of the eggshell (Figure 2). Further preparation includes equilibration and removal of the eggshell membrane to gain access to the chorioallantoic membrane. This membrane is then cut carefully to provide access to a suitable blood vessel for blood collection. Preferably, the large vessel originating from the abdomen of the embryo is placed on a plastic pH-strip. To avoid dilution of the blood during collection, the vessel and pH-strip is patted dry with filter paper. The blood vessel is then cut and blood leaking onto the pH-strip is collected for analysis using a pipette.
Effects of selected herbal extracts on blood glucose levels:
With a recently established GLUT4-translocation quantitation-based primary screen, we are able to identify substances with an insulin-mimetic characteristic10. Screening of hundreds of water-soluble herbal extracts resulted in the identification of several hits. These extracts were tested in our in-ovo model. Extracts prepared from Combretum indicum (Rangoon creeper) and a non-disclosed extract (termed extract 0845) were used for these experiments. We dissolved the extracts in HBSS buffer at 300 mg/L, a concentration that turned out to be appropriate in previous studies12,13. Substances were applied to the embryos incubated for 11 days. As indicated in Figure 3, the extract from Rangoon creeper did not result in a significant blood glucose reduction in-ovo. However, the blood glucose concentration was successfully reduced with extract 0845, with a minor decrease after 60 min, which is comparable to the effect observed using a commercially available insulin analog. A significant effect was observed when the incubation time of the eggs was prolonged.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57237/57237fig1.jpg" /> 
Figure 1: Influence of toxic compounds on embryo vitality. Eggs were incubated for 11 days (A and C) or treated with a toxic herbal extract on day 10 for another 24 h (B and D). Application of the extract led to severe lesions of blood vessels in the chorioallantoic membrane (D) and the embryo (B).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57237/57237fig2.jpg" /> 
Figure 2: Description of the in-ovo model. From left to right: Opening and removal of the eggshell (1-2); equilibration and removal of the eggshell membrane with chorioallantoic membrane exposed (3); prepared vessel on a pH-strip (4); the collection of blood and measurement of glucose concentration using a glucose meter (5). Adapted from Haselgruebler et al., 201712. <a href="https://cloudflare.jove.com/files/ftp_upload/57237/57237fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57237/57237fig3.jpg" /> 
Figure 3: Effect of herbal extracts on blood glucose levels compared to untreated or insulin-treated eggs. Eggs were incubated for 11 days and treated with the indicated substances (commercially available insulin analog: 3.3 U/mL; extracts: 300 mg/L) dissolved in HBSS buffer (300 &#181;L volume) for up to 3 h. Blood glucose levels were determined using a blood glucose meter. Results are shown without the effect of the buffer. Error bars are based on the standard error of the mean. *P &#60;0.05 and ****P &#60;0.0001, significant decrease with respect to HBSS-treated eggs of the same incubation time. Adapted from Haselgruebler et al., 201712. <a href="https://cloudflare.jove.com/files/ftp_upload/57237/57237fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

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9.7K Views.  University of Applied Sciences Upper Austria. In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

Video: An In Ovo Model for Testing Insulin-mimetic Compounds

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

Bioengineering

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.

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.

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

Biology

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Medicine

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

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.

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

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

Acclimation Prior to an Intraperitoneal Insulin Tolerance Test to Mitigate Stress-Induced Hyperglycemia in Conscious Mice

The insulin tolerance test is commonly used in metabolic studies to assess whole body insulin sensitivity in rodents. It is a relatively simple test that involves measurement of blood glucose levels over time following a single intraperitoneal injection of insulin. Given that it is performed in the conscious state and blood is often collected via a tail snip, it has the potential to elicit a stress response from animals due to anxiety associated with handling and blood collection. As such, a stress-induced rise in blood glucose can occur, making it difficult to detect and interpret the primary endpoint measure, namely an insulin-mediated reduction in blood glucose. This has been seen in many mouse strains, and is quite common in diabetic db/db mice, where glucose levels can increase, rather than decrease, after insulin administration. Here, we describe a method of acclimating mice to handling, injections and blood sampling prior to performing the insulin tolerance test. We find that this lowers stress-induced hyperglycemia and results in data that more accurately reflects whole body insulin sensitivity.

Stress-induced elevations in glucose levels can confound interpretation of data derived from a conscious intraperitoneal insulin tolerance test in mice. In this article, we describe a method to acclimate the mice to handling, injections and blood sampling prior to performing the insulin tolerance test in order to limit stress-induced hyperglycemia.

The insulin tolerance test is commonly used in metabolic studies to assess whole body insulin sensitivity in rodents. It is a relatively simple test that ...

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.

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

Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, depending on the specific assay. Secreted luciferase assays expedite results and lower the assay cost per sample substantially. Here we present a relatively underused approach to gauge insulin secretory activity from pancreatic &#946; cells by using Gaussia luciferase genetically inserted within the C-peptide. During proteolytic processing of proinsulin, the C-peptide is excised releasing the luciferase within the insulin secretory vesicle where it is co-secreted with insulin. Results can be obtained within minutes after sample collection because of the speed of luciferase assays. A limitation of the assay is that it is a relative measurement of insulin secretion and not an absolute quantitation. However, this protocol is economical, scalable, and can be performed using most standard luminescence plate readers. Analog and digital multichannel pipettes facilitate multiple steps of the assay. Many different experimental variations can be tested simultaneously. Once a focused set of conditions are decided upon, insulin concentrations should be measured directly using antibody-based assays with standard curves to confirm the luciferase assay results.

This protocol describes how to perform rapid low-cost luciferase assays at medium-throughput using an insulin-linked Gaussia luciferase as a proxy for insulin secretion from beta cells. The assay can be performed with most luminescence plate readers and multichannel pipettes.

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, ...

An In Ovo Model for Testing Insulin-mimetic Compounds

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Sustained Administration of β-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

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

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

A High-content In Vitro Pancreatic Islet β-cell Replication Discovery Platform

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

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

Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Acclimation Prior to an Intraperitoneal Insulin Tolerance Test to Mitigate Stress-Induced Hyperglycemia in Conscious Mice

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

Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate