This work was supported by a pilot project grant from CREH center sponsored by NIEHS and by National Natural Science Foundation of China (No. 31572626).

All animal experiments were executed in compliance with all relevant guidelines, regulations and regulatory agencies. The protocol being demonstrated was performed under the guidance and approval of the Institutional Animal Care and Use Committee (IACUC) of the Texas A&#38;M Institute for Genomic Medicine.
1. Solution Preparation
<ol>
	<li>Dilute 10x Hank&#39;s balanced salt solution to 1x with double distilled H2O, and store at 4 &#176;C.</li>
	<li>Prepare the isolation solution by adding HEPES (10 mM), MgCl2 (1 mM), and glucose (5 mM). Adjust the pH to 7.4 and keep on ice.</li>
	<li>Prepare the collagenase solution from collagenase Type 4. 
	Note: Specifically, collagenase Type 4 contains 160 U per mg of dry weight. 1 g of collagenase Type 4 is diluted in 5 mL double distilled H2O with a concentration of 200 mg/mL. 2 mL of the diluted Collagenase is added to 30 mL ice cold isolation solution, and 6 mL of collagenase solution is transferred to a 50 mL tube for each mouse.</li>
	<li>Prepare the wash solution by adding 1 mM CaCl2 to the isolation solution.</li>
	<li>Prepare the purification solution with cold polysucrose/sodium diatrizoate solution (1.1119 g/mL, 5 mL).</li>
	<li>Prepare 500 mL of islet culture medium by adding L-glutamine (20 mM), penicillin (100 U/mL), streptomycin (100 &#181;g/mL), and fetal bovine serum (FBS) (10%) into RPMI medium.</li>
	<li>Prepare the DCFH-DA stock solution by dissolving the powder in distilled water at 200 &#181;M and store at -20 &#176;C. 
	Note: The ROS staining solution is freshly made by diluting DCFH-DA stock solution to the final concentration of 5 &#181;M in RPMI. This reagent should be protected from light.</li>
</ol>
2. Surgical Preparation
<ol>
	<li>Intraperitoneally inject (I.P.) a mouse with avertin (2.5% avertin, 0.4 mL/20 g). After the mouse is completely anesthetized and no longer responds to hind foot pinches, euthanize the mouse and move to the biological hood.</li>
	<li>Place the euthanized mouse with the abdominal side facing up and spray the skin with 70% ethanol for sterilization.</li>
	<li>Open the abdominal cavity with a 1 cm U-incision upon the upper abdomen and retract the skin in the rostral direction over the chest to expose the abdominal cavity.</li>
</ol>
3. Pancreas Perfusion and Removal
<ol>
	<li>As illustrated in Figure 1, find the location of the ampulla. Use a pair of curved forceps to clamp the duodenum close to the position of sphincter of Oddi. Use another pair of curved forceps to clamp the duodenum wall to block the bile pathway in the duodenum. 
	Note: The ampulla has not been inserted in this step. This step is meant to block the bile pathway in the duodenum.</li>
	<li>Reposition the mouse with the head proximal and the feet distal with regards to the researcher.</li>
	<li>Find the common bile duct and slowly inject 3 mL of collagenase solution with a 30 G needle and 5 mL syringe. Make sure the common bile duct and the pancreas are inflated after the injection.</li>
	<li>Remove the distended pancreas using the curved forceps and fine scissors to separate it from the descending colon, intestines, stomach, and spleen. Place the pancreas in a 50-mL tube containing 3 mL of collagenase solution and keep it on ice.</li>
</ol>
4. Pancreas Digestion
<ol>
	<li>Place each 50-mL tube into a 37 &#176;C water-bath. Incubate for 15 - 20 min (time varies with the strain and age of the mice).</li>
	<li>Vibrate the tube using a vortex.</li>
	<li>Pool 4 - 5 digested pancreas in a large sterile sink strainer and use a scoop to make the pancreas fall apart thoroughly. Use a 23 G needle and 10 mL syringe containing wash solution to forcefully pipette the digested cells off the screen. Collect the wash solution in a 50-mL tube.</li>
	<li>Centrifuge the tubes at 97 x g at 4 &#176;C for 1 min and decant the supernatant.</li>
	<li>Add 25 mL of wash solution to tubes and re-suspend the tissue by gentle vortexing. Spin down the tissues at 97 x g at 4 &#176;C for 1 min and decant the supernatant. Repeat for 3 times. Be careful not to dislodge the pelleted tissue.</li>
</ol>
5. Purification of Islets
<ol>
	<li>Add 10 mL of purification solution to the washed tissue pellet and gently resuspend the contents.</li>
	<li>Gently overlay 5 mL of isolation solution on the purification solution. Be careful to keep a sharp liquid interface between the two solutions.</li>
	<li>Centrifuge the tube at 560 x g at 4 &#176;C for 15 min with slow acceleration and no brake. Insert the pipette into the interface and collect the islets layer into new 50 mL tubes.</li>
	<li>Wash the islets as described in step 3.5.</li>
</ol>
6. Plating and Treatment of Islet Cells
<ol>
	<li>Resuspend the islets in 10 mL of islets culture solution, gently mix well, and place 100 &#181;L/well with a multichannel pipette to a 96-well plate. Place approximately 5 islets per 96 well.</li>
	<li>Place the plate in the tissue culture incubator for 12 h.</li>
	<li>Centrifuge (112 x g) the 96-well plate and remove the culture medium.</li>
	<li>Dilute the environmental chemicals in the islets culture medium. Add 100 &#181;L of each of the chemical diluents into separate wells of the plate. Resuspend the islets in each well and incubate for 12 h at 37 &#176;C (the concentration varies for the different environmental chemicals).</li>
</ol>
7. ROS Staining and Measurement
<ol>
	<li>&#160;After islets treatment, wash with 1x PBS (200 &#181;L for each 96-well). 
	Note: The concentration of the chemicals is as follow: AFB1: 3 &#181;M, Atrazine: 100 &#181;M, BaP: 10 &#181;M, BPA: 100 &#181;M, &#946;-estrogen, PCB126: 10 &#181;M, Nonylphenol, and BPA: 100 &#181;M.</li>
	<li>Use N-Acetyl Cysteine (NAC) as an antioxidant to quench ROS. Add 5 mM of NAC to the ROS-induced chemical diluents and incubate for 12 h at 37 &#176;C.</li>
	<li>Centrifuge the 96-well plate at 112 x g and incubate with 5 &#181;M solution of fluorescent probe (H2-DCFH-DA dissolved in RPMI media) at 37 &#176;C for 60 min.</li>
	<li>Wash the treated islet cells 3 times with PBS and measure the fluorescence with a microplate reader at 488 nm. 
	Note: The accumulation of DCF in cells may be measured by an increase in fluorescence at 530 nm when the sample is excited at 488 nm.</li>
	<li>Perform the glucose-stimulated insulin secretion (GSIS) assay9 on ROS-inducing xenobiotic treated islets and untreated islets to measure the xenobiotic effects on the physiological function of the pancreatic islets.</li>
</ol>

<ol>
	<li>Maruthur, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+prevalence+of+type+2+diabetes:+increased+incidence+or+improved+survival?.">The growing prevalence of type 2 diabetes: increased incidence or improved survival?.</a> Current diabetes reports. 13 (6), 786-794 (2013).</li><li>Houstis, N., Rosen, E. D., Lander, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+have+a+causal+role+in+multiple+forms+of+insulin+resistance.">Reactive oxygen species have a causal role in multiple forms of insulin resistance.</a> Nature. 440 (7086), 944-948 (2006).</li><li>Ma, Z. A., Zhao, Z., Turk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+and+beta-cell+failure+in+type+2+diabetes+mellitus.">Mitochondrial dysfunction and beta-cell failure in type 2 diabetes mellitus.</a> Exp Diabetes Res. , 703538 (2012).</li><li>Valavanidis, A., Vlahogianni, T., Dassenakis, M., Scoullos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biomarkers+of+oxidative+stress+in+aquatic+organisms+in+relation+to+toxic+environmental+pollutants.">Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants.</a> Ecotoxicology and environmental safety. 64 (2), 178-189 (2006).</li><li>Robertson, R. P., Harmon, J., Tran, P. O., Tanaka, Y., Takahashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+toxicity+in+&#946;-cells:+type+2+diabetes,+good+radicals+gone+bad,+and+the+glutathione+connection.">Glucose toxicity in &#946;-cells: type 2 diabetes, good radicals gone bad, and the glutathione connection.</a> Diabetes. 52 (3), 581-587 (2003).</li><li>Kaneto, H., 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=Oxidative+stress+induces+p21+expression+in+pancreatic+islet+cells:+possible+implication+in+beta-cell+dysfunction.">Oxidative stress induces p21 expression in pancreatic islet cells: possible implication in beta-cell dysfunction.</a> Diabetologia. 42 (9), 1093-1097 (1999).</li><li>Maechler, P., Jornot, L., Wollheim, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+alters+mitochondrial+activation+and+insulin+secretion+in+pancreatic+beta+cells.">Hydrogen peroxide alters mitochondrial activation and insulin secretion in pancreatic beta cells.</a> Journal of Biological Chemistry. 274 (39), 27905-27913 (1999).</li><li>Gao, 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=The+effects+of+palmitate+on+hepatic+insulin+resistance+are+mediated+by+NADPH+Oxidase+3-derived+reactive+oxygen+species+through+JNK+and+p38MAPK+pathways.">The effects of palmitate on hepatic insulin resistance are mediated by NADPH Oxidase 3-derived reactive oxygen species through JNK and p38MAPK pathways.</a> Journal of Biological Chemistry. 285 (39), 29965-29973 (2010).</li><li>Efendi&#263;, S., 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=Pancreastatin+and+islet+hormone+release.">Pancreastatin and islet hormone release.</a> Proceedings of the National Academy of Sciences. 84 (20), 7257-7260 (1987).</li><li>Tian, Y., Ke, S., Denison, M. S., Rabson, A. B., Gallo, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ah+receptor+and+NF-&#954;B+interactions,+a+potential+mechanism+for+dioxin+toxicity.">Ah receptor and NF-&#954;B interactions, a potential mechanism for dioxin toxicity.</a> Journal of Biological Chemistry. 274 (1), 510-515 (1999).</li><li>Cui, H., 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=Pregnane+X+receptor+regulates+the+AhR/Cyp1A1+pathway+and+protects+liver+cells+from+benzo-[&#945;]-pyrene-induced+DNA+damage.">Pregnane X receptor regulates the AhR/Cyp1A1 pathway and protects liver cells from benzo-[&#945;]-pyrene-induced DNA damage.</a> Toxicology Letters. 275, 67-76 (2017).</li><li>Li, L. A., Wang, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCB126+induces+differential+changes+in+androgen,+cortisol,+and+aldosterone+biosynthesis+in+human+adrenocortical+H295R+cells.">PCB126 induces differential changes in androgen, cortisol, and aldosterone biosynthesis in human adrenocortical H295R cells.</a> Toxicological Sciences. 85 (1), 530-540 (2005).</li><li>Asahi, 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=Bisphenol+A+induces+endoplasmic+reticulum+stress-associated+apoptosis+in+mouse+non-parenchymal+hepatocytes.">Bisphenol A induces endoplasmic reticulum stress-associated apoptosis in mouse non-parenchymal hepatocytes.</a> Life sciences. 87 (13), 431-438 (2010).</li><li>Szot, G. L., Koudria, P., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+pancreatic+islet+isolation.">Murine pancreatic islet isolation.</a> JoVE (Journal of Visualized Experiments). (7), e255 (2007).</li><li>Kirstetter, P., Lagneau, F., Lucas, O., Krupa, Y., Marty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+endothelium+in+the+modulation+of+isoflurane-induced+vasodilatation+in+rat+thoracic+aorta.">Role of endothelium in the modulation of isoflurane-induced vasodilatation in rat thoracic aorta.</a> British journal of anaesthesia. 79 (1), 84-87 (1997).</li><li>Brown, E., Umino, Y., Loi, T., Solessio, E., Barlow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+can+cause+sustained+hyperglycemia+in+C57/BL6J+mice.">Anesthesia can cause sustained hyperglycemia in C57/BL6J mice.</a> Visual neuroscience. 22 (5), 615-618 (2005).</li><li>Vaupel, D., McCoun, D., Cone, 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=Phencyclidine+analogs+and+precursors:+rotarod+and+lethal+dose+studies+in+the+mouse.">Phencyclidine analogs and precursors: rotarod and lethal dose studies in the mouse.</a> Journal of Pharmacology and Experimental Therapeutics. 230 (1), 20-27 (1984).</li><li>Neuman, J. C., Truchan, N. A., Joseph, J. W., Kimple, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+mouse+pancreatic+islet+isolation+and+intracellular+cAMP+determination.">A method for mouse pancreatic islet isolation and intracellular cAMP determination.</a> Journal of visualized experiments: JoVE. (88), (2014).</li></ol>

The authors declare that they have no competing financial interests.

Accumulating evidence suggests that exposure to environmental chemicals plays an important role in the development of T2DM. Xenobiotics-induced ROS has been recognized as a potential etiological factor contributing to the development of T2DM. Humans are exposed to a wide range of xenobiotic chemicals and there is a great need for novel research techniques to effectively identify the pancreatic toxicants and to investigate the mechanism of toxicity specific to the pancreatic cells.
In this study, based on published procedures14, we have developed a protocol to isolate pancreatic islets from mouse and screen the cells with xenobiotic chemicals for oxidative stress-induced damage to the pancreatic cells. We also use this procedure to investigate the pancreatic specific toxic responses induced by the xenobiotic compounds. To obtain the pancreas from mouse, we prefer to use avertin over other anesthesia because it does not alter the blood glucose levels and does not affect the vasculature of the pancreas15,16,17. We found that the density gradient centrifugation step is critical for isolation of the islet with high purity. Care should be taken to obtain the distinct layer between the polysucrose/sodium and isolation buffer phase to obtain a pure islets sample, devoid of debris and exocrine cells/tissues. The method can be used for in vitro analysis of environmental chemical effects on pancreatic islet physiology, such as the GSIS assay (as shown in Figure 3). The islets should be carefully dispersed with repeated pipetting into separate cells to ensure uniformed cell number in the 96-cell culture plate. The method described here combined the isolation of the islet and a rapid assay for ROS generation and therefore, is a simple and effective initial screen for potential pancreas-damaging chemicals.
We used insulin secretion in response to glucose challenge to confirm the identity of the isolated pancreatic islets. The procedure is highly adaptable for analysis of changes in parameters, including insulin secretion (Figure 3) and cAMP generation, in response to xenobiotic treatments18.

Increases in the prevalence of T2DM have become a global health crisis in recent years posing a serious threat to public health1. Many factors have been found to be causally linked to the development of T2DM, among which, recurring findings suggest that one common convergent point for these factors is the induction of oxidative stress which leads to the generation of excessive ROS2,3. 
A wide spectrum of environmental chemicals including PCBs, dioxins, and BaP has been found to induce oxidative stress, which may impair the function of pancreatic &#946; cells and lead to insulin resistance and T2DM4. Although the physiological level of ROS plays an important role in cellular functions, exposure to ROS that exceeds the capacity of the antioxidant system results in the damage to cells/tissues and leads to diseases5. Pancreatic &#946; cells express a low level of antioxidant enzyme, and thus are a sensitive target for the oxidative stress-mediated damage6,7. Chronic exposure to high levels of ROS has been shown to cause stress-induced pancreatic cell dysfunction5 as well as insulin resistance in the liver and adipose tissue8. 
The overall goal of this project is to develop a cell-based assay to screen chemicals for their diabetogenic potentials based on their induction of ROS in pancreatic cells. The pancreas lacks metabolic detoxification and is a sensitive target for xenobiotic-induced damage6,7. Therefore, by directly measuring the ROS generated in the pancreatic cells, this assay should provide a direct approximation of the chemical-induced injury in the pancreas. To develop this method, we isolated mouse pancreatic islets, cultured the isolated islet under cell culture condition with chemicals, and utilized the chemical-induced ROS generation as the readout. This procedure is simple and effective in identifying ROS-inducing chemicals in the isolated islet; it can be further developed for investigation of the mechanisms of toxicity that are specific to the pancreas in vitro.

<table><tbody><tr><td>10&amp;times;Hank&amp;rsquo;s balanced salt solution&amp;nbsp;</td><td>GIBCO</td><td>14185-052</td><td></td></tr><tr><td>Collagenase Type 4</td><td>Worthington Biochemical Corporation</td><td>CLS-4</td><td></td></tr><tr><td>polysucrose/sodium diatrizoate solution&amp;nbsp;</td><td>Sigma</td><td>10771</td><td></td></tr><tr><td>2&amp;rsquo;,7&amp;rsquo;-dichlorofluorescein (DCFH-DA)</td><td>Sigma</td><td>D6883-50MG</td><td></td></tr><tr><td>fluorescence microplate reader&amp;nbsp;</td><td>Biotek</td><td></td><td></td></tr><tr><td>L-glutamine</td><td>Sigma</td><td>G8540-25G</td><td></td></tr><tr><td>streptomycin</td><td>GIBCO</td><td>15140148</td><td></td></tr><tr><td>FBS</td><td>GIBCO</td><td>26140079</td><td></td></tr><tr><td>RPMI 1640</td><td>GIBCO</td><td>11875-085</td><td></td></tr><tr><td>avertin</td><td>Sigma</td><td>T48402-25G</td><td></td></tr><tr><td>Rat/Mouse Insulin ELISA Kit</td><td>Millipore</td><td>EZRMI-13K</td><td></td></tr><tr><td>Centrifuge</td><td>Sorval</td><td>Sorval RT7</td><td>for 96-well plate centrifuge</td></tr><tr><td>Microplate reader</td><td>Biotek</td><td>Epoch 2</td><td>for fluorescence reading</td></tr></tbody></table>

a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the &#946; cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

A micrograph of the healthy Isolated islet is shown in Figure 2, in which islets have a round or oval&#160;shape with relatively uniform size (although size uniformity can vary from strain to strain). We next investigated the pancreatic islet functions in an in vitro assay by isolating the islet and stimulating the insulin secretion in the culture islets. Figure 3 shows our typical analysis of the GSIS assay from C57BL/6 mouse isolated islets induced by 3.3 mM and 16.7 mM glucose9.
Using the plate format screen method, we have identified several xenobiotics that caused significant induction of ROS in islet cells (Figure 4). We treated the isolated islets with different chemicals for 12 h and found that Atrazine, BPA, BaP, and PCB126, and &#946;-estrogen could cause significant induction of ROS in the isolated islets10,11,12,13.
Although ROS is not equal to the diabetogenic induction, there is ample evidence indicating the important role of ROS in causing damage to the physiological functions of pancreatic &#946; cells, which leads to T2DM8. Therefore, the value of the work is to identify these ROS inducing chemical as the initial screen. Furthermore, the compounds identified can be further analyzed in vitro in the isolated pancreatic islet cell culture system described in the manuscript. We have shown the effects of ROS-inducing xenobiotics on glucose-stimulating insulin secretion of the isolated islets, which briefly provide diabetogenic data for these tested compounds. Pancreatic islets pre-treated with PCB126 and BPA showed a significant decrease in insulin secretion than the control group (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57327/57327fig1v3.jpg" /> 
Figure 1: Procedure of mouse pancreas perfusion. The procedure consists of the following steps: (1) Find the ampulla and use two pairs of curved forceps to clamp the ampulla. (2) Insert the needle through the common bile. (3) Slowly inject 3 mL of collagenase solution stock in the 5 mL syringe. (4) Collect the distended pancreas starting from the duodenum. 
&#160;
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57327/57327fig2v3.jpg" /> 
Figure 2: Isolated mouse islets. Isolated mouse pancreatic islets in culture medium are shown. Scale bar of 500 &#181;m.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57327/57327fig3v3.jpg" /> 
Figure 3: Glucose-stimulated insulin secretion of isolated pancreatic islets. Dose-dependent glucose-stimulated insulin secretion (1, 3.3, and 16.7 mM, treated for 15 min) was measured in isolated islets (average 5 islets per well), using a mouse insulin ELISA kit. Error bars show standard deviations. *p &#60;0.05.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57327/57327fig4v3.jpg" /> 
Figure 4: Induction of ROS by xenobiotics in isolated islets from C57BL/6 mice. (A) Isolated islets were treated for 12 h and stained with fluorescence dye 2&#39;,7&#39;-dichlorofluorescein (DCFH-DA) for 1 h. The fluorescence was determined by a fluorescence plate reader. (B) The chemical-induced ROS were quenched by NAC (5 mM). (AFB1: 3 &#181;M, Atrazine: 100 &#181;M, BaP: 10 &#181;M, BPA: 100 &#181;M,&#160;PCB126: 10 &#181;M, Nonylphenol, BPA: 100 &#181;M, and NAC: 5 mM). Error bars show standard deviations. *p &#60;0.05. <a href="/files/ftp_upload/57327/57327fig4v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57327/57327fig5v3.jpg" /> 
Figure 5: Xenobiotic effects on glucose-stimulated insulin secretion of isolated islets. Isolated islets were pre-treated with PCB126, &#946;-estrogen, and BPA (4 h) and kept in islet culture medium for 1 h. The supernatant of the islets was collected for measurement of insulin by a commercially available mouse insulin ELISA assay. Error bars show standard deviations. *p &#60;0.05.

Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

a-murine-pancreatic-islet-cell-based-screening-for-diabetogenic

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.2K Views.  Hunan Agriculture University. Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

Video: A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Murine Pancreatic Islet Isolation

Hi, I am Greg Zott. I'm with the UCSF Diabetes Center in the Bluestone Laboratory, and today I'm gonna show you the process for isolating mouse eyelets. The procedure involves the injection of a collagenase solution into the pancreas of a C3 H donor mouse, where we will then remove that pancreas, digest it away, purify the eyelets on a fial gradient, and then handpick them for ultimate transplant into the kidney capsule of a diabetic NOD mouse, Which will be our recipient.Before we start this procedure, we diluted our ATE solution in a modified Hanks buffer and we have adjusted the concentration specific for the strain of mouse that we're using the C3 H.We found digests better at a 0.8 mgs per mil collagenase solution, and we will use that to inject into the common bile duct to distend the pancreas.Performing. The procedure today Will be Pavo codre of the diabetes center. He will inject, open up the mouse, expose the the intestines, and then inject into the common bile duct.We will spray down the mouse with ethanol to keep the hair down, and then we will also verify that the mouse has been sacrificed properly with a toe pinch. Pavo will now open up the mouse and exposing the intestine and the pancreas with a midline incision. The diaphragm will also be cut to verify that the mouse will be sacrificed.Before we get started, I'd like to point out some of the important structures. The stomach is lying right here with the pancreas attached underneath into the duodenum and above. This is the liver where we'll find the gallbladder and the common bile duct, which you can see right here.So what Pavo will do next is he will isolate the small intestine, find where the common bile duct enters the small intestine. Once the intestine is exposed, we clamp on either side of the common bile duct with mosquito clamps. We find that this allows more collagenates to enter the pancreas while other groups try to direct the clamp on top of the common bile duct.We find that this is a better technique in allowing enzyme to enter the pancreas. This is a gentle procedure. You don't want to tear the bowel.You wanna allow the clamps to lie gently on either side of the common bile duct. Now we will find a gallbladder, and once the gallbladder's exposed, we will use that as a guide to injecting the collagenase into the common bile duct. Powell's using a 30 gauge needle with a five cc syringe containing three mills of collagenase solution.As he's entering the common bile duct, he's distending that duct with collagenase solution and then he travels down the common bile duct till he reaches near the intestine. And as he's going down, the enzyme solutions being pumped into the injected into the pancreas. Now that the pancreas is inflated with collagenase solution, Powell's gonna remove the hemostats and then remove the pancreas from the the mouse carefully tearing it away from the intestine.He removes it from the stomach. Okay, and then using the spleen as a handle, he lifts up the pancreas and then slowly snips it away in the common bile duct, and that's it. Pancreas is laid out and then the spleen is removed, and there he's removing the meso enteric tissue, and then the pancreas is placed in a two mill collagenase solution and a sterilized glass vial and it's placed back on Ice.The isolated pancreas that we put into the glass vials will now be placed into a 37 degree water bath for approximately 17 minutes. We placed them into a 50 mil conical rack so that they don't float up. Once during the bath, the timer starts and the digestion begins.Once the digestion time is completed, vials are removed, and then they are shaken and disrupted gently. And once the tissue starts falling apart, the vial is then placed on ice. A sterile screen is placed over a 400 mil beaker that contains washing buffer.The pancreas digest is then carefully poured over the screen into the wash buffer. For demonstration purposes, the hood has been turned off and the glass of the hood has been raised so we could see what we're doing. Once the pancreas digest is poured into the beaker, the vials are rinsed with the same washing buffer and the wash buffer placed over the screen.We have a syringe filled with washing buffer. We will use that to forcefully wash the tissue through the screen. The idea is to release any loose eyelets that are unattached to the matrix, but leaving the membranes and the undigested tissue on the screen.And when you're finished washing a tissue, this is what you usually See at the end of the procedure. The Pancreas tissue that's in the bottom of the tube will now be transferred to two 50 mil conical, and the tissue will be washed, the tissue's mixed evenly, and then distributed evenly so that we have a equal pellet volume in each tube. The beaker is now rinsed with more washing buffer.The sides are rinse so that you get all tissue that is entered the beaker again. Then it's equally distributed between each two, both 50 mil. Conical then are topped off with wash buffer.The tissue is inverted several times and then placed into a centrifuge. This is a quick spin. The centrifuge is allowed to reach a thousand RPM and then is turned off.The tissue at this time is very S eyelets are very sensitive, and you don't want to over compact them. The tubes are removed and we will do this wash step three more times. This is the end of the third wash.The components of the 50 mil conical tube are transferred to the 15 conical tube and centrifuge for a thousand RP M and then stopped. The supernat will be removed and the first part of the FI Call will be started. We are at the step where we Have our pelleted di pancreas digest.We'll resuspend that tissue so it's nice and loose to the pellets. We'll add the first density gradient, which is 1 1 0 8. We'll add five mils to each tube.The pellet is then mixed thoroughly in each of the gradients. It's important that you get good tissue distribution in your heaviest gradient. Now we will layer two mils of 1 0 9 6 density.We will then on top of that, layer two mils of 1 0 6 9 density, and then on top of that 1 0 3 7 density. It's important while you're layering the densities that you don't mix. Then all the layers have been added to the tube.And you can see this is the top of 1 1 0 8, the top of 9 1 0 9 6, the top of 1 0 6 9 and the top of 1 0 3 7. And you could see the tissue is already starting to move to its proper density even without centri fusion. The fial gradients are brought up to speed at 1800 RPM for 15 minutes, and remember to keep the brake off after the tubes are removed from the centrifuge.You can see between the 0 0 9 6 and 0 0 7 layer, there's a nice band of eyelet tissue Pablo's. Now removing the 0 3 7 layer to remove any tissue debris and DNA that might have been released during the processing. Using a sterile plastic pasture pipette, he will now remove the islet layer and transfer them to a 50 mil conical containing 25 mils of wash buffer.You want do this step quickly because fol is toxic to eyelets. We actually removed the entire 0 6 9 and 0 9 6 layer. Pavo cuts the top of the plastic pasture pipette and rinses it with wash buffer.The eyelets are then centrifuge like we centrifuge previously. Centrifuge is brought up to a Thousand RPM and then we stop it and We aspirate off. The supernatant eyelets will be washed three times with wash media.So we just completed the eyelid Isolation process we took from the digest to the fol gradient to the purified eyelets after our third wash with wash buffer, we resuspended the eyelets in our RPMI media and we played it in a tissue culture plate. These eyelets are now ready for in vitro assays or in vivo preps like eyelet Transplant.

Biology

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Developmental Biology

Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate the measurement of islet oxygen consumption using a 96-well microplate designed for the measurement of oxygen consumption in spheroids. In this assay, spheroid microplates are coated with a cell and tissue adhesive on the day prior to the assay. We utilize a small volume of adhesive solution to encourage islet adherence to only the bottom of the well. On the day of the assay, 15 islets are loaded directly into the base of each well using a technique that ensures optimal positioning of islets and accurate measurement of oxygen consumption. Various aspects of mitochondrial respiration are probed pharmacologically in non-human primate islets, including ATP-dependent respiration, maximal respiration, and proton leak. This method allows for consistent, reproducible results using only a small number of islets per well. It can theoretically be applied to any cultured spheroids of similar size.

This protocol demonstrates the accurate and reproducible measurement of oxygen consumption in non-human primate pancreatic islets. The islet loading techniques and coating of the microplate provide a framework for efficient measurement of respiration in other types of cultured spheroids.

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Medicine

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 ...

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.

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.

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

Neuroscience

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Murine Pancreatic Islet Isolation

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption

Lepr^db Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

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

An In Ovo Model for Testing Insulin-mimetic Compounds

Intraportal Transplantation of Pancreatic Islets in Mouse Model

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells