Glucose metabolism and lipolysis in rat islets in the absence or presence of GLP-1.

<p>Islets were processed as described for insulin secretion (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006221#pone.0006221-Prentki1" target="_blank">[Fig. 1]</a>) and after the pre-incubation step they were incubated in 70 µl KRBH/0.25% defatted BSA medium containing 2.8, 8.3 or 16.7 mM glucose plus or minus 20 nM GLP-1 in presence of D-[U-¹⁴C]-glucose (for oxidation) (A) and D-[5-³H]-glucose (for utilization) (B). Incubations were stopped after 90 min as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006221#s2" target="_blank">Methods</a>. Glucose oxidation was measured as ¹⁴CO₂ released, and glucose utilization was determined by measuring the amount of released³H₂O. Results are means±SE of 15 determinations in 3 separate experiments. *p<0.05, **p<0.01, ***p<0.001 when compared to the corresponding 2.8 mM glucose group; #p<0.05 when compared to the corresponding ‘minus GLP-1’ group. For lipolysis determinations (C) overnight-cultured rat islets were washed in KRBH/0.07% BSA medium with 2.8 mM glucose and were transferred into 0.2 mL KRBH/0.07% BSA medium with 2.8, 8.3 or 16.7 mM glucose with or without 20 nM GLP-1. After incubation for 3 h at 37°C, glycerol released into the media and the islet protein content were determined. Means±SE from 4 independent experiments with pentaplicates.</p

Oxygen consumption of rodent islets in the absence or presence of Ex-4.

<p>Single rat (A) and mouse (B) islets were adhered on glass coverslips inside a 35 mm dish using CellTak adhesive. After 30 min equilibration at 4 mM glucose, oxygen consumption was measured in response to 4, 7.5 and 16.6 mM glucose with and without 10 nM Ex-4 (10 nM). Data are means±SE of 3 experiments.</p

Exendin-4 increases [Ca²⁺]_i and cAMP content in mouse β-cells.

<p>A, A single fura-2 loaded and Ad-MtLuc-RFP-infected mouse β-cell was imaged to determine the [Ca²⁺]_I, at 5.6 mM glucose in KRBH. After establishment of a stable baseline [Ca²⁺]_i, 10 nM Ex-4 was applied for 25 sec (indicated by horizontal bars). Note that a repeatable increase of [Ca²⁺]_i was measured. B, Population study conducted at the single cell level in which the action of Ex-4 to increase [Ca²⁺]_i was evaluated in β-cells not infected (open bars) or infected with Ad-MtLuc-RFP (filled bars). For these experiments, the KRB contained 5.6 or 7.5 mM glucose, as indicated. A response to Ex-4 was defined as a >100 nM increase of [Ca²⁺]_i occurring in a single β-cell. C, Ex-4 caused a dose-dependent increase in cAMP content in mouse islet cells in KRB containing 7.5 mM glucose without or with Ad-MtLuc-RFP infection.</p

Mitochondrial ATP levels of rodent islet cells in the absence or presence of Ex-4.

<p>Dispersed rat (A,B,C) and mouse (D,E,F) islet cells were transduced with Ad-MtLuc-RFP. ATP levels in the presence of 4, 7.5 and 16.6 mM glucose with and without 10 nM Ex-4 were determined as photoemission resulting from luciferase-catalyzed oxidation of luciferin, in populations of approximately 250,000 single islet cells at 5, 15 and 30 min. Data are means±SE of 3 experiments.</p

Acute effects of GLP-1 and Ex-4 on GSIS in rat (A) and mouse (B) islets.

<p>Pancreatic islets were isolated and cultured overnight prior to use as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006221#s2" target="_blank">Methods</a>. Islets were incubated for 1 h (A) or 30 min (B) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006221#s2" target="_blank">Methods</a> for examining insulin secretion at indicated concentrations of glucose and GLP-1 (20 nM) or Ex-4 (20 nM) in A, or 10 nM Ex-4 in B, in the absence or presence of 0.3 mM palmitate. Insulin released into the media and the total islet insulin content were measured. Results shown are mean±SE from 3 independent experiments with quadruplicates (n = 12). For A, *p<0.05, **p<0.01, ***p<0.001 when compared with corresponding 2.8 mM glucose group. #p<0.05, ##p<0.01, ###p<0.001 when compared with corresponding groups without GLP-1 or Ex-4 treatment. For B, †p<0.01 when compared to corresponding minus Ex-4 group. Insulin secretion in mouse islets at basal glucose levels (4 mM) was 1.56±0.3 ng insulin/10 islets/30 min.</p

Palmitate β-oxidation and esterification into different lipids in rat islets in the absence or presence of GLP-1.

<p>Islets were processed as described for insulin secretion (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006221#pone.0006221-Prentki1" target="_blank">[Fig. 1]</a>) and after the pre-incubation step they were incubated for 2 h (FA oxidation) or 4 h (FA esterification) in 1 ml KRBH/0.25% defatted BSA containing medium with 1 mM carnitine and 1 µCi/ml [9,10(n)-³H] palmitate (51 Ci/mmol), at 2.8, 8.3 or 16.7 mM glucose in the presence or absence of 20 nM GLP-1. Cold palmitate (pal) was present at 0.1 mM for oxidation and 0.2 mM for esterification experiments. A, Palmitate oxidation; B—H, palmitate incorporation into diacylglycerol, DAG (B), triacylglycerol, TG (C), monoacylglycerol, MAG (D), non-esterified fatty acids, NEFA (E), cholesterol esters, CE (F), phospholipids, PL (G) and total glycerolipids, GL (H). Means±SE of 6–8 separate incubations in 3 independent experiments.</p

Automated, Resin-Based Method to Enhance the Specific Activity of Fluorine-18 Clicked PET Radiotracers

Radiolabeling of substrates with 2-[¹⁸F]­fluoroethylazide exploits the rapid kinetics, chemical selectivity, and mild conditions of the copper-catalyzed azide–alkyne cycloaddition reaction. While this methodology has proven to result in near-quantitative labeling of alkyne-tagged precursors, the relatively small size of the fluoroethylazide group makes separation of the ¹⁸F-labeled radiotracer and the unreacted precursor challenging, particularly with precursors >500 Da (e.g., peptides). We have developed an inexpensive azide-functionalized resin to rapidly remove unreacted alkyne precursor following the fluoroethylazide labeling reaction and integrated it into a fully automated radiosynthesis platform. We have carried out 2-[¹⁸F]­fluoroethylazide labeling of four different alkynes ranging from <300 Da to >1700 Da and found that >98% of the unreacted alkyne was removed in less than 20 min at room temperature to afford the final radiotracers at >99% radiochemical purity with specific activities up to >200 GBq/μmol. We have applied this technique to label a novel cyclic peptide previously evolved to bind the Her2 receptor with high affinity, and demonstrated tumor-specific uptake and low nonspecific background by PET/CT. This resin-based methodology is automated, rapid, mild, and general allowing peptide-based fluorine-18 radiotracers to be obtained with clinically relevant specific activities without chromatographic separation and with only a minimal increase in total synthesis time