Chronic Antidiabetic Sulfonylureas In Vivo: Reversible Effects on Mouse Pancreatic β-Cells

In a mouse study aiming to understand why long-term treatment for type 2 diabetes with sulfonylureas eventually fails, Colin Nichols and Maria Remedi suggest that slow restoration of insulin secretion may be possible after a drug-resting period

Acute and Chronic Effect of Glibenclamide on Glucose Tolerance and Blood Glucose Levels

<div><p>In each graph, asterisks indicate control group is significantly different (<i>p</i> < 0.05) from the test group at the specified time point.</p> <p>(A) GTTs on 6-wk-old WT mice (<i>n</i> = 10) fasted for 12 h and intraperitoneally injected with 1.5 g/kg glucose and glibenclamide simultaneously.</p> <p>(B) Blood glucose response of fed 6-wk-old mice (<i>n</i> = 10) to acute injection of glibenclamide.</p> <p>(C) Fed blood glucose from WT and Kir6.2 knockout (Kir6.2KO) mice implanted with placebo or glibenclamide pellets (0.0001 or 2.5 mg/pellet) over time.</p> <p>(D) Individual values of fed glucose (plus mean and SEM) from WT control and glibenclamide-pelleted mice, as well as Kir6.2-KO and SUR1-KO mice implanted with 2.5 mg pellets, at 1 and 42 d after implantation.</p> <p>Each group in (C) and (D) contained ten to 15 mice.</p></div

Drug and Insulin Responsivity In Vivo

<div><p>In each graph, asterisks indicate control group is significantly different (<i>p</i> < 0.05) from the test group at the specified time point.</p> <p>(A) Individual fed plasma insulin (plus mean and SEM) in glibenclamide-treated mice, 2 d and 42 d after glibenclamide implantation.</p> <p>(B) Individual values (plus mean and SEM) of plasma insulin 30 min after glucose injection (normalized to control unpelleted) at either 2 d or 42 d.</p> <p>(C and D) WT mice 5 wk implanted with different doses of glibenclamide were intraperitoneally injected with glibenclamide (C) or with insulin (D) and blood glucose was assessed over time.</p> <p>(E and F) Control and high-dose glibenclamide-pelleted mice were intraperitoneally injected with 20 mg/kg diazoxide, and blood glucose was assessed over time. In (F), glucose is normalized to the precontrol value (indicated by broken line).</p></div

Enhancement and Crossover of Glucose Tolerance in Mice Implanted with Low Dose Slow-Release Glibenclamide Pellets

<div><p>In each panel, asterisks indicate control group is significantly different (<i>p</i> < 0.05) from the test group at the specified time point.</p> <p>GTTs performed on WT mice before (A), 2 d (B), 7 d (C), 42 d (D), 95 d (E), and 118 d (F) after implantation of pellets containing high doses of glibenclamide. Groups contained ten mice each, except five in (E) and (F). Note: The lowest-dose pellets (0.0001 mg) were examined only at 14 and 42 d postimplant.</p></div

Impaired Glucose-Dependence of Insulin Secretion in Fresh Isolated, but Not in 24-h Incubated Islets from Mice Chronically Treated with Glibenclamide

<div><p>(A) Insulin content from glibenclamide-pelleted mice.</p> <p>(B and C) Glucose-sensitive insulin secretion from freshly isolated islets (B) and from islets incubated in 5.6 mM glucose for 24 h (C). Insulin release and content was measured by radioimmunoassay. Each group contained ten mice, and samples were assayed in triplicate. Significant differences between glibenclamide-implanted and control are indicated for each assay condition by asterisk.</p> <p>There were no significant differences in (A).</p></div

Impaired Glucose Tolerance in Mice Implanted with High Dose Slow-Release Glibenclamide Pellets

<p>GTTs on WT mice before (A), 2 d (B), 7 d (C), 42 d (D), 95 d (E), and 118 d (F) after implantation of pellets containing high doses of glibenclamide. Each group contained ten mice, except five in (E) and (F). Mice were injected intraperitoneally with glucose (1.5 g/kg). Blood was taken at times indicated and assayed for glucose concentration. In each graph, asterisks indicate control group is significantly different (<i>p</i> < 0.05) from the test group at the specified time point.</p

Morphological and Physiological Response to Chronic Hyperexcitability, and Proposed “Inverse U” Model Response for Enhanced β-Cell Excitability

<div><p>(A) Hematoxylin-eosin staining (left), glucagon (middle), and insulin (right) immunostaining of pancreatic paraffin sections from WT control and glibenclamide-implanted mice.</p> <p>(B) Insulin (green) and TUNEL (red) staining of pancreatic sections from control and glibenclamide-pelleted mice. Left images show images from untreated sections; right images show islets from paraffin sections treated with recombinant DNAase I (TUNEL-positive controls).</p> <p>(C) Proposed “Inverse U” model response for enhanced β-cell excitability: normal islets (white circle) secrete normally, but following a high-fat diet (HFD; grey dashed arrow) progress to insulin hypersecretion. Both Kir6.2[AAA] and heterozygous Kir6.2- and SUR1-KO mice (50%–70% decreased K_ATP activity with increased excitability) hypersecrete (grey circles, solid line) and are positioned on the “ascending limb” of the curve [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0050206#pmed-0050206-b013" target="_blank">13</a>,<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0050206#pmed-0050206-b014" target="_blank">14</a>,<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0050206#pmed-0050206-b043" target="_blank">43</a>]. HFD causes further enhancement of excitability, beyond the threshold driving those islets “over the top” (dashed) to an undersecretory phenotype. Conversely, Kir6.2- and SUR1-KO islets (zero K_ATP channel activity), which have maximally enhanced excitability, hypersecrete as neonates (grey circle on solid line), but rapidly progress to an undersecretory phenotype (black circle on solid line), and are positioned on the “descending limb” [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0050206#pmed-0050206-b010" target="_blank">10</a>–<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0050206#pmed-0050206-b012" target="_blank">12</a>]. Glibenclamide-treated WT mice rapidly progress from normal insulin secretion to undersecretion (white circle on “ascending limb” is converted to black circle on “descending limb”). This phenotype can be completely reversed when hyperexcitability is removed (small black dashed line). Finally, mice expressing mutant β-cell K_ATP channels with enhanced activity (Kir6.2[ΔN30]) [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0050206#pmed-0050206-b041" target="_blank">41</a>] have a severely undersecreting phenotype, extending the ascending limb beyond the position of normal animals.</p></div