Stephen A. Smith

Antagonism of the stimulatory effects of efaroxan and glibenclamide in rat pancreatic islets by the imidazoline, RX801080.

1 The imidazoline α2‐adrenoceptor antagonist, efaroxan, stimulates insulin secretion from rat isolated islets and antagonizes the ability of diazoxide to inhibit glucose‐induced insulin secretion. These effects result from closure of ATP‐sensitive potassium channels although the mechanisms involved have not been elucidated. 2 In the present work, we have examined the effects of a close structural analogue of efaroxan, RX801080, in rat isolated islets of Langerhans. RX801080 was found to be ineffective as a stimulator of insulin secretion and did not prevent the inhibition of insulin secretion mediated by diazoxide. 3 RX801080 acted as an antagonist of the actions of several imidazolines (efaroxan, phentolamine and midaglizole) in rat islets. It dose‐dependently inhibited the ability of efaroxan to antagonize the effects of diazoxide in islets and also completely inhibited the direct stimulation of insulin secretion mediated by efaroxan. 4 RX801080 also antagonized the effects of the non‐imidazoline, ATP‐sensitive potassium channel blocker, glibenclamide, in rat islets. It inhibited both the capacity of glibenclamide to stimulate insulin secretion and the ability of glibenclamide to overcome the inhibitory effects of diazoxide in rat islets. 5 Antagonism of glibenclamide responses by RX801080 was not due to inhibition of binding of the sulphonylurea to its receptor on the pancreatic β‐cell. 6 The results suggest that imidazoline compounds and sulphonylureas interact with distinct binding sites on islet cells, but that these sites can interact functionally to control islet cell ATP‐sensitive potassium channel activity and insulin secretion.

Stimulation of insulin secretion by imidazoline compounds is not due to interaction with non-adrenoceptor idazoxan binding sites

1 The potency of interaction of several imidazoline compounds with non‐adrenoceptor idazoxan binding sites (NAIBS) in rat liver membranes was compared with their ability to alter insulin secretion from rat pancreatic islets. 2 NAIBS could be labelled specifically with [3H]‐idazoxan in both rat liver membranes and in rat islet homogenates. Liver binding sites exhibited a KD for [3H]‐idazoxan of 24 nm and a Bmax of 264 fmol mg−1 protein. 3 Binding of [3H]‐idazoxan to NAIBS in rat liver membranes was displaced effectively by unlabelled idazoxan (IC50 0.1 μm) and by UK14304 (IC50 0.5 μm). However, two other imidazoline compounds efaroxan and RX821002, which are related in structure to idazoxan, were much less effective as displacers. 4 In insulin secretion experiments, the ATP‐sensitive potassium channel agonist diazoxide (250 μm) was able to suppress the rise in insulin secretion induced by 20 mm glucose. Both efaroxan and RX821002 (100 μm) antagonized the inhibitory effect of diazoxide on glucose‐induced insulin secretion. By contrast, neither idazoxan (100 μm) nor UK14304 (50 μm), was able to overcome significantly the inhibitory effect of diazoxide. 5 The ability of 100 μm efaroxan to antagonize the suppression of insulin secretion mediated by diazoxide, was not prevented by idazoxan (up to 100 μm) or by UK14304 (up to 50 μm). 6 The results indicate that the stimulatory effects of imidazoline compounds on insulin secretion are not due to interaction with NAIBS similar to those present in rat liver.

Interactions between imidazoline compounds and sulphonylureas in the regulation of insulin secretion

Imidazoline α2‐antagonist drugs such as efaroxan have been shown to increase the insulin secretory response to sulphonylureas from rat pancreatic B‐cells. We have investigated whether this reflects binding to an islet imidazoline receptor or whether α2‐adrenoceptor antagonism is involved. Administration of (±)‐efaroxan or glibenclamide to Wistar rats was associated with a transient increase in plasma insulin. When both drugs were administered together, the resultant increase in insulin levels was much greater than that obtained with either drug alone. Use of the resolved enantiomers of efaroxan revealed that the ability of the compound to enhance the insulin secretory response to glibenclamide resided only in the α2‐selective‐(+)‐enantiomer; the imidazoline receptor‐selective‐(−)‐enantiomer was ineffective. In vitro, (+)‐efaroxan increased the insulin secretory response to glibenclamide in rat freshly isolated and cultured islets of Langerhans, whereas (−)‐efaroxan was inactive. By contrast, (+)‐efaroxan did not potentiate glucose‐induced insulin secretion but (−)‐efaroxan induced a marked increase in insulin secretion from islets incubated in the presence of 6 mM glucose. Incubation of rat islets under conditions designed to minimize the extent of α2‐adrenoceptor signalling (by receptor blockade with phenoxybenzamine; receptor down‐regulation or treatment with pertussis toxin) abolished the capacity of (+)‐and (±)‐efaroxan to enhance the insulin secretory response to glibenclamide. However, these manoeuvres did not alter the ability of (±)‐efaroxan to potentiate glucose‐induced insulin secretion. The results indicate that the enantiomers of efaroxan exert differential effects on insulin secretion which may result from binding to effector sites having opposite stereoselectivity. Binding of (−)‐efaroxan (presumably to imidazoline receptors) results in potentiation of glucose‐induced insulin secretion, whereas interaction of (+)‐efaroxan with a second site leads to selective enhancement of sulphonylurea‐induced insulin release.

Multiple effector pathways regulate the insulin secretory response to the imidazoline RX871024 in isolated rat pancreatic islets

When isolated rat islets were cultured for 18u2003h prior to use, the putative imidazoline binding site ligand, RX871024 caused a dose‐dependent increase in insulin secretion at both 6u2003mM and 20u2003mM glucose. By contrast, a second ligand, efaroxan, was ineffective at 20u2003mM glucose whereas it did stimulate insulin secretion in response to 6u2003mM glucose. Exposure of islets to RX871024 (50u2003μM) for 18u2003h, resulted in loss of responsiveness to this reagent upon subsequent re‐exposure. However, islets that were unresponsive to RX871024 still responded normally to efaroxan. The imidazoline antagonist, KU14R, blocked the insulin secretory response to efaroxan, but failed to prevent the stimulatory response to RX871024. By contrast with its effects in cultured islets, RX871024 inhibited glucose‐induced insulin release from freshly isolated islets. Efaroxan did not inhibit insulin secretion under any conditions studied. In freshly isolated islets, the effects of RX871024 on insulin secretion could be converted from inhibitory to stimulatory, by starvation of the animals. Inhibition of insulin secretion by RX871024 in freshly isolated islets was prevented by the cyclo‐oxygenase inhibitors indomethacin or flurbiprofen. Consistent with this, RX871024 caused a marked increase in islet PGE2 formation. Efaroxan did not alter islet PGE2 levels. The results suggest that RX871024 exerts multiple effects in the pancreatic β‐cell and that its effects on insulin secretion cannot be ascribed only to interaction with a putative imidazoline binding site.

Effector systems involved in the insulin secretory responses to efaroxan and RX871024 in rat islets of Langerhans.

One component of the mechanism by which imidazoline compounds promote insulin secretion involves closure of ATP-sensitive K+ channels in the beta-cell plasma membrane. Recently, however, it has also been proposed that these compounds may exert important effects on more distal effector systems. In the present work, we have investigated the contribution played by protein kinases A and C to the insulin secretory responses of isolated rat islets of Langerhans treated with efaroxan and RX871024 (1-phenyl-2-(imidazolin-2-yl) benzimidazole). Removal of extracellular Ca2+ or blockade of voltage-sensitive Ca2+ channels prevented stimulation of insulin secretion by efaroxan, confirming a critical role for increased Ca2+ influx in the secretory response. By contrast, inhibition of protein kinases A or C failed to alter efaroxan-induced insulin secretion. RX871024 dose-dependently increased insulin secretion from cultured islets incubated with 20 mM glucose. This effect was unaffected by modulation of protein kinase C, but was significantly attenuated by a selective inhibitor of protein kinase A (Rp-cAMPs). Measurements of cAMP revealed that RX871024 increased the islet cAMP content by more than 3-fold; reaching values similar in magnitude to those elicited by 50 microM 3-isobutyl-1-methyl xanthine. The results reveal that neither protein kinase A nor protein kinase C is obligatory for stimulation of insulin secretion by imidazolines. However, they suggest that a rise in cAMP may contribute to the amplified secretory response observed when cultured islets are incubated with RX871024 in the presence of a stimulatory glucose concentration.

Expression and functional activity of PPARγin pancreaticβcells

Rosiglitazone is an agonist of peroxisome proliferator activated receptor‐γ (PPARγ) and ameliorates insulin resistance in type II diabetes. In addition, it may also promote increased pancreatic β‐cell viability, although it is not known whether this effect is mediated by a direct action on the β cell. We have investigated this possibility. Semiquantitative real‐time reverse transcription–polymerase chain reaction analysis (Taqman®) revealed that freshly isolated rat islets and the clonal β‐cell line, BRIN‐BD11, express PPARγ, as well as PPARα and PPARδ. The levels of expression of PPARγ were estimated by reference to adipose tissue and were found to represent approximately 60% (islets) and 30% (BRIN‐BD11) of that found in freshly isolated visceral adipose tissue. Western blotting confirmed the presence of immunoreactive PPARγ in rat (and human) islets and in BRIN‐BD11 cells. Transfection of BRIN‐BD11 cells with a PPARγ‐sensitive luciferase reporter construct was used to evaluate the functional competence of the endogenous PPARγ. Luciferase activity was modestly increased by the putative endogenous ligand, 15‐deoxy‐Δ12,14 prostaglandin J2 (15dPGJ2). Rosiglitazone also caused activation of the luciferase reporter construct but this effect required concentrations of the drug (50–100 μM) that are beyond the expected therapeutic range. This suggests that PPARγ is relatively insensitive to activation by rosiglitazone in BRIN‐BD11 cells. Exposure of BRIN‐BD11 cells to the lipotoxic effector, palmitate, caused a marked loss of viability. This was attenuated by treatment of the cells with either actinomycin D or cycloheximide suggesting that a pathway of programmed cell death was involved. Rosiglitazone failed to protect BRIN‐BD11 cells from the toxic actions of palmitate at concentrations up to 50 μM. Similar results were obtained with a range of other PPARγ agonists. Taken together, the present data suggest that, at least under in vitro conditions, thiazolidinediones do not exert direct protective effects against fatty acid‐mediated cytotoxicity in pancreatic β cells.