Loss of SUR1 subtype KATP channels alters antinociception and locomotor activity after opioid administration.

Opioid signaling can occur through several downstream mediators and influence analgesia as well as reward mechanisms in the nervous system. KATP channels are downstream targets of the µ opioid receptor and contribute to morphine-induced antinociception. The aim of the present work was to assess the role of SUR1-subtype KATP channels in antinociception and hyperlocomotion of synthetic and semi-synthetic opioids. Adult male and female mice wild-type (WT) and SUR1 deficient (KO) mice were assessed for mechanical and thermal antinociception after administration of either buprenorphine, fentanyl, or DAMGO. Potassium flux was assessed in the dorsal root ganglia and superficial dorsal horn cells in WT and KO mice. Hyperlocomotion was also assessed in WT and KO animals after buprenorphine, fentanyl, or DAMGO administration. SUR1 KO mice had attenuated mechanical antinociception after systemic administration of buprenorphine, fentanyl, and DAMGO. Potassium flux was also attenuated in the dorsal root ganglia and spinal cord dorsal horn cells after acute administration of buprenorphine and fentanyl. Hyperlocomotion after administration of morphine and buprenorphine was potentiated in SUR1 KO mice, but was not seen after administration of fentanyl or DAMGO. These results suggest SUR1-subtype KATP channels mediate the antinociceptive response of several classes of opioids (alkaloid and synthetic/semi-synthetic), but may not contribute to the "drug-seeking" behaviors of all classes of opioids.

Hypoglycaemic effects of glimepiride in sulfonylurea receptor 1 deficient rat.

BACKGROUND AND PURPOSE: Sulfonylureas (SUs) have been suggested to have an insulin-independent blood glucose-decreasing activity due to an extrapancreatic effect. However, a lack of adequate in vivo evidence makes this statement controversial. Here, we aimed to evaluate whether glimepiride has extrapancreatic blood glucose-lowering activity in vivo. EXPERIMENTAL APPROACH: Sulfonylurea receptor 1 deficient (SUR1-/- ) rats were created by means of transcription activator-like effector nucleases (TALEN)-mediated gene targeting technology. Type 2 diabetic models were established by feeding a high-fat diet and administering a low-dose of streptozotocin. These rats were then randomly divided into four groups: glimepiride, gliclazide, metformin and saline. All rats were treated for 2 weeks. KEY RESULTS: Glimepiride decreased blood glucose levels and increased insulin sensitivity without elevating insulin levels. Gliclazide showed similar effects as glimepiride. Both agents were weaker than metformin. Further mechanistic investigations revealed that glimepiride increased hepatic glycogen synthesis and decreased gluconeogenesis, which were accompanied by the activation of Akt in the liver. Moreover, glimepiride increased both total and membrane glucose transporter 4 (GLUT4) levels in muscle and fat, which might be attributed to insulin receptor-independent IRS1/Akt activation. CONCLUSION AND IMPLICATIONS: Glimepiride possesses an extrapancreatic blood glucose-lowering effect in vivo, which might be attributed to its direct effect on insulin-sensitive tissues. Therefore, the combination of glimepiride with multiple insulin injections should not be excluded per se.

Modeling Congenital Hyperinsulinism with ABCC8-Deficient Human Embryonic Stem Cells Generated by CRISPR/Cas9.

Congenital hyperinsulinism (CHI) is a rare genetic disorder characterized by excess insulin secretion, which results in hypoglycemia. Mutation of sulfonylurea receptor 1 (SUR1), encoded by the ABCC8 gene, is the main cause of CHI. Here, we captured the phenotype of excess insulin secretion through pancreatic differentiation of ABCC8-deficient stem cells generated by the CRISPR/Cas9 system. ABCC8-deficient insulin-producing cells secreted higher insulin than their wild-type counterparts, and the excess insulin secretion was rescued by nifedipine, octreotide and nicorandil. Further, we tested the role of SUR1 in response to different potassium levels and found that dysfunction of SUR1 decreased the insulin secretion rate in low and high potassium environments. Hence, pancreatic differentiation of ABCC8-deficient cells recapitulated the CHI disease phenotype in vitro, which represents an attractive model to further elucidate the function of SUR1 and to develop and screen for novel therapeutic drugs.

Chronic ß-Cell Depolarization Impairs ß-Cell Identity by Disrupting a Network of Ca2+-Regulated Genes.

We used mice lacking Abcc8, a key component of the ß-cell KATP-channel, to analyze the effects of a sustained elevation in the intracellular Ca2+ concentration ([Ca2+]i) on ß-cell identity and gene expression. Lineage tracing analysis revealed the conversion of ß-cells lacking Abcc8 into pancreatic polypeptide cells but not to α- or δ-cells. RNA-sequencing analysis of FACS-purified Abcc8-/- ß-cells confirmed an increase in Ppy gene expression and revealed altered expression of more than 4,200 genes, many of which are involved in Ca2+ signaling, the maintenance of ß-cell identity, and cell adhesion. The expression of S100a6 and S100a4, two highly upregulated genes, is closely correlated with membrane depolarization, suggesting their use as markers for an increase in [Ca2+]i Moreover, a bioinformatics analysis predicts that many of the dysregulated genes are regulated by common transcription factors, one of which, Ascl1, was confirmed to be directly controlled by Ca2+ influx in ß-cells. Interestingly, among the upregulated genes is Aldh1a3, a putative marker of ß-cell dedifferentiation, and other genes associated with ß-cell failure. Taken together, our results suggest that chronically elevated ß-cell [Ca2+]i in Abcc8-/- islets contributes to the alteration of ß-cell identity, islet cell numbers and morphology, and gene expression by disrupting a network of Ca2+-regulated genes.

Sulphonylurea receptor-1, sulphonylureas and amplification of insulin secretion by Epac activation in ß cells.

Amplification of insulin secretion by cyclic AMP involves activation of protein kinase A (PKA) and Epac2 in pancreatic ß cells. Recent hypotheses suggest that sulphonylurea receptor-1 (SUR1), the regulatory subunit of ATP-sensitive potassium channels, is implicated in Epac2 effects and that direct activation of Epac2 by hypoglycaemic sulphonylureas contributes to the stimulation of insulin secretion by these drugs. In the present experiments, using islets from Sur1KO mice, we show that dibutyryl-cAMP and membrane-permeant selective activators of Epac or PKA normally amplify insulin secretion in ß cells lacking SUR1. In contrast to Epac activator, sulphonylureas (glibenclamide and tolbutamide) did not increase insulin secretion in Sur1KO islets, as would be expected if they were activating Epac2 directly. Furthermore, glibenclamide and tolbutamide did not augment the amplification of insulin secretion produced by Epac activator or dibutyryl-cAMP. Collectively, the results show that SUR1 is dispensable for amplification of insulin secretion by Epac2 activation and that direct activation of Epac2 is unimportant for the action of therapeutic concentrations of sulphonylureas in ß cells.

Effect of fluoroquinolones on mitochondrial function in pancreatic beta cells.

Hyper- and hypoglycaemias are known side effects of fluoroquinolone antibiotics, resulting in a number of fatalities. Fluoroquinolone-induced hypoglycaemias are due to stimulated insulin release by the inhibition of the KATP channel activity of the beta cell. Recently, it was found that fluoroquinolones were much less effective on metabolically intact beta cells than on open cell preparations. Thus the intracellular effects of gatifloxacin, moxifloxacin and ciprofloxacin were investigated by measuring NAD(P)H- and FAD-autofluorescence, the mitochondrial membrane potential, and the adenine nucleotide content of isolated pancreatic islets and beta cells. 100 µM of moxifloxacin abolished the NAD(P)H increase elicited by 20mM glucose, while gatifloxacin diminished it and ciprofloxacin had no significant effect. This pattern was also seen with islets from SUR1 Ko mice, which have no functional KATP channels. Moxifloxacin also diminished the glucose-induced decrease of FAD-fluorescence, which reflects the intramitochondrial production of reducing equivalents. Moxifloxacin, but not ciprofloxacin or gatifloxacin significantly reduced the effect of 20mM glucose on the ATP/ADP ratio. The mitochondrial hyperpolarization caused by 20mM glucose was partially antagonized by moxifloxacin, but not by ciprofloxacin or gatifloxacin. Ultrastructural analyses after 20 h tissue culture showed that all three compounds (at 10 and 100 µM) diminished the number of insulin secretory granules and that gatifloxacin and ciprofloxacin, but not moxifloxacin induced fission/fusion configurations of the beta cell mitochondria. In conclusion, fluoroquinolones affect the function of the mitochondria in pancreatic beta cells which may diminish the insulinotropic effect of KATP channel closure and contribute to the hyperglycaemic episodes.