Elsevier

Neuropharmacology

Volume 111, December 2016, Pages 212-222
Neuropharmacology

Chronic exposure to KATP channel openers results in attenuated glucose sensing in hypothalamic GT1-7 neurons

https://doi.org/10.1016/j.neuropharm.2016.09.008Get rights and content
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Highlights

  • Chronic exposure to NN414 blunts hypoglycaemic responses in GT1-7 neurons.

  • NN414 does not cause adaptations in glucose metabolism.

  • The availability of KATP to open is greatly diminished by NN414.

  • Preventing NN414 binding or conformation change to KATP rescues channel activity.

Abstract

Individuals with Type 1 diabetes (T1D) are often exposed to recurrent episodes of hypoglycaemia. This reduces hormonal and behavioural responses that normally counteract low glucose in order to maintain glucose homeostasis, with altered responsiveness of glucose sensing hypothalamic neurons implicated. Although the molecular mechanisms are unknown, pharmacological studies implicate hypothalamic ATP-sensitive potassium channel (KATP) activity, with KATP openers (KCOs) amplifying, through cell hyperpolarization, the response to hypoglycaemia. Although initial findings, using acute hypothalamic KCO delivery, in rats were promising, chronic exposure to the KCO NN414 worsened the responses to subsequent hypoglycaemic challenge. To investigate this further we used GT1-7 cells to explore how NN414 affected glucose-sensing behaviour, the metabolic response of cells to hypoglycaemia and KATP activity. GT1-7 cells exposed to 3 or 24 h NN414 exhibited an attenuated hyperpolarization to subsequent hypoglycaemic challenge or NN414, which correlated with diminished KATP activity. The reduced sensitivity to hypoglycaemia was apparent 24 h after NN414 removal, even though intrinsic KATP activity recovered. The NN414-modified glucose responsiveness was not associated with adaptations in glucose uptake, metabolism or oxidation. KATP inactivation by NN414 was prevented by the concurrent presence of tolbutamide, which maintains KATP closure. Single channel recordings indicate that NN414 alters KATP intrinsic gating inducing a stable closed or inactivated state. These data indicate that exposure of hypothalamic glucose sensing cells to chronic NN414 drives a sustained conformational change to KATP, probably by binding to SUR1, that results in loss of channel sensitivity to intrinsic metabolic factors such as MgADP and small molecule agonists.

Keywords

NN414
Diazoxide
KATP
Glucose sensing
Hypoglycemia
Type 1 diabetes

Abbreviations

CRR
counterregulatory responses
DMEM
Dulbecco's modified Eagle medium
FBS
fetal bovine serum
FCCP
carbonyl cyanide-p-trifluoromethoxyphenylhydrazone
HBS
hepes buffered saline
ICV
intracerebroventricular
KATP
ATP-sensitive potassium channel
KIR6
inward rectifier potassium channel family 6
NN414 (tifenazoxide)
6-chloro-N-(1-methylcyclopropyl)-1,1-dioxo-4H-thieno[3,2-e][1,2,4]thiadiazin-3amine
OCR
oxygen consumption rate
SUR1/2
sulphonylurea receptor 1/2
T1D
Type 1 Diabetes
TTA
Tris base
TES (N-tris(hydroxylmethyl)methyl-1-l-aminoethine sulphonic acid
glacial acetic acid
VMH
ventromedial hypothalamus
2DG
2-deoxy-D-[3H]glucose
ΔVm
membrane potential change

Chemical compounds

Diazoxide (PubChem CID: 3019)
NN414 (Tifenazoxide) (PubChem CID 219048)

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