Dysregulation of Glucagon Secretion by Hyperglycemia-Induced Sodium-Dependent Reduction of ATP Production

© 2018 The Author(s). Published by Elsevier Inc.Diabetes is a bihormonal disorder resulting from combined insulin and glucagon secretion defects. Mice lacking fumarase (Fh1) in their β cells (Fh1βKO mice) develop progressive hyperglycemia and dysregulated glucagon secretion similar to that seen in diabetic patients (too much at high glucose and too little at low glucose). The glucagon secretion defects are corrected by low concentrations of tolbutamide and prevented by the sodium-glucose transport (SGLT) inhibitor phlorizin. These data link hyperglycemia, intracellular Na+ accumulation, and acidification to impaired mitochondrial metabolism, reduced ATP production, and dysregulated glucagon secretion. Protein succination, reflecting reduced activity of fumarase, is observed in α cells from hyperglycemic Fh1βKO and β-V59M gain-of-function KATP channel mice, diabetic Goto-Kakizaki rats, and patients with type 2 diabetes. Succination is also observed in renal tubular cells and cardiomyocytes from hyperglycemic Fh1βKO mice, suggesting that the model can be extended to other SGLT-expressing cells and may explain part of the spectrum of diabetic complications.Peer reviewe

Altered glycolysis triggers impaired mitochondrial metabolism and mTORC1 activation in diabetic β-cells

Chronic hyperglycaemia causes a dramatic decrease in mitochondrial metabolism and insulin content in pancreatic β-cells. This underlies the progressive decline in β-cell function in diabetes. However, the molecular mechanisms by which hyperglycaemia produces these effects remain unresolved. Using isolated islets and INS-1 cells, we show here that one or more glycolytic metabolites downstream of phosphofructokinase and upstream of GAPDH mediates the effects of chronic hyperglycemia. This metabolite stimulates marked upregulation of mTORC1 and concomitant downregulation of AMPK. Increased mTORC1 activity causes inhibition of pyruvate dehydrogenase which reduces pyruvate entry into the tricarboxylic acid cycle and partially accounts for the hyperglycaemia-induced reduction in oxidative phosphorylation and insulin secretion. In addition, hyperglycaemia (or diabetes) dramatically inhibits GAPDH activity, thereby impairing glucose metabolism. Our data also reveal that restricting glucose metabolism during hyperglycaemia prevents these changes and thus may be of therapeutic benefit. In summary, we have identified a pathway by which chronic hyperglycaemia reduces β-cell function

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

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.We thank Novo Nordisk for providing NN414. This study was funded by grants from Diabetes UK (to MLJA, RJM) and the Juvenile Diabetes Research Foundation (postdoctoral fellowship 3-576-2010 to CB and 1-2008-728 to RJM)

Beta Cell Hubs Dictate Pancreatic Islet Responses to Glucose

N.R.J. was supported by a Diabetes UK RW and JM Collins Studentship (12/0004601). J.B. was supported by a European Foundation for the Study of Diabetes (EFSD) Albert Renold Young Scientist Fellowship and a Studienstiftung des deutschen Volkes PhD Studentship. D.T. was supported by an Advanced Grant from the European Research Commission (268795). G.A.R. was supported by Wellcome Trust Senior Investigator (WT098424AIA) and Royal Society Wolfson Research Merit Awards, and by MRC Programme (MR/J0003042/1), Biological and Biotechnology Research Council (BB/J015873/1), and Diabetes UK Project (11/0004210) grants. G.A.R. and M.W. acknowledge COST Action TD1304 Zinc-Net. D.J.H. was supported by Diabetes UK R.D. Lawrence (12/0004431), EFSD/Novo Nordisk Rising Star and Birmingham Fellowships, a Wellcome Trust Institutional Support Award, and an MRC Project Grant (MR/N00275X/1) with G.A.R. D.J.H and G.A.R. were supported by Imperial Confidence in Concept (ICiC) Grants. J.F. was supported by an MRC Programme grant (MR/L02036X/1). L.P. provided human islets through collaboration with the Diabetes Research Institute, IRCCS San Raffaele Scientific Institute (Milan), within the European islet distribution program for basic research supported by JDRF (1-RSC-2014-90-I-X). P.M. and M.B. were supported by the Innovative Medicine Initiative Joint Undertaking under grant agreement no. 155005 (IMIDIA), resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies in kind contribution, and by the Italian Ministry of University and Research (PRIN 2010-2012). D.B. and E.B. provided human islets through the European Consortium for Islet Transplantation sponsored by JDRF (1-RSC-2014-100-I-X)

Binding of sulphonylureas to plasma proteins – A K_ATP channel perspective

<div><p>Sulphonylurea drugs stimulate insulin secretion from pancreatic β-cells primarily by inhibiting ATP sensitive potassium (K_ATP) channels in the β-cell membrane. The effective sulphonylurea concentration at its site of action is significantly attenuated by binding to serum albumin, which makes it difficult to compare <i>in vitro</i> and <i>in vivo</i> data. We therefore measured the ability of gliclazide and glibenclamide to inhibit K_ATP channels and stimulate insulin secretion in the presence of serum albumin. We used this data, together with estimates of free drug concentrations from binding studies, to predict the extent of sulphonylurea inhibition of K_ATP channels at therapeutic concentrations <i>in vivo</i>. K_ATP currents from mouse pancreatic β-cells and <i>Xenopus</i> oocytes were measured using the patch-clamp technique. Gliclazide and glibenclamide binding to human plasma were determined in spiked plasma samples using an ultrafiltration-mass spectrometry approach. Bovine serum albumin (60g/l) produced a mild, non-significant reduction of gliclazide block of K_ATP currents in pancreatic β-cells and <i>Xenopus</i> oocytes. In contrast, glibenclamide inhibition of recombinant K_ATP channels was dramatically suppressed by albumin (predicted free drug concentration <0.1%). Insulin secretion was also reduced. Free concentrations of gliclazide and glibenclamide in the presence of human plasma measured in binding experiments were 15% and 0.05%, respectively. Our data suggest the free concentration of glibenclamide in plasma is too low to account for the drug’s therapeutic effect. In contrast, the free gliclazide concentration in plasma is high enough to close K_ATP channels and stimulate insulin secretion.</p></div

Effect of BSA on glibenclamide stimulated insulin release.

<p>Insulin secretion was evaluated in islets from 8–10 week-old male mice in response to 60–120 minute stimulation with various glucose and glibenclamide concentrations, as indicated, in the presence of (A) 15μmol/l BSA or (B) 0.9mmol/l BSA (n = 3 animals, 3 technical replicates per condition). Insulin secretion is expressed as a percentage of the insulin content. Insulin secretion in the presence of glucose and glibenclamide was significantly affected by the BSA concentration (<i>F(5</i>,<i>12)</i> = 10.77; <i>p</i> = 0.004). For both incubation times and for all drug concentrations tested, insulin secretion was significantly lower in the presence of 0.9mmol/l BSA than 15μM BSA (<i>p</i><0.05).</p

Effect of BSA on gliclazide block of the β-cell K_ATP current.

<p>(A,B) Representative whole-cell K_ATP currents recorded from mouse pancreatic β-cells in response to alternating ±20mV steps from a holding potential of -70mV. Gliclazide (1μmol/l) was added (as indicated by the bars) in the absence (A) or presence (B) of 0.9mmol/l BSA. The dotted line indicates the zero current level. (C) Concentration-response relationships for gliclazide inhibition of whole-cell K_ATP currents in mouse β-cells in the absence (○, n = 6) or presence (●, n = 5) of 0.9mmol/l BSA. Current is expressed relative to that in the absence of gliclazide. The solid lines are the best fit of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197634#pone.0197634.e002" target="_blank">Eq 2</a> to the mean data: (○) <i>IC</i>_<i>50</i> = 190nmol/l, <i>h</i> = 0.97, <i>a</i> = 0.05; (●) <i>IC</i>_<i>50</i> = 320nmol/l, <i>h</i> = 0.90, <i>a</i> = 0.03. This shift predicts 60% of drug is bound and a dissociation constant (<i>K</i>_<i>d</i>) for drug binding to BSA of ~1.3mmol/l (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197634#pone.0197634.e003" target="_blank">Eq 3</a>). The dotted line is the estimated gliclazide block in the presence of human plasma (HP) assuming gliclazide binds to plasma proteins with a <i>K</i>_<i>d</i> of 155μmol/l. The width of the grey bar indicates the mean C_SS±SEM (steady-state total plasma concentrations of sulphonylurea drugs) of the total gliclazide concentration in the plasma estimated from a daily dose of 80mg and an AUC (the area under the plasma concentration against time curve) of 44μg.h/ml [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197634#pone.0197634.ref026" target="_blank">26</a>].</p

Effect of BSA on glibenclamide and gliclazide block of recombinant β-cell K_ATP channels.

<p>(A,B) Representative Kir6.2/SUR1 currents recorded at -60mV from cell-attached patches on <i>Xenopus</i> oocytes. Currents were recorded in the presence of 3mmol/l Na-azide (top trace), 3mmol/l Na-azide plus 1μmol/l glibenclamide (A, middle) or 3mmol/l Na-azide plus 1μmol/l gliclazide (B, middle), and 3mmol/l Na-azide, 1μmol/l glibenclamide and 0.9mmol/l BSA (<b>A</b>, bottom) or 3mmol/l Na-azide, 1μmol/l gliclazide and 0.9mmol/l BSA (<b>C</b>, bottom). The dotted line indicates the zero current level. (C) Concentration-response relationships for glibenclamide inhibition of Kir6.2/SUR1 currents in the absence (○, n = 10) and presence (●, n = 10) of 0.9mmol/l BSA. Open probability (<i>P</i>_<i>O</i>) was recorded in the cell-attached configuration and is expressed relative to that in the absence of glibenclamide. The lines are the best fit of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197634#pone.0197634.e002" target="_blank">Eq 2</a> to the mean data: (○) <i>IC</i>_<i>50</i> = 1.2nmol/l, <i>h</i> = 1.1; (●) <i>IC</i>_<i>50</i> = 1.6μmol/l, <i>h</i> = 0.95. <i>a</i> was set at 0 in both cases. The dotted line is the estimated glibenclamide block in the presence of human plasma (HP) assuming the drug binds to plasma proteins with a <i>K</i>_<i>d</i> of 0.44μmol/l. The width of the grey bar indicates the mean C_SS±SEM of the total glibenclamide concentration in the plasma of patients with type 2 diabetes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197634#pone.0197634.ref028" target="_blank">28</a>]. (D) Concentration-response relationships for gliclazide inhibition of Kir6.2/SUR1 currents in the absence (○, n = 10) and presence (●, n = 10) of 0.9mmol/l BSA. Open probability (<i>P</i>_<i>O</i>) was recorded in the cell-attached configuration and is expressed relative to that in the absence of gliclazide. The lines are the best fit of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197634#pone.0197634.e002" target="_blank">Eq 2</a> to the mean data: <i>IC</i>_<i>50</i> = 128nmol/l, <i>h</i> = 1.3 (○); <i>IC</i>_<i>50</i> = 217nmol/l, <i>h</i> = 1.3 (●). <i>a</i> was set at 0. The <i>IC</i>_<i>50</i> obtained in the absence of BSA (128nmol/l) is similar to that previously reported for whole-cell Kir6.2/SUR1 currents in oocytes (108nmol/l; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197634#pone.0197634.ref027" target="_blank">27</a>]). The width of the grey bar indicates the mean C_SS±SEM of the total gliclazide concentration in the plasma estimated from a daily dose of 80mg and an AUC of 44μg.h/ml [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197634#pone.0197634.ref026" target="_blank">26</a>].</p