br Acknowledgments This work was supported by a grant

Acknowledgments This work was supported by a grant CIHR-NSFC China-Canada Joint Health Research from the National Science Foundation of China (Grant Number 81061120525) and the Canadian Institutes for Health Research (Grant number CCI-109605). The authors thank two anonymous reviewers for helpful suggestions.
Introduction By catalyzing the rate-limiting SB 290157 trifluoroacetate salt synthesis phosphorylation step in β-cell glucose metabolism, glucokinase (GCK), functions as the glucose sensor within β-cells for regulating glucose-stimulated insulin secretion (GSIS). GCK, through its central role in glucose metabolism, also indirectly regulates β-cell proliferation [1], [2]. As such, mutations resulting in changes in GCK activity can have profound effects on β-cell function. Mutations resulting in GCK deficiency causes of maturity onset diabetes of the young (GCK-MODY) while activating GCK mutations cause hyperinsulinemic hypoglycemia (GCK-HH) [3]. Patients with GCK-MODY generally do not require treatment [3], [4], [5]. However treatment for patients with GCK-HH depends on the severity of hypoglycemia and can range from diazoxide to partial pancreatectomy [6], [7]. The GCK mutations in GCH-HH activates GCK by increasing GCK’s affinity for glucose [6], [7], [8], [9], [10], [11], [12]. This enhances β-cell glycolytic flux and increases GSIS. However, excessive glycolytic flux has also been shown to increase β-cell toxicity [13], [14]. Indeed, pancreas samples from a patient with the V91L GCK-HH mutation also showed increased TUNEL staining in β-cells. However, while Tornovsky-Babeay et al. demonstrated that the Y214C GCK-HH mutation induces apoptosis in β-cells via p53 activation [14], it remains unclear if other GCK-HH mutations would share the same mechanisms of β-cell toxicity.
Materials and methods
Results
Discussion To date, 15 GCK-HH mutations have been identified [3], [7], [15], [16]. However, only the V91L GCK mutation has been associated with β-cell death in humans [7]. Pancreas sections from the patient with the V91L GCK mutation showed enlarged islets, enhanced β-cell proliferation, and increased β-cell TUNEL staining [7]. The enlarged islets is likely the result of the enhanced β-cell proliferation from increased glycolytic flux [7], as glucose has been shown to be a potent regulator of β-cell proliferation [1], [2], [17]. The mechanism of β-cell death in β-cells carrying the V91L GCK mutation remains unknown. Our results in vitro propose a mechanism of cell death via necrosis in these cells. Our results showed that β-cell expressing V91L GCK undergo glucose phosphorylation-dependent necrosis coincident with ATP depletion, suggesting that, in the presence of excess D-glucose, the hyperactive V91L GCK consumed intracellular ATP for glucose phosphorylation faster than ATP can be replenished via downstream glucose metabolism. This excess glucose phosphorylation likely results in accumulation of glucose 6-phosphate, and its conversion product fructose 6-phosphate, similar to what was observed by Wang and Iynedjian in INS-r3GK27 cells expressing wild-type GCK 20-fold above normal [13]. However, our results showed a more complete depletion of intracellular ATP (90% vs. 75%) and a more rapid morphological change (<1 h vs. 16 h) compared to what was observed by Wang and Iynedjian [13], likely due to the higher enzymatic kinetics of the V91L GCK mutant [7]. It is possible the accumulation of glucose 6-phosphate and fructose 6-phosphate may result in osmotic stress-induced necrosis due to the osmotic intake of water. However, previous studies from another cell line suggest against this possibility. Similar, rapid morphological changes upon ATP depletion have been observed the ROC-1 glial cell line by Jurkowitz‐Alexander et al. [18]. Inhibition of the Na+, K+-ATPase pump in ROC-1 cells results in accumulation of Na+ and Cl- ions and promotes osmotic swelling [18]. However, osmotic swelling following inhibition of the Na+, K+-ATPase pump, though inducing similar morphological changes as ATP depletion, did not result in necrosis [18]. The level of ATP depletion we observed in β-cell expressing V91L GCK is also consistent with previously reported level of ATP depletion required for induction of necrosis. ATP depletion, therefore, is likely the primary cause of necrosis in β-cell expressing V91L GCK.