While TUNEL staining is traditionally thought to be

While TUNEL staining is traditionally thought to be an apoptosis marker, studies have shown that TUNEL can stain necrotic IL-10, human recombinant protein [19], [20], [21]. Furthermore, DNA degradation has also been observed in necrotic cells [22]. TUNEL staining alone is therefore insufficient to distinguish apoptosis from necrosis. To distinguish apoptosis from necrosis in our in vitro model, we conducted kinetic analyses of Annexin V staining, cell permeability, and caspase 3/7 activation. Based on these combined parameters, our results indicate that β-cells expressing V91L GCK primarily undergo cell death via necrosis. Clinical presentations of the patient with V91L GCK mutation were similar to those of the patient with Y214C GCK mutation. Both patients showed increased β-cell area and severe hypoglycemia requiring partial pancreatectomy [6], [7] While β-cell death was not assessed in the patient with Y214C GCK mutation [6], transgenic mice carrying the Y214C GCK mutation had increased β-cell apoptosis from DNA damage and subsequent p53 activation [14]. Our results in vitro using the SV40 large T antigen suggests a mechanism of cell death independent of p53 with the V91L GCK mutant. This implies that the mechanism of cell death observed with the V91L GCK mutation may be different from those observed with the Y214C GCK mutation. The difference in cell death mechanism may be due to the severity of ATP depletion. Intracellular ATP is required for apoptosis [23], [24], and ATP depletion greater than 85% has been shown to divert the cell death pathway from apoptosis to necrosis [25], [26], [27]. As such, necrosis induced by the V91L GCK mutant is likely not specifically due to the V91L mutation, but rather the resulting high level of GCK activity. It should be noted, however, that further studies with the V91L GCK mutant are required to verify our findings. Particularly, our results remain to be validated in an in vivo model.
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Acknowledgements
Introduction Hexokinases are ubiquitously expressed and have a central role in metabolism phosphorylating hexoses (e.g. glucose), raising the hexose free-energy content and facilitating subsequent reactions central to metabolic energy production and biosynthesis via NADPH production. Hexokinases have undergone extensive evolution resulting in highly specialized enzymes and isoenzymes that have distinct intracellular location, substrate specificity and kinetics [1], [2]. This has resulted in highly specialized functions and accessory actions such as the key components of the glucose sensor in the pancreas and brain [3], [4]. The majority of hexokinases studied use ATP exclusively as the phosphoryl donor, though a novel mammalian hexokinase, expressed in rat and man, with a different evolutionary origin uses ADP [5]. Many bacteria and archaea also synthesise and use a variety of polyphosphate substrates [6], [7] which are considered evolutionary starting points for the ATP-dependent enzymes. Polyphosphate chains may contain 1000 inorganic phosphates joined by high energy phospho-anhydride bonds; they are readily formed under high temperatures, or by dehydration of inorganic phosphate, and would have been an available source of energy before ATP became ubiquitous [8]. Polyphosphate is present at varying concentration in all mammalian cells studied, although no enzymes have been identified to date responsible for either its synthesis or utilization [9]. Two organisms, a phosphate accumulating bacterium, Microlunatus phosphovorous[10] and a nitrogen-fixing cyanobacterium, Anabaena sp. PCC 1720, express a hexokinase which exclusively uses inorganic polyphosphate and, importantly, cannot phosphorylate hexoses with ATP or ADP [11]. We now report on a mammalian hexokinase which phosphorylates glucose exclusively using inorganic polyphosphate, and which is inhibited by ATP. Inorganic polyphosphate is often concentrated in the hepatocyte nucleus [12], with a variety of roles proposed, such as acting as a chaperone for nuclear proteins, but until now it has not been shown to be of metabolic significance [12]. The enzyme reported here is the first mammalian enzyme shown to utilize polyphosphate in a biochemical reaction. The enzyme is expressed predominantly in the nucleus of hepatocytes, but is also present in cardiac and striated muscle. Because of the enzyme's kinetics we have named it polyphosphate dependent GlucoKinase, mammalian, or PPGKm.