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    • Nuclear export of GK has been reported

    2022-05-11

    Nuclear export of GK has been reported to be dependent on a GK nuclear export signal (NES), mapped to residues 300–310 (Shiota et al., 1999). The assignment of a nuclear export signal common to both the liver and pancreatic GK isoform sequence suggests a possible reversible translocation of GK across the nuclear membrane also in pancreatic beta-cells. In the 3D structure of GK (Supplementary Fig. 4), the NES signal (in helix-8) is located in close proximity to the SUMOylation sites previously identified in pancreatic GK (Lys12,13,15 of helix-1 and Lys346 of helix-10) (Aukrust et al., 2013). Considering the size of SUMO proteins (∼12 kDa), the attachment of one or more SUMO proteins at Lys12,13,15 and 346 may mask the NES and prevent nuclear export of GK while SUMOylated. In conclusion, we demonstrate for the first time to our knowledge a nuclear localization of GK in human and mouse isolated islets, as well as in beta-cells in human and mouse pancreatic sections. Furthermore, our results suggest that import of GK to the nucleus in MIN6 cells take place by a redundant mechanism involving a NLS and is modulated by the SUMOylation machinery. Although glucose-phosphorylating activity has been confirmed for nuclear GK (Kaminski et al., 2014), the purpose for the nuclear retention of the enzyme in pancreatic beta-cells is still unclear. The functional implications of GK in the nucleus represents a future challenge.
    Conflicts of interest
    Author contribution
    Funding This study was supported by grants from the University of Bergen, Western Norway Regional Health Authority, The Novo Nordisk Foundation, The Research Council of Norway, The Norwegian Diabetes Association, The Stiftelsen Kristian Gerhard Jebsen and The National Institutes of Health.
    Acknowledgments
    Introduction The glucokinase (GK, EC 2.7.1.1), also known as hexokinase (HK) IV (or D), is one of the four glucose-phosphorylating isoenzymes described initially in the vertebrate liver. The other three are called HK I (or A), II (or B) and III (or C) (Panserat et al., 2014). All of them catalyze the ATP-dependent phosphorylation of glucose to glucose-6-phosphate as the first step, and the first rate-limiting step, in the glycolytic pathway (Kawai et al., 2005). GK, a molecular weight of 50kDa, is characterized by a low affinity for glucose and by a lack of product inhibition by glucose-6-phosphate compared with the hexokinases I to III (Cheng et al., 2012). GK is widely expressed in liver, brain, pancreas, and intestine of humans and most other vertebrates. The function of GK protein in these tissues is not to convert excess glucose to glycogen or lipids (through lipogenesis) as in the liver but more to act as a glucose sensor to help the organism regulate glucose homeostasis (Polakof et al., 2012). Glucokinase also has an important role as glucose sensor and metabolic signal generator in pancreatic β-cells and hepatocytes (Egea et al., 2008). GK in the fak pathway can also be a glucose sensor in fish. Its activation in the brain could be related to modulation of the decrease in food intake with hyperglycaemia in fish (Polakof et al., 2008a, Polakof et al., 2008b), as in mammals. Moreover, spontaneous mutations of the GK gene are manifested in a wide range of pathologies of glucose homeostasis, including hypoglycaemias, milder forms of persistent hyperinsulinaemic hypoglycaemia of infancy, borderline and mild hyperglycaemias of maturity-onset diabetes of the young type 2 (MODY2), and life-threatening permanent neonatal diabetes mellitus requiring intensive and lifelong insulin therapy (Matschinsky, 2009, Panserat et al., 2014). Carbohydrates are an excellent source of energy and carbon, but in principle as in other vertebrates, fish can survive and grow when fed diets without carbohydrates because of their ability to efficiently synthesize glucose from non-carbohydrate precursors such as pyruvate, lactate and amino acids (council, 2011, Kamalam et al., 2017). Nevertheless, optimal inclusion of carbohydrates in the diet of cultured fish can increase retention of protein and lipid by preventing the catabolism of these expensive nutrients for energy needs; provide metabolites for biological syntheses; support feed formulations that maintain growth at a lower cost per unit gain; help pellet binding, stability and floatability; and facilitate the removal of faeces through their binding properties (Hardy, 2010, Honorato et al., 2010, Panserat et al., 2014). Previous studies have shown that carbohydrate digestibility can be affected by various biological, nutritional and environmental factors influencing carbohydrate use, such as feeding habit based evolutionary hardwiring, genotypic differences, sustained swimming exercise, influence of other dietary components, carbohydrate source characteristics, gelatinization, meal timing and changes in thermal regime (Kamalam et al., 2017). However, studies on gene expression and activities of the key carbohydrate metabolic enzymes which can be used as indicators of carbohydrate digestibility in fish are limited (Metón et al., 1999). Recently, the studies on the hepatic GK activity and gene expression are more and more meaningful, especially about advancing the relative inability of fish to efficiently utilize dietary carbohydrates (Panserat et al., 2000, Panserat et al., 2001, Soengas et al., 2006). High dietary carbohydrate levels lead to activations of enzymes of the anabolic pathways of glucose disposal (glycolysis, such as glucokinase), glycogen synthesis from glucose (glycogenesis) and de novo synthesis of lipids (lipogenesis) are activated (Kahn, 1997, Pilkis and Granner, 1992, Postic et al., 2004). The studies related to GK have been conducted in mammals, but little in fish. Previous studies have shown GK activity can be regulated by dietary carbohydrates in rainbow trout (Panserat et al., 2000, Skiba-Cassy et al., 2013), gilhead seabream (Egea et al., 2007, Panserat et al., 2000), common carp (Panserat et al., 2000), Dicentrarchus labrax (Pérez-Jiménez et al., 2007). However, other researchers have demonstrated that the above results has not been observed in theirstudies (Cowey et al., 1977, Nagayama and Oshima, 1974, Panserat et al., 2000). Further studies are thus needed to clarify this discrepancy in fish. Currently, GK had only been cloned in some fish. Until now the complete coding sequences of Danio rerio (GenBank: BC122359), Oncorhynchus mykiss (GenBank: AF053331), Sparus aurata (AF053330), Cyprinus carpio (AF053332) and Ctenopharyngodon idella (GU065314) have been reported. As a consequence, it is important and urgent to research the features and modulation of GK in fish.