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  • Blebbistatin mg The subcellular redistribution of some metab

    2021-10-16

    The subcellular redistribution of some metabolic enzymes is dynamic and responsive to metabolic conditions. In the absence of glucose, Glucokinase (GK) is bound to its regulatory protein in the nucleus of the hepatocyte and translocates to the cytoplasm when sugar levels increase [12]. Muscle glycogen synthase (GS) is also concentrated in the nucleus at low glucose and translocates to the cytosol, where it adopts a particulate pattern at high glucose concentrations [13], [14]. In contrast, liver GS presents a cytosolic distribution in the absence of glucose and concentrates at the periphery of the hepatocytes when the hexose concentration Blebbistatin mg increases [15].
    Materials and methods
    Results We studied the effect of glucose and DHA (substrates) and insulin on the intracellular distribution of liver FBPase by immunofluorescence and confocal analysis in cultured rat hepatocytes. We concomitantly analyzed the subcellular distribution patterns of GK and GS and compared them with that of FBPase. In addition, we evaluated the effect of glucose, DHA and insulin on the intracellular Glu-6-P concentration and glycogen content. Hepatocytes were maintained for 16 h in a medium devoid of substrates and then incubated for 6 h with 25 mM glucose or 10 mM DHA in the absence or presence of 10 nM insulin. In the hepatocytes incubated in the absence of substrates, the level of glycogen and Glu-6-P was very low (Table 1). In this condition, FBPase was observed in the cytoplasm of the hepatocytes, showing a strong immunostaining in the cell periphery around the group of the cells. Low immunoreaction was detected in the nuclei (Fig. 1B and H). When the Blebbistatin mg were incubated with 25 mM glucose, the enzyme changed its subcellular localization and concentrated within the nucleus (Fig. 2E and K). As previously reported, GK was present in the nucleus in the absence of glucose and translocated to the cytosol in the presence of the sugar, and showed a visible juxtanuclear localization (Fig. 1A and D). Detailed co-localization analysis of FBPase with GK showed that these two enzymes translocated in opposite directions in response to glucose (Fig. 1A and D, respectively). Additionally, in response to high glucose concentration, FBPase was recruited to the cell periphery (Fig. 1D–F and J–L). As previously described, GS changed its cellular distribution from a uniform cytoplasmatic distribution to an accumulation at the cell periphery (Fig. 1G and J), where it showed a high degree of co-localization with FBPase (Fig. 1J–L). In the hepatocytes incubated with 25 mM glucose, there was an 8- and 12-fold increase in the intracellular concentration of glycogen and Glu-6-P, respectively, when compared with those incubated in the absence of substrates (Table 1). DHA is an effective gluconeogenic substrate, thus we analyzed its effect on the intracellular distribution of FBPase. DHA (10 mM) induced the same pattern of FBPase and GS localization as glucose. DHA stimulus produced the recruitment of the two enzymes in peripheral compartments of the hepatocytes. Moreover, DHA induced FBPase translocation into the nucleus, but to a lesser extent than that produced by glucose. By contrast, this gluconeogenic precursor was unable to elicit any change in GK subcellular distribution and the enzyme remained in the nucleus (data not shown). Incubation with this substrate led to a marked increase in Glu-6-P levels, which were about 22-fold higher than in control cells (Table 1). Under these conditions, the hepatocytes produced the same amount of glycogen as those incubated with 25 mM glucose (Table 1). We next analyzed the effect of insulin in the absence and in the presence of glucose. In hepatocytes incubated with 10 nM insulin in the absence of glucose, FBPase translocated largely into the nucleus (Fig. 2D–F), although it presented a less significant peripheral distribution than that observed when cells were incubated with glucose alone. In this condition, there was a slight increase in glycogen content (Table 1). When incubated with 10 nM insulin plus 25 mM glucose, FBPase showed a similar accumulation at the cell periphery but was clearly more concentrated in the nucleus of the hepatocytes than when incubated with glucose alone (Fig. 2G–L). The same distribution pattern was observed in hepatocytes co-incubated with 10 mM DHA and 10 nM insulin (data not shown). Insulin alone produced a limited effect on GS distribution; nevertheless, this hormone potentiated the glucose-induced recruitment of GS towards the cell periphery (Fig. 2G and J). Interestingly, hepatocytes co-incubated with 10 mM DHA and 10 nM insulin used this substrate in the gluconeogenic production of Glu-6-P and glycogen.