Functional analysis of the 5′ flanking region of the human G6PC3 gene: regulation of promoter activity by glucose, pyruvate, AMP kinase and the pentose phosphate pathway

G6PC3 is a widely expressed isoform of glucose-6-phosphatase, found in many foetal and adult tissues. Mutations in this gene cause developmental abnormalities and severe neutropenia due to abolition of glucose recycling between the cytoplasm and endoplasmic reticulum. Low G6PC3 expression as a result of promoter polymorphisms or dysregulation could produce similar outcomes. Here we investigated the regulation of human G6PC3 promoter activity. HeLa and H4IIE cells were transiently transfected with G6PC3 promoter coupled to the firefly luciferase gene, and promoter activity was measured by dual luciferase assay. Activity was highest in a 453 bp segment of the G6PC3 promoter, from − 455 to − 3 relative to the transcriptional start site. This promoter was unresponsive to glucostatic hormones. Its activity increased significantly between 1 and 5.5 mM glucose, and was not elevated further by glucose concentrations up to 25 mM. Pyruvate increased its activity, but β-hydroxybutyrate and sodium acetate did not. Promoter activity was reduced by inhibitors of hexokinase, glyceraldehyde phosphate dehydrogenase and the oxidative branch of the pentose phosphate pathway, but not by a transketolase inhibitor. Deletion of two adjacent Enhancer-boxes (− 274 to − 279 and − 299 to − 304) reduced promoter activity and abolished the glucose effect, suggesting they could function as a glucose response element. Deletion of an additional downstream 140 bp (− 140 to − 306) restored activity, but not the glucose response, suggesting the presence of repressor elements in this region. 5-Aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) reduced promoter activity, showing dependence on AMP-kinase. Regulation of the G6PC3 promoter is thus radically different to that of the hepatic isoform, G6PC. It is sensitive to carbohydrate, but not to fatty acid metabolites, and at much lower physiological concentrations. Based on these findings, we speculate that reduced G6PC3 expression could occur during hypoglycemic episodes in vivo, which are common in utero and in the postnatal period. If such episodes lower G6PC3 expression they could place the foetus or infant at risk of impaired immune function and development, and this possibility requires further examination both in vitro and in vivo.

Effects of a 12-week aerobic exercise intervention on eating behaviour, food cravings and 7-day energy intake and energy expenditure in inactive men

This study examined effects of 12 weeks of moderate-intensity aerobic exercise on eating behaviour, food cravings and weekly energy intake and expenditure in inactive men. Eleven healthy men (mean ± SD: age, 26 ± 5 years; body mass index, 24.6 ± 3.8 kg/m2; maximum oxygen uptake, 43.1 ± 7.4 mL/kg/min) completed the 12-week supervised exercise programme. Body composition, health markers (e.g. blood pressure), eating behaviour, food cravings and weekly energy intake and expenditure were assessed before and after the exercise intervention. There were no intervention effects on weekly free-living energy intake (p=0.326, d=-0.12) and expenditure (p=0.799, d=0.04), or uncontrolled eating and emotional eating scores (p>0.05). However, there was a trend with a medium effect size (p=0.058, d=0.68) for cognitive restraint to be greater after the exercise intervention. Total food cravings (p=0.009, d=-1.19) and specific cravings of high-fat foods (p=0.023, d=-0.90), fast-food fats (p=0.009, d=-0.71) and carbohydrates/starches (p=0.009, d=-0.56) decreased from baseline to 12 weeks. Moreover, there was a trend with a large effect size for cravings of sweets (p=0.052, d=-0.86) to be lower after the exercise intervention. In summary, 12 weeks of moderate-intensity aerobic exercise reduced food cravings and increased cognitive restraint, however, these were not accompanied by changes in other eating behaviours and weekly energy intake and expenditure. The results indicate the importance of exercising for health improvements even when reductions in body mass are modest.