Paola Marcolongo

Genotype/phenotype correlation in glycogen storage disease type 1b: a multicentre study and review of the literature

We studied the genotype/phenotype correlation in a cohort of glycogen storage disease type (GSD) 1b patients. A total of 25 GSD1b patients, 13 females and 12 males, age range: 4.3–28.4 years, mean:14.6±6.8 years; median: 15 years, representing the entire case load of Italian GSD1b patients, were enrolled in the study. Molecular analysis of the glucose 6-phosphate translocase (G6PT1) gene was performed in all patients. We analysed the presence of a correlation among both the clinical features associated with GSD1b (neutropenia, frequency of admission to the hospital for severe infections) and the presence of systemic complications (liver adenomas, nephropathy, bone mineral density defect, polycystic ovaries, short stature, inflammatory bowel disease) and the mutations detected in each patient. Nine patients were homozygous or compound heterozygous for mutations causing stop codons. In particular, three patients were homozygous for the same mutation (400X); of these patients, one showed chronic neutropenia with severe and frequent infections and severe inflammatory bowel disease, another patient cyclic neutropenia associated with rare bacterial infections and mild bowel involvement and the last one normal neutrophil count. Two patients were homozygous for the mutation 128X; one of these patients did not show neutropenia, whereas the other one had severe neutropenia needing frequent hospital admission and was under granulocyte-colony stimulating factor treatment. In three patients no mutations were detected. Conclusion:no correlation was found between individual mutations and the presence of neutropenia, bacterial infections and systemic complications. These results suggest that different genes and proteins modulate neutrophil differentiation, maturation and apoptosis and thus the severity and frequency of infections. The absence of detectable mutations in three patients could suggest that a second protein plays a role in microsomal phosphate transport.

Structure and mutation analysis of the glycogen storage disease type 1b gene

Glycogen storage disease (GSD) 1b is the deficiency of endoplasmic reticulum glucose‐6‐phosphate (G6P) transport. We here report the structure of the gene encoding a protein likely to be responsible for G6P transport, and its mapping to human chromosome 11q23.3. The gene is composed of nine exons spanning a genomic region of approximately 4 kb. Primers based on the genomic sequence were used in single strand conformation polymorphism (SSCP) analysis and mutations were found in six out of seven GSD 1b patients analysed.

Mutations in the glucose-6-phosphate transporter (G6PT) gene in patients with glycogen storage diseases type 1b and 1c

Glycogen storage diseases type 1 (GSD 1) are a group of autosomal recessive disorders characterized by impairment of terminal steps of glycogenolysis and gluconeogenesis. Mutations of the glucose‐6‐phosphatase gene are responsible for the most frequent form of GSD 1, the subtype 1a, while mutations of the glucose‐6‐phosphate transporter gene (G6PT) have recently been shown to cause the non 1a forms of GSD, namely the 1b and 1c subtypes. Here, we report on the analysis by single‐stranded conformation polymorphism (SSCP) and/or DNA sequencing of the exons of the G6PT in 14 patients diagnosed either as affected by the GSD 1b or 1c subtypes. Mutations in the G6PT gene were found in all patients. Four of the detected mutations were novel mutations, while the others were previously described. Our results confirm that the GSD 1b and 1c forms are due to mutations in the same gene, i.e. the G6PT gene. We also show that the same kind of mutation can be associated or not with evident clinical complications such as neutrophil impairment. Since no correlation between the type and position of the mutation and the severity of the disease was found, other unknown factors may cause the expression of symptoms, such as neutropenia, which dramatically influence the severity of the disease.

GULONOLACTONE OXIDASE ACTIVITY-DEPENDENT INTRAVESICULAR GLUTATHIONE OXIDATION IN RAT LIVER MICROSOMES

The orientation of gulonolactone oxidase activity was investigated in rat liver microsomes. Ascorbate formation upon gulonolactone addition resulted in higher intravesicular than extravesicular ascorbate concentrations in native microsomal vesicles. The intraluminal ascorbate accumulation could be prevented or the accumulated ascorbate could be released by permeabilising the vesicles with the pore‐forming alamethicin. The formation of the other product of the enzyme, hydrogen peroxide caused the preferential oxidation of intraluminal glutathione in glutathione‐loaded microsomes. In conclusion, these results suggest that the orientation of the active site of gulonolactone oxidase is intraluminal and/or the enzyme releases its products towards the lumen of the endoplasmic reticulum.

Subcellular compartmentation of ascorbate and its variation in disease states

Beyond its general role as antioxidant, specific functions of ascorbate are compartmentalized within the eukaryotic cell. The list of organelle-specific functions of ascorbate has been recently expanded with the epigenetic role exerted as a cofactor for DNA and histone demethylases in the nucleus. Compartmentation necessitates the transport through intracellular membranes; members of the GLUT family and sodium-vitamin C cotransporters mediate the permeation of dehydroascorbic acid and ascorbate, respectively. Recent observations show that increased consumption and/or hindered entrance of ascorbate in/to a compartment results in pathological alterations partially resembling to scurvy, thus diseases of ascorbate compartmentation can exist. The review focuses on the reactions and transporters that can modulate ascorbate concentration and redox state in three compartments: endoplasmic reticulum, mitochondria and nucleus. By introducing the relevant experimental and clinical findings we make an attempt to coin the term of ascorbate compartmentation disease.

Contribution of Fructose-6-Phosphate to Glucocorticoid Activation in the Endoplasmic Reticulum: Possible Implication in the Metabolic Syndrome

Both fructose consumption and increased intracellular glucocorticoid activation have been implicated in the pathogenesis of the metabolic syndrome. Glucocorticoid activation by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) depends on hexose-6-phosphate dehydrogenase (H6PD), which physically interacts with 11β-HSD1 at the luminal surface of the endoplasmic reticulum (ER) membrane and generates reduced nicotinamide adenine dinucleotide phosphate for the reduction of glucocorticoids. The reducing equivalents for the reaction are provided by glucose-6-phosphate (G6P) that is transported by G6P translocase into the ER. Here, we show that fructose-6-phosphate (F6P) can substitute for G6P and is sufficient to maintain reductase activity of 11β-HSD1 in isolated microsomes. Our findings indicate that the mechanisms of F6P and G6P transport across the ER membrane are distinct and provide evidence that F6P is converted to G6P in the ER lumen, thus yielding substrate for H6PD-dependent reduced nicotinamide adenine dinucleotide phosphate generation. Using the purified enzyme, we show that F6P cannot be directly dehydrogenated by H6PD, and we also excluded H6PD as a phosphohexose isomerase. Therefore, we postulate the existence of an ER luminal hexose-phosphate isomerase different from the cytosolic enzyme. The results suggest that cytosolic F6P promotes prereceptor glucocorticoid activation in white adipose tissue, which might have a role in the pathophysiology of the metabolic syndrome.

Caffeine releases a glucose-primed endoplasmic reticulum Ca2+ pool in the insulin secreting cell line INS-1

Caffeine mobilized an intracellular Ca2+ pool in intact fura‐2‐loaded INS‐1 cells in suspension exposed to high (16 mM) [glucose], while a minor effect was observed with low (2 mM) [glucose]. Cells were kept in a medium containing diaxozide or no Ca2+ to prevent the influx of extracellular Ca2+. The caffeine‐sensitive intracellular Ca2+ pool was within the endoplasmic reticulum since it was depleted by the inhibitor of the reticular Ca2+ pumps thapsigargin and the InsP3‐dependent agonist carbachol. No effect of caffeine was observed in the parent glucose‐insensitive RINmF5 cells. In microsomes from INS‐1 but not RINmF5 cells, the type 2 ryanodine receptor was present as revealed by Western blotting. It was concluded that the endoplasmic reticulum of INS‐1 cells possesses caffeine‐sensitive type 2 ryanodine receptors Ca2+ channels.

Maintenance of luminal NADPH in the endoplasmic reticulum promotes the survival of human neutrophil granulocytes

The present study demonstrates the expression of hexose‐6‐phosphate dehydrogenase and 11β‐hydroxysteroid dehydrogenase type 1 in human neutrophils, and the presence and activity of these enzymes in the microsomal fraction of the cells. Their concerted action together with the previously described glucose‐6‐phosphate transporter is responsible for cortisone–cortisol interconversion detected in human neutrophils. Furthermore, the results suggest that luminal NADPH generation by the cortisol dehydrogenase activity of 11β‐hydroxysteroid dehydrogenase type 1 prevents neutrophil apoptosis provoked by the inhibition of the glucose‐6‐phosphate transporter. In conclusion, the maintenance of the luminal NADPH pool is an important antiapoptotic factor in neutrophil granulocytes.

Metyrapone prevents cortisone-induced preadipocyte differentiation by depleting luminal NADPH of the endoplasmic reticulum

Preadipocyte differentiation is greatly affected by prereceptorial glucocorticoid activation catalyzed by 11beta-hydroxysteroid dehydrogenase type 1 in the lumen of the endoplasmic reticulum. The role of the local NADPH pool in this process was investigated using metyrapone as an NADPH-depleting agent. Metyrapone administered at low micromolar concentrations caused the prompt oxidation of the endogenous NADPH, inhibited the reduction of cortisone and enhanced the oxidation of cortisol in native rat liver microsomal vesicles. However, in permeabilized microsomes, it only slightly decreased both NADPH-dependent cortisone reduction and NADP(+)-dependent cortisol oxidation. Accordingly, metyrapone administration caused a switch in 11beta-hydroxysteroid dehydrogenase activity from reductase to dehydrogenase in both 3T3-L1-derived and human stem cell-derived differentiated adipocytes. Metyrapone greatly attenuated the induction of 11beta-hydroxysteroid dehydrogenase type 1 and the accumulation of lipid droplets during preadipocyte differentiation when 3T3-L1 cells were stimulated with cortisone, while it was much less effective in case of cortisol or dexamethasone. In conclusion, the positive feedback of glucocorticoid activation during preadipocyte differentiation is interrupted by metyrapone, which depletes NADPH in the endoplasmic reticulum. The results also indicate that the reduced state of luminal pyridine nucleotides in the endoplasmic reticulum is important in the process of adipogenesis.

Glucose 6-phosphate transport in fibroblast microsomes from glycogen storage disease type 1b patients: evidence for multiple glucose 6-phosphate transport systems.

In liver endoplasmic reticulum the intralumenal glucose-6-phosphatase activity requires the operation of a glucose 6-phosphate transporter (G6PT1). Mutations in the gene encoding G6PT1 cause glycogen storage disease type 1b, which is characterized by a loss of glucose-6-phosphatase activity and impaired glucose homoeostasis. We describe a novel glucose 6-phosphate (G6P) transport activity in microsomes from human fibroblasts and HeLa cells. This transport activity is unrelated to G6PT1 since: (i) it was similar in microsomes of skin fibroblasts from glycogen storage disease type 1b patients homozygous for mutations of the G6PT1 gene, and in microsomes from human control subjects; (ii) it was insensitive to the G6PT1 inhibitor chlorogenic acid; and (iii) it was equally active towards G6P and glucose 1-phosphate, whereas G6PT1 is highly selective for G6P. Taken together, our results provide evidence for the presence of multiple transporters for G6P (and other hexose phosphoesters) in the endoplasmic reticulum.