A test-retest multisite reproducibility study of cardiovascular four-dimensional flow MRI without respiratory gating.

AIM: To systematically investigate the multisite reproducibility, test-retest reliability, and observer variability of non-respiratory-gated four-dimensional (4D) flow magnetic resonance imaging (MRI) in the thoracic great vessels for the assessment of blood flow and peak velocity. MATERIALS AND METHODS: Electrocardiogram (ECG)-gated 4D flow MRI data were acquired without respiratory gating in 10 healthy volunteers. To analyse multisite reproducibility, 4D flow was scanned at three different sites using a 3 T GE MRI machine with identical protocols for the group of participants. In addition, to evaluate test-retest reliability, the same volunteers were scanned in each centre during a second visit. Data analysis included calculation of peak systolic velocity and time-resolved and total flow of both the ascending aorta and pulmonary artery. Two observers conducted the above measurements to assess the interobserver variability. RESULTS: Multisite, test-retest, interobserver agreement were good for the calculation of total flow and peak systolic velocity (mean differences <10% of the average flow parameter). CONCLUSION: Non-respiratory-gated 4D MRI-based assessment of aortic and pulmonary blood flow can be performed with good reproducibility. It may facilitate the potential clinical application of this technique.

Abnormal spontaneous brain activity in type 2 diabetic retinopathy revealed by amplitude of low-frequency fluctuations: a resting-state fMRI study.

AIM: To explore the altered spontaneous cerebral activity patterns and impaired functional regions in patients with diabetic retinopathy (DR) using resting-state functional magnetic resonance imaging (rs-fMRI) based on the amplitude of low-frequency fluctuations (ALFF) algorithm. MATERIALS AND METHODS: Twenty-one patients with DR (mean age, 54.9±9.9 years; 11 females) and 17 healthy control subjects (54.8±5.7 years; 9 females) were prospectively studied. The DR patients underwent laboratory tests. All individuals underwent a neuropsychological test. The differences in the ALFF values between the two groups were compared. The relationships between ALFF values and clinical measurements were analysed using a multiple-factor analysis. RESULTS: Compared to the controls, the DR group showed significantly increased ALFF values in the bilateral occipital gyrus, right lingual gyrus, and precuneus, and decreased values in the right posterior/anterior cerebellar lobe and the parahippocampal, fusiform, superior temporal, inferior parietal, and angular gyrus. Furthermore, the Montreal Cognitive Assessment (MoCA) scores were negatively correlated with decreased ALFF values in the right occipital lobe of the DR group, while increased ALFF values in the right precuneus and lingual gyrus were found to be positively correlated with glycosylated haemoglobin (HbA1c) levels. CONCLUSIONS: Patients with DR showed spontaneous cerebral activity abnormalities in many cerebral regions that were associated with cognitive impairments. HbA1c levels altered spontaneous cerebral activity in DR patients.

Evaluation of nasal patency by visual analogue scale/nasal obstruction symptom evaluation questionnaires and anterior active rhinomanometry after septoplasty: a retrospective one-year follow-up cohort study.

OBJECTIVE: To assess the efficacy of septoplasty and the correlation between the subjective evaluations of a visual analogue scale (VAS) and the Nasal Obstruction Symptom Evaluation (NOSE) questionnaire and active anterior rhinomanometry of the nasal airway after septoplasty. DESIGN: A retrospective, individual cohort study. SETTING: Ear, Nose and Throat Department, Taipei City Hospital, Taipei, Taiwan. PARTICIPANTS: Fifty patients with chronic nasal obstruction were enrolled in the study. All 50 patients underwent septoplasty because of nasal septal deviation. Another 28 patients without nasal symptoms served as controls. MAIN OUTCOME MEASURES: VAS, NOSE and active anterior rhinomanometry were used to measure the sensation of nasal obstruction. All measurements were performed in both groups preoperatively and then repeated on three postoperative visits (3, 6 and 12 months). RESULTS: The mean VAS score, NOSE score and the nasal resistance in the narrow side of the nose in the study group showed reduced symptoms at 3, 6 and 12 months postoperatively compared with the respective preoperative measurements (P < 0.001, all). The VAS and NOSE scores did not significantly correlate with total nasal resistance preoperatively or postoperatively. The VAS and nasal resistance in the obstructed nasal cavity correlated significantly preoperatively (P < 0.05), but not postoperatively. CONCLUSIONS: The subjective and objective symptoms of nasal obstruction had improved 1 year after septoplasty. A significant correlation between VAS scores and nasal resistance in the narrow side of the nose was found before surgery. The subjective and objective measurements of nasal obstruction lacked significant correlation postoperatively.

Equatorial atmospheric Kelvin waves during El Niño episodes and their effect on stratospheric QBO.

Equatorial atmospheric Kelvin waves are investigated during a positive El Niño Southern Oscillation (ENSO) episode using temperature data retrieved from GPS Radio Occultation (RO) observations of FORMOSAT-3/COSMIC during the period from August 2006 to December 2013. Enhanced Kelvin wave amplitudes are observed during the El Niño episode of 2009-2010 and it is also observed that these amplitudes correlate with the Niño 3.4 index and also with outgoing longwave radiation and trade wind index. This study indicates that the enhanced equatorial atmospheric Kelvin wave amplitudes might be produced by geophysical processes that were involved in the onset and development of the El Niño episode. Further, easterly winds above the tropopause during this period favored the vertically upward propagation of these waves that induced a fast descending westerly regime by the end of 2010, where the zero-wind line is observed to take only 5 months to descend from 10 to 50 hPa. The current study presents observational evidence of enhanced Kelvin wave amplitudes during El Niño that has affected the stratospheric quasi-biennial oscillation (QBO) through wave-mean flow interactions. Earlier El Niño episodes of 1987 and 1998 are also qualitatively investigated, using reanalysis data. It is found that there might have been an enhancement in the equatorial Kelvin wave amplitudes during almost all El Niño episodes, however, an effect of a fast descending westerly is observed in the QBO only when the ambient zonal winds in the lower stratosphere favor the upward propagation of the Kelvin waves and consequently they interact with the mean flow. This study indicates that the El Niño and QBO are not linearly related and wave mean flow interactions play a very important role in connecting these two geophysical phenomena.

Angiotensin mediates renal fibrosis in the nephropathy of glycogen storage disease type Ia.

Patients with glycogen storage disease type Ia (GSD-Ia) develop renal disease of unknown etiology despite intensive dietary therapies. This renal disease shares many clinical and pathological similarities to diabetic nephropathy. We studied the expression of angiotensinogen, angiotensin type 1 receptor, transforming growth factor-beta1, and connective tissue growth factor in mice with GSD-Ia and found them to be elevated compared to controls. While increased renal expression of angiotensinogen was evident in 2-week-old mice with GSD-Ia, the renal expression of transforming growth factor-beta and connective tissue growth factor did not increase for another week; consistent with upregulation of these factors by angiotensin II. The expression of fibronectin and collagens I, III, and IV was also elevated in the kidneys of mice with GSD-Ia, compared to controls. Renal fibrosis was characterized by a marked increase in the synthesis and deposition of extracellular matrix proteins in the renal cortex and histological abnormalities including tubular basement membrane thickening, tubular atrophy, tubular dilation, and multifocal interstitial fibrosis. Our results suggest that activation of the angiotensin system has an important role in the pathophysiology of renal disease in patients with GSD-Ia.

Preparation and characterization of rapamycin-loaded PLGA coating stent.

In this study, using polylactic acid-co-glycolic acid (PLGA) with a molecular weight of 95,800 Da as drug carrier, three dose (low, moderate, high) rapamycin-eluting stents and the corresponding coating films were prepared. The pre- and post-expansion morphology of the rapamycin-eluting stent was examined by scanning electron microscopy (SEM), indicating that the coating was very smooth and uniform. The coating had the ability to withstand the compressive and tensile strains imparted without cracking from the stent during expansion process. There were many voids on stent coating surface after released for 18 days in release medium. The thermodynamics data of the stent coating film measured by differential scanning calorimetry (DSC) showed a lack of measurable solubility of rapamycin in the PLGA matrix. The release behavior of rapamycin from stent surface had a two phase release profile with a burst release period of about 2 days, followed by a sustained and slow release phase. The mass loss behavior of PLGA appeared linear throughout most of the degradation period, corresponding to an approximately constant mass loss rate. The platelet adhesion tests showed that the rapamycin-eluting films may have a good blood compatibility compared with control samples. Take into these results account, this novel rapamycin-eluting may be a good candidate to resolve in-stent restenosis.

Improved blood compatibility of rapamycin-eluting stent by incorporating curcumin.

This paper dealt with improving the blood compatibility of the rapamycin-eluting stent by incorporating curcumin. The rapamycin- and rapamycin/curcumin-loaded PLGA (poly(d,l-lactic acid-co-glycolic acid)) coatings were fabricated onto the surface of the stainless steel stents using an ultrasonic atomization spray method. The structure of the coating films was characterized by Fourier transform infrared spectroscopy (FTIR). The optical microscopy and scanning electron microscopy (SEM) images of the drug-eluting stents indicated that the surface of all drug-eluting stents was very smooth and uniform, and there were not webbings and "bridges" between struts. There were not any cracks and delaminations on stent surface after expanded by the angioplasty balloon. The in vitro platelet adhesion and activation were investigated by static platelet adhesion test and GMP140 (P-selection), respectively. The clotting time was examined by activated partially prothromplastin time (APTT) test. The fibrinogen adsorption on the drug-loaded PLGA films was evaluated by enzyme-linked immunosorbent assay (ELISA). All obtained data showed that incorporating curcumin in rapamycin-loaded PLGA coating can significantly decrease platelet adhesion and activation, prolong APTT clotting time as well as decrease the fibrinogen adsorption. All results indicated that incorporating curcumin in rapamycin-eluting coating obviously improve the blood compatibility of rapamycin-eluting stents. It was suggested that it may be possible to develop a drug-eluting stent which had the characteristics of not only good anti-proliferation but also improved anticoagulation.

In vitro studies of platelet adhesion, activation, and protein adsorption on curcumin-eluting biodegradable stent materials.

A major complication of coronary stenting is in-stent restenosis (ISR) due to thrombus formation. We hypothesized that locally released curcumin from coronary stent surface would inhibit ISR due to thrombus formation because of antithrombosis of curcumin. In the present work, curcumin-eluting polylactic acid-co-glycolic acid (PLGA) films were fabricated and their properties in vitro were investigated. The in vitro platelet adhesion and activation, as well as protein adsorption on curcumin-loading PLGA films were investigated to evaluate the blood compatibility of curcumin-eluting films. The structure of curcumin-eluting PLGA film and control was examined by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy indicating that the peaks of curcumin did not shift in curcumin-eluting films. The results of contact angle and surface free energy indicated that loading curcumin in PLGA would make PLGA become more hydrophilic, which contributed to the increase of polar fraction of surface free energy. With the increase of curcumin in films, platelets adhering to the curcumin-eluting films decreased significantly. The number of activation platelets decreased after incorporating curcumin in PLGA films. Loading curcumin in PLGA film can markedly reduce the fibrinogen adsorption. All results indicated that incorporating curcumin in PLGA film can improve the blood compatibility of PLGA films. It can be used to fabricate drug-eluting stent to prevent thrombosis formation.

Glucose-6-phosphate transporter gene therapy corrects metabolic and myeloid abnormalities in glycogen storage disease type Ib mice.

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT), an endoplasmic reticulum-associated transmembrane protein that is ubiquitously expressed. GSD-Ib patients suffer from disturbed glucose homeostasis and myeloid dysfunctions. To evaluate the feasibility of gene replacement therapy for GSD-Ib, we have infused adenoviral (Ad) vector containing human G6PT (Ad-hG6PT) into G6PT-deficient (G6PT(-/-)) mice that manifest symptoms characteristics of the human disorder. Ad-hG6PT infusion restores significant levels of G6PT mRNA expression in the liver, bone marrow and spleen, and corrects metabolic as well as myeloid abnormalities in G6PT(-/-) mice. The G6PT(-/-) mice receiving gene therapy exhibit improved growth; normalized serum profiles for glucose, cholesterol, triglyceride, uric acid and lactic acid; and reduced hepatic glycogen deposition. The therapy also corrects neutropenia and lowers the elevated serum levels of granulocyte colony-stimulating factor. The development of bone and spleen in the infused G6PT(-/-) mice is improved and accompanied by increased cellularity and normalized myeloid progenitor cell frequencies in both tissues. This effective use of gene therapy to correct metabolic imbalances and myeloid dysfunctions in GSD-Ib mice holds promise for the future of gene therapy in humans.

Long-term correction of murine glycogen storage disease type Ia by recombinant adeno-associated virus-1-mediated gene transfer.

Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in glucose-6-phosphatase-alpha (G6Pase-alpha), a nine-transmembrane domain, endoplasmic reticulum-associated protein expressed primarily in the liver and kidney. Previously, we showed that infusion of an adeno-associated virus (AAV) serotype 2 vector carrying murine G6Pase-alpha (AAV2-G6Pase-alpha) into neonatal GSD-Ia mice failed to sustain their life beyond weaning. We now show that neonatal infusion of GSD-Ia mice with an AAV serotype 1-G6Pase-alpha (AAV1-G6Pase-alpha) or AAV serotype 8-G6Pase-alpha (AAV8-G6Pase-alpha) results in hepatic expression of the G6Pase-alpha transgene and markedly improves the survival of the mice. However, only AAV1-G6Pase-alpha can achieve significant renal transgene expression. A more effective strategy, in which a neonatal AAV1-G6Pase-alpha infusion is followed by a second infusion at age one week, provides sustained expression of a complete, functional, G6Pase-alpha system in both the liver and kidney and corrects the metabolic abnormalities in GSD-Ia mice for the 57 week length of the study. This effective use of gene therapy to correct metabolic imbalances and disease progression in GSD-Ia mice holds promise for the future of gene therapy in humans.

In islet-specific glucose-6-phosphatase-related protein, the beta cell antigenic sequence that is targeted in diabetes is not responsible for the loss of phosphohydrolase activity.

AIMS/HYPOTHESIS: There are three members of the glucose-6-phosphatase (G6Pase) family: (1) the liver/kidney/intestine G6Pase-alpha (encoded by G6PC), which is a key enzyme in glucose homeostasis; (2) the ubiquitous G6Pase-beta (encoded by G6PC3); and (3) the islet-specific G6Pase-related protein (IGRP, encoded by /G6PC2). While G6Pase-alpha and G6Pase-beta are functional glucose-6-phosphate hydrolases, IGRP possesses almost no hydrolase activity. This was unexpected since G6Pase-alpha is more closely related to IGRP than G6Pase-beta. Recently, amino acids 206-214 in IGRP were identified as a beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes, suggesting that this peptide confers functional specificity to IGRP. We therefore investigated the molecular events that inactivate IGRP activity and the effects of the beta cell antigen sequence on the stability and enzymatic activity of G6Pase-alpha. METHODS: Studies were performed using site-directed mutagenesis and transient expression assays. Protein stability was evaluated by Western blotting, proteasome inhibitor studies and in vitro transcription-translation. RESULTS: We showed that the residues responsible for G6Pase activity are more extensive than previously recognised. Introducing the IGRP antigenic motif into G6Pase-alpha does not completely destroy activity, although it does destabilise the protein. The low hydrolytic activity in IGRP is due to the combination of multiple independent mutations. CONCLUSIONS/INTERPRETATION: The loss of catalytic activity in IGRP arises from the sum of many sequence differences. G6Pase-alpha mutants containing the beta cell antigen sequence are preferentially degraded in cells, which prevents targeting by pathogenic CD8+ T cells. It is possible that IGRP levels in beta cells could dictate susceptibilities to diabetes.

Mechanisms of nitrogen oxide-mediated disruption of metalloprotein function: an examination of the copper-responsive yeast transcription factor Ace1.

Nitric oxide (NO) has been found to inhibit the copper-responsive yeast transcription factor Ace1 in an oxygen-dependent manner. However, the mechanism responsible for NO-dependent inhibition of Ace1 remains unestablished. In the present study, the chemical interaction of nitrogen oxide species with Ace1 was examined using a yeast reporter system. Exposure of yeast to various nitrogen oxides, under a variety of conditions, revealed that the oxygen-dependent inhibition of Ace1 is due to the reaction of NO with O(2). The nitrosating nitrogen oxide species N(2)O(3) is likely to be the disrupter of Ace1 activity. Considering the similarity of metal-thiolate ligation in Ace1 with other mammalian metalloproteins such as metallothionein, metal chaperones, and zinc-finger proteins, these results help to understand the biochemical interactions of NO with those mammalian metalloproteins.

Discriminant responses of the catalytic unit and glucose 6-phosphate transporter components of the hepatic glucose-6-phosphatase system in Ehrlich ascites-tumor-bearing mice.

The effect of Ehrlich ascites tumor cells, in vivo, on the hepatic glucose-6-phosphatase (G6Pase) system was examined. The V(max) for glucose 6-phosphate hydrolysis by G6Pase was reduced by 40% and a greater than 15-fold decrease in mRNA encoding the catalytic unit of the G6Pase system was observed 8 days after injection with tumor cells. Blood glucose concentration was decreased from 169 +/- 17 to 105 +/- 9 mg/dl in tumor-bearing mice. There was no change in the G6P transporter (G6PT) mRNA level. However, there was a significant decrease in G6P accumulation into hepatic microsomal vesicles derived from tumor-bearing mice. Decreased G6P accumulation was also associated with a decrease in G6Pase hydrolytic activity in the presence of vanadate, a potent catalytic-unit inhibitor. In addition, G6P accumulation was nearly abolished in microsomes treated with N-bromoacetylethanolamine phosphate, an irreversible inhibitor of the G6PT. These results demonstrate that the catalytic unit and G6PT components of the G6Pase system can be discriminantly regulated, and that microsomal glucose 6-phosphate uptake is dependent on catalytic unit activity as well as G6PT action.

A molecular link between the common phenotypes of type 1 glycogen storage disease and HNF1alpha-null mice.

The clinical manifestations of type 1 glycogen storage disease (GSD-1) in patients deficient in the glucose-6-phosphatase (G6Pase) system (e.g. growth retardation, hepatomegaly, hyperlipidemia, and renal dysfunction) are shared by Hnf1alpha(-/-) mice deficient of a transcriptional activator, hepatocyte nuclear factor 1alpha (HNF1alpha). However, the molecular mechanism is unknown. The G6Pase system, essential for the maintenance of glucose homeostasis, is comprised of glucose 6-phosphate transporter (G6PT) and G6Pase. G6PT translocates G6P from the cytoplasm to the lumen of the endoplasmic reticulum where it is metabolized by G6Pase to glucose and phosphate. Deficiencies in G6Pase and G6PT cause GSD-1a and GSD-1b, respectively. Hnf1alpha(-/-) mice also develop noninsulin-dependent diabetes mellitus caused by defective insulin secretion. In this study, we sought to determine whether there is a molecular link between HNF1alpha deficiency and function of the G6Pase system. Transactivation studies revealed that HNF1alpha is required for transcription of the G6PT gene. Hepatic G6PT mRNA levels and microsomal G6P transport activity are also markedly reduced in Hnf1alpha(-/-) mice as compared with Hnf1alpha(+/+) and Hnf1alpha(+/-) littermates. On the other hand, hepatic G6Pase mRNA expression and activity are up-regulated in Hnf1alpha(-/-) mice, consistent with observations that G6Pase expression is increased in diabetic animals. Taken together, the results strongly suggest that metabolic abnormalities in HNF1alpha-null mice are caused in part by G6PT deficiency and by perturbations of the G6Pase system.

Structural requirements for the stability and microsomal transport activity of the human glucose 6-phosphate transporter.

Deficiencies in glucose 6-phosphate (G6P) transporter (G6PT), a 10-helical endoplasmic reticulum transmembrane protein of 429 amino acids, cause glycogen storage disease type 1b. To date, only three missense mutations in G6PT have been shown to abolish microsomal G6P transport activity. Here, we report the results of structure-function studies on human G6PT and demonstrate that 15 missense mutations and a codon deletion (delta F93) mutation abolish microsomal G6P uptake activity and that two splicing mutations cause exon skipping. While most missense mutants support the synthesis of G6PT protein similar to that of the wild-type transporter, immunoblot analysis shows that G20D, delta F93, and I278N mutations, located in helix 1, 2, and 6, respectively, destabilize the G6PT. Further, we demonstrate that G6PT mutants lacking an intact helix 10 are misfolded and undergo degradation within cells. Moreover, amino acids 415-417 in the cytoplasmic tail of the carboxyl-domain, extending from helix 10, also play a critical role in the correct folding of the transporter. However, the last 12 amino acids of the cytoplasmic tail play no essential role(s) in functional integrity of the G6PT. Our results, for the first time, elucidate the structural requirements for the stability and transport activity of the G6PT protein.

Correction of glycogen storage disease type 1a in a mouse model by gene therapy.

Glycogen storage disease type 1a (GSD-1a), characterized by hypoglycemia, liver and kidney enlargement, growth retardation, hyperlipidemia, and hyperuricemia, is caused by a deficiency in glucose-6-phosphatase (G6Pase), a key enzyme in glucose homeostasis. To evaluate the feasibility of gene replacement therapy for GSD-1a, we have infused adenoviral vector containing the murine G6Pase gene (Ad-mG6Pase) into G6Pase-deficient (G6Pase(-/-)) mice that manifest symptoms characteristic of human GSD-1a. Whereas <15% of G6Pase(-/-) mice under glucose therapy survived weaning, a 100% survival rate was achieved when G6Pase(-/-) mice were infused with Ad-mG6Pase, 90% of which lived to 3 months of age. Hepatic G6Pase activity in Ad-mG6Pase-infused mice was restored to 19% of that in G6Pase(+/+) mice at 7-14 days post-infusion; the activity persisted for at least 70 days. Ad-mG6Pase infusion also greatly improved growth of G6Pase(-/-) mice and normalized plasma glucose, cholesterol, triglyceride, and uric acid profiles. Furthermore, liver and kidney enlargement was less pronounced with near-normal levels of glycogen depositions in both organs. Our data demonstrate that a single administration of a recombinant adenoviral vector can alleviate the pathological manifestations of GSD-1a in mice, suggesting that this disorder in humans can potentially be corrected by gene therapy.

Human variant glucose-6-phosphate transporter is active in microsomal transport.

Glycogen storage disease type lb (GSD-lb) is caused by deficiencies in the glucose-6-phosphate transporter (G6PT), which works together with glucose-6-phosphatase to maintain glucose homeostasis. In humans, there are two alternatively spliced transcripts, G6PT and variant G6PT (vG6PT), differing by the inclusion of a 66-bp exon-7 sequence in vG6PT. We have previously shown that the G6PT protein functions as a microsomal glucose-6-phosphate (G6P) transporter, which is anchored to the endoplasmic reticulum by ten transmembrane helices. Here, we demonstrate that vG6PT is also active in microsomal G6P transport. The additional 22 amino acids in vG6PT is predicted to constitute a part of the luminal loop 4. Our data indicate that this loop plays no vital role in microsomal G6P transport. Further, we show that G6PT mRNA is expressed in all organs and tissues examined, but that the vG6PT transcript is expressed exclusively in the brain, heart, and skeletal muscle. These results raise the possibility that mutations in exon-7 of the G6PT gene, which would not perturb glucose homeostasis, might have other deleterious effects.

Type-1c glycogen storage disease is not caused by mutations in the glucose-6-phosphate transporter gene.

Glycogen storage disease type 1 (GSD-1) is a group of autosomal recessive disorders caused by deficiencies in glucose-6-phosphatase (G6Pase) and the associated substrate/product transporters. Molecular genetic studies have demonstrated that GSD-1a and GSD-1b are caused by mutations in the G6Pase enzyme and a glucose-6-phosphate transporter (G6PT), respectively. While kinetic studies of G6Pase catalysis predict that the index GSD-1c patient is deficient in a pyrophosphate/phosphate transporter, the existence of a separate locus for GSD-1c remains unclear. We have previously shown that the G6Pase gene of the index GSD-1c patient is intact; we now show that the G6PT gene of this patient is normal, strongly suggesting the existence of a distinct GSD-1c locus.

Transmembrane topology of human glucose 6-phosphate transporter.

Glycogen storage disease type 1b is caused by a deficiency in a glucose 6-phosphate transporter (G6PT) that translocates glucose 6-phosphate from the cytoplasm to the endoplasmic reticulum lumen where the active site of glucose 6-phosphatase is situated. Using amino- and carboxyl-terminal tagged G6PT, we demonstrate that proteolytic digestion of intact microsomes resulted in the cleavage of both tags, indicating that both termini of G6PT face the cytoplasm. This is consistent with ten and twelve transmembrane domain models for G6PT predicted by hydropathy analyses. A region of G6PT corresponding to amino acid residues 50-71, which constitute a transmembrane segment in the twelve-domain model, are situated in a 51-residue luminal loop in the ten-domain model. To determine which of these two models is correct, we generated two G6PT mutants, T53N and S55N, that created a potential Asn-linked glycosylation site at residues 53-55 (N53SS) or 55-57 (N55QS), respectively. N53SS or N55QS would be glycosylated only if it is situated in a luminal loop larger than 33 residues as predicted by the ten-domain model. Whereas wild-type G6PT is not a glycoprotein, both T53N and S55N mutants are glycosylated, strongly supporting the ten-helical model for G6PT.

Inactivation of the glucose 6-phosphate transporter causes glycogen storage disease type 1b.

Glycogen storage disease type 1b (GSD-1b) is proposed to be caused by a deficiency in microsomal glucose 6-phosphate (G6P) transport, causing a loss of glucose-6-phosphatase activity and glucose homeostasis. However, for decades, this disorder has defied molecular characterization. In this study, we characterize the structural organization of the G6P transporter gene and identify mutations in the gene that segregate with the GSD-1b disorder. We report the functional characterization of the recombinant G6P transporter and demonstrate that mutations uncovered in GSD-1b patients disrupt G6P transport. Our results, for the first time, define a molecular basis for functional deficiency in GSD-1b and raise the possibility that the defective G6P transporter contributes to neutropenia and neutrophil/monocyte dysfunctions characteristic of GSD-1b patients.