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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 1 224-229
Copyright © 1998 by The Endocrine Society

Original Studies

Liver Microsomal Transport of Glucose-6-Phosphate, Glucose, and Phosphate in Type 1 Glycogen Storage Diseases1

P. Marcolongo, G. Bánhegyi2, A. Benedetti, C. J. Hinds and A. Burchell3

Istituto di Patologia Generale (P.M., G.B., A.Be.), Univesità di Siena, 53100 Siena, Italy; and Department of Obstetrics and Gynaecology (C.J.H., A.Bu.), Ninewells Hospital and Medical School, University of Dundee, DD1 9SY Scotland

Address all correspondence and requests for reprints to: Prof. A. Benedetti, Istituto di Patologia Generale, Univesità di Siena, Viale Aldo Moro, 53100 Siena, Italy. E-mail: benedetti{at}unisi.it


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The transport of glucose-6-phosphate (G6P), glucose, and orthophosphate into liver microsomes, isolated from six patients with various subtypes of type 1 glycogen storage disease (GSD), was measured using a light-scattering method. We found that G6P, glucose, and phosphate could all cross the microsomal membrane, in four cases of type 1a GSD. In contrast, liver microsomal transport of G6P and phosphate was deficient in the GSD 1b and 1c patients, respectively. These results support the involvement of multiple proteins (and genes) in GSD type 1. The results obtained with the light-scattering method are in accordance with conventional kinetic analysis of the microsomal glucose-6-phosphatase system. Therefore, this technique could be used to directly diagnose type 1b and 1c GSD.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
LIVER glucose-6-phosphatase (G6Pase; EC 3.1.3.9) catalyses the terminal reaction of glycogenolysis and gluconeogenesis (1) and plays a key role in the maintenance of blood glucose homeostasis (1, 2, 3). The importance of this enzyme in the regulation of blood glucose levels is clear from the debilitating effects of the absence of the enzyme activity in type 1a glycogen storage disease (GSD) (4, 5). Topological studies indicate that the G6Pase enzyme is situated with its active site inside the lumen of the endoplasmic reticulum (ER; 6–8). Therefore, at least 3 transport systems (termed T1, T2, and T3) also are needed to transport, respectively, the substrate glucose-6-phosphate (G6P) and the products phosphate, inorganic orthophosphate (Pi) and glucose across the liver ER membrane (3, 8, 9). T2, the Pi transport protein, has been isolated from hepatic rat microsomes (10). It also transports PPi, which is hydrolyzed by the G6Pase enzyme; thus, in the test tube, PPi hydrolysis by (intact) microsomes is assumed to reflect T2 capacity (3). Subsequently, evidence has been provided also for a second microsomal Pi transport protein that does not transport PPi (11). A rat liver microsomal 52-kDa protein, clearly a new member of the facilitative glucose transport family of plasma membrane proteins (GLUT family), has been identified (and termed GLUT 7) (12, 13). The putative liver microsomal G6P transport protein(s) has not yet been purified or even identified; however, G6P uptake into microsomal vesicles has been demonstrated (14, 15). Genetic deficiencies of the enzyme, T1, T2, and T3 cause GSD type 1a, 1b, 1c, and 1d, respectively (5). The type 1 GSDs are severe metabolic disorders, which usually present early in childhood; and the most severe form is GSD 1b, in which G6P transport across ER membranes is defective (5). A human liver G6Pase enzyme complementary DNA has been cloned (16), and the human G6Pase gene has been isolated (17). To date, 26 different mutations in the G6Pase enzyme gene have been found in patients with type 1a GSD (16, 18, 19, 20). No mutations in the G6Pase enzyme gene, however, have been found in patients with GSD 1b who are deficient in liver microsomal G6P transport (19, 20), confirming that additional protein(s) is needed for ER G6P transport. In addition, a recent report of family studies in type 1b GSD (21) indicates that the gene(s) associated with G6P transport is not located on human chromosome 17, the location of the human G6Pase enzyme gene (17).

The human G6Pase system transport proteins have not yet been cloned; therefore, the only GSD subtype that it is possible to diagnose, using DNA probes, is type 1a. Moreover, though mutation analysis of the G6Pase enzyme gene is informative in many patients with GSD type 1a, in others no mutations have been found, even after sequencing of all the 5 exons plus intron-exon boundaries (19). Therefore, the only method of unequivocal diagnosis for the different GSD type 1 subtypes, in the patients where type 1a mutations cannot be found, is still a complex kinetic analysis of G6Pase activity in microsomes isolated from liver biopsy samples (5, 22, 23, 24). However, there are several drawbacks of using such kinetic analysis: 1) it is time consuming and difficult to carry out in routine diagnostic laboratories; 2) it is not suitable for assessing transport protein function in the absence of enzyme activity, i.e. in GSD type 1a; and 3) it cannot distinguish all possible defects from each other; for example, some forms of partial 1b may kinetically mimic type 1d. Therefore, a direct method to measure transport protein function is required to aid diagnosis and characterization of GSD 1 subtypes. Current radioactive filtration-based transport assays (25, 26) cannot be easily done in routine diagnostic laboratories. Some (radioactive) substrates, in fact, are not currently commercially available, or require labeling with 32P (32P-labeled substrates have a short half-life, are expensive, and not always readily available). In addition, some methods require expensive sophisticated equipment for rapid filtration (26). Another complication of filtration-based assays is that, although they may work well on normal microsomal samples, the excessive glycogen content in GSD microsomal samples tends to block the filters, and it increases the variation in both blank and test measurements. Because of the obviously limited amount of liver tissue from GSD patients, microsomal preparation, in fact, cannot be easily separated from glycogen.

Here, we have used a different type of assay (based on light-scattering) to measure transport of G6P, glucose, and Pi into liver microsomes isolated from six patients with type 1 GSD. We show that G6P, glucose, and Pi can all cross the microsomal membrane in the four type 1a GSD cases, whereas liver microsomal transport of G6P and Pi was found to be deficient in the GSD 1b and 1c patient, respectively. These results support the involvement of multiple proteins (and genes) in GSD type 1, and the method used clearly has the potential to be very useful for diagnosing type 1 GSD caused by defects in ER G6Pase system transport proteins.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patients and samples

The patients (four type 1a, one type 1b, and one type 1c GSD) all had classical symptoms of type 1 GSD and were initially diagnosed by kinetic analysis of the G6Pase system in microsomes isolated from liver biopsy samples (see Table 1Go). The control adult human liver samples were small portions of wedge or needle-biopsy samples obtained for the investigation or treatment of the original condition for which the patient was referred. All control liver samples were graded by a pathologist on routine histochemistry on a scale of 1–5, and only liver samples graded 1 (1 being apparently normal and 5 severely diseased) were used as controls in this study. The study of the G6Pase system in human liver samples was approved by the Ethics Committee of Tayside Health Board. Rat livers were obtained from fed adult male Wistar rats.


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  		Table 1. Kinetic parameters of glucose-6-phosphatase in liver microsomes from different sources

 
Microsomal preparation

Microsomes were prepared from liver samples in 0.25 mol/L sucrose/5 mmol/L Hepes (pH 7.4) by differential centrifugation, as previously described (27, 28), resuspended (5–15 mg protein/mL) in the above medium, and maintained under liquid N2 until used. Isolated microsomes are a mixture of intact and disrupted vesicles. The proportion of intact microsomes was determined by assays of low KM mannose-6-phosphatase activity, which is only expressed in disrupted microsomes (9, 29). Intactness of liver microsomes from GSD 1a patients was checked by measuring the latency of p-nitrophenol UDP-glucuronosyltransferase activity (30). All of the microsomal preparations used in this paper were more than 90% intact. Latency is defined as the percentage of activity in disrupted microsomes that is not expressed in intact microsomes. Protein concentrations were estimated by the method of Lowry, as modified by Peterson (31).

Enzymatic assays

G6Pase, mannose-6-phosphatase, and pyrophosphatase activities were assayed and calculated as in (22) and are expressed as nanomoles of substrate hydrolyzed per minute per milligram of microsomal protein. All assays were linear, with respect to incubation time. The concentrations of the substrate G6P used to calculate the kinetic constants were 1.0, 1.4, 2.0, 2.6, 5.0, and 30 mmol/L; and 0.5, 1.0, 1.4, 2.0, 2.6, and 5.0 mmol/L were the concentrations of pyrophosphate used as a substrate. All Vmax and KM values given in this paper were calculated using a BBC computer program of nonlinear multiple regression analysis.

Light-scattering measurements

Osmotically induced changes in microsomal vesicle size and shape were monitored at 400 nm at a right angle to the incoming light beam, using a fluorometer (Perkin-Elmer model 650–10S) equipped with a recorder, a temperature-controlled cuvette holder (22 C), and a magnetic stirrer, as described elsewhere (32, 33). Briefly, microsomal vesicles (30–35 µg protein/mL) were equilibrated in a hypotonic medium (5 mmol/L K-Pipes, pH 7.0) and the osmotically induced changes in light scattering were measured after the addition of a small volume (1/10–1/20 of the reaction volume) of the concentrated solutions of different compounds to be tested.

Chemicals

G6P (dipotassium salt), mannose-6-phosphate (dipotassium salt), cacodylic acid (recrystallised from 95% ethanol), alamethicin and histone type IIA, and type II-AS were obtained from Sigma, Poole, UK. SDS (especially purified for biochemical work) and tetrasodium pyrophosphate were purchased from BDH, Poole, UK. All other chemicals were of analytical grade.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Type 1a GSD

First, the permeability of the investigated compounds was tested on rat liver and human control liver microsomal vesicles. As can be seen in the first and second lines of Fig. 1Go, sucrose (which is a poor permeant) caused a sustained shrinking of vesicles, as revealed by the increase in light-scattering intensity (trace b). On the other hand, permeant compounds, e.g. glucose (trace c), Pi (trace d), or G6P (trace e), caused a transient shrinking, followed by a swelling phase (decrease in light-scattering), whose rate depends on the rate of entry of these compounds into the vesicles. Addition of the pore-forming agent alamethicin to the system (empty arrowheads) resulted in a fast decrease of the signal to a basal value. When alamethicin was added first, it completely prevented the shrinking effect (data not shown), indicating that the vesicles were rendered fully permeable to the investigated compounds. Alamethicin by itself caused a moderate decrease in light-scattering signal (Fig. 1Go, trace a ), which probably reflects a minor vesicle swelling caused by the entry of Pipes into the vesicles (5 mmol/L K-Pipes, pH 7.0, was present in the hypoosmotic medium). The organic anion Pipes per se does not easily permeate the microsomal membrane (30). The addition of K-Pipes (to a final concentration of 25 mmol/L) to microsomes resuspended in the hypoosmotic medium, caused a permanent shrinking of the microsomal vesicles (not shown). The permeabilization of microsomal vesicles by alamethicin, however, was still compatible with a relatively high light-scattering signal (Fig. 1Go, trace a), which indicated that microsomes maintained their vesicular structure (30). This signal could only be minimalized by the addition of detergents (e.g. Triton X-100; data not shown), which fully destroyed the vesicular structure.



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  		Figure 1. Influx of G6P, glucose, and phosphate into rat and human liver microsomal vesicles. Osmotically induced changes in light-scattering intensity of liver microsomal vesicles were detected as described in Subjects and Methods. Concentrated solutions (1 mol/L) were added, where indicated by black arrowheads, giving the following final concentrations: 100 mmol/L sucrose (Sucr); 100 mmol/L glucose (Glc); 50 mmol/L G6P (G-6-P); 50 mmol/L mannose-6-phosphate (M-6-P); 50 mmol/L potassium phosphate (pH 7.2, Pi). Two to 4 µL of an alamethicin solution (5 mg/mL in ethanol) were then added to give the final concentration of 10 µg/mL in the reaction mixture and thus fully permeabilize the microsomal vesicles (empty arrowheads). The shrinking phase (dotted lines) has been reconstructed graphically by taking into account the loss of light-scattering intensity caused by dilution of microsomal suspension caused by solute additions (see first traces of each set of experiments). Traces are representative of three individual measurements on two to five separate microsomal preparations.

 
In human liver microsomes obtained from four GSD 1a patients (one typical set is shown in the third line of Fig. 1Go.), the absence of the hydrolytic activity (Table 1Go) did not influence the permeation of glucose, Pi, or G6P in a qualitative way. The permanent shrinking in the presence of sucrose shows the intactness of the vesicular structure. For the sake of better comparison, the half-time of transport for each compound also was calculated. Major differences were not found in the transport velocity of the investigated compounds between GSD 1a and control (human or rat) microsomes (Table 2Go).


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  		Table 2. Half-time of transport of glucose-6-phosphate, glucose, and orthophosphate in liver microsomal vesicles

 
Type 1b and 1c GSD

Microsomes from the liver of a GSD 1b patient showed normal G6Pase enzyme activity, with G6P as substrate in disrupted microsomes (Table 1Go) but no activity in intact microsomes, i.e. G6Pase activity with G6P as substrate was 100% latent. In contrast, G6Pase enzyme activity, with pyrophosphate as substrate, was normal in both intact and disrupted microsmal vesicles (Table 1Go). In accordance with this, addition of G6P to the microsomal vesicles resulted in a sustained shrinking (Fig. 2Go, trace b), which was comparable with that caused by the nonpermeant mannose-6-phosphate (Fig. 2Go, trace a), indicating the absence of G6P transport. The alteration of transport was selective; the permeation of Pi (Fig. 2Go, trace c) or glucose (Fig. 2Go, trace d; see also Table 2Go) was not affected.



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  		Figure 2. Influx of G6P, glucose, and phosphate into human liver microsomal vesicles from patients with GSD 1b and 1c subtypes. For experimental details, see the legend to Fig. 1Go. Traces are representative of three individual measurements on the two microsomal preparations.

 
Microsomes from the liver of a GSD 1c patient showed normal enzyme activity in disrupted microsomes, with both G6P and pyrophosphate as substrates (Table 1Go). In contrast, in intact microsomes, there was no activity with pyrophosphate as substrate; i.e. G6Pase activity, with pyrophosphate as substrate, was 100% latent (Table 1Go). In intact microsomes with G6P as substrate, the activity had an abnormally high KM (Table 1Go). In hepatic microsomes from a GSD 1c patient, the addition of Pi resulted in a rapid shrinking, followed by a swelling phase that was slower and only partial (Fig. 2Go, trace d), as compared with microsomes from rat, human control, GSD 1a (Fig. 1Go, trace d) and GSD 1b (Fig. 2Go, trace c; see also Table 2Go). This indicates a marked reduction in Pi transport. The slow and minor Pi transport, still present in microsomes of the GSD 1c patient, might be caused by Pi transporters other than those transporting Pi and PPi and involved in most of the GSD 1c cases (3, 11). In this respect, evidence has been provided for a microsomal Pi transporter that does not transport PPi (11). In the GSD 1c patient, the microsomal transport of glucose (Fig. 2Go, trace b) or G6P (Fig. 2Go, trace c) was preserved (see also Table 2Go).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The data in Fig. 1Go and Table 1Go clearly show that G6P, glucose, and Pi are taken up into human microsomes from both control and type 1a GSD livers. These results are in agreement with previous reports on G6P uptake into GSD type 1a microsomes obtained using radioactive filtration assays by other authors (34) and by ourselves (15). They also are consistent with the result of a filtration assay in G6Pase enzyme knockout mice, where G6P uptake into microsomes was low but measurable (35). Our results, however, are in contrast with a recent report on a case of GSD type 1a, where microsomal G6P transport could not be detected but Pi transport was observed (36). The possibility that their result was caused by a double deficiency of both the G6Pase enzyme and T1 exists, but it is very unlikely. To our knowledge, such a double deficiency has never been reported and, in the above reported case, was not supported by the typical clinical symptoms of a defect in microsomal G6P transport (e.g. neutropenia; Ref.5). A reasonable explanation for the observed lack of G6P transport in this study may be that the liver sample tested contained transformed (hepatoma) cells, instead of or besides hepatocytes. The sample was, in fact, from a liver that had been transplanted because of an enlarging mass. Many GSD type 1(a) patients need liver transplants because enlarging masses (or hepatomas) are detected in their livers, which also contain multiple foci of much smaller adenomas. Adenomas and hepatomas do not express the G6Pase system and can contain elevated levels of other nonspecific phosphatases. Consistent with this explanation, a relatively high nonspecific phosphatase activity was found in the liver microsomes in the above study (approximately 10% of G6Pase activity), whereas normal human liver microsomes contain less than 3% nonspecific phosphatase activity.

The rates of uptake of G6P into the type 1a GSD microsomes (Fig. 1Go, Table 2Go) are not identical to those in control microsomes. However, until many more control and GSD liver microsomal samples are analyzed by our light-scattering method, it will not be possible to determine whether this is caused by the interindividual variations in T1 expression in human liver microsomes or whether the lack of either G6Pase enzyme function or alterations in enzyme structure can effect G6P uptake. Kinetic analysis of control human liver microsomes has shown that the latency of the G6Pase system with G6P as substrate can vary (11, 23, 25, 27, 37), suggesting that interindividual variations in T1 expression in human liver microsomes may well occur. In any case, in our experiments, neither the presence nor absence of the enzyme protein nor its hydrolytic activity affected qualitatively the permeation of G6P. Among our 1a patients, case 1 (Fig. 1Go) expresses G6Pase enzyme protein (as judged by immunoblot analysis), and the mutation is C-to-T change at base 326, resulting in a R83C change in the G6Pase protein (the second allele contains a mutation that results in a stop codon early in the protein). We do not know what mutations are present in the G6Pase enzyme in cases 2 and 4, but they express G6Pase protein of normal molecular weight, whereas in case 3, there is no protein (as judged by immunoblot analysis). The swelling profile of microsomes from the GSD 1a case 3 upon G6P addition is shown in an accompanying paper (15); it seems qualitatively similar to that of microsomes from GSD 1a case 1 (Fig. 1Go).

This is the first direct demonstration for G6P, glucose, and Pi uptake into microsomes from type 1a GSD patients, and very similar results were obtained in all the patients studied (Table 2Go). The data clearly demonstrate that G6P, glucose, and Pi uptake takes place in the absence of the function and/or presence of the G6Pase enzyme protein.

Similarly, this method gave consistent results, compared with the data gained with the kinetic analysis of the G6Pase system in liver microsomes from type 1b and 1c GSD patients. The complete absence of the function of a single transport protein component of the G6Pase system in these cases did not influence the transport of the other compounds. This demonstrates that G6P, glucose, and Pi are taken up into microsomes by different transport proteins. Clearly, this method can be used to show the function of the three transport systems in type 1a GSD; and, in addition, it has the potential to allow further characterization of the transport, e.g. the kinetics or effects of inhibitors in conditions that are not complicated by potential effects of alterations in enzyme activity.

In addition, the fact that the data obtained with the light-scattering method are completely consistent with the results of kinetic analysis of the G6Pase system means that this relatively simple, cheap, and easily adaptable method, which could be simply carried out in routine diagnostic laboratories, is suitable for the diagnosis of GSD 1b and 1c.


   Footnotes
 
1 The financial support of Telethon-Italy (Grant E.271 to A.Be.) is gratefully acknowledged. This work was also supported by grants from the Medical Research Council and Scottish Home and Health Department (to A.Bu.) and by a grant from the Ciba Fellowship Trust (to A.Bu. and A.Be.). Back

2 Recipient of a Research Fellowship in Siena from the European Science Foundation Programme of Fellowships in Toxicology. Back

3 A Lister Institute Research Fellow. Back

Received April 22, 1997.

Revised September 26, 1997.

Accepted October 6, 1997.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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