The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 1 224-229
Copyright © 1998 by The Endocrine Society
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
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Abstract
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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.
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Introduction
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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.
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Subjects and Methods
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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 1
). 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.
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.
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Results
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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. 1, 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. 1, 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. 1, 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.
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In human liver microsomes obtained from four GSD 1a patients (one
typical set is shown in the third line of Fig. 1
.), the absence of the
hydrolytic activity (Table 1) 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 2).
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 1) 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 1). In accordance with
this, addition of G6P to the microsomal vesicles resulted in a
sustained shrinking (Fig. 2, trace b),
which was comparable with that caused by the nonpermeant
mannose-6-phosphate (Fig. 2, trace a), indicating the absence of G6P
transport. The alteration of transport was selective; the permeation of
Pi (Fig. 2, trace c) or glucose (Fig. 2, trace d; see also
Table 2) 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. 1. Traces
are representative of three individual measurements on the two
microsomal preparations.
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Microsomes from the liver of a GSD 1c patient showed normal enzyme
activity in disrupted microsomes, with both G6P and pyrophosphate as
substrates (Table 1). 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 1).
In intact microsomes with G6P as substrate, the activity had an
abnormally high KM (Table 1). 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. 2, trace d), as compared with microsomes from rat, human
control, GSD 1a (Fig. 1, trace d) and GSD 1b (Fig. 2, trace c; see also
Table 2). 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. 2, trace b) or G6P (Fig. 2, trace c) was preserved (see also Table 2).
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Discussion
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The data in Fig. 1 and Table 1 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. 1, Table 2) 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. 1) 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. 1).
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 2). 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.
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Footnotes
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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.). 
2 Recipient of a Research Fellowship in Siena from the European
Science Foundation Programme of Fellowships in Toxicology.
3 A Lister Institute Research Fellow.
Received April 22, 1997.
Revised September 26, 1997.
Accepted October 6, 1997.
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Abstract
Introduction
