(Received for publication, January 17, 1995; and in revised form, June 20, 1995)
From the
In search for a nonmetabolized, superior glucose analogue to
study the mechanism of glucose-induced glycogen synthesis, we have
tested 2-deoxy-2-fluoro--D-glucopyranosyl fluoride, which
inhibits muscle phosphorylase b 10-fold better than does
glucose (Street, I. P., Armstrong, C. R., and Withers, S. G. (1986) Biochemistry 25, 6021-6027). In a gel-filtered liver
extract, 0.6 mM analogue and 10 mM glucose equally
accelerated the inactivation of phosphorylase and shortened the latency
before the activation of glycogen synthase. The analogue was not
measurably defluorinated or phosphorylated by intact hepatocytes, as
monitored by
F NMR. When added to isolated hepatocytes, 10
mM analogue inactivated phosphorylase more extensively than
did 50 mM glucose, but unlike glucose, it did not result in
the activation of glycogen synthase. Therefore, the binding of glucose
to phosphorylase a can account for the inactivation of
phosphorylase, but the metabolism of glucose (probably to Glc-6-P)
appears to be required to achieve activation of glycogen synthase.
The livers of overnight-fasted, anesthetized mice contained appreciable amounts of both phosphorylase a and glycogen synthase a, without net glycogen accumulation. Likewise, hepatocytes isolated from fasted rats and incubated with 10 mM glucose contained 41% of phosphorylase and 32% of glycogen synthase in the a form, and these values remained stable for 1 h, while glycogen accumulated at only 22% of the rate expected from the glycogen synthase activity. The addition of 10 mM analogue decreased phosphorylase a to 10% without significant change in glycogen synthase a (38%), but with a 4-fold increased rate of glycogen accumulation. These findings imply that synthase a is fully active in the liver of the fasted animal and that the absence of net glycogen synthesis is due to continuous glycogenolysis by phosphorylase a.
The rates of glycogen synthesis and glycogenolysis in the liver
are mainly controlled by the phosphorylation state of glycogen synthase
and phosphorylase, respectively(1) . In the fed state, there is
a tight, inverse coupling between the activation states of the two
enzymes(1, 2) . A key element in this control is the
potent allosteric inhibition that phosphorylase a (phosphoenzyme) exerts on the hepatic glycogen-associated
glycogen-synthase phosphatase (protein phosphatase 1G), which converts
glycogen synthase b to the a form(1, 3, 4, 5) . This
mechanism appears to explain the sequential inactivation of
phosphorylase and activation of glycogen synthase in the liver after
the administration of glucose(2) : binding of glucose to
phosphorylase a renders the enzyme a better substrate for
phosphorylase phosphatase; the conversion of phosphorylase a to b switches off glycogenolysis and relieves
glycogen-synthase phosphatase from inhibition by phosphorylase a, thus allowing the phosphatase to activate glycogen
synthase. Hence, little or no ``glycogen cycling'' (Glc-1-P
glycogen
Glc-1-P) is observed during glucose-induced
hepatic glycogen synthesis in man (6) and during glycogen
synthesis and subsequent glycogenolysis in cultured rat
hepatocytes(7) .
A minimal concentration of glycogen is required for the inhibition of glycogen-synthase phosphatase by phosphorylase a(8) . Depletion of glycogen explains the anomalous situation in the liver of the fasted animal, which contains appreciable amounts of both phosphorylase a and glycogen synthase a, obviously without net glycogen synthesis(9, 10, 11) . However, the question then arises whether the absence of net glycogen synthesis reflects a full-blown substrate cycle or whether the synthase a measured in such liver homogenates is not catalytically active in the hepatocyte. Part of the present work was aimed at solving this dilemma, which has been a tantalizing problem for many years.
Our proposal (1, 2) that the mere removal of phosphorylase a would explain the glucose-induced activation of glycogen synthase has been challenged by Carabaza et al.(12) in a study on the effects of glucose analogues on isolated hepatocytes: while high concentrations of several analogues (e.g. 50 mM 6-deoxyglucose) promoted the inactivation of phosphorylase, only glucose, 5-thioglucose, and 2-deoxyglucose, which can be phosphorylated on carbon 6, were also able to activate glycogen synthase. More recently, while exploring the glycogenic action of 5-iodotubercidin, we also concluded that the activation of glycogen synthase could not be explained merely by the inactivation of phosphorylase(13) . Another part of the present work deals with the question of whether phosphorylation of glucose is essential for the glucose-induced activation of glycogen synthase.
Hepatocytes were prepared
in the morning from the livers of male, overnight-fasted Wistar rats
weighing 250 g(14) . The cells (5
10
/ml) were incubated at 37 °C as described (14) in a Krebs-Henseleit medium supplemented with 13.5
mM lactate, 1.5 mM pyruvate, 0.2 mM glycerol, and (unless indicated otherwise) 10 mM glucose.
Samples for the assays of glycogen synthase and phosphorylase were
transferred into tubes containing an ice-cold buffer with inhibitors of
protein kinases and phosphatases and were immediately frozen in liquid
nitrogen(14) . Samples for glycogen determination were mixed
with KOH (1 M final concentration), heated for 20 min at 90
°C, and neutralized with acetic acid. For the assay of Glc-6-P, the
cells were separated from the incubation medium by centrifugation for 5
s at 10,000
g and deproteinized with cold 1 M
HClO
, and the mixture was centrifuged. The supernatant was
neutralized with KOH and KHCO
and clarified by
centrifugation.
Glucose uptake by hepatocytes was measured in
hepatocyte suspensions incubated at 20 °C with or without 10
mM glucose analogue for 5 min before the addition of 10 mM [U-C]glucose. After 1 and 5 min, aliquots
were deposited in microcentrifuge tubes on a layer of ice-cold 0.15 M NaCl containing 50 mM glucose, and the hepatocytes
were pelleted at once by centrifugation as described above and frozen
in liquid N
until deproteinization and determination of
radioactivity.
For the preparation of gel-filtered liver extracts,
fed rats were injected intraperitoneally with 70 µg of glucagon 10
min before decapitation to activate phosphorylase fully and to
inactivate glycogen synthase. Their livers were homogenized in a
Potter-Elvehjem tube in 3 volumes of an ice-cold solution containing
0.25 M sucrose, 50 mM glycylglycine, pH 7.4, and 1
mM dithiothreitol. The homogenate was centrifuged for 10 min
at 8000 g, and 2.5 ml of supernatant (liver extract)
was filtered in the cold as recommended by the manufacturer (Pharmacia
Biotech Inc.) through a Sephadex G-25 column (10
1.5 cm)
equilibrated with 50 mM glycylglycine, pH 7.4, and 1 mM dithiothreitol.
Results are means ± S.E. for the indicated number (n) of observations. Statistical differences were calculated with Student's t test for independent random samples and are considered as significant if p < 0.05.
Figure 1:
F NMR spectra for
synthetic F
-Glc as such (A) and after incubation
with hexokinase (B). A,
H-decoupled
spectrum for preparation I (2 mM). The insets with an
expanded frequency scale show the F-F coupling (bottom) and
imposed H-F couplings (top) in the
H-coupled
spectrum. B, spectrum for the same preparation (final
concentration of 2 mM) after incubation for 4 h at 30 °C
in 1.5 ml of 40 mM Tris, pH 7.8, containing 100 µg of
hexokinase and 2.5 mM each ATP and magnesium acetate, and
subsequent heating for 3 min at 90 °C. See ``Experimental
Procedures'' and ``Results and Discussion'' for
assignments of the numbered peaks.
The H-decoupled
F NMR spectra of both preparations were virtually
identical. As illustrated in Fig. 1A for preparation I,
the spectra contained two major doublets of equal intensity (doublets 2
and 3, centered at -72.02 and -125.70 ppm, respectively),
which accounted for 82 and 83% of the total
F signal in
preparations I and II, respectively. These ratios were independent of
the degree of saturation, tested at repetition times ranging between 1
and 5 s. The chemical shift values, F-F coupling constants (19.7 Hz),
and the
H-coupled spectra allow us to identify doublets 2
and 3 as the F-1 and F-2 signals, respectively, of
F
-Glc(19) . The glucosidic bond of
F
-Glc was acid-labile; after boiling for 10 min in 1 M HClO
, 39% of the compound had been converted to
2-deoxy-2-fluoroglucose, with equivalent production of inorganic
fluoride (data not shown).
An unidentified impurity gave rise to two
minor doublets (Fig. 1A; doublets 1 and 4, centered at
-65.54 and -130.77 ppm, respectively) of equal intensity
and reciprocal F-F coupling (18 Hz), which amounted to 18% of the total F signal. The spectral parameters differ from those
reported for the
-anomer of F
-Glc and for both anomers
of F
-mannose(19) .
Figure 2:
Levels of phosphorylase a and
glycogen synthase a in the livers of overnight-fasted,
anesthetized mice injected with saline () or glucose (
). The
mice were injected via a tail vein either with glucose (1 mg/g of body
weight) or with an equivalent volume of saline 5 min before part of the
liver was quick-frozen in situ between aluminium tongs cooled
in liquid N
. The hatchedarea covers the
results obtained previously with
120 fed mice injected for various
times with saline, glucose, or glucagon, as shown individually in Fig. 3of (2) .
Figure 3:
Effects of glucose and F-Glc
on the inactivation of phosphorylase and on the activation of glycogen
synthase in a gel-filtered liver extract. A gel-filtered liver extract,
prepared as described under ``Experimental Procedures,'' was
incubated at 25 °C in the presence of 10 mM
(NH
)
SO
only (
) or plus 10
mM glucose (
) or 0.6 mM F
-Glc
(▴). At the indicated times, samples were taken for the assays of
phosphorylase and glycogen synthase.
The major
problem with the glucose analogues that have been tested biologically (12) is their poor efficiency, as reflected by their low
affinity for phosphorylase(21, 22) . Better candidates
emerged from a study by Street et al.(22) of
deoxyfluoro derivatives of glucose, which yielded several compounds
that were superior to glucose as inhibitors of phosphorylase (at least
the b form from skeletal muscle). One such compound is
-D-glucopyranosyl fluoride(22) , which had to be
rejected because it is a good substrate for
-1,4-glucosidases as
well as for amylo-1,6-glucosidase(23, 24) ; obviously,
we could not tolerate intracellular production of fluoride, which is a
potent inhibitor of, for example, protein phosphatases(25) .
Another good inhibitor of phosphorylase, 2-deoxy-2-fluoroglucose, can
be phosphorylated on carbon 6 and further metabolized beyond the
UDP-sugar stage(26) . Hence, we decided to explore the
double-fluorinated derivative F
-Glc, which was also the
most potent phosphorylase inhibitor synthesized(22) , with a K
of 0.2 mM for muscle
phosphorylase b, i.e. 10 times better than glucose.
A quantitative comparison conducted at a much lower
concentration of hexokinase indicated, however, that F-Glc
was phosphorylated at least 5000-fold more slowly than glucose. This
finding agrees with that of Bessell et al.(28) , who
found that 2-deoxy-2-fluoroglucose was a good substrate for hexokinase,
whereas
-D-glucopyranosyl fluoride was neither a
substrate nor an inhibitor.
Subsequent work indicated that
F-Glc (10 mM) was neither phosphorylated nor
defluorinated when incubated with hepatocytes. For this purpose, we
analyzed the
F spectra of neutralized HClO
extracts of both the cell pellet and the medium after incubation
of hepatocytes with F
-Glc for up to 4 h (data not shown).
Neither F
-Glc-6-P nor inorganic fluoride could be detected.
The former observation is in agreement with the absence of hexokinase
(and the presence of the more specific glucokinase) in differentiated
hepatocytes(29) . Clearly, F
-Glc is not a substrate
for hepatic
-glucosidases; it has been reported as an inhibitor of
yeast
-glucosidase(23) .
We have checked that
F-Glc did not measurably interfere with the transport and
phosphorylation of glucose. Preincubation of hepatocytes for 5 min with
10 mM F
-Glc influenced neither the uptake of 10
mM glucose by hepatocytes (measured after 1 and 5 min at 20
°C) nor the intracellular concentration of Glc-6-P during
incubation at 37 °C with 10 or 50 mM glucose for up to 10
min (data not shown).
This suggests that liver phosphorylase a has a much higher affinity for F-Glc than for glucose,
as experimentally confirmed (Fig. 4): 1 mM
F
-Glc sufficed to achieve 50% inactivation of phosphorylase
after 3 min of incubation, whereas
20 mM glucose was
required to achieve the same effect.
Figure 4:
Effect of the concentrations of glucose
and F-Glc on the inactivation of phosphorylase in
gel-filtered liver extracts. A gel-filtered liver extract was prepared
and incubated as described in the legend to Fig. 3with the
indicated concentrations of either glucose or F
-Glc. After
3 min, a sample was taken for the assay of phosphorylase. Results are
the means ± S.E. for five liver
preparations.
Figure 5:
Effects of glucose and F-Glc
on the activation states of phosphorylase and glycogen synthase in
hepatocytes isolated from fasted rats. Freshly isolated hepatocytes
were incubated in the presence of the indicated concentrations of
glucose only (
,
) or plus the indicated concentrations of
F
-Glc (
, ▴). At the times shown, samples were
taken for the assays of phosphorylase (--) and glycogen
synthase(- - - ). Results are the means ± S.E.
for five to eight hepatocyte preparations.
Taken together, the data in Fig. 5(B and C) illustrate clear discrepancies between the extent of inactivation of phosphorylase and the extent of activation of glycogen synthase. They are compatible with a (partial) inactivation of phosphorylase as a prerequisite for the activation of glycogen synthase, but they show that the mere inactivation of phosphorylase is not sufficient to elicit the activation of glycogen synthase. This conclusion, based on the use of a superior glucose analogue, corroborates the proposal of Carabaza et al.(12) , who were limited by the choice of 1-deoxyglucose and 6-deoxyglucose as nonphosphorylatable glucose analogues. In comparison with glucose, 1-deoxyglucose binds to phosphorylase with some 4-fold lower affinity(21, 22) , while 6-deoxyglucose is a very poor ligand(22) . In our hands, 50 mM 1-deoxyglucose decreased the concentration of phosphorylase a in hepatocytes by only 25%, and 50 mM 6-deoxyglucose was ineffective (data not shown).
Figure 6:
Effect of the glucose concentration on
the intracellular concentration of Glc-6-P in hepatocytes incubated in
the presence of F-Glc. Freshly isolated hepatocytes were
incubated in the presence of either 10 mM glucose plus 10
mM F
-Glc (
; cf.Fig. 5B) or 50 mM glucose plus 5 mM F
-Glc (▴; cf.Fig. 5C).
At the indicated times, samples were taken for the determination of the
intracellular concentration of Glc-6-P. Results are the means ±
S.E. for four to five hepatocyte
preparations.
First, some glucose derivatives that are phosphorylated on carbon 6 but not further metabolized have been shown to elicit the activation of glycogen synthase (besides inactivation of phosphorylase). This was initially discovered in studies on polymorphonuclear leukocytes, where an extensive and long-lasting activation of glycogen synthase could be elicited by the addition of 0.5-1 mM 2-deoxyglucose (32) or glucosamine(33) . Similar results were obtained more recently upon incubation of hepatocytes with 2-deoxyglucose and 5-thioglucose(12) .
Second, Glc-6-P is a well known ligand of glycogen synthase, and binding could alter the enzyme conformation so as to expose phosphorylated site(s) to the action of protein phosphatases or to shield such site(s) from synthase kinases. There is in fact evidence in vitro that the addition of Glc-6-P can enhance the dephosphorylation and activation of purified (muscle) glycogen synthase by the catalytic subunits of protein phosphatases 1 and 2A(34) . However, Glc-6-P had also been shown many years ago to block in a crude liver extract the inactivation of glycogen synthase elicited by MgATP plus cAMP(35) . The mechanism has recently been investigated with purified muscle glycogen synthase(36) . The inhibition is limited to the action of cAMP-dependent protein kinase on glycogen synthase, but at least in vitro, it operates at physiological concentrations of Glc-6-P.
Further work will obviously be required to delineate the importance of Glc-6-P in the activation of glycogen synthase and the relative importance of synthase phosphatases and synthase kinases in substrate-directed effects of Glc-6-P. To explore these issues, we plan to test on isolated hepatocytes a series of newly synthesized glucose analogues (37) in combination with inhibitors of protein kinases and protein phosphatases(13, 38) .
For the sake of comparison, other glycogenic agents (50
mM glucose, 0.1 mM Proglycosyn) have also been used,
alone and in combination (Table 1). Proglycosyn is a phenylacyl
imidazolium compound that causes the inactivation of phosphorylase and
(in contrast to F-Glc) also the activation of glycogen
synthase (Table 1)(39, 40) . Intracellular
glucuronidation of Proglycosyn is an essential step in the generation
of the active compound(40) , whose action mechanism remains
currently unknown. Using these various agents, a wide range of values
for glycogen accumulation was obtained, and the actual rates of
glycogen synthesis have been compared with the concurrent levels of
glycogen synthase a (Fig. 7). In this graph, a line has
been drawn from the origin to the result obtained with 50 mM glucose plus 5 mM F
-Glc plus 0.1 mM Proglycosyn, yielding the highest rate of glycogen accumulation,
elicited by 89% synthase a and with barely 5% phosphorylase a. Results significantly below this line would indicate that
either synthase a is catalytically less efficient than
expected or that glycogen is being degraded simultaneously. One
observes a clear deviation only in the few instances where 15% or more
of phosphorylase was present in the a form and most
prominently in the cells incubated in the presence of 10 mM glucose only. Fig. 7shows that the addition of 5 and 10
mM F
-Glc to the latter cells caused an almost
purely upward movement of the experimental result, i.e. net
glycogen deposition occurred as a result of a selective inactivation of
phosphorylase; one notices also that, in the presence of 10 mM each glucose and F
-Glc (10.5% phosphorylase a), the substrate cycle had largely been suppressed. In fact,
the data in Fig. 7allow us to calculate (since 1 g of
hepatocytes contains 220 mg of protein) that the cells incubated with
10 mM glucose only should have synthesized
230 nmol of
glycogen/min/g (wet weight) in the absence of substrate cycling. Since
the actual rate of glycogen accumulation was merely 50 nmol/min/g, the
cycling must have consumed
180 nmol of ATP/min/g. Upon addition of
10 mM F
-Glc, the rate of glycogen accumulation
rose to 210 nmol/min/g, while the cycling decreased to 60 nmol of
ATP/min/g. In conclusion, our data indicate that, in the liver of the
fasted animal (Fig. 2), glycogen synthase a is indeed
fully active and that the absence of net glycogen accumulation is due
to continuous glycogenolysis by phosphorylase a.
Figure 7:
Comparison between the concentration of
glycogen synthase a and the rate of glycogen synthesis in
hepatocytes isolated from fasted rats. Freshly isolated hepatocytes
were incubated in the presence of 10 mM glucose (opensymbols) or 50 mM glucose (closedsymbols), either as such (,
) or in the
presence of F
-Glc (5 mM (
, ▴) and 10
mM (
)), 0.1 mM Proglycosyn (PGS;
, ▾), or 5 mM F
-Glc plus 0.1 mM Proglycosyn (both; ▪). The rate of glycogen
synthesis is plotted as a function of the activation state of glycogen
synthase (data from Table 1). Horizontal and verticalbars represent ±S.E. Values to the
left or right of the horizontalbars represent the
mean value for phosphorylase a as a percent of the total
(±S.E. is indicated in Table 1).