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Localization of glycogen synthase activity in liver of fasted normal and adrenalectomized rats after injection of dexamethasone.

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THE ANATOMICAL RECORD 236:486-492 11993)
Localization of Glycogen Synthase Activity in Liver of Fasted Normal
and Adrenalectomized Rats After Injection of Dexamethasone
Department of Anatomy and Cell Biology, University of Cincinnati College of Medicine,
Cincinnati, Ohio
Hepatic glycogen synthase activity was localized in normal
and adrenalectomized (ADX) rats after fasting overnight and in fasted ADX
rats after injection of dexamethasone (DEX) ZS h prior to sacrifice to stimulate glycogen synthesis. Cryostat sections were incubated in medium containing substrate to demonstrate glycogen synthase activity as indicated by
glycogen synthesized during incubation. Sections from fasted normal rats
showed limited dispersed glycogen synthase activity in both periportal and
centrilobular regions. In contrast, activity for glycogen synthase in hepatocytes from fasted ADX rats appeared as large aggregates in random hepatocytes throughout the lobule. Two hours after injection of DEX the reaction product appeared as aggregates in some hepatocytes, but other cells
revealed dispersed enzyme activity. Glycogen synthase activity was evident in more hepatocytes after 4 h treatment with DEX and after 8 h virtually all hepatocytes contained abundant reaction product. The results
suggest that synthase activity becomes concentrated in limited regions of
selected hepatocytes in fasted ADX rats. DEX stimulation of glycogen synthesis for 4-8 h results in increased enzyme activity. o 1993 Wiley-Liss, IUC.
Key words: Liver glycogen, Glycogen synthase, Histochemistry, Adrenalectomy-fasting-dexamethasone
Glycogen synthase (UDP-g1ucose:glycogen4-a-glucosyltransferase, EC is the rate-limiting enzyme in glycogen synthesis in the liver and occurs in
two forms: the D form which requires the presence of
glucose 6-phosphate for activity and the I form which is
generally regarded as independent of glucose 6-phosphate although it is stimulated by the presence of the
compound (Friedman and Larner, 1963). Previously,
glycogen synthase activity has been localized in normal liver of fed and fasted rats (e.g., Sasse et al., 1975).
In the current study changes in the patterns of glycogen synthase activity occurring within the lobule during the early stages of glycogen synthesis were examined in the adrenalectomized rat stimulated to
synthesize glycogen by injection of dexamethasone.
Glycogen synthase activity was localized in sections of
rat liver by a histochemical procedure proposed by
Smith (1970) and modified by Denizot (1978).
Cardell (1977) previously reported that fasting adrenalectomized rats overnight reduced liver glycogen
content to minimal levels (< 0.1%). These animals became depleted of glycogen to such a degree that p particles were virtually absent from sections of hepatocytes after careful and extensive observation by
electron microscopy. Subsequent injection of a pharmacological dose of dexamethasone resulted in a prolonged stimulation of glycogen synthesis that has been
demonstrated within liver lobules by radioautographic
localization of a tritiated glycogen precursor incorporated into the newly synthesized glycogen. Prior to
stimulation, only a few random hepatocytes were
heavily labeled (Michaels et al., 1984). The percentage
of heavily labeled cells increased after administration
of dexamethasone and the percentage of glycogen per
liver wet weight increased.
Jungermann and coworkers (1989) have suggested
that different metabolic processes in which the liver
participates are not uniformly distributed throughout
the liver lobule. The adrenalectomized fasted rat stimulated to synthesize glycogen by administration of
DEX provided a relatively controlled system for following changes in the distribution of glycogen synthase
activity in the liver lobule during early glycogen synthesis.
Eighteen young male Wistar rats weighing approximately 100-150 gm at the time of sacrifice were used
in this study. Fifteen of the rats were adrenalectomized
(ADX) 7-10 days before the experiment and all of the
animals were fed ad libitum until the night prior to
experimentation. The ADX rats were allowed contin-
Received February 20, 1992;accepted October 21,1992.
Address reprint requests to Dr. John E. Michaels, Department of
Anatomy and Cell Biology, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267-0521.
ued access to water containing 0.75% NaCl. Rats were
provided with a 12:12 hours light-dark cycle. They
were housed singly in wire-bottomed cages the night
prior to the experiment and fasted in order to deplete
their glycogen reserves. The following morning three
fasted ADX rats and the three fasted normal rats were
sacrificed without further treatment. The remaining
twelve fasted ADX rats were separated into groups of
four and injected with 2 mg of dexamethasone (DEX),
2, 4, and 8 h prior to sacrifice.
The liver was removed following laparotomy of the
anesthetized rat. One lobe was removed, diced into 3
mm cubes, and plunged into a bath of isopentane (2methyl butane) that was contained in a 300 ml steel
cup suspended and cooled in a bath of liquid nitrogen.
After freezing for 30 seconds to a minute the tissue was
placed on dry ice and then maintained at -70°C until
sectioned on a cryostat. Frozen sections 10 Fm thick
were mounted by brief thawing onto glass slides that
had been subbed with gelatin chrome alum.
The sections were fixed in absolute alcohol at 4"C,
dried for 10 min, and then incubated for 2 or 3 h at 37°C
in medium as described by Smith (1970) and modified
by Denizot (1978) to evaluate the distribution of glycogen synthase activity. For each incubation control
slides were incubated in medium without substrate.
The incubation mixture consisted of 50 mg disodium
uridine 5' diphosphoglucose(UDP-glucose),50 mg glucose 6-phosphate, 28 mg disodium ethylenediamine tetraacetate dihydrate (EDTA), 10 mg glycogen, 10 mg
sodium fluoride, 10 ml 0.1 M Tris maleate buffer, pH
7.5, 1 ml ethanol, and 14 ml H 2 0 containing 250 mg
gelatidl00 ml.
The reaction product produced by activity of glycogen synthase during incubation was glycogen that was
made visible by staining with iodine or the periodic
acid-Schiff technique (PAS). Some sections were
stained with iodine alone (Smith, 1970). Iodine staining is regarded as differentially staining short and long
carbohydrate chains that occur in starch and glycogen
(Swanson, 1948). In our experiments, most control sections showed no iodine staining, but minimal staining
was evident in some of the sections from rats that had
been stimulated with DEX for 4 or 8 h; this staining
was very pale. Other sections stained with iodine were
counterstained. These sections were washed in H 2 0 after incubation, then stained 30 seconds in an aqueous
1:4 dilution of light green S yellowish (Color Index
#42095) prepared from a stock solution that consisted
of 200 mg light green S yellowish, 0.2 ml glacial acetic
acid, and 100 ml H20. Sections were rinsed in H 2 0 and
stained 5 min in iodine solution consisting of 1 gm
iodine, 2 gm potassium iodide in 100 ml H,O. The sections were drained and rinsed in the following solutions: 95% alcohol, 2 min; 100%alcohol, 2 min; xylene,
2 min; xylene, 5 min; each solution contained 0.1% iodine. Sections were then mounted in Histoclad containing 0.2% iodine.
The second method used for visualizing reaction
product for glycogen synthase was to stain the sections
by the PAS technique. All glycogen within hepatocytes, whether newly formed by the enzyme during incubation or pre-existing, was stained by the PAS
method. In the ADX rats that received no DEX and in
those that were injected with DEX 2 h prior to sacrifice,
minimal amounts of glycogen were present in hepatocytes prior t o incubation for glycogen synthase activity.
Therefore, almost all glycogen present in these hepatocytes after incubation in medium containing substrate was formed by the activity of the enzyme during
incubation. However, 4 and 8 h after injection of DEX,
a substantial amount of glycogen was present in hepatocytes prior to incubation in medium; therefore, PAS
staining was less useful for evaluating synthase activity in sections from these rats.
Amylase Incubation
Tissue sections were incubated in amylase; removal
of the reaction product by amylase confirmed that the
reaction product was glycogen. The amylase incubation
solution consisted of 1gm malt amylase in 100 ml phosphate buffered saline. Sections were incubated at 37°C
for 15 min to 1h and then stained with iodine or PAS
to determine if glycogen was present.
After incubation in medium containing UDP-glucose
and glucose 6-phosphate sections of liver from normal
fasted rats showed minimal amounts of reaction product for glycogen synthase. Iodine staining of newly
formed glycogen resulting from glycogen synthase activity was variable and sparse. When stained glycogen
was present it appeared dispersed around periportal
(Fig. 1)and centrilobular (Fig. 2) regions of the lobule.
These observations were confirmed by staining incubated sections with PAS (not shown) which displayed a
similar distribution, but slightly more glycogen was
evident since both pre-existing glycogen and glycogen
formed by glycogen synthase activity during incubation were stained. Staining with either procedure
showed that adjacent hepatocytes often had different
levels of glycogen synthase activity including hepatocytes with little or no activity. Glycogen synthase reaction product was evenly dispersed within the cytoplasm of the hepatocytes displaying enzyme activity
(Fig. 1).
In the sections of liver from fasted ADX rats, many
hepatocytes showed glycogen synthase activity (Fig. 3)
and its distribution was striking. Reaction product for
glycogen synthase was limited to random hepatocytes
scattered within the lobule, and enzyme activity for
glycogen synthase was lacking in many cells adjacent
to cells containing reaction product. The reaction product within the hepatocytes occurred primarily as a
large granule or aggregate. Occasionally, other cells
contained small deposits that were distributed
throughout the cytoplasm. PAS staining showed that
the glycogen levels were minimal in these sections
prior to incubation and reflected glycogen synthase activity during incubation. Therefore, after incubation in
medium with substrate, the glycogen that appeared in
the tissue was primarily reaction product for glycogen
synthase formed during incubation. The PAS staining
revealed a distribution of glycogen as the reaction
product for glycogen synthase (Fig. 4) similar to that
obtained with iodine. Control sections in which substrate was omitted were negative for the large aggregates (not shown).
To confirm the nature of these large deposits as glycogen, sections were treated with amylase after incu-
bation with substrate. Large aggregates were not apparent after amylase treatment (not shown). Glycogen
was a constituent of the incubation medium (with and
without substrate); however, even when glycogen was
omitted from the medium, large aggregates continued
to occur in the liver sections from the fasted ADX animals.
In the livers from the four rats that received DEX 2
h prior to sacrifice (Figs. 5,6) there was more variation
with regard to distribution and amount of glycogen
synthase activity than in animals that did not receive
DEX. Reaction product for glycogen synthase was dispersed or appeared as large aggregates in hepatocytes
throughout the lobules. Since little native glycogen
was present after 2 h stimulation with DEX, PAS
staining (Figs. 5, 6) could be interpreted to reflect the
distribution of glycogen synthase activity. Results from
iodine staining were similar (not shown) to those obtained with PAS staining. Control sections confirmed
that little glycogen was present prior to incubation (not
After 4 h treatment with DEX (Fig. 7) liver sections
showed greater distribution and intensity of glycogen
synthase activity than after 2 h treatment. The individual glycogen deposits tended to be small and dispersed throughout the cells. Considerable variation
was evident between adjacent cells: some hepatocytes
displayed no glycogen synthase activity; others showed
aggregated or dispersed reaction product for glycogen
synthase. In control sections glycogen deposits were
minimal when stained with iodine. However, control
sections as well as tissue that had not been incubated
in medium were positive for glycogen after PAS staining since many hepatocytes had commenced glycogen
synthesis at this interval. Sections incubated in medium containing substrate showed a substantial increase in glycogen content when stained with PAS, indicative of activity of glycogen synthase. It was evident
that reaction product for glycogen synthase was more
concentrated near the periportal regions than in the
centrilobular hepatocytes.
Eight hours after injection of DEX, sections incubated with substrate showed substantially more reaction product for glycogen synthase than sections from
rats 4 h after DEX treatment (Fig. 8). Virtually all
hepatocytes demonstrated some iodine positive glycogen and most cells contained large quantities of glycogen synthase reaction product. Occasionally, control
sections revealed faint purple staining of pre-existing
glycogen (not shown). PAS staining of the 8 h tissue
prior to incubation showed substantial amounts of preexisting glycogen. After incubation in substrate containing medium, glycogen was much more abundant in
the sections when stained with PAS. The reaction product appeared more dispersed in hepatocytes in the centrilobular regions, whereas larger aggregates occurred
in the periportal cells.
Localization of glycogen synthase activity in normal
liver has been demonstrated histochemically by several authors (e.g., Sie et al., 1966; Grillo et al., 1964;
Sasse et al., 1975). Most investigators have used histochemical methods that were based on the technique
described by Takeuchi and Glenner (1960, 1961) al-
though substantial modifications have been suggested
(Sie et al., 1966). Histochemical localization of glycogen synthase activity also has been used to demonstrate metabolic changes due to pathological conditions
(e.g., Hacker et al., 1982; Enzmann et al., 1989).
Sasse et al. (1975) found that glycogen synthase activity was greater in periportal vs. centrilobular regions in fed rats as Takeuchi and Glenner (1961) had
suggested earlier. In the same study it was demonstrated that glycogen synthase activity decreased in
rats that were fasted for 24 h. It was concluded (Sasse
et al., 1975) that hepatocytes showing different enzyme
activities histochemically reflected differences in metabolic functions. A decrease in liver glycogen synthase
activity was determined biochemically in fasted mice
(Sie et al., 1964). In the chick liver (Grillo et al., 1964),
glycogen synthase activity was shown to increase for
several days prior to hatching; concurrently, glycogen
Subsequent to the study with Sasse (Sasse et al.,
1975) Jungermann and coworkers (1989) suggested
that many enzymes are distributed in liver lobules according to a pattern of metabolic zonation. With regard
to carbohydrate metabolism gluconeogenic pathways
are more concentrated periportally, whereas activities
of glycolytic enzymes are more concentrated near the
central vein. Our previous radioautographic studies indicated that 3H-glycogen precursors were not incorporated uniformly in all hepatocytes throughout the
lobule as glycogen synthesis was stimulated by administration of DEX to adrenalectomized rats. However,
definitive periportal or centrilobular patterns were not
In the current study of glycogen synthase activity in
the rat liver, the distribution of enzyme activity was
compared in fasted normal rats and fasted ADX rats
with and without DEX treatment. Enzyme activity was
demonstrated using a modification (Smith, 1970; Denizot, 1978) of previous methods. The histochemical procedures including the incubation mixture and time of
incubation were kept constant for the results shown. In
liver sections from fasted normal rats the histochemical method used for these studies generated a weak and
variable signal for activity of glycogen synthase. Recently, Giffin et al. (1991) demonstrated by immunocytochemistry that glycogen synthase was present in hepatocytes throughout the liver lobules in fasted normal
Figs. 1, 2. These sections demonstrate that glycogen synthase activity is limited and dispersed in hepatocytes (arrows) of fasted normal rats in both periportal (Fig. 1) and centrilobular (Fig. 2) regions.
Only random, scattered cells show activity. Sections stained with iodine. Contrast was photographically enhanced to demonstrate cells
containing enzyme activity. C, central vein; P, portal canal. x 235.
Fig. 3. This micrograph includes a portal canal (P) and a central
vein (C) and shows the distribution of enzyme activity in hepatocytes
of fasted ADX rats. Large aggregates of glycogen (reaction product for
glycogen synthase) are distributed throughout the lobule (arrows).
Iodine stained. x 235.
Fig. 4. Glycogen synthase activity (glycogen)in a portal region of a
fasted ADX rat is demonstratedby PAS. The presence of large aggregates of glycogen (arrows) is corroborated by this method (P portal
canal). x 235.
Figs. 1-4.
Figs. 5, 6. These micrographs show the distribution of activity for glycogen synthase in a fasted ADX
rat injected with DEX 2 h prior to sacrifice. Hepatocytes in both periportal (Fig. 5) and centrilobular(Fig.
6) regions reveal aggregated (arrows) and dispersed sites of enzyme activity. Other hepatocytes appear
unresponsive. The glycogen was stained with PAS. C, central vein, P, portal canal. x 235.
rats. It is evident that the histochemical technique employed in our study reflects active and not total glycogen synthase in the hepatocytes. Activity shown by
this method apparently is limited to enzyme in a
“primed condition,” perhaps in some intermediate state
of phosphorylation such as that suggested by Nuttall
and coworkers (Nuttall et al., 1988; Nuttall and Gannon, 1989).
After fasting overnight the ADX rat has minimal
hepatic glycogen content. Glycogen particles become
virtually undetectable by electron microscopy (Cardell,
1977). However, it was shown previously that most hepatocytes still incorporated limited amounts of labeled
precursor into glycogen (Michaels et al., 1984). Our
histochemical observations showed that some of the hepatocytes from fasted ADX rats contained hot spots for
glycogen synthase activity, suggesting that exposure of
sections to the incubation medium containing substrate could induce discretely localized activity of glycogen synthase in certain cells. These sites of glycogen
synthase activity may correspond to small active sites
in which incorporation of tritiated glycogen precursor
was detected by electron microscopic radioautography
in a previous study (Cardell et al., 1985). Although the
distribution of glycogen synthase activity was very different in the fasted normal and ADX rats, it was evident that in both cases there was a range of enzyme
activities in the hepatocytes, even between adjacent
cells. The distribution of reaction product for glycogen
synthase indicates that adjacent cells were not in the
same physiological state and these differences persisted throughout the lobule.
The patterns of synthase activity after 2 h treatment
with DEX represented a transition with cells having
reaction product that was aggregated as well as dispersed. In the earlier radioautography study (Michaels
et al., 1984), labeling of glycogen from a radioactive
precursor began to increase at the 2 h interval. After 4
and 8 hours DEX treatment, hepatocytes showed progressively more glycogen synthase activity which correlated well with the distribution and increase of label
observed in our radioautographic studies (Michaels et
al., 1984). However, prior to the 8 h interval, many
cells remained without observable glycogen synthase
activity indicating that the heterogeneity in enzyme
activity persisted in hepatocytes within both the periportal and centrilobular regions. Increased periportal
concentration of enzyme activity was clearly evident in
the rats stimulated for 4 hours with DEX correlating
with earlier observations (Sasse et al., 1975).
Several laboratories have raised antibodies to glycogen synthase from canine brain (Inoue et al., 1988) and
from rabbit skeletal muscle (Kaslow and Lesikar, 1984;
Lane et al., 1989). The distribution of glycogen synthase in brain tissue, revealed by immunocytochemistry, was similar to earlier histochemical localization of
enzyme activity (Ibrahim et al., 1973). Ibrahim et al.
(1973) had shown that the distribution of glycogen syn-
Fig. 7. This micrograph shows liver from a fasted ADX rat injected
with DEX 4 h prior to sacrifice. Most hepatocytes in both periportal
(P) and centrilobular (C) regions show glycogen synthase activity.
Reaction product for the enzyme was stained with iodine. X 235.
Fig. 8. A section of liver from a fasted ADX rat injected with DEX
8 h prior to sacrifice. Virtually all hepatocytes show some reaction
product for glycogen synthase. There are substantial differences in
enzyme activity even between adjacent cells. Reaction product for the
enzyme was stained with iodine. P, portal canal. x 477.
glycogen synthase and glycogen phosphorylase in livers of fed
thase activity in the brain resembled the distribution
and fasted rats. J. Cell Biol., 115:450a.
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T., G. Okuno, S. Price, and P. FoL 1964 The activity of uridine
clonal antibodies which did not cross-react with liver
diphosphate glucose-glycogen synthetase in some embryonic tisglycogen synthase. Immunostaining coincided with
sues. J. Histochem. Cytochem., 12:275-280.
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Michaels, J.E., J.T. Hung, S.A. Garfield, R.R. Cardell, Jr. 1984 Lob-
We thank Mrs. Thelma Shepard for technical assistance, Ms. Petrina Aquino for photographic assistance,
and Mrs. Barbara Burch for typing the manuscript.
This work was supported by U.S.P.H. grant DK27097.
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adrenalectomized, injections, dexamethasone, localization, activity, live, norman, rats, synthase, faster, glycogen
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