Protective Effects of Active Hexose Correlated
Compound (AHCC) on the Onset of Diabetes Induced by Streptozotocin
in the Rat
Koji Wakame, PhD
Department of Biochemistry, Dokkyo University School
of Medicine, Mibu, 321-0293 Tochigi, and Research and Development
Department, Amino UP Chemical Co. Ltd., Sapporo, 004-0839 Hokkaido,
Biomedical Research 20 (3) 145-152, 1999
Effects of Active Hexose Correlated Compound (AHCC) on the onset
of diabetes were studied in rats treated with Streptozotocin (STZ).
AHCC was given to male rats at 4% in drinking water. A single
i.v. injection of STZ (40mg/kg body weight) to rats resulted in
an increase in blood glucose levels, a decrease in serum insulin
levels, suppression of body weight gain, and an increase in serum
GOT and GPT activities and serum levels of lipid peroxides. Treatment
of AHCC restored these parameters to normal. Insulin immunoreactive
B-cells in Langerhans islets reduced in number after treatment
with STZ, while insulin immunoreactivity in the islets was normalized
when AHCC was administered to STZ-treated rats. These results
show that AHCC treatment is effective on the prevention of diabetes
onset induced by STZ.
Crude extracts derived from fungi of Basidiomycetes family such
as lingzhi (Ganoder-malucidum, reishi) and zhuling (Polyporus
umbellatus, chorei) are still used as components of Chinese traditional
medicine. These fungi contain polysaccharides as well as other
bioactive substances whose, actions include regulation of the
immune system, antitumor action, hypo-glycemic effect, improvement
of lipid metabolism and diuretic effect (26).
Recent advances in culture techniques have enabled us to culture
various species of Basidiomycetes. Active Hexose Correlated Compound
(AHCC, Amino UP Chemical Co; Ltd., Sappro) is a mixture
of polysaccharides, amino acids and minerals derived from fungi.
It is obtained by hot water extraction after culturing mycelia
of several basidiomycetes in a liquid culture tank and then treating
them with some enzymes.
AHCC has been successfully used as a biological response modifier
(BRM) in various disorders but only a little is known about its
mechanism of actions. Recently we have reported that AHCC has
hepatoprotective and detoxicating effects due to induction of
hepatic enzymes and antioxidant action (23). It was also found
that AHCC prolonged the life span and stimulated cytokine secretion
from macrophages in mice bearing SST-2 tumor when treated together
with 5-fluorouracil (5-FU)(14). Furthermore, it was shown that
AHCC was effective in preventing lung metastasis in breast cancer-bearing
mice when treated simultaneously with 5-FU (14). Sun et al. (13,
22) described that AHCC diminished the side effects caused by
antitumor agents such as cyclophosphamide, 5-FU and cytosine arabinoside.
In other words, the disorders in hematopoietic function, depilation
and he-patotoxicity induced by these pharmaceuticals were all
ameliorated by AHCC. These results suggest that AHCC restores
the depression of the immune system and reduces side effects caused
by antitumor agents, resulting in the prolongation of life span
in experimental animals.
It is reported that AHCC treatment for 2 years of patients with
hepatoma who had undergone hepatectomy suppressed the remission
and prolonged their life span significantly when compared with
control patients (10). Yagita et al. (28) found that oral administration
of AHCC to patients with malignant tumors resulted in an increase
in serum levels of tumor necrosis factor (TNF)-a, interferon-
g, and interleukin-12 and a decrease in serum immunosupressive
acidic protein (IAP) and tumor growth factor (TGF)-b levels. AHCC
has clinically shown to be beneficial for the treatment of other
diseases. In addition to patients with malignant tumors (28),
diabetic patients have been shown to respond to AHCC (11). Polysaccharides
which lower elevated blood glucose levels were found in several
fungi. For example, ganoderan A, B and C, polysaccharides derived
from Ganoderma lucidum have hypo-glycemic activity in alloxan-treated
mice (24). It is not known whether or not AHCC contain such anti-diabetogenic
In diabetes, the metabolism of glucose, proteins and lipids is
abnormal due to the deficit in glucagon and insulin secretion,
leading to various metabolic disorders (4) and onset of complications
(8, 21). It is also reported that diabetics are highly sensitive
to oxidative stress (1, 17, 25).
The objective of the present study is to examine the preventive
effects of AHCC on the onset of diabetes in rats treated with
streptozotocin (STZ). This chemical is thought to induce type
I diabetes possibly by stimulating xanthine oxidase (XOD) reaction
in b-cells of Langerhans islets to increase O2 radicals, which
in turn destroy the cells (9). Thus, the STZ-induced diabetes
can be regarded as an experimental model of type I diabetes. The
present study deals with the preventive effects of AHCC on the
impairment of islet cells.
Materials and Methods
Materials: STZ was obtained from Sigma Chemical Co. (St.
Louis, MO, USA). It was dissolved in 0.05 M citrate buffer, pH
4.5 at a Final concentration of 40 mg/mL. The solution was stored
in darkness at 4C until use. Lyophilized AHCC was obtained from
Amino UP Chemical Co. Ltd. (Sapporo, Japan).
Animals: Male Wistar rats of 4 weeks old were purchased
from Charles River Japan Inc. (Kanagawa, Japan). The animals were
housed in a room kept at 22±2¡C with 12h light and dark cycle
(light on 8 : 00-20 : 00 h) and kept free access to food and water.
The general conditions of the rats were observed every day. The
amount of water intake was measured by subtracting the amount
of residual of water from that measured the previous day. A group
of rats were given only AHCC at 4% in their drinking water for
3 weeks (AHCC-treated group). STZ at a dose of 40 mg per kg body
weight was injected through the tail vein to 2 groups of rats
(STZ-treated group). One of the STZ-treated groups received 4%
AHCC in drinking water for one week prior to the treatment and
for 2 weeks thereafter (STZ-AHCC treated group).
Blood sampling and organ preparation. Blood was collected
via the tail vein of the rats into EDTA-coated blood sampling
tubes at 10:00 a.m. on the same day immediately after STZ treat-ment
and at 7 and 14 days after STZ treatment. At 14 days after STZ
treatment the rats were sacrificed by decapitation, and their
livers and pancreases were removed. These organs were quickly
rinsed in ice-cold saline solution. Pan creases were immediately
fixed in 10% formalin.
Biochemical analyses. Blood samples were centrifuged at
4¡C for 15 min at 3,000 rpm and the sera thus obtained were used
for biochemical analyses. Serum GOT and GPT, blood glucose, lipid
peroxides (LPO) and insulin were measured by Henry's method (5),
glucose oxidase method, Yagi's method (27) and Insulin-EIA test
(Wako, Osaka), respectively.
Immunocytochemistry. The pancreatic tissue was fixed in
10% buffered formalin for 2 days, and embedded in parafin according
to the conventional procedure. Paraffin sections were serially
cut at 5 um in thickness and immumostained. Localization of insulin
in pancreatic islets was visualized by the streptavidin-biotinylated
horse-radish peroxiase complex immunoenzymatic technic using Histofine
SAB-PO (M) kits (Nichirei, Tokyo, Japan) according to the protocol
of the manufacturer. Briefly, a monoclonal antibody against rat
insulin was employed as the first antibody. Reaction products
were formed with 3, 3'-diaminobenzidine tetra-hydrochloride in
the presence of hydrogen peroxide. The immumoreaction specifity
was determined using normal serum instead of the specific antiserum.
Statistical analysis. All the data were expressed as mean±SD. Statistical analysis was performed by Dunnett's multiple comparison
test to control and non parametric method by Scheffe's multiple
comparison test. A P value less than 0.05 was considered statistically
Poor general conditions such as depilation and deterioration
in quality of fur were noticed in the STZ-treated group. AHCC
treatment resulted in an improvement of these conditions. AHCC
alone showed no effect on body weight changes. A significant decrease
in body weight gain was observed during 14 days after STZ treatment
when compared with intact controls. The body weight gain of the
STZ-AHCC treated rats was nearly the same as that of the intact
An increase in the daily water consumption was noticed in the
STZ-treated group (Fig. 3). AHCC given alone increased somewhat
the water consumption but decreased the increased water intake
when given to the STZ-treated rats.
A significant rise in blood glucose levels was observed in the
STZ-treated group up to 14th day after the drug treatment, reaching
its maximum on 7 days after the treatment (Fig. 4). The blood
glucose level at 14 days after STZ treatment was more than twice
that in the control group. AHCC treatment nearly normalized the
elevated levels of blood glucose in STZ-treated rats. The serum
insulin levels decreased following STZ treatment and remained
low till 14 days (Fig. 5). AHCC treatment restored the decreased
insulin levels to normal.
The serum levels of GOT, GPT and LPO were greater in the STZ-treated
group than in the intact control and STZ-AHCC treated group at
14 days after the treatments.
Histology of the pancreas
A prominent decrease in the number of b-cells in the Islets of
Langergans was observed in the STZ-treated group when compared
with control group. The immunoreactivity of insulin in b-cells
of the STZ- Paraffin sections obtained from the control and treated
rats were immunostained with anti-insulin antibody. A prominent
decrease in the number of insulin immunoreactive cells was recognized
in STZ-treated group, while appreciable insulin cells were found
in the STZ-AHCC treated group.
Streptozotocin (STZ) was originally discovered as a metabolite
of Streptomyces achromogenes var. streptozotics by a research
group in Upjohn Co. Ltd. in 1959. At first, there were expectations
over possible clinical applications of its antibacterial and antitumor
activities but it was soon found that diabetes was induced as
a side-effect of STZ treatment. This chemical has widely been
used as a diabetogenic agent there-after. Okamoto suggested that
STZ induces diabetes through damaging DNA in the nuclei of pancreatic
b-cells by alkylation, leading to an increase in poly (ADP-ribose)
synthase. The increase in the enzyme activity results in a drastic
decrease in nicotinamide adenine dinu-cleotide (NAD) concentrations
of the yff-cells and then a decrease in the number of b-cells
and death of the cells. All these changes may induce dysfunction
of the pancreas.
According to Kawada, STZ transported into b-cells through glucose
transporter GLUT-2 located on their cell membranes is activated
inside the cells and injures their mitochondria. This inevitably
leads to a reduction of ATP generation through electron transport
system and an increase in ADP concentrations. Subsequent degradation
of ADP provides hypoxanthine, a substrate of xanthine oxidase
(XOD). When XOD reaction takes place in the b-cells where XOD
activity is intrinsically very high, O2 radicals are produced,
resulting in cell damage and the onset of diabetes. STZ also activates
XOD directly and augments 02" generation.
The present study showed that the body weight of the STZ-treated
rats decreased soon after STZ administration. This finding was
in close agreement with that of other investigators (2, 3, 7,
12). In the STZ-AHCC treated group, however, no significant decrease
in body weight was observed, suggesting that AHCC normalized the
weight change caused by STZ.
A significant increase in water intake, one of the general signs
of diabetes, was observed in the STZ-treated rats. The water consumption
was a little greater in the AHCC-treated groups than in the control,
presumably because the rats had preference to AHCC-containing
water which is rich in polysaccharides, amino acids, lipids and
minerals. The water consumption of the STZ-treated group is much
greater than that of the AHCC-treated groups. AHCC nearly normalized
the water consumption and body weight gain of STZ-treated rats,
suggesting that AHCC may have prevented the onset of diabetes.
In the STZ-treated group, blood glucose rose immediately after
the treatment and reached a quite high level at 14th day. The
serum insulin levels decreased significantly in the STZ-treated
group, indicating that their pancreatic b-cells were damaged.
On the other hand, the blood glucose levels in the STZ-AHCC treated
group were much lower than in STZ-treated group but slightly higher
than in the control group. These results showed that mild diabetes
did occur in the STZ-AHCC-treated rats, even though the serum
insulin levels were restored to normal in STZ-AHCC treated rats.
The serum levels of GOT, GPT and LPO increased in the STZ-treated
group, while they were normalized by AHCC treatment. Rhee et ai
(20) reported that the increase in the serum levels of GOT, GPT
and LPO was due to the oxidative damage in the pancreas, liver,
kidney and other organs caused by STZ through the increase in
the free radical production. They suggested that the production
of free radicals was augmented by the increase in arachidonate
concentrations and the activity of lipoxygenase and cyclooxygenase.
Furthermore, they observed the suppressive effects of antioxidants
such as vitamin E on the onset of diabetes. The results of our
present study suggest that AHCC suppresses the production of free
radicals induced by STZ, whereby symptoms of diabetes were diminished.
Diabetogenic substances such as STZ preferentially destroy nuclei
of b-cells. Apoptosis has recently been suggested to be involved
in STZ-induced degeneration of islet cells (15). Since b-cells
of the islets decreased in number, one can expect that apoptosis
may have occurred following STZ administration. The STZ-induced
decrease in insulin immunoreactivity in islet b-cells was restored
by AHCC. These findings suggest that AHCC prevents cellular damage
induced by STZ and preserve the capability of insulin secretion.
In conclusion, the results of our present study show that AHCC
can prevent the onset of STZ-induced diabetes by protecting b-cells
from degeneration and by diminishing oxidative injuries of cells
in various organs. It remains to be clarified the mechanism by
which AHCC acts as an antioxidant.
The author would like to thank Professor S. Matsuzaki (Dokkyo
University School of Medicine) for his critical review of the
manuscript. Thanks are also due to Amino UP Chemical Co. Ltd for
the supply of AHCC.
Fig. 1 Effects of AHCC and STZ on the body weight. Single
intravenous injection of STZ reduced significantly the body weight
gain. The body weight in the STZ-AHCC treated group was significantly
larger than that in the STZ-treated group. Values are the means
±SD of 6 determinations in each group. * P<0.01 vs. control
group. Ê P<0.01 vs. STZ-AHCC group.
Fig. 2 Effects of AHCC and STZ on water intake. Single
intravenous injection of STZ caused significant increase in the
water intake. Treatment of the diabetic rats with AHCC nearly
normalized the water intake. The values are the means ±SD of 6
determinations in each group. * P<0.01 vs. control group. Ê
¤ P<0.01 vs. AHCC-STZ group.
Fig. 3 Effects of STZ and AHCC on serum glucose levels..
Single intravenous injection of STZ caused marked elevation of
the blood glucose, reaching its maximum at 7 days of the treatment..
Treatment of the diabetic rats with AHCC nearly normalized the
elevated serum glucose level. The values are the means ± SD of
6 determinations in each group. * P<0.01 vs. control group.
P<0.01 vs. AHCC-STZ group.
Fig. 4 Effects of STZ and AHCC on serum insulin levels..
Single intravenous injection of STZ caused a significant decrease
in the immunoreactive insulin level. AHCC restored the decreased
serum insulin in diabetic with rats. Values are the means ± SD
of 6 determinations in each group.
* P<0.01 vs. control group. P<0.01 vs. AHCC-STZ group.
Fig. 5 Effects of STZ and AHCC on the activity of serum
GOT and GPT. Rats were sacrificed at 14 days after STZ treatment
and their sera were taken. The activity of the transaminases (GOT
and GPT) was assayed as described in the text. Values are the
means ± SD of 6 determinations in each group. * P<0.01 vs.
control group. P<0.01 vs. AHCC-STZ group.
Table 1: Effects of STZ and AHCC on serum lipid peroxides
After 14 days
Lipid peroxides (LPO) (nmol MDA/ml)
* P<0.01 vs. control group. P<0.01 vs. AHCC-STZ group.
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