Journal of Ethnopharmacology, 7 (1983) 205-234 Elsevier Scientific Publishers Ireland Ltd. 205 ENZYME CHANGES AND GLUCOSE U’I’ILISATION IN DIABETIC RABBITS: THE EFFECT OF GYMNEMA SYL VESTRE, R.Br. K.R. SHANMUGASUNDARAM’, C. PANNEERSELVAMa, P. SAMUDRAMb and E.R.B. SHANMUGASUNDARAMb aDepartment of Biochemistry, Post Graduate Institute of Basic Medical Sciences, University of Madras, Tammani Campus, Madras-600 113 and bDepartment of Bio- chemistry, University of Madras, Guindy Campus, Madras-600 025 (India) (Accepted June 29,1982) The administration of the dried leaf powder of Gymnema sylvestre regulates the blood sugar levels in alloxan diabetic rabbits. G. sylvestre therapy not only produced blood glucose homeostasis but also increased the activities of the enzymes affording the utilisation of glucose by insulin dependent pathways: it controlled phosphorylase levels, gluconeogenic enzymes and sorbitol dehydrogenase. The uptake and incorporation of [“Cl glucose into the glycogen and protein are increased in the liver, kidney and muscle in G. sylvestre administered diabetic animals when compared to the untreated diabetic animals. Pathological changes initiated in the liver during the hyperglycemic phase are reversed by controlling hyperglycemia by G. sylvestre. G. sylvestre, a herb used for the control of diabetes mellitus in several parts of India, appears to correct the metabolic derangements in diabetic rabbit liver, kidney and muscle. Introduction Diabetes was classified as a urinary disorder by Sushruta (6th century B.C.a), an Indian physician and surgeon, who lived in Varanasi (Benares). In his treatise, he states, “It may be prognosticated that an idle man, who indulges in day sleep or follows sedentary pursuits or is in the habit of taking sweet liquids or cold and fattening food, will ere long fall an easy victim to this disease.” The appearance of abscess, increased urine volume, sweet taste of urine, perspiration and sputum are recorded as the premoni- tory symptoms of Madhumeha (meaning Honey Urine in Sanskrit) or diabetes mellitus. It may be recalled that in Europe, the first record of sweetness of 0 1983 Elsevier Scientific Publishers Ireland Ltd. -Published and printed in Ireland
ENZYME CHANGES AND GLUCOSE U’I’ILISATION IN DIABETIC RABBITS: THE EFFECT OF GYMNEMA SYL VESTRE, R.Br.
K.R. SHANMUGASUNDARAM’, C. PANNEERSELVAMa, P. SAMUDRAMb and E.R.B. SHANMUGASUNDARAMb
aDepartment of Biochemistry, Post Graduate Institute of Basic Medical Sciences, University of Madras, Tammani Campus, Madras-600 113 and bDepartment of Bio- chemistry, University of Madras, Guindy Campus, Madras-600 025 (India)
(Accepted June 29,1982)
The administration of the dried leaf powder of Gymnema sylvestre regulates the blood sugar levels in alloxan diabetic rabbits. G. sylvestre therapy not only produced blood glucose homeostasis but also increased the activities of the enzymes affording the utilisation of glucose by insulin dependent pathways: it controlled phosphorylase levels, gluconeogenic enzymes and sorbitol dehydrogenase. The uptake and incorporation of [“Cl glucose into the glycogen and protein are increased in the liver, kidney and muscle in G. sylvestre administered diabetic animals when compared to the untreated diabetic animals. Pathological changes initiated in the liver during the hyperglycemic phase are reversed by controlling hyperglycemia by G. sylvestre. G. sylvestre, a herb used for the control of diabetes mellitus in several parts of India, appears to correct the metabolic derangements in diabetic rabbit liver, kidney and muscle.
Introduction
Diabetes was classified as a urinary disorder by Sushruta (6th century B.C.a), an Indian physician and surgeon, who lived in Varanasi (Benares). In his treatise, he states, “It may be prognosticated that an idle man, who indulges in day sleep or follows sedentary pursuits or is in the habit of taking sweet liquids or cold and fattening food, will ere long fall an easy victim to this disease.” The appearance of abscess, increased urine volume, sweet taste of urine, perspiration and sputum are recorded as the premoni- tory symptoms of Madhumeha (meaning Honey Urine in Sanskrit) or diabetes mellitus. It may be recalled that in Europe, the first record of sweetness of
0 1983 Elsevier Scientific Publishers Ireland Ltd. -Published and printed in Ireland
206
the diabetic urine was documented only in 1675 A.D. by Thomas Willis (Marble, 1973).
Sushruta (6th century B.C.b) also describes the two types of diabetes, one of which is congenital or inherited from the parents (juvenile diabetes in today’s parlance) which is characterised by emaciation and dryness of the body, diminished capacity for eating, excessive thirst and restlessness. Diabetics with the second type of disease are obese, pursue sedentary habits and are gluttons. The latter category should control themselves by fasting and physical exercise. For this type (maturity onset diabetes) Sushruta (6th century B.C.b) recommended several remedies including the intake of medicines prepared using plants of “SalaSaradi” group.
One such plant (belonging to the “SalaSaradi” group) used by several Indian medical practitioners (both Ayurvedic and Sidha disciplines) is “meshashringi” meaning ram’s horn. The botanical name for this plant is Gymnema sylvestre, R.Br. belonging to the Asclepiadaceae (Milk Weed family). G. sylvestre is a woody branched vine growing in the wild forests of central India and the Western ghats. The widespread use of the plant is obvious when one looks into the different names by which it is known in different Indian languages. Sastri (1956) has pointed out that it is known as Meshashringi, Meshavalli, Medashrangi and $arpadarushtrika in Sanskrit; Meshasingi and Memsingi in Hindi; Kalikordori in Marathi; Kogilam and Shirukurinja in Tamil and Podapatri in Telugu. He also reported that the plant is known as Periploca of the woods in English and Waldschlinge in German.
G. syluestre has been included in the Indian Materia Medica (Nadkarni, 1954). Its root is used as a remedy for snake bite. Leaves of G. syluestre have the peculiar property of abolishing the sense of sweet taste of sugar which was confirmed by Hooper (1887). This property has been known from ancient times and hence the plant is also known as Sarkaraikolli in Tamil and Malayalam and Gurmar in Hindi meaning sugar destroyer. On analysis, Hooper (1889) observed that the leaves contain a brittle, black, acidic resinous substance which was called gymnemic acid. It was observed by Berthold (1888) that the gymnemic acid fraction was responsible for the action on taste, Gymnemic acid has been resolved into four components by Sinsheimer et al. (1968) and their antiviral activity was investigated. The chemical structure of the acid remains unknown.
Power and Tutin (1904) found that the leaves of 0. sylvestre contain hentriacontane, a glucoside and gymnemic acid with antisaccharine properties. Gharpurey (1926) discovered that the leaves contain a substance with hydrolytic action on glucose but with no effect on blood sugar when given by subcutaneous injection to rabbits.
Mhaskar and Caius (1930) observed that the leaves contain chlorophyll, xanthophylls, carotene, phytol, hentriacontane, pentatriacontane, lime salts and gymnemic acid. The pharmacological action of G. sylvestre leaf powder, its alcohol extract, diethyl ether extract and gymnemic acid fraction (crude
207
acidic fraction) were investigated. They observed that the gymnemic acid fraction increases oxygen uptake and blood pressure and increases the secre- tions of liver and pancreas. These physiological effects were not observed in pancreatectomised rabbits or dogs showing that the action of G. syluestre is mediated through the pancreas. The effect of G. syluestre leaf powder on diabetic patients was attempted by Mhaskar and Caius (1930). They observed that at thedosage of 4 g/day the blood glucose levels are significantly lowered and total urinary glucose excretion was also lowered. The herb was ineffective for some patients and the drop out rate during the trial was high.
Use of oral insulinotropic agents like sulphonylureas in diabetic therapy can be useful in not only regulating the blood glucose levels, but also ef- fectively regulating the utilisation of glucose by stimulating the insulin- dependent pathways like glycogenesis and glycolysis, and inhibiting lipo- lysis. In this study, the usefulness of G. sylvestie therapy in alloxan diabetic rabbits in correcting the abnormal accumulation of lipids, glycogen and protein depletion in the liver, kidney and muscle was investigated. The secondary changes in the kidney of diabetic animals and their regeneration by G. syluestre were also studied.
Materials and methods
Albino rabbits (lo-12 weeks old) of both sexes weighing between 650 g an 800 g were obtained from the inbred stock of animals from the King’s Institute of Preventive Medicine, Madras. They were maintained on Hindustan Lever rabbit feed and water, given ad libitum.
Chemicals used in this investigation were obtained as follows: alloxan monohydrate from E. Merck, F.R.G.; anthrone from Hopkins and Williams, U.K.; sodium salts of DL-glyceraldehyde 3-phosphate, phosphoenol pyruvate, UDP, UDPG, Sephadex G-200, dithiothreitol, cholesterol and tripalmitin from Sigma Chemical Company, New Jersey, U.S.A. ; sodium salts of glucose 6-phosphate, fructose-l ,6diphosphate, NAD+, NADP+ and NADH from Wessex Biochemicals Ltd., Castle Road, Boumemouth, U.K.; ATP (sodium salt) from SD’S Lab. Chem., India, and also from the C.S.I.R. Centre for Biochemicals, India; sodium salts of glucose l-phosphate, glycogen (rabbit liver) and some UDPG and UDP from ICN Pharmaceuticals Inc., Life Sciences Group, Cleveland, OH, U.S.A.; [14C]glucose from Isotope Division, BARC, Bombay.
G. syluestre, R.Br. was collected from the hillocks (600 m above main sea level) near the village of Mambedu, in the Chitoor District of Andra Pradesh, India. The samples were checked by an expert Sidha medical practitioner.
During the long summer months the plant sheds its leaves, and the leaves sprout after the onset of rain. This area is a comparatively arid region and gets a rainfall of not more than 100 cm a year which is spread over the period from July to November. The plant collection was undertaken between October and February.
208
The leaves were air dried indoors along with the stem, and stored in teak- wood chambers until used. These dried leaves have been found to retain their hypoglycemic action for at least 1 year. Not more than 100 g of dried leaves were taken at a time, ground into powder in a hand operated coffee grinder and stored in glass stoppered bottles for administration and tests.
Induction of diabetes in mbbits and the experimental design Diabetes was produced in albino rabbits (700-900 g body wt.) by in-
jecting intravenously 90 mg/kg body wt. of alloxan monohydrate in sterile distilled water after overnight fasting. Animals with a fasting blood sugar of 150-230 mg/dl were included in the group of diabetics for the experi- ment. The drug (powder of G. sylvestre leaves) dosage of 250 mg/kg body wt. was arrived at after a preliminary study. The leaf powder was mixed in carrot slices and was administered once a day in the morning.
The animlas were grouped as follows: Group I, normal rabbits; Group IIa, un~ntro~~ diabetics maintained hy~glycemic with food and water given ad libitum for 12 weeks and then killed; Group IIb, uncontrolled diabetics maintained for 24 weeks before killing; Group IIIa, controlled diabetics - diabetic animals left hyperglycemic for the first 24 weeks followed by G. sylvestre administration for the next 12 weeks before killing; Group IIIb, controlled diabetics - diabetic animals left hyperglycemic for the first 24 weeks followed by G. sylvesti a~in~~ation for the next 24 weeks before killing.
The animals were included in the experiment not all at the same time, but in several continuous batches. Glucose tolerance tests (GTT) were carried out on not more than 4 animals on any particular day. Since mortality rates among the diabetics were high not all the animals survived throughout the experimental period.
The study is designed as a sequential analysis of a cohort. Diabetic animals, left untreated, show very poor survival after 24 weeks due to increased deterioration of glucose homeostasis and the greater susceptibility to in- fections. The observations on the untreated diabetics at 36 and 48 weeks are not reported because of the complexities involved due to poor survival. No instance of reversal of the diabetic state was observed in any of these un- treated animals. Fasting blood sugar was estimated every 4 weeks.
At the end of the experimental period the animals were killed; liver, kidney and muscles were removed quickly into icecold containers. Small fragments of the tissues were used for the estimation of protein (Lowry et al., 1951), glycogen {Morales et al., 1973), total lipids (Folch et al., 1957), total cholesterol (Leffler and McDougald, 1963) and triglycerides (Brixova and Dzurikova, 1968). An aliquot of lipid fraction was digested with perchloric acid and the phosphate was estimated (Fiske and Subbarow, 1926) and the phospholipid levels obtained.
Enzyme assays All the samples for enzyme assays were stored in an ice-bath at 4% in
209
a cold room and assays were made within 24 h of killing the animals. The tissues were homogenised in 0.1 M Tris-HCl buffer (pH 7.4) and centri- fuged at 20,000 X g for 20 min. The supernatants were used for enzyme assays.
Hexokinase (EC 2.7.1.1) was assayed after Brandstrup et al. (1957); glyceraldehydeS-phosphate dehydrogenase (EC 1.2.1.12) activity (Kirk and Ritz, 1967), glycogen synthetase (Leloir and Goldemberg, 1962), total phosphorylase (Lederer and Stalmans, 1976), phosphorylase a (EC 2.4.1.1) (Comblath et al., 1963), glucose&phosphate dehydrogenase (EC 1.1.1.49) (Ells and Kirkman, 1961), sorbitol dehydrogenase (EC 1 .l .1.14) and glucose- 6-phosphatase (EC 3.1.3.9) (King, 1965), fructose-l,(idiphosphatase (EC 3.1.3.11) (Gancedo and Gancedo, 1971), glutamate oxaloacetate trans- aminase, GOT (EC 2.6.1.1) and glutamate pyruvate transaminase, GPT (EC 2.6.1.2) (King, 1965) were assayed in the tissue extracts. Lactate dehydrogenase, LDH (EC 1.1.1.27) was assayed by the method of King (1965); acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) were assayed by the method of Moog (1946) as modified by King (1965) and ATPase (EC 3.6.1.3) by the method of Evans (1969).
Blood glucose was estimated by the method of Sasaki and Sanae (1972) using o-toluidine.
Up take and incorpom tion of [ “C]glucose One set of Group IIb and Group IIIb animals together with sex- and age-
matched controls were given an oral dose of 28,300 cpm/g body wt. of uniformly labelled [ 14C] glucose with 1 .O g of unlabelled glucose as carrier, after overnight fasting. The animals were killed after 4 h and the liver, kidney and muscle were dissected out. The incorporation of radioactivity into theglycogen, lipids, protein and nucleic acids were studied by measuring these different fractions in a liquid scintillation counter (Nuclear Chicago, Unilux II). For this, the tissue homogenate in water was subjected to the fractionation procedure of LePage (1959). The homogenate was incubated for 30 min with 5% TCA at 4°C and centrifuged. This supernatant is the cold TCA fraction. The residue was extracted successively with ethanol, and ethanol (7O%)/ether (1: 1, v/v) mixture and centrifuged. The supernatants were pooled to obtain the lipid fraction. The residue was bolied with 5% TCA for 30 min and centrifuged to obtain the nucleic acids in the super- natant and proteins in the residue. A separate portion of the tissue was extracted with KOH and glycogen, estimated by the method of Morales et al. (1973), was counted for radioactivity. The scintillation fluid was a mixture of toluene and Triton X-100 (2 : 1, v/v) containing PPO and POPOP as fluors. The incorporation of [ 14C] glucose in each fraction was expressed as dpm/g tissue.
Histological examination A small portion of each tissue was fixed in a 10% solution of formalin
TA
BL
E
1
FA
STIN
G
BL
OO
D
GL
UC
OSE
L
EV
EL
A
ND
C
EL
LU
LA
R
CO
NST
ITU
EN
TS
IN N
OR
MA
L,
DIA
BE
TIC
A
ND
C
ON
TR
OL
LE
D
DIA
BE
TIC
R
AB
BIT
S
The
dia
beti
c an
imal
s re
mai
ned
hype
rgly
cem
ic
for
a pe
riod
of
24
wee
ks
befo
re
the
init
iati
on
of G
. sy
lues
tre
adm
inis
trat
ion.
T
he a
naly
ses
wer
e m
ade
12 a
nd
24 w
eeks
af
ter
allo
xani
sati
on
in t
he
untr
eate
d di
abet
ics
and
12 a
nd
24 w
eeks
af
ter
the
init
iati
on
of a
dmin
istr
atio
n.
Val
ues
are
expr
esse
d as
mea
n f
S.D
. (f
or
n nu
mbe
r of
ani
mal
e).
For
ass
essi
ng
the
stat
isti
cal
sign
ific
ance
fo
r th
e va
riat
ions
re
port
ed,
grou
ps
IIa
and
IIb
are
com
pare
d w
ith
I, w
hile
II
Ia a
nd I
IIb
are
com
pare
d w
ith
IIb.
Nor
mal
an
imal
s (?
I =
7)
Unt
reat
ed
diab
etic
s C
ontr
olle
d di
abet
ics
IIa
IIb
IIIa
II
Ib
12 w
eeks
24
wee
ks
36 w
eeks
48
wee
ks
(n=5
) (n
=5)
(n=5
) (n
=5)
Fas
ting
bl
ood
gluc
ose
80.2
f
6.5
210.
0 f
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5d
150.
0 f
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11
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5d
(mg/
lOO
m
l)
Gly
coge
n (m
g/g
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h ti
ssue
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iver
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Pro
tein
(m
g/g
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y M
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e
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0 f
8.0
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15
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3d
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ld
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1.3’
12
.2
f l.
la
10.0
f l
.lb
9.
1 +
1.2
=
17.7
f
1.7
24.3
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1.3
27.2
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22
.6
f l.
ld
20.6
*
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Lip
ids
kid
ney
(m
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sh ti
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tal
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gly
ceri
de
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lip
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+
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1=
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+
5.2
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.6 f
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b
4.3
f 0.
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scle
(mg
/g f
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212
(formaldehyde) in 0.9% saline. These tissues were processed for paraffin embedding and sections were stained with (i) haematoxylin-eosin (H and E) and (ii) periodic acid-Schiff’s (PAS) reagent and examined for intracellular changes and alterations in lipid and glycogen deposition.
Results
Table 1 gives the overall picture of fasting blood glucose levels in the experimental animals together with glycogen, protein and lipids of liver, kidney and muscle. Administration of G. syluestre (Groups IIIa and IIIb) brings down the fasting blood glucose levels, together with considerable lowering of tissue lipids, while raising the tissue glycogen and proteins.
Glycogen levels in the liver and muscle show a decrease of about 80% in the diabetic state. Twelve weeks of treatment with G. syluestre (I&) significantly increased the glycogen level in the liver and after 24 weeks of treatment (IIIb) the levels were near normal, indicating that the defective glycogen storage of the diabetic state had been corrected by the herb. Changes in muscle parallel those seen for liver and were not statistically significant.
Protein content of the diabetic tissues is less than that of the healthy normal rabbits, and G. syluestre administration raises the protein levels in all three tissues investigated.
Total lipids are increased progressively in the diabetic animals (IIa and IIb), and G. syluestre administration reverses this trend.
Activities of some of the enzymes involved in the metabolism of glucose are given in Table 2. Table 3 gives the levels of transaminases, LDH and phosphatases in the normal, diabetic and G. syluestre controlled diabetic rabbit liver. Table 2 shows that hexokinase activity is reduced in the liver by over 20% in the la-week hyperglycemic state (IIa) and to 40% at the end of 24 weeks (IIb). This enzyme is considerably enhanced by G. syluestre administration.
Glyceraldehyde-3-phosphate dehydrogenase is reduced markedly in diabetics and G. syluestre increases the activity only marginally. This enzyme is an index of glycolytic activity. Glycogen synthetase and glucose-6-phos- phate dehydrogenase, two other insulindependent reactions are also lowered in the diabetic animals (IIa and IIb) and are increased after control by G. syluestre. Glycogen synthetase is the rate-limiting enzyme in glycogen synthesis from UDPG (Larner, 1971). The activity of this enzyme falls very rapidly and the activities at 12 and 24 weeks (Groups IIa and IIb) are not very different, while the fall in the activities of hexokinase and glyceral- dehyde&phosphate dehydrogenase is less rapid and more gradual. In the same manner, the increase in activity observed during G. syluestre adminis- tration is also very rapid indicating that G. syluestre might favour glycogen synthesis as one of the earliest steps in correcting the metabolic derangements caused by insulin deficiency in alloxan diabetic rabbits.
TA
BL
E
2
AC
TIV
ITY
O
F SO
ME
KE
Y E
NZ
YM
ES
IN G
LU
CO
SE
ME
TA
BO
LIS
M
IN T
HE
LIV
ER
O
F N
OR
MA
L,
DIA
BE
TIC
A
ND
CO
NT
RO
LL
ED
D
IAB
ET
IC
RA
BB
ITS
The
dia
betic
an
imal
a re
mai
ned
hype
rgly
cem
ic
for
a pe
riod
of
24
wee
ks b
efor
e th
e in
itiat
ion
of 0
. sy
lues
tre
adm
inis
trat
ion.
T
he a
naly
ses
wer
e m
ade
12 a
nd
24 w
eeks
aft
er
allo
xani
satio
n in
the
unt
reat
ed
diab
etic
s an
d 12
and
24
wee
ks a
fter
th
e in
itiat
ion
of a
dmin
istr
atio
n.
Enzy
me
activ
ities
ar
e ex
pres
sed
as r
un01
pro
duct
fo
rmed
&
prot
ein
unde
r in
cuba
tion
cond
ition
s.
For
glyc
eral
dehy
de-3
-ph~
phat
e de
hydr
ogen
ase
and
gluc
ose&
-pho
spha
te
dehy
drog
enas
e,
the
enzy
me
activ
ities
ar
e ex
pres
sed
as A
O.D
./min
/mg
prot
ein.
V
alue
s ar
e ex
pres
sed
as m
ean
* S.
D.
(for
n n
umbe
r of
ani
mal
s).
For
asse
ssin
g th
e st
atis
tical
si
gnif
ican
ce
for
the
vari
atio
ns
repo
rted
, gr
oups
II
a an
d II
b ar
e co
mpa
red
with
I,
whi
le
IIIa
and
III
b ar
e co
mpa
red
with
IIb
.
Enz
yme
Con
trol
(‘
117)
U
ntre
ated
di
abet
es
Con
trol
led
diab
etes
IIa
IIb
IIIa
II
Ib
12 w
eeks
24
wee
ks
36 w
eeks
48
wee
ks
(n=5
) (n
-5)
(n-5
) (n
= 5
)
Hex
okin
ase
1067
*
127
767
* 92
c 60
9 f
78
a52
+ 62
’ 86
8 f
7oc
Gly
cera
ldeh
yde-
3-ph
os-
22
f 3.
0 18
*
1.2b
17
f
1.0”
19
*
1.3a
20
f
1.2c
ph
ate
dehy
drog
enas
e G
lyco
gen
synt
heta
se
56.4
f
1.5
27.1
*
4.16
22
.6
f 5.
2d
43.2
f
4.7b
48
.5
f 4J
d T
otal
ph
osph
oryl
ases
60
8 f
104
1173
f
128d
12
81
* 18
1d
965
f 1O
Ob
618
f 10
5d
Phos
phor
ylas
e a
206
f 65
67
6 f
78d
698
j: 92
6 25
8 f
65d
209
f 74
d G
luco
se&
phos
phat
e 0.
43
f 0.
03
0.22
*
0.06
d 0.
13
f 0.
04d
0.29
f
0.05
O
0.40
f:
0.
02d
dehy
drog
enaa
e So
rbito
l de
hydr
ogen
ase
312
f 37
51
9 f
29d
553
f 33
d 38
1 f
36’
322
i: 32
e G
luco
se
6-ph
osph
atas
e 12
70
f 16
8 22
51
* 24
3d
2689
f
357d
17
36
* 20
1=
1605
f.
109
d Fr
ucto
se-1
,6di
phos
phat
ase
305
f 14
82
7 f
113d
10
57
f 1O
ld
527
f 44
d 48
4 f
94d
aP <
0.0
6.
bP <
0.0
2.
cP <
0.0
1.
dP <
0.0
01.
TA
BL
E
3
AC
TIV
ITY
D
IAB
ET
IC ‘O
F R
EL
AT
ED
E
NZ
YM
ES
IN
GL
UC
OS
E M
ET
AB
OL
ISM
IN
TH
E L
IVE
R
OF
NO
RM
AL
, D
IAB
ET
IC
AN
D C
ON
TR
OL
LE
D
RA
BB
ITS
Th
edia
beti
c an
imai
s re
mai
ned
hyp
ergl
ycem
ic
for
a pe
riod
of
24 w
eek
s be
fore
th
e in
itia
tion
of
G
. sy
lves
tre
adm
inis
trat
ion
. T
he
anal
yses
w
ere
mad
e 12
an
d 24
wee
ks
afte
r ai
loxa
nis
atio
n
in t
he
un
trea
ted
diab
etic
s an
d 12
an
d 24
wee
ks
afte
r th
e in
itia
tion
of
adm
inis
trat
iolr
E
nzy
me
acti
viti
es a
re e
xpre
ssed
as
nm
ol
prod
uct
fo
rmed
/mg
prot
ein
un
der
incu
bati
on
con
diti
ons.
V
alu
es a
re e
xpre
ssed
as
mea
n +
S.D
. (f
or n
nu
mbe
r of
an
imal
s).
For
ass
essi
ng
the
stat
isti
cal
sign
ific
ance
fo
r th
e va
riat
ion
s re
port
ed,
grou
ps I
Ia a
nd
IIb
are
com
pare
d w
ith
I,
wh
ile
IIIa
an
d II
Ib a
re c
ompa
red
wit
h I
Ib.
En
zym
es
Con
trol
U
ntr
eate
d di
abet
es
Con
trol
led
diab
etes
GO
T
GP
T
LD
H
Aci
d ph
osph
atas
e A
lkal
ine
phos
phat
ase
Ade
nos
ine
trip
hos
phat
ase
. ,
IIa
IIb
IIIa
II
Ib
12 w
eek
s 24
wee
ks
36 w
eek
s 48
wee
ks
(n=
5)
(n=
5)
(n=
5)
(n =
5)
978
f 14
3 13
54
f 12
gp
1562
*
187s
11
54
f 11
5a
1113
f
15ga
88
1 f
67
1161
*
75b
1299
f
1Olb
98
3 *
108a
91
6 +
122
a 82
6 f
37
1637
f
263’
17
25
i 18
0b
1057
f
135s
94
9 f
54b
167
f 32
22
5 f
16’
250
f 21
a 21
3 16
+
20
2 f
21a
389
f 29
63
7 f
86b
661
f 59
b 42
1 5g
a *
392
f 32
a
a57
f 48
13
33
f 96
’ 14
06
* 14
4b
1213
+
73
’ 10
23
+ 1
05b
aP <
0.0
1.
bP <
0.0
01.
215
In diabetes, total phosphoryl~ activity (active + inactive) is doubled and the activity of the a (active) form is increased three-fold over the control. Treatment of diabetic animals with G. syluestre reduces the total activity and that of the a form considerably. The rapidity with which phosphorylase activity is elevated runs antipamllel to the changes in glycogen synthetase activity. The active form of the ezyme reaches the normal level after 24 weeks ad~is~tion of G. syluestre.
Glucose-6-phosphate dehydrogenase levels are lowered significantly in the diabetic state. G. syluestre administration induces moderate reversal of this effect and a great increase in the levels is observed in the 24-week treatment. The reduced activity of this enzyme in the diabetic state shows that glucose does not enter into the pentose phosphate pathway to a greater extent.
Sorbitol dehydrogenase activity of the liver is increased by 80% in the diabetic condition (Group IIb). Treatment of diabetic animals with leaf powder of G. syluestre reduces the enzyme activity considerably and brings it to normal levels. Sorbitol dehydrogenase aids the synthesis of polyols and is known to be accelerated in insulin deficiency in rat lens (Collins and Corder, 1977) and in Chinese hamsters (Holcomb et al,, 1974).
Glucose-6-phosphatase of liver is almost doubled in diabetes and is con- siderably lowered by G. syluestre administration. Fructose 1,6diphosphatase and glucose Gphosphatase are the two key enzymes involved in gluconeo- genesis (Weber et al., 19’71). Fructose 1,6-diphosphatase activity is increased nearly three-fold in the 24-week diabetic animals. A~in~~tion of the herbal medicine markedly reduces the activity of the enzyme and partially restores it to normal levels,
Hepatic LDH activity is almost doubled in the diabetic state (Group IIa). The hypoglycemic drug reduces the enzyme activity significantly in 24 weeks of treatment.
Aspartate and alanine transaminases (GOT and GPT, res~~ely) in the liver are increased in diabetes and animals of group IIb exhibit 50% increase in their levels. The lowering of these enzymes by G. syluestie administration might indicate a partial reversal of enhanced gluconeogenic activity in the diabetic tissues.
Acid phosphatase is known to be enhanced in the diabetic state (Begum and Sh~rnu~~nd~rn, 1978). G. syluestre adm~~~ation lowers the enzyme level. The membrane bound alkaline phosphatase and ATPase which are involved in transport phenomena and in the maintenance of membrane integrity are elevated in diabetes. This is also partially reversed by G. syluestre administration.
Table 4 gives the activities of enzymes of glucose metabolism in the kidney and Table 5 presents the levels of transaminases, LDH and phos- phatases in the kidney of normal, diabetic and G. SyZuestre treated diabetic animals. The enzymes of the insulindependent pathways, namely, hexo- kinase and glyceraldehyde-3-phosphate dehydrogenase are lowerd in dia-
TA
BL
E
4
AC
TIV
ITY
O
F
SOM
E
KE
Y
EN
ZY
ME
S IN
GL
UC
OSE
M
ET
AB
OL
ISM
IN
TH
E
KID
NE
Y
OF
NO
RM
AL
, D
IAB
ET
IC
AN
D
CO
N-
TR
OL
LE
D
DIA
BE
TIC
R
AB
BIT
S
The
dia
beti
c an
imal
a re
mai
ned
hype
rgly
cem
ic
for
a pe
riod
of
24
wee
ka
befo
re
the
init
iati
on
of G
. sy
lues
tre
adm
inis
trat
ion.
T
he
anal
yses
w
ere
mad
e 12
and
24
wee
ks
afte
r ai
ioxa
nisa
tion
in
the
un
trea
ted
diab
etic
s an
d 12
and
24
wee
ke
afte
r th
e in
itia
tion
of
adm
inis
trat
ion.
E
nzym
e ac
tivi
ties
ar
e ex
pres
sed
88 m
nol
prod
uct
form
ed/m
gpro
tein
un
der
incu
bati
on
cond
itio
ns.
For
giy
cera
ideh
yde-
3-ph
osph
ate
dehy
drog
enaa
e an
d gl
ucos
e&ph
osph
ate
dehy
drog
enas
e,
the
enzy
me
acti
viti
es
are
expr
esse
d as
AO
.D./m
in/m
g pr
otei
n.
Val
ues
are
expr
esse
d m
mea
n f
S.D
. (f
or
n nu
mbe
r of
ani
mal
e).
For
ass
essi
ng
the
stat
isti
cal
sign
ific
ance
fo
r th
e va
riat
ions
re
port
ed,
grou
ps
IIa
and
IIb
are
com
pare
d w
ith
I, w
hiie
II
Ia a
nd I
IIb
are
com
pare
d w
ith
IIb.
EnZ
ynW
S C
ontr
ol
(n-7
)
Unt
reat
ed
diab
etes
C
ontr
olle
d di
abet
es
IIa
IIb
IIIa
II
Ib
12 w
eeks
24
wee
ks
36 w
eeks
48
wee
ks
(n-5
) (n
=5)
(n=5
) (n
=5)
Hex
okin
ase
464
* 62
G
lyce
rald
ehyd
e-3p
hoe-
24
f
1.3
phat
e de
hydr
ogen
ase
Gly
coge
n qm
thet
ase
23.1
f
2.3
Tot
al
phos
phor
yias
es
315
f 45
P
hosp
hory
iase
(I
23
4 f
28
Glu
cose
-G-p
hosp
hate
0.
29
f 0.
03
dehy
drog
enaa
e So
rbit
ol
dehy
drog
enas
e 31
7 f
42
Glu
cose
6-
phos
phat
ase
1035
f
124
Fru
ctos
e-1,
6dip
hosp
hata
se
299
f 18
332
f 26
’ 29
3 f
41=
21
f
l.4c
20
f l.l
d
21.3
*
2.0
329
* 50
24
1 f
30
0.19
f
o.04
c
406
f 14
c 19
62
f 13
4d
679
f 53
d
20.1
f
2.5
330
f 55
24
8 f
28
0.16
*
o.05
c
436
f 19
d 20
66
f 23
8d
664
f 49
d
353
* 26
a 22
f
1.3*
22.6
f
1.5
320
f 45
23
9 f
28
0.21
f
0.03
390
f 19
b 11
65
f 12
96
526
f 53
b
423
f 11
0=
24
f 0.
4d
22.8
f
1.3
317
f 35
23
6 f
24
0.22
f
0.03
355
f 44
b 10
49
* 6g
d 42
4 f
43c
*P <
0.0
5.
bP <
0.0
2.
cP <
0.0
1.
dP <
0.0
01.
TA
BL
E
5
AC
TIV
ITIE
S O
F R
EL
AT
ED
E
NZ
YM
ES
IN G
LU
CO
SE
ME
TA
BO
LIS
M
IN T
HE
K
IDN
EY
O
F N
OR
MA
L,
DIA
BE
TIC
A
ND
CO
N-
TR
OL
LE
D
DL
AB
EX
’IC
RA
BB
ITS
The
dia
betic
an
imal
s re
mai
ned
hype
rgly
cem
ic
for
a pe
riod
of
24
wee
ks b
efor
e th
e in
itiat
ion
of G
. sy
lves
tre
adm
inis
trat
ion.
T
he a
naly
ses
wer
e m
ade
12 a
nd
24 w
eeks
aft
er
allo
xani
satio
n in
the
un
trea
ted
diab
etic
s an
d 12
and
24
wee
ks
afte
r th
e in
itiat
ion
of a
dmin
istr
atio
n.
Enz
yme
activ
ities
ar
e ex
pres
sed
as n
mol
pr
oduc
t fo
rmed
/mg
prot
ein
unde
r in
cuba
tion
cond
ition
s.
Val
ues
are
expr
esse
d as
mea
n f
SD.
(for
n
num
ber
of a
nim
als)
. Fo
r as
sess
ing
the
stat
istic
al
sign
ific
ance
fo
r th
e va
riat
ions
re
port
ed,
grou
ps
Ha
and
IIb
are
com
pare
d w
ith
I, w
hiie
II
Ia a
nd
IIIb
ar
e co
mpa
red
with
II
b.
Enz
ymes
GO
T
GPT
L
DH
A
cid
phos
pbat
ase
Alk
alin
e ph
osph
atas
e A
deno
sine
tr
ipho
spha
tsse
Con
trol
(n
=7)
967
* 12
1 78
5 f
76
778*
60
32
8 f
30
2132
*
390
845
f 75
Unt
reat
ed
diab
etes
C
ontr
olle
d di
abet
es
Ha
IIb
IIIa
II
Ib
12 w
eeks
24
wee
ks
36 w
eeks
48
wee
ks
(n=5
) (n
=6)
(n=
S)
(n”5
5)
1448
f
125d
16
36
i 14
8a
1196
f
14gb
11
11
* 17
1=
1107
f
946
1234
f
94d
1026
f
58’
928
f 43
’ 16
58
f 24
gd
1757
f
113c
10
12
f 12
6d
975
i 53
d 41
6*
40’
456
f 45
’ 39
3 f
23s
366
* 36
b 27
31
i 25
3b
2809
f
184’
22
08
f 17
1=
2175
f
116’
17
23 l 27
1d
1873
i
310d
13
06
* 13
2b
1039
f
120c
aP <
0.0
6.
bP <
0.0
2.
OP <
0.0
1.
dP
< 0.
001.
218
betes and this is reversed by G. syluestre administation. While the levels of liver glycogen synthetase in groups IIa and IIb animals were less than half of the normal rabbits no significant changes are observed in the kidney. This has to be considered in the light of the observations made in Table 1, about the glycogen content of these two tissues. Since liver stores glycogen, liver glycogen synthetase assumes greater importance in glucose utilisation in this organ. In the kidney, catabolism of glucose for energy production assumes greater importance than glycogenesis.
The enzymes involved in the insulin-independent pathways, sorbitol dehydrogenase, glucose 6-phosphatase and fructose 1,6diphosphatase are considerably elevated in diabetic kidney. With G. syluestre therapy, these enzyme activities are significantly lowered. While glucose 6-phosphatase and sorbitol dehydrogenase activities of the treated diabetics (IIIb) are very near to the normal levels, fructose 1,6diphosphatase levels are not completely reversed during treatment.
Kidney exhibits more than two-fold increase in the level of LDH in diabetes and G. syluestre therapy induces reduction in the level and partially restores it to normal.
Transamines of the kidney show nearly 50% elevation in diabetes (IIb). Administration of G. syluestre to these animals results in significant lowering of transaminase levels showing that the drug decreases gluconeogenesis in the kidney after 24 weeks of treatment.
Kidney acid phosphatase levels show moderate increases in the diabetic state. Administration of G. syluestre reduces the levels. Increased lysosomal enzymatic activity observed in the diabetic state is corrected by the herb.
In diabetes, increase in membrane transport is evidenced by enhanced activities of membrane bound enzymes, alkaline phosphatase and ATPase in the kidney. G. syluestre therapy causes marked decreases in these enzyme levels after 24 weeks of treatment. Kidney, which is a very rich source of alkaline phosphatase, shows ATPase activity to the same extent as liver. The effects of diabetes mellitus on the kidney phosphatases are also very similar to the liver enzymes and G. syluestre administration brings about a reduction in their levels.
Table 6 gives the activities of enzymes involved in glucuse metabolism and Table 7 depicts the levels of transaminases, LDH and phosphatases in the muscle of normal, diabetic and controlled diabetic animals. Among the in- sulin-dependent enzymes hexokinase, glyceraldehyde-3-phosphate dehydro- genase, glucose8-phosphate dehydrogenase and glycogen synthetase activities are reduced in diabetes and are increased by G. syluestre.
Phosphorylase, sorbitol dehydrogenase, glucose 6-phosphatase, fructose l,S- diphosphatase and LDH activities are increased two- to three-fold in the diabetic condition. G. syluestre therapy reduces the enzyme activities to near normal levels.
TA
BL
E
6
AC
TIV
ITIE
S O
F SO
ME
KE
Y E
NZ
YM
ES
IN G
LU
CO
SE
ME
TA
BO
LIS
M
IN T
HE
MU
SCL
E
OF
NO
RM
AL
, D
IAB
ET
IC
AN
D C
ON
- T
RO
LL
ED
D
IAB
ET
IC
RA
BB
ITS
The
dia
betic
anim
ais
rem
aine
d hy
perg
lyce
mic
fo
r a
peri
od
of 2
4 w
eeks
bef
ore
the
initi
atio
n of
G.
sylu
estr
e ad
min
istr
atio
n.
The
an
alys
es
wer
e m
ade
12 a
nd
24 w
eeks
aft
er
aiIo
xani
satio
n in
the
un
trea
ted
diab
etic
s an
d 12
and
24
wee
ks a
fter
th
e in
itiat
ion
of a
dmin
istr
atio
n.
Enz
yme
activ
ities
ar
e ex
pres
sed
aa n
mol
pr
oduc
t fo
nned
/mg
prot
ein
unde
r in
cuba
tion
cond
ition
s.
For
~yce
rald
ehyd
e-3-
ph~p
hate
de
hydr
ogen
ase
and
gluc
ose-
6-ph
osph
ate
dehy
drog
enas
e,
the
enzy
me
activ
ities
ar
e ex
pres
sed
as A
C.D
./min
/mg
prot
ein.
V
alue
s ar
e ex
pres
sed
as m
ean
f S.
D.
(for
n n
umbe
r of
ani
mal
s).
For
asse
ssin
g th
e st
atis
tical
si
gnif
ican
ce
for
the
vari
atio
ns
repo
rted
, gr
oups
II
a an
d II
b ar
e co
mpa
red
with
I,
whi
Ie I
IIa
and
IIIb
ar
e co
mpa
red
with
II
b.
Enz
ymes
C
ontr
ol
I U
ntre
ated
di
abet
es
Con
trol
led
diab
etes
(n
-7)
IIa
IIb
IIIa
II
Ib
12 w
eeks
24
wee
ks
36 w
eeks
48
wee
ks
(n=5
) (n
= 5
) (n
=5)
(n =
5)
Hex
okin
aae
1101
f
110
855
f 80
b 65
6 f
70=
84
1 +
soa
999
* 9o
c G
lyce
rald
ehyd
e-3-
phos
- 28
.9
* 2.
15
22.8
f
2.0S
b 18
.8
f 1.
2sc
21.9
*
1.21
a 27
.0
t 2.
01C
ph
ate
dehy
drog
enas
e G
lyco
gen
synt
heta
se
65.3
f
3.7
40.5
*
3.2’
30
.5
f 2.
7’
42.2
f
2.gb
53
.6
f 4.
1’
Tot
al
phos
phor
yiaa
es
1025
9 *
660
1355
8 f
580’
16
968
f 60
0’
1412
5 f
480’
11
256
f 35
0c
Phos
phor
ylas
e a
3594
f
250
4589
f
310c
67
95
* 40
0e
4851
f
310b
37
15
f 28
1’
Glu
cose
+ph
osph
ate
0.30
*
0.03
0.
25
* 0.
02b
0.20
f
o.02
c 0.
25
f 0.
02n
0.29
*
o.02
c de
hydr
ogen
aae
Sorb
itol
dehy
drog
enas
e 16
050
* 71
0 18
750
f 74
oc
2556
0 f
910=
20
550
f 85
0’
1655
0 f:
750
=
Glu
cose
6-
phos
phat
ase
26.5
*
2.0
32.0
f
2.5
45.5
f
3.0
33.6
f
2.5
28.9
*
2.1
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221
Muscle transaminases (GOT and GPT) are increased in diabetes and the animals of Group IIb exhibit 50% increase in their levels (like liver). The administration of hypoglycemic drug reduces it significantly.
Acid phosphatase, alkaline phosphatase and ATPase are increased in diabetic muscle and G. syluestre therapy reduces it to near normal level.
Incorporation of orally administered [14C] glucose into various cellular
TABLE 8
INCORPORATION OF 14C FROM ORALLY ADMINISTERED UNIFORMLY LABELLED [ 14C ] GLUCOSE INTO CELLULAR COMPONENTS IN THE LIVER AND MUSCLE OF NORMAL (I), DIABETICS (IIb) AND CONTROLLED DIABETIC (IIIb) RABBITS
Values are expressed as dpm/mg tissue f S.D. 28,300 dpm/g body wt. was given orally. The number of animals used are given in parentheses. For assessing the statistical signifi- cance for the values reported, group IIb is compared with I while IIIb is compared with IIb.
components in the liver, kidney and muscle of normal, diabetic and diabetic animals treated with G. syluestre is given in Table 8.
Uptake of [ 14C] glucose into the glycogen fraction of diabetic animals shows 50% reduction in liver and muscle when compared to the controls. G. syluestre treated diabetic animals exhibit an uptake higher than the untreated diabetics, suggesting that the metabolic imbalances in diabetes are being corrected. This is in accordance with the variation observed in the glycogen levels estimated chemically (Table 1) and glycogen synthetase
223
Fig. 1. (a) Section of liver from normal rabbit. Haematoxylin, X 100. (b) Section of liver from diabetic rabbit (24 weeks) showing degenerative changes in some loci with mild periportal infiltration. H and E, x 100. (c) Section of liver from controlled diabetic rabbit with G. syloestre (24 + 24 weeks) resembles normal. H and E, x100.
activity (Tables 2 and 6). In kidney, i4C uptake into glycogen is not affected to the same degree.
Parallel to the lipid deposition observed in Table 1, in the diabetic con- dition 14C is incorporated to a greater extent in the diabetic liver, kidney and muscle. G. syluestre treatment brings down the incorporation of [14C] - glucose into lipids, although accelerated catabolism of the lipid cannot be ruled out. The incorporation of 14C into lipids is greatest in liver while in kidney and muscle only half the label is observed in the normal animals. Comparing the lipid levels of these tissues (Table l), liver and kidney have nearly the same percentage of lipids (34.2 * 6.4 and 33.4 f 5.1 mg/g tissue, respectively) while muscle lipid levels are low (17.7 + 2.5 mg/g). But during the short duration of the experiment (4 h) the rate of incorporation in the liver is double that of kidney, confirming that this organ plays a vital role not only in lipogenesis, but also in glucose disposal.
Uptake of label by the nucleic acid fraction of the liver and muscle are decreased by 25% in diabetes and G. syluestre administration restores the incorporation to that of controls. Kidney shows reduced incorporation into nucleic acid fraction in diabetes, and G. syluestre appears to have very little effect in increasing the uptake into nucleic acids.
Incorporation of [ 14C] glucose into the protein fraction in the liver is decreased in diabetes. G. syluestre treated animals show incorporation at a higher rate than the diabetic animals. Incorporation of 14C into the protein components is very much retarded in diabetic kidney and muscle when compared to the liver and the reversal by G. syluestre is significant.
224
The cold TCA fraction includes glucose, hexose derivatives, nucieotides, amino acids, fatty acids, intermediates of glucose metabolism and formate. The incorporation of labelled carbon into this fraction is lower in the diabetic liver and muscle, when compared to the controls, and this is reversed by G. syluestre therapy. The low level of 14C in this fraction in the untreated diabetic rabbits may be due to the loss of glucose in the urine, and also in the reduced uptake and utilisation of glucose by the tissues due to insulin deficiency. It may be noted that the uptake of glucose by the kidney is not
225
Fig. 2. (a) Section of kidney from normal rabbit. H and E, X100. (b) Section of kidney from diabetic rabbit (24 weeks), showing intact glomerular apparatus, with vascular degeneration of the renal tubules. H and E, x100. (c) Section of kidney from controlled diabetic rabbit (24 + 24 weeks) with G. sylvestre, showing vascular degeneration in very small isolated loci and regeneration of the kidney parenchyma. H and E, X100.
insulin dependent and the levels in the kidney remain unaffected in the diabetic state.
Figure la-c shows the sections of liver from normal, diabetic (24 weeks) and controlled diabetics (24 + 24 weeks) stained with H and E. In the diabetic rabbit liver, some isolated areas showing degenerative changes and necrosis in small loci are observed. Further, mild periportal infiltration of the round cells is also observed. In the controlled diabetic liver, the picture is similar to that of the normal, indicating that the degenerative changes initiated by diabetes (Group IIb) are partially reversed by G. syluestre administration.
Figure 2a-c shows the architecture of kidney cells of normal and experi- mental animals stained with haematoxylin-eosin. In the alloxan diabetic animals, glomerular apparatus of the kidney appears to be intact, while there is widespread vascular degeneration of the renal tubules. In the con- trolled diabetic kidney, the vascular degeneration is limited to very small isolated loci, again indicating that there may be a regeneration of the kidney parenchyma during G. sylvestre administration.
Figure 3b shows the PAS stained diabetic kidney section in which vacuo- lation is observed throughout the organ. However, the controlled diabetic kidney (Fig. 3c) appears to be normal compared to the normal rabbit kidney (Fig. 3a).
The muscle section shows no lesions in diabetic and controlled diabetic rabbits.
226
These observations indicate that G. syluestre administration to diabetic animals not only brings about blood glucose homeostasis but also reverses the metabolic and pathological changes taking place in the liver, kidney and muscle.
Discussion
From the observations made in Table 1 it is clear that the administration of G. s&es&e brings down the blood glucose levels considerably in alloxan
227
Fig. 3. (a) Section of kidney from normal rabbit. PAS, X100. (b) Section of kidney from diabetic rabbit (24 weeks), showing vacuolation throughout. PAS, X100. (c) Section of kidney from controlled diabetic rabbit (24 + 24 weeks) with G. sylvestre showing no pathological variations - appears to be normal. PAS, x100.
induced diabetic animals. The hypoglycemic effect of G. syluestre may be mediated through stimulation of insulin release resembling the oral hypo- glycemic sulfonylureas (Colwell and Zuckerman, 19’72; Hecht et al., 1973) or through inhibition of intestinal absorption of glucose as observed with biguanides (Kruger et al., 1970) or through stimulation of one of the in- sulinogenic signals promoting insulin release. In this context, we have ob- served the ins~~otropic activity of G. syiuestre in experimental diabetes (Shanmugasundaram et al., 1981).
The glycogen and protein depletions and lipid accumulation in the diabetic animals are reversed by G. syluestre administration. Insulin is reported to exert a similar effect on glycogen, protein and lipids in streptozotocin diabetic rats (Chang and Schneider, 1971) and in alloxan induced diabetic rabbits (Begum and Shanmugasund~am, 1978). Inve~~ations made in our laboratory (Shanmugasundaram et al., 1981) have demonstrated that the serum insulin levels are increased both in the fasting and post-prandial state in G. syluestre administered diabetic rabbits and the changes observed here can be attributed to the elevated insulin levels in these animals.
Cholesterol metabolism in alloxan induced diabetic rats has been studied by several workers. Sadahiro et al. (1970) observed that the liver cholesterol levels in diabetic rats were higher than the non-diabetics as early as the 10th day of induction of the disease. Further, in diabetic rats the excretion of cholesterol and bile acids was decreased and isotope tracer studies revealed
228
that in diabetes the rate of conversion of cholesterol to cholic acid is altered (Sadahiro et al., 1970).
The suggestion that the hypoglycemic effect of G. sylvestre may be mediated through increased levels of circulating insulin is further strengthened by the alterations observed in the liver, kidney and muscle enzyme activities reported in Tables 2-7. The insulin dependent enzymes, hexokinase, glycogen synthetase glyceraldehyde3-phosphate dehydrogenase and glucose&phos- phate dehydrogenase have a lowered activity in the diabetic tissues and a higher activity in the G. sylvestre controlled diabetics, while the activities of enzymes catalysing insulin independent pathways are grossly increased in the untreated diabetics and are reversed during the administration of the herb. The enzymes of this group which have been studied are glycogen phosphorylase, gluconeogenic enzymes glucose 6-phosphatase, fructose-1,6- diphosphatase and sorbitol dehydrogenase of the polyol pathway.
Studies by Germanyuk and Gula (1970) in diabetic rabbit liver and by Storey and Bailey (1978) in streptozotocin diabetic rat liver indicate a de- creased level of glucose&phosphate dehydrogenase in diabetes. Insulin is reported to increase the activity of glucose&phosphate dehydrogenase (Weber and Convery, 1966) in a dosedependent manner. In our studies, G. sylvestre administration increased its activity considerably. Beloff-Chain et al. (1956) and Winegrad and Renold (1958) showed that insulin stimulates oxidation of glucose by the hexose monophosphate shunt, predominantly in the fat laden cells. A number of investigators have suggested that the increased supply of NADPH produced by the two dehydrogenases (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) in this pathway may itself be a promoter of lipid synthesis. Weber et al. (1971) after an analysis of the observations made in diabetes and in insulin therapy have concluded that the impact of insulin in the liver is to increase gly- colysis and decrease gluconeogenesis, “i.e. predominance’ of glycolysis over gluconeogenesis”. Further, insulin integrates hepatic carbohydrate metabolism by increasing the biosynthesis of enzymes of glycolysis, glycogenesis, pentose oxidative pathway and lipogenesis while inhibiting gluconeogenesis. This picture is obtained in the study of cellular consti- tuents and enzymes of liver, kidney and muscle of diabetic animals under G. sylvestre administration. This indicates that the above changes may be a sequel to elevated levels of circulating insulin following herbal adminis- tration.
Glycogen synthetase levels (Tables 2,4 and 6) of liver, kidney and muscle are decreased in the diabetic animals. Decrease in liver glycogen synthetase has been reported by Chang (1972) and Stearns and Benzo Camillo (1977) in the diabetic condition. This may be the result of low levels of circulating insulin which is an inducer of glycogen synthetase (Villar-Palasi and Lamer, 1961; Witters and Auruch, 1978; Hauguel and Cedard, 1979). Administra- tion of G. sylvestre restored the enzyme activities to normal levels in 24 weeks of treatment of diabetic rabbits.
229
Phosphorylase activity in diabetic rabbit tissues shows increased levels of both total and the active a form. This is parallel to the finding of Witters and Auruch (19’78) in which insulin administration to fed rats increased the glycogen synthetase activity and decreased glycogen phosphorylase activity. The increase in glycogen is accounted for mainly by the decrease in glycogen phosphorylase activity. Under conditions of insulin deficiency the enzyme is elevated, as observed in our studies. G. syluestre administration favoured a decrease in phosphorylase, an effect closely. resembling insulin.
The effect of insulin in streptozotocin diabetic rats was to decrease the activities of gluconeogenic enzymes (Chang and Schneider, 1971). G. syl- uestre administration also produces a similar effect suggesting that this may be mediated through increased levels of insulin. Oral hypoglycemics (biguanides) also act by ~h~ition of gluconeogenesis in the liver (Searle and Cavalieri, 1968; Lloyd et al., 1975).
The transaminases of tissues (Tables 3,5 and 7) are found to be elevated in diabetes. Many workers have reported increases in transaminase activities in the liver and serum (Pogliaro and Notarbartola, 1961). A decrease in cy-ketoglutarate level was reported by Deptula (1970) in alloxan diabetic rabbits. The increased levels of transaminases which are active in the absence of insulin because of increased availability in the blood of amino acids in diabetes (Bondy et al., 1949; Felig et al., 1970) are responsible for the in- creased gluconeogenesis and ketogenesis observed in diabetes.
The increase in sorbitol dehydrogenase activity in diabetes is due to the increase in polyol pathway operating in diabetes and serves as a route for the disposal of rapidly ac~mulat~g i&race&&u glucose (Collins and Corder, 1977). The sorbitol pathway is responsible for the accumulation of sorbitol and other polyols resulting in senile cataract in diabetes. G. syluestre ad- ministration alters the sorbitol pathway by reducing sorbitol dehydrogenase levels in tissues.
Acid phosphatase activities are increased in diabetes in all the three tissues (Tables 3,6 and 7) and the increases are seen early in the disease. Mishima (1967) has reported increased acid phosphatase levels in alloxan diabetic rats. Begum and Shanmugasundaram (1978) have reported similar increases in acid phosphatase level in diabetes. The elevated levels were corrected by G. syZ~s~e adrn~~~tion, The effect is not significant in the liver while kidney and muscle show significant reduction in their activities.
Elevation of alkaline phosphatase has been reported in diabetic rats (Mishima, 1967) and in alloxan diabetic rabbits (Begum and Shanmuga- sundaram, 1978). The increased activities of alkaline phosphatase were decreased by the administration of G. syluestre in the 24-week therapy and they reached the control values (Group I animals).
ATPase activities (Tables 3,5 and 7) of all the tissues show great elevation in the diabetic state. ATPase is a membrane bound enzyme and its activity has been reported to increase in starvation (Gutman and Glushevitsky, 1973). Bqum ind Shanmugasundaram (1978) have reported that tissue
230
phosphatases are increased in diabetes. The uptake of Na+, water and amino acids are mediated through ATPase and the increased activities observed in our studies, suggest a metabolic alteration of the cell membrane. G. sylvestre admin~ation to diabetic animals normal&es this also,
However, it has to be noted that in the kidney the phosphorylase (total as well as active) and glycogen synthetase activities are not greatly altered in the diabetic state. G. syluestre administration also does not produce any change in these enzymes showing that the action of G. syluestre on the regulation of carbohydrate metabolism is not directly on any activities of these two enzymes, but may be through the circulating levels of insulin.
The effect of the postulated elevated levels of circulating insulin in the G. syluestre controlled diabetic rabbits is also seen by the studies made on the uptake of [ 14C] glucose into the cellular constituents reported in Table 8. In the liver, [14C]glucose is incorporated equally between glycogen and lipids in the diabetic rabbit, while in the normal, the distribution of radio- activity between glycogen and lipids is highly in favour of the former. However, the incorporation of [ 14C] glucose into the liver lipids of normal and diabetic animals are not significantly different. The difference is in the incorporation of labelled carbon into glycogen and protein. In the diabetic animal, incorporation into the cold TCA fraction is also lowered and on the whole a siginficant part of labelled glucose is not accounted for. Although a quanti~tive assessment of the excretion of carbon has not been made (CO, expired, urine and faeces), glucose is lost in the urine in the diabetic animals and at the time of experiment (4 h after feeding) a significant part of ingested glucose may be lost in the urine.
In the kidney, while no difference is observed in the proportion of label in glycogen between normal and diabetic animals, a significant increase of radioactivity in the lipid and a reduction into proteins is observed. In the muscle, incorporation of [ 14C] glucose into glycogen is strongly impaired. These changes in glycogen radioactivity run parallel to the enzyme changes (glycogen synthetase, phosphorylase, etc.) reported earlier. In the G. syluestre administered diabetics, the incorporation of [“Cl glucose into the tissue ~~ituents runs parallel to that of normal animals.
A decrease in hepatic and muscle glycogen content in diabetes has been observed (Loser% et al., 1966; William et al., 1967; Whitton and Hems, 1975). The decrease observed in our studies is probably due to the lack of insulin in the diabetic state which results in the inactivation of glycogen synthetase system. In mammalian tissues, the amount and route of glucose utilisation are geared to the different metabolic characteristics of different tissues. Beloff-Chain et al. (1956) and Pocchiari and Agnolo (1977) have found that in muscle and liver radioactivity from [i4C] glucose was incorporated into glycogen and oligosaccharides. Kipnets (1970) has observed that following an oral glucose load, approximately 70% is disposed of by the liver, with only 30% reaching the extra hepatic tissues and the inadequacy of insulin in diabetes results in the diminished glycogen storage.
231
The results of our experiments show that the incorporation of 14C into glycogen was very much decreased in the liver and muscle in diabetes. Treat- ment of these animals with G. syluestre resulted in an enhanced incorporation of [14C] glucose into glycogen. This may be due to increase in glucose uptake by the tissues following G. syhestre administration or increased glycogen synthesis or a combination of both probably mediated through the action of insulin, The total radioactivity measured at the end of 4 h in the tissues of normal and G. syluestre controlled diabetics are the same, and it may be inferred that G. syluestre does not bring about any inhibition of intestinal glucose uptake.
Distribution of radioactivity into the nucleic acid fraction is decreased in diabetes which may be the direct effect of non-availability of pentoses, pentose phosphate being required for nucleic acid synthesis, due to the decreased pentose oxidative pathway operating in insulin deficiency (Weber and Convery, 1966). The incorporation was partially increased by the admonition of G. syZues~e, possibly by the activation of this pathway or by augmented entry of [14C] glucose into the tissues.
The diabetic animals show reduced incorporation of [r4C] glucose into the cold TCA fraction, when compared to the controls, showing reduced glycolytic rates observed in diabetes. Some glucose may also be lost in the urine. G. syluestre treated animals show enhanced incorporation compared to the diabetic animals which may be due to increased glycolytic pathway directly stimulated by G. syluestre administration or through the stimu- lation of insulin release by the pancreas. The insulin release may also be mediated through the gastrointestinal tract factors.
Gastric inhibitory polypeptide, an inhibitor of gastric juice secretion has been found to be a powerful stimulator of insulin secretion in man and dog (Brown et al., 1975). Turner (1969) suggested the existence of another poly- peptide called insulin releasing polypeptide, which may regulate insulin secretion after glucose load. Pancreatic glucagon stimulates insulin secretion both in vivo and in vitro. The evidence by Unger et al. (1968) has established the existence of glucagon-like immunoreactive material in the gut which stimu- lates insulin secretion. Higher serum insulin levels observed in the diabetic animals after treatment with G. syiuestre (Sha~uga~nd~ et al., 1981) may be due to the stimulation of one or more enteric hormones which have insulinotropic activity.
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