273s
Biochemical SocietyTransactions ( 1 993) 21
Quantitation of type I and 111 collagen of liver in alloxaninduced diabetic rabbits
MAHMQUD DJAVANI, GULDAL KlRKALl, SEDEF YENICE,
GUL GUNER and HUSEYIN T. SESSIZ
kDa
1 2 3 4 5 6 7 8
-Y
Department of Biochemistry, Faculty of Medicine, Dokuz
Eylul University, 35340 Izmir, Turkey
Collagen is the most abundant protein in the body and
it is the major macromolecular constituent of extracellular
matrix, representing afamily of genetically distinct molecules
[I]. Soft organs such as pancreas and liver contain only a
small amount of collagen, whereas in tissues such as skin
and tendon, collagen account for over 70 per cent of the dry
weight [2]. In vertebrates most tissues are composed of type
I and 111 collagen. Connective tissue disorders are apparent
in diabetics, and diabetes mellitus involves metabolic
processes and produces profound changes in the biochemical
cornposition and biochemical properties of the affected
tissue [3]. Our interest has centered on hepatic tissue
architecture and how mild experimental diabetes influence
total collagen content and the relative proportions of major
collagen types I and 111 in rabbit liver.
Twenty two male albino rabbits aged 1.0-1.5 years,
weighing 1.4-2.0y were divided into two groups: a) Control
group (1 2 rabbits) and b) alloxan-induced diabetic group (1 0
rabbits). The animals were fasted for 48 h before alloxan
treatment and the diabetic group was treated once a week for
four weeks with alloxan at a dose of 120 mgikg dissolved in
0.5 ml sterile saline. Blood glucose levels were measured
before and after treatment for four weeks. After decapitation
of the rabbits of both groups, the tissue samples were
removed and defatted with acetone and ether. The dry
defatted liver samples were hydrolyzed in 6 N HCI at 105' C
for 16 h. Following hydrolysis, the hydroxyproline contents
of the neutralized samples were measured by the
spectrophotometric method of Tougaard [4]. Values for
collagen content were derived from the hydroxyproline values
by multiplying 7.46. Collagen types I and 111 were prepared
from liver by solubilization with pepsin, followed by three
times repeated differential salt precipitation with crystalline
NaCl and dissolution. The dry defatted tissue samples were
suspended in 0.5 M acetic acid and pepsin added at a ratio
of 1 :lo0 (10 mgig dry tissue weight). Digestion was allowed
to proceed for 24 h at room temperature. The insoluble
residue was extracted a second time under similar conditions.
The collagen was precipitated from the combined supernatant
by addition of solid NaCl to achieve a 0.9 M concentration.
The precipitate was redissolved in 0.15 M NaCI, 0.05 M TrisHCI, pH 7.4 and collagens sequentially precipitated at 1.7 M
(Type Ill) and 2.6 M NaCl (Type I). The precipitation at 1.7 M
and 2.6 M NaCl and dissolution were repeated three times
[ 5 ] . SDS-polyacrylamide gel electrophoresis was carried
out, in principle, according to the method of Hayashi et al [6]
Liver samples were diluted 1 : 10 with 0.05 M Tris buffer at pH
6.8toobtain afinal concentration 15 !ig/50 111of collagen. The
concentration of urea was 3.6 M and the running gel
concentration was 5 %. Electrophoresis was performed
PharmaciaiFine Chemicals, starting with 30 mA at 120 V. 50
pl of samples were applied, and electrophoresiswas terminated
at 7 hours 30 minutes.The mean blood glucose levels of
experimental group measured before and after four week
alloxan treatment were found as 106.2 F5.5 mgidl and 366.0
+ 9.3 mg/dl, respectively and the increase was found to be
significant (p<O.OOl ). Dry defatted weight of the liver samples
of alloxan-induced diabetic rabbits did not differ from those
of the control group. Alloxan diabetes did not induce a
significant change (p>0.05) in total hepatic collagen and the
relative amounts of collagen types I and 111, after four weeks
of treatment (Table 1).
Table 1.
Group
Control
Diabetic
Percentaqe of collaclen types in liver samdes
Total Collagen
(maia dry weight)
8.1
8.7
Type I Type 111
5.3
6.1
2.3
21
Type lilll
Ratio
2.3
2.9
18011684-
-15,
I
-13,
I
-#I
(I).,,
(ill)
-(I
(I)
58.548.536.F
26.6-
Fig. 1 . Electrophoretic pattern of liver t w e I and 111 collaqen
on SDS- polyacrylamide qel.
Lane.1:Type 111 marker
3: M. Wt markers
5: Control type I
7: Control type 111
2: Type I marker
4: Type I + 111 marker
6: Diabetic type I
8: Diabetic tvoe 111
Total collagen content of non-diabetic and diabetic rabbits
liver were found to be 0.79 % 2 0.14 and 0.84 YO-+ 0.08.
respectively.
SDS-PAGE revealed hepatic type I and also type 111
collagen to separate into three protein bands ( ( 1 , 1-1, y) in both
groups, whereas type I collagen of the other tissues generally
divided into five protein bands ( a l ,( t P , (ill,
PI, and y). SDS
polyacrylamide gel electrophoretogram showed type I and
111 collagen to have homogenous protein bands corresponding
to molecular weights of 95,000-100,000 Daltons for both al
and a,chains [Fig. I ] . The obtained data provide a quantitative
evaluation of collagen composition in diabetic animals which
may be of interest in studies of connective tissue diseases
involving the hepatocellular degeneration.
This work was supported part by a grant (No. 923-8703-03) from Dokuz Eylul University Research Project
Administration.
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Kittelberger, R.. Davis, P.F., Flynn, D.W. & Greenhill,
N.S. (1 990) Connect. Tissue Res. 24, 303-318.
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Mays, P.K., McAnulty, R. & Laurent. G.J. (1 991)
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McCullagh, K.G., Duance, V.C. & Bishop. K.A. (1980)
Am. J.Pathol. 130, 45-55.
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