479
L-Arginine Reduces Heart Collagen
Accumulation in the Diabetic db/db Mouse
A. Khaidar; M. Marx, MD; B. Lubec, MD; G. Lubec, MD
Downloaded from http://circ.ahajournals.org/ by guest on September 29, 2016
Background Diabetic cardiomyopathy presents with significant collagen accumulation; decreased solubility, increased
glucose-mediated abnormal cross-linking, free radical crosslinking, or glucose-induced increased transcription of collagen
is incriminated. In a previous study, we reduced collagen
accumulation in the kidneys of diabetic mice by treatment with
oral arginine. This observation led us to examine the effect of
arginine on cardial fibrosis.
Methods and Results Twenty-nine db/db spontaneously diabetic mice were used in the experiments. Sixteen were given
L-arginine (free base, in tap water, 50 mg/kg body wt per day)
for 4 months. At the end of the experiment, we determined
total collagen content of total ventricular tissue, acid solubility,
carboxymethyllysine, O-tyrosine, glutathione, blood glucose,
and fructosamine as parameters for glycemic control. Heart
collagen level was significantly (P=.0001) reduced in the
experimental group (mean, 0.24±0.05) compared with the
control group (mean, 0.49±0.10 ,mol hydroxyproline per 100
mg heart tissue). Significantly more collagen could be eluted
from heart samples of the experimental group (P=.02). Carboxymethyllysine and O-tyrosine did not differ when related to
heart weight. Glutathione level was significantly higher in the
untreated group (P=.003). Parameters of glycemic control did
not differ between the groups.
Conclusions Our findings clearly indicate that L-arginine reduced total heart collagen and increased acid solubility of heart
collagen. Both findings are compatible with the cross-linking
hypothesis. The data for carboxymethyllysine, O-tyrosine, and
glutathione would rule out the glycoxidation hypothesis and,
therefore, free radical cross-linking. The postulated mechanism
of action is most likely the blocking of reactive carbonyl functions
by L-arguiine in analogy to aminoguanidine activity. Correlations
of collagen with glycemic control, however, point to an association of glucose with collagen metabolism, a phenomenon documented in cell cultures at the transcriptional level. (Circukation.
yocardial fibrosis is a main feature of diabetic cardiomyopathy. The mechanisms
leading to collagen accumulation, however,
are not fully elucidated. Several factors have been
incriminated, but nonenzymatic glycosylation is a major pathogenetic factor for connective tissue changes
in the diabetic state. Advanced-stage nonenzymatic
glycosylation products (AGEs) are known to lead to
increased abnormal glucose-mediated cross-linking of
collagen and other connective tissue proteins.1 Reactive carbonyl species (eg, deoxyglucosone or methylglyoxal) are reactive aldehydes mediating cross-linking
through reaction with free amino groups of proteins.
According to other modern concepts, glycoxidation
appears to be the underlying chemical process for
collagen accumulation and long-term diabetic complications.2 Glycoxidation is the free radical-mediated
oxidation of glycosylated proteins with AGEs (eg,
N-epsilon-carboxymethyllysine3).
Previously, we reported the reduction of kidney
collagen accumulation by L-arginine in the db/db
mouse model for diabetes.4 Based on this observation,
we used L-arginine for the experimental reduction of
heart collagen.
Methods
1994;90(479-483.)
Key Words * hydroxyproline * free oxygen radicals cross-linking * diabetes * collagen
M
Received February 25, 1994; revision accepted March 23, 1994.
From the Department of Paediatrics, University of Vienna,
Vienna, Austria.
Correspondence to G. Lubec, FRSC, Prof of Paediatrics, University of Vienna, Wahringer Gurtel 18, Austria.
01994 American Heart Association, Inc.
Animals
Twenty-nine db/db mice (Shaw's Farm) were used in the
experiment (Fig 1). The db/db mice used in the study have
diabetes that resembles type 2 adult-onset diabetes in humans.
During the first months of life, these animals are hyperphagic
and rapidly become obese. The initial metabolic abnormality is
hyperinsulinemia, which persists until 4 to 5 months when
degenerative changes in the islets also occur.5 Severe hyperglycemia occurs along with severe glycosuria between weeks 6
and 8.6 Collagen accumulation increases steadily between 5
weeks and 4 months.7 Treatment was started at the age of 3
months.
All animals had free access to mouse ration (Altromin), and
the control group also had free access to tap water. The
experimental group (n=16) was given 50 mg L-arginine PO
(free base, per kg body wt per day) for 4 months.
Body weight and food and fluid uptake did not differ
statistically between the groups at the start of the experiment
and at death. The animals were maintained at a day/night
rhythm at 23°C.
At the end of the experiment, animals were killed by neck
dislocation, and necropsies were performed. The hearts (total
ventricular tissue) were taken for biochemical investigations.
All experiments were conducted in accordance with the guiding principles of the American Physiological Society.
Determination of Total Ventricular Hydroxyproline
For determination of total heart hydroxyproline, aliquots of
total ventricular tissue were weighed and hydrolyzed with 6 N
HCl at 105°C for 16 hours. Hydrolyzed tissue was evaporated
on a Pierce reactitherm and redissolved in distilled water.
After centrifugation to remove insoluble material, the samples
were analyzed for presence of 4-trans-hydroxyproline by the
method of Woessner.8
480
Circulation Vol 90, No 1 July 1994
FIG 1. One of 29 spontaneously diabetic db/db mice used in
the experiment.
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For determination of eluted heart hydroxyproline, heart
collagen was eluted to obtain information on collagen solubility, which in turn provides evidence of collagen cross-linking.
Aliquots of heart tissue were homogenized and eluted by
pepsin (1 mg/100 mg heart tissue) in 0.05 mol/L acetic acid
containing 0.005 mol/L EDTA for 72 hours at 25°C. The eluate
was hydrolyzed, evaporated, and redissolved as described
above. This solution was also subject to analysis by Woessner's
method.
Determination of Total Ventricular Collagen
Aliquots of ventricular tissue were homogenized by a Potter
in an ice bath in a solution used for collagen elution as
described above, incubated for 72 hours at 25°C, and spun
down in a centrifuge at 4°C at 400Qg. The supernatant was used
for the Sircol collagen assay (Sircol Collagen Assay Kit, Oubis
Ltd) and sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (PAGE).
The principle of the Sircol collagen assay is the binding of a
dye to collagen. The collagen-bound dye is recovered by
centrifugation, eluted with alkali, and measured using a spectrophotometer at 540 nm. The intensity of color measurement
is proportional to the collagen concentration in a sample.
FIG 2. Representative chromatogram of carboxymethyllysine
determination in heart tissue hydrolyzates.
SDS-PAGE
Collagen extracted from total ventricular tissue was characterized on SDS-PAGE following the principle of Limmli.9
This method provides semiquantitative information about
collagen degradation products.
Determination of N-Epsilon-carboxymethyllysine
N-Epsilon-carboxymethyllysine was determined in tissue
hydrolyzates as previously determined'0 and expressed as
N-epsilon-carboxymethyllysine per 100 mg of heart tissue or as
N-epsilon-carboxymethyllysine per hydroxyproline. Briefly,
samples were derivatized with O-phthalaldehyde and run on
high-performance liquid chromatography using a gradient
system. A typical chromatogram is given in Fig 2.
Determination of O-Tyrosine
O-Tyrosine was determined in heart hydrolyzates by highperformance liquid chromatography as determined
previously. 11
Determination of Glutathione
Glutathione was determined in pepsin eluates (extracts) as
described above and assayed following the enzymatic method
of Anderson12 using a commercially available kit (GSH 400,
Bioxytech). Serum glucose was determined following a glucose
oxidase standard method on a Greiner autoanalyzer. Serum
fructosamine was determined using a commercially available
spectrophotometric method (fructosamine kit, Hoffmann La
Roche).
Statistical Analysis
Mann-Whitney U test for comparison of the groups and the
linear regression coefficient r were used in evaluating the
results.'3
Results
The determination of total heart hydroxyproline expressed in micromoles per 100 mg of heart tissue reflecting collagen accumulation revealed significantly higher
concentrations in the untreated group (P=.0001), as
described in Table 1.
In determination of eluted heart collagen, as shown in
Table 1, no significant differences in amount of collagen
eluted from heart tissue could be detected (P=.62). If,
however, the ratios of eluted to total collagen were
calculated, significantly more collagen could be extracted from treated heart (P=.02).
As shown in Table 1, total ventricular collagen was
significantly (P<.01) reduced in the treated group.
Khaidar et al L-Arginine Reduces Heart Collagen
481
TABLE 1. Mean and SD Values and Range of Parameters Evaluated
L-Arginine-Treated Animals
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Total heart collagen hydroxyproline, ,umol/L
per 100 mg heart tissue
Total ventricular tissue collagen (Sircol assay),
mg collagen/i 00 mg heart tissue
Eluted collagen hydroxyproline, ;tmol/L per
100 mg heart tissue
Ratio of eluted to total collagen
Carboxymethyllysine, nmol/L per 100 mg
heart tissue
Carboxymethyllysine, nmol/L per nmol/L
hydroxyproline
O-Tyrosine, nmol/L per 100 mg heart tissue
O-Tyrosine, nmol/L per nmol/L hydroxyproline
Glutathione, nmol/L per 100 mg heart tissue
Serum glucose, mg/dL
Serum fructosamine, rmol/L
Untreated Animals
Mean
0.24
SD
0.05
Range
0.17-0.35
Mean
0.49
SD
0.10
Range
0.35-0.65
1.24
0.31
1.01-1.32
2.06
0.41
1.71-2.34
0.22
0.11
0.14-0.50
0.22
0.05
0.15-0.32
0.96
0.54
0.56
0.26
0.54-2.54
0.10-0.98
0.46
0.40
0.13
0.29
0.29-0.67
0.09-1.09
0.002
0.001
0.0004-0.004
0.001
0.001
0.0002-0.002
0.80-9.30
0.003-0.05
20.83-27.93
260-412
3.23-15.15
3.63
0.008
28.63
313
8.34
5.09
0.02
24.53
314
7.49
2.21
0.01
2.13
34
3.31
On SDS-PAGE, no collagen split products could be
revealed, and no different patterns were observed in the
groups studied.
The results of carboxymethyllysine per 100 mg of
heart weight and carboxymethyllysine related to collagen content are listed in Table 1. Comparison of the
groups showed no significant difference in carboxymethyllysine per 100 mg of heart weight (P=.24). The results
expressed as carboxymethyllysine related to collagen
contents, however, clearly indicated significantly higher
ratios in the treated group (P=.0003).
As shown in Table 1, no difference between O-tyrosine in the treated group and in the untreated group
could be evaluated in expressing O-tyrosine per 100 mg
heart tissue (P=.36). When O-tyrosine was expressed as
related to collagen contents, a significantly higher 0-tyrosine content was found in the treated group (P=.03).
5.57
0.01
2.79
17
7.04
0.30-20.30
0.001-0.05
22.16-32.77
287-345
1.62-21.88
Table 1 presents the outcome of glutathione determinations indicating significantly higher levels in the untreated panel (P=.003).
As listed in Table 1, neither serum fructosamine
(P=.60) nor serum glucose (P=.70) differed significantly between the groups.
.c
s
Discussion
As shown in "Results," L-arginine treatment for 4
months significantly reduced collagen accumulation in
total ventricular tissue of spontaneously diabetic mice.
We propose that the mechanism of action is the inhibition of glucose-mediated collagen cross-links4 through
blockade of reactive carbonyl species. Our findings of
increased solubility of collagen in the hearts of treated
mice support this hypothesis as collagen solubility is a
reliable marker for cross-linking. The possibility that
TABLE 2. Significant Correlations of Laboratory Findings
L-ArginineTreated
Animals
Untreated
Animals
P
.02
r
.28
P
.33
.1
.95
.21
vs
CML, nmol/L per 100 mg heart tissue
r
.54
gmol/L OHP/100 mg
vs
Serum fructosamine
Serum glucose
.73
.49
.0012
.05
.37
CML, nmol/L per 100 mg heart tissue
vs
CML, nmol/L per nmol/L OHP
vs
.86
.71
.87
.0001
.001
.0001
.96
.07
.99
.0001
Serum fructosamine
O-Tyrosine, nmol/L per nmol/L OHP
Serum fructosamine
Eluted collagen
CML, nmol/L per 100 mg heart tissue
.09
.90
.23
.72
.0001
.55
.7
.38
.57
.04
.007
.03
Total heart collagen, /Lmol/L
OHP/100 mg heart tissue
Eluted collagen,
heart tissue
O-Tyrosine, nmol/L per 100 mg heart
tissue
Serum glucose
Ratio of eluted to total collagen
vs
vs
OHP indicates hydroxyproline; CML, carboxymethyllysine.
.81
.0001
482
Circulation Vol 90, No 1 July 1994
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differences in glycemic control may have led to differences in the collagen content was ruled out by comparable serum fructosamine levels. In testing the current
concept of glycoxidation as a pathogenetic mechanism,
we determined parameters for the involvement of free
oxygen radicals, ie, carboxymethyllysine, O-tyrosine,
and glutathione. In contrast to studies on diabetic
kidneys in db/db mice showing decreased kidney carboxymethyllysine after arginine treatment, we could not
find any differences in heart carboxymethyllysine when
expressed as carboxymethyllysine per heart weight. Correlating carboxymethyllysine per weight with the ratio
of eluted to total collagen showed a significant positive
correlation (in the untreated group only), ruling out free
radical cross-linking. This finding is confirmed by an
increased ratio of carboxymethyllysine to hydroxyproline in the treated group.
O-Tyrosine, a parameter for free hydroxyl radical
attack," failed to differ between the groups if expressed
as O-tyrosine per 100 mg of heart weight. In addition,
no significant correlation to collagen content or eluted
collagen was found, ruling out a role of hydroxyl attack
in the postulated oxidative stress in diabetes mellitus
and free radical cross-linking of collagen as the cause of
collagen accumulation. When O-tyrosine was related to
total hydroxyproline content, the unexpected result of
higher values in the treated group was found. No
correlation was found with total collagen content,
eluted collagen, or ratio of eluted to total collagen.
In the glutathione-system heart, glutathione was significantly higher in the untreated panel. This again is
further evidence against the oxidative stress hypothesis.
Glutathione is a potent free oxygen radical scavenger
and would be expected to be higher in the treated
animals, where oxidative stress compounds and intermediates should have already been blocked by arginine
therapy. One explanation could be that the sulfhydryl
residues responsible for scavenging of free oxygen radicals are less reactive with aldehydes abundant in the
diabetic system than reactive guanidino and amino
groups of L-arginine free base. No correlation of glutathione with total collagen, eluted collagen, or ratio of
eluted to total collagen was observed. Three parameters
for oxidative stress did not indicate that glycoxidation is
responsible for collagen accumulation in the diabetic
heart. The findings also postulate that L-arginine, which
clearly reduced heart collagen in our study and led to
increased solubility and therefore reduced abnormal
collagen cross-linking, did not act by scavenging free
oxygen radicals but rather by blocking carbonyl residues
of reactive aldehydes as cross-linking aldehydes from
collagen lysines and deoxyglucosones as reactive species
observed in diabetes.14
Major organ differences (in terms of free oxygen
radical handling by superoxide dismutase, glutathionemetabolizing enzymes, and other redox systems) between heart and kidney, where N-epsilon-carboxymethyllysine correlation with collagen reduction was
described, also could be responsible for our findings.
The fact that free oxygen radical parameters showed
oxidative stress in the treated group rather than in the
untreated group could be explained by the effect of
nitric oxide formation from arginine in the endothelium.15 Nitric oxide from arginine, however, could not
have been involved in free radical cross-linking because
the increased solubility by arginine treatment clearly
showed decreased cross-linking. At present, nothing is
known about collagen turnover in the presence of nitric
oxide.
Recent publications described in vitro evidence of the
direct role of glucose in collagen synthesis. Using molecular biological methods in cell cultures, authors
described increased mRNA for procollagen by glucose
in a dose-dependent manner.16 We found no difference
in glucose serum concentrations between the groups, no
significant correlation between glucose and total heart
collagen, but a significant correlation between glucose
levels and eluted collagen in the arginine-treated group.
No significant correlation of glucose and the quotient of
ratio of eluted to total heart collagen was found, but
there was a trend toward correlation. In testing longterm integrated glucose values as expressed by levels of
serum fructosamine, we found no differences between
the groups, but serum fructosamine turned out to be
significantly and impressively correlated with total heart
collagen in the treated panel. Collagens are the major
extracellular matrix proteins, making up approximately
80%. Collagen types and their relevance in diabetic
cardiomyopathy were described recently. In addition to
an increase in collagen types I and III, the switch and
increase of collagen type VI can be observed.'7 Collagen accumulation or myocardial fibrosis is an unequivocal consistent finding in diabetic cardiomyopathy,18-21
although it is not known whether increased collagen
synthesis or decreased catabolism by increased glucosemediated abnormal cross-linking (expressed by decreased solubility) is the underlying cause. Both increased synthesis and abnormally cross-linked collagen
could be responsible for the observed increase in stiffness of the myocardial tissue.
Arginine led to increased solubility and therefore
decreased cross-linking by a mechanism described for
arginine and aminoguanidine on kidney collagen.1'4,22
Other mechanisms also could have been active; arginine
is known to release insulin, but this mechanism is highly
improbable as no differences were detected in fructosamine. Another mechanism could have been responsible as it is well documented that arginine stimulates
macrophage activities23 and that macrophages present
receptors for AGEs on their surfaces,24 an indicator that
AGEs could have been cleared from tissues and/or
circulation. Because arginine is known to activate interleukin-l a and this cytokine induces collagenase activity,
increased collagenolysis also could have been
involved.25
Our findings on SDS-PAGE did not show the presence of collagen split products, and therefore the mechanism of increased collagenolysis is highly improbable.
We successfully reduced collagen accumulation in the
diabetic heart, but it remains to show whether this
finding has functional benefits.
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L-arginine reduces heart collagen accumulation in the diabetic db/db mouse.
A Khaidar, M Marx, B Lubec and G Lubec
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Circulation. 1994;90:479-483
doi: 10.1161/01.CIR.90.1.479
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1994 American Heart Association, Inc. All rights reserved.
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