Accumulation of
fructosyl-lysine and advanced glycation end products in the kidney,
retina and peripheral nerve of streptozotocin-induced diabetic rats
N. Karachalias, R. Babaei-Jadidi, N. Ahmed
and P.J. Thornalley
Department of Biological Sciences
University of Essex, Central Campus
Wivenhoe Park, Colchester
Essex CO4 3SQ, U.K.
e-mail thorp@essex.ac.uk
Abstract
The accumulation of AGEs (advanced
glycation end products) in diabetes mellitus has been implicated in the
biochemical dysfunction associated with the chronic development of
microvascular complications of diabetes nephropathy, retinopathy and
peripheral neuropathy. We investigated the concentrations of
fructosyl-lysine and AGE residues in protein extracts of renal
glomeruli, retina, peripheral nerve and plasma protein of
streptozotocin-induced diabetic rats and normal healthy controls.
Glycation adducts were determined by LC with tandem MS detection. In
diabetic rats, the fructosyl-lysine concentration was increased markedly
in glomeruli, retina, sciatic nerve and plasma protein. The
concentrations of Ne-carboxymethyl-lysine and Ne-carboxyethyl-lysine
were increased in glomeruli, sciatic nerve and plasma protein, and
Ne-carboxymethyl-lysine also in the retina. Hydroimidazolone AGEs
derived from glyoxal, methylglyoxal and 3-deoxylglucosone were major
AGEs quantitatively. They were increased in the retina, nerve, glomeruli
and plasma protein. AGE accumulation in renal glomeruli, retina,
peripheral nerve and plasma proteins is consistent with a role for AGEs
in the development of nephropathy, retinopathy and peripheral neuropathy
in diabetes. High-dose therapy with thiamine and
Benfotiamine suppressed the accumulation of
AGEs, and is a novel approach to preventing the development of diabetic
complications.
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Introduction
Microvascular disease (nephropathy, retinopathy and neuropathy) develops
in human substects with diabetes mellitus over 1015 years. It is a
common and disabilitating complication of diabetes mellitus, with no
effective therapy. Diabetic nephropathy is characterized by the
development of proteinuria, culminating in end-stage renal disease with
a particularly high risk of cardiovascular morbidity and mortality. The
initial stage of development of nephropathy, incipient nephropathy, is
characterized by the onset of persistent microalbuminuria and
hyperfiltration [1]. Diabetic retinopathy is characterized by early loss
of pericytes, vessel weakening and endothelial dysfunction that leads to
the development of acellular capillaries, microaneurysms, capillary
closure and ischaemia. In the later stages, proliferative retinopathy,
there is angiogenesis, macular oedema and severe visual impairment [2].
Diabetic neuropathy is a spectrum of damage to peripheral nerves in
diabetes mellitus. It arises as a result of progressive damage to the
peripheral sensory and autonomic nervous systems [3]. Hyperglycaemia is
a risk factor for the development of microvascular complications in both
Type I and Type II diabetic substects [4,5]. Tight control of blood
glucose (and blood pressure) decreases the risk of developing
microvascular complications, but this is not always achievable because
of limitations of current drug therapy [6].
The link between hyperglycaemia and the development of microvascular
complications is explained by the effect of plasma glucose concentration
on vascular cells with GLUT1 glucose transporter expression. A high
plasma glucose concentration leads to a high cytosolic glucose
concentration in capillary endothelial cells and pericytes, with
consequent biochemical dysfunction, including increased formation and
accumulation of AGEs (advanced glycation end products) [7]. Increased
concentrations of triose phosphate glycolytic intermediates
(glyceraldehyde 3-phosphate and dihydroxyacetone phosphate) is the
trigger for these processes [8,9]. A pharmacological strategy that
countered triose phosphate accumulation in hyperglycaemia would suppress
multiple pathogenic pathways and prevent the development of diabetic
microvascular complications. Activation of the reductive pentose
phosphate pathway by high-dose thiamine therapy may achieve this by
increasing transketolase activity and stimulating the conversion of
glyceraldehyde 3-phosphate and fructose 6-phosphate into ribose
5-phosphate (Scheme 1).
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Scheme 1 Shunting of glycolytic intermediates from the
EmbdenMeyerhof pathway (dotted enclosure) to the reductive pentose
phosphate pathway in anaerobic glycolysis
G-6-P, glucose 6-phosphate; F-6-P, fructose 6-phosphate; F-1,6-bis-P,
fructose 1,6-bisphosphate; TPI, triose phosphate isomerase; DHAP,
dihydroxyacetone phosphate; GA3P, glyceraldehyde 3-phosphate; PG,
phosphoglycerate; PEP, phosphoenolpyruvate; R-5-P, ribose 5-phosphate.
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Concentrations of AGEs in renal glomeruli, retina, peripheral nerve and
plasma proteins of STZ (streptozotocin)-diabetic rats and normal healthy
controls
The effects of high-dose thiamine and
Benfotiamine (7 and 70
mg/kg) therapy on the accumulation of AGEs in renal glomeruli, retina,
sciatic nerve and plasma proteins in the STZ-induced diabetic rat model
with moderate insulin therapy were investigated after 24 weeks of
diabetes (Table 1). Incipient nephropathy developed in the STZ-diabetic
controls over a 24-week period, as judged by hyperfiltration and
microalbuminuria, and both high-dose thiamine and
Benfotiamine therapy prevented this [3].
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Table 1 AGEs in proteins of renal glomeruli, retina, sciatic nerve and
blood plasma of control and STZ-induced diabetic rats
- denotes that the concentration was below the limit of detection.
Significance: *P<0.05, **P<0.01, ***P<0.001 with respect to normal
healthy controls. NS, not significant.
| Analyte |
Study group |
Renal glomeruli |
Retina |
Sciatic nerve |
Plasma protein |
| CML (mmol/mol of Lys) |
Control |
0.269±0.111 |
0.172±0.051 |
0.151±0.087 |
0.033±0.004 |
| |
Diabetic |
0.501±0.186* |
0.451±0.291 |
0.437±0.077** |
0.062±0.008*** |
| CEL (mmol/mol of Lys) |
Control |
0.329±0.102 |
0.339±0.091 |
0.115±0.069 |
0.008±0.003 |
| |
Diabetic |
0.706±0.047*** |
NS |
0.519±0.286** |
0.017±0.006** |
| G-H1 (mmol/mol of Arg) |
Control |
0.044±0.029 |
0.552±0.103 |
0.517±0.238 |
0.275±0.041 |
| |
Diabetic |
NS |
1.39±0.89** |
1.22±0.55* |
0.565±0.206** |
| MG-H1 (mmol/mol of Arg) |
Control |
2.30±0.25 |
1.88±0.51 |
4.75±2.74 |
1.45±0.39 |
| |
Diabetic |
6.79±0.19*** |
5.24±2.34*** |
10.03±0.66** |
2.24±0.38** |
| 3DG-H (mmol/mol of Arg) |
Control |
3.23±0.90 |
0.20±0.09 |
2.85±1.24 |
2.26±0.89 |
| |
Diabetic |
4.87±0.32** |
0.42±0.15* |
5.73±0.72** |
NS |
| FL (mmol/mol of Lys) |
Control |
0.233±0.015 |
0.72±0.21 |
0.49±0.09 |
1.77±0.36 |
| |
Diabetic |
0.974±0.098*** |
2.59±1.23** |
3.69±0.72*** |
7.35±1.59*** |
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The diabetic rats had the characteristics of the diabetic state:
increased plasma glucose concentration and increased glycated
haemoglobin HbA1. After 24 weeks of diabetes, HbA1 was 17.7±1.6%, which
was increased with respect to normal controls (9.0±0.8%; P<0.001). Most
HbA1 reflects the presence of the fructosamine residues
Na-fructosylvaline and FL (Ne-fructosyl-lysine) [10]. FL residues were
detected in protein extracts of rat tissues and plasma protein. The
concentration of FL residues was highest in renal glomeruli (4.13
mmol/mol of Lys). The FL residue concentration was increased markedly in
diabetic rats in renal glomeruli (6-fold), retina (3-fold), sciatic
nerve (7-fold) and plasma protein (3-fold). There were, therefore,
marked increases in early-glycation adduct concentrations in tissue and
plasma proteins of diabetic rats, with respect to normal controls. This
was suggested previously by immunoblotting detection of FL [11].
There were significant increases in diabetic rats in the levels of CML
(Ne-carboxymethyl-lysine) residues in protein extracts of renal
glomeruli (86%), retina (189%), sciatic nerve (216%) and plasma protein
(64%). There were also significant increases in diabetic rats of CEL
[Ne-(1-carboxyethyl)lysine] residues in protein extracts of renal
glomeruli (115%), sciatic nerve (351%) and plasma protein (112%), but no
significant increase in the retina. There were tissue-specific increases
in hydroimidazolone residue concentrations for hydroimidazolones derived
from methylglyoxal {MG-H1
[Nd-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine]}, 3-deoxygluocosone
{3DG-H [Nd-(5-hydro-5-(2, 3,
4-trihydroxybutyl)-4-imidazolon-2-yl)-ornithine]} and glyoxal {G-H1
[Nd-(5-hydro-4-imidazolon-2-yl)ornithine]} in diabetic rats with respect
to normal controls. G-H1 was increased in the retina (152%), nerve
(136%) and plasma protein (105%); MG-H1 was increased in renal glomeruli
(195%), retina (279%), nerve (111%) and plasma protein (54%); and 3DG-H
was increased in renal glomeruli (51%), retina (110%) and nerve (50%).
High-dose therapy with both thiamine and the thiamine prodrug
Benfotiamine at doses of 7 and 70 mg/kg
respectively prevented the accumulation of AGEs in renal glomeruli,
retina, sciatic nerve and plasma protein in a dose-dependent manner
without reversing the increase in FL, as exemplified by the decreases in
MG-H1 and CEL in renal glomeruli [3].
This accumulation of AGEs and FL residues may be linked to the
development of retinopathy, nephropathy, neuropathy and generalized
angiopathy. STZ-diabetic rats on insulin maintenance therapy for 24
weeks developed incipient nephropathy [12] (as was found in the present
study), but they do not usually develop retinopathy or neuropathy in
this period more severe, untreated diabetes is required. AGE
accumulation generally precedes the development of complications in this
model of diabetes (including overt nephropathy), and AGEs may be
causally linked to the development of complications rather than being
indicators of complications status. The lack of accumulation of CML and
CEL residues in the retina may be due to the high proteasomal
proteolysis activity in the retina [13], such that only AGEs with the
highest flux of formation increase significantly. Overall, the
quantitative screening of glycation adducts in STZ-diabetic rats and
controls has shown a marked increase in FL, hydroimidazolones, CML and
CEL residues at the sites of development of microvascular complications
and in blood plasma, consistent with a role for glycation in the
development of vascular complications of diabetes.
The prevention of AGE accumulation in the glomeruli, retina, peripheral
nerve and plasma protein by high-dose thiamine and
Benfotiamine suggests that high-dose thiamine supplementation may
prevent the development of diabetic complications. Indeed, evidence is
now emerging that high-dose thiamine and
Benfotiamine prevent the development of microvascular
complications of diabetes [3,14,15]. The primary intervention by these
agents in the STZ-diabetic rat model was the prevention of thiamine
deficiency and induction of transketolase expression, with consequent
activation of the reductive pentose phosphate pathway shunt [3]. It is
remarkable that these effects were achieved by increasing the dietary
availability of thiamine to diabetic rats by as little as 20 times the
minimum daily allowance although this was sufficient to prevent
thiamine deficiency. Thiamine deficiency exacerbated the development of
diabetic nephropathy. We therefore propose that clinically diabetic
substects should avoid becoming thiamine deficient, even weakly so, and
that high-dose thiamine repletion should be considered for therapy to
prevent the development of clinical microvascular complications of
diabetes.
We thank the Wellcome Trust (U.K.) for support of our LC-MS/MS-related
protein biomarker research at the University of Essex. We thank the
Juvenile Diabetes Research Foundation International (New York, NY,
U.S.A.) for support of glycation-related research.
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