X-Message-Number: 27873
Date: Wed, 26 Apr 2006 20:51:53 -0700 (PDT)
From: Doug Skrecky <>
Subject: elucidating age-associated stiffness

[Glucosepane may be the primary driving force behind hardened arteries,
stiff joints, presbyopia, and a host of other afflications associated with
old age. Glucosepane also affords a partial explanation for why the free
radical theory of aging has lost credibility.]

Ann N Y Acad Sci. 2005 Jun;1043:533-44.
Cross-linking of the extracellular matrix by the maillard reaction in
aging and diabetes: an update on "a puzzle nearing resolution".
  The aging extracellular matrix is characterized by an age-related
increase in insolubilization, yellowing, and stiffening, all of which can
be mimicked by the Maillard reaction in vitro. These phenomena are
accelerated in metabolic diseases such as diabetes and end-stage renal
disease, which have in common with physiological aging the accumulation of
various glycation products and cross-links. Eight years ago we concluded
that the evidence favored oxidative cross-linking in experimental
diabetes [Monnier, V.M. et al. 1996. The mechanism of collagen
cross-linking in diabetes: a puzzle nearing completion. Diabetes
45(Suppl. 3): 67-72] and proposed a major role for a putative non-UV
active cross-link derived from glucose. Below, we provide an update of
the field that leads to the conclusion that, while oxidation might be
important for Maillard reaction-mediated cross-linking via Strecker
degradation and allysine formation, the single most important collagen
cross-link known to date in diabetes and aging is glucosepane, a
lysyl-arginine cross-link that forms under nonoxidative conditions.

J Biol Chem. 2005 Apr 1;280(13):12310-5. Epub 2005 Jan 26.
Glucosepane is a major protein cross-link of the senescent human
extracellular matrix. Relationship with diabetes.
  The extracellular matrix in most tissues is characterized by
progressive age-related stiffening and loss of proteolytic digestibility
that are accelerated in diabetes and can be duplicated by the
nonenzymatic reaction of reducing sugars and extracellular matrix
proteins. However, most cross-links of the Maillard reaction described so
far are present in quantities too low to account for these changes. Here
we have determined in human skin and glomerular basement membrane
(GBM) collagen the levels of the recently discovered lysine-arginine
cross-links derived from glucose, methylglyoxal, glyoxal, and
3-deoxyglucosone, i.e. glucosepane, MODIC, GODIC, and DOGDIC,
respectively. Insoluble preparations of skin collagen (n = 110) and
glomerular basement membrane (GBM, n = 28) were enzymatically digested,
and levels were measured by isotope dilution technique using liquid
chromatography/mass spectrometry. In skin, all cross-links increased with
age (p < 0.0001) except DOGDIC (p = 0.34). In nondiabetic controls,
levels at 90 years were 2000, 30, and 15 pmol/mg for glucosepane, MODIC,
and GODIC, respectively. Diabetes, but not renal failure, increased
glucosepane to 5000 pmol/mg (p < 0.0001), and for all others, increased it
to <60 pmol/mg (p < 0.01). In GBMs, glucosepane reached up to 500 pmol/mg
of collagen and was increased in diabetes (p < 0.0001) but not old
age. In conclusion, glucosepane is the single major cross-link of the
senescent extracellular matrix discovered so far, accounting for up to
>120 mole% of triple helical collagen modification in diabetes. Its
presence in high quantities may contribute to a number of structural and
cell matrix dysfunctions observed in aging and diabetes.

[Neither vitamins nor green tea help with cross-links.]

Diabetes. 2005 Feb;54(2):517-26.
Paradoxical effects of green tea (Camellia sinensis) and antioxidant
vitamins in diabetic rats: improved retinopathy and renal mitochondrial
defects but deterioration of collagen matrix glycoxidation and
cross-linking.
  We tested the hypothesis that green tea prevents diabetes-related
tissue dysfunctions attributable to oxidation. Diabetic rats were treated
daily with tap water, vitamins C and E, or fresh Japanese green tea
extract. After 12 months, body weights were decreased, whereas glycated
lysine in aorta, tendon, and plasma were increased by diabetes (P <
0.001) but unaffected by treatment. Erythrocyte glutathione and plasma
hydroperoxides were improved by the vitamins (P < 0.05) and green tea (P
< 0.001). Retinal superoxide production, acellular capillaries, and
pericyte ghosts were increased by diabetes (P < 0.001) and improved by
green tea and the vitamins (P variable). Lens crystallin fluorescence at
370/440 nm was ameliorated by green tea (P < 0.05) but not the
vitamins. Marginal effects on nephropathy parameters were noted. However,
suppressed renal mitochondrial NADH-linked ADP-dependent and
dinitrophenol-dependent respiration and complex III activity were
improved by green tea (P variable). Green tea also suppressed the
methylglyoxal hydroimidazolone immunostaining of a 28-kDa mitochondrial
protein. Surprising, glycoxidation in tendon, aorta, and plasma was
either worsened or not significantly improved by the vitamins and green
tea. Glucosepane cross-links were increased by diabetes (P < 0.001), and
green tea worsened total cross-linking. In conclusion, green tea and
antioxidant vitamins improved several diabetes-related cellular
dysfunctions but worsened matrix glycoxidation in selected tissues,
suggesting that antioxidant treatment tilts the balance from oxidative to
carbonyl stress in the extracellular compartment.

[Even so called AGE-breakers find glucosepane too tough a nut to crack.]

Arch Biochem Biophys. 2003 Apr 1;412(1):42-6.
AGE-breakers cleave model compounds, but do not break Maillard crosslinks
in skin and tail collagen from diabetic rats.
  Advanced glycation end products (AGE), formed by nonenzymatic Maillard
reactions between carbohydrate and protein, contribute to the increase in
chemical modification and crosslinking of tissue proteins with
age. Acceleration of AGE formation in collagen during hyperglycemia, with
resultant effects on vascular elasticity and basement membrane
permeability, is implicated in the pathogenesis of diabetic
complications. AGE-breakers, such as N-phenacylthiazolium (PTB) and
N-phenacyl-4,5-dimethylthiazolium (PMT) halides, have been proposed as
therapeutic agents for reversing the increase in protein crosslinking in
aging and diabetes. We have confirmed that these compounds, as well as
the AGE-inhibitor pyridoxamine (PM), cleave the model AGE crosslink,
phenylpropanedione, and have studied the effects of these compounds in
reversing the increased crosslinking of skin and tail collagen
isolated from diabetic rats. Crosslinking of skin collagen, measured as
the half-time for solubilization of collagen by pepsin in 0.5M acetic
acid, was increased approximately 5-fold in diabetic, compared to
nondiabetic rats. Crosslinking of tail tendon collagen, measured as
insolubility in 0.05 N acetic acid, was increased approximately
10-fold. Collagen preparations were incubated in the presence or absence
of AGE-breakers or PM in phosphate buffer, pH 7.4, for 24h at 37 degrees
C. These treatments did not decrease the half-time for solubilization of
diabetic skin collagen by pepsin or increase the acid solubility of
diabetic tail tendon collagen. We conclude that, although AGE-breakers
and PM cleave model crosslinks, they do not significantly cleave AGE
crosslinks formed in vivo in skin collagen of diabetic rats.

[Glucosepane breakers may be in the works...]

Mol Cell Biol Res Commun. 2000 Jun;3(6):360-6.
Novel inhibitors of advanced glycation endproducts (part II).
  Enhanced formation and accumulation of advanced glycation endproducts
(AGEs), have been implicated as a major pathogenesis process leading to
diabetic complications, normal aging, atherosclerosis, and Alzheimer's
Disease. Several potential drug candidates as AGE inhibitors have been
reported recently. The aim of this study was to develop classes of novel
inhibitors of glycation, AGE formation, and AGE-crosslinking and to
investigate their effects through in vitro chemical and immunochemical
assays. A total of 92 compounds were designed and synthesized. The first
63 compounds were reported before. Nearly half of the 29 novel inhibitors
reported here are benzoic acid derivatives and related molecules, and
found to be potent inhibitors of multistage glycation, AGE formation, and
AGE-protein crosslinking. All 29 compounds show some degrees of
inhibitory activities as detected by the four assay methods, 9 compounds
demonstrated high percent inhibition (PI) in all tests, 30 to 40 times
stronger than aminoguanidine.

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