Diabetes mellitus and its complications such as cataract are major health problem of developing countries (Ivancic et al. 2005
; Kokiwar et al. 2007
). In Pakistan, 6.9 million people are affected by diabetes (Rathur and Boulton, 2007
) and approximately 570, 000 adults are blind as a result of cataract (Jadoon et al. 2007
). The earlier onset of cataract in diabetic individuals is suggested to be a consequence of non-enzymatic glycation. Glycation of long-lived proteins such as lens crystallins in aging and diabetes has been identified as the most important post-translational modification leading to cross-linking, aggregation, and insolubilization (Monnier and Cerami, 1981
; Kumar et al. 2007
). Direct correlation between AGEs formation and glycemic level has been reported. The AGEs hypothesis proposes that chemical modification of proteins by glucose during hyperglycemia plays a pivotal role in pathogenesis of diabetic complications (Yamagishi et al. 2007
We have investigated levels of AGEs and CEL, a derivative of CML in lenses from diabetic and senile lens samples. It is reported in previous studies that level of AGEs is higher in diabetic cataractous lenses than in non-diabetic cataractous lenses (Zarina et al. 2000
; Pokupec et al. 2003
). As expected, levels of CEL were found to be greater in diabetic lens samples than in non-diabetic samples. Similar results have been observed for Pentosidine (Hashimoto et al. 1997
), CML, and immidazolone (Franke et al. 2003
Lens crystallins can be divided in to α, β and γ-crystallins. α and β-crystallins are found in lens cortex and both crystallins being rich in lysine are more likely to undergo the process of glycation (Swamy and Abraham 1991
). γ-crystallins, on the other hand, is another extremely vulnerable target for glycation and localized in nucleus. Earlier studies indicated that human α-crystallin undergoes post translational modifications with aging and cataract. Glycation of α-crystallin may cause protein unfolding, while in hyperglycemic condition the same process has shown to be increased many folds (Kumar et al. 2007
). α and γ-crystallins have been studied extensively with reference to alteration due to glycation. In the current study, we investigated the probability of isolated β-crystallin to be glycated by glucose, fructose, and galactose upon incubation at different time intervals. Initially, increased concentration of AGEs was observed in diabetic samples as compared to non-diabetic subjects and the levels of AGEs increased in time-dependant manner. AGEs formation is a consequence of normal aging process but in case of diabetes, level of glycation is higher (Harding, 2002
). Our incubated sample showed the same pattern. Before incubation, the level of glycation was greater in diabetic sample as expected. Incubation with glucose, galactose (data not shown), and fructose resulted in a gradual increase in level of glycation in both non-diabetic and diabetic samples. After 10 days incubation, the level of AGEs became constant in diabetic samples presumably due to the fact that in diabetic samples, all possible glycation sites were already saturated. In aging cells, the level of AGEs and CEL increases gradually but the process of glycation is very slow. The rate of glycation in cataract patients with diabetes is accelerated due to higher sugar levels in hyperglycemic condition (Gul et al. 2009
; Duhaiman, 1995
). Hence, β-crystallin from diabetic samples being already glycated has less capacity to bind with incubated sugars. It was observed that, among all sugars (glucose, fructose, and galactose) fructose showed a higher affinity to bind with β-crystallin demonstrating that the rate of fructation is faster as compared to glucation and galactation. It has been suggested that fructose, due to its acyclic structure is a more potent glycating agent (Vinson, 2006
). Structural variation of the β-crystallins was also analyzed before and after alteration with glucose, galactose, and fructose by measuring absorption spectra. We observed time-dependent structural variation as a consequence of glycation induced by incubated sugars as compared to control samples. The variation was more obvious from 270 to 290 nm. Nagaraj and Sady (1996
) reported that incubation of α-crystallin with 3-deoxyglucose causes an alteration at 289 nm which is particular for pyrraline. Our results showed that introduction of high sugar level to lens can alter the structure of lens proteins as well as its function. This model may not reflect true in-vivo picture, however, it may still serve to be an indicator of changes that may occur in cell.
From the above facts and results, it can be proposed that non-enzymatic glycation of β-crystallin by various sugars promotes the alteration in protein structure. Our results also support the hypothesis that AGEs may play essential role in degenerative changes in lens, which occur much earlier in diabetic patients than in non-diabetics.
AGEs formation and cataract progression both are extremely slow processes and both are triggered in presence of reactive oxygen species. Oxidative insult along with AGEs may integrate resulting in acceleration of cataract formation. Approaches towards keeping normal antioxidant levels, utilizing low AGEs-content food and keeping normoglycemia may be beneficial to delay cataract formation.