Diabetes is a major risk factor for producing lens opacity called cataract and ultimately loss of vision (Harding 1981; Brian and Taylor 2001). The process of cataractogenesis in diabetic patients is more accelerated as compared with senile subjects with at least 20 years earlier onset. Diabetes is characterized by excessive sugar levels and there is substantial evidence suggesting involvement of these sugars in formation of AGEs through non-enzymatic glycation. The process of glycation is instigated by the formation of Schiff’s base adduct between the carbonyl group of reducing sugars and the free amino group of proteins (Maillard reaction; Stitt 2005). The initial Schiff’s base is then followed by structural Amadori rearrangement or isomerisation producing an amino-ketose derivative (Groenen et al. 1994). These derivatives then undergo series of degradation reactions giving rise to highly reactive dicarbonyl compounds such as glucosone and 3-deoxyglucosone. Dicarbonyls act as protein cross-linking agents and react with free amino group of proteins, producing additional Schiff’s bases. The eventual effect of glycation therefore is the generation of protein aggregates. It has also been observed that production of these AGEs is associated with the generation of chemiluminescence, suggesting the role of reactive oxygen species (Namiki et al. 1993).

AGEs have been observed in normal aging process while excessive levels have been reported in diabetes mellitus (Peppa et al. 2003). Non-enzymatic glycation of lens proteins by reducing sugars (glucose, galactose, etc.) may cause conformational changes resulting in the formation of protein aggregates that precipitate in the lens (Monnier et al. 1979) leading towards lens opacity. AGEs formation is also suggested to be accelerated by oxidative stress as reactive oxygen species enhance cross-linking between carbohydrate moiety and amino group of protein (Peppa et al. 2004). This phenomenon has generated a new term glucoxidation which indicates that the two processes are integrated. Recently, we reported an inverse relationship between levels of antioxidants and glycation (Hashim et al. 2009). There are several AGEs which have been identified structurally (Ahmed et al. 1997). In human lens, numerous AGEs have been discovered including N-(carboxymethyl)-L-lysine (CML), pentosidine, fluorophore LM-1, pyrraline, N-carboxyethyl-lysine (CEL) to name a few (Nagaraj and Monnier 1992; Nagaraj and Sady 1996; Ahmed et al. 1997; Nagaraj et al. 1991; Dunn et al. 1989).

Immunohistochemical analyses were performed using human AGEs and CEL antibodies as shown in Figs. 2 and ​and3,3, respectively. The nuclear region of the lens comprises of columnar cells which form lens fibers. Positive immunostaining was observed in the nucleus where antibodies reacted strongly.

Figure 4 (a, b) illustrates the western blot analysis of human cataractous lenses from diabetic and non-diabetic individuals for AGEs and CEL detection, respectively.

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.