Immunochemical detection of glycated beta- and gamma-crystallins in lens and their circulating autoantibodies (IgG) in streptozocin induced diabetic rat.
OBJECTIVE
This study used an immunochemical approach aimed to detect the glycated crystallins (beta- and gamma-crystallin) in rat lens and their circulating specific autoantibodies in serum during the course of cataractogenesis.


METHODS
Streptozocin (STZ; 55 mg/kg body mass) induced diabetic male Wistar/NIN rats (2-3 months old) and control nondiabetic rats were used for this study. Plasma glucose, glycated hemoglobin and body weight were evaluated on day zero, and at the interval of every two weeks up to the eighth week of post-injection in both the groups. Other biochemical parameters, such as the levels of nonprotein sulfhydryl (-SH) groups and the activity of gamma-glutamyl transpeptidase (gamma-GT) in lens proteins were also estimated. Cataract progress was monitored by measuring the advanced glycation end product (AGE)-like fluorophores in both intact lens as well as in lens homogenate employing digital based image analysis and spectrofluorimetric methods. Similarly, the polyclonal antibodies specific to beta-glycated-, gamma-glycated-, beta-, and gamma-crystallins were used to determine the concentration of respective immunogens in lens by noncompetitive ELISA and their respective circulating antibodies by antibody capture assay. The profile of glycated lens protein (soluble and insoluble fractions) during the course of cataractogenesis was assessed by the western blot technique.


RESULTS
STZ induced diabetic rats showed typical signs of diabetes (hyperglycemia, increased water and food intake with no increase in body weight). Biochemical analysis of total lens protein showed a significant (p = <0.001) decrease in the levels of nonprotein -SH groups. The activity of lenticular gamma-GT in diabetic rats was found to be unaltered as compared to the control group. Digital analysis of intact lens illustrated a positive correlation (r(2)=0.888) with the formation of AGE-like fluorophores during the course of cataractogenesis. A similar trend was also observed in the levels of AGE-like fluorophores in the total lens homogenate of diabetic animals during the course of cataractogenesis. The concentration of beta- and gamma-glycated-crystallins in the rat lens (soluble and insoluble fractions) was analyzed by non-competitive ELISA. The concentration of beta- and gamma-glycated-crystallins were found to be enhanced by the end of week eight, as compared to the control group. Concomitantly, crystallin-specific (beta- and gamma-glycated-crystallin) autoantibodies were also detected in the serum of the diabetic rats from week two onwards. Western blot analysis indicated the formation of enhanced glycated lens crystallins (beta- and gamma-crystallin) in the insoluble fraction.


CONCLUSIONS
The following was observed during the course of cataractogenesis: (1) there was an enhanced formation of AGEs-like fluorophores in intact lens; (2) beta- and gamma-glycated-crystallin levels increased in the rat lens (insoluble fraction) by the end of week eight; and (3) release of these glycated lens proteins into peripheral circulation resulted in the production of autoantibodies to beta- and gamma-glycated-crystallins that could be detected as early as week two, after induction of diabetic status in experimental rats.
Ranjan
Mala
M
Department of Biochemistry, University College of Science, Osmania University, Hyderabad, India. mala.alok@yahoo.co.in <mala.alok@yahoo.co.in>
Nayak
Sujatha
S
Rao
Beedu Sashidhar
BS
D000818:Animals; D001323:Autoantibodies / Q000097:blood*; D015153:Blotting, Western; D002386:Cataract / Q000209:etiology*; D003921:Diabetes Mellitus, Experimental / Q000150:complications / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005470:Fluorometry; D017127:Glycation End Products, Advanced / Q000378:metabolism; D006031:Glycosylation; D007074:Immunoglobulin G / Q000097:blood; D007158:Immunologic Techniques; D007908:Lens, Crystalline / Q000378:metabolism*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D038204:beta-Crystallins / Q000276:immunology / Q000378:metabolism*; D038222:gamma-Crystallins / Q000276:immunology / Q000378:metabolism*
D001323:Autoantibodies; D017127:Glycation End Products, Advanced; D007074:Immunoglobulin G; D038204:beta-Crystallins; D038222:gamma-Crystallins