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Increased risk of cataract development in WNIN-obese rats due to accumulation of intralenticular sorbitol
Article first published online: 18 MAR 2013
Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.
Volume 65, Issue 5, pages 472–478, May 2013
How to Cite
Reddy, P. Y., Giridharan, N. V., Balakrishna, N., Validandi, V., Pullakhandam, R. and Reddy, G. B. (2013), Increased risk of cataract development in WNIN-obese rats due to accumulation of intralenticular sorbitol. IUBMB Life, 65: 472–478. doi: 10.1002/iub.1163
- Issue published online: 22 APR 2013
- Article first published online: 18 MAR 2013
- Manuscript Accepted: 31 JAN 2013
- Manuscript Revised: 26 JAN 2013
- Manuscript Received: 12 JAN 2013
- protein aggregation
Epidemiological studies have reported an association between obesity and increased incidence of ocular complications including cataract, yet the underlying biochemical and molecular mechanisms remained unclear. Previously we had demonstrated accumulation of sorbitol in the lens of obese rats (WNIN/Ob) and more so in a related strain with impaired glucose tolerance (WNIN/GR-Ob). However, only a few (15–20%) WNIN/Ob and WNIN/GR-Ob rats develop cataracts spontaneously with age. To gain further insights, we investigated the susceptibility of eye lens proteins of these obese rat strains to heat- and UV-induced aggregation in vitro, lens opacification upon glucose-mediated sorbitol accumulation ex vivo, and onset and progression of cataract was followed by galactose feeding and streptozotocin (STZ) injection. The results indicated increased susceptibility toward heat- or UV-induced aggregation of lens proteins in obese animals compared to their littermate lean controls. Further, in organ culture studies glucose-induced sorbitol accumulation was found to be higher and thus the lens opacification was faster in obese animals compared to their lean littermates. Also, the onset and progression of galactose- or STZ-induced cataractogenesis was faster in obese animals compared to lean control. These results together with our previous observations suggest that obesity status could lead to hyperaccumulation of sorbitol in eye lens, predisposing them to cataract, primarily by increasing their susceptibility to environmental and/or physiological factors. Further, intralenticular sorbitol accumulation beyond a threshold level could lead to cataract in WNIN/Ob and WNIN/GR-Ob rats. © 2013 IUBMB Life, 65(5):472–478, 2013
Overweight and obesity are highly prevalent life-style disorders worldwide and are associated with various comorbidities (1–3), with a plethora of complications such as diabetic retinopathy, high intraocular pressure, cataracts, macular degeneration, and exophthalmos (4, 5). Among these disorders, cataract is the leading cause of blindness worldwide and accounts for estimated cases of 18 million, half of all these cases originating in developing countries (6, 7). Several large-scale population-based epidemiological studies consistently revealed strong links between obesity and cataract (5, 8–12). It is widely accepted that oxidative stress, osmotic stress, and nonenzymatic glycation of lens proteins are the primary mechanisms leading to cataract (13), and obesity might influence any or all of these physiological processes (14, 15). Although the underlying pathophysiology remains elusive (either one or all the above), stress-related mechanisms might contribute to the development of cataract in obesity. However, there are yet no studies to explain the molecular basis for cataract associated with obesity.
Earlier, we had reported 15–20% prevalence of cataracts in a spontaneous obese rat model without (WNIN/Ob) or with impaired glucose tolerance (WNIN/GR-Ob) (16), a case very similar to the observation in humans. Impaired glucose tolerance (IGT) is defined as a condition with plasma glucose concentration of 140–199 mg/dl up on a 2-h oral glucose tolerance test. Further, we also showed accumulation of sorbitol in the eye lens, and sorbitol accumulation beyond a threshold level resulted in cataract in WNIN/Ob and WNIN/GR-Ob rats (16). Although the exact mechanism by which sorbitol accumulates in obese rat lens remains to be understood, based on the above evidence, we hypothesize that intralenticular accumulation of sorbitol could cause subtle changes in lens proteins, rendering them susceptible to aggregation when subjected to physiological/environmental insults, which in turn predisposes the animal to cataract.
We carried out the following investigations to test our hypothesis: (i) susceptibility to heat and UV radiation induced aggregation of total soluble lens proteins, (ii) effect of glucose-mediated sorbitol accumulation on lens transparency, and (iii) effect of galactose feeding and streptozotocin (STZ)-induced diabetes on onset and progression of cataract in WNIN/Ob and WNIN/GR-Ob rats.
Methods and Procedures
STZ, galactose, glucose, modified TC-199 medium, penicillin, streptomycin, bovine serum albumin, sorbitol, Nicotinamide adenine dinucleotide, sorbitol dehydrogenase, Tris buffer, ethylenediamminetetraacetate were procured from Sigma-Aldrich (St. Louis, MO). All other chemicals and solvents were of analytical grade and were obtained from local companies.
In Vitro Studies
Isolation of Total Soluble Protein from Rat Lens.
WNIN/Ob and WNIN/GR-Ob rats were obtained from National Center for Laboratory Animal Sciences (NCLAS), National Institute of Nutrition. Eye balls were collected and lenses were dissected. Total soluble protein (TSP) fraction from 6- and 12-month-old WNIN/Ob and WNIN/GR-Ob rat lens and their respective lean littermates was isolated as described previously (17).
Heat-Induced Aggregation of Crystallins.
Heat-induced aggregation of TSP was carried out as described previously (17). Briefly, the TSP (1 mg/ml) in 50 mM phosphate buffer, pH 7.4 containing 100 mM NaCl, was heated at 62 °C, and light scattering due to aggregation was monitored at 360 nm as a function of time in a spectrophotometer (Perkin Elmer, Lambda-35) attached with a peltier control and circulating water bath.
The UV-induced aggregation of TSP was performed as described previously (17, 18). Briefly, 1 mg/ml lens TSP in 50 mM phosphate buffer, pH 7.4 containing 100 mM NaCl was irradiated at 300 nm (UV-B radiation) in a quartz cuvette in a final volume of 1 ml. The protein samples were subjected to constant and very gentle stirring during irradiation. The light scattering due to aggregation was monitored at 360 nm using a spectrophotometer (Perkin Elmer, Lambda-35) at intervals of 1 h over a period of 10 h.
Lens Organ Culture Studies Under Ex Vivo Conditions.
Isolated whole lens organ culture experiments were done as described previously (19, 20). Briefly, lenses of 6-month-old WNIN/Ob and WNIN/GR-Ob rats and their corresponding lean controls were cultured in 2 ml modified TC-199 (supplemented with 0.1 mg/ml penicillin and streptomycin) at 37° C under 95% air and 5% CO2 with 5.5 mM or 55 mM glucose for a period of 18 h (for sorbitol estimation) or 4–7 days (for lens morphology).
Estimation of Sorbitol in the Lens.
The sorbitol from rat lens was extracted and estimated as described previously by fluorometric method (16).
In Vivo Studies
WNIN/Ob and WNIN/GR-Ob rats (3 months old) with an average body weight of 160 ± 20 g (lean) and 290 ± 30 g (obese) were obtained from the NCLAS. Animals were housed in individual cages in a temperature and humidity controlled room having a 12 h light/dark cycle. All of the animals had free access to water. All the rats were kept on AIN-93 diet. The experiments were carried out for a period of 10 weeks.
Diabetes was induced in 16-h fasted animals by single intraperitoneal injection of STZ (35 mg/kg bw) in 0.1 M citrate buffer, pH 4.5. As there was 100% mortality in the obese strain of WNIN/Ob and WNIN/GR-Ob rats, the dose was optimized to 30 mg/kg bw for WNIN/Ob and 27 mg/kg bw for WNIN/GR-Ob rats. Rats which received only vehicle (0.1 M citrate buffer) served as control. There were 6 animals in control and 10 animals in diabetic groups.
Blood Glucose Levels.
Fasting blood glucose levels were measured 72 h after STZ injection using commercial kits (Ozone Biomedicals, New Delhi, India). Animals having blood glucose levels >145 mg/dl were considered as diabetic.
While a group of control rats (both lean and obese phenotype of WNIN/Ob and WNIN/GR-Ob strain) were fed with AIN-93 diet, the rats in experimental group were fed on AIN-93 diet supplemented with 30% galactose. There were six animals in control and eight animals in galactose groups.
Slit Lamp Examination and Cataract Classification.
The onset and progression of the cataract during experiment was monitored using a slit lamp biomicroscope (Kowa Portable, Japan) as described by us previously (21). Initiation, progression, and maturation of lenticular opacity were graded as described previously.
Animal care and protocols were in accordance with and approved by the Institutional Animal Ethics Committee and conform to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Results were expressed as means with their standard errors. Data were analyzed using SPSS version 15.0 software (SPSS, Chicago, IL). “t test” was used to compare the mean values between the groups for given strain. Level of significance was considered as 0.05.
In Vitro Studies
Heat- and UV-Induced Aggregation.
The light scattering due to heat- (Fig. 1A) and UV radiation (Fig. 1B)-induced aggregation of TSP of WNIN/Ob eye lens was higher compared to their lean counter parts both at 6 and 12 months of age. The heat- and UV-induced aggregation of TSP was higher in older animals (12 months) compared to younger animals (6 months) irrespective of the phenotype studied (Fig. 1). Similar results were obtained with respect to WNIN/GR-Ob rat strain as well (data not shown).
Effect of Glucose on Lens Sorbitol Levels and Opacification.
High glucose-induced lens opacification in organ cultures was seen in both WNIN/Ob (Fig. 2A) and WNIN/GR-Ob rats (Fig. 2B) by 4 days, in contrast to 7 days in lean control (data not shown). Appearance of lens opacification under high glucose conditions is associated with remarkable accumulation of sorbitol in obese lens of both the strains compared to the levels in respective lean transparent lens under same conditions (Table 1). As reported earlier sorbitol levels were higher under normal conditions in WNIN/GR-Ob rat lens compared to WNIN/Ob and thus accumulation of sorbitol under high glucose was further higher in WNIN/GR-Ob rat lens compared to WNIN/Ob (Table 1).
In Vivo Studies
Effect of Galactose Feeding on Cataract Progression.
The onset and progression of cataract due to galactose feeding was much faster in WNIN/Ob (Fig. 3A) and WNIN/GR-Ob (Fig. 3B) over their respective lean controls. Interestingly, the onset and progression of galactose-induced cataract was much faster in WNIN/GR-Ob rats compared to WNIN/Ob rats (Fig. 3).
Effect of STZ-Induced Diabetes on Cataract Progression.
Diabetes was induced in WNIN/Ob and WNIN/GR-Ob rats by using a dose of STZ (35 mg/kg bw) that was effective for parental Wistar strain, WNIN (21). However, there was 100% mortality in the obese strain of WNIN/Ob and WNIN/GR-Ob rats with this dose. Thus, the dose was gradually decreased and was optimized to 30 mg/kg bw for WNIN/Ob and 27 mg/kg bw for WNIN/GR-Ob rats (Table 2). The dose of STZ required to induce diabetes without significant mortality was lower for WNIN/Ob and WNIN/GR-Ob rats compared to their respective lean rats (Table 2), and also the onset and progression of cataract in WNIN/Ob (Fig. 4A) and WNIN/GR-Ob (Fig. 4B) was faster than lean controls.
The interplay of obesity status and cataractogenesis in mammals has so far not been fully investigated. Against this backdrop, we felt that an animal model that develops cataract following the onset of obesity (akin to obesity associated cataract in patients) would aid in understanding the molecular nexus between obesity and cataract. We had earlier observed that 15–20% of the animals develop cataract in WNIN/Ob rats—a colony of rats that spontaneously developed an obesity phenotype and are housed at our in-house animal facility (16, 22–25). Subsequent biochemical investigations revealed that except for the polyol pathway, all the other principal pathways of lens remained unaltered in these rats (16). As a consequence, sorbitol levels were found to be high in the normal eye lens of obese rats (both WNIN/Ob and WNIN/GR-Ob) compared to their lean controls from 3 months of age onward. Between WNIN/Ob and WNIN/GR-Ob the levels of sorbitol were higher in the latter suggesting a synergistic effect of IGT along with obesity in the activation of sorbitol pathway. Activation of sorbitol pathway correlated with cataract formation in these mutant rats.
Although accumulation of sorbitol was seen in almost all the WNIN/Ob and WNIN/GR-Ob rats compared to their respective lean littermates, only 15–20% of WNIN/Ob and WNIN/GR-Ob rats develop cataract. We surmised that obesity in synergy with additional physiological and environmental factors might instigate cataract formation in an osmotically predisposed eye lens. We also hypothesized that sorbitol levels should cross a threshold level (>650 nmoles/g tissue) to develop cataract in these animals (16) and convergence of various stressors could aid in reaching this threshold level of sorbitol. In addition, chronic exposure of lens proteome to sorbitol renders it more susceptible to subsequent environmental and physiological stress.
The transparency of eye lens is maintained by the exquisite packing of lens proteins and chaperone-like activity of α-crystallin (26, 27). It is believed that oxidative stress, osmotic stress, and nonenzymatic glycation of lens proteins result in altered structural and functional integrity either due to direct modification and/or due to reduced α-crystallin chaperone activity, resulting in lens protein aggregation and thus opacification (13–15, 28). Heat, chemical- or UV-induced aggregation of lens proteins in vitro has been used to study the factors leading to cataract formation in vivo, as in vitro observations are well correlated with that of cataract progression in vivo. Hence, we investigated the susceptibility of TSP to the heat- and UV-induced aggregation. These results indicate that being obese could bring adverse impact on the lens proteins and may increase their susceptibility to aggregation by other convergent factors such as IGT and age.
To further correlate the vulnerability of sorbitol-loaded lens with opacification, lenses were cultured in high glucose conditions that resulted in a sevenfold and eightfold increase in sorbitol in WNIN/Ob and WNIN/GR-Ob, respectively. In tune with higher sorbitol accumulation, lens opacification was also faster in WNIN/Ob and WNIN/GR-Ob rats (4 days) compared to their lean counterparts (7 days), suggesting a correlation with the increased sorbitol levels and lens opacification. These results imply that lenses of obese rats are highly prone to cataract due to high lens sorbitol content, particularly when challenged with environmental (heat and UV) or physiological stress such as hyperglycemia or diabetes.
The in vivo results with experimental animals further lend support to the observations obtained from in vitro and ex vivo studies. As expected, galactose feeding or STZ injection induced onset and progression of cataract both in lean and obese rat phenotypes. But the onset and progression of cataract was faster in obese phenotype, in either of the physiological challenges studied. However, the differences in onset and progression of cataract between obese and their lean controls was marked with galactose feeding, but not with STZ. It should be noted that the mechanism of inducing cataractogenesis in these modes of treatment is different. For instance, galactose feeding leads to accumulation of galactitol (isomer of sorbitol) in the lens leading to osomotic stress and cataract formation (20, 29). We have already demonstrated high levels of lenticular sorbitol accumulation induced by glucose, corroborating our previous observations (16). Therefore, it is possible that galactose feeding further enhances the lens galactitol levels, which along with high sorbitol levels in turn leads to crossing the threshold levels and thus faster cataract progression. Whereas hyperglycemia induced by STZ is similar in both lean and obese counterparts might severely mask the differences if any during the experimental time course. However, it is important to note that the STZ dose required to induce cataract is lower in obese phenotype, more so in WNIN/GR-Ob strain, compared to their lean counterparts, suggesting inherent predisposition to diabetes induced cataractogenesis during obesity.
A study reported no change in sorbitol even in the presence of hyperglycemia (due to STZ-induction) in ob/ob (both lean and obese) mice (30). Most importantly cataracts were not reported in ob/ob mice. Interestingly, there was an increase in lens sorbitol in db/db upon STZ induction, but they too did not develop cataract (30). Therefore, WNIN-Ob rat model is unique in terms of sorbitol accumulation and appearance of cataracts in some obese but not lean rats under normal conditions. Because of sorbitol overload WNIN-obese rat are predisposed to cataract and they develop cataracts due to other (physiological and/or environmental) factors. Although, the factors other than the increased intracellular glucose concentrations that cause sorbitol accumulation needs to be identified, these findings are in line with similar observations made previously in models with different health anomalies other than diabetes and aging (31, 32).
It is likely that chronic exposure of lens proteins to high sorbitol renders them more susceptible to aggregation by other stressors such as UV radiation and temperature. Further, intralenticular sorbitol accumulation beyond a threshold level could predispose WNIN/Ob and WNIN/GR-Ob rats to cataract. In short, these results together with our previous work demonstrate that obesity is a predisposing factor that can lead to intralenticular accumulation of sorbitol, which exacerbates cataract, primarily by increasing the lens protein susceptibility to aggregation by environmental or physiological stress such as diabetes. These results open up new vistas of intervention research, where in the objective will be to prevent or reduce intracellular accumulation of sorbitol that can have a bearing on obesity associated complication of eye disorders.
The authors acknowledge Ms. M. Satyavani, Mr. N. Yadagiri, and Mr. E. Ganesh from National Center for Laboratory Animal Sciences for their help in breeding and maintenance of rats. This work was supported by grants from Indian Council of Medical Research and Department of Biotechnology, Government of India.
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