Research Article Evaluation of Neonatal Streptozotocin ...downloads.hindawi.com/journals/omcl/2014/463264.pdf · Research Article Evaluation of Neonatal Streptozotocin Induced Diabetic

Research ArticleEvaluation of Neonatal Streptozotocin Induced DiabeticRat Model for the Development of Cataract

Madhoosudan A Patil1 Palla Suryanarayana1 Uday Kumar Putcha2

Myadara Srinivas2 and G Bhanuprakash Reddy1

1 Department of Biochemistry National Institute of Nutrition Jamai-Osmania Hyderabad 500007 India2Department of Pathology National Institute of Nutrition Hyderabad 500007 India

Correspondence should be addressed to G Bhanuprakash Reddy geereddyyahoocom

Received 3 March 2014 Accepted 2 August 2014 Published 19 November 2014

Academic Editor Rajesh N Gacche

Copyright copy 2014 Madhoosudan A Patil et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Type 2 diabetes (T2D) generally follows prediabetes (PD) conditions such as impaired fasting glucose (IFG) andor impairedglucose tolerance (IGT) Although studies reported an association of IGT or IFG with cataract the experimental basis for PDassociated cataract is not known Hence we evaluated neonatal streptozotocin (nSTZ) induced rat model to study PD associatedcataractogenesis by injecting STZ to two-day old rats While majority (70) of nSTZ injected pups developed IGT (nSTZ-PD)by two months but not cataract even after seven months remaining (30) nSTZ rats developed hyperglycemia (nSTZ-D) bytwo months and mature cataract by seven months Lens biochemical analysis indicated increased oxidative stress as indicatedby increased SOD activity lipid peroxidation and protein carbonyl levels in nSTZ-D cataractous lens There was also increasedpolyol pathway as assessed by aldose reductase activity and sorbitol levels Though nSTZ-PD animals have not shown any signsof lenticular opacity insolubilization of proteins along with enhanced polyol pathway was observed in the lens Further there wasincreased oxidative stress in lens of IGT animals These results suggest that oxidative stress along with increased polyol pathwaymight play a role in IGT-associated lens abnormalities In conclusion nSTZ-PD rat model could aid to investigate IGT-associatedlens abnormalities

1 Introduction

Diabetes mellitus (DM) is a group of metabolic disorderscharacterized by circulating hyperglycemia Type 2 diabetesis more prevalent in humans and accounts for 90ndash95 of alldiabetic cases worldwide [1] It is caused by a combinationof resistance to insulin action and impaired insulin secretion[2] The prevalence of diabetes for all age groups worldwidewas found to be 382 million which may rise to 592 million by2035 [3] whereas in India the current prevalence of diabetes is409million and is expected to rise to 699million by 2025 [4ndash6] Without proper management diabetes can lead to variouscomplications such as cataract retinopathy nephropathyneuropathy and cardiovascular problems Cataract charac-terized by the opacification of the eye lens that interferes withtransmission of light onto the retina is one of the earliest

secondary complications of diabetes Cataract is the leadingcause of visual impairment across the world and accounts for478 of the blind people in the world [7] Studies indicatethat the incidence of cataract is much higher in diabetic thanin nondiabetic individuals [8 9]

Though the etiology of cataract is not fully understoodoxidative damage to the constituents of the eye lens isconsidered to be a major mechanism in the initiation andprogression of various types of cataracts including diabeticcataract [10] Many studies indicate the role of oxidativestress in the development of diabetic complications includingcataract due to glucose autoxidation nonenzymatic glycationof proteins leading to advanced glycation end products(AGE) and enhanced glucose flux through polyol pathway[11ndash13] More recent studies indicate that the polyol pathwayis likely a major contributor to oxidative stress at least in the

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2014 Article ID 463264 10 pageshttpdxdoiorg1011552014463264

2 Oxidative Medicine and Cellular Longevity

lenses and nerves of diabetic mice and it was reported thataldose reductase inhibitor reducesoxidative stress enhancementin sugar cataract [14] The polyol pathway may be relatedto hyperglycemia induced oxidative stress and there maybe a metabolic connection between the polyol pathway andoxidative stress Furthermore carbonyl stress has been foundto be the cause for advanced glycation end product toxicityindicating the involvement of nonenzymatic glycation in thedevelopment of oxidative stress

Type 2 diabetes (T2D) always precedes with prediabeticconditions such as impaired glucose tolerance (IGT) orimpaired fasting glucose (IFG) [6] Increased oxidative stresswas found in subjects with IGT and IFG [15] Interestinglyseveral recent large population studies reported that IGT orprediabetes is also associated with various types of cataracts[16 17]There are three principle mechanisms through whichlens can be damaged resulting in the development of cataract(oxidative stress osmotic stress andnonenzymatic glycation)and IGT might influence any or all of these physiologicalprocesses However no experimental studies have explainedthe association between IGT and cataract along with theirplausible pathophysiological mechanisms Previous studiesfrom our group have shown that obesity [18 19] as well asprediabetes [20] is associated with increased susceptibility tocataract formation due to the activation of sorbitol pathway inexperimental animalsThus there is a need to understand themechanism of cataractogenesis during IGT as well as T2D Inthis context availability of a suitable animalmodel that showsincreased susceptibility to IGT-induced cataractogenesis is ofimmense value

There are several animal models available to study themechanisms of diabetic complications including diabeticcataract However most of the studies on diabetic cataractin animal models are largely restricted to type 1 diabetic(T1D) conditions [21ndash23] In this context streptozotocin(STZ) [21 24 25] or alloxan-induced diabetic models [26ndash28] are extensively used to study the diabetic cataract andboth these models mimic the T1D in humans Studies fromour laboratory and elsewhere indicate that oxidative stressappears to be a major factor in the development of cataractin T1D along with the activation of polyol pathway andnonenzymatic glycation [21 22 29]

Neonatal-STZ (nSTZ) induced rat model is one of themost frequently used T2D-likemodels [30ndash33] whichmimicshuman diabetes [34] Previous studies on this model werefound to have characteristics of pancreatic beta-cell destruc-tion followed by beta-cell regeneration and glucose intoler-ance [35 36] Subsequently other authors confirmed thesefindings and showed that nSTZ treated rats in adulthooddisplay the typical characteristics of T2D [37ndash40]This animalmodel is mainly used to screen hypoglycemic or antidiabeticagents [32 41 42] and also for hypolipidemic and oxidativestress related studies [43] However no studies attempted touse this model for investigating the biochemical abnormal-ities in the lens and development of cataract Therefore inthe present study we evaluated nSTZ model for developmentof cataract due to IGT or T2D and associated biochemicalalterations in lens in rodent model

2 Materials and Methods

21 Materials Streptozotocin (STZ) NADPH DL-glyceral-dehyde lithium sulfate 120573-mercaptoethanol bovine serumalbumin sorbitol andsorbitol dehydrogenase tetraethoxypro-pane thiobarbituric acid NAD and pyrogallol were pur-chased from Sigma Chemical Company (St Louis USA) Allother chemicals were of analytical grade and were obtainedfrom local companies

22 Experimental Design Two-day-old male Sprague Daw-ley (SD) rat pups obtained from the National Center forLaboratory Animal Sciences National Institute of Nutri-tion Hyderabad India were injected intraperitoneally with90mgkg body weight STZ dissolved in 01M citrate bufferpH 45 (119899 = 23) Control pups (119899 = 7) received only vehicleThe pups were weaned after 21 days and maintained on AIN-93GM diet in individual cages throughout the experimentalperiod

23 Animal Care Animal care and protocols were in accor-dance with and approved by the Institutional Animal EthicsCommittee (IAEC) Animals were housed in individual cagesin a temperature and humidity controlled room with a 12 hL D cycle All these animals had free access to water Asso-ciation for Research in Vision and Ophthalmology (ARVO)statement for the use of animals in ophthalmic and visionresearch was followed

24 Oral Glucose Tolerance Test Oral glucose tolerance test(OGTT) was performed at the age of two months on ratsfasted for 16 h by administering glucose orally as a bolus at adose of 20 gkg body wt Blood samples were collected fromretroorbital plexus at 0 30 60 and 120min for determiningplasma glucose and insulin concentrations for assessingimpaired glucose tolerance (IGT) and insulin resistance

25 Homeostasis Model Assessment (HOMA) Insulin resis-tance was assessed by homeostasis model assessment HOMAas described earlier for rats [20] using the following equationHOMA-IR = [fasting plasma glucose (mgdL) times fastingplasma insulin (120583UmL)2430] To assess insulin response ofthese rats to a challenge of glucose load the area under thecurve (AUC) for insulin and glucose during the OGTT wascomputed

26 Clinical Parameters Plasma glucose was measured bythe glucose oxidase-peroxidase (GOD-POD) kit method(Biosystems Barcelona Spain) and plasma insulin by RIAkit (BRIT-DAE Mumbai India) method as per the manufac-turerrsquos instructions Blood glucose was measured weekly andinsulin levels were measured at the age of two months and atthe end of the experimental period of seven months

27 Slit-Lamp Examination and Cataract Classification Eyeswere examined every week using a slit-lamp biomicroscope(Kowa SL-15 Portable Japan) on dilated pupils Initiationand progression of lenticular opacity was graded into fourcategories as reported earlier [21]

Oxidative Medicine and Cellular Longevity 3

28 Collection of Lens and Pancreas At the end of sevenmonths experiment was terminated Pancreas and eyeballswere collected Eyeballs were dissected using the posteriorapproach and lenses were frozen at minus80∘C until furtheranalysis Pancreas was collected and fixed in Bouinrsquos solutionfor 24 h and then washed with 70 ethanol and stored in 70ethanol until further analysis

29 Immunohistochemistry of Pancreas The tissues werelater paraffin embedded and blocks were prepared usingHistocentre 3 (UK) The sections were deparaffinized byincubating the sections with xylene for 10min each forthree times Then sections were dehydrated with decreasinggrades of alcohol (90 70 and 50) Antigen retrieval wasdone by boiling the slides in 01M citrate buffer pH 6 for20min The endogenous peroxidase activity was quenchedby incubating the slides in 3 H

2O2for 30min followed by

twowasheswith PBS up to 5min each To prevent nonspecificbinding of the antibody blocking was done by incubating theslides in 10 normal goat serum in PBS at room temperaturefor 1 h Slides were incubated overnight at 4∘C with anti-rat polyclonal insulin primary antibody raised in rabbit inPBS containing 1 normal goat serum Slides were washedwith PBS and incubated further with biotinylated anti-rabbitsecondary antibody for 30min followed by incubation withVECTASTAIN Elite ABC reagent (three-layer streptavidin-biotin peroxidase complex staining method Vector Lab-oratories) Sections were developed using 3 H

2O2and

diaminobenzidine tetrahydrochloride (DAB) and observedunder microscope Results were expressed in the form ofaverage percent of insulin positive cells in the islets

210 Lens Weight and Protein Content A 10 homogenatewas made from 4 to 5 pooled lenses in 50mM phosphatebuffer pH 74 Before centrifugation a set of aliquots weremade for estimation of total protein lipid peroxidation andsorbitol The remaining total homogenate was centrifuged at10000timesg for 30min at 4∘C The supernatant was referredto as the soluble fraction The pellet was washed twice with50mM phosphate buffer pH 74 and the wash was pooledwith the supernatant Total and soluble protein content wasestimated with the Lowry method and the percentage ofsoluble protein was calculated

211 Measurement of Polyol Pathway Intermediates Thestatus of the polyol pathway in the eye lens of controlexperimental rats was assessed by analyzing the activity ofaldose reductase (AR) and sorbitol levels The activity of ARand sorbitol levels were assayed according to the methodsreported earlier [21 22]

212 Oxidative Stress Lens lipid peroxidation was mea-sured as thiobarbituric acid reacting substances (TBARS)and protein carbonyl content was determined based ontheir reactivity with 24-dinitrophenylhydrazine accordingto reported methods [21 22 44] In brief malondialdehydeis the end product of lipid peroxidation of polyunsaturatedfatty acids Malondialdehyde was estimated by utilizing itsreactivity with 2-thiobarbituric acid resulting in a pink

coloured condensation product a trimethinewhichwasmea-sured spectrophotometrically at 533 nm The carbonyls reactwith 24-dinitrophenylhydrazine to formprotein hydrozoneswhich can be detected and quantified spectrophotometricallyat 365 nm

213 Antioxidant Enzymes The activities of antioxidantenzymes superoxide dismutase (SOD) glutathione S-trans-ferase (GST) and glutathione peroxidase (GPx) were assayedspectrophotometrically according to the reported methods[21 22 44] The assay of SOD is based on the ability of theenzyme to inhibit the autoxidation of pyrogallol The rate ofautoxidation of pyrogallol is measured following the changein absorbance at 420 nm GST catalyzes the conjugation oftoxic electrophilic compounds with glutathione During thisassay formation of 1-chloro-24-dinitrobenzene-glutathioneconjugate was monitored at 340 nm GPx catalyzes theoxidation of reduced glutathione by hydrogen peroxide orlipid peroxides to oxidized glutathione (GSSG) The rateof GSSG formation a measure of enzyme activity wasmonitored coupling with the glutathione reductase reactionwhere NADPH oxidation was followed at 340 nm

214 Statistical Analysis One-way ANOVA was used fortesting statistical significance between groups of data andindividual pair difference was tested by means of Duncanrsquosmultiple-range test Heterogeneity of variance was testedby the nonparametric Mann-Whitney test 119875 lt 005 wasconsidered significant

3 Results

31 Induction of IGT orDiabetes After 8weeks experimental(STZ injected) rats were assessed for the development ofhyperglycemia by estimating fasting blood glucose nSTZrats were subdivided into two groups depending upontheir fasting plasma glucose levels Those without fastinghyperglycemia (lt110mgdL) were considered as prediabetic(nSTZ-PD 119899 = 16) and those with fasting hyperglycemia(gt110mgdL) were considered as diabetic (nSTZ-D 119899 = 7)The experiment was continued for another 5 months (total 7months) for development of cataract

32 General Characteristics The characteristics of controland nSTZ animals are shown in Table 1 There was anincreased food intake in nSTZ-D rats when compared withcontrol Despite increased food intake the body weight ofnSTZ-D animals was decreased when compared with thecontrol rats Though there was no significant difference infood intake of nSTZ-PD rats the body weights of these ani-mals were decreased when compared with control (Table 1)

33 Blood Glucose and Insulin Fasting blood glucose levelsof nSTZ-PD as well as nSTZ-D animals were comparable atthe age of one month However at the age of two monthsonly 30 of the all STZ injected rats (7 out of 23) developedhyperglycemia and they continued to be in hyperglycemic


Table 1 Characteristics of control nSTZ-PD and nSTZ-D group rats

Parametergroup Control (119899 = 7) nSTZ-PD (119899 = 16) nSTZ-D (119899 = 7)Food intake (gdayrat) 2069 plusmn 221 1908 plusmn 184 2605 plusmn 468

Body weight (grat) 448 plusmn 4039 394 plusmn 4736 364 plusmn 8600lowast

HOMA-IR 0740 plusmn 0218 0376 plusmn 0243lowast 0582 plusmn 0176

Glucose AUC (mmolh) 884 plusmn 085 2840 plusmn 298lowast 3638 plusmn 418lowast

Insulin AUC (120583molh) 6925 plusmn 1307 4045 plusmn 996lowast 4061 plusmn 898lowast

Glucose AUCinsulin AUC 0130 plusmn 0015 0744 plusmn 0269lowast 0944 plusmn 0315lowast

Values are expressed as mean plusmn SD The asterisk denotes that data are significantly different from control group and the hash mark denotes that data aresignificantly different from nSTZ-PD group

state throughout the experiment Interestingly the remaining70 (17) rats showed normal fasting glucose at the age oftwo months and they maintained the normal fasting glucoseeven up to the age of seven months (Figure 1) As expected asignificant (119875 lt 0001) decrease in insulin levelswas observedin nSTZ-D rats when compared to controls Interestinglyin spite of significantly (119875 lt 0001) low levels of insulinnSTZ-PD rats exhibited IGT or prediabetic characteristics(Figure 1) However the levels of insulin in nSTZ-PD ratsare higher than that of nSTZ-D rats particularly at 7-month period Further these results are in accordance withpercentage of insulin positive cells in pancreas of theseanimals as evidenced by immunohistochemistry at the endof the seven months While there is a 50 reduction in theinsulin levels as well as percentage of insulin positive cells innSTZ-PD rats insulin levels and insulin positive cells wereabout 20 in nSTZ-D rats as compared to controls (Figures 1and 2)

34 Postprandial Glucose Postprandial glucose is an earlyindicator of prediabetes and impaired glucose metabolismPostprandial glucose was measured at two months and mostof the nSTZ-PD rats had high postprandial glucose levels(227 plusmn 103mgdL) when compared with control rats (140 plusmn416mgdL) indicating that majority of these rats are at IGTstate

35 Glucose Tolerance and Insulin Response Impairment ofglucose tolerance in animals was assessed by OGTT con-ducted at the age of two months nSTZ-PD group animalshave shown the development of IGT at the age of twomonthsas evidenced by significantly (119875 lt 0001) high levels of plasmaglucose at two-hour time point during OGTT Also thegreater area under curve for glucose in OGTT test comparedto control further support the IGT characteristic of nSTZ-PDanimals However in case of nSTZ-D animals the two-hourglucose levels as well as glucose area under curve were morethan that of control as a consequence of its higher fastingglucose levels (Figure 3) There is a decrease in the AUC forinsulin in both nSTZ-PD and nSTZ-D rats (Figure 3)

36 Insulin Resistance and Type 2 Diabetes Insulin insen-sitivity is a hallmark of T2D [45] and degree of insulinresistance was calculated by HOMA-IR A significant (119875 lt001) decrease in HOMA-IR index was observed in nSTZ-PD rats when compared with controls indicating that these

0

4

8

12

16

1 2 3 4 5 6 7

(mm

olL

)

Duration (months)

ControlnSTZ-PDnSTZ-D

lowast lowast

(a)

0

10

20

30

40

50

Control nSTZ-PD nSTZ-D2 months7 months

lowast

lowastlowast

(120583U

mL)

(b)

Figure 1 Plasma glucose and insulin levels during experimentFasting glucose levels (mgdL)were estimated once in aweek Valuesrepresent mean plusmn SD (119899 = 7ndash16) The asterisk denotes that data aresignificantly different from control group and hash mark denotessignificantly different from nSTZ-PD group

rats did not develop insulin resistance As the fasting glucoselevels are more in nSTZ-D rats these rats exhibited slightlyhigher HOMA-IR index than nSTZ-PD animals Howeverthis increased HOMA-IR for an nSTZ-D animal was notbecause of increased insulin resistance but because HOMA-IR is not a valid index during hyperglycemic stage Further


Control nSTZ-PD nSTZ-DNegative control

100x

400x

(a)

0

20

40

60

80

()

100

Control nSTZ-PD nSTZ-D

lowast

lowast

(b)

Figure 2 Immunohistochemistry of insulin positive cells in pancreas Pancreatic beta cells were identified by insulinmonoclonal antibody (a)and the insulin positive cells were counted by Leica Laser Microdissection microscope (b) Arrow indicates isletThe values are mentioned aspercentage beta cells against total islets cells (b) Values represent mean plusmn SD (119899 = 3) The asterisk denotes that data are significantly differentfrom control group and hash mark denotes significantly different from nSTZ-PD group

there was a significant (119875 lt 0001) increase in glucose toinsulin ratio in nSTZ groups when compared with controlanimals (Table 1) supporting that the nSTZ-PD group ani-mals did not develop insulin resistance However as indicatedby glucose tolerance test nSTZ-PD animals developed IGT

37 Slit-Lamp Examination and Cataract Progression Eyeswere examined weekly for the development of lens opacityfrom the age of one month till the end of the experimentAs expected the onset of cataract was observed at 9 weeksin nSTZ-D rats However it should be noted that theprogression of cataract was very slow when compared to ourprevious observations with T1D rats upon injection of STZto adult rats where the onset of cataract was between 3 and 4weeks after diabetes induction and developedmature cataractby 8-9 weeks [21 22 29] Though nSTZ-PD rats showed IGTthey did not develop lens opacification even at the age ofseven months (Figure 4)

38 LensWeight and Protein Content Aggregation of solublelens proteins and increased insolubilization of proteins isone of the major biochemical alterations leading to catarac-togenesis Therefore we determined the total soluble andpercentage of soluble protein content in the lens (Table 2)

There were a significant (119875 lt 005) reduction in lens weightin nSTZ-PD and further reduction in nSTZ-D animals whencompared with controls While soluble protein content innSTZ-PD rats was marginally decreased it was found tobe significantly increased in nSTZ-D rats due to maturedcataract as compared to control (Table 2)

39 Polyol Pathway Activation of polyol pathway has beenlinked to several diabetic complications Aldose reductase(AR) a key enzyme of polyol pathway converts excessglucose to sorbitol the accumulation of which is associatedwithmany secondary complications including cataract In thepresent study while the activity of AR was marginally highin the lens of nSTZ-PD rats the activity was significantlyhigh in nSTZ-D rats compared to control rats (Table 3)As expected significantly increased levels of sorbitol wereobserved in nSTZ-D rat lens due to hyperglycemia and highARactivity (Table 3)Though therewas amarginal increase inAR activity in nSTZ-PD rats when compared with controlsthere was a significant increase of sorbitol accumulation inthe lens indicating that IGT state also leads to accumulationof intracellular sorbitol which further supported our previousresults [20]


0

5

10

15

20

25

30

35

0 30 60 120

Glu

cose

(mm

olL

)

Time (min)


lowast

lowastlowast

(a)


0

10

20

30

40

50

60

70

0 30 60 120

Time (min)

lowast

lowast

lowast

Insu

lin (120583

Um

in)

(b)

Figure 3 Glucose and insulin response during OGTT at twomonths of age Plasma glucose (a) and insulin (b) levels in responseto an oral glucose challenge in 2-month-old STZ injected (119899 = 7ndash16)and control (119899 = 7) SD rats following a 16 h fast Values representmean plusmn SD The asterisk denotes that data are significantly differentfrom control group

310 Oxidative Stress Oxidative stress is known to play animportant role in the development of various complicationsduring diabetic and prediabetic states In the present studymarginal increase in lipid peroxidation was evidenced byMDA levels whereas significant increase in protein carbonylcontent was observed in nSTZ-PD rats as compared tocontrol indicating an increased oxidative stress in formergroup However as expected both these parameters werefound to be significantly high in case of nSTZ-D animals(Figure 5)

311 Antioxidant Parameters There was a marginal increasein the activity of antioxidant enzymes such as superox-ide dismutase (SOD) glutathione peroxidase (GPx) andglutathione S-transferase (GST) in nSTZ-PD rats whencompared to controls (Table 3) This indicates that IGT isassociated with increased oxidative stress in lens However

0

1

2

3

4

1 2 3 4 5 6

Stag

es o

f cat

arac

t

Duration (months)


Control nSTZ-PD nSTZ-Dlowast

Figure 4 Cataract development in nSTZ induced diabetic ratsCataract formation was monitored weekly by slit-lamp microscopeand the stage of cataract was scored according to the classificationdescribed in Section 2 Stages of cataract in each group wereaveraged at the given time and plotted as a function of timeRepresentative photographs of lens from each group at the end ofexperiment are shown in the inset Values represent the mean plusmn SDThe asterisk denotes that data are significantly different from controlgroup

nSTZ-D rats exhibited significantly higher levels of GPx andGST activities (Figure 6) This indicates that oxidative stresswas further enhanced by hyperglycemia

4 Discussion

Theprogression of T2Dbeginswith an impairment of glucosetolerance [37 46] and is often associatedwith a state of insulinresistance Several animal models have been developed toinvestigate T2D or IGT associated pathophysiology nSTZmodel is one of the frequently used animal models of T2Dor IGT [37ndash40 47] and associated complications particularlyCVD [30 48] In the present study we evaluated this modelfor development of cataract and mechanisms responsiblefor cataractogenesis According to the previous reports STZtreated neonatal rats exhibit slightly elevated plasma glucoselevels and low pancreatic insulin content at adulthood [4749ndash52] Though in our previous study we did not observethe development of diabetic characteristics in nSTZ inducedWistar-NIN rats [20] the present study with SD rats hasshown the development of IGT in majority (70) of animalsInterestingly though all the nSTZ-PD exhibited lower fastinginsulin levels they showed normal fasting glucose through-out the experimental period

The development of IGT insulin resistance or diabeticcharacteristics in rats depends on the STZdose age durationand strain of the rats [20 30 47 48 52ndash57] Earlier ithas been reported that the intraperitoneal injection of STZ(90mg kgminus1) in two-day-old SD rat pups developed hyper-glycemia and insulin resistance at adult age [53] Howeverin the present study none of the animals developed insulinresistance butmajority of animals showed the development of


Table 2 Lens weight and protein content

Parametergroup Control (119899 = 7) nSTZ-PD (119899 = 16) nSTZ-D (119899 = 7)Lens weight (mglens) 5228 plusmn 402 4820 plusmn 297lowast 4277 plusmn 254lowast

Total protein (mgg lens) 435 plusmn 72 489 plusmn 100 459 plusmn 1455

Soluble protein (mgg lens) 305 plusmn 32 329 plusmn 16lowast 279 plusmn 583lowast

Percent of soluble protein 7011 6728 6078Values are expressed as mean plusmn SD The asterisk denotes that data are significantly different from control group and the hash mark denotes that data aresignificantly different from nSTZ-PD group

0

3

6

9

12

15

18


(nm

olg

lens

)

MDA

0

2

4

6

8

10


(nm

olm

g pr

otei

n)

Protein carbonyls

lowast

lowast lowast

Figure 5 Lipid peroxidation (MDA) and protein carbonyl contentin rat lens Values are mean plusmn SD (119899 = 3) The asterisk denotes thatdata are significantly different from control group

Table 3 Polyol pathway parameters

Parametergroup Control (119899 = 7) nSTZ-PD (119899 = 16) nSTZ-D (119899 = 7)

AR activity 3820 plusmn 1037 3840 plusmn 1045 4291 plusmn 874

Sorbitol 0310 plusmn 0026 0533 plusmn 0155lowast 8738 plusmn 0131lowast

AR activity was expressed as 120583moles of NADPH oxidizedh100mg proteinSorbitol was expressed as 120583molesg lens Values are expressed as mean plusmn SDThe asterisk denotes that data are significantly different from control andhashmark denotes that data are significantly different from nSTZ-PD group

IGT whereas the remaining developed frank hyperglycemiaThese results correlated with previous reports [47 49ndash52]The major findings from this study are that though 30of rats developed hyperglycemia around 2 months of ageand continued to be in hyperglycemic state throughout theexperimental period remaining 70 of rats developed IGTand remained in IGT state till the end of the experimentwithout developing hyperglycemia Indeed this situation has

0

10

20

30

40

50


SOD

0

10

20

30

40

50

60


GST

0

5

10

15

20

25

30


GPx

lowast

lowast

(Uni

tsm

in100

mg

prot

ein)

(120583m

ol o

f NA

DPH

oxi

dise

dh

100

mg

prot

ein)

(120583m

ol o

f CD

NB-

GSH

oxi

dise

dh

100

mg

prot

ein)

Figure 6 Activities of antioxidant enzymes superoxide dismutase(SOD) glutathione peroxidase (GPx) and glutathione S-transferase(GST) in lens protein Values are mean plusmn SD (119899 = 3) The asteriskdenotes that data are significantly different from control group

provided us an opportunity to investigate the impact ofprediabetes or IGT on lens biochemistry vis-a-vis cataractdevelopment

Thus we have evaluated the development of cataract andassociated biochemical alterations in these rats The onset of


cataract in nSTZ-D animals was observed after eight weeks ofage and itmatured by 24weeksThe interesting finding of thisstudywas that the progression of cataract in these animalswasvery slow when compared to our previous reports on T1D ratmodels [21 22] However the remaining 70 animals withIGT did not develop any lenticular opacification during thestudy period and their lenses were apparently clear

In general activation of polyol pathway nonenzymaticglycation and oxidative stress are three major pathwaysthat contribute significantly in the pathogenesis of diabeticcomplications including cataract [58] We have also analyzedthe lenses of these rats to study the possible associationof IGT with lens abnormalities As expected there was anincreased activity of AR along with increased accumulationof polyols in the nSTZ-D lens indicating the activationof polyol pathway Similarly there were an increased lipidperoxidation protein carbonyls and altered SOD activityindicating the involvement of oxidative stress in lenses ofnSTZ-D rats when compared with controls However we didnot observe nonenzymatic glycation as assessed bymeasuringAGE fluorescence in lens protein fraction (data not shown)We also evaluated the pathways in prediabeticIGT (nSTZ-PD) rat lens Interestingly in accordance with our previ-ous study [20] we observed a marginal increase in polyoland SOD indicating the activation of polyol pathway andincreased oxidative stress in these animals However thismarginal increase of polyol may not be sufficient to developlens opacification in these nSTZ-PD rats but may be respon-sible for early changes in lens biochemical parameters whichmay subsequently lead to cataract onset and progressionHowever long term studies are needed to study the actualimpact of IGT on eye lens

In conclusion though one-fourth of nSTZ injected ratsdeveloped diabetes and subsequently cataract majority ofthem developed prediabetic (IGT) state and also showedearly lens biochemical changes which in due course maylead to cataractogenesisOxidative stress alongwith increasedpolyols was found to play a major role in the developmentof early lens abnormalities even in the IGT state HencenSTZ-PD rat model can be used to study IGT associatedcomplications and to develop preventive strategies

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Madhoosudan A Patil and Palla Suryanarayana contributedequally to this paper

Acknowledgment

Part of this work is supported by the Department of Sci-ence and Technology (DST) and Indian Council of MedicalResearch (ICMR) Government of India Madhoosudan APatil received ICMR Senior Research Fellowship

References

[1] N Bottini T Vang F Cucca and T Mustelin ldquoRole of PTPN22in type 1 diabetes and other autoimmune diseasesrdquo Seminars inImmunology vol 18 no 4 pp 207ndash213 2006

[2] R E Warren ldquoThe stepwise approach to the management oftype 2 diabetesrdquoDiabetes Research and Clinical Practice vol 65supplement 1 pp S3ndashS8 2004

[3] IDF Diabetes Atlas International Diabetes Federation 6thedition 2013

[4] A Coughlon D J McCorty L N Jorgensen and P ZimmetldquoThe epidemic of NIDDM in Asian and Pacific Island popu-lations prevalence and risk factorsrdquo Hormone and MetabolicResearch vol 29 no 7 pp 323ndash331 1997

[5] P S Quoc M A Charles N H Cuong et al ldquoBlood glucosedistribution and prevalence of diabetes in Hanoi (Vietnam)rdquoAmerican Journal of Epidemiology vol 139 no 7 pp 713ndash7221994

[6] A Ramachandran C Snehalatha P Shyamala V Vijay andM Viswanathan ldquoHigh prevalence of NIDDM and IGT inan elderly south Indian population with low rates of obesityrdquoDiabetes Care vol 17 no 10 pp 1190ndash1192 1994

[7] S Resnikoff D Pascolini D Etyarsquoale et al ldquoGlobal data onvisual impairment in the year 2002rdquoBulletin of theWorldHealthOrganization vol 82 no 11 pp 844ndash851 2004

[8] N G Congdon D S Friedman and T Lietman ldquoImportantcauses of visual impairment in the world todayrdquo Journal of theAmerican Medical Association vol 290 no 15 pp 2057ndash20602003

[9] P E Stanga S R Boyd andAM PHamilton ldquoOcularmanifes-tations of diabetesmellitusrdquoCurrent Opinion inOphthalmologyvol 10 no 6 pp 483ndash489 1999

[10] A Spector ldquoReview oxidative stress and diseaserdquo Journal ofOcular Pharmacology and Therapeutics vol 16 no 2 pp 193ndash201 2000

[11] M Brownlee ldquoBiochemistry and molecular cell biology ofdiabetic complicationsrdquo Nature vol 414 no 6865 pp 813ndash8202001

[12] M M Gabir R L Hanson D Dabelea et al ldquoPlasma glucoseand prediction of microvascular disease and mortality evalu-ation of 1997 American Diabetes Association and 1999 WorldHealth Organization criteria for diagnosis of diabetsrdquo DiabetesCare vol 23 no 8 pp 1113ndash1118 2000

[13] A Y W Lee and S S M Chung ldquoContributions of polyolpathway to oxidative stress in diabetic cataractrdquo The FASEBJournal vol 13 no 1 pp 23ndash30 1999

[14] S S M Chung E C M Ho K S L Lam and S K ChungldquoContribution of polyol pathway to diabetes-induced oxidativestressrdquo Journal of the American Society of Nephrology vol 14 no3 pp S233ndashS236 2003

[15] Y Su X-M Liu Y-M Sun et al ldquoThe relationship betweenendothelial dysfunction and oxidative stress in diabetes andprediabetesrdquo International Journal of Clinical Practice vol 62no 6 pp 877ndash882 2008

[16] W N Dugmore and K Tun ldquoGlucose tolerance tests in 200patients with senile cataractrdquo British Journal of Ophthalmologyvol 64 no 9 pp 689ndash692 1980

[17] S Saxena P Mitchell and E Rochtchina ldquoFive-year incidenceof cataract in older persons with diabetes and pre-diabetesrdquoOphthalmic Epidemiology vol 11 no 4 pp 271ndash277 2004


[18] P Y Reddy N V Giridharan N Balakrishna V ValidandiR Pullakhandam and G B Reddy ldquoIncreased risk of cataractdevelopment in WNIN-obese rats due to accumulation ofintralenticular sorbitolrdquo IUBMB Life vol 65 no 5 pp 472ndash4782013

[19] P Y Reddy N V Giridharan and G B Reddy ldquoActivation ofsorbitol pathway in metabolic syndrome and increased suscep-tibility to cataract in Wistar-Obese ratsrdquo Molecular Vision vol18 pp 495ndash503 2012

[20] P Suryanarayana M A Patil and G B Reddy ldquoInsulinresistance mediated biochemical alterations in eye lens ofneonatal streptozotocin-induced diabetic ratrdquo Indian Journal ofExperimental Biology vol 49 no 10 pp 749ndash755 2011

[21] P Suryanarayana M Saraswat T Mrudula T P Krishna KKrishnaswamy andG B Reddy ldquoCurcumin and turmeric delaystreptozotocin-induced diabetic cataract in ratsrdquo InvestigativeOphthalmology andVisual Science vol 46 no 6 pp 2092ndash20992005

[22] P Suryanarayana M Saraswat M Petrash and G B ReddyldquoEmblica officinalis and its enriched tannoids delay strepto-zotocin-induced diabetic cataract in ratsrdquoMolecular Vision vol13 pp 1291ndash1297 2007

[23] P Suryanarayana A Satyanarayana N Balakrishna P UKumar and G B Reddy ldquoEffect of turmeric and curcuminon oxidative stress and antioxidant enzymes in streptozotocin-induced diabetic ratrdquoMedical ScienceMonitor vol 13 no 12 ppBR286ndashBR292 2007

[24] S Zhang F Y Chai H Yan Y Guo and J J Harding ldquoEffectsof N-acetylcysteine and glutathione ethyl ester drops on strep-tozotocin-induced diabetic cataract in ratsrdquo Molecular Visionvol 14 pp 862ndash870 2008

[25] V R Drel W Xu J Zhang et al ldquoPoly(adenosine 51015840-diphos-phate-ribose) polymerase inhibition counteracts multiple man-ifestations of experimental type 1 diabetic nephropathyrdquoEndocrinology vol 150 no 12 pp 5273ndash5283 2009

[26] T A Siddiqui Z Shadab I Nishat N Ayasha Z Zehraand S H Alavi ldquoAnticataract activity of Kohl-Chikni Dawamdasha compound ophthalmic formulation of Unani medicine inalloxan-diabetic ratsrdquo Journal of Ethnopharmacology vol 86no 1 pp 109ndash112 2003

[27] V Vats S P Yadav and J K Grover ldquoEthanolic extract ofOcimum sanctum leaves partially attenuates streptozotocin-induced alterations in glycogen content and carbohydratemetabolism in ratsrdquo Journal of Ethnopharmacology vol 90 no1 pp 155ndash160 2004

[28] A Preet B L Gupta M R Siddiqui P K Yadava and NZ Baquer ldquoRestoration of ultrastructural and biochemicalchanges in alloxan-induced diabetic rat sciatic nerve on treat-ment with Na

3

VO4

and Trigonellamdasha promising antidiabeticagentrdquoMolecular and Cellular Biochemistry vol 278 no 1-2 pp21ndash31 2005

[29] M Saraswat P Suryanarayana P Y Reddy M A Patil NBalakrishna and G B Reddy ldquoAntiglycating potential of Zin-giber officinalis and delay of diabetic cataract in ratsrdquoMolecularVision vol 16 pp 1525ndash1537 2010

[30] SW Schaffer andG LWilson ldquoInsulin resistance andmechan-ical dysfunction in hearts of Wistar rats with streptozotocin-induced non-insulin-dependent diabetes mellitusrdquo Diabetolo-gia vol 36 no 3 pp 195ndash199 1993

[31] J Takada M A Machado S B Peres et al ldquoNeonatal strepto-zotocin-induced diabetes mellitus a model of insulin resistance

associated with loss of adipose massrdquoMetabolism vol 56 no 7pp 977ndash984 2007

[32] T Anwer M Sharma K K Pillai andM Iqbal ldquoEffect ofWith-ania somnifera on insulin sensitivity in non-insulin- dependentdiabetes mellitus ratsrdquo Basic and Clinical Pharmacology andToxicology vol 102 no 6 pp 498ndash503 2008

[33] M Abdollahi A B Zuki Y M Goh A Rezaeizadeh andM M Noordin ldquoEffects of Momordica charantia on pancre-atic histopathological changes associated with streptozotocin-induced diabetes in neonatal ratsrdquo Histology and Histopathol-ogy vol 26 no 1 pp 13ndash21 2010

[34] J Movassat and B Portha ldquoEarly administration of ker-atinocyte growth factor improves120573-cell regeneration in rat withstreptozotocin-induced diabetesrdquo Journal of Endocrinology vol195 no 2 pp 333ndash340 2007

[35] B Portha C Levacher L Picon andG Rosselin ldquoDiabetogeniceffect of streptozotocin in the rat during the perinatal periodrdquoDiabetes vol 23 no 11 pp 889ndash895 1974

[36] G C Weir E T Clore C J Zmachinski and S Bonner-WeirldquoIslet secretion in a new experimental model for non-insulin-dependent diabetesrdquo Diabetes vol 30 no 7 pp 590ndash595 1981

[37] M Iwase M Kikuchi K Nunoi et al ldquoA new model of type2 (non-insulin-dependent) diabetes mellitus in spontaneouslyhypertensive rats diabetes induced by neonatal streptozotocintreatmentrdquo Diabetologia vol 29 no 11 pp 808ndash811 1986

[38] V Grill M Westberg and C-G Ostenson ldquoB cell insensitivityin a rat model of non-insulin-dependent diabetes Evidencefor a rapidly reversible effect of previous hyperglycemiardquo TheJournal of Clinical Investigation vol 80 no 3 pp 664ndash669 1987

[39] N Welsh and C Hellerstrom ldquoIn vitro restoration of insulinproduction in islets from adult rats treated neonatally withstreptozotocinrdquo Endocrinology vol 126 no 4 pp 1842ndash18481990

[40] S J Hemmings and D Spafford ldquoNeonatal STZ model of typeII diabetes mellitus in the Fischer 344 rat characteristics andassessment of the status of the hepatic adrenergic receptorsrdquoTheInternational Journal of Biochemistry amp Cell Biology vol 32 no8 pp 905ndash919 2000

[41] M Farswan P M Mazumder and V Percha ldquoProtective effectof Cassia glauca Linn on the serum glucose and hepaticenzymes level in streptozotocin induced NIDDM in ratsrdquoIndian Journal of Pharmacology vol 41 no 1 pp 19ndash22 2009

[42] J-O Kim G-D Lee J-H Kwon and K-S Kim ldquoAnti-diabeticeffects of new herbal formula in neonatally streptozotocin-induced diabetic ratsrdquoBiological amp Pharmaceutical Bulletin vol32 no 3 pp 421ndash426 2009

[43] L Pari and N Ashokkumar ldquoEffect of N-benzoyl-119863-phenylalanine on lipid profile in liver of neonatal streptozotocindiabetic ratsrdquo Fundamental amp Clinical Pharmacology vol 19no 5 pp 563ndash568 2005

[44] P Suryanarayana K Krishnaswamy and G BhanuprakashReddy ldquoEffect of curcumin on galactose-induced cataractoge-nesis in ratsrdquoMolecular Vision vol 9 pp 223ndash230 2003

[45] P Song YWu J Xu et al ldquoReactive nitrogen species induced byhyperglycemia suppresses Akt signaling and triggers apoptosisby upregulating phosphatase PTEN (phosphatase and tensinhomologue deleted on chromosome 10) in an LKB1-dependentmannerrdquo Circulation vol 116 no 14 pp 1585ndash1595 2007

[46] P Zimmet and C R Thomas ldquoGenotype obesity and car-diovascular diseasemdashhas technical and social advancementoutstripped evolutionrdquo Journal of Internal Medicine vol 254no 2 pp 114ndash125 2003


[47] D K Arulmozhi A Veeranjaneyulu and S L BodhankarldquoNeonatal streptozotocin-induced rat model of type 2 diabetesmellitus a glancerdquo Indian Journal of Pharmacology vol 36 no4 pp 217ndash221 2004

[48] U A Shinde and R K Goyal ldquoEffect of chromiumpicolinate onhistopathological alterations in STZ and neonatal STZ diabeticratsrdquo Journal of Cellular and Molecular Medicine vol 7 no 3pp 322ndash329 2003

[49] O Blondel D Bailbe and B Portha ldquoRelation of insulindeficiency to impaired insulin action inNIDDMadult rats givenstreptozocin as neonatesrdquo Diabetes vol 38 no 5 pp 610ndash6171989

[50] K Inoue M Cetkovic-Cvrlje D L Eizirik and V GrillldquoIrreversible loss of normal beta-cell reguation by glucose inneonatally streptozotocin diabetic ratsrdquoDiabetologia vol 37 no4 pp 351ndash357 1994

[51] C Tourrel D Bailbe M-J Meile M Kergoat and B PorthaldquoGlucagon-like peptide-1 and exendin-4 stimulate beta-cellneogenesis in streptozotocin-treated newborn rats resultingin persistently improved glucose homeostasis at adult agerdquoDiabetes vol 50 no 7 pp 1562ndash1570 2001

[52] M-H Giroix A B Nadi A Sener B Portha and W JMalaisse ldquoMetabolism of D-[3-3H]glucose D-[5-3H]glucoseD-[U-14C]glucose D-[1-14C]glucose and D-[6-14C]glucose inpancreatic islets in an animal model of type-2 diabetesrdquo Inter-national Journal ofMolecularMedicine vol 9 no 4 pp 381ndash3842002

[53] B RGoyal K Parmar R KGoyal andAAMehta ldquoBeneficialrole of telmisartan on cardiovascular complications associatedwith STZ-induced type 2 diabetes in ratsrdquo PharmacologicalReports vol 63 no 4 pp 956ndash966 2011

[54] C A M de Oliveira E Luciano and M A R de Mello ldquoTherole of exercise on long-term effects of alloxan administered inneonatal ratsrdquo Experimental Physiology vol 90 no 1 pp 79ndash862005

[55] C Ribeiro C A M De Oliveira E Luciano and M A R DeMello ldquoDiabetes evolution in rats after neonatal treatment withalloxanrdquo Research Communications in Molecular Pathology andPharmacology vol 117-118 pp 29ndash46 2005

[56] J L Sartoretto G A N Melo M H C Carvalho et al ldquoMet-formin treatment restores the altered microvascular reactivityin neonatal streptozotocin-induced diabetic rats increasingNOS activity but not NOS expressionrdquo Life Sciences vol 77 no21 pp 2676ndash2689 2005

[57] S W Schaffer C Ballard and M S Mozaffari ldquoIs there a linkbetween impaired glucose metabolism and protein kinase Cactivity in the diabetic heartrdquoMolecular and Cellular Biochem-istry vol 176 no 1-2 pp 219ndash225 1997

[58] M Polak R S Newfield P Fioretto P Czernichow and RMarchase ldquoPathophysiology of diabetic complicationsrdquo Dia-betologia vol 40 no 3 pp B65ndashB67 1997

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


lenses and nerves of diabetic mice and it was reported thataldose reductase inhibitor reducesoxidative stress enhancementin sugar cataract [14] The polyol pathway may be relatedto hyperglycemia induced oxidative stress and there maybe a metabolic connection between the polyol pathway andoxidative stress Furthermore carbonyl stress has been foundto be the cause for advanced glycation end product toxicityindicating the involvement of nonenzymatic glycation in thedevelopment of oxidative stress

Type 2 diabetes (T2D) always precedes with prediabeticconditions such as impaired glucose tolerance (IGT) orimpaired fasting glucose (IFG) [6] Increased oxidative stresswas found in subjects with IGT and IFG [15] Interestinglyseveral recent large population studies reported that IGT orprediabetes is also associated with various types of cataracts[16 17]There are three principle mechanisms through whichlens can be damaged resulting in the development of cataract(oxidative stress osmotic stress andnonenzymatic glycation)and IGT might influence any or all of these physiologicalprocesses However no experimental studies have explainedthe association between IGT and cataract along with theirplausible pathophysiological mechanisms Previous studiesfrom our group have shown that obesity [18 19] as well asprediabetes [20] is associated with increased susceptibility tocataract formation due to the activation of sorbitol pathway inexperimental animalsThus there is a need to understand themechanism of cataractogenesis during IGT as well as T2D Inthis context availability of a suitable animalmodel that showsincreased susceptibility to IGT-induced cataractogenesis is ofimmense value

There are several animal models available to study themechanisms of diabetic complications including diabeticcataract However most of the studies on diabetic cataractin animal models are largely restricted to type 1 diabetic(T1D) conditions [21ndash23] In this context streptozotocin(STZ) [21 24 25] or alloxan-induced diabetic models [26ndash28] are extensively used to study the diabetic cataract andboth these models mimic the T1D in humans Studies fromour laboratory and elsewhere indicate that oxidative stressappears to be a major factor in the development of cataractin T1D along with the activation of polyol pathway andnonenzymatic glycation [21 22 29]

Neonatal-STZ (nSTZ) induced rat model is one of themost frequently used T2D-likemodels [30ndash33] whichmimicshuman diabetes [34] Previous studies on this model werefound to have characteristics of pancreatic beta-cell destruc-tion followed by beta-cell regeneration and glucose intoler-ance [35 36] Subsequently other authors confirmed thesefindings and showed that nSTZ treated rats in adulthooddisplay the typical characteristics of T2D [37ndash40]This animalmodel is mainly used to screen hypoglycemic or antidiabeticagents [32 41 42] and also for hypolipidemic and oxidativestress related studies [43] However no studies attempted touse this model for investigating the biochemical abnormal-ities in the lens and development of cataract Therefore inthe present study we evaluated nSTZ model for developmentof cataract due to IGT or T2D and associated biochemicalalterations in lens in rodent model

2 Materials and Methods

21 Materials Streptozotocin (STZ) NADPH DL-glyceral-dehyde lithium sulfate 120573-mercaptoethanol bovine serumalbumin sorbitol andsorbitol dehydrogenase tetraethoxypro-pane thiobarbituric acid NAD and pyrogallol were pur-chased from Sigma Chemical Company (St Louis USA) Allother chemicals were of analytical grade and were obtainedfrom local companies

22 Experimental Design Two-day-old male Sprague Daw-ley (SD) rat pups obtained from the National Center forLaboratory Animal Sciences National Institute of Nutri-tion Hyderabad India were injected intraperitoneally with90mgkg body weight STZ dissolved in 01M citrate bufferpH 45 (119899 = 23) Control pups (119899 = 7) received only vehicleThe pups were weaned after 21 days and maintained on AIN-93GM diet in individual cages throughout the experimentalperiod

23 Animal Care Animal care and protocols were in accor-dance with and approved by the Institutional Animal EthicsCommittee (IAEC) Animals were housed in individual cagesin a temperature and humidity controlled room with a 12 hL D cycle All these animals had free access to water Asso-ciation for Research in Vision and Ophthalmology (ARVO)statement for the use of animals in ophthalmic and visionresearch was followed

24 Oral Glucose Tolerance Test Oral glucose tolerance test(OGTT) was performed at the age of two months on ratsfasted for 16 h by administering glucose orally as a bolus at adose of 20 gkg body wt Blood samples were collected fromretroorbital plexus at 0 30 60 and 120min for determiningplasma glucose and insulin concentrations for assessingimpaired glucose tolerance (IGT) and insulin resistance

25 Homeostasis Model Assessment (HOMA) Insulin resis-tance was assessed by homeostasis model assessment HOMAas described earlier for rats [20] using the following equationHOMA-IR = [fasting plasma glucose (mgdL) times fastingplasma insulin (120583UmL)2430] To assess insulin response ofthese rats to a challenge of glucose load the area under thecurve (AUC) for insulin and glucose during the OGTT wascomputed

26 Clinical Parameters Plasma glucose was measured bythe glucose oxidase-peroxidase (GOD-POD) kit method(Biosystems Barcelona Spain) and plasma insulin by RIAkit (BRIT-DAE Mumbai India) method as per the manufac-turerrsquos instructions Blood glucose was measured weekly andinsulin levels were measured at the age of two months and atthe end of the experimental period of seven months

27 Slit-Lamp Examination and Cataract Classification Eyeswere examined every week using a slit-lamp biomicroscope(Kowa SL-15 Portable Japan) on dilated pupils Initiationand progression of lenticular opacity was graded into fourcategories as reported earlier [21]






2O2and








3 Results

















0

4

8

12

16

1 2 3 4 5 6 7

(mm

olL

)

Duration (months)


lowast lowast

(a)

0

10

20

30

40

50


lowast

lowastlowast

(120583U

mL)

(b)





100x

400x

(a)

0

20

40

60

80

()

100


lowast

lowast

(b)








0

5

10

15

20

25

30

35

0 30 60 120

Glu

cose

(mm

olL

)

Time (min)


lowast

lowastlowast

(a)


0

10

20

30

40

50

60

70

0 30 60 120

Time (min)

lowast

lowast

lowast

Insu

lin (120583

Um

in)

(b)




0

1

2

3

4

1 2 3 4 5 6

Stag

es o

f cat

arac

t

Duration (months)





4 Discussion









0

3

6

9

12

15

18


(nm

olg

lens

)

MDA

0

2

4

6

8

10


(nm

olm

g pr

otei

n)

Protein carbonyls

lowast

lowast lowast








0

10

20

30

40

50


SOD

0

10

20

30

40

50

60


GST

0

5

10

15

20

25

30


GPx

lowast

lowast

(Uni

tsm

in100

mg

prot

ein)

(120583m

ol o

f NA

DPH

oxi

dise

dh

100

mg

prot

ein)

(120583m

ol o

f CD

NB-

GSH

oxi

dise

dh

100

mg

prot

ein)












Acknowledgment


References






























3

VO4







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2O2and








3 Results

















0

4

8

12

16

1 2 3 4 5 6 7

(mm

olL

)

Duration (months)


lowast lowast

(a)

0

10

20

30

40

50


lowast

lowastlowast

(120583U

mL)

(b)





100x

400x

(a)

0

20

40

60

80

()

100


lowast

lowast

(b)








0

5

10

15

20

25

30

35

0 30 60 120

Glu

cose

(mm

olL

)

Time (min)


lowast

lowastlowast

(a)


0

10

20

30

40

50

60

70

0 30 60 120

Time (min)

lowast

lowast

lowast

Insu

lin (120583

Um

in)

(b)




0

1

2

3

4

1 2 3 4 5 6

Stag

es o

f cat

arac

t

Duration (months)





4 Discussion









0

3

6

9

12

15

18


(nm

olg

lens

)

MDA

0

2

4

6

8

10


(nm

olm

g pr

otei

n)

Protein carbonyls

lowast

lowast lowast








0

10

20

30

40

50


SOD

0

10

20

30

40

50

60


GST

0

5

10

15

20

25

30


GPx

lowast

lowast

(Uni

tsm

in100

mg

prot

ein)

(120583m

ol o

f NA

DPH

oxi

dise

dh

100

mg

prot

ein)

(120583m

ol o

f CD

NB-

GSH

oxi

dise

dh

100

mg

prot

ein)












Acknowledgment


References






























3

VO4







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























0

4

8

12

16

1 2 3 4 5 6 7

(mm

olL

)

Duration (months)


lowast lowast

(a)

0

10

20

30

40

50


lowast

lowastlowast

(120583U

mL)

(b)





100x

400x

(a)

0

20

40

60

80

()

100


lowast

lowast

(b)








0

5

10

15

20

25

30

35

0 30 60 120

Glu

cose

(mm

olL

)

Time (min)


lowast

lowastlowast

(a)


0

10

20

30

40

50

60

70

0 30 60 120

Time (min)

lowast

lowast

lowast

Insu

lin (120583

Um

in)

(b)




0

1

2

3

4

1 2 3 4 5 6

Stag

es o

f cat

arac

t

Duration (months)





4 Discussion









0

3

6

9

12

15

18


(nm

olg

lens

)

MDA

0

2

4

6

8

10


(nm

olm

g pr

otei

n)

Protein carbonyls

lowast

lowast lowast








0

10

20

30

40

50


SOD

0

10

20

30

40

50

60


GST

0

5

10

15

20

25

30


GPx

lowast

lowast

(Uni

tsm

in100

mg

prot

ein)

(120583m

ol o

f NA

DPH

oxi

dise

dh

100

mg

prot

ein)

(120583m

ol o

f CD

NB-

GSH

oxi

dise

dh

100

mg

prot

ein)












Acknowledgment


References






























3

VO4







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















100x

400x

(a)

0

20

40

60

80

()

100


lowast

lowast

(b)








0

5

10

15

20

25

30

35

0 30 60 120

Glu

cose

(mm

olL

)

Time (min)


lowast

lowastlowast

(a)


0

10

20

30

40

50

60

70

0 30 60 120

Time (min)

lowast

lowast

lowast

Insu

lin (120583

Um

in)

(b)




0

1

2

3

4

1 2 3 4 5 6

Stag

es o

f cat

arac

t

Duration (months)





4 Discussion









0

3

6

9

12

15

18


(nm

olg

lens

)

MDA

0

2

4

6

8

10


(nm

olm

g pr

otei

n)

Protein carbonyls

lowast

lowast lowast








0

10

20

30

40

50


SOD

0

10

20

30

40

50

60


GST

0

5

10

15

20

25

30


GPx

lowast

lowast

(Uni

tsm

in100

mg

prot

ein)

(120583m

ol o

f NA

DPH

oxi

dise

dh

100

mg

prot

ein)

(120583m

ol o

f CD

NB-

GSH

oxi

dise

dh

100

mg

prot

ein)












Acknowledgment


References






























3

VO4







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

20

25

30

35

0 30 60 120

Glu

cose

(mm

olL

)

Time (min)


lowast

lowastlowast

(a)


0

10

20

30

40

50

60

70

0 30 60 120

Time (min)

lowast

lowast

lowast

Insu

lin (120583

Um

in)

(b)




0

1

2

3

4

1 2 3 4 5 6

Stag

es o

f cat

arac

t

Duration (months)





4 Discussion









0

3

6

9

12

15

18


(nm

olg

lens

)

MDA

0

2

4

6

8

10


(nm

olm

g pr

otei

n)

Protein carbonyls

lowast

lowast lowast








0

10

20

30

40

50


SOD

0

10

20

30

40

50

60


GST

0

5

10

15

20

25

30


GPx

lowast

lowast

(Uni

tsm

in100

mg

prot

ein)

(120583m

ol o

f NA

DPH

oxi

dise

dh

100

mg

prot

ein)

(120583m

ol o

f CD

NB-

GSH

oxi

dise

dh

100

mg

prot

ein)












Acknowledgment


References






























3

VO4







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















0

3

6

9

12

15

18


(nm

olg

lens

)

MDA

0

2

4

6

8

10


(nm

olm

g pr

otei

n)

Protein carbonyls

lowast

lowast lowast








0

10

20

30

40

50


SOD

0

10

20

30

40

50

60


GST

0

5

10

15

20

25

30


GPx

lowast

lowast

(Uni

tsm

in100

mg

prot

ein)

(120583m

ol o

f NA

DPH

oxi

dise

dh

100

mg

prot

ein)

(120583m

ol o

f CD

NB-

GSH

oxi

dise

dh

100

mg

prot

ein)












Acknowledgment


References






























3

VO4







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























Acknowledgment


References






























3

VO4







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























3

VO4







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

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