Pharmacology, Biochemistry and Behavior 114–115 (2013) 43–51
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Pharmacology, Biochemistry and Behavior
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Amelioration of diabetes-induced neurobehavioral and neurochemicalchanges by melatonin and nicotinamide: Implication of oxidativestress–PARP pathway
Ashok Jangra, Ashok Kumar Datusalia, Shriya Khandwe, Shyam Sunder Sharma ⁎Molecular Neuropharmacology Laboratory, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar 160062,Punjab, India
Pharmacology and Toxicology, National Institute of PResearch (NIPER), Sector 67, S.A.S. Nagar (Mohali) 16006221 4682 to 87x2040, +91 172 229 2040; fax: +91 172
Diabetes associated hyperglycemia results in generation of reactive oxygen species which induces oxidativestress and initiate massive DNA damage leading to overactivation of poly (ADP-ribose) polymerase (PARP). Inthis study, we have elucidated the involvement of oxidative stress–PARP pathway using pharmacologicalinterventions (melatonin, as an anti-oxidant and nicotinamide, as a PARP inhibitor) in diabetes-induced neuro-behavioral and neurochemical alterations. Sprague–Dawley rats were rendered diabetic by a single intraperito-neal injection of streptozotocin. Behavioral and cognitive deficits were assessed after 8 weeks of diabetesinductionusing a functional observation battery, passive avoidance and rotarod test. Acetylcholinesterase activitywas significantly decreased in hippocampus of diabetic rats as compared to control rats. Diabetic animals showedsignificant increase inmalondialdehyde levels and reduction inNAD levels in hippocampus. Glutamate andGABAlevels were also altered in hippocampus of the diabetic animals. Two week treatment with melatonin (3 and10 mg/kg) and nicotinamide (300 and 1000 mg/kg) alone and in combination significantly improved the neuro-behavioral parameters which were altered in diabetes. Neurotransmitter (glutamate and GABA) levels wereimproved by these interventions. Our results emphasize that simultaneous inhibition of oxidative stress–PARPoveractivation cascade can be beneficial in treatment of diabetes associated CNS changes.
Diabetes mellitus is a heterogeneous metabolic disorder character-ized by hyperglycemia resulting from defective insulin secretion, resis-tance to insulin action or both. It is estimated that approximately371 million people worldwide, within the age group of 20–79 yearswere affected by diabetes in 2011. By 2030, diabetes is predicted toaffect 537 million people worldwide, which would be about 10% ofthe adult population. According toWHO, neurological and psychologicalcomplications are developed in 15–40% of diabetic patients (Bernsteinet al., 2013; Guariguata, 2012). Hyperglycemia leads to severe diabetic
otocin; PARP, poly (ADP-ribose)al Observational Battery; AChE,ogen-activated protein kinase;MDA, malondialdehyde.logy Laboratory, Department ofharmaceutical Education and2, Punjab, India. Tel.: +91 172221 [email protected] (S.S. Sharma).
ghts reserved.
complications, such as cardiovascular disease, retinopathy, nephropa-thy, peripheral and autonomic neuropathy through oxidative stress.Diabetes associated cognitive dysfunction has been recognized in themedical literature since 1922 (Richardson, 1991).
In diabetes, acute and chronic metabolic and vascular disturbancescan impair the functional and structural integrity of the brain. Hypergly-cemia activates multiple pathways such as polyol pathway, myo-inositol depletion, increased sorbitol accumulation disturbed Ca2+
homeostasis, increased advanced glycation end products, increasedreactive oxygen species, and alteration of protein kinase C activity(Ryle et al., 1998; Levy et al., 1994; Knudsen et al., 1989; Flier et al.,1987; Vlassara et al., 1983). Cerebrovascular changes like reducedblood flow to the brain, impaired vascular reactivity and neurotropicchanges like reduced level of IGF (insulin growth factor) indicate thatthese abnormalities precede functional cognitive impairments andapoptotic neuronal loss in hippocampus (Li et al., 2002, 2005). Inhumans, diabetes mellitus is associated with moderate impairments incognitive function, a high risk of affective disorders, dementia andAlzheimer's disease (Brismar et al., 2007; Northam et al., 2006; Ristow,2004; Sima, 2004; Ott et al., 1999; Biessels et al., 1994).
Animal models of diabetes, including the streptozotocin-induceddiabetic rats, have been proved to be very useful to determine theunderlying cause of CNS complications. Streptozotocin (STZ)-induced
Table 1Behavioral parameters of functional observational battery (Rajput et al., 2009; Irwin,1968).
Spontaneous activity level (home cage): (1 = no movement and asleep/2 = slightmovement of head or body/3 = moderate movement in the cage/4 = activemovement around cage/5 = rapid movements in the cage)
Spontaneous activity level (open field): (1 = no body movement/2 = little orsluggish movement/3 = little movement with exploratory activity/4 = walkingwith very little or no running/5 = exploratory movements, including walking andrunning/6 = highly active with darting/running)
Posture: (normal/flattened, pelvis flat on surface/pelvis dragging/hunched, backraised up)
Respiration: [1 = normal/2 = breathing either fast and shallow or slow
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diabetes produces hippocampal apoptosis and astrogliosis, decreasesproliferation rate in the dentate gyrus resulting in poor neurogenesisand reduces number of hilar neurons (Beauquis et al., 2006, 2008;Revsin et al., 2005; Saravia et al., 2004). Cognitive deficit might beconnected to neurotoxic effects of hyperglycemia and changes in neuro-transmission and neuronal functionality. The increased oxidative stressin diabetes produces oxidative damage in many regions of rat brainincluding the hippocampus (Kuhad and Chopra, 2008). Oxidativestress and related pathways have a major contributory role in thedevelopment of diabetic complications, whereas antioxidant therapymay protect or restore physiological function (Kuhad and Chopra,2008). Melatonin as well as its metabolites are well established antiox-idants (Galano et al., 2011, 2013). Melatonin has been found to beneuroprotective in streptozotocin-induced rat model of diabetes (Negiet al., 2010a, 2011; Saravia et al., 2004). Quenching of free radicals bymelatonin is the central mechanism for counteracting oxidative stress-induced neurotoxicity (Rosales-Corral et al., 2012; Tuzcu and Baydas,2006).
Hyperglycemia also causes overproduction of nitric oxide (NO)which reacts with superoxide resulting in the generation of highly reac-tive peroxynitrite which induces massive DNA damage. Downstream ofthis DNA damage, the enzyme poly (ADP-ribose) polymerase (PARP, EC2.4.2.30) is activatedwhich is a nuclear enzyme that acts as a nicksensorand facilitates DNA repair induced by oxidative stress, ionizing radia-tions and cytotoxic agents. However, in case of extensive DNA damage,PARP-1 overactivation induces a decrease in NAD+ and ATP levels,leading to energy failure and cell necrosis. PARP overactivation mayrelease apoptosis inducing factor (AIF) from the mitochondria andfacilitates AIF translocation to the nucleus, thus activating a caspase-independent type of apoptosis (Chaitanya et al., 2010). Moreover, thebeneficial effects of PARP inhibitors like 3-aminobenzamide and5-aminoisoquinolinone have been shown in spinal cord trauma(Genovese et al., 2005). PARP inhibition using nicotinamide can beviewed as an important target to delineate manifestation inducedby diabetes and its CNS complication. We have earlier demonstratedthe protective effect of melatonin and nicotinamide and their combina-tion in diabetic neuropathy (Negi et al., 2010b, 2011). However,their effects on diabetes-induced neurobehavioral and cognitivedysfunctions have not been investigated. Therefore, in the presentstudy we investigated the neuroprotective role of melatonin and nico-tinamide on neurobehavioral and neurochemical alterations occurringin diabetes.
(bradypnea)/3 = breathing very fast shallow or very shallow and labored inappearance/4 = wheezing or breathing with mouth open/5 = weak breathing(breathing very little)]
Excitation: (1 = no resistance, easy to hold or prick up/2 = slight resistance/3 = squirming or moving around/4 = excited, squirming, twisting/5 = aggressive actions like biting, tail and throat rattling)
Salivation: (0 = dryness of mouth/1 = normal salivation/2 = wetness around themouth and chin/3 = drooling of saliva)
Lacrimation: (0 = dryness of eye/1 = normal eye/2 = wetness around the eyes)Muscle tone: (normal/increase/decrease)Gait: [1 = normal/2 = slight abnormality (uncoordinated, staggering, wobbly gait,exaggerated, overcompensated, or splayed moving hind limbs)/3 = moderateabnormality (hind limb point outward from body, forelimbs, drag or showabnormal positioning and walking on toes)/4 = severe abnormality (nomovement)]
Arousal: [1 = very low (stupor, coma, or prostrate)/2 = low (sluggish, only somemovements)/3 = somewhat low (slightly sluggish, some exploratorymovements)/4 = moderate (alert, exploratory behavior)]
Auditory response (1–5 scale), somatosensory response (1–5 scale), visual approachresponse (1–5 scale), olfactory response (1–5 scale)[1 = no reaction or response/2 = slight or sluggish reaction (flinch or startle asevidence of perception)/3 = obvious reaction (locomotor orientation as evidenceof perception)/4 = clear reaction or response (more intense startle or locomotion)/5 = exaggerated reaction (may jump, bite, or attack)]
2. Material and methods
2.1. Induction of diabetes and experimental design
The experiments were performed in accordance with regulationsspecified by the Institutional Animal Ethics Committee (IAEC), NIPER.Male Sprague–Dawley rats (250–270 g) were used for the study andwere fed on a standard rat diet and water ad libitum. Diabetes wasinduced by streptozotocin at a dose of 55 mg/kg (i.p.). Blood sampleswere collected from tail vein ~72 h after STZ administration. Rats withplasma glucose level more than 250 mg/dl were considered diabeticand were further considered for study. The experimental groups werecomprised of non-diabetic control group (ND), diabetic control rats(STZ-D), and diabetic rats treated with melatonin (D + M3 andD + M10, respectively, for melatonin 3 and 10 mg/kg, p.o.), nicotin-amide (D + N300 and D + N1000, respectively, for nicotinamide 300and 1000 mg/kg, p.o.) and a combination of melatonin 3 mg/kg andnicotinamide 300 mg/kg (D + M3 + N300). The experimental groupsconsisted of 6 animals each. The treatment was started 6 weeks afterdiabetes induction and continued for two weeks. The behavioral andbiochemical experiments were performed 24 h after administration ofthe last dose.
2.2. Behavioral parameters
2.2.1. Functional observational battery (FOB)This test was performed to evaluate the effect of diabetes on
behavior and physiological function using scores as described else-where with slight modification as per Table 1. The open field activitiesand sensory responses were observed in open field cages for 5 min(Columbus Instruments, OH, USA). The body temperature was mea-sured by inserting rectal probes (Harvard apparatus, MA, USA). Theprobe was left in place until a stable temperature was achieved. Mostsymptomswere evaluated by their presence or absence. Some parame-ters were rated at 5-point scale like spontaneous activity, excitability,and auditory response (Mattsson et al., 1996; Irwin, 1968).
2.2.2. Motor coordination testMotor coordinationwas assessed using a rotarod apparatus (IITC Life
Science, CA, USA). Rotating speed and cut off time were fixed at 15 rpmand 180 s respectively. Ratswere acclimatized for twodays and on thirdday test session were carried out. The time taken for the animal to dropoff the rod, or the number of animals remaining on the rod over a setduration was measured. The incidence of ataxia i.e., the ability of therat to fall was recorded in control, diabetic and drug treated animals(Sharma et al., 2011).
2.2.3. Passive avoidance testIt was carried out using Shuttle Box Apparatus (Columbus Instru-
ments, OH, USA). The apparatus consists of a two compartment dark/light shuttle box with a guillotine door separating the compartments.The dark compartment had a stainless steel shock grid floor. During
Table 2Body weight and plasma glucose levels characteristic of experimental groups.
Numerical values were expressed as mean ± SEM (n = 6). ***p b 0.001 as compared toND (ND: Non-diabetic; STZ-D: Diabetic; D + N300, D + N1000: Diabetic group treatedwith nicotinamide 300 and 1000 mg/kg respectively; D + M3, D + M10: group treatedwith melatonin 3 and 10 mg/kg respectively; D + M3 + N300: Diabetic group treatedwith melatonin 3 mg/kg and nicotinamide 300 mg/kg).
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the acquisition trial, each rat was placed in the lighted chamber. After a60 s habituation period, the guillotine door was opened, and latency ofanimals to enter the dark chamber was recorded. Immediately after therat entered the dark chamber, the guillotine door was closed and anelectric foot shock (0.6 mA) was delivered to the floor grids for 2 s.Five seconds later, the rat was removed from the dark chamber andreturned to its home cage. After 24 h, retention latency time wasmeasured in the same way as in the acquisition trial, but the footshock was not delivered and latency time was recorded to a maximumof 300 s. Shorter latencies indicate poor retention (Kumar et al., 2009).
2.3. Biochemical parameters
2.3.1. Plasma glucose levelsBlood was collected from the tail vein in microcentrifuge tubes con-
taining heparin. Plasmawas separated and blood glucosewas estimatedby the GOD–POD method (Accurex, India) as per manufacturer'sinstructions.
2.3.2. Lipid peroxidationBrains were isolated on a Petri dish on ice. Hippocampus was
removed immediately and homogenized in ice cold phosphate buffersaline (pH 7.4). After homogenization, samples were sonicated andcentrifuged at 10,000 rpm for 5 min (4 °C). Supernatant was separatedand kept under −80 °C. MDA estimation was carried out according tothe method of Ohkawa (1979) with slight modification. Briefly, 0.1 mlof sample was added to 0.1 ml of 8.1% SDS, 0.75 ml of 20% acetic acidsolution adjusted to pH 3.4with NaOH and 0.75 ml of 0.8% thiobarbitu-ric acid. The mixture was made up to 3 ml with distilled water. Themixture was then heated on a water bath at 95 °C for 60 min, cooledand centrifuged at 10,000 rpm for 5 min. Absorbance of the supernatantwas measured at 532 nm. Protein estimation was performed accordingto Lowry method. Results were expressed as μM/mg of protein concen-tration (Lowry et al., 1951).
homogenate was measured using an enzyme cycling assay (Nisselbaumand Green, 1969). In brief, hippocampus was isolated bilaterally andhomogenized in nine volume potassium phosphate buffer and kept inboiling water bath for 5 min. The homogenate was centrifuged at1000 g on 4 °C and supernatants were stored at −20 °C until furtherused. The NAD content was analyzed using an enzyme cycling mixturecontaining alcohol dehydrogenase and absorbance was measured at556 nm using a Beckmen DU 7400 spectrophotometer (Negi et al.,2010a).
2.3.4. Measurement of acetylcholinesterase (AChE) activityThe hippocampus was isolated bilaterally and homogenized in
0.1 M phosphate buffer (pH 8). 0.4 ml aliquot of the homogenate wasadded to a cuvette containing 2.6 ml phosphate buffer (0.1 M, pH 8)and 100 μl of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). The contentsof the cuvette were mixed thoroughly by bubbling. 20 μl of substrate(acetylthiocholine) was added and change in absorbance was recordedfor a period of 10 min at intervals of 2 min. The enzyme activity wascalculated using the formula; R = 5.74 × 10−4 × A / CO; where, R =rate in moles of substrate hydrolyzed/minute/g tissue; A = change inabsorbance/min; CO = original concentration of the tissue (mg/ml)(Lakshmana et al., 1998).
2.3.5. Measurement of neurotransmittersBrain GABA and glutamate levels were measured by HPLC-EC
method. Briefly, brainswere isolated on ice cold Petri dish. Immediatelyhippocampus were removed and homogenized in ice cold perchloricacid (0.05 M) containing L-Tyrosine (0.1 mg/ml or 5.5 μmol/ml). Ho-mogenized sampleswere centrifuged and supernatantwere derivatized
with o-phthalaldehyde (OPA). Derivatizing agent was prepared by dis-solving 100 mg of o-phthalaldehyde in 2 ml of HPLC grade methanoland added into 200 ml of NaHCO3 (0.5 M, pH: 9.5). Finally, 20 μl ofβ-mercaptoethanol was added into it and the solution was kept indark and cold (2–8 °C). Samples were derivatized with 30:50 ratios ofsample and derivatizing reagent with excessive shaking for 2 min.Mobile phase was prepared by dissolving 10.92 g of Na2HPO4 and148.8 mg of EDTA in 500 ml of ultra pure Millipore water. Then 1.5 mltetrahydrofuran and 450 ml of HPLC grade methanol were added.After adjusting the pH to 5.25 with o-phosphoric acid, volume wasmade up to 1000 ml. 50 μl derivatized samples were injected usingan auto sampler (Waters, 717) into column (Nucleosil®: RP18, 5 μm,4.6 × 250, 30 °C). The sample was run for 30 min at a flow of1 ml/min (Pump: Waters 515) and components present in samplewere detected by an electrochemical detector (Waters 2465: Mode:DC, Ec: +0.80 V, 30 °C). Glutamate and GABA were identified usingcorresponding standards and quantified using internal standardmethod (Rao et al., 1998).
2.4. Statistical analysis
Results were expressed as mean ± SEM. Jandel Sigma Stat Version3.5 software was used for statistical analysis. Significance of the differ-ence between the two groups was evaluated using Student's t-test. Forthe multiple comparisons, one way analysis of variance (ANOVA) wasused. In case ANOVA showed a significant difference, post hoc analysiswas performed with Tukey's test or Dunnet test. p b 0.05 was consid-ered statistically significant.
3. Results
3.1. Biochemical parameters
Rats injected with STZ (55 mg/kg, i.p.) showed a significant(p b 0.001) rise in plasma glucose and a significant decline in bodyweight as compared to age matched normal control rats (p b 0.001).Treatment with melatonin and nicotinamide alone and in combinationdid not produce any significant effect on blood glucose level or bodyweight of treated rats compared to diabetic rats (Table 2).
3.2. Behavioral and functional observation battery test
3.2.1. Effect of melatonin and nicotinamide alone and in combination inFOB test in diabetic rats
The effect of diabeteswas studied on CNS using the functional obser-vation battery. Treatment with melatonin and nicotinamide alone andin combination in diabetic rats did not produce any effect on homecage observation except the presence of polyuria and diarrhea indiabetic rats. Treatment with nicotinamide (1000 mg/kg) has onlyimproved the condition of diarrhea in diabetic treated rats. Piloerection,fur appearance, ptosis and exophthalmia were absent in all groups.
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During open field observation diabetic rats showed a significantincrease in supported and unsupported rears as compared to controlgroup. Treatment with melatonin (10 mg/kg) improved only theunsupported rears. Whereas, nicotinamide 300 and 1000 mg/kg andin combination with melatonin showed significant improvement insupported and unsupported rears as compared to diabetic control rats(Fig. 1). No significant changes were observed in neuromuscularmeasurements (abdominal tone). In sensory responses, there were nosignificant changes in auditory response, somatosensory response,olfactory response and visual response as compared to control rats.Pinna reflex, extensor thrust reflex, palpebral reflex, visual placing,surface righting, aerial righting, pupil reaction and tail pinch responseswere present in all the groups. No significant changes were found inbody temperature in all groups.
3.2.1. Effect of melatonin and nicotinamide alone and in combination inrotarod test in diabetic rats
Diabetes significantly reduced the rotarod performance in diabeticanimals as compared to control group (Fig. 2A). Melatonin treatmentat lower dose (3 mg/kg) did not show significant effect on fall of timein diabetic animals, but at a dose of 10 mg/kg (p b 0.05) it showedsignificant improvement. Nicotinamide (300 and 1000 mg/kg) aloneand in combination with melatonin showed significantly increased fallof time in the rotarod test as compared to diabetic group.
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Fig. 1. Effect of melatonin and nicotinamide alone and in combination in 8 week diabetic animamean ± SEM (n = 6). ###p b 0.001 as compared to non-diabetic group; ***p b 0.001 as comtreated with melatonin 3 and 10 mg/kg respectively; D + N300, D + N1000: Diabetic groupgroup treated with melatonin 3 mg/kg and nicotinamide 300 mg/kg).
3.2.2. Effect of melatonin and nicotinamide alone and in combination inpassive avoidance test in diabetic rats
Diabetic animals (8th week) showed significant decrease in avoid-ance response (transfer latency 29.4 ± 6.3 s) compared to controlnon diabetic animals (transfer latency 254.7 ± 45.3 s). Melatonin didnot show a significant change in transfer latency at 3 mg/kg dose com-pared to diabetic animals but showed a significant effect at 10 mg/kgdose (170.4 ± 47.4 s). Treatment with nicotinamide with the doses(300 and 1000 mg/kg) produced significant increase in the avoidanceresponse to 130.5 ± 54.3 s (p b 0.05) and 158.7 ± 48.7 s (p b 0.05)respectively as compared to diabetic rat values (Fig. 2B). A combinationof melatonin (3 mg/kg) with nicotinamide (300 mg/kg) producedsynergistic effects in avoidance response test.
3.3. Neurochemical analysis
3.3.1. Effect of melatonin and nicotinamide alone and in combination onhippocampal MDA and NAD levels in diabetic rats
Diabetic animals showed a significant increase in hippocampalMDA levels, indicating oxidative stress. Two weeks of treatment withmelatonin (10 mg/kg) and nicotinamide (1000 mg/kg) significantly(p b 0.01) reduced the hippocampal MDA levels. The combination reg-imen showed decrease in oxidative stress as manifested by decreasedMDA levels (p b 0.01) (Fig. 3A). Diabetic rat (8 weeks) showed a signif-icant reduction in hippocampal NAD levels as compared to agematched
+M10 D+N300 D+N1000 D+M3+N300
+M10 D+N300 D+N1000 D+M3+N300
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ls on (A) supported rears and (B) unsupported rears. Numerical values were expressed aspared to diabetic group. (ND: Non-diabetic; STZ-D: Diabetic; D + M3, D + M10: grouptreated with nicotinamide 300 and 1000 mg/kg respectively; D + M3 + N300: Diabetic
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Fig. 2. Effect of melatonin and nicotinamide alone and in combination in 8 week diabetic animals on (A) motor coordination test (B) passive avoidance test. Numerical valueswere expressed as mean ± SEM (n = 6). ###p b 0.001 as compared to non-diabetic group; *p b 0.05, **p b 0.01, ***p b 0.001 as compared to diabetic group (ND: Non-diabetic;STZ-D: Diabetic; D + M3, D + M10: group treated with melatonin 3 and 10 mg/kg respectively; D + N300, D + N1000: Diabetic group treated with nicotinamide 300 and1000 mg/kg respectively; D + M3 + N300: Diabetic group treated with melatonin 3 mg/kg and nicotinamide 300 mg/kg).
47A. Jangra et al. / Pharmacology, Biochemistry and Behavior 114–115 (2013) 43–51
normal control rats (p b 0.001) (Fig. 3B). Significant protection inmelatonin treated (10 mg/kg) diabetic animals was observed but notwith lower dose of melatonin (3 mg/kg). Nicotinamide (300 mg/kg)did not show significant protection against the decreased NAD levelbut higher dose showed (1000 mg/kg) a significant increase in NADlevel compared to diabetic animals (p b 0.01). The combination regi-men significantly (p b 0.001) enhanced protection against reductionin NAD levels in hippocampus.
3.3.2. Effect of melatonin and nicotinamide alone and in combination onhippocampal ache activity in diabetic rats
Eight weeks of diabetes resulted in significant reduction in hippo-campal AChE activity levels as compared to agematched normal controlrats. Two week treatment with melatonin (10 mg/kg), nicotinamide(300 and 1000 mg/kg) and the combination regimen significantly(p b 0.01) ameliorated the reduction in hippocampal AChE level (Fig. 4).
3.3.3. Effect of melatonin and nicotinamide alone and in combination onhippocampal glutamate and GABA levels in diabetic rats
Diabetic rats showed a significant reduction in glutamate levels inhippocampus as compared to control rats. Two week treatment withnicotinamide (1000 mg/kg) and the combination regimen therapysignificantly ameliorated the reduction in hippocampal glutamatelevel. Similarly, GABA levels were found to be significantly increased
in hippocampus of diabetic rats as compared to control rats. GABA levelsin hippocampus of diabetic animals were significantly reduced bymelatonin (10 mg/kg), nicotinamide (300 and 1000 mg/kg) and com-bination regimen (Fig. 5A & B).
4. Discussion
This study demonstrates the protective effect of melatonin (antioxi-dant) and nicotinamide (PARP inhibitor) on diabetes-induced neurobe-havioral and neurochemical alterations. Chronic diabetes of 8 weeksproduced cognitive impairment, motor incoordination and an increasein both supported and unsupported rears in rats. Neurobehavioralalterations were coupled with a marked effect on AChE activity, GABA,glutamate, and MDA and NAD levels in the hippocampus of diabeticrats. There are ample evidences that diabetes induced hyperglycemiahas disruptive effects on the central nervous system. Inprevious reports,STZ induced diabetic rats showed cognitive impairment which wasreversed by insulin treatment (Sima and Li, 2005; Brands et al., 2004;Biessels et al., 1998). These studies also revealed that cognitive andbehavioral abnormalities in STZ-induced diabetic rats are not attributedto per se effect/direct neurotoxicity of STZ. The major pathwaysinvolved diabetic complications include polyol pathway activation, pro-tein kinase C activation, hexosamine pathway, aldose reductase path-way, MAPK pathway and advanced glycation formation (Brands et al.,
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Fig. 3.Effect ofmelatonin and nicotinamide alone and in combination in 8 week diabetic animals on (A)MDA level and (B) NAD level in hippocampus. Numerical valueswere expressed asmean ± SEM (n = 6). ###p b 0.001 as compared to non-diabetic group; *p b 0.05, **p b 0.01, ***p b 0.001 as compared to diabetic group (ND: Non-diabetic; STZ-D: Diabetic; D + M3,D + M10: group treated with melatonin 3 and 10 mg/kg respectively; D + N300, D + N1000: Diabetic group treated with nicotinamide 300 and 1000 mg/kg respectively;D + M3 + N300: Diabetic group treated with melatonin 3 mg/kg and nicotinamide 300 mg/kg).
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Fig. 4. Effect of melatonin and nicotinamide alone and in combination in 8 week diabetic animals on AChE activity in hippocampus. Numerical values were expressed as mean ± SEM(n = 6). ###p b 0.001 as compared to non-diabetic group; *p b 0.05, **p b 0.01 as compared to diabetic group (ND: Non-diabetic; STZ-D: Diabetic; D + M3, D + M10: group treatedwith melatonin 3 and 10 mg/kg respectively; D + N300, D + N1000: Diabetic group treated with nicotinamide 300 and 1000 mg/kg respectively; D + M3 + N300: Diabetic grouptreated with melatonin 3 mg/kg and nicotinamide 300 mg/kg).
48 A. Jangra et al. / Pharmacology, Biochemistry and Behavior 114–115 (2013) 43–51
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Fig. 5. Effect ofmelatonin andnicotinamide alone and in combination in 8 week diabetic animals on (A) glutamate level; (B) GABA level in hippocampus. Numerical valueswere expressedas mean ± SEM (n = 6). ##p b 0.01, ###p b 0.001 as compared to non-diabetic group; *p b 0.05, **p b 0.01 as compared to diabetic group (ND: Non-diabetic; STZ-D: Diabetic;D + M3, D + M10: group treated with melatonin 3 and 10 mg/kg respectively; D + N300, D + N1000: Diabetic group treated with nicotinamide 300 and 1000 mg/kg respectively;D + M3 + N300: Diabetic group treated with melatonin 3 mg/kg and nicotinamide 300 mg/kg).
49A. Jangra et al. / Pharmacology, Biochemistry and Behavior 114–115 (2013) 43–51
2004; Vlassara et al., 1983). These processes occur via inhibitionof many regulatory proteins and enzymes like glyceraldehyde-3-phosphate dehydrogenase activity which is likely to be involved inpoly ADP ribosylation by PARP. PARP overactivation has also beenshown to trigger pathogenesis of neurodegenerative conditions(Chaitanya et al., 2010). Oxidative stress has shown to overactivatePARP leading to neuronal death. Studies on peripheral diabetic neurop-athy have already provided substantial evidence about the neuropro-tection offered by PARP inhibitors and various antioxidants (Kumaret al., 2011; Negi et al., 2010a, 2010b, 2011). Moreover, PARP-1 inhibi-tion has been implicated in providing defense against tissue injury ina variety of oxidative stress related conditions. It is an important cofac-tor in the activation of transcriptional factors such as NF-κB and AP-1which aremainly involved in the expression of inflammatorymediators(Hassa and Hottiger, 2002; Kameoka et al., 2000). Hence, the treatmentwith an antioxidant and a PARP inhibitor in diabetes-induces CNSchanges were evaluated.
For melatonin monotherapy, two doses 3 and 10 mg/kg and fornicotinamide monotherapy, two doses 300 and 1000 mg/kg wereselected which were based on earlier studies (Negi et al., 2010a,2010b; Feng et al., 2006; Yang et al., 2002). Two week treatment withmelatonin (10 mg/kg) resulted in a significant protection againstneurobehavioral changes and improvement in NAD levels. Melatonintreatment also showed significant reduction in MDA levels. Both doses
of nicotinamide showed improvement in behavioral functions likerotarod performance, FOB parameters, and avoidance response. Reduc-tion of hippocampal NAD levels, confirms interference of melatonin inPARP activation. Moreover, melatonin is reported for its protectiveeffect related to interference with DNA damage and PARP activationby increasing cleavage of PARP protein in cancer, methotrexate inducedintestinal damage and STZ induced β-cell damage (Jung et al., 2013;SÃnchez-Hidalgo et al., 2012; Wang et al., 2012; Yavuz et al., 2003).Further, an improvement in biochemical deficits like MDA, NAD, andAChE levels was observed on nicotinamide treatment at higher doses.Subtherapeutic doses of nicotinamide 300 mg/kg and melatonin3 mg/kg were used for the combination study. This provided a bettereffect onmotor coordination performance, cognitive ability, NAD levels,MDA levels and AChE levels as compared to monotherapy.
Increasing evidence indicates that factors such as oxidative andnitrosative stress, glutathione depletion and impaired protein metabo-lism can interact in a vicious cycle which is central to the pathogenesisof behavioral and other neurodegenerative disorders (Andersen, 2004;Emerit et al., 2004). In previous studies on diabetic neuropathy, increasein lipid peroxidation andNADdepletionwas observed in sciatic nerve ofdiabetic rats (Chaitanya et al., 2010; Negi et al., 2010a). Similarly, NADlevel was reduced in the hippocampus of diabetic rats in the presentstudy. This might be due to PARP overactivation that causes NAD deple-tion in the hippocampus (Kaundal et al., 2006; Strosznajder et al., 2005).
Diabetes
Supeoxide O2- iNOS activation
Nitric oxide NO
Peroxynitrite ONOO-
Melatonin
Neurotransmitter
levels change
NAD/ATP
consumption
Nicotinamide
DNA damage
Oxidative stress
Proinflammatory signals
AP1, MAPK, NF-κB
Neurobehavioral alterations
PARP overactivation↑Cleavage of PARP
protein
Fig. 6. Role of melatonin and nicotinamide in protection against CNS changes induced bydiabetes. Diabetes-induced hyperglycemia may lead to oxidative stress. Oxidative stressin diabetes can induceDNAdamage leading to overactivation of PARP. PARP overactivationin turn can activate pro-inflammatory signals, neurotransmitter changes and depletionof energy (NAD/ATP) leading to neurobehavioral changes. Melatonin and nicotinamideacts on oxidative stress and PARP pathway, thereby ameliorate diabetes-induced CNSalterations.
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Treatment with nicotinamide improved MDA and NAD levels to theircontrol values. Melatonin and nicotinamide monotherapy and theircombination resulted in reduction in MDA level and improvement inNAD level. Melatonin, as a free radical scavenger, also regulate the activ-ity and gene expression of antioxidant and pro-oxidant enzymes whichmay be another possible mechanism of protection offered bymelatoninagainst diabetes-induced neurobehavioral changes (Negi et al., 2011;Tan et al., 1993). Even melatonin metabolites have shown antioxidantactivity (Reiter et al., 2007; Ressmeyer et al., 2003).
Cholinergic transmission dysfunction is evidently observed in neu-rological disorders and dementia. Acetylcholine, a neurotransmitterassociated with learning and memory, is degraded by the enzymeacetylcholinesterase, terminating the physiological action of the neuro-transmitter. The changes in AChE activity might reflect impairment inits biosynthesis, degradation, or insertion into the plasma membrane.Diabetic state is known to cause membrane alterations that can affectthe kinetic properties of membrane-bound cholinesterases (Khandkaret al., 1995; Suhail and Rizvi, 1989). The activity of AChE was evidentlyaffected in diabetes and varies in brain region. Increase in AChE activitywas observed in the cerebral cortex of diabetic rats (Kuhad and Chopra,2007). Contrary, AChE activity has been reported to decrease in brain ofdiabetic rats bymany investigators (Ghareeb andHussen, 2008; Kambojet al., 2008; Ashok kumar et al., 2006). Both increase and decrease inAChE activity have been reported in memory impairments (Saleset al., 2010; Udayabanu et al., 2008; Das et al., 2005). Furthermore,AChE activity was significantly decreased in hippocampus of 8 weekdiabetic animals in this study. Itmight be due to an increase in oxidativestress which leads to changes in the membrane microenvironment ordirect effect of free radicals on the AChE enzyme. AChE activity hasbeen reported to be affected by oxidative stress induced alteration inmembrane fluidity (Sandhir et al., 2009). Hydroxyl radicals producedduring the Fenton reaction through H2O2/Fe2+ system have beenshown to damage the active site of the AChE molecule which mayresponsible for the decrease in hippocampal AChE activity (Ashokkumar et al., 2006; Tsakiris, 2001). Nicotinamide andmelatonin provideprotection by maintaining the AChE activity through modulation ofdiabetes induced oxidative stress in the brain.
Neurotransmitter levels like GABA and glutamate were estimated indiabetic rats. GABA was found to increase and glutamate was found todecrease significantly in hippocampus of diabetic rats as compared toage matched control animals. Glutamate plays a key role in synapticplasticity and cognition (Riedel and Reymann, 2003). In diabetic rats,the hyperglycemic state could be responsible for the increased glucoseutilization with continued ATP or energy consumption. ATP maintainsthe ion pumps, hence less ATP leads to a reduction in the amount ofglutamate released due to depolarization (Cousin et al., 1995). PARPinhibitor alone and in combination with an antioxidant effectivelyrestored the glutamate level by preventing the energy depletion in thebrain. Many studies support the concept of inhibition of memoryformation by GABAergic system. Drugs, which modulate the GABAergicsystem at different stages like synthesis or binding of GABA or at chlo-ride channels, significantly affect the memory and cognitive ability(Brioni, 2004). Similarly, GABA antagonists and some GABA inhibitingsteroids like pregnenolone sulfate shows significant improvement inlearning and memory (Cruz-Morales et al., 1993; Flood et al., 1992).We observed an increase in GABA level in the hippocampus of diabeticrats. Alteration of GABA-glutamate homeostasis may be the possiblereason of impaired avoidance response and cognitive impairment indiabetic animals (Sickmann et al., 2011). GABA levels were significantlyreduced by nicotinamide, melatonin and their combination.
Overall, the result of the study postulates that oxidative stress indiabetes can induce DNA damage leading to overactivation of PARPwhich, in turn can activate pro-inflammatory signals and neurotrans-mitter changes leading to neurobehavioral alteration and memorydeficits. The involvement of oxidative stress–PARP pathway in diabeticinduced alteration in CNS is evident from the protective effects of
melatonin (antioxidant) and nicotinamide (PARP inhibitor) (Fig. 6).The protective effect of melatonin and nicotinamide may also be attrib-uted through maintaining the GABA-glutamate homeostasis.
Disclosure/conflict of interest
The authors have no conflict of interest.
Acknowledgments
Wewould like to thank theDepartment of Pharmaceuticals,Ministryof Chemicals and Fertilizers, Government of India for financial support.Mr. Ashok Kumar Datusalia is a recipient of University Grant Commis-sion (UGC), New Delhi research fellowship.
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