ETM Experimental and Therapeutic Medicine 1792-0981 1792-1015 D.A. Spandidos 10.3892/etm.2015.2317 ETM-0-0-2317 Articles Adenosine monophosphate-activated protein kinase activation mediates the leptin-induced attenuation of cognitive impairment in a streptozotocin-induced rat model ZHUBIN1 JIANGRI-YUE2 YANGCHUN3 LIUNING1 Department of Critical Care Medicine, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu 213003, P.R. China Department of Radiotherapy, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu 213003, P.R. China Department of Anesthesiology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu 213003, P.R. China Correspondence to: Dr Chun Yang, Department of Anesthesiology, The Third Affiliated Hospital of Soochow University, 185 Juqian Street, Changzhou, Jiangsu 213003, P.R. China, E-mail: yangchuntz@sina.com 05 2015 25 02 2015 9 5 1998 2002 25062014 16022015 Copyright © 2015, Spandidos Publications 2015

Several lines of evidence have shown that the incidence of cognitive impairment in diabetic patients is significantly higher than that in healthy individuals, but the exact pathogenesis has not been fully elucidated. Furthermore, it has been suggested that leptin may have a therapeutic effect in cognitive dysfunction. The aim of the present study was to observe the effect of leptin on cognitive dysfunction in streptozotocin (STZ)-induced diabetic rats, and to explore whether adenosine monophosphate-activated protein kinase (AMPK) activation was involved in any potential therapeutic effect of leptin. Compared with control rats, STZ rats exhibited decreased levels of AMPK and a poor performance in the Morris water maze, while these changes were reversed by leptin. Furthermore, Compound C, an AMPK antagonist, significantly attenuated the leptin-induced cognitive function improvement in the STZ rats. In conclusion, these results suggest that AMPK activation may play a critical role in the leptin-induced attenuation of STZ-induced cognitive impairment.

 leptin cognitive impairment adenosine monophosphate-activated protein kinase streptozotocin diabetes Introduction

Diabetes mellitus is a common disease in older individuals, affecting ∼20% of the population aged >65 years (1,2). It has been reported that diabetes has a close association with the reduced performance in numerous domains of cognitive function (1,3–5). A clinical study by Arvanitakis et al (1) has indicated that diabetes may be associated with an increased risk of Alzheimer's disease development and may eventually affect cognitive function; however, the underlying mechanisms of diabetes-induced cognitive dysfunction have not been fully elucidated.

Numerous studies have focused on diabetes-induced cognitive impairment, while the majority have focused on fat metabolism and vascular dementia (6–8). Jafari Anarkooli et al (9) demonstrated that Bcl-2 family gene expression and caspase-3 activity were altered in the streptozotocin (STZ)-induced diabetic rat hippocampus. Furthermore, Revsin et al (10) found that a glucocorticoid receptor antagonist normalized hippocampal alterations and cognitive impairment in STZ-induced diabetic mice. Our previous study (11), however, found that intraperitoneal injection of a single dose of 60 mg/kg STZ could cause cognitive dysfunction and increase inflammatory cytokine levels and oxidative activity in the rat hippocampus. Notably, leptin levels in the rat hippocampus significantly decreased.

Leptin, which is a hormone-like protein secreted from fat cells, plays a critical role in regulating food intake and energy metabolism (12,13). It has been reported that leptin is a potential cognitive enhancer (14). Several studies have shown that leptin is the upstream activator of the adenosine monophosphate-activated protein kinase (AMPK) pathway (15,16). Yi et al (17) observed that acute and chronic exercise could indirectly activate the leptin-AMPK signaling pathway, while a study by Namkoong et al (18) showed that leptin and insulin deficiencies in diabetes led to increased hypothalamic AMPK activity.

The aim of the present study was to investigate the effect of leptin on cognitive dysfunction in STZ-induced diabetic rats and to explore whether AMPK activation was involved in any potential therapeutic effects of leptin. An AMPK antagonist, Compound C, was selected to investigate the potential involvement of AMPK in the leptin-induced therapeutic effect in cognitive impairment.

 Materials and methods <sec> <title>Animals

Male Wistar rats weighing 220–300 g were purchased from the Shanghai Animal Center (Shanghai, China). Six rats were housed per cage with food and water available ad libitum and maintained under a 12-h light/dark cycle (lights on at 7:00 a.m.). Animal care was approved by the Animal Use and Protection Committee of Soochow University (Changzhou, China). Thirty-six rats were randomly divided into three groups (n=12 each). The rats were intraperitoneally pretreated with either a single injection of saline or STZ (Sigma-Aldrich, St. Louis, MO, USA) at a dose of 60 mg/kg. One month later, the rats were either intracerebroventricularly injected with saline or 10 µg leptin (Sigma-Aldrich) dissolved in 5 µl Tris-HCl. For the second experiment, a further 36 rats were randomly divided into three groups (n=12 each). The rats were intraperitoneally pretreated with either a single injection of saline or STZ at a dose of 60 mg/kg according to the first protocol. One month later, the rats were either intracerebroventricularly injected with saline, 10 µg leptin or 10 µg leptin plus intraperitoneal injection of 1 mg/kg Compound C (Tocris Bioscience, Bristol, UK).

 Morris water maze test

According to our previous study (11), cognitive function was assessed using the Morris water maze test system between 9:00 a.m. and 3:00 p.m. The maze was 80 cm deep and 100 cm in diameter and was filled with water to a depth of 30 cm. The maze was divided into four quadrants of equal size on the monitor screen of a computer, and the water temperature was maintained at 23–24°C. The swimming paths of the rats were recorded by a video camera and analyzed by VideoMot software (Huaibei Zhenghua Biologic Apparatus Facilities Co., Ltd., Huaibei, China). The trials were conducted for four consecutive days in order to observe the escape latency of the rats and the time spent in each quadrant. The escape latency and the proportion of time spent in the target quadrant were recorded.

 Leptin and tumor necrosis factor-α (TNF-α) measurement

Leptin and TNF-α levels were determined using ELISA kits. According to the manufacturer's instructions (Wuhan Huamei Bioengineering Co., Ltd., Wuhan, China), microtiter plates were coated for overnight incubation with the samples diluted 1:2 in sample diluent. The plates were then washed three times with sample diluent, and the primary antibody (monoclonal anti-leptin or -TNF-α), diluted 1:1,000 in sample diluent, was added to each well and incubated for 3 h at room temperature. Subsequent to washing, a peroxidase-conjugated anti-rabbit antibody (diluted 1:10,000; Nanjing Sunshine Biotechnology Co., Ltd.) was added to each well and incubated at room temperature for 1 h. Streptavidin enzyme, substrate and stop solution were then added, and the leptin and TNF-α levels were determined by measuring the absorbance at 450 nm. In addition, total protein was measured using the Lowry method with bovine serum albumin as a standard.

 Amyloid-β (Aβ) measurement

The animals were sacrificed immediately by decapitation and the protein concentrations were determined using a bicinchoninic acid assay kit (cat. no. P0012S; Beyotime Institute of Biotechnology, Haimen, China). The samples were then centrifuged at 3,000 × g at 4°C for 30 min to obtain the supernatants. The proteins were separated using SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Subsequent to blocking with 5% non-fat milk, the membranes were incubated with rabbit anti-Aβ primary antibody (1:200; Sigma-Aldrich). The membranes were then incubated for 1 h at room temperature with anti-rabbit horseradish peroxidase-conjugated immunoglobulin G secondary antibody (1:20,000; CWBIO, Beijing, China). The labeled protein was detected using enhanced chemiluminescence reagents (GE Healthcare Life Sciences, Little Chalfont, UK) and the band intensity was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

 Malondialdehyde (MDA) measurement

According to our previous study (11), the samples were mixed with 1 ml 10% trichloroacetic acid and 1 ml 0.67% thiobarbituric acid, and were then heated in a boiling water bath for 30 min. MDA equivalents were determined in the tissue and submitochondrial particles of the rat brain using a spectrophotometer at an absorbance of 532 nm.

 Blood glucose measurement

The rats were anesthetized with 10% chloral hydrate (0.4 ml/100 g, intraperitoneal) and blood samples were then collected from the carotid artery. Blood glucose levels were determined with a blood glucometer (OneTouch® UltraEasy®; Johnson & Johnson, Inc., New Brunswick, NJ, USA).

 Statistical analysis

Data are expressed as the mean ± standard deviation. Statistical analyses were performed by one-way analysis of variance, and post hoc analyses were conducted using Fisher's least significant difference tests. All statistical analyses were carried out using SPSS version 17.0 software (SPSS, Inc., Chicago, IL, USA). In the Morris water maze test, the percentage of time spent in the target quadrant was evaluated using χ2 tests. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Effect of STZ and/or leptin on the escape latency of the rats and the proportion of time spent in the target quadrant in the Morris water maze test

The results of the Morris water maze test demonstrated that STZ administration significantly increased the escape latency as compared with the control group, and decreased the percentage of time spent in the fourth quadrant (P<0.05 or 0.01). Compared with the STZ group, however, rats in the STZ plus leptin group exhibited significantly decreased escape latencies, and an increased percentage of time spent in the fourth quadrant (P<0.05) (Fig. 1).

 Effect of STZ and/or leptin on the expression of AMPK in the rat hippocampus

A single injection of STZ significantly decreased the protein expression of AMPK in the rat hippocampus (P<0.05). By contrast, leptin administration could abrogate the STZ-induced decrease in AMPK expression in the hippocampus (P<0.05) (Fig. 2).

 Effect of Compound C, an AMPK inhibitor, on the escape latency of STZ-induced diabetic rats and the percentage of time spent in the target quadrant in the Morris water maze test

Compound C, which acts as an AMPK inhibitor, significantly increased the escape latency as compared with the leptin group, and decreased the percentage of time spent in the fourth quadrant (P<0.05 or 0.01) (Fig. 3A and B).

 Effect of Compound C on the leptin-induced changes in the expression of TNF-α, Aβ and MDA and the blood glucose levels

Compared with the saline group, leptin administration caused a significant decrease in the serum TNF-α and Aβ levels in the rat hippocampus (P<0.05), and a significant increase in the MDA levels (P<0.01). Compared with the leptin group, Compound C abrogated the leptin-induced changes in the expression of TNF-α, Aβ and MDA (P<0.05), but did not affect the blood glucose levels (P>0.05) (Fig. 4A-D).

 Discussion

In the present study, the results demonstrated that leptin could attenuate STZ-induced cognitive impairment, which was characterized by the poor performance of the rats in the Morris water maze test. Compound C, an AMPK inhibitor, significantly abrogated the protective effects of leptin against the STZ-induced rat cognitive impairment. The findings of these results suggested, therefore, that AMPK activation contributed to the underlying mechanism of the therapeutic effect of leptin in STZ-induced cognitive impairment.

Numerous studies have shown that the mechanism of cognitive dysfunction in STZ-induced diabetic rats is associated with the vascular lesions caused by diabetes (8,19). Denver et al (12), however, proposed that diabetes is simply a trigger in the pathogenesis of diabetes-induced cognitive dysfunction, rather than the main mechanism. Zhu et al (20) suggested that oxidative stress, the inflammatory response and Aβ formation comprised the primary mechanism in diabetes-induced cognitive dysfunction, which was also consistent with the results of our previous study (11). In the present study, the results supported the above-mentioned hypothesis that diabetes-induced cognitive dysfunction manifested as a result of the abnormal expression of TNF-α, Aβ and MDA. Notably, the blood glucose levels did not show any significant change following leptin treatment. Although it has previously been demonstrated that leptin acts to reduce blood glucose levels (21), the present study utilized an injection of exogenous leptin, not endogenous; therefore, the leptin was not able to rapidly activate the leptin receptors, which prevented it from exerting its biological effects.

AMPK is an enzyme that plays a role in cellular energy homeostasis. Numerous studies have highlighted the important role of AMPK in the pathogenesis of Alzheimer's disease, which is characterized by cognitive impairment (22,23). An in vitro study by Greco et al (24) showed that leptin increased cellular metabolism by activating AMPK to reduce Aβ expression in neurons. A different study also performed by this group (25) demonstrated that leptin directly regulated Aβ through the AMPK pathway. The results of the present study showed that STZ administration significantly decreased the AMPK levels in the rat hippocampus, while leptin abrogated this effect. Notably, it was observed that Compound C, an AMPK inhibitor, significantly reversed the effects of leptin, suggesting that AMPK activation is likely to be involved in the mechanism underlying the protective effect of leptin against cognitive impairment.

The activation of oxidative stress has been considered to be an important inducing factor for the incidence of cognitive impairment. Praticò et al (26) demonstrated that increased oxidative stress could be used as a predictor for the onset of cognitive impairment. A different study suggested that oxidative activation has harmful effects on the rat synapses in the cerebral cortex and hippocampus, which may result in cognitive impairment (27). Notably, a clinical study found that the successful treatment of patients with cognitive impairment could restore the oxidative cytokine levels in the peripheral blood of the patients to a normal levels (28). In the present study, it was observed that an AMPK inhibitor could abrogate the effect of leptin on cognitive impairment and change hippocampal MDA levels. This result indirectly showed that the control of cognitive function by AMPK signaling potentially occurs through the regulation of oxidative stress. Similarly, the inflammatory response plays a critical role in the incidence of diabetes-related cognitive impairment. The present results showed that TNF-α levels decreased following leptin treatment, while the AMPK inhibitor reversed this change. This result was consistent with our expected hypothesis.

In conclusion, AMPK activation may contribute to the therapeutic effect of leptin in cognitive impairment in an STZ-induced rat model. It is likely that oxidative stress and the inflammatory response are also involved.

 References ArvanitakisZWilsonRSBieniasJLEvansDABennettDADiabetes mellitus and risk of Alzheimer disease and decline in cognitive functionArch Neurol61661666200410.1001/archneur.61.5.66115148141 EngelgauMMGeissLSSaaddineJBThe evolving diabetes burden in the United StatesAnn Intern Med140945950200410.7326/0003-4819-140-11-200406010-0003515172919 van den BergEReijmerYDde BresserJUtrecht Diabetic Encephalopathy Study Group: A 4 year follow-up study of cognitive functioning in patients with type 2 diabetes mellitusDiabetologia535865201010.1007/s00125-009-1571-919882137 HuberJDDiabetes, cognitive function, and the blood-brain barrierCurr Pharm Des1415941600200810.2174/13816120878470544118673200 StrachanMWPriceJFFrierBMDiabetes, cognitive impairment, and dementiaBMJ3366200810.1136/bmj.39386.664016.BE18174567 PeilaRRodriguezBLLaunerLJHonolulu-Asia Aging Study: Type 2 diabetes, APOE gene and the risk for dementia and related pathologies: The Honolulu-Asia Aging StudyDiabetes5112561262200210.2337/diabetes.51.4.125611916953 BretelerMMVascular risk factors for Alzheimer's disease: An epidemiologic perspectiveNeurobiol Aging21153160200010.1016/S0197-4580(99)00110-410867200 GispenWHBiesselsGJCognition and synaptic plasticity in diabetes mellitusTrends Neurosci23542549200010.1016/S0166-2236(00)01656-811074263 AnarkooliJafari ISankianMAhmadpourSVarastehARHaghirHEvaluation of Bcl-2 family gene expression and Caspase-3 activity in hippocampus STZ-induced diabetic ratsExp Diabetes Res2008638467200818923682 RevsinYRekersNVLouweMCGlucocorticoid receptor blockade normalizes hippocampal alterations and cognitive impairment in streptozotocin-induced type 1 diabetes miceNeuropsychopharmacology34747758200910.1038/npp.2008.13618784648 YangCZhuBDingJWangZGIsoflurane anesthesia aggravates cognitive impairment in streptozotocin-induced diabetic ratsInt J Clin Exp Med7903910201424955160 DenverRJBonettRMBoorseGCEvolution of leptin structure and functionNeuroendocrinology942138201110.1159/00032843521677426 SinghAWirtzMParkerNLeptin-mediated changes in hepatic mitochondrial metabolism, structure and protein levelsProc Natl Acad Sci USA1061310013105200910.1073/pnas.090372310619622746 HarveyJShanleyLJO'MalleyDIrvingAJLeptin: A potential cognitive enhancer?Biochem Soc Trans3310291032200510.1042/BST2005102916246038 MinokoshiYKimYBPeroniODLeptin stimulates fatty-acid oxidation by activating AMP-activated protein kinaseNature415339343200210.1038/415339a11797013 MinokoshiYKahnBBRole of AMP-activated protein kinase in leptin-induced fatty acid oxidation in muscleBiochem Soc Trans31196201200310.1042/BST031019612546684 YiXCaoSChangBEffects of acute exercise and chronic exercise on the liver leptin-AMPK-ACC signaling pathway in rats with type 2 diabetesJ Diabetes Res2013946432201310.1155/2013/94643224455748 NamkoongCKimMSJangPGEnhanced hypothalamic AMP-activated protein kinase activity contributes to hyperphagia in diabetic ratsDiabetes546368200510.2337/diabetes.54.1.6315616011 SharmaBSinghNAttenuation of vascular dementia by sodium butyrate in streptozotocin diabetic ratsPsychopharmacology (Berl)215677687201110.1007/s00213-011-2164-021225418 ZhuXRainaAKLeeHGCasadesusGSmithMAPerryGOxidative stress signalling in Alzheimer's diseaseBrain Res10003239200410.1016/j.brainres.2004.01.01215053949 DavisCMuddJHawkinsMNeuroprotective effects of leptin in the context of obesity and metabolic disordersNeurobiol Dis726171201410.1016/j.nbd.2014.04.01224780498 SalminenAKaarnirantaKHaapasaloASoininenHHiltunenMAMP-activated protein kinase: A potential player in Alzheimer's diseaseJ Neurochem118460474201110.1111/j.1471-4159.2011.07331.x21623793 CaiZYanLJLiKQuaziSHZhaoBRoles of AMP-activated protein kinase in Alzheimer's diseaseNeuromolecular Med14114201210.1007/s12017-012-8173-222367557 GrecoSJSarkarSJohnstonJMTezapsidisNLeptin regulates tau phosphorylation and amyloid through AMPK in neuronal cellsBiochem Biophys Res Commun38098104200910.1016/j.bbrc.2009.01.04119166821 GrecoSJHamzelouAJohnstonJMSmithMAAshfordJWTezapsidisNLeptin boosts cellular metabolism by activating AMPK and the sirtuins to reduce tau phosphorylation and β-amyloid in neuronsBiochem Biophys Res Commun414170174201110.1016/j.bbrc.2011.09.05021945934 PraticòDClarkCMLiunFRokachJLeeVYTrojanowskiJQIncrease of brain oxidative stress in mild cognitive impairment: a possible predictor of Alzheimer diseaseArch Neurol59972976200210.1001/archneur.59.6.97212056933 FukuiKOmoiNOHayasakaTCognitive impairment of rats caused by oxidative stress and aging and its prevention by vitamin EAnn NY Acad Sci959275284200210.1111/j.1749-6632.2002.tb02099.x11976202 PadurariuMCiobicaAHritcuLStoicaBBildWStefanescuCChanges of some oxidative stress markers in the serum of patients with mild cognitive impairment and Alzheimer's diseaseNeurosci Lett469610201010.1016/j.neulet.2009.11.03319914330

Effect of STZ and/or leptin on (A) the escape latency of the rats and (B) the proportion of time spent in the target quadrant in the Morris water maze test. *P<0.05 and **P<0.01 vs. control; #P<0.05 vs. STZ. STZ, streptozotocin.

Effect of STZ and/or leptin on the expression of AMPK in the rat hippocampus. *P<0.05 vs. control; #P<0.05 vs. STZ. STZ, streptozotocin; AMPK, adenosine monophosphate-activated protein kinase.

Effect of Compound C on (A) the escape latency of the STZ-induced diabetic rats and (B) the proportion of time spent in the target quadrant in the Morris water maze test. *P<0.05 and **P<0.01 vs. STZ; #P<0.05 vs. leptin. STZ, streptozotocin.

Effect of Compound C on the leptin-induced changes in the expression of (A) TNF-α, (B) Aβ, (C) MDA and (D) blood glucose. *P<0.05 and **P<0.01 vs. saline; #P<0.05 vs. leptin. STZ, streptozotocin; TNF-α, tumor necrosis factor-α; Aβ, amyloid-β; MDA, malondialdehyde.