Sex Differences in High-fat Diet-induced Obesity, Metabolic Alterations and Learning, and Synaptic Plasticity Deficits in Mice

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1 nture publishing group Sex Differences in High-ft Diet-induced Obesity, Metbolic Altertions nd Lerning, nd Synptic Plsticity Deficits in Mice Ling-Ling Hwng 1,2, Chien-Hu Wng 3, Tzu-Ling Li 2, Shih-Dr Chng 4, Li-Chun Lin 3, Ching-Ping Chen, Chiung-Tong Chen, Keng-Chen Ling 4,6, Ing-Kng Ho 7, Wei-Shiung Yng 8,9 nd Lih-Chu Chiou 3,6 Obesity is potentil risk fctor for cognitive deficits in the elder humns. Using high-ft diet (HFD) induced obese mouse model, we investigted the impcts of HFD on obesity, metbolic nd stress hormones, lerning performnce, nd hippocmpl synptic plsticity. Both mle nd femle C7BL/6J mice fed with HFD (3 weeks to 9 12 months) gined significntly more weights thn the sex-specific control groups. Compred with the obese femle mice, the obese mles hd similr energy intke but developed more weight gins. The obese mle mice developed hyperglycemi, hyperinsulinemi, hypercholesterolemi, nd hyperleptinemi, but not hypertriglyceridemi. The obese femles hd less hyperinsulinemi nd hypercholesterolemi thn the obese mles, nd no hyperglycemi nd hypertriglyceridemi. In the contextul fer conditioning nd step-down pssive voidnce tsks, the obese mle, but not femle, mice showed poorer lerning performnce thn their norml counterprts. These lerning deficits were not due to sensorimotor impirment s verified by the open-field nd hot-plte tests. Although, bsl synptic trnsmission chrcteristics (input output trnsfer nd pired-pulse fcilittion (PPF) rtio) were not significntly different between norml nd HFD groups, the mgnitudes of synptic plsticity (long-term potentition (LTP) nd long-term depression (LTD)) were lower t the Schffer collterl-ca1 synpses of the hippocmpl slices isolted from the obese mle, but not femle, mice, s compred with their sex-specific controls. Our results suggest tht mle mice re more vulnerble thn the femles to the impcts of HFD on weight gins, metbolic ltertions nd deficits of lerning, nd hippocmpl synptic plsticity. Obesity () 18, doi:.38/oby Introduction Obesity, in ddition to being risk fctor for crdiovsculr nd metbolic diseses, hs been implicted in degrding cognitive opertion (1). Elis et l. (2) found tht obesity ws ssocited with lower cognitive functioning in lte middleged nd elderly ( 88 yers) men, but not women. This ssocition is independent of other crdiovsculr risk fctors. Gustfson et l. (3) lso found tht overweight t older ges (79 88 yers) is risk fctor for dementi, prticulrly the Alzheimer s disese. Additionlly, obesity in the midlife ( 4 yers) is risk fctor for dementi nd Alzheimer s disese in Americn (both sexes) (4) nd in Swedish men (). Impired sptil memory nd hippocmpl synptic plsticity were lso reported in geneticlly obese niml models (6,7). However, insted of genetic fctors, consumption of high-ft diet (HFD) is conceived to be common cuse of obesity in humns (8). Therefore, the niml models of HFD-induced obesity could better mimic the pthologicl chnges in obese humns. The impirment of cognitive functions hs been observed in severl rodent models of HFD-induced obesity but most of these studies were limited to using one sex of nimls, mostly the mles (9 12), or short-term (<6 months) HFD feeding (13). In n obese mouse model with long-term (up to 9 12 months) HFD feeding, we hve previously reported tht the 1 Deprtment of Physiology, College of Medicine, Tipei Medicl University, Tipei, Tiwn; 2 Grdute Institute of Medicl Sciences, Tipei Medicl University, Tipei, Tiwn; 3 Deprtment of Phrmcology, College of Medicine, Ntionl Tiwn University, Tipei, Tiwn; 4 Deprtment of Psychology, College of Science, Ntionl Tiwn University, Tipei, Tiwn; Division of Biotechnology nd Phrmceuticl Reserch, Ntionl Helth Reserch Institutes, Zhunn, Mioli, Tiwn; 6 Neurobiology nd Cognitive Science Center, Ntionl Tiwn University, Tipei, Tiwn; 7 Division of Mentl Helth nd Addiction Medicine, Institute of Popultion Helth Sciences, Ntionl Helth Reserch Institutes, Zhunn, Mioli, Tiwn; 8 Grdute Institute of Clinicl Medicine, College of Medicine, Ntionl Tiwn University, Tipei, Tiwn; 9 Deprtment of Internl Medicine, Ntionl Tiwn University Hospitl, Tipei, Tiwn. Correspondence: Lih-Chu Chiou (lcchiou@ntu.edu.tw) Received 16 Jnury 9; ccepted 9 July 9; published online 3 September 9. doi:.38/oby obesity VOLUME 18 NUMBER 3 mrch 463

2 hippocmpl synptic plsticity of obese mle mice ws impired (14). In this study, we further chrcterized this obese mouse model in both sexes nd compred the sex differences in the impcts of HFD on the development of obesity nd the impirments in metbolic regultion, lerning performnce, nd synptic plsticity. Methods nd Procedures C7BL/6J mice of both sexes were rndomly divided into two groups fed with norml chows or HFD fter wening until 9 12 month old, unless stted otherwise, nd their body weights nd diet consumption were monitored. The plsm levels of fsting glucose, triglycerides, nd totl cholesterol in the mice were mesured by spectrophotometry. The serum levels of insulin, leptin, diponectin, nd corticosterone were mesured with enzyme-linked immunosorbnt ssy (see Supplement I in Supplementry Methods nd Procedures online). The lerning nd memory functions of the mice were evluted by their performnces in two tsks, step-down pssive voidnce nd single-trining tril contextul fer conditioning tsks. The ltter is hippocmpus-sensitive tsk () nd not subjected to functionl compenstion becuse compenstion requires dditionl trining trils (16). The locomotor ctivity nd nociceptive sensitivity were ssessed by the open-field nd hot-plte tests, respectively (see Supplement II in Supplementry Methods nd Procedures online). Severl forms of synptic plsticity, including long-term potentition (LTP), post-tetnic potentition (PTP), depotentition, nd long-term depression (LTD), t the Schffer collterl-ca1 synpses of the hippo cmpl slices isolted from the four groups of mice were exmined nd compred. The chrcteristics of bsl synptic trnsmission, including the input output trnsfer nd pired-pulse fcilittion (PPF) rtio, in the hippocmpl slices of different groups were lso compred (see Supplement III in Supplementry Methods nd Procedures online). Dt re expressed s men ± s.e.m. Sttisticl nlyses were performed by Student s t-tests for the prmetric dt or Mnn Whitney tests for the nonprmetric dt. Synptic plsticity between groups (tretments or sexes) ws compred by two-wy (time tretment/sex) ANOVA of the filed excittory postsynptic potentils recorded during the time period described. Results Estblishment of long-term HFD-induced obesity model in C7BL/6J mice Growth curves. To estblish mouse model of HFD-induced obesity, C7BL/6J mice of both sexes were fed with HFD immeditely fter wening (3 week-old). The groups fed with HFD gined more weights thn those with norml chow in both sexes. The HFD-induced weight gins ppered t n erlier time in the mles, s compred with the femles (fter 46- vs. 129-dy old, rrows in Figure 1), nd were much greter in the mles (filled squres) thn those in the femles (filled circles) (Figure 1). The mice on HFD consumed less mounts of chow thn those on norml diet in both mle (17.9 ± 1. vs ±.1 g/week) nd femle (19. ± 1.2 vs ± 2.8 g/week) mice. However, the energy intkes were not different between the HFD nd norml groups in either mle (84. ± 7.1 vs. 9.7 ±.3 kcl/ week) or femle (92.1 ±.7 vs. 9.1 ± 9.6 kcl/week) mice. There ws no sex difference in the diet intke either in the norml or obese group in terms of mounts or clories. In mle group fed with HFD from 8 to 3 weeks of ge (filled tringles in Figure 1), the weight gin ws not significntly different from tht of the ge-mtched norml group. Therefore, the mice fed with norml chows nd HFD immeditely fter wening until 9 12 months of ge were used in the Body weight (g) 3 High ft (8 weeks) Norml (wening) High ft (wening) Norml (wening) High ft (wening) Age (months) Figure 1 Growth curves of mle nd femle mice fed with norml or HFD. Body weights of mle (squres) nd femle mice (circles) fed with norml (open symbols, n = ) or HFD (filled symbols, n = 11) immeditely fter wening were monitored until they were subjected to experiments t 9 12-month old. Initilly, one group of mle mice (filled tringles, n = 12) ws fed with HFD t the ges from 8 to 3 weeks. However, there ws no significnt increse of body weight in this group. P <. vs. the sex-specific control group from the ges indicted by rrows (Student s t-test). HFD, high-ft diet. Tble 1 Blood levels of glucose, lipids, nd hormones in norml nd obese mice of both sexes Concentrtion Mle Femle Norml High ft Norml High ft Glucose (mg/dl) 112 ± () 169 ± 16 (6) 122 ± (8) 123 ± 9.2 (8) # Triglycerides (mg/dl) 72.6 ± 8. () 71.4 ±.4 (6) 6.1 ± 3.4 (8) 76.4 ±.9 (8) Cholesterol (mg/dl) 83.9 ± 7.2 () ± 24.4 (6) 98.2 ± 7.6 (8) ± 6. (8), ## Insulin (ng/ml) b 1.8 ±.2 (6) 9.9 ± 1.1 (6). ±.1 (6) ## 2.3 ±.2 (8), ## Leptin (ng/ml) b 12.6 ±.9 (6) 9. ± 13. (6) 3.7 ±.8 (6) ## 89.4 ± 8.9 (8) Adiponectin (μg/ml) b 6. ±.9 () 7. ±. (6) 8. ±.8 (6).6 ±.4 (8), ## Corticosterone (ng/ml) b 28. ± 7.3 (6) 48.7 ±.8 (6) 37.9 ±. (6) 66. ± 12.9 (8) Dt re men ± s.e.m. nd the n numbers of smples re shown in prentheses. Plsm levels. b Serum levels. P <., P <.1 vs. the norml group. # P<., ## P <.1 vs. the mle group (Student s t-test). 464 VOLUME 18 NUMBER 3 mrch

3 Number of footshocks Norml High ft present study nd referred to s the norml nd obese groups, respectively. Ltency of step-down (s) 12 C D E 16 b A Number of crossings C A Number of footshocks Number of crossings Norml High ft Norml Norml High ft Number of rering High ft D Number of rering B 7 2 Norml High ft B Ltency of step-down (s) Norml High ft Dy 1 Dy 2 Response ltency of hot-plte test(s) E Norml High ft 8 6 Norml High ft Dy 1 Dy 2 Response ltency of hot-plte test (s) Norml High ft 8 6 Norml High ft Figure 2 Step-down pssive voidnce lerning, locomotor ctivity, nd nociceptive response in norml nd obese mice of both sexes. The responses in () mle nd (b) femle mice fed with norml diet (open brs) or HFD (filled brs) were compred. In step-down pssive voidnce lerning tsk, the performnce of cquisition nd retention were evluted, respectively, by (A) the number of footshocks required for the mice to sty on the pltform nd (B) the ltency to step down fter trining session. Locomotor ctivity ws ssessed by the number of (C) crossings nd (D) rering obtined in the open-field test. (E) Nociceptive response ws exmined by the ltency of foot withdrw/ licking in the hot-plte test. Dt re expressed s men ± s.e.m. P <., P <.1 vs. the norml group. The numbers of mice re s follows: norml mles (12), high-ft mles (13), norml femles (17), nd high-ft femles (12). HFD, high-ft diet. Freezing (%) 8 6 Mle Femle Blood levels of glucose, lipids, nd metbolic nd stress hormones. To exmine whether the obese mice mimic the metbolic syndrome usully observed in obese humns cliniclly, the plsm levels of glucose, lipids (triglycerides nd cholesterol), nd the serum levels of metbolic hormones (insulin, leptin, nd diponectin), nd corticosterone were mesured in the four groups (Tble 1). The obese mle mice, but not the femles, developed significnt hyperglycemi. The triglyceride levels were not significntly chnged in the obese groups of both sexes. The obese mice of both sexes developed hypercholesterolemi, hyperinsulinemi, nd hyperleptinemi, nd the obese mles, s compred with the obese femles, hd more severe hypercholesterolemi nd hyperinsulinemi but similr degree of hyperleptinemi. Conversely, the obese femles, but not mles, developed hyperdiponectinemi. The corticosterone levels in the obese mice of both sexes tended to be higher, though not significntly, s compred to their sex-specific counterprts. Interestingly, in the norml groups, the femles hd lower levels of insulin nd leptin thn the mles. Lerning tests Step-down pssive voidnce tsk. In the step-down pssive voidnce tsk, the obese mle mice required more footshocks to keep them stying on the pltform in the trining session thn the norml mles (Figure 2-A), suggesting tht the obese mle mice lerned less well thn the norml mles. However, no difference existed between the obese nd norml femle mice (Figure 2b-A). In the retention session, the step-down ltency ws not significntly different between the obese nd norml mice in both sexes (Figure 2-B,2b-B). The locomotor ctivities (Figure 2-C,2-D) mesured in the open-field test, nd the nociceptive sensitivity detected in the hot-plte test (Figure 2-E) were not significntly different between the obese nd norml mle mice. Nevertheless, obese femle mice displyed higher locomotor ctivity (Figure 2b-C) nd shorter hot-plte withdrwl ltency (Figure 2b-E) thn norml femles. Contextul fer conditioning tsk. In contextul fer conditioning with single-trining tril, the obese mle mice showed less conditioned fer, expressed by the percentge of freezing, thn norml mles (Figure 3). Norml femle mice expressed less freezing thn norml mles (open brs). However, no difference ws observed between the norml nd obese femles. This negtive finding is unlikely due to floor effect in norml femles becuse their freezing scores were higher thn the no-shock group, which ws exposed to the sme context but not receiving electroshocks (see Supplement IV in Supplementry Methods nd Procedures online). Hippocmpl synptic plsticity nd trnsmission LTP nd PTP induced by high-frequency stimultion. The mgnitude of LTP t Schffer collterl-ca1 synpses of the hippocmpl slices isolted from obese mle mice ws significntly ## Norml High ft Figure 3 Contextul fer conditioning lerning in norml nd obese mice of both sexes. The contextul conditioned fer in the mice fed with norml diet (open brs) or HFD (filled brs) were determined by exposing the mouse to the context previously pired with footshock nd recording the percentge of its freezing behvior in the 6-min exposure period. Dt re expressed s men ± s.e.m., n = in ech group. P <.1 vs. the norml group, ## P <.1 vs. the mle group (Student s t-test). HFD, high-ft diet. obesity VOLUME 18 NUMBER 3 mrch 46

4 fepsp (% of bseline) b fepsp (% of bseline) c fepsp (% of bseline) HFS 2 Norml mle High-ft mle HFS HFS 2 Norml mle Norml femle lower thn tht from norml mles (Figure 4). Conversely, the LTP in femle slices ws not significntly different between the norml nd obese groups (Figure 4b). The PTP in the Norml femle High-ft femle Figure 4 LTP, PTP, nd depotentition t Schffer collterl-ca1 synpses in the hippocmpl slices isolted from norml nd obese mice of both sexes. LTP nd PTP induced by HFS (rrows), consisting of three trins, s prt, of θ bursts ( Hz, ms, per ms), nd depotentition induced by (1 Hz, min, horizontl brs) were compred in slices from () mle nd (b) femle mice fed with norml (open symbols) or HFD (filled symbols), nd in slices from norml mle (open squres) nd femle (open circles) mice (c). fepsp slopes were verged every minute nd expressed s the percentge of bseline fepsp which ws the men fepsp slope recorded min before HFS. The results of two-wy ANOVA for the LTP mesured 6 min fter HFS between groups re () F (1, 26) = 139, P <.1, (b) F (1, 2) = 2.69, P =.3, nd (c) F (1, 2) = 24.9, P <.1. For the PTP mesured 1 4 min fter HFS, they re () F (1, 2) = 2.7, P =.6, (b) F (1, 48) = 3.983, P =.2, nd (c) F (1, 44) =.9749, P =.329. For the fepsps mesured 6 min fter, they re () F (1, 26) = 117, P <.1, (b) F (1, 2) = 3.37, P =.62, nd (c) F (1, 2) = 13.8, P <.1. Dt re men ± s.e. The numbers of slices: norml mles (7), high-ft mles (8), norml femles (6), nd high-ft femles (8). fepsp, field excittory postsynptic potentil; HFS, high-frequency stimultion;, low-frequency stimultion; LTP, long-term potentition; PTP, post-tetnic potentition. obese groups of both sexes were not significntly different from their sex-specific counterprts (Figure 4,b). It is notble tht in the norml group, the LTP in slices from mle mice ws significntly higher thn tht from the femles (Figure 4c). Further ppliction of low-frequency stimultion () 6 min fter high-frequency stimultion did not induce significnt depotentition in ll four groups regrdless of their different mgnitudes of LTP before (Figure 4). It remins to be elucidted whether depotentition cn be induced nd is different mong groups by giving erlier, e.g., min fter high-frequency stimultion. LTD induced by. induced significnt LTD in the norml, but not obese, mle group (Figure ). No LTD ws induced in either norml or obese femle slices (Figure b). The mgnitudes of LTD were significntly different between norml mle nd femle groups (Figure c). Bsl synptic trnsmission. The input output trnsfer of synptic trnsmission, clculted by the rtio of filed excittory postsynptic potentil to presynptic volley, ws not significntly different between the norml nd obese slices from either mle (8.16 ±.81 vs. 9.1 ±.83, P =.466) or femle (6.98 ±.62 vs..64 ±.66, P =.14) mice. The filed excittory postsynptic potentils evoked by pired-pulse stimultion displyed PPF, phenomenon originted from clcium ccumultion in the presynptic terminls (17), in ll four groups of slices. The PPF rtios were not significntly different mong the four groups (norml mles: 1.21 ±.; obese mles: 1.31 ±.3; norml femles: 1.3 ±.3; obese femles: 1.32 ±.). Discussion In this study, we found tht mle mice re more vulnerble thn the femles to the impcts of long-term HFD feeding on body weight gins nd the impirments in energy regultion, lerning nd memory, nd hippocmpl synptic plsticity. This is the first study, to the best of our knowledge, providing evidence to support sex differences in the impcts of long-term HFD on lerning functions nd synptic plsticity. Mle nd bby mice re more vulnerble to HFD-induced weight gins Our finding tht mle mice were more vulnerble to HFDinduced weight gins in terms of onset or mgnitude grees with the observtion in rts (13). This sex difference might be ttributed to the ntiobese effects of estrogen in the femles (18), through estrogen receptor α (19). In the rt model (13), lower estrdiol levels were observed in the obese group of both sexes. The drmtic body weight gins in mle mice could be consequence of less-energy expenditure (), s compred to the femles on HFD with similr energy intke. The finding tht bby mice (fter wening) fed with HFD re more vulnerble to obesity thn young dults (8 weeks) is interesting. Erly postntl period hs been demonstrted to be criticl for the hypothlmic feeding circuit development, 466 VOLUME 18 NUMBER 3 mrch

5 fepsp (% of bseline) b fepsp (% of bseline) c fepsp (% of bseline) Norml mle High-ft mle Norml femle High-ft femle Norml mle Norml femle 6 8 Figure LTD t Schffer collterl-ca1 synpses in hippocmpl slices isolted from norml nd obese mice of both sexes. LTD evoked by (1 Hz, min, horizontl brs) in nive slices were recorded, nlyzed, nd expressed in the four groups s described in Figure 4. The results of two-wy ANOVA for the LTD mesured 6 min fter between groups re () F (1, 26) = 12, P <.1, (b) F (1, 3) = 3.7, P =., nd (c) F (1, 3) = 161, P <.1. The numbers of slices: norml mles (8), high-ft mles (7), norml femles (9), nd high-ft femles (). LTD, long-term depression. influenced by intrinsic nd environmentl fctors (21). Our dt lso support tht the nutritionl condition in erly life predisposes mice to future vulnerbility to HFD-induced weight gins. In humns, the dipocyte number ws found to increse in childhood nd remin constnt in dulthood, wheres the lipid storge in lredy developed dipocytes determines the ft mss in dulthood (22). Therefore, the obesity model estblished in this study cn mimic the sex nd ge differences in the impcts of high-ft consumption on weight gin. It would be interesting to further elucidte the ontogenic ssocition between obesity susceptibility nd the development of feeding nd lerning circuits. Mle mice re more vulnerble to HFD-induced metbolic ltertions Glucose, lipids, nd insulin. The findings of hyperglycemi, hypercholesterolemi, hyperinsulinemi (insulin resistnce), nd hyperleptinemi in the obese mle mice suggest tht they hve developed the chnges resembling the metbolic syndrome of obese humns. The obese femles developed less hypercholesterolemi nd hyperinsulinemi, nd no hyperglycemi. This sex difference might be due to the less weight gins in the femles. In -week HFD feeding mouse model, the femles lso developed less hypercholesterolemi thn the mles (23). In rt model on high-ft/high-sugr diet, the femles lso hd less hypertriglyceridemi thn the mles (13). Adipokines. Compring the findings in the blood levels of dipokines in the rt model (high-ft/high-sugr diet, 4 9 month old) of Mrtin et l. (13) nd our mouse model (HFD, 3 week old to 9 month old), we found severl interesting inferences relevnt to the reports in humns. In the norml groups, femle rts nd mice hd lower leptin levels nd tended to hve higher diponectin levels thn the mles. Women hve been reported to hve higher diponectin levels thn men (24). In norml mice, the femles hd lower insulin levels thn the mles, but this sex difference ws not observed in norml rts. Women were found to hve lower fsting insulin levels thn men, possibly owing to their lower BMI (2). In the HFD groups of rts nd mice, similr degree of hyperleptinemi ws developed in both sexes, but the femles showed less/no chnges in the weight gins nd the blood levels of glucose, cholesterol, nd insulin (13). This suggests tht leptin ppers to be more sensitive mrker for obesity. Hyperleptinemi hs been reported to be n erly sign of juvenile obesity (26). The serum levels of diponectin, n dipoctye-derived hormone, were slightly elevted in the obese groups of both mice nd rts, but this chnge ws significnt only in the femle mice. Mle mice on high-ft/high-sucrose diet lso hd elevted circulting diponectin (27). These findings support the notion tht circulting diponectin might not be good biomrker for obesity becuse diponectin levels re lower in dibetic ptients including those nonobese ones (28,29). Corticosterone. Elevted corticosterone levels nd impired hippocmpl neurogenesis were reported in mle, but not femle, rts on HFD for 4 weeks (11) in mnner independent of diposity. However, unchnged corticosterone levels nd impired hippocmpl neurogenesis were found in mle rts on high-ft/ high-sugr diet for 8 months (12). Our finding tht the corticosterone levels were not significntly higher in the obese groups of both sexes further rgues ginst direct ssocition between obesity nd stress. obesity VOLUME 18 NUMBER 3 mrch 467

6 Sex differences in hippocmpus-relted lerning nd synptic plsticity in norml mice It is worth noting tht hippocmpus-dependent memory (single-trining tril contextul fer conditioning) nd synptic plsticity in norml mle mice t the ge (9 12 months) studied were better thn the ge-mtched femles. This grees with the previous reports tht the mles perform better thn the femles in severl rodent models of sptil lerning nd memory, which is hippocmpus relevnt (3,31) nd tht men hve better memory in sptil nvigtion, but not object recognition, thn women (32). It remins to be elucidted whether this sex difference is due to tht the femle mice hd lower insulin nd leptin (Tble 1), both of which positively regulte hippocmpl synptic plsticity nd memory. Impired hippocmpl synptic plsticity in obese rodents Our finding of less LTP in the hippocmpl CA1 region of obese mle mice grees with the findings using other obese mle rt models (6,7,12). Impired presynptic function ws suggested to prtly contribute to the impirment of LTP in obese Zucker rts becuse PTP nd PPF, two forms of presynptic events, were lso impired (7). However, in our obese mouse model, presynptic relese mchinery nd trnsmission efficiency of bsl synptic trnsmission were intct becuse the input output trnsfer, PPF, nd PTP were not significntly ltered. We lso noted tht LTD, which nd LTP re both N-methyld-sprtte receptor-dependent plsticity in the hippocmpl CA1 region (33), ws impired in the obese mle mice. Sex differences in HFD-induced deficits in lerning nd synptic plsticity Impired lerning nd cognitive functions hve been reported in severl obese niml models, mostly using single sex (6,7,9,,12). We, for the first time, demonstrted tht the mle mice re more vulnerble to HFD-induced impirments of lerning functions nd synptic plsticity. Sex differences in the genes ltered by HFD feeding were lso observed in the sme mouse strin (34) nd rt model (3). The sex differences in HFD-induced deficits in lerning nd synptic plsticity might be ttributed to the more severe obesity nd metbolic dysfunction in mle mice. Higher insulin resistnce developed in the obese mle mice might mke them more vulnerble to HFD-induced impirment in lerning nd synptic plsticity. Insulin resistnce chrcterized with elevted peripherl insulin levels in dibetic or obese ptients is ccompnied by lower insulin levels nd ctivity in the brin (36). Insulin hs been known to ply positive role in mintining synptic plsticity nd memory. It cn rescue the LTP impirment in the dibetic niml model (37) nd, through nsl dministrtion, improve verbl memory in ptients with erly Alzheimer s disese (38). Leptin lso plys positive role in mintining synptic plsticity nd memory (6,7,39). It cn be trnsported cross the blood brin brrier through substrte concentrtion dependent trnsporter nd ply n importnt role in the development of hypothlmic feeding circuits (21). The serum levels of leptin nd food-intke mounts were similr between mle nd femle obese mice. It remins to be further elucidted whether their centrl levels of leptin re lso similr nd contribute to the sex difference in HFD-induced impirment in lerning nd synptic plsticity. The estrus stte of the mice is unlikely to be confounding fctor for the nonimpct observed in the femles becuse % of 9-month-old C7BL/6J mice re cyclic () nd the experiments were conducted in continuous period, covering ll the estrus sttes, if ny, in both norml nd obese femles. In conclusion, mle mice re more vulnerble thn the femles to HFD-induced weight gins, metbolic ltertions nd deficits in lerning, nd synptic plsticity. The norectic ction of estrogen could protect femle mice from the impct of HFD nd mke them gin less weights nd less insulin resistnce, leding to less chnge in lerning nd synptic plsticity. Supplementry mteril Supplementry mteril is linked to the online version of the pper t Acknowledgments This study ws supported by grnts NSC9-23-B2-9-MY3 (L.C.C.) nd NSC H-2-6-PAE (K.C.L.) from Ntionl Science Council, Tiwn, nd grnts NHRI-EX98-96NI (L.C.C.), NHRI-EX98-96NI (L.L.H.) nd NHRI-BP-93-PP-11 (C.T.C.) from Ntionl Helth Reserch Institutes, Tiwn. Disclosure The uthors declred no conflict of interest. 9 The Obesity Society REFERENCES 1. Beydoun MA, Beydoun HA, Wng Y. Obesity nd centrl obesity s risk fctors for incident dementi nd its subtypes: systemtic review nd met-nlysis. Obes Rev 8;9: Elis MF, Elis PK, Sullivn LM, Wolf PA, D Agostino RB. Lower cognitive function in the presence of obesity nd hypertension: the Frminghm hert study. Int J Obes Relt Metb Disord 3;27: Gustfson D, Rothenberg E, Blennow K, Steen B, Skoog I. An 18-yer follow-up of overweight nd risk of Alzheimer disese. Arch Intern Med 3;163: Whitmer RA, Gunderson EP, Brrett-Connor E, Quesenberry CP, Yffe K. 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