Liraglutide improves hippocampal synaptic plasticity ... - Nature

International Journal of Obesity (2013) 37, 678–684 & 2013 Macmillan Publishers Limited All rights reserved 0307-0565/13 www.nature.com/ijo

ORIGINAL ARTICLE

Liraglutide improves hippocampal synaptic plasticity associated with increased expression of Mash1 in ob/ob mice WD Porter, PR Flatt, C Ho¨lscher and VA Gault OBJECTIVE: Consumption of high-fat diet exerts adverse effects on learning and memory formation, which is linked to impaired hippocampal function. Activation of glucagon-like peptide-1 (GLP-1) signalling ameliorates detrimental effects of obesity-diabetes on cognitive function; however, mechanisms underlying these beneficial actions remain unclear. This study examined effects of daily subcutaneous treatment with GLP-1 mimetic, Liraglutide, on synaptic plasticity, hippocampal gene expression and metabolic control in adult obese diabetic (ob/ob) mice. RESULTS: Long-term potentiation (LTP) induced by area CA1 was completely abolished in ob/ob mice compared with lean controls. Deleterious effects on LTP were rescued (Po0.001) with Liraglutide. Indeed, Liraglutide-treated mice exhibited superior LTP profile compared with lean controls (Po0.01). Expression of hippocampal brain-derived neurotropic factor and neurotrophic tyrosine kinase receptor-type 2 were not significantly different, but synaptophysin and Mash1 were decreased in ob/ob mice. Treatment with Liraglutide over 21 days increased expression of Mash1 in ob/ob mice (2.0-fold; Po0.01). These changes were associated with significantly reduced plasma glucose (21% reduction; Po0.05) and markedly improved plasma insulin concentrations (2.1- to 3.3-fold; Po0.05 to Po0.01). Liraglutide also significantly reduced the glycaemic excursion following an intraperitonal glucose load (area under curve (AUC) values: 22%; Po0.05) and markedly enhanced the insulin response to glucose (AUC values: 1.6-fold; Po0.05). O2 consumption, CO2 production, respiratory exchange ratio and energy expenditure were not altered by Liraglutide therapy. On day 21, accumulated food intake (32% reduction; Po0.05) and number of feeding bouts (32% reduction; Po0.05) were significantly reduced but simple energy restriction was not responsible for the beneficial actions of Liraglutide. CONCLUSION: Liraglutide elicits beneficial effects on metabolic control and synaptic plasticity in mice with severe obesity and insulin resistance mediated in part through increased expression of Mash1 believed to improve hippocampal neurogenesis and cell survival. International Journal of Obesity (2013) 37, 678–684; doi:10.1038/ijo.2012.91; published online 5 June 2012 Keywords: cognitive function; GLP-1; ob/ob mice; Liraglutide; long-term potentiation

INTRODUCTION The hippocampus is a component of the brain directly involved in aspects of complex learning and memory processes.1 In particular, the hippocampus is directly involved in short- and long-term memory consolidation, spatial memory and navigation.2 In animal studies, hippocampal lesions or age-related changes in hippocampal function lead to impaired learning during spatial memory tasks.3,4 Importantly, studies in rodent models have consistently shown that consumption of a high-fat diet adversely affects cognitive function when assessed via a range of hippocampal-dependent behavioural tasks and paradigms.5–10 The deleterious effects of increased consumption of high-fat diet have been linked with impaired hippocampal function6,9,10 and, interestingly, adverse effects of high-fat diet and obesity on cognitive function are reversed following administration with glucagon-like peptide-1 (GLP-1) mimetics.9,10 The precise mechanisms underlying these positive actions are not yet known but evidence points to beneficial actions in improving hippocampal synaptic plasticity.11,12 Although numerous signalling molecules are involved in regulating aspects of hippocampal synaptic plasticity, two molecules which have a key role are brain-derived neurotropic

factor (BDNF) and synaptophysin.13,14 BDNF is a neurotrophin involved in regulating dendritic and axonal growth, hippocampal synaptic transmission and long-term potentiation (LTP).14–16 Hippocampal mRNA expression of BDNF is reduced in mice consuming a diet rich in saturated fat and refined sugar.17 Intracerebroventricular administration of BDNF improves cognition, prevents impairments in LTP and enhances hippocampal synaptic density in a mouse model of Alzheimer’s disease.18 BDNF induces its effects by activating neurotrophic tyrosine kinase receptor-type 2 (NTRK2).19 Furthermore, synaptophysin is a synaptic vesicle membrane protein that determines synaptic strength and modulates efficacy of learning and memory and is thought to be a ubiquitous marker of synaptic integrity.13,20,21 Although the precise role of synaptophysin in neuronal synaptic transmission is unclear, mice with genetic deletion of synaptophysin exhibit impairments in learning and memory.21 Mammalian achaete-scute homologue 1 (Mash1) is a basic helix-loop-helix protein broadly expressed in neuronal progenitor cells, which has an important role in neuronal differentiation.22,23 Mash1 immunoreactivity has been observed in subventricular zone and the subgranular zone of the dentate gyrus in adult

Diabetes Research Group, The SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine, UK. Correspondence: Dr VA Gault, Diabetes Research Group, The SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine BT52 1SA, Northern Ireland, UK. E-mail: [email protected] Received 28 November 2011; revised 20 March 2012; accepted 22 April 2012; published online 5 June 2012

Liraglutide improves neuronal synaptic plasticity WD Porter et al

679 rodents;24,25 these are areas where neurogenesis of neural stem/ progenitor cells and astrocytes occur.26,27 A recent paper has shown that chronic treatment with the GLP-1 mimetic, Exendin-4, upregulated mRNA expression of Mash1 and improved hippocampus-associated cognitive function in normal adult rats.28 Given the adverse effects of obesity and related insulin resistance on cognitive parameters and that increasingly more diabetes patients are receiving incretin-based therapies, we sought to explore potential mechanisms underlying the beneficial actions of incretin hormones. As such, this study examined actions of twice-daily administration of Liraglutide in obese diabetic (ob/ ob) mice on hippocampal synaptic plasticity via measurement of LTP induction and maintenance, and the expression of key genes involved in neuronal signalling. Additionally, measurement of locomotor activity, indirect calorimetry, energy expenditure and metabolic parameters were evaluated. Observations in ob/ob mice placed on restricted diet indicate that the central benefits of Liraglutide are attributable to the peptide itself, rather than to a reduction in energy intake.

MATERIALS AND METHODS Animals and study design Male ob/ob mice (n ¼ 16; 14–16 weeks old) derived from breeding pairs of the Aston colony29 were age-matched, divided into groups and housed individually in an air-conditioned room at 22±2 1C with a 12-h light:12-h dark cycle (08:00–20:00 hours). Mice (n ¼ 8) received twice-daily subcutaneous injections (10:30 and 16:00 hours) of Liraglutide (50 nmol kg 1 body weight (bw); GL Biochem, Shanghai, China) or saline vehicle (0.9% (w/v), NaCl) for 21 days. Body weight and non-fasting plasma glucose and insulin concentrations were monitored at regular intervals. Glucose tolerance test (18 mmol kg 1 bw; intraperitoneally), non-fasting insulin sensitivity test (50 U kg 1 bw; intraperitoneally) and in vivo electrophysiological LTP of neurotransmission were performed at the end of the study as described below. No adverse effects were observed following peptide administration. In a separate series of experiments to investigate possible effects of change in energy intake on the parameters measured, two groups of ob/ob mice (18–20 weeks old) were employed with one group (n ¼ 5) having free access to food and the other group (n ¼ 5) placed on a restricted diet, corresponding to 20% less food than eaten by the ad libitum group. A 20% reduction of energy intake over 10 days was chosen to represent a small but significant decrease of food intake at the level possibly observable in Liraglutide-treated ob/ob mice. All experiments were conducted according to UK Home Office Regulations (UK Animals Scientific Procedures Act, 1986) and the ‘Principles of Laboratory Animal Care’ (NIH publication No. 86–23, revised 1985).

Biochemical analyses Blood samples were collected from the cut tip of the tail vein of conscious mice at the times indicated in the Figures and immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, Galway, Ireland) for 30 s at 13 000 g. Plasma glucose was assayed by an automated glucose oxidase procedure30 using a Beckman Glucose Analyzer II (Beckman Instruments). Plasma insulin was assayed using a modified dextran-coated charcoal radioimmunoassay as described previously.31

Measurement of indirect calorimetry, energy expenditure, locomotor activity and food intake using an automated Oxymax complete laboratory animal monitoring system (CLAMS) system On day 21 mice were placed in CLAMS metabolic chambers (Columbus Instruments, Columbus, OH, USA) following injection of respective peptides at 12:00 hours. Following a 2 h acclimatisation period in the metabolic chambers, consumption of O2 and production of CO2 were measured for 30 s at 15 min intervals for a total period of 22 h. Respiratory exchange ratio (RER) was calculated by dividing VCO2 by VO2. Energy expenditure was calculated using RER with the following equation (3.815 þ 1.232 RER) VO2. Ambulatory locomotor activity of each mouse was measured simultaneously using the optical beams (Opto M3, Columbus Instruments). Consecutive photo-beam breaks were scored as an ambulatory movement. Activity counts in X- and Z- axes were & 2013 Macmillan Publishers Limited

recorded every minute for 22 h. Food intake was measured every minute over the 22 h period.

In vivo electrophysiological recording of LTP in CA1 region of the hippocampus The technique used for measuring LTP in the hippocampus was similar to that described previously.9 Briefly, after 21 days of treatment, animals were anaesthetised with urethane (ethyl carbamate, 1.8 g kg 1; intraperitoneally) and electrodes implanted at coordinates: 1.2 mm posterior and 1.5 mm lateral to midline for recording electrode; 2.5 mm posterior to bregma and 2 mm lateral to midline for stimulating electrode. Electrodes were slowly lowered through the cortex and upper layers of the hippocampus into the CA1 region until the appearance of a negative deflecting excitatory postsynaptic potential (EPSP) with a latency of approximately 10 ms was observed. Recordings of field EPSPs (fEPSP) were made from stratum radiatum in CA1 region of the right hippocampal hemisphere in response to stimulation of the Schaffer collateral/commissural pathway. fEPSPs were recorded on a PowerLab computerised stimulating and recording unit. Sampling speed was set at 20 kHz for recordings of fEPSPs. The high-frequency stimulation protocol for inducing long-term potentiation (LTP) consisted of three trains of 200 stimuli; interstimulus interval of 5 ms (200 Hz); and inter-train interval of 2 s.9

RNA extraction, cDNA synthesis and quantitative PCR Hippocampus tissue (n ¼ 6) was excised following measurement of LTP and immediately snap-frozen in liquid nitrogen and stored at 80 1C before RNA extraction for gene expression analysis. Briefly, total RNA was isolated and purified using QIAzol lysis reagent (Qiagen, West Sussex, UK) and RNA concentration determined from the absorbance at 260 nm. Firststrand cDNA was synthesised using 2 mg of total RNA at 42 1C for 50 min in the presence of 0.5 mg oligo dT(12–18) primer, 10 mM dNTP and 200 U Superscript II reverse transcriptase (Invitrogen, Paisley, UK) in a final volume of 20 ml using a GeneStorm GS1 Thermal Cycler (Gene Technologies Ltd, Essex, UK). Genes were amplified using specific primers for BDNF (sense: 5’-GGA GAG CAG AGT CCA TTC AGC ACC T-30 and antisense: 5’-TCC AGC CCC GAT CTC GGT GTG-30 ); NTRK-2 (sense: 5’-GGCCGGCACTGTCCTGCT ACC-30 and antisense: 5’-CGC AGA ACC GCT AAA CCG GC-30 ); Synaptophysin (sense: 5’-CCC CAT TCA TGC GCG CAC CT30 and antisense: 5’-CCC TGC CCA TAG CCC GCA TC-30 ), Mash1 (sense: 5’GGG GGC GGT CAC AAG TCA GC-30 and antisense: 5’-ACT TGA CCC GGT TGC GCT CG-30 ) and HPRT (sense: 5’-AAG CTT GCT GGT GAA AAG GA-30 and antisense: 5’-TTG CGC TCA TCT TAG GCT TT-30 ). The DNA-denaturing step was carried out at 95 1C for 5 min in a Roche LightCycler 1.5 carousel-based thermal cycler (Roche Diagnostics, West Sussex, UK). cDNA amplification then commenced for 40 cycles with 95 1C denaturation for 30 s, 58 1C annealing for 30 s and 72 1C elongation for 30 s with SYBR green fluorescence being read after each cycle and recorded by Roche LightCycler Software (Version 3.5) to construct an amplification curve. Gene expression was calculated from 2DCt values normalised to mouse HPRT control primer.

Presentation of data and statistical analysis Data were analysed using repeated measures one-way or two-way analysis of variance (ANOVA) with Bonferroni or Student–Newman–Keuls post hoc tests or repeated measure two-tailed t-tests using PRISM 3.0 (GraphPad Software Inc., San Diego, CA, USA). Data are expressed as mean±s.e.m. and a P-value o0.05 was considered statistically significant.

RESULTS Effects of Liraglutide on metabolic parameters in ob/ob mice The ob/ob mice exhibited increased bodyweight and elevated concentrations of glucose and insulin, impaired glucose tolerance and insulin resistance compared with respective values for agematched littermate controls (Po0.05 to Po0.001; data not shown). Daily administration of Liraglutide to ob/ob mice had no significant effect on body weight compared with saline-treated controls over the 21-day period (Figure 1a). However, by day 21 non-fasting plasma glucose concentrations were significantly decreased (21% reduction; Po0.05; Figure 1b). In agreement with this, non-fasting plasma insulin levels were significantly elevated International Journal of Obesity (2013) 678 – 684


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Figure 1. Effects of Liraglutide on body weight (a), plasma glucose (b), plasma insulin (c), glucose tolerance (d), plasma insulin response to glucose (e) and insulin sensitivity (f ) in ob/ob mice. Animals received twice-daily injections of Liraglutide (50 nmol kg 1 bw; subcutaneously (sc)) or saline vehicle for 21 days. Metabolic parameters (a–c) were measured at 2–3-day intervals and glucose tolerance and insulin sensitivity tests were conducted at the end of the study. Values are means±s.e.m. for eight mice. *Po0.05 and **Po0.01 compared with saline-treated ob/ob mice.

(2.1 to 3.3-fold; Po0.05 to Po0.01) in Liraglutide-treated mice compared with ob/ob saline controls (Figure 1c). Treatment with Liraglutide significantly improved the glycaemic response following a glucose load with individual 15, 30 and 60 min post-injection glucose values significantly decreased (34%, 22% and 17% reduction, respectively; Po0.05 to Po0.01; Figure 1d) compared with saline controls. Similarly, plasma glucose AUC (0–60 min) values integrating overall glycaemic excursion was significantly reduced (22% reduction; Po0.05; Figure 1d) for Liraglutide-treated mice. Individual plasma insulin responses were not significantly changed for Liraglutide-treated mice, although values were close to statistical significance (Figure 1e). However, overall plasma insulin response as measured by integrated AUC (0–60 min) was significantly elevated (1.6-fold; Po0.05; Figure 1e) for Liraglutide-treated mice. Furthermore, following exogenous administration of insulin, the percentage plasma glucose change from basal did not show any significant difference between Liraglutide-treated and control groups (Figure 1f). Effects of Liraglutide on indirect calorimetry and energy expenditure in ob/ob mice Daily administration of Liraglutide had no significant effect on O2 consumption (Figures 2a and d) or CO2 production (Figures 2b and 2e) in ob/ob mice compared with saline controls. However, as shown in Figures 2d and e, lean littermate animals displayed significantly elevated O2 consumption and CO2 production compared with Liraglutide-treated (1.9 and 2.2-fold, respectively; Po0.001) and saline control ob/ob mice (2.0 and 2.6-fold, respectively; Po0.001). RER for Liraglutide-treated mice was unchanged compared with saline control ob/ob controls, but was significantly elevated in lean mice (1.3-fold; Po0.001; Figures 2c and f). Lean mice exhibited increased energy expenditure International Journal of Obesity (2013) 678 – 684

(13.85±0.51 Kcal h 1 kg 1; Po0.001) compared with Liraglutide and saline control ob/ob mice (7.02±0.32 and 6.39±0.67 Kcal h 1 kg 1, respectively). Effects of Liraglutide on locomotor activity and feeding in ob/ob mice Daily administration of Liraglutide had no significant effect on locomotor activity compared with ob/ob control mice (Figure 3). Both saline control and Liraglutide-treated ob/ob mice exhibited decreased general activity (X beams broken; Figure 3a; Po0.001), ambulatory activity (different X beams broken; Figure 3b; Po0.001) and exploratory behaviour (Z beams broken; Figure 3c; Po0.01) compared with lean mice. Liraglutide-treated ob/ob mice demonstrated significantly decreased number of feeding bouts (32% reduction; Po0.05; Figure 3d) and accumulated food intake (32% reduction; Po0.05; Figure 3e) compared with saline control mice with levels similar to that of lean littermate animals. Effects of Liraglutide on LTP in hippocampal CA1 region in ob/ob mice In the analysis of in vivo LTP induced by high-frequency stimulation in area CA1 of the hippocampus, LTP was completely abolished in saline-treated ob/ob mice (Figure 4). Daily treatment with Liraglutide ameliorated LTP (Figure 4) with a two-level twoway ANOVA, indicating a significant difference between ob/ob saline controls and Liraglutide-treated mice (DF1,10; F ¼ 19.1; Po0.001) and over time (DF1,119; F ¼ 1.5; Po0.001). Interaction between factors was not significant. Similarly, a two-level two-way ANOVA showed a significant difference in LTP between ob/ob saline controls and lean littermate controls (DF1,10; F ¼ 16.2; Po0.001) and over time (DF1,119; F ¼ 1.38; Po0.005). Interaction between factors was not significant. Finally, there was a significant & 2013 Macmillan Publishers Limited


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Figure 2. Effects of Liraglutide on O2 consumption, CO2 production and RER in ob/ob mice. Parameters were measured after twice-daily treatment with Liraglutide (50 nmol kg 1 bw; sc) or saline vehicle for 21 days. Lean control mice were included for comparative purposes. Mice were placed in CLAMS metabolic chambers and O2 consumption or CO2 production were measured for 30 s at 15 min intervals (a, b, d, e). Respiratory exchange rate (RER) (c, f) was calculated by dividing VCO2 by VO2. Values are means±s.e.m. for eight mice. ***Po0.001 compared with saline-treated ob/ob mice. DDDPo0.001 compared with Liraglutide-treated ob/ob mice.

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Figure 3. Effects of Liraglutide on locomotor and feeding activity in ob/ob mice. Parameters were measured after twice-daily treatment with Liraglutide (50 nmol kg 1 bw; sc) or saline vehicle for 21 days. Lean control mice were included for comparative purposes. Mice were placed in CLAMS metabolic chambers and locomotor activity was measured using optical beams. Activity in X- and Z-axes was recorded every minute for 22 h (a–c). Accumulated food intake and feeding bouts were measured every minute over the 22 h period (d, e). Values are means±s.e.m. for eight mice. *Po0.05, **Po0.01 and ***Po0.001 compared with saline-treated ob/ob mice. DDPo0.01 and DDDPo0.001 compared with Liraglutide-treated ob/ob mice.

difference noted between Liraglutide-treated ob/ob mice and lean littermate controls as found by two-level two-way ANOVA, (DF1,10; F ¼ 9.2; Po0.01). No difference over time or interaction between factors was found. Effects of Liraglutide on hippocampal gene expression in ob/ob mice There were no significant differences in hippocampal mRNA expression of BDNF, NTRK2 or synaptophysin between salinetreated ob/ob mice and Liraglutide-treated mice (Figure 5). However, there was a significant enhancement (2.0-fold; Po0.01) in Mash1 expression for Liraglutide-treated ob/ob mice & 2013 Macmillan Publishers Limited

compared with saline-treated ob/ob controls (Figure 5d). Furthermore, there was a significant increase in mRNA expression of synaptophysin (1.5-fold; Po0.05) and Mash1 (2.2-fold; Po0.01) in lean mice compared with ob/ob saline-treated controls (Figures 5b and d). Effects of food restriction on metabolic parameters, hippocampal LTP and expression of Mash1 in ob/ob mice Food restriction was used to establish whether decreased energy intake has any role in the above actions of Liraglutide. The ob/ob mice placed on a restricted diet did not exhibit significant differences in body weight (80.7±7.1 vs 84.5±3.5 g), plasma International Journal of Obesity (2013) 678 – 684


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Figure 4. Effects of Liraglutide on LTP in hippocampal CA1 region in ob/ob mice. Mice received twice-daily injections of Liraglutide (50 nmol kg 1 bw; sc) or saline vehicle for 21 days. Lean littermate control mice were included for comparative purposes (a). (b) Mice placed on a 20% restricted diet or given food ad libitum. A two-level two-way ANOVA found a significant difference between ob/ob controls and Liraglutide-treated ob/ob groups (Po0.001). Similarly, a two-level two-way ANOVA showed a significant difference in LTP between ob/ob control group and lean littermate controls (Po0.001). There was a significant difference noted between Liraglutide-treated ob/ob mice and lean littermate controls by twolevel two-way ANOVA, (Po0.01). Values are means±s.e.m. for six mice.

glucose (19.5±6.9 vs 24.1±2.1 mM) and plasma insulin (2.3±0.2 vs 2.0±0.3 ng ml 1) compared with ob/ob mice on unrestricted diet. Similarly, there were no significant differences in glucose tolerance (AUC 0–60 min, 894.5±110.3 vs 859.0±99.3 mM.min) or insulin response to glucose (AUC 0–60 min, 77.8±15.2 vs 85.3±16.6 ng ml 1.min) compared unrestricted controls. A twolevel two-way ANOVA showed no significant difference in LTP between ob/ob mice on restricted diet compared with ob/ob mice on unrestricted diet (Figure 4b). Similarly, hippocampal mRNA levels of Mash1 were not significantly different for mice on restricted vs unrestricted diet (Figure 5e).

DISCUSSION Obesity is a major contributor to development of insulin resistance, impaired glucose tolerance and progression to type 2 diabetes mellitus.32,33 Recent observations in animal and human studies have shown that high-fat diet, obesity and related insulin resistance have profound adverse effects on cognitive function.34–37 Moreover, there is now emerging evidence to suggest that the negative effects of high-fat diet and obesity on brain performance is reversed by incretin hormones.9,10,38 In this study, we assessed the impact of obesity and insulin resistance on metabolic control and probed potential mechanisms underlying these adverse effects on brain dysfunction. Furthermore, we evaluated the potential of the GLP-1 mimetic Liraglutide to reverse these detrimental effects. We chose to use ob/ob mice, which represent a well-established animal model of spontaneous obesity and type 2 diabetes.29 These mice have a homozygous recessive mutation in the ob gene, which encodes the hormone International Journal of Obesity (2013) 678 – 684

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Figure 5. Effects of Liraglutide on hippocampal gene expression in ob/ob mice. Mice received twice-daily injections of Liraglutide (50 nmol kg 1 bw; sc) or saline vehicle for 21 days. Following excision of the hippocampus gene expression of BDNF (a), Synaptophysin (b), NTRK-2 (c) and Mash1 (d) were examined and expression quantified using quantitative PCR and values normalised to the levels of the internal control gene HPRT. Lean littermate control mice were included for comparative purposes. (e) Mice placed on a 20% restricted diet or given food ad libitum. Values are means±s.e.m. for five mice *Po0.05 and **Po0.01 compared with saline-treated ob/ob mice.

leptin, a 167 amino-acid protein expressed primarily in adipocytes.39–41 The ob/ob mice lack functional leptin, which leads to a marked increase in food consumption, reduced energy expenditure and deposition of excess fat in adipose tissue.42 Consequently, ob/ob mice exhibit an extreme phenotype and represent one of the most robust animal models for obesityrelated diabetes.43 In-line with previous observations, ob/ob mice exhibited gross obesity, hyperglycaemia and hyperinsulinaemia when compared with age-matched lean control mice.29 Consistent with previous studies, twice-daily administration of Liraglutide significantly reduced plasma glucose concentrations and enhanced plasma insulin in ob/ob mice.44,45 Liraglutide did not result in any significant decrease in body weight, which could be due to the relatively short duration of the present study. Similar effects have previously been noted where Liraglutide dose-dependently reduced glycaemic excursion in ob/ob mice independent of effects on body weight.45 Similarly, another GLP-1 agonist, Exendin-4, significantly reduced plasma glucose levels over a 14-day period in ob/ob mice independent of changes on body weight and food intake.46 However, in harmony with known actions of Liraglutide as a glucoregulatory hormone, Liraglutide-treated ob/ ob mice exhibited markedly improved glucose tolerance, which was associated with significantly improved plasma insulin responses.44 Insulin sensitivity was not improved, suggesting that the glucoselowering actions of Liraglutide, similar to other incretin mimetics, is attributed largely to its insulinotropic action. & 2013 Macmillan Publishers Limited


683 When examined using CLAMS, ob/ob mice exhibited significantly lower O2 consumption, CO2 production, RER and energy expenditure compared with age-matched lean littermates. The differences between obese and lean animals most likely stems, at least in part, from decreased locomotor activity, in that, the less active the mice the lower the oxygen consumption and energy expended. Furthermore, 21 days of Liraglutide treatment did not alter rates of O2 consumption, CO2 production, RER and energy expenditure. Saline-treated ob/ob mice exhibited marked hyperphagia as shown by a notable increase in cumulative food intake and feeding bouts. Interestingly, Liraglutide therapy decreased food intake in ob/ob mice, indicative of the signalling of GLP-1 receptor pathways in the central nervous system, which promote satiety.47 However, both body weight and food intake were not significantly decreased over the entire 21-day study, suggesting that the benefits of Liraglutide are not consequent to a reduction of energy intake. Previous observations have noted that hippocampal LTP induction and synaptic plasticity are severely compromised in mice maintained on a high-fat diet.9,10 Importantly though, this marked impairment was ameliorated by chronic administration of the GLP-1R mimetics Exenatide and Liraglutide. Furthermore, a direct enhancing effect of GLP-1R signalling is also supported by studies in GLP-1R knockout mice that exhibit impairment of recognition and spatial learning and almost complete abolition of LTP in area CA1 of the hippocampus.48 Consistent with these findings, LTP was abolished in ob/ob mice, whereas, Liraglutide treatment rescued the impairment in LTP. Furthermore, our data clearly show that treatment with Liraglutide resulted in a superior LTP profile than lean controls. This result is consistent with our previous findings that Liraglutide protects learning abilities and enhances LTP in a mouse model of Alzheimer’s disease.49 Furthermore, Liraglutide also protected synapses from degeneration in the hippocampus, which is most likely the basis for the preserved cognitive abilities and LTP.49 Thus, the cognitiveenhancing properties of Liraglutide in modulating neurotransmitter release and LTP formation in the hippocampus are important and further highlight a cardinal role for GLP-1 in mediating neuroprotective effects in specific brain regions associated with learning and memory.11,12 In an attempt to elucidate possible underlying mechanisms that may cause this impairment in LTP and its reversal by Liraglutide, hippocampal gene expression was performed for several key genes involved in neuronal synaptic transmission and progenitor cell differentiation. There were no significant alterations in the gene expression of BDNF between any of the groups tested. This is somewhat surprising, given that BDNF is known to exhibit cognitive-enhancing properties50,51 and its expression is reduced in STZ-induced diabetic rats.52,53 In support of this, we did not observe any changes in the expression of NTRK2, through which BNDF mediates its neurotrophic effects.19 Despite no changes in BDNF or NTRK2 expression, we observed that the expression of synaptophysin was reduced in ob/ob mice compared with lean littermates. Comparable to the findings in this study, an agedependent reduction in hippocampal synaptophysin immunoreactivity was reported in aged rats54 and in other APP/PS1 mouse models of Alzheimer’s disease.49 In contrast, increased synaptophysin immunoreactivity was observed in the tg2576 APP mouse model of Alzheimers disease, which was described as a compensatory process reflecting the onset of cognitive decline.55 Moreover, this study would also suggest that LTP enhancement demonstrated in Liraglutide-treated ob/ob mice is independent of changes in synaptophysin, whose levels of mRNA expression were similar to control ob/ob mice. As ob/ob mice are deficient in leptin signalling, and as leptin has an important role in synaptic plasticity in the hippocampus,56 it appears that synaptophysin expression is affected by the lack of leptin. However, it has been reported that synaptophysin is not & 2013 Macmillan Publishers Limited

essential for synaptic transmission as related synaptic vesicle proteins like synaptoporin/syn II and the synaptogyrins that interact with synaptophysin may compensate for any reduction in synaptophysin levels.57 Whether or not this is the case is unclear, as the exact mechanism of action and function of synaptophysin remain unknown. Further examination of the role of synaptophysin is required through protein analysis, enzymelinked immunosorbent assay and immunohistochemical staining to determine the function of synaptophysin in synaptic transmission. Interestingly, there were significant differences observed in mRNA expression of Mash1 between the Liraglutide-treated and saline control ob/ob mice. The upregulation of mRNA expression of Mash1 indicates that Liraglutide is involved in proliferation and neuronal differentiation in the hippocampus. In agreement with this, the GLP-1 agonist, Exendin-4, has also been shown to increase Mash1 mRNA expression as well as increase progenitor cell proliferation in the dentate gyrus in rodents.28,58 Furthermore, a recent study in our laboratory has shown that daily Exendin-4 and Liraglutide administration increased the number of progenitor cells in the dentate gyrus of three mouse models of diabetes: ob/ob mice, db/db mice and high-fat-fed mice.38 Given that the observed neural effects in this study do not appear to be dependent on insulin action, Liraglutide is likely to exert its effects directly by entry across the blood–brain barrier or through activation of GLP-1 receptors in the intestine–hepatic portal liver area to communicate via vagal afferents. Interestingly, these actions of Liraglutide were not dependent on reduction of food intake as illustrated by lack of effect of restricted feeding in ob/ob mice. Overall, these studies suggest GLP-1 agonists act to improve cognition, neurogenesis and cell survival in the hippocampal dentate gyrus.

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS These studies were supported by an EFSD/GSK grant and University of Ulster Strategic Research Funding.

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