Exendin-4 improves long-term potentiation and neuronal 1 dendritic growth in high-fat diet mice and neurons under 2 metabolic imbalance conditions

31 Metabolic syndrome, which increases the risk of obesity and type 2 diabetes, has emerged as a 32 significant issue worldwide. Metabolic syndrome can occur due to diverse factors such as 33 genetic background, lifestyle changes, food intake, and aging. Recent studies have highlighted 34 the relationship between metabolic imbalance and neurological pathologies, such as synaptic 35 dysfunction and memory loss. Glucagon-like peptide 1 (GLP-1) secreted from gut L-cells, and 36 specific brain nuclei play multiple roles, including glucose metabolism, regulation of insulin 37 sensitivity, inflammation control, synaptic plasticity improvement, and neuronal protection. 38 Even though GLP-1 and GLP-1 receptor agonists (GLP-1RA) appear to have neuroprotective 39 functions, the specific mechanisms of GLP-1 and GLP-1RA in brain function have remained 40 unclear. Here, we investigated whether exendin-4 improves cognitive function and brain 41 insulin resistance in metabolic imbalanced high-fat diet mice brain as a GLP-1RA, using 42 electrophysiological experiments. Further, we identified the neuroprotective effect of exendin- 43 4 in primary cultured hippocampal and cortical neurons under an in vitro metabolic imbalance 44 condition, including neuronal structure improvement. This study provides significant findings 45 on the effects of exendin-4 in synaptic plasticity, long-term potentiation (LTP), 46 neuroinflammation, and neural structure. We suggest that GLP-1 may be vital to treating 47 neuropathology caused by metabolic imbalance. 48

Introduction glucagon level 11 . A clinical study proved that the administration of GLP-1RA could improve 78 diabetic pathology in patients with type 2 diabetes 12 . 79 Furthermore, the treatment of GLP-1RA could effectively suppress increased glycemic 80 parameters such as hemoglobin A1c (HbA1c) and fasting glucose, reduce body weight, and 81 suppress inflammatory cytokine secretion 13 . In addition, GLP-1RA could improve the lipid 82 profile, promote hypothalamic connectivity in the CNS, and improve the limbic system circuit 83 in the CNS involved in feeding behavior 14 . 84 At a cellular level, GLP-1 has multiple functions, including the activation of protein kinase A 85 (PKA) and 3',5'-cyclic adenosine monophosphate (cAMP) signaling, the cytoplasmic Ca 2+ 86 pathway 15 , and various mitogen-activated protein kinase (MAPK) pathways 16 . 87 In the CNS, GLP-1Rs are observed in diverse brain areas, including the hypothalamus. GLP-88 1 signaling is involved in several brain functions, including feeding satiety, reward circuit, and 89 stress response 17 . In particular, GLP-1Rs expressed in the hippocampus region are related to 90 learning and memory function 18 . 91 Recently, GLP-1 and GLP-1RA have been highlighted in the CNS field as they may protect 92 neurons against oxidative stress and ultimately protect the process and onset of neuronal 93 diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis 94 through diverse CNS mechanisms 19 . 95 One study reported that the intracerebroventricular injection of GLP-1 resulted in the dramatic 96 improvement of memory function dependent on the hippocampus in rodents 20 . 97 Previous study has shown that GLP-1 attenuates the incidence of cognitive deficit in patients 98 with type 2 diabetes 21 . Furthermore, GLP-1 neuritogenesis changes (long-term potentiation 99 [LTP]) are improved by GLP-1 treatment and enhance cognitive deficit 22 . 100 As mentioned above, GLP-1 and GLP-1RA have multiple beneficial effects in neuropathology. 101 In this study, we investigated whether exendin-4 as GLP-1RA rescues LTP and promotes 102 6 synaptic plasticity in the high-fat diet (HFD) mice brain. Moreover, our in vitro study examines 103 which mechanisms are involved in the effect of exendin-4 in hippocampal neurons under a 104 metabolic imbalance condition. Our findings demonstrate the significant potential of GLP-1, 105 which may improve cognitive decline in the HFD brain by attenuating neuroinflammation, 106 enhancing the neural structure, and enhancing LTP.   Furthermore, we have tried to develop an in vitro neuron model to mimic insulin resistance in 144 HFD mice brains. The primary hippocampal and cortical neurons were exposed to a 145 combination of tumor necrosis factor-alpha (TNF-α), insulin, glucose, and palmitate. We 146 observed that the metabolic imbalance alleviated insulin sensitivity in the primary hippocampal 147 (Fig. 3a) and cortical neurons (Fig. 3b). The levels of phosphorylated protein related to insulin 148 sensitivity such as IRS-1, AKT, and GSK-3β decreased under the metabolic imbalance 149 condition compared to the normal state. However, exendin-4 increased the phosphorylation of 150 proteins in the metabolic imbalance hippocampal and cortical neurons, indicating that exendin-151 4 enhances insulin sensitivity. Besides, a protein related to both neuroinflammation and insulin 152 8 resistance, p65 NF-κB, was highly phosphorylated in the metabolic imbalance condition 153 compared to the normal condition. However, exendin-4 recovered the degree of p65 NF-κB 154 phosphorylation in the metabolic imbalanced hippocampal and cortical neurons. We concluded 155 that exendin-4 improves insulin sensitivity in HFD mice and in vitro metabolic imbalance 156 neurons from these protein phosphorylation analyses. 157 We observed that exendin-4 improves synaptic plasticity in HFD mice (Fig. 5). Altogether, 158 these results suggest that the metabolic imbalances observed in HFD mice exacerbated neuron 159 growth, neurite generation, and complexity of neurons in the brain, resulting in the depression  the dendritic spine's shape depending on the degree of maturation. The dendritic spine's shape 174 was classified and measured depending on the length and width (Fig. 2b). We observed a 175 remarkable decrease in the diversity of the dendritic spine shape in neurons under the metabolic 176 imbalance condition compared to the normal condition ( Fig. 2a and 2c). While the dendritic 9 spine's overall shape was partially reduced, the number of stubby, mushroom, and branched 178 dendritic spines markedly decreased in the metabolic imbalanced condition compared to the 179 normal condition. However, exendin-4 changed the spine spread pattern in in vitro neurons 180 under the metabolic imbalance condition. Overall, exendin-4 increased the number of spines.

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In particular, the filopodia-like spine shape was dramatically formed in the exendin-4 treated 182 metabolic imbalanced neurons. Exendin-4 also augmented the number of stubby, mushroom, 183 and branched spines in metabolic imbalanced neurons, suggesting that exendin-4 increased 184 dendritic spine morphogenesis and maturation after neuronal damages by metabolic imbalance. 185 We found that protein expression related to PSD-95 was remarkably downregulated in 186 metabolic imbalanced neurons compared to under normal conditions. However, exendin-4 187 increased the expression of PSD-95 protein, as shown in dendritic spine morphology (Fig. 2d). 188 These results indicate that metabolic imbalance alleviates the diversity of the neural structure the p-p65 NF-κB (S536) protein levels increased in the mice exposed to HFD. However, 200 exendin-4 specifically decreased the p-p65 NF-κB (S536) protein levels in the hippocampus 201 (n=4, P < 0.01 vs. control; Fig. 4a). An assessment of the total p65 NF-κB protein levels showed     in the HFD-fed mice hippocampus (n=4, P < 0.05 vs. control; Fig. 5d). These results revealed 228 that exposure to HFD impairs high-frequency stimulation-triggered LTP in the hippocampus. 229 However, the presence of exendin-4 reverses the LTP deficiency, suggesting that exendin-4 230 improves HFD-impaired hippocampal synaptic plasticity. In the present study, we investigated whether GLP-1 contributes to the neural structure, 234 synaptic plasticity, LTP improvement, neuroinflammation, and insulin sensitivity in the HFD 235 mouse hippocampus and primary cortical neurons under metabolic imbalance conditions. 236 Previous study mentioned that the HFD-induced obesity mouse brain showed brain insulin 237 resistance, synaptic failure, impaired neurogenesis, neurotransmitter imbalance, severe 238 neuroinflammation, and memory loss 25 .

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Here, we suggest significant and novel findings regarding the therapeutic effects of GLP-1 240 in the obese brain hippocampus and metabolic imbalance stress exposure in primary cortical 241 and hippocampal neurons.  Next, we found that exendin-4 could ameliorate brain insulin resistance in mice brain, and 272 cortical and hippocampal neurons despite metabolic imbalance conditions. Insulin has a 273 neuroprotective effect and promotes energy homeostasis and neuronal differentiation in the 274 CNS 41 . In the diabetic and obese brain, insulin cannot act normally in neuronal cells, called 275 "brain insulin resistance," leading to neuronal cell death, synaptic failure, and impaired glucose 276 metabolism in the brain 42 . A recent study found that brain insulin resistance can damage  Considering these results and previous findings of the impairment of insulin resistance in the 292 obese brain 51 , we assume that GLP-1 improves brain insulin resistance, and has a potential to 293 enhance cognitive decline through the IRS-1/AKT signal pathway in the obese brain.

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Finally, we observed that exendin-4 treatment contributes to the improvement of LTP in the 295 HFD mouse brain hippocampus. LTP has been measured in several brain regions, including reported that brain insulin resistance leads to memory loss and inhibits the activity of the IRS-  In the type 2 diabetes rat model brain, low LTP in the hippocampus region has been found 55 302 and is linked to insulin signaling defects and the inappropriate secretion of neurotransmitters 303 such as GABA 56 . One study reported that GLP-1 receptor knockout mouse showed memory 304 loss in the hippocampus compared to the controls 20 . Another study mentioned that GLP-1 305 administration could rescue memory loss and synaptic dysfunction and decrease GSK-3β 306 activity in the AD mouse model 57 .

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GSK-3β activity is an important sign in the brain because GSK-3β regulates synaptic and 308 mitochondrial function as well as amyloid β toxicity in the brain 58 .  Considering that spine maturation, synaptic plasticity, and neuronal connectivity are reduced 315 in metabolic imbalance conditions such as diabetes and obesity 60 , we assumed that exendin-4 316 may recover the neural structure and synaptic plasticity in the obese brain. Therefore, GLP-1 317 may be vital to the treatment of the diverse neuropathology caused by metabolic imbalance.     To analyze the neural structure and protein phosphorylation related to insulin signaling, all 351 drugs were treated at 2-day intervals until the primary neurons became day 7 in vitro (DIV 7).

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To analyze the dendritic spine shape and PSD-95 protein expression, all drugs were treated at 353 2-day intervals from 4 days before the primary neurons became day 16 in vitro (DIV 16). The mice were sacrificed between 9:00 and 10:00 a.m. by dislocating the cervical vertebrae.