Intermittent Fasting Reduces Neuroinflammation and Cognitive Impairment in High-Fat Diet-Fed Mice by Downregulating Lipocalin-2 and Galectin-3

Intermittent Fasting Reduces Neuroinflammation and Cognitive Impairment in High-Fat Diet-Fed Mice by Downregulating Lipocalin-2 and Galectin-3 1 2 * † Nutrients 2024 , 16 (1), 159; https://doi.org/10.3390/nu16010159 Abstract : 1. Introduction 2. Materials and Methods 2.1. Animals and IF Mouse Model 2.2. Echo MRI 2.3. Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT) 2.4. Enzyme-Linked Immunosorbent Assay (ELISA) 2.5. Hematoxylin and Eosin (H&E) Staining 2.6. Terminal Deoxynucleotidyl Transferase Dutp Nick end Labeling (TUNEL) Assay 2.7. Western Blot Analysis 2.8. Double or Triple Immunofluorescences 2.9. Morris Water Maze (MWM) 2.10. Statistical Analysis 3. Results 3.1. IF Attenuates Adipocyte Death and Macrophage Infiltration in the WAT of HFD Mice 3.2. IF Reduces Circulating and WAT LCN2 Protein Levels in HFD Mice 3.3. IF Reduces Circulating and WAT GAL3 Protein Levels in HFD Mice 3.4. IF Improves Memory Deficits in HFD Mice 3.5. IF Inhibits BBB Leakage in the Hippocampus of HFD Mice 3.6. IF Reduces Microglial GAL3 and Astrocytic LCN2 in the Hippocampus of HFD Mice 3.7. IF Reduces Hippocampal Inflammation in HFD Mice 4. Discussion 5. Conclusions Supplementary Materials Author Contributions Funding Institutional Review Board Statement Informed Consent Statement Data Availability Statement Acknowledgments Conflicts of Interest References Blüher, M. Obesity: Global epidemiology and pathogenesis. Nat. Rev. Endocrinol. 2019 , 15, 288–298. [Google Scholar] [CrossRef] [PubMed] Lee, Y.S.; Li, P.; Huh, J.Y.; Hwang, I.J.; Lu, M.; Kim, J.I.; Ham, M.; Talukdar, S.; Chen, A.; Lu, W.J.; et al. Inflammation is necessary for long-term but not short-term high-fat diet-induced insulin resistance. Diabetes 2011 , 60, 2474–2483. [Google Scholar] [CrossRef] [PubMed] Kim, K.E.; Jeong, E.A.; Lee, J.Y.; Yi, C.O.; Park, K.A.; Jin, Z.; Lee, J.E.; Horvath, T.L.; Roh, G.S. 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Scale bar, 100 μm. ( F ) Quantification of CLSs in H&E-stained sections. ( G ) Quantification of TUNEL-positive cells in TUNEL-stained sections. ( H ) Western blot analysis of Bcl-2 and Bax proteins in WAT lysates (n = 3–4). Quantification of Bax-to-Bcl-2 ratio. ( I ) Representative images of double immunofluorescence staining of F4/80 (green) and perilipin-1 (red) in WAT sections. Nuclei were stained with DAPI. Scale bar, 100 µm. Significance was determined by one-way ANOVA. * p < 0.05 versus ND. † p < 0.05 versus HFD. Figure 2. Effects of IF on serum and WAT LCN2 protein levels in HFD mice. ( A ) Serum LCN2 levels (n = 6) as assessed using ELISA. ( B ) Western blot and quantitative analysis of LCN2 protein in WAT lysates (n = 3–4). Protein levels were normalized to α-tubulin from the same immunoblot. ( C ) Representative images of triple immunofluorescence staining of LCN2 (red), MPO (green), and F4/80 (purple) in WAT sections. Nuclei were stained with DAPI. Scale bar, 50 µm. Significance was determined by one-way ANOVA. * p < 0.05 versus ND. † p < 0.05 versus HFD. Figure 3. Effects of IF on serum and WAT GAL3 protein levels in HFD mice. ( A ) Serum GAL3 levels (n = 6–8) as assessed using ELISA. ( B ) Western blot and quantitative analysis of GAL3 protein in WAT lysates (n = 3–4). Protein levels were normalized to α-tubulin from the same immunoblot. ( C ) Representative images of GAL3 (green) and LCN2 (red) double immunofluorescence staining in WAT sections. Nuclei were stained with DAPI. Scale bar, 50 µm. Significance was determined by one-way ANOVA. * p < 0.05 versus ND. † p < 0.05 versus HFD. Figure 4. Effect of IF on cognitive impairment in HFD mice. ( A ) Latency to reach the target platform over 4 days of MWM training (n = 7). ( B , C ) Swimming speed ( B ) and swimming distance ( C ) on the test day (n = 7). ( D ) Representative images of swimming paths without the platform during testing. Red lines indicate the swimming path. Numbers of target zone quadrant ( E ) and platform ( F ) crossings on the test day (n = 7). Black dashed circles indicate the location of the hidden platform. Significance was determined by one-way ANOVA. * p < 0.05 versus ND. † p < 0.05 versus HFD. Figure 5. Effect of IF on BBB leakage in the hippocampus of HFD mice. ( A ) Western blot and quantitative analysis of claudin-5, ZO-1, ICAM-1, and MMP9 in hippocampal lysates (n = 3–4). Protein levels were normalized to β-actin from the same immunoblot. ( B ) Serum MMP9 level (n = 6). ( C ) Representative images of double immunofluorescence staining of AQP4 (green) and albumin (red) in hippocampal sections. White arrows indicate AQP4-positive astroglial endfeet. Yellow arrow indicates extravascular albumin. Nuclei were stained with DAPI. Scale bar, 50 µm. ( D ) Quantitative analysis of albumin from ( C ). Significance was determined by one-way ANOVA. * p < 0.05 versus ND. † p < 0.05 versus HFD. Figure 6. Effects of IF on GAL3 and LCN2 protein in the hippocampus of HFD mice. ( A ) Western blot and quantitative analysis of GAL3 and LCN2 in hippocampal lysates (n = 3–4). Protein levels were normalized to β-actin. ( B ) Representative images of double immunofluorescence staining of GAL3 (green) and Iba-1 (red), GAL3 (green) and GFAP (purple), or LCN2 (red) and GFAP (green) in hippocampal sections. Arrows indicate co-localized GAL3 and Iba-1-positive microglia. Nuclei were stained with DAPI. Scale bar, 50 µm. Significance was determined by one-way ANOVA. * p < 0.05 versus ND. † p < 0.05 versus HFD. Figure 7. Effects of IF on neuroinflammation in the hippocampus of HFD mice. ( A , B ) Western blot ( A ) and quantitative analysis ( B ) of TNF-α, TNFR1, IL-6, HMGB1, and RAGE in hippocampal lysates (n = 3–4). Protein levels were normalized to β-actin from the same immunoblot. Significance was determined by one-way ANOVA. * p < 0.05 versus ND. † p < 0.05 versus HFD. 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Nutrients 2024 , 16 , 159. https://doi.org/10.3390/nu16010159 AMA Style Lee J, An HS, Shin HJ, Jang HM, Im CO, Jeong Y, Eum K, Yoon S, Lee SJ, Jeong EA, et al. Intermittent Fasting Reduces Neuroinflammation and Cognitive Impairment in High-Fat Diet-Fed Mice by Downregulating Lipocalin-2 and Galectin-3. Nutrients . 2024; 16(1):159. https://doi.org/10.3390/nu16010159 Chicago/Turabian Style Lee, Jaewoong, Hyeong Seok An, Hyun Joo Shin, Hye Min Jang, Chae Oh Im, Yeonjun Jeong, Kibaek Eum, Sejeong Yoon, So Jeong Lee, Eun Ae Jeong, and et al. 2024. "Intermittent Fasting Reduces Neuroinflammation and Cognitive Impairment in High-Fat Diet-Fed Mice by Downregulating Lipocalin-2 and Galectin-3" Nutrients 16, no. 1: 159. https://doi.org/10.3390/nu16010159