High-Fat-Diet-Induced Metabolic Disorders: An Original Cause for Neurovascular Uncoupling Through the Imbalance of Glutamatergic Pathways

Figures

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  Figure 1. Overview of the experimental design. This schematic representation shows 2 sessions of 16 mice divided into 2 diet groups. The first session stops at 6 months of the diet, the second at 12 months of the diet.

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  Figure 2. HFD induces a disturbance in lipid and glucose homeostasis. (A) Body weight gain (g) over 12 months as a function of time in male C57Bl6/J mice fed a normal diet (ND) or a high-fat diet (HFD). (B) Visceral adipose tissue accumulation (VAT, in g) after 6 and 12 months of ND (nND = 69) or HFD (nHFD = 61). (C) Lipid infiltration in the liver of ND and HFD mice, stained with oil red O (x20). Representative illustrations of liver sections from ND and HFD mice after 6 months of diet. (D) Quantification of lipid accumulation in the livers of ND (nND = 64) and HFD (nHFD = 51) mice at M6 and M12. (E,F) Oral glucose tolerance test. (E) Representative kinetics of blood glucose values obtained in ND and HFD mice shown at M6. (F) Quantification of area under the curve (AUC) (mg min dL(−1) of ND (n = 64) and HFD (n = 56) mice at M6 and M12. Data are presented as mean ± S.E.M. Statistical analysis was performed using a two-way ANOVA, §§§ p < 0.001 for the effect of diet factor. Student’s t-test was used to compare the weight of mice at each time point, *** p < 0.001. Šídák’s multiple comparisons test was performed for the HFD vs. ND comparison, ** p < 0.01, *** p < 0.001.

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  Figure 3. The HFD alters the myogenic tone of the basilar artery and the maximal vasodilatory effect of glutamate. (A). Representative traces of the basilar artery diameter in response to increasing pressure steps in the absence (black line) or presence (red line) of papaverine to assess the myogenic tone. (B). Basilar artery myogenic tone is shown at M6 (left panel) and M12 (right panel) for mice in the normal (ND) or high-fat diet (HFD) groups. (C). The maximal effect of glutamate was measured using an ex vivo brain slice model in ND and HFD mice at M6 (nND = 19, nHFD = 19) and M12 (nND = 19, nHFD = 11). (D). Percentage contributions of vasoactive enzymes involved in the glutamate response. COX I, COX II, and NOS were successively inhibited by SC560 (100 nM), NS398 (25 µM), and L-NNA (10 µM), respectively, after application of glutamate (100 µM). The charts represent the cumulative percentages of COX I, COX II, and NOS in the ND and HFD groups at M6 and M12. Data are presented as mean ± S.E.M. Statistical analysis was performed using a two-way ANOVA (for myogenic tone and for maximal glutamate response) §§§ p < 0.001 and § p < 0.05 for the effect of the diet factor, and two-way ANOVA…

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  Figure 4. HFD induces changes in glutamatergic pathways. Specific inhibition of vasoactive enzymes involved in the glutamate response. (A) Probable astrocytic pathway involving COX I (left), specific contribution of COX I assessed by SC560 100 nM in glutamate-induced diameter change (right panel nND = 4; nHFD = 7 at M6 and nND = 11; nHFD = 16 at M12). (B) Probable neuronal pathway involving NOS (left), specific contribution of NOS assessed by 10 µM L-NNA in glutamate-induced diameter change (right panel nND = 10; nHFD = 13 at M6 and nND = 16; nHFD = 20 at M12). (C) Probable neuronal pathway involving COX II (left), specific contribution of COX II assessed by NS398 25 µM in glutamate-induced diameter change (right panel nND = 15; nHFD = 11 at M6 and nND = 15; nHFD = 14 at M12). Data are presented as mean ± S.E.M for ND and HFD mice at M6 and M12. Statistical analysis was performed using one-way ANOVA, ** p < 0.01, * p < 0.05 for ND versus HFD at M6. # p < 0.05 for M6 versus M12. $ p < 0.05 for HFD at M6 versus ND M12, $$ p < 0.01 for ND M6 versus HFD M12.

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  Figure 5. HFD and obesity increase the expression of COX II and GFAP. (A,C) Representative illustrations of immunohistochemistry tests performed on COX II (A) and GFAP (C). (B,D) Number of cells expressing COX II (B) and GFAP (D) in the ND and HFD groups at M6 and M12. Data are presented as mean ± S.E.M. Statistical analysis was performed using a two-way ANOVA, §§ p < 0.01, § p < 0.05, for the effect of the “diet” factor. Šídák’s multiple comparison test was performed: ** p < 0.01, * p < 0.05 for HFD vs. ND and ## p < 0.01 for M6 vs. M12.

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  Figure 6. HFD does not alter the availability of glutamate and glutamine in the brain. (A). Estimated (upper trace) and original (lower trace) representative traces extracted from the quantitative analysis of MRS spectra performed from a large 6 mm3 voxel located in the anterior region of the brain. (B). Relative magnitudes (in arbitrary units) of glutamate and glutamine levels in the anterior brain region for the normal diet (nND = 7) and the high-fat diet (n(HFD)) = 11) at M6 and the normal diet (nND = 8) and the high-fat diet (nHFD = 9) at M12. Data are presented as mean ± S.E.M. for ND and HFD mice at M6 and M12. Statistical analysis was performed using one-way ANOVA and no significant differences were found between groups.