Journal: Molecular metabolism

Article Title: Dietary sugars, not lipids, drive hypothalamic inflammation.

doi: 10.1016/j.molmet.2017.06.008

Figure Lengend Snippet: Figure 3: RAGE and ALCAM are expressed on non-neuronal cell populations. (A) CML binds to proteins of RAGE (molecular weight z 75 KDa) and ALCAM (molecular weight z 105 KDa); the vehicle does not bind to protein of either receptor, as illustrated by no band detected in binding of ALCAM. (B) RAGE and ALCAM gene expression in mediobasal hypothalami of chow or HCHF mice (n ¼ 6 for chow or for HCHF, P ¼ 0.019 for RAGE, P ¼ 0.006 for ALCAM). (C) RAGE is intensely expressed by microglia (iba1-ir, indicated by white arrowheads). Higher magnifications of the areas framed by dashed lines are presented in D. (E) RAGE is intensely expressed on endothelial cells (laminin-ir, indicated by white arrows). Higher magnifications of the areas framed by dashed lines are presented in F. (G) CML stimulates TNFa, but not PDGF-B, gene expression in cultured primary microglia (n ¼ 6 wells of cells for vehicle, n ¼ 5 for TNFa treatments, P ¼ 0.04 for TNFa). (H & I) CML stimulates microglial reactivity in the mediobasal hypothalamic area, arrowheads point to the areas where the tip of the infusion probes located. (J) Iba1-ir cell number and cell coverage in H & I (n ¼ 4 mice for vehicle, n ¼ 5 for CML). (K) ALCAM is expressed on part of the vasculature (laminin-ir, indicated by white arrows, two pericytes are indicated by white arrowheads); higher magnifications of the areas framed by dashed lines are presented in L. (M) ALCAM is expressed on pericytes (PDGFRb-ir, white arrowheads). Higher magnifications of the areas framed by dashed lines are presented in N. Scale bar: 30 mm in C, E, K and M, 7.5um in D, F, L and N. Data are presented as means s.e.m. *P < 0.05, **P < 0.01. P values for unpaired comparisons were analyzed by two-tailed Student’s t test.

Article Snippet: Briefly, 2 mg recombinant CD166/ALCAM protein (Recombinant Mouse ALCAM/CD166 Fc Chimera; R&D Systems) and RAGE protein (Recombinant Mouse RAGE Fc Chimera R&D Systems) were separated on 10% SDS gels (Bio-rad cat.567-1033) and transferred to PVDF membranes.

Techniques: Molecular Weight, Binding Assay, Gene Expression, Cell Culture, Two Tailed Test

Journal: Molecular metabolism

Article Title: Dietary sugars, not lipids, drive hypothalamic inflammation.

doi: 10.1016/j.molmet.2017.06.008

Figure Lengend Snippet: Figure 4: Deletions of RAGE or ALCAM genes improve metabolic symptoms induced by a HCHF diet and exert diverse impacts on microglia, pericytes, and vasculature in the arcuate nucleus. (A & B) Daily caloric intake (in wk10) and weekly BW gain of chow or HCHF diet-fed WT versus RAGE/ mice (n ¼ 5e8 per group); For weekly BW gain, in all time points, WT and RAGE/ mice have less BW gain on chow diet than on HCHF diet (P < 0.0001); from wk14 to wk16, BW gain on HCHF of RAGE/ mice is significantly less than WT mice. (C & D) Daily caloric intake (in wk10) and weekly BW gain of chow or HCHF diet fed WT mice versus ALCAM/ mice (n ¼ 5e8 per group). For weekly BW gain, from wk2 on, WT and ALCAM/ mice have less BW gain in chow than in HCHF; from wk12, wk14 to wk16, BW-gain on HCHF of ALCAM/ mice is significantly less than WT mice. (E & F) Quantification of the number of iba1-ir microglia and the PDGFRb-ir pericytes in the ARC in chow or HCHF diet fed WT mice versus RAGE/ mice (n ¼ 5e9 per group). (G & H) Quantification of vessel length and vascular density in the ARC from chow or HCHF diet fed WT mice versus RAGE/ mice (n ¼ 5e7 per group). (I & J) Quantification of the number of iba1-ir microglia and the PDGFRb-ir pericytes in the ARC in chow or HCHF diet fed WT mice versus ALCAM/ mice (n ¼ 5e6 per group). (K & L) Quantification of vessel length and vascular density in the ARC from chow or HCHF diet fed WT mice versus ALCAM/ mice (n ¼ 6 per group). Data are presented as means s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Asterisks in B and D indicate significance between WT and RAGE/ or ALCAM/ mice on HCHF diet. Two-way ANOVA followed by Bonferroni multiple comparisons for post-hoc analysis was performed to detect significant interaction between genotype and diet on each parameter.

Article Snippet: Briefly, 2 mg recombinant CD166/ALCAM protein (Recombinant Mouse ALCAM/CD166 Fc Chimera; R&D Systems) and RAGE protein (Recombinant Mouse RAGE Fc Chimera R&D Systems) were separated on 10% SDS gels (Bio-rad cat.567-1033) and transferred to PVDF membranes.

Techniques:

Journal: Molecular metabolism

Article Title: Dietary sugars, not lipids, drive hypothalamic inflammation.

doi: 10.1016/j.molmet.2017.06.008

Figure Lengend Snippet: Figure 5: Deletion of RAGE and ALCAM genes improves metabolic symptoms induced by a HCHF diet. (A & B) Daily caloric intake (in wk10) and weekly body weight gain of chow or HCHF diet-fed WT versus RAGE-ALCAM/ mice (n ¼ 6e11 per group); for weekly BW gain, from wk5 on, WT and RAGE-ALCAM/ mice have less BW gain in chow than in HCHF; From wk1 to wk4 and from wk9 to wk16, there are significant effect of genotype on BW gain on HCHF. (C) Body composition of WT versus RAGE-ALCAM/ mice fed HCHF diet (n ¼ 4e8 per group). (D) Glucose tolerance of WT versus RAGE-ALCAM/ mice fed chow or HCHF diet (n ¼ 5e7 per group). (E) Glucose tolerance of RAGE-ALCAM/

Article Snippet: Briefly, 2 mg recombinant CD166/ALCAM protein (Recombinant Mouse ALCAM/CD166 Fc Chimera; R&D Systems) and RAGE protein (Recombinant Mouse RAGE Fc Chimera R&D Systems) were separated on 10% SDS gels (Bio-rad cat.567-1033) and transferred to PVDF membranes.

Techniques:

Journal: Molecular metabolism

Article Title: Dietary sugars, not lipids, drive hypothalamic inflammation.

doi: 10.1016/j.molmet.2017.06.008

Figure Lengend Snippet: Figure 6: Deletion of RAGE and ALCAM genes reduces microglial reactivity and neovasculature formation in the arcuate nucleus (A, B & C) Quantification of iba1-ir microglial number, coverage and the PDGFRb-ir pericytes number in the ARC from chow or HCHF diet fed WT (n ¼ 5e8 per group) versus RAGE-ALCAM/ mice (n ¼ 7e 10 per group). (D & E) Quantification of vessel length and vascular density in the ARC from chow or HCHF diet fed WT (n ¼ 5e7 per group) versus RAGE-ALCAM/ mice (n ¼ 5e 7 per group). (FeH) Illustrations of the iba1-ir microglia, PDGFRb-ir pericytes and FITC-albumin labeled vessels in WT versus RAGE-ALCAM/ mice fed chow or HCHF diet, with a frame of 0.2 mm 0.2 mm for quantifications in the ARC. (I) Illustration of the skeletonization of vessel in H for vascular density analysis. III: third cerebral ventricle. Scale bar: 50um in F and G, 100um in F. Data are presented as means s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Two-way ANOVA followed by Bonferroni multiple comparisons for post-hoc analysis was performed to detect significant interaction between genotype and diet on each parameter.

Article Snippet: Briefly, 2 mg recombinant CD166/ALCAM protein (Recombinant Mouse ALCAM/CD166 Fc Chimera; R&D Systems) and RAGE protein (Recombinant Mouse RAGE Fc Chimera R&D Systems) were separated on 10% SDS gels (Bio-rad cat.567-1033) and transferred to PVDF membranes.

Techniques: Labeling