Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function

Abstract

Diets rich in saturated fat produce inflammation, gliosis, and neuronal stress in the mediobasal hypothalamus (MBH). Here, we show that microglia mediate this process and its functional impact. Although microglia and astrocytes accumulate in the MBH of mice fed a diet rich in saturated fatty acids (SFAs), only the microglia undergo inflammatory activation, along with a buildup of hypothalamic SFAs. Enteric gavage specifically with SFAs reproduces microglial activation and neuronal stress in the MBH, and SFA treatment activates murine microglia, but not astrocytes, in culture. Moreover, depleting microglia abrogates SFA-induced inflammation in hypothalamic slices. Remarkably, depleting microglia from the MBH of mice abolishes inflammation and neuronal stress induced by excess SFA consumption, and in this context, microglial depletion enhances leptin signaling and reduces food intake. We thus show that microglia sense SFAs and orchestrate an inflammatory process in the MBH that alters neuronal function when SFA consumption is high.

Figure 1. High-fat diet (HFD) induces the inflammatory activation of microglia specifically in the hypothalamic ARC

(A) Increased ARC staining of Iba1 in hypothalamic sections from mice fed a HFD for 4 weeks (3V: third ventricle). (B) Quantification of ARC microglial number and size in A (n=5/group *p< 0.05 vs. chow). (C) Representative immunostained ARC sections, illustrating diet-induced induction of TNF-α and its strong co-localization with Iba1 (see also inset), but not GFAP (D). (E) qPCR analysis, showing increased mRNA levels of M1 mediators, cytokines, and chemokines in whole hypothalami and microglial fractions of mice fed a 4-week HFD (n=5/group *p< 0.05 vs. chow). (F) Fold change in individual FA levels (LC-MS) in the cerebral cortices (CTX) and hypothalami (HYPO) of mice following 4 weeks of HFD (n=5/group *p< 0.05 vs. CTX). See also Figure S1.

Figure 2. SFA gavage reproduces diet-induced hypothalamic inflammation and neuronal stress in mice

(A) Increased mRNA levels of M1 markers in the MBH of mice receiving isocaloric OA (C18:1; olive oil), lauric acid (C12:0; coconut oil), or PA (C16:0; milk fat) vs. saline by enteric gavage twice daily for 3 days (n=4/group *p< 0.05 vs. control). (B) Increased Iba1 staining in the ARC of mice receiving milk fat (vs. olive oil) by gavage as above. (C) Immunostaining, showing specific TNF-α induction in the ARC by milk fat gavage, and co-localization with Iba1, but not GFAP (D). (E) Induction of Hsp72 in NeuN⁺ cells specifically in the ARC by feeding mice a 4-week high-SFA diet or by milk fat by gavage for 3 days (vs. chow diet and olive oil gavage, respectively; n=4/group).

Figure 3. Long-chain dietary SFAs specifically stimulate the inflammatory activation of primary microglia

(A) M1 cytokine secretion (ELISA) by primary murine microglia treated for 24 hours with 100 μM of the listed FAs (n=3/group; *P<0.05 vs. BSA). Representative immunoblots, showing phosphorylation of (B) IKK-β and (C) p65 (Rel-A) induced specifically by treatment with SFAs between 100-300 μM (total protein and GAPDH as loading controls). (D) Immunostaining for GFAP (green), CD11b (red) and DAPI (blue) in primary astrocyte cultures treated for 72 hours with a control liposome (Lip-C) or Lip-CLO (200ng/ml), showing microglial elimination. (E) Differential M1 cytokine secretion by microglia and astrocytes treated for 24 hours with PA (100μM). LPS and OA were positive and negative controls, respectively (n=3/group, *P<0.05 or **P<0.01 vs. astrocytes). See also Figure S3.

Figure 4. Microglial depletion abolishes the inflammatory response of hypothalamic slice cultures to SFA treatment

(A) Immunostaining for Iba1 (green) and NeuN (red) in 5 day-old hypothalamic slices cultures treated with either Lip-C or Lip-CLO (3 days), showing profound microglial depletion. qPCR analysis of (B) Lip-C and Lip-CLO-treated WT slice cultures and (C) DT-treated CD11b-DTR and WT slice cultures, showing reduced mRNA levels of microglial markers (Iba1, CD68, Itgam), but not astrocyte (Gfap) or neuronal (Agrp, Npy) markers. Microglial depletion by (D) Lip-CLO treatment (WT) or (E) DT treatment (CD11b-DTR) specifically abolishes M1 cytokine secretion by hypothalamic slices treated with 100μM PA or OA. BSA (negative control); LPS (positive control) (n=3/group, *P<0.05 or **P<0.01 vs. control or as indicated). See also Figure S4.

Figure 5. Selectively and stably depleting microglia in the MBH of DTR^BMT mice

(A) Hypothalamic sections 24 hours after BM-transplanted WT and DTR^BMT mice were given DT (10ng/g I.P.) daily for 3 days, showing depletion of Iba1⁺ cells (green) in DT-treated DTR^BMT mice along with abnormal microglial morphology (inset). Reduction in (B) hypothalamic microglial number and (C) microglial mRNA levels (Iba1, Itgam), but not the astrocyte marker Gfap in DT-treated DTR^BMT mice (n=3/group, *P<0.05 vs. control). (D) Stabilization of microglial depletion in the MBH of DTR^BMT mice by preventing post-depletive microglial proliferation [Iba1+/BrdU+ cells] with Ki-20227 treatment (30mg/kg/day for 3 days post-DT). (E) Quantification of D (n = 5/group). (F) Reduced microglial mRNA levels in hypothalami of DT-treated DTR^BMT mice also treated with Ki20227 (*P<0.05 vs. control). See also Figure S5.

Figure 6. Hypothalamic microglial content modulates inflammatory response and neuronal injury to dietary SFAs

(A) Protocol for administering DT (10ng/g I.P.) followed by enteric SFA gavage under conditions where post-depletive microglial proliferation in the MBH was allowed to occur, or in which Ki20227-treatment sustained microglial depletion. (B) Analysis of M1 markers in DT-treated DTR^BMT mice, showing that the inflammatory response to SFA gavage is enhanced by increasing microglial content in the MBH and abolished by depleting microglia from the MBH (DT-treated WT and vehicle-treated CD11b-DTR mice as reference). (C) Immunostaining of ARC sections, illustrating induction of Hsp72 in NeuN⁺ cells by SFA gavage, the accentuation of SFA-induced neuronal stress by increasing basal microglial content in the MBH, and the complete prevention of SFA-induced neuronal stress by local depletion of microglia. Quantification of C, showing reduced (D) Hsp72 intensity and (E) percentage of Hsp72⁺/NeuN⁺ cells induced by microglial depletion in the MBH (n=6/group, *P<0.05 vs. matched control or as indicated and **P<0.05 vs. DTR control). See also Figure S6.

Figure 7. Microglial depletion enhances leptin signaling and decreases food intake in response to excess dietary SFAs

A) Hypothalamic sections from mice fed a chow diet or a matched diet compounded with PLX5622 for 7 days followed by continuation of the respective regimens with milk fat (twice-daily gavage) supplementation for 10 more days, showing pSTAT3 staining 45 min after injection with I.P. leptin (3mg/kg) or vehicle B) pSTAT3 quantification, showing enhanced leptin responsiveness in the ARC of mice consuming excess milk fat in the setting of microglial depletion. C) Equivalent food intake in PLX5622-treated and control mice consuming chow alone. D) Reduced food intake in PLX5622-treated mice receiving SFA gavage for 8 days. E) Reduced food intake measured by metabolic cages in PLX5622-treated mice with excess SFA intake. F) Area under the curve (AUC) analysis for E. G) RER (CO₂ exhaled/O₂ inhaled) and H) AUC analysis of RER during light and dark cycles for G. I) Energy expenditure (heat) and J) AUC analysis of energy expenditure during light and dark cycles for I. (n=6 mice/group, *P< 0.05). See also Figure S7.