Hormones and diet, but not body weight, control hypothalamic microglial activity

Abstract

The arcuate nucleus (ARC) of the hypothalamus plays a key role in sensing metabolic feedback and regulating energy homeostasis. Recent studies revealed activation of microglia in mice with high-fat diet (HFD)-induced obesity (DIO), suggesting a potential pathophysiological role for inflammatory processes within the hypothalamus. To further investigate the metabolic causes and molecular underpinnings of such glial activation, we analyzed the microglial activity in wild-type (WT), monogenic obese ob/ob (leptin deficient), db/db (leptin-receptor mutation), and Type-4 melanocortin receptor knockout (MC4R KO) mice on either a HFD or on standardized chow (SC) diet. Following HFD exposure, we observed a significant increase in the total number of ARC microglia, immunoreactivity of ionized calcium binding adaptor molecule 1 (iba1-ir), cluster of differentiation 68 (CD68-ir), and ramification of microglial processes. The ob/ob mice had significantly less iba1-ir and ramifications. Leptin replacement rescued these phenomena. The db/db mice had similar iba1-ir comparable with WT mice but had significantly lower CD68-ir and more ramifications than WT mice. After 2 weeks of HFD, ob/ob mice showed an increase of iba1-ir, and db/db mice showed increase of CD68-ir. Obese MC4R KO mice fed a SC diet had comparable iba1-ir and CD68-ir with WT mice but had significantly more ramifications than WT mice. Intriguingly, treatment of DIO mice with glucagon-like peptide-1 receptor agonists reduced microglial activation independent of body weight. Our results show that diet type, adipokines, and gut signals, but not body weight, affect the presence and activity levels of hypothalamic microglia in obesity.

Keywords: high calorie diet; leptin; obesity.

FIGURE 1

High-fat diet (HFD) increases the total number of microglia and the number of activated iba1-positive microglia in the arcuate nucleus of the hypothalamus. Wild-type mice fed a standard chow (SC) diet have a modest number of microglia (expressing eGFP (green) under the control of the endogenous Cx3cr1 locus) (B), including some iba1-positive cells (red, arrows in A and C). After 6 weeks of HFD diet, both the total number of microglia (E) and the number of iba1-positive microglia (D) are increased. Under these conditions, only a proportion of the total microglial population is still iba1-positive (F). III indicates third ventricle; scale bar = 25 μm.

FIGURE 2

Iba1-ir microglia in the hypothalamic arcuate nucleus (ARC) of diet induced and monogenic obese mice. The numbers of iba1-positive microglia in the ARC of ob/ob mice (B) is reduced compared with wild-type (WT) mice (A) on standard chow (SC) diet as well as to WT mice on high-fat diet (HFD) (D, H), and the microglial processes are less extensively branched (high-magnification images for the cells pointed by white arrows are in the upper right panel) (I). The ARC of ob/ob mice fed a HFD contains an increased number of iba1-ir positive microglia, which also have more extensively branched processes (C). When body weight is matched to the ob/ob group (D), similar numbers of iba1-positive microglia are observed in db/db (E) and WT mice. Ramification of microglia in db/db mice is significantly higher than in SC diet fed WT mice and lower than in HFD fed WT mice. After feeding with 2 weeks HFD (F), the number of iba1-ir positive microglia is increased in the ARC, while ramification is not changed. In obese MC4R KO mice (G) with body weight matched to ob/ob and db/db mice and 10 weeks HFD-fed mice, the number of iba1-positive microglia are significantly lower than in WT HFD-fed controls, while ramification did not differ between MC4R KO and WT HFD mice. WT mice fed for 10 weeks with HFD have the largest number of iba1-positive microglia with the most extensive ramification (D). P < 0.05 for *: vs. WT SC group, ^^: vs. ob/ob SC group, §: vs. db/db SC group, #: vs. MC4 KO group, ^: vs. db/db HFD group; scale bar = 100 μm.

FIGURE 3

CD68-ir microglia in the hypothalamic arcuate nucleus of diet induced and monogenic obese mice. In comparison to iba1-ir, which stains both the soma and processes of microglia, CD68-ir is mainly visible inside the soma of the microglia. P < 0.05 for *: vs. WT SC group, ^^: vs. ob/ob SC group, §: vs. db/db SC group, ^: vs. db/db HFD group; scale bar = 100 μm.

FIGURE 4

Total microglial population in the hypothalamic arcuate nucleus (visualized by eGFP expression under the control of the endogenous Cx3cr1 locus) does not differ between WT mice (A) and ob/ob (B) mice. III indicates third ventricle; scale bar = 100 μm.

FIGURE 5

Effect of anti-obesity pharmacotherapy on hypothalamic microglial activation in obese mice. Compared with vehicle control (A), no effects on iba1-ir and ramification were observed in WT mice treated with 5 days of leptin or pair-fed (high-magnification images for the cells pointed by white arrows are in the upper right panel) (A–E); 5 days of leptin treatment significantly increased the iba1-ir microglial number and ramification in the arcuate nucleus (ARC) of ob/ob mice in comparison with the vehicle group. The pair-fed (to leptin) group also had significantly increased microglial activation but with less-extensive ramification (F–J). Exendin-4 treatment in mice with diet-induced obesity significantly reduced the number of iba1-positive microglia and ramification in the ARC compared with vehicle control and pair-fed control groups (L–P). P < 0.05 for #: vs. vehicle in (I), (J), (O) and (P); *: vs. leptin group; scale bar = 100 μm.

FIGURE 6

In cultured primary hypothalamic microglia, leptin treatment stimulates IL-1β and TNFα, but not iba1 gene expression (A), whereas HFD-fed mice serum treatment stimulates iba1, IL-1β, and TNFα genes expression compared with SC diet fed mice serum (B). P < 0.05 for *: vs. vehicle group or Chow - serum group.

FIGURE 7

Microglial functional related gene expression profiles in the hypothalamus of diet-induce obese and monogenic obese mice. *P < 0.05 vs. WT SC, ^#P < 0.05 vs. both WT SC and WT HFD groups.