Obesity is associated with hypothalamic injury in rodents and humans

Abstract

Rodent models of obesity induced by consuming high-fat diet (HFD) are characterized by inflammation both in peripheral tissues and in hypothalamic areas critical for energy homeostasis. Here we report that unlike inflammation in peripheral tissues, which develops as a consequence of obesity, hypothalamic inflammatory signaling was evident in both rats and mice within 1 to 3 days of HFD onset, prior to substantial weight gain. Furthermore, both reactive gliosis and markers suggestive of neuron injury were evident in the hypothalamic arcuate nucleus of rats and mice within the first week of HFD feeding. Although these responses temporarily subsided, suggesting that neuroprotective mechanisms may initially limit the damage, with continued HFD feeding, inflammation and gliosis returned permanently to the mediobasal hypothalamus. Consistent with these data in rodents, we found evidence of increased gliosis in the mediobasal hypothalamus of obese humans, as assessed by MRI. These findings collectively suggest that, in both humans and rodent models, obesity is associated with neuronal injury in a brain area crucial for body weight control.

Figure 1. Time course of hypothalamic inflammation after the onset of HFD feeding.

(A and B) Quantification of mRNA encoding proinflammatory cytokines (Il1b, Il6, Tnfa) and NF-κB pathway genes (Nfkbia, Ikbkb) in (A) hypothalamus and (B) liver of rats fed either standard chow (white bars) or HFD (gray bars) for 20 weeks (n = 6/group). *P < 0.05 versus chow-fed controls. (C–E) Effect of 4 weeks of HFD feeding (gray bars) on proinflammatory cytokine gene expression in rat (C) hypothalamus, (D) liver, and (E) white adipose tissue compared with that in chow-fed controls (white bars) (n = 6/group). *P < 0.05 versus chow-fed controls. (F) Total weight gain (black bars), fat mass gain (white bars), and (G) average (avg) daily food intake (kcal/d) of rats (n = 6/group) fed chow for 2 weeks or HFD for up to 28 days. *P < 0.05 versus chow-fed controls. (H) Comparison of daily food intake (kcal) in rats (n = 6/group) fed chow (gray) or HFD (black) for 14 days. *P < 0.05 versus chow-fed controls. (I) Time course of induction of mRNA encoding inflammatory mediators, including proinflammatory cytokine (Il1b, Il6, Tnfa), cytokine pathway (Socs3), and NF-κB pathway (Nfkbia, Ikbkb, Ikbke) gene expression in the hypothalamus of rats fed chow or HFD for up to 28 days (n = 6/group). All mRNA species were quantified relative to 18S and Gapdh housekeeping gene expression (by ΔΔCT method) and presented as fold change relative to chow-fed controls [fold chow]. The dashed line in I represents the level of expression equal to chow-fed controls. *P < 0.05 versus chow.

Figure 2. Effect of HFD feeding on hypothalamic microglial markers in rats.

(A) Time course of hypothalamic microglia-specific (Cd68 and Emr1) and astrocyte-specific (Gfap) gene expression in rats fed chow or HFD for up to 28 days (n = 6/group). *P < 0.05 versus chow. (B) Correlation of hypothalamic Emr1 mRNA level (linearized using difference in CT count between the Gapdh and the Emr1 gene [ΔCT]) with change in fat mass (g) over 4 weeks of HFD feeding (r = 0.70; P < 0.001).

Figure 3. Histochemical analysis of HFD-induced microglial accumulation in rat ARC.

Immunohistochemical detection of Iba1 protein, a microglial marker (25), in coronal sections of rat hypothalamus (14 μm) from animals fed either (A and B) chow or (C–F) HFD for up to 14 days. (A) Low-magnification view (original magnification, ×10) of microglia distributed throughout the MBH. The dashed box indicates the region used for quantification of ARC microglial number and size in G and H. 3V, third ventricle. Scale bar: 100 μm. (B–F) Higher-magnification view (original magnification, ×20) of Iba1 immunohistochemistry in the ARC of rats fed (B) chow or (C) HFD for 1 day, (D) 3 days, (E) 7 days, or (F) 14 days. Scale bar: 50 μm. (G and H) Quantification of (G) mean ARC microglial cell number (per field defined in A) and (H) microglial cell size (average number of pixels in 10 largest cells) from rats fed either chow or HFD (n = 6/group). *P < 0.05 versus chow. (I and J) Comparison of microglial fine structure in hypothalamus of rats fed (I) chow or (J) HFD for 7 days. Microglia from HFD-fed rats manifest a more “ameboid” morphology, characterized by larger cell bodies with thickened and shortened processes. Scale bar: 10 μm. (K) Correlation of microglial cell number and fat mass gain (g) over 2–8 weeks of HFD consumption, with indicated linear regression line. (L) Correlation of microglial cell size (no. pixels in 10 largest cells/ARC) and fat mass gain (g) over 2 to 8 weeks of HFD consumption, with indicated linear regression line. Each symbol in K and L represents an animal.

Figure 4. Time course of the effect of HFD feeding on hypothalamic astrocytes.

(A–D) Representative images of astrocytes identified by immunohistochemical detection of GFAP protein in coronal sections of hypothalamus (10 μm) obtained from 10-week-old mice fed either (A) chow or HFD for (B) 1 week, (C) 2 weeks, or (D) 3 weeks. (E and F) GFAP staining of hypothalamic sections from 8-month-old mice fed (E) chow or (F) HFD. Scale bar: 50 μm. (G) Quantification of GFAP staining intensity (mean ± SEM) in the region of the ARC from mice fed either chow or HFD for 1 to 3 weeks (n = 6/group). (H) Quantification of GFAP staining intensity (mean ± SEM) in mice fed chow or HFD for 8 months. *P < 0.05 versus chow. The dashed boxes indicate the region used for quantification in G and H.

Figure 5. Effect of HFD feeding on ARC astrocyte morphology.

High-magnification (original magnification, ×100) examination of astrocyte processes by GFAP immunohistochemistry of sections through mouse ARC. (A) Astrocyte processes in the ARC of mice fed chow remain separated into discrete areas. (B) One week of HFD feeding is accompanied by the apparent formation of a syncytium of astrocytic processes. (C) This astrocyte response is partially resolved by 2 weeks of HFD feeding, with only a few scattered overlapping processes, and, (D) by 3 weeks of HFD, glial morphology appears to be fully normalized. (E) Mice fed chow for 8 weeks show increased astrocyte number but no overlap of processes. (F) Mice fed HFD for 8 months exhibit severe astrocytosis suggestive of syncytium formation. Scale bar: 10 μm.

Figure 6. Effect of HFD feeding on hypothalamic markers of neuronal injury and on POMC cell number.

(A–D) Immunohistochemical analysis of the neuronal injury marker Hsp72 in the ARC of rats fed (A and C) chow or (B and D) HFD for 7 days. Immunofluorescence in C and D shows colocalization of Hsp72 (green) with POMC peptide (red). (E and F) Electron micrograph of a POMC neuron from a mouse fed (E) chow or (F) HFD for 20 weeks. In E, no pathological changes in cytoplasm or mitochondria (white arrows) are observed, whereas, in F, mitochondrial integrity is disrupted (white arrows), and mature and developing autophagosomes are present (black arrows). The inset in F is a higher-magnification example of an autophagosome. (G) Additional high-magnification examples of autophagosomes (black arrows). (H) High-magnification images of POMC neuron mitochondria from mice fed chow (parallel-oriented cristae and regular structure) or HFD (nonparallel cristae and irregular shape). (I) Quantification of percentage of POMC neurons with autophagosomes (n = 5 cells examined in each of 5 mice). *P < 0.05 versus chow. (J and K) Representative images of POMC neurons in the ARC of mice fed either (J) chow or (K) HFD for 8 months. (L) Quantification of POMC neuron number in the hypothalamus of mice fed chow or HFD for 8 months (mean ± SEM; n = 8/group). *P < 0.05 versus chow. Scale bar: 50 μm (A–D, J, and K); 1 μm (E and F); 500 nm (F, inset); 400 nm (G and H).

Figure 7. Radiologic evidence of gliosis in the MBH of obese humans.

Representative coronal T2-weighted images through the hypothalamus from (A) a normal weight and (B) an obese subject. Insets show the placement of right and left ROIs (green circles) in the MBH and amygdala (AMY). In the MBH (thick arrows) of the obese subject, signal ratios demonstrated subtle hyperintensity (brightness) relative to the amygdala (thin arrows). Scale bar: 20 mm; 10 mm (insets). (C) Correlation of BMI with MBH hyperintensity as measured by left (L) MBH/amygdala signal ratio (n = 34 subjects; r = 0.38; P = 0.027).