High-fat diet feeding causes rapid, non-apoptotic cleavage of caspase-3 in astrocytes

Abstract

Astrocytes respond to multiple forms of central nervous system (CNS) injury by entering a reactive state characterized by morphological changes and a specific pattern of altered protein expression. Termed astrogliosis, this response has been shown to strongly influence the injury response and functional recovery of CNS tissues. This pattern of CNS inflammation and injury associated with astrogliosis has recently been found to occur in the energy homeostasis centers of the hypothalamus during diet-induced obesity (DIO) in rodent models, but the characterization of the astrocyte response remains incomplete. Here, we report that astrocytes in the mediobasal hypothalamus respond robustly and rapidly to purified high-fat diet (HFD) feeding by cleaving caspase-3, a protease whose cleavage is often associated with apoptosis. Although obesity develops in HFD-fed rats by day 14, caspase-3 cleavage occurs by day 3, prior to the development of obesity, suggesting the possibility that it could play a causal role in the hypothalamic neuropathology and fat gain observed in DIO. Caspase-3 cleavage is not associated with an increase in the rate of apoptosis, as determined by TUNEL staining, suggesting it plays a non-apoptotic role analogous to the response to excitotoxic neuron injury. Our results indicate that astrocytes in the mediobasal hypothalamus respond rapidly and robustly to HFD feeding, activating caspase-3 in the absence of apoptosis, a process that has the potential to influence the course of DIO.

Figure 1

Body composition of rats fed HFD. A, fat mass in rats after feeding chow or HFD for 1, 3, 7, or 14 days. B, change in fat mass (final minus initial) in rats fed HFD for 1, 3, 7, or 14 days. C, lean mass in rats after chow or HFD feeding for 1, 3, 7 or 14 days. n = 5, 6, 6, 4 rats, respectively. *, p < 0.05; **, p < 0.01; ***, p < 0.001 relative to chow or 1 d HFD by one-way ANOVA with Tukey post-test.

Figure 2

Cleaved caspase-3 immunoreactivity in ARC and S1 cortex (coronal sections). Low-magnification representative images. A and B, ARC and S1 cortex with blocking peptide. C and D, ARC and S1 cortex in chow-fed rats. E and F, ARC and S1 cortex in 1 day HFD-fed rats. G and H, ARC and S1 cortex in 3 day HFD-fed rats. I and J, ARC and S1 cortex in 7 day HFD-fed rats. K and L, ARC and S1 cortex in 14 day HFD-fed rats. M, high-magnification of ARC in chow-fed rat. N, high-magnification of ARC in 3d HFD-fed rat. Scale bars represent 100 µM.

Figure 3

Quantification of cleaved caspase-3 immunoreactivity in coronal sections of ARC, VMH and S1 cortex on day 1, 3, 7 or 14 of HFD feeding. A, ARC. B, VMH. C, S1 cortex. n = 5, 6, 6, 4 rats, respectively. *, p < 0.05; **, p < 0.01; ***, p < 0.001 relative to 1d HFD by ANOVA with Tukey post-test.

Figure 4

Double immunohistochemical localization of NeuN and GFAP immunoreactivity with cleaved caspase-3 immunoreactivity in ARC of 3 day HFD-fed rats (coronal sections). A, low magnification image of cleaved caspase-3 (green) and NeuN (red). B, high magnification image of cleaved caspase-3 (green) and NeuN (red). C, low magnification image of cleaved caspase-3 (green) and GFAP (red). D, high magnification image of cleaved caspase-3 (green) and GFAP (red). E, quantification of double immunohistochemical localization of cleaved caspase-3 immunoreactivity with NeuN and GFAP (p < 0.001 by paired t-test). n = 5, 6, 6, 4 rats, respectively. Scale bar: A and C, 20 µM; B and D, 5µM.

Figure 5

TUNEL staining. A, DNase I-treated positive control section from a chow-fed rat, showing ARC. Area of ARC is outlined; median eminence and third ventricle indicated. Note large TUNEL-positive nuclei, which are characteristic of this positive control method, but distinct from the condensed (pyknotic) morphology observed in true apoptotic nuclei. B, rat spleen positive control, showing characteristic small pyknotic nuclei. C, representative ARC image from chow group. D, representative ARC image from 3 day HFD group. E, representative ARC image from 7 day HFD group. F, representative ARC image from 3 month group. Scale bars represent 100 µM. All images are coronal sections with the ventral surface to the left, and the third ventricle and median eminence marked (3V; ME).