In Vivo Autophagy Up-Regulation of Small Intestine Enterocytes in Chinese Soft-Shelled Turtles during Hibernation

Abstract

Many studies have focused on how autophagy plays an important role in intestinal homeostasis under pathological conditions. However, its role in the intestine during hibernation remains unclear. In the current study, we characterized in vivo up-regulation of autophagy in enterocytes of the small intestine of Chinese soft-shelled turtles during hibernation. Autophagy-specific markers were used to confirm the existence of autophagy in enterocytes through immunohistochemistry (IHC), immunofluorescence (IF), and immunoblotting. IHC staining indicated strong, positive immunoreactivity of the autophagy-related gene (ATG7), microtubule-associated protein light chain (LC3), and lysosomal-associated membrane protein 1 (LAMP1) within the mucosal surface during hibernation and poor expression during nonhibernation. IF staining results showed the opposite tendency for ATG7, LC3, and sequestosome 1 (p62). During hibernation ATG7 and LC3 showed strong, positive immunosignaling within the mucosal surface, while p62 showed strong, positive immunosignaling during nonhibernation. Similar findings were confirmed by immunoblotting. Moreover, the ultrastructural components of autophagy in enterocytes were revealed by transmission electron microscopy (TEM). During hibernation, the cumulative formation of phagophores and autophagosomes were closely associated with well-developed rough endoplasmic reticulum in enterocytes. These autophagosomes overlapped with lysosomes, multivesicular bodies, and degraded mitochondria to facilitate the formation of autophagolysosome, amphisomes, and mitophagy in enterocytes. Immunoblotting showed the expression level of PTEN-induced kinase 1 (PINK1), and adenosine monophosphate-activated protein kinase (AMPK) was enhanced during hibernation. Furthermore, the exosome secretion pathway of early-late endosomes and multivesicular bodies were closely linked with autophagosomes in enterocytes during hibernation. These findings suggest that the entrance into hibernation is a main challenge for reptiles to maintain homeostasis and cellular quality control in the intestine.

Keywords: ATG7; Chinese soft-shelled turtle; LC3; autophagosome; autophagy; enterocytes; hibernation; p62.

Figure 1

Immunohistochemistry and immunofluorescence staining of ATG7 in the mucosal surface of the small intestine of Pelodiscus sinensis. During hibernation (A,B) and nonhibernation (C,D), there were strong, positive (black arrow) and weak expressions (white arrow). Scale bar = 50 µm (A,C) and 20 µm (B,D).

Figure 2

Immunohistochemistry and immunofluorescence staining of LC3 in the mucosal surface of the small intestine of P. sinensis. During hibernation (A,B) and nonhibernation (C,D), there was a strong, positive expression (black arrow), a strong, positive expression in the lumen of the intestine (red arrow), and weak expression (white expression). Scale bar = 50 µm (A,C) and 20 µm (B,D).

Figure 3

Immunofluorescence staining of p62 and immunohistochemistry of LAMP1 in the mucosal surface of the small intestine of P. sinensis. During hibernation (B,C) and nonhibernation (A,D), there were strong, positive (black arrow) and weak expressions (white arrow). Scale bar = 50 µm (A,B) and 20 µm (C,D).

Figure 4

(A) Fluorescence intensity quantification of ATG7, LC3, and p62 (n = 10 turtle/group and 20 villi/section of each turtle). The values represent mean ± SEM. * indicates a statically significant difference between hibernation and nonhibernation. (B) Immunoblotting protein expression levels of LC3, p62, PINK1, and AMPK in the small intestine of P. sinensis during hibernation and nonhibernation. Experiments were repeated three times, with similar results in each.

Figure 5

Formation of phagophores in enterocytes of the small intestine in P. sinensis during hibernation (A,B) and nonhibernation periods (C,D). E, enterocyte; GC, goblet cell; M, mitochondria; ER, endoplasmic reticulum; ID, interdigitation; multivesicular bodies (white arrow head), phagophore (white bold arrow). Scale bar = 1 µm (A,C), 400 nm (B), and 200 nm (D).

Figure 6

Formation of autophagosomes in enterocytes of the small intestine in P. sinensis during hibernation (A–D) and nonhibernation periods (E,F). E, enterocyte; GC, goblet cell; MRC, mitochondria-rich cells; M, mitochondria; autophagosome (white arrow); interdigitation (black arrow head); ee, early endosome; Le, late endosome; multivesicular bodies (white arrow head); exo, exosome. Scale bar = 1 µm (A,E), 200 nm (B,D), and 400 nm (C,F).

Figure 7

Autophagolysosome and amphisome formation in enterocytes of the small intestine in P. sinensis during hibernation (A–D) and nonhibernation periods (E,F). E, enterocyte; GC, goblet cell; M, mitochondria; Lu, lumen; Mv, microvilli; rb, residual bodies; autophagosome (white arrow), lysosome (black arrow); amphisome (curve black arrow). Scale bar = 1 µm (A,B,E) and 200 nm (C,D, F).

Figure 8

Luminal formation of autophagosomes and mitophagy in enterocytes of the small intestine in P. sinensis during hibernation (A–I). E, enterocyte; GC, goblet cell; M, mitochondria; ER, endoplasmic reticulum; rb, residual bodies; autophagosome (white arrow); mitophagy (curve white arrow); ee, early endosome; Le, late endosome; Lu, lumen. Scale bar = 1 µm (A,C,G) and 200 nm (B,D,E–I).

Figure 9

Schematic diagram of autophagy in enterocytes of the small intestine in P. sinensis during hibernation.