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.

Integrated analysis of transcriptome and metabolome data reveals insights for molecular mechanisms in overwintering Tibetan frogs, Nanorana parkeri

“…In this study, expression of most genes involved in autophagy was significantly upregulated in winter, such as ATG9A, ATG14, ATG13, and ATG4B. Similar results were found in Chinese soft-shelled turtles (P. sinensis), where the autophagy-related gene (ATG7), lysosomal-associated membrane protein 1 (LAMP1), and microtubule-associated protein light chain (LC3) were up-regulated significantly during hibernation (Vistro et al, 2019). Fibrinogen is involved in many biochemical processes in living organisms.…”

Mechanisms of Cadmium-Induced Testicular Injury: A Risk to Male Fertility

“…Autophagy is an evolutionary conserved catabolic process that degrades the cytoplasmic components in lysosomes and plays an important role in many physiological and pathological processes [ 113 , 114 , 115 , 116 ]. This degrading pathway supports cells in getting rid of unwanted and damaging substances, which plays a significant role in the complex process of spermatogenesis [ 117 ].…”

Pathological Roles of Mitochondrial Oxidative Stress and Mitochondrial Dynamics in Cardiac Microvascular Ischemia/Reperfusion Injury

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