Determination of Expression and Relationships of Nedd4-Related E3 Ubiquitin Ligase-2 (NEDL2) and Ubiquitin Proteasome System Proteins in Developing Rat Pancreatic Tissue

Abstract

Nedd4-associated E3 ubiquitin ligase 2 (NEDL2) and p97/valosin-containing protein (p97/VCP) are critical regulators of ubiquitin-mediated proteostasis and cellular homeostasis. However, their age-dependent expression patterns in pancreatic tissue during postnatal development and senescent decline remain uncharacterized. This study aimed to investigate the histological alterations alongside the expression profiles of NEDL2 and p97/VCP in rat pancreatic tissue across the postnatal lifespan and ageing. Pancreatic tissues obtained from male Wistar albino rats at postnatal Weeks 1, 4, 10, 36 and 72 were evaluated using histochemical, immunohistochemical and immunofluorescence analyses. Haematoxylin and eosin (H&E) staining were performed to assess morphological changes in endocrine and exocrine pancreas. Immunohistochemistry and semi-quantitative H-score analyses were utilized to evaluate NEDL2 and p97/VCP expression levels. Double immunofluorescence staining was conducted to determine cellular co-localization of NEDL2 with p97/VCP, insulin and glucagon in pancreatic islet cells. Histological analysis showed that the islets of Langerhans progressively enlarged and hypertrophic changes during postnatal ageing, while the exocrine acinar structures maintained its morphological integrity. Immunohistochemical findings revealed dynamic and age-dependent expression of NEDL2 and p97/VCP. Both proteins demonstrated a progressive increase in expression from Week 1 through Week 4. NEDL2 expression peaked significantly at Week 10 (p < 0.001) followed by a gradual decline, whereas p97/VCP expression reached its maximum level at Week 36 (p < 0.001). A significant decrease in expression was observed at Week 72. Immunofluorescence analyses confirmed that NEDL2 strongly co-localized with p97/VCP, insulin-positive beta cells and glucagon-positive alpha cells, particularly during young and mature adulthood (Weeks 10 and 36). Conversely, a marked reduction in fluorescence intensity and co-localization was observed in senescent pancreatic tissue (Week 72). These findings demonstrate that NEDL2 and p97/VCP exhibit coordinated and developmentally regulated expression patterns within pancreatic islet cells. Their elevated expression and prominent co-localization during adulthood suggest a key role for these proteins in pancreatic maturation, endocrine function and cellular proteostasis. Conversely, their down-regulation in senescent tissue may reflect an age-related impairment of protein quality control systems during pancreatic ageing.