Salsalate Prevents β-Cell Dedifferentiation in OLETF Rats with Type 2 Diabetes through Notch1 Pathway
Fei Han1, Xiaochen Li1, Juhong Yang1, Haiyi Liu1, Yi Zhang1, Xiaoyun Yang2, Shaohua Yang1, Bai Chang1, Liming Chen1, Baocheng Chang1,*
1NHC Key Laboratory of Hormones and Development (Tianjin Medical University), Tianjin Key Laboratory of Metabolic Diseases, Tianjin Medical University Metabolic Diseases Hospital & Tianjin Institute of Endocrinology, Tianjin, China 2Tianjin Medical University General Hospital, Tianjin Medical University, Tianjin, China
A strategic approach is urgently needed to curb the growing global epidemic of diabetes. In this study, we investigated the effects and mechanisms of salsalate (SAL), an anti-inflammatory drug with anti-diabetic properties, assessing its potential to prevent diabetes in Otsuka Long-Evans Tokushima Fatty rats (OLETF). All animals in our placebo group developed diabetes, whereas none in the SAL test group did so, and only 25% of SAL-treated rats displayed impaired glucose tolerance (IGT). SAL lowered levels of glucagon and raised levels of insulin in plasma, while improving both insulin sensitivity and β-cell function. The protective effect of SAL is likely due to diminished β-cell dedifferentiation, manifested as relative declines in Neurogenin 3+/insulin- cells and synaptophysin+/islet hormone- cells and increased expression of β-cell-specific transcription factor Foxo1. Both Notch1-siRNA and N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester (DAPT; an indirect inhibitor of the Notch1 pathway) were shown to prevent β-cell dedifferentiation. Similar to DAPT, SAL effectively reduced β-cell dedifferentiation, significantly suppressing Notch1 pathway activation in INS-1 cells. The inhibitory role of SAL in β-cell dedifferentiation may thus be attributable to Notch1 pathway suppression.
Figure 1. SAL prevents onset of diabetes in OLETF rats
(A) Serial changes in food intake; (B) Serial changes in body weight; (C-F) OGTT results in rats at ages 8 (C), 24 (D), 40 (E) and 56 (F) weeks. Data are reported as mean ± SEM. *P < 0.05 vs. LETO rats, #P < 0.05 vs. OLETF rats; (G) AUCg values determined by OGTT. Data are reported as mean ± SEM. *P < 0.05; H and I: Ratios of IGT and diabetes in rats at ages 40 (H) and 56 (I) weeks.
Figure 2. SAL improves islet-cell function and insulin resistance in OLETF rats
Fasting plasma glucagon (A) and insulin (B) concentrations; (C) HOMA-β values in rats. (D) HOMA-IR values in rats. (E) GIRs in rats. Data are reported as mean ± SEM. *P < 0.05.
Figure 3. SAL restores morphology and architecture of islets in OLETF rats
(A) H&E staining of islets in rats. The scale bar represents 100 μm. (B) Immunofluorescence images of islets stained for insulin (Ins) (red) and glucagon (Ggc) (green). The scale bar represents 100 μm. Data are reported as mean ± SEM. *P < 0.05. (C) EM details of α and β cells in various groups. The scale bar represents 1 μm.
Figure 4. SAL prevents β-cell dedifferentiation in OLETF rats
(A) Immunofluorescence images of rat pancreatic tissue stained for Syn (red), Ins, Ggc, and Ssn (green); (B) Expression levels of Ngn3 and Foxo1 proteins in isolated islet tissue samples; (C) Quantified Ngn3 protein expression levels; (D) Quantified Foxo1 protein expression levels; (E) Immunofluorescence images of rat pancreatic tissue stained for Ins (red) and Ngn3 (green). (F) Immunofluorescence images of rat pancreatic tissue stained for Ins (red) and Foxo1 (green). The scale bar represents 100 μm. Data are reported as mean ± SEM. *P < 0.05.
Figure 5. SAL inhibits Notch1 pathway in islets of OLETF rats
(A) Immunofluorescence images of islets stained for Ins (red) and Notch1 (green); (B) Western blot images of Notch1 pathway-related protein expression; (C) Western blot results of the expression of Notch1. (D) Western blot results of the expression of Nicd. (E) Western blot results of the expression of Hes1. The scale bar represents 100 μm. Data are reported as mean ± SEM. *P < 0.05.
Figure 6. SAL inhibition of β-cell dedifferentiation attributable to suppression of Notch1 pathway
(A) Western blot images of Notch pathway-related protein expression in cells transfected by Notch1-siRNA; (B-D) Quantification of results in A; (E) Western blot images of Ngn3 and Foxo1 expression levels in cells transfected with Notch1-siRNA. (F-G) Quantification of results in E; H: Western blot images of protein expression in INS-1 cells treated for 48 h. I: Quantification of results in H. Data are reported as mean ± SEM. *P < 0.05.
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