Effect of aging on islet beta-cell function and its mechanisms in Wistar rats

Abstract

Type 2 diabetes mellitus is characterized by islet β-cell dysfunction and its incidence increases with age. However, the mechanisms underlying the effect of aging on islet β-cell function are not fully understood. We characterized β-cell function in 4-month-old (young), 14-month-old (adult), and 24-month-old (old) male Wistar rats, and found that islet β-cell function decreased gradually with age. Old rats displayed oral glucose intolerance and exhibited a decrease in glucose-stimulated insulin release (GSIR) and palmitic acid-stimulated insulin release (PSIR). Furthermore, total superoxide dismutase (T-SOD), CuZn superoxide dismutase (CuZn-SOD), and glutathione peroxidase (GSH-Px) activity decreased, whereas serum malondialdehyde (MDA) levels increased in the older rats. Moreover, we detected a significant reduction in β-cell proliferation and an increase in the frequency of apoptotic β-cells in the islets of rats in the old group. Finally, Anxa1 expression in the islets of old rats was significantly upregulated. These data provide new insights into the development of age-related β-cell dysfunction in rats.

Fig. 1

Glucose infusion rate (GIR). The GIR of each group of rats was determined using a hyperinsulinemic–euglycemic clamp test. During the test, the rats were maintained at a CV_BG of 3.85–4.95%, CV_GIR of 0.52–1.71%, and a steady-state blood glucose level of 5.03–5.06 mmol/l. Data are expressed as the mean ± SD of each group (n = 6 per group) of rats from three separate experiments. *P < 0.05 vs. young rats; ^#P < 0.05 vs. adult rats

Fig. 2

Oral glucose tolerance. Individual rats were challenged with oral glucose and the concentrations of blood glucose and plasma insulin were measured longitudinally. a Dynamic changes in blood glucose levels. b Dynamic changes in plasma insulin concentrations. Data are expressed as the mean ± SD of each group of rats (young, n = 6; adult, n = 7; and old, n = 6) from three separate experiments

Fig. 3

Immunohistochemical analysis of islets. Pancreatic tissue was harvested from the different age groups and subjected to an immunohistochemical analysis using specific antibodies. a Double immunostaining for insulin (brown) and glucagon (blue). b Double immunostaining of the islets with anti-insulin (brown) and DNA fragmentation using the TUNEL assay (blue). Black arrows positive cells for TUNEL staining. c Immunohistochemical staining of the pancreatic tissue sections with anti-proliferating cell nuclear antigen (PCNA, in brown). d Immunohistochemical staining of the pancreatic tissue sections with anti-insulin (brown) in the same islet as in c. e and f Quantitative analysis: data are expressed as the mean ± SD of the relative percentage of TUNEL-positive or PCNA-positive β-cells from at least 60 islets from each group of six rats. The frequency of TUNEL-positive or PCNA-positive β-cells in young rats was designated as 100% (2.3% TUNEL-positive β-cells and 17.6% PCNA-positive β-cells in the young group of rats). *P < 0.05 vs. young rats. ^#P < 0.05 vs. adult rats

Fig. 4

Quantitative RT-PCR analysis of mRNA transcripts in islets. The relative level of Anxa1 mRNA transcripts to control β-actin in the islets of the three age groups was determined using RT-PCR. Data are expressed as the mean ± SD of the relative levels of Anxa1 to β-actin in the islets of the three groups (n = 6 per group) from three separate experiments. *P < 0.05 vs. young rats. ^#P < 0.05 vs. adult rats

Fig. 5

Western blot analysis of Anxa1 expression in islets. The relative level of Anxa1 to β-actin expression in the islets of young, adult, and old age groups was determined using Western blot assays with specific antibodies. Data shown are representative images or expressed as mean ± SD of the relative levels of the target protein in islets of the three age groups (n = 6 per group) from three separate experiments. a Western blot analysis; b Quantitative analysis. *P < 0.05 vs. young rats. ^#P < 0.05 vs. adult rats