Age-associated loss of Sirt1-mediated enhancement of glucose-stimulated insulin secretion in beta cell-specific Sirt1-overexpressing (BESTO) mice

Abstract

The Sir2 (silent information regulator 2) family of NAD-dependent deacetylases regulates aging and longevity across a wide variety of organisms, including yeast, worms, and flies. In mammals, the Sir2 ortholog Sirt1 promotes fat mobilization, fatty acid oxidation, glucose production, and insulin secretion in response to nutrient availability. We previously reported that an increased dosage of Sirt1 in pancreatic beta cells enhances glucose-stimulated insulin secretion (GSIS) and improves glucose tolerance in beta cell-specific Sirt1-overexpressing (BESTO) transgenic mice at 3 and 8 months of age. Here, we report that as this same cohort of BESTO mice reaches 18-24 months of age, the GSIS regulated by Sirt1 through repression of Ucp2 is blunted. Increased body weight and hyperlipidemia alone, which are observed in aged males and also induced by a Western-style high-fat diet, are not enough to abolish the positive effects of Sirt1 on beta cell function. Interestingly, plasma levels of nicotinamide mononucleotide (NMN), an important metabolite for the maintenance of normal NAD biosynthesis and GSIS in beta cells, are significantly reduced in aged BESTO mice. Furthermore, NMN administration restores enhanced GSIS and improved glucose tolerance in the aged BESTO females, suggesting that Sirt1 activity decreases with advanced age due to a decline in systemic NAD biosynthesis. These findings provide insight into the age-dependent regulation of Sirt1 activity and suggest that enhancement of systemic NAD biosynthesis and Sirt1 activity in tissues such as beta cells may be an effective therapeutic intervention for age-associated metabolic disorders such as type 2 diabetes.

Fig. 1

Intraperitoneal glucose tolerance tests (IPGTTs), plasma insulin levels, glucose-stimulated insulin secretion assays, and ATP levels in male BESTO and control mice and islets at 18–24 months of age. (A) Intraperitoneal glucose tolerance tests were conducted in male BESTO transgenic mice (closed circles) compared to controls (open circles) at 18–24 months of age in two independent transgenic lines. Following a 15 h fast, 2 g of 50% glucose (w/v) per kg body weight was injected intraperitoneally, and blood glucose values were assessed at 0, 15, 30, 60, and 120 min post-injection (line 431-2, n = 13–15; line 431-4, n = 6–7). (B) Plasma insulin levels in male BESTO mice (black bars) and controls (white bars) were measured at the 0 and 30 min time points during the IPGTTs (line 431-2, n = 13–15; line 431-4, n = 6–7). (C) Insulin secreted (ng mL⁻¹ h⁻¹) and (D) ATP levels (pmol islet⁻¹) from transgenic (black bars) and control (white bars) islets were measured at the indicated glucose concentrations (line 431-2, n = 11; line 431-4, n = 9–12). Insulin secretion and ATP assays were performed on triplicate groups of 10 islets for each mouse. All results are expressed as mean ± standard error.

Fig. 2

Sirt1 and uncoupling protein 2 (Ucp2) protein levels and stearoyl-CoA desaturase 1 (Scd1) and dipeptidyl peptidase IV (Dpp4) mRNA levels in islets isolated from aged BESTO and control mice. (A) Western blot analysis of Sirt1 and Ucp2 expression in transgenic (Tg) and control (Con) islet extracts from lines 431-2 and 431-4. Actin was used as a loading control for normalization. (B) Quantitation of the Ucp2 expression levels normalized to actin in BESTO transgenic islets (black bars) relative to control islets (white bars). (C) Quantitative real-time reverse transcriptase–polymerase chain reaction analysis of Scd1 and Dpp4 expression in BESTO transgenic islets (black bars) relative to control islets (white bars). The 3- to 5-month-old mice were considered young, while 18- to 19-month-old mice were considered old. All results are expressed as mean ± standard error. *P ≤ 0.05; **P ≤ 0.01.

Fig. 3

Insulin secretion in aged BESTO transgenic and control islets and mice in response to other insulin secretagogues. (A) Insulin secreted (ng mL⁻¹ h⁻¹) from transgenic (black bars) and control (white bars) islets was measured after stimulation with either 2 mM glucose or 20 mM KCl. Insulin secretion assays were performed on islets isolated from 18- to 24-month-old male BESTO and control mice from line 431-2 using triplicate groups of 10 islets for each mouse (n = 11). (B) Insulin secretion in response to arginine stimulation was measured in male BESTO mice (closed circles) compared to controls (open circles) from line 431-2 at 18–24 months of age. Following a 15 h fast, 1.5 g of arginine per kg body weight was injected intraperitoneally, and plasma insulin levels were assessed at 0, 2, and 5 min post-injection (n = 3–6). All results are expressed as mean ± standard error. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 4

Plasma glucose, insulin, and lipid levels in BESTO transgenic and control mice fed a Western-style, high-fat diet (HFD). (A) Fed and fasted plasma glucose levels in male (M) and female (F) BESTO (black bars) and control (white bars) mice from line 431-4 before (0) and after (12 weeks) a HFD regimen. For fed glucose levels, n = 12–15. For fasted glucose levels, n = 5–15. (B) Fed and fasted plasma insulin levels in male (M) and female (F) BESTO (black bars) and control (white bars) mice before (0) and after (12) a HFD regimen. For fed insulin levels, n = 4–12. For fasted insulin levels, n = 5–15. a, P ≤ 0.001; b, P ≤ 0.01; c, P ≤ 0.05, compared to glucose or insulin levels before HFD feeding. (C–E) Fed and fasted plasma (C) cholesterol (D) triglycerides, and (E) total free fatty acid levels in male (M) and female (F) BESTO (black bars) and control (white bars) mice from line 431-4 before (0) and after (12 weeks) an HFD regimen (n = 6–15). a, P ≤ 0.001; b, P ≤ 0.01, compared to values before HFD feeding. All results are expressed as mean ± standard error. *P ≤ 0.05.

Fig. 5

Intraperitoneal glucose tolerance tests (IPGTTs) in BESTO transgenic and control mice fed a Western-style high-fat diet (HFD). (A) IPGTTs were conducted in male and female BESTO transgenic (closed circles) and control (open circles) mice following 12 weeks on a HFD. Following a 15 h fast, 2 g of 50% glucose (w/v) per kg body weight was injected intraperitoneally, and blood glucose values were assessed at 0, 15, 30, 60, and 120 min post-injection (n = 9–14). (B) Plasma insulin levels in male and female transgenic mice (black bars) and controls (white bars) were measured at the 0, 15, and 30 min time points during the IPGTTs shown in (A). All results are expressed as mean ± standard error. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 6

The effect of NMN administration on IPGTTs and plasma insulin levels in aged BESTO and control female mice. (A) NAD content in 100 cultured pancreatic islets isolated from young (Y, 5 months old) and old (O, 19 months old) BESTO mice. The measurements were conducted in duplicate or triplicate of primary islets pooled from three individual mice. (B) Plasma NMN levels in young (Y, 5 months old) and old (O, 19 months old) BESTO mice. The measurements were conducted with four individual mice for each sex and age. (C) IPGTTs were conducted in 20-month-old female BESTO transgenic mice (closed circles) and controls (open circles) from line 431-4 after mice were injected with PBS or NMN (500 mg kg⁻¹ body weight) and fasted for 14 h (n = 10–15). (D) Plasma insulin levels were measured at 0 and 30 min post-glucose injection. #P ≤ 0.05; §P ≤ 0.01, compared to insulin levels 30 min after glucose injection in PBS-injected aged BESTO and control mice. All results are expressed as mean ± standard error. *P ≤ 0.05; ***P ≤ 0.001.

Fig. 7

A model for the age-associated loss of the glucose-responsive phenotypes in aged BESTO mice. Sirt1 promotes insulin secretion through two independent mechanisms in pancreatic β cells. In one mechanism, Sirt1 mediates the repression of Ucp2 expression and thereby increases ATP content, resulting in the enhancement of GSIS. In the other mechanism, Sirt1 regulates the expression of an unidentified factor that acts downstream of β cell depolarization and possibly stimulates insulin granule exocytosis, resulting in the enhancement of (KCl)-stimulated insulin secretion (KSIS). Aging affects systemic NAD biosynthesis, perhaps at the level of biosynthesis of NMN in plasma, and decreases Sirt1 activity in pancreatic β cells of aged BESTO mice.