We previously proposed a model whereby Sirt1 regulates insulin secretion by at least two independent mechanisms, one of which acts upstream of depolarization through Ucp2, while the other acts downstream of depolarization (Moynihan et al., 2005). In this current study, we found that there is differential regulation of these two pathways with advanced age: while the Sirt1-promoted GSIS was completely blunted as the BESTO mice reached 18–24 months of age, the Sirt1-mediated mechanism that acts downstream of depolarization remained partially intact (Fig. 3). Although the molecular target of Sirt1 in the mechanism acting downstream of depolarization is still unclear, we currently suspect that Sirt1 might regulate the expression of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex proteins, based on our recent microarray analyses (unpublished findings). SNARE complex components are known to be critical for insulin granule exocytosis (Lam et al., 2005). Therefore, it will be interesting to examine whether Sirt1 indeed affects the function of SNAREs or SNARE-associated proteins in pancreatic β cells, resulting in the enhancement of KCl-stimulated insulin granule exocytosis (Fig. 7).
One of the greatest risk factors for the development of type 2 diabetes in humans is age (Wilson et al., 1986). Type 2 diabetes is characterized by a combination of defective insulin secretion and insulin resistance that results from a progressive age-associated decline in β cell function, increased visceral fat accumulation, and decreased physical activity, among other alterations (Iozzo et al., 1999; Basu et al., 2003; Moller et al., 2003). An age-associated decline in β cell function has also been reported in rat (Reaven et al., 1979, 1983, 1987; Muzumdar et al., 2004). Although the molecular mechanism for this age-dependent β cell defect is still unknown, the findings presented here suggest the possibility that a reduction in Sirt1 activity with age, likely caused by a decline in plasma NMN levels, contributes to this age-related impairment of β cell function (Fig. 7). If this is the case, it will be of great interest to examine whether administration of NMN can restore normal β cell function in the elderly. While further understanding of the precise molecular mechanisms by which Sirt1 activity is regulated with advanced age is necessary, our findings provide new insights into the age-associated pathophysiology of β cell function. Furthermore, our results suggest a novel approach for the development of an effective therapeutic strategy to activate Sirt1 in β cells by increasing systemic NAD biosynthesis in the treatment of age-associated metabolic disorders, such as impaired glucose tolerance and type 2 diabetes.
Experimental procedures
Animal experimentation
The BESTO transgenic mice have been described previously (Moynihan et al., 2005). BESTO mice (18- to 24-month-old) were used as the aged cohort of mice, while 3- to 5-month-old BESTO mice were considered the young cohort. Non-transgenic litter-mates were used as controls. The regular chow [PicoLab Rodent Diet 20 (Lab Diets, St Louis, MO, USA), 5053] contained protein (20%), fat (ether extract, 5.0%), far (acid hydrolysis, 5.6%), crude fiber (4.7%), nitrogen-free extract (52.9%), ash (6.1%), and vitamins, and 13.2% of total calories came from fat. The HFD consisted of chow containing 42% calories from fat (TD 88137; Harlan Teklad, Madison, WI, USA). The precise dietary composition of TD88137 is: casein, 195; DL-methionine, 3.0; sucrose, 341.46; cornstarch, 150.0; anhydrous milk fat, 210.0; cholesterol, 1.5; cellulose (fiber), 50.0; mineral mix (AIN-76), 35.0; calcium carbonate, 4.0; vitamin mix, 10.0; ethoxyquin (antioxidant), 0.04 (g kg−1). All animal procedures were approved by the Washington University Animal Studies Committee.
Fed and fasted glucose, insulin, and lipid measurements
Fed glucose, insulin, and lipid levels were measured between 09:00 and 10:00 hours, while fasted glucose, insulin, and lipid levels were measured after 15 h of overnight fasting. Glucose levels were determined using the Accu-Chek II glucometer (Roche Diagnostics, Indianapolis, IN, USA) with blood collected from the tail vein. For insulin and lipid measurements, blood was collected from the tail vein into chilled heparinized capillary tubes, plasma was separated by centrifugation, and samples were stored at −80 °C. Insulin levels were determined on 10 μL aliquots using rat insulin enzyme-linked immunosorbent assay (ELISA) kits with mouse insulin standards (Crystal Chem Inc., Downers Grove, IL, USA) at the Washington University Radio-immunoassay (RIA) Core facility. Cholesterol and triglycerides were measured by the Washington University Clinical Nutrition Research Unit (CNRU) Core facility using reagents from Thermo Electron Corporation (Waltham, MA, USA), while nonesterified free fatty acid levels were measured using reagents from Wako (Richmond, VA, USA).
IPGTTs and arginine stimulation
For the IPGTTs, mice were fasted for 15 h before being given an intraperitoneal injection of 50% dextrose (2 g kg−1 body weight). Blood was collected from the tail vein at 0, 15, 30, 60, and 120 min post-injection for determination of glucose values. Plasma for insulin measurements was also collected at 0, 15, and 30 min after glucose injection and stored at −80 °C. For the arginine stimulation experiments, mice were fasted for 15 h before being given an intraperitoneal injection of arginine (1.5 g kg−1 body weight). Blood was collected from the tail vein at 0, 2, and 5 min post-injection for both glucose and insulin measurements. For the NMN administration experiments, mice were injected with either PBS or NMN (500 mg kg−1 body weight) and fasted for 14 h prior to performing the IPGTTs as described earlier. All insulin samples were submitted to the Washington University RIA Core facility for ELISA analyses.
GSIS and ATP measurements from isolated islets
Islets were isolated by collagenase digestion as described previously (Moynihan et al., 2005). Briefly, after clamping off the pancreatic ducts prior to their entry site to the duodenum, pancreata were inflated with isolation buffer (10× Hank’s buffered salt solution, 10 mM 4-2-hydroxyethyl-1-piperazineethanesulfonic acid, 1 mM MgCl2, 5 mM glucose, pH 7.4) containing 0.375 mg mL−1 collagenase (Sigma, St Louis, MO, USA). The inflated pancreata were then removed, incubated at 37 °C for 15 min, and shaken vigorously. Islets were separated from acinar tissue after a series of washes and passages through a 70 μm nylon BD Falcon Cell Strainer (BD Biosciences, San Jose, CA, USA). Handpicked islets were cultured overnight in RPMI media containing 5 mM glucose, 2 mM L-glutamine, penicillin/streptomycin, and 10% fetal bovine serum (Gibco, Invitrogen, Carlsbad, CA, USA). The islets were then preincubated in Krebs–Ringer bicarbonate (KRB) buffer containing 2 mM glucose for 1 h at 37 °C. Islets of similar size were handpicked into groups of 10 islets in triplicate and incubated with 1 mL KRB buffer containing either 2 mM glucose, 20 mM glucose, or 20 mM KCl plus 2 mM glucose for 1 h at 37 °C. The supernatant was stored at −20 °C prior to insulin measurements. Islets were then washed two times with PBS, followed by extraction with extraction buffer (0.1 M NaOH, 0.5 mM ethylenediaminetetraacetic acid). After neutralizing the samples with 0.1 M HCl, ATP levels were measured using the ATP Bioluminescent Assay Kit (Sigma) according to the manufacturer’s instructions. Insulin levels of all samples were measured by radio-immunoassay at the Washington University RIA Core facility.
Western blotting
Protein extracts prepared from islets were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto Immobilon-P membranes (Millipore, Bedford, MA, USA). Membranes were blocked in Tris–buffered saline with 0.1% Tween-20 (TBS-T) and 5% dry milk (w/v). The primary antibodies used were affinity-purified polyclonal rabbit anti-mouse Sirt1 against an N-terminal fragment of mouse Sirt1 (1 : 5000), anti-Ucp2 [1 : 100, Santa Cruz Biotechnology (Santa Cruz, CA, USA) sc-6525], and anti-actin [1 : 5000, Calbiochem (La Jolla, CA, USA) #CP01]. Secondary antibodies included the horseradish peroxidase-conjugated anti-rabbit IgG (1 : 10 000, Amersham), anti-mouse IgM (1 : 2000, Calbiochem), and anti-goat IgG (1 : 10 000, Santa Cruz Biotechnology). Signals were visualized using the ECL Advance detection system (Amersham, Pittsburgh, PA, USA) and quantitated with the ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA).
Quantitative real-time reverse transcriptase–polymerase chain reaction (RT–PCR)
Total RNA samples were purified from isolated islets as described previously (Moynihan et al., 2005). cDNA synthesis and quantitative real-time RT–PCR were conducted as previously described (Revollo et al., 2004). Primer sequences are available upon request.
Islet NAD and plasma NMN measurements
The measurements of islet NAD and plasma NMN levels were conducted as described previously (Revollo et al., 2007b). Briefly, for NAD measurements in young and old BESTO islets, primary islets (~100 per sample) were isolated from 5- and 19-month-old BESTO mice, cultured in RPMI media containing 1 μM nicotinamide in 6 cm dishes, and harvested 24 h later in 800 μL of ice-cold PBS. Islets were lysed with 100 μL of 1 M perchloric acid on ice for 15 min. Lysates were cleared by centrifugation and neutralized by adding 33 μL of 3 M K2CO3 and incubating on ice for 10 min. After centrifugation, 100 μL of the supernatant was mixed with 300 μL of buffer A (50 mM K2PO4/KHPO4, pH 7.0) and loaded onto the column. The high performance liquid chromatography (HPLC) was run, and the amounts of NAD were quantitated based on the peak areas compared to a standard curve. The measurements were conducted in duplicate or triplicate of primary islets pooled from three individual mice. For NMN measurements, the HPLC was run at a flow rate of 0.7 mL min−1 in an isocratic condition. Then, 10 μL of freshly collected mouse plasma was extracted with 100 μL of 1 M perchloric acid, and the extracts were neutralized by adding 33 μL of 3 M K2CO3 and incubating on ice for 10 min. After clearing the extracts, 25 μL of the plasma extract was mixed with 275 μL of buffer and water. NMN levels were quantitated based on the peak areas compared to a standard curve. The measurements were conducted with four individual mice for each sex and age.
Statistical analysis
Unless otherwise indicated, all values are expressed as mean ± standard error, and statistical analyses were carried out using an unpaired Student’s t-test. Differences were considered to be statistically significant when P ≤ 0.05.
Acknowledgments
We would like to thank Jeffrey Gordon, Kerry Kornfeld, M. Alan Permutt, Kenneth Polonsky, Kelle Moley, and David Beebe for their helpful suggestions and discussions. We would also like to acknowledge the Diabetes Research and Training Center and the CNRU at Washington University for insulin and lipid measurements, respectively. Shin-ichiro Imai is an Ellison Medical Foundation Scholar in Aging, and is also supported by grants from the National Institute on Aging (AG024150), American Diabetes Association, Juvenile Diabetes Research Foundation, the Washington University CNRU (DK56341), and the National Center for Research Resources (C06RR015502).
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