Sirtuin 6 regulates glucose-stimulated insulin secretion in mouse pancreatic beta cells
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Sirtuin 6 (SIRT6) has been implicated in ageing, DNA repair and metabolism; however, its function in pancreatic beta cells is unclear. The aim of this study is to elucidate the role of SIRT6 in pancreatic beta cells.
To investigate the function of SIRT6 in pancreatic beta cells, we performed Sirt6 gene knockdown in MIN6 cells and generated pancreatic- and beta cell-specific Sirt6 knockout mice. Islet morphology and glucose-stimulated insulin secretion (GSIS) were analysed. Glycolysis and oxygen consumption rates in SIRT6-deficient beta cells were measured. Cytosolic calcium was monitored using the Fura-2-AM fluorescent probe (Invitrogen, Grand Island, NY, USA). Mitochondria were analysed by immunoblots and electron microscopy.
Sirt6 knockdown in MIN6 beta cells led to a significant decrease in GSIS. Pancreatic beta cell Sirt6 knockout mice showed a ~50% decrease in GSIS. The knockout mouse islets had lower ATP levels compared with the wild-type controls. Mitochondrial oxygen consumption rates were significantly decreased in the SIRT6-deficient beta cells. Cytosolic calcium dynamics in response to glucose or potassium chloride were attenuated in the Sirt6 knockout islets. Numbers of damaged mitochondria were increased and mitochondrial complex levels were decreased in the SIRT6-deficient islets.
These data suggest that SIRT6 is important for GSIS from pancreatic beta cells and activation of SIRT6 may be useful to improve insulin secretion in diabetes.
KeywordsBeta cell Calcium Glucose metabolism Insulin secretion Mitochondria SIRT6
Sirt6 beta cell-specific knockout
Sirt6 pancreas-specific knockout
Extracellular acidification rate
Glucose-stimulated insulin secretion
Histone H3 at lysine 9
Insulin tolerance test
National Institute of Diabetes and Digestive and Kidney Diseases
National Institutes of Health
Oxygen consumption rate
Short hairpin RNA
Transmission electron microscopy
Transient receptor potential cation channel subfamily M, member 2
The pathogenesis of type 2 diabetes is multifactorial, but impaired insulin secretion from pancreatic beta cells is one of the critical factors . Glucose-stimulated insulin secretion (GSIS) is a complex process that involves glucose sensing, transport and metabolism (glycolysis and mitochondrial oxidation), plasma membrane depolarisation, and calcium signalling and exocytosis, among other things .
Sirtuins belong to a conserved family of proteins, and mammals have seven members (SIRT1–7) . SIRT6 is a chromatin-associated enzyme that deacetylates histone H3 at lysine 9 (H3K9) and lysine 56 residues [4, 5, 6]. Some non-histone substrates, such as forkhead box O1 (FoxO1), general control of amino acid synthesis protein 5 (GCN5), and CTBP-interacting protein (CtIP), have also been reported [7, 8, 9]. SIRT6 can also remove long-chain fatty acyl groups from lysine residues of its substrates such as TNF-α [10, 11]. SIRT6-deficient mice exhibit accelerated ageing and die of hypoglycaemia by 4 weeks of age [12, 13]. SIRT6 has been implicated in a variety of metabolic processes, including glycolysis, gluconeogenesis, hepatic lipid and cholesterol metabolism, neuroendocrine regulation and circadian regulation of metabolism [7, 14, 15, 16, 17, 18, 19, 20, 21]. Interestingly, high-fat diet (HFD)-treated Sirt6 transgenic mice secrete more insulin in response to a bolus of glucose than their wild-type (WT) counterparts . These data suggest that SIRT6 is probably required for insulin secretion and beta cell function. In this work, we generated both pancreas- and beta cell-specific Sirt6 knockout mice to illustrate the role of SIRT6 in the pancreatic beta cells.
Pancreas-specific deletion of Sirt6 was generated by crossing Sirt6 floxed mice (Sirt6 Tm1.1Cxd, provided by C. Deng, Mammalian Genetics Section, National Institute of Diabetes and Digestive and Kidney Disease [NIDDK], Bethesda, MD, USA) with Tg(Pdx1-Cre)6Tuv mice from the Jackson Laboratory (Bar Harbor, ME, USA) [16, 23]. Beta cell-specific Sirt6 deletion was generated by crossing Sirt6 floxed mice with MIP-Cre/ERT mice (Tg(Ins1-Cre/ERT)1Lphi, provided by L. Philipson, Department of Medicine, University of Chicago, Chicago, IL, USA) and tamoxifen administration (oral gavage at a dose of 4 mg/mouse in corn oil for four consecutive days) . These mice were on the mixed background (FVB/NJ:129S6/Sv:C57BL/6J). Mice were fed either regular chow (18% energy from fat) or a HFD (42% energy from fat, Harlan Teklad, Indianapolis, IN, USA). Blood glucose levels were measured under ad libitum or overnight 16 h fasting conditions. GTT and insulin tolerance test (ITT) were performed in mice fasted for 6 or 4 h before injection of glucose (2 g/kg, i.p. or oral gavage) or insulin (0.5 U/kg for chow-fed mice and 0.75 U/kg for HFD-fed mice, i.p.), respectively. GSIS and l-arginine-stimulated insulin secretion were performed in mice fasted for 16 h before injection of glucose (2 g/kg, i.p.) or l-arginine (1 g/kg, i.p.), respectively. Tail-vein blood samples were collected for insulin measurements using an ELISA kit (ALPCO, Salem, NH, USA). All animal procedures were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee. Samples were not randomised and the experimenters were not blind to group assignment and outcome assessment. No data were excluded for the report.
MIN6 cells (provided by D. Thurmond, Department of Pediatrics, Indiana University, Indianapolis, IN, USA) were cultured and transduced with adenoviruses as previously described [25, 26]. The cell line was verified in our laboratory and it did not have mycoplasma contamination.
Islets were isolated from mouse pancreases at the Islet Core of the Indiana Diabetes Research Center as previously described .
Insulin secretion analysis
Insulin secretion analysis in MIN6 cells or mouse islets was performed as previously described . Glucose, potassium chloride (KCl), α-ketoisocaproate (KIC, Sigma-Aldrich, St Louis, MO, USA) and ionomycin (IM, Cayman, Ann Arbor, MI, USA) were used for the experiments. Insulin was analysed using an ELISA kit (ALPCO).
Total RNA samples were prepared and analysed as previously described .
Protein extracts from mouse islets and other tissues or MIN6 cells were prepared and analysed as previously described . The following antibodies were used: SIRT6 (Abcam, Cambridge, MA, USA, and Sigma-Aldrich, 1:1,000 dilution), actinin (Santa Cruz Biotechnology, Dallas, TX, USA, 1:1,000 dilution), Ac-H3K9 and cleaved caspase 3 (Cell Signaling Technology, Beverly, MA, USA, 1:1,000 dilution) and total OXPHOS antibody cocktail (Abcam, 1:250 dilution). Antibodies were validated through confirmation of protein molecular weight and their known characteristics according to existing knowledge.
Pancreases were fixed and processed as previously described . The following antibodies were used for immunostaining: glucagon (Sigma-Aldrich, 1:5,000 dilution), insulin and Ki67 (Cell Signaling Technology, 1:500 dilution). Beta cell areas were determined by analysing 10–15 pancreatic sections stained for insulin per genotype using NIH ImageJ software (http://rsb.info.nih.gov/ij/download.html). To examine proliferating beta cells, 15–20 islets were analysed for each genotype. The number of Ki67-positive beta cells per islet was normalised to the total beta cell number in each islet.
Pancreatic insulin content analysis
To measure total pancreatic insulin content, whole pancreas was dissected and processed as previously described . Insulin was measured using an ELISA kit (ALPCO).
ATP was extracted from mouse islets using 2.5% trichloroacetic acid (wt/vol.) and measured using a bioluminescent assay kit (Sigma-Aldrich).
Cellular bioenergetics analysis
The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured in MIN6 cells using an XF24 Analyzer (Seahorse Bioscience, North Billerica, MA, USA).
Cytoplasmic Ca2+ analysis
Cytosolic Ca2+ dynamics in mouse islets was measured using the Fura-2-AM (Invitrogen) calcium probe as previously described .
Isolated mouse islets were fixed with 2% glutaraldehyde (wt/vol.) and processed as previously described . Numbers of total mitochondria and damaged mitochondria from each cell were counted from a randomly selected group of 30 cells. The cytosolic area of each cell was determined using SPOT software (Sterling Heights, MI, USA), which was used to normalise the numbers of total and damaged mitochondria.
All data are presented as means ± SEM. Two-group comparisons were performed using two-tailed unpaired Student’s t test, and multiple-group comparisons were performed using ANOVA and Turkey’s post hoc test. A p value of <0.05 was considered as significant.
SIRT6 regulates insulin secretion from pancreatic beta cells
Pancreatic SIRT6 deficiency leads to insulin secretory impairment and glucose intolerance
Deletion of Sirt6 in pancreatic beta cells causes insulin secretory defects
SIRT6 deficiency impairs mitochondrial glucose oxidation
To identify the potential causes for the GSIS defects in SIRT6-deficient beta cells, we first examined glucose metabolism in MIN6 cells using an extracellular flux analyser (Seahorse; mouse islets could not be examined because of technical issues). Glycolysis and mitochondrial respiration can be monitored by measuring ECAR and OCR, respectively. Under high glucose conditions, Sirt6 knockout significantly reduced the levels of ECAR and OCR in MIN6 cells, indicating a decrease of glucose metabolism (Fig. 4b, c). Next, we measured ATP production, which is the final product of mitochondrial glucose metabolism and controls insulin secretion via closure of KATP channels. At low glucose levels (2.5 mmol/l), bMko and control islets had comparable levels of ATP; however, the ATP production in response to 16.7 mmol/l glucose was decreased by ~20% in the bMko islets relative to controls (Fig. 4d). To further confirm the defect in mitochondrial energy metabolism, we used KIC, a mitochondrial substrate that can be directly metabolised by the tricarboxylic acid cycle and bypass glycolysis to perform insulin secretion assays in isolated bMko and control islets. As predicted, bMko islets secreted 58% less insulin after incubation with 12.5 mmol/l KIC compared with controls (Fig. 4e), suggesting a defect in mitochondrial oxidation.
Ablation of SIRT6 in pancreatic beta cells leads to mitochondrial defects
SIRT6 deficiency causes aberrant calcium flux in beta cells
In pancreatic beta cells, an increase in intracellular calcium ([Ca2+]i) is critical for secretagogue-induced insulin release [33, 34]. To examine whether the reduction in the insulin secretory response to glucose or KCl may be associated with reduced calcium influx, we measured [Ca2+]i (indicated by the fluorescence ratio of 340 nm/380 nm) of isolated islets loaded with Fura-2-AM fluorescent probes. Consistent with the observation that bMko mice had normal basal insulin secretion, the islets from bMko and control mice showed similar resting [Ca2+]i levels (Fig. 7c, d). However, in response to 16.7 mmol/l glucose or 30 mmol/l KCl, [Ca2+]i was significantly lower in the bMko islets compared with the control group (Fig. 7c, d), suggesting the presence of a defect in calcium flux regulation. To further verify that the reduction of [Ca2+]i is critical for the impaired insulin secretion in SIRT6-deficient beta cells, we performed insulin secretion assays in bMko and control islets in the presence of a Ca2+ ionophore, ionomycin. Remarkably, 30 μmol/l ionomycin rescued the insulin secretion deficiency in the bMko islets when stimulated with 16.7 mmol/l glucose (Fig. 7e). These data suggest that SIRT6 regulates calcium flux in the pancreatic beta cells.
Pancreatic beta cell SIRT6 is required to protect mice against obesity-induced glucose intolerance
Impairment of GSIS is one of the early clinical manifestations in the development of type 2 diabetes ; however, the underlying mechanisms are not well understood. In this work, we have shown that SIRT6 is required for proper insulin secretion in response to glucose stimulation. Our data suggest that SIRT6 regulates GSIS through mitochondrial glucose oxidation, plasma membrane depolarisation and calcium dynamics (and possibly other mechanisms).
SIRT6 is expressed in multiple tissues, albeit at different levels with high expression in the skeletal muscle, thymus and brain in mice . Our data show here that SIRT6 is readily detectable in mouse islets. Data from a previous transcriptomic analysis of purified mouse pancreatic alpha and beta cells also reveal that Sirt6 gene expression ranks in approximately the 75th and 80th percentiles among 23,406 genes in alpha and beta cells, respectively . By contrast, another SIRT family member, SIRT1, which has been implicated in the regulation of insulin secretion from pancreatic beta cells [37, 38, 39, 40], only ranks in the 68th and 64th percentiles in alpha and beta cells, respectively . These data suggest that SIRT6 is likely to play a role in beta cell function.
SIRT6 has been shown to suppress glycolysis in mouse embryonic stem (ES) cells, fibroblasts and hepatocytes [16, 21]. However, this does not seem to be the case in pancreatic beta cells because glycolysis was decreased in SIRT6-deficient MIN6 cells in response to high glucose. Interestingly, the ECAR was decreased in both WT and SIRT6-deficient MIN6 cells in the presence of oligomycin, which usually inhibits mitochondrial OCR and promotes ECAR. This finding suggests that those beta cells might have low capacity to convert pyruvate to lactate compared with other cell types. Our data are consistent with a recent report in mouse primary islets . It is well known that an increase in ATP levels or the ATP/ADP ratio from glucose metabolism is a critical trigger in GSIS . Significantly, in our study, ATP production in the bMko islets upon glucose stimulation was lower than that in the WT islets. This result can be attributed to the compromised mitochondrial oxidation as indicated by reduced OCR in the SIRT6-deficient beta cells. Interestingly, levels of mitochondrial Complexes III and IV were decreased in the Sirt6 knockout beta cells. The electron microscopy analysis also reveals an increase in mitochondrial damage in the SIRT6-deficient beta cells. However, the cause of the mitochondrial defects is unclear. Consistent with these findings, mitochondrial defects have been also observed in Sirt6-knockout mouse ES cells and Sirt6-knockout breast cancer cells [21, 42]. In SIRT6-deficient mouse ES cells, mitochondrial respiration and a number of intermediate metabolites in the tricarboxylic acid cycle, including citrate, isocitrate, succinate, fumarate and malate, are decreased . In Hs578t breast cancer cells, overexpression of SIRT6 increases OCR and knockdown of SIRT6 decreases it . Together, these data suggest that SIRT6 promotes mitochondrial respiration. However, further study is required to elucidate how SIRT6 regulates mitochondrial function.
A high concentration of KCl can cause depolarisation of the beta cell plasma membrane, which subsequently triggers Ca2+ influx and insulin granule exocytosis . The reduction in Ca2+ influx and insulin secretion from the bMko mouse islets in response to 30 mmol/l KCl seen in the current study suggests that SIRT6 may regulate insulin secretion at membrane depolarisation and/or downstream of the depolarisation event. According to our data, aberrant Ca2+ flux is one of the potential downstream defects in SIRT6-deficient beta cells. The first evidence is that cytosolic [Ca2+] was lower in bMko islets in response to high glucose or KCl compared with the WT islets. Second, the increase of cytosolic [Ca2+] caused by the Ca2+ ionophore ionomycin normalised GSIS in the bMko islets. Since ionomycin has multiple actions that increase cytosolic [Ca2+], including store-operated Ca2+ entry and calcium-induced Ca2+ release , the precise mechanism of the regulation by SIRT6 is not clear yet. In addition, transient receptor potential cation channel, subfamily M, member 2 (TRPM2) has been suggested to play a role in insulin secretion [44, 45], and SIRT6 can modulate TRPM2 activity through its byproduct O-acetyl-ADP ribose (OAADPR) and its derivative ADP ribose (ADPR) , which can activate TRPM2 . It would be interesting to see that to what extent the OAADPR/ADPR–TRPM2 pathway contributes to the SIRT6 effect on GSIS.
In summary, this work characterises the role of SIRT6 in pancreatic beta cells and reveals its importance in insulin secretion and glucose homeostasis. Specifically, our data demonstrate that SIRT6 activity is necessary to regulate insulin secretion by maintaining mitochondrial function and modulating Ca2+ dynamics. Therefore, it is important to further investigate the mechanisms by which SIRT6 regulates beta cell function. Pharmacological activation of SIRT6 may be useful to enhance insulin secretion and it has potential for the development of effective drugs to treat type 2 diabetes.
We thank C. Deng (NIDDK, Bethesda, MD, USA) for providing the Sirt6 floxed mice, L. Philipson (University of Chicago, Chicago, IL, USA) for providing the MIP-Cre mice, D. Thurmond (Indiana University School of Medicine, Indianapolis, IN, USA) for providing the MIN6 cells, and P. Fueger, B. Maier and E. Oh (Indiana University School of Medicine, Indianapolis, IN, USA) for technical discussions.
This work was supported by grant no. R01DK091592 (XCD) from the NIDDK.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
XX carried out the study, interpreted and analysed the data, and wrote the manuscript. GW, RT, PW, KL, TK, XT and SAT contributed to data collection and manuscript preparation and revision. RAH, CE-M and RGM participated in the experimental design, data interpretation, and manuscript preparation and revision. W-XD contributed to data collection and interpretation and manuscript writing. XCD conceived the hypothesis, designed the experiments, analysed and interpreted the data and wrote the manuscript. XCD is the guarantor of this work. All authors approved the manuscript.
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