Sirt1 and mir-9 expression is regulated during glucose-stimulated insulin secretion in pancreatic b-islets Deepti Ramachandran*, Upasana Roy*, Swati Garg, Sanchari Ghosh, Sulabha Pathak and Ullas Kolthur-Seetharam Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, India Introduction MicroRNAs (mirs) regulate protein expression due to their ability to target the 3¢UTRs of mRNAs [1]. Although, in the recent past, there have been numer- ous studies reporting mir targets and their physiologi- cal implications, we still do not understand fully the mechanisms that regulate their expression. This is cru- cial as they are now known to play diverse roles and are being considered as potential therapeutic targets. Mirs have also been found to be important modulators of changes in metabolic response, including endocrine functions [2]. Several mirs involved in the control of pancreatic development and insulin secretion have been discovered recently [3,4]. Mir-375 was one of the first mirs to be identified as a key factor affecting insu- lin secretion by inhibiting glucose-stimulated insulin secretion (GSIS) [4]. Another mir that has been impli- cated in the control of insulin secretion is mir-9 [5]. Plaisance et al. [5] indicated a possible role for mir-9 in insulin secretion by showing that mir-9 targets Onecut-2 (OC-2) mRNA and down-regulates its expression in insulin-secreting cells. This decrease in OC-2 consequently leads to an increase in the levels of its target gene, granuphilin. Granuphilin has been well characterized as a key player in insulin secretion and is known to negatively regulate insulin exocytosis [6]. Therefore, on the basis of these results in INS-1E cells, using exogenously expressed human growth hormone, mir-9 has been proposed to negatively regulate insulin exocytosis [5]. However, it is unclear whether altera- tions in mir-9 levels and targeting are physiologically Keywords glucose-stimulated insulin secretion; mir-9; Sirt1; b-islets Correspondence U. Kolthur-Seetharam, B-306, Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005, India Fax: +91 22 2280 4610 Tel: +91 22 2278 2721 E-mail: ullas@tifr.res.in *These authors contributed equally to this work (Received 17 November 2010, revised 5 January 2010, accepted 31 January 2011) doi:10.1111/j.1742-4658.2011.08042.x MicroRNA mir-9 is speculated to be involved in insulin secretion because of its ability to regulate exocytosis. Sirt1 is an NAD-dependent protein deacetylase and a critical factor in the modulation of cellular responses to altered metabolic flux. It has also been shown recently to control insulin secretion from pancreatic b-islets. However, little is known about the regu- lation of Sirt1 and mir-9 levels in pancreatic b-cells, particularly during glu- cose-dependent insulin secretion. In this article, we report that mir-9 and Sirt1 protein levels are actively regulated in vivo in b-islets during glucose- dependent insulin secretion. Our data also demonstrates that mir-9 targets and regulates Sirt1 expression in insulin-secreting cells. This targeting is relevant in pancreatic b-islets, where we show a reduction in Sirt1 protein levels when mir-9 expression is high during glucose-dependent insulin secre- tion. This functional interplay between insulin secretion, mir-9 and Sirt1 expression could be relevant in diabetes. It also highlights the crosstalk between an NAD-dependent protein deacetylase and microRNA in pancre- atic b-cells. Abbreviations ARBP, acidic ribosomal binding protein; GSIS, glucose-stimulated insulin secretion; LNA, locked nucleic acid; mir, microRNA; OC-2, Onecut-2; pS, pSuper vector; pS9, pSuper mir-9 vector. FEBS Journal 278 (2011) 1167–1174 ª 2011 The Authors Journal compilation ª 2011 FEBS 1167 relevant, particularly under conditions in which insulin secretion is regulated in vivo. The Sir2 family of proteins (sirtuins) are NAD- dependent protein deacetylases that have been impli- cated in several physiological processes [7]. Mamma- lian Sirt1, one of the most well-studied members of the family, is a nuclear protein. It is known to deacetylate histones, transcription factors and co-regulators in an NAD-dependent manner [8–10]. Interestingly, levels of Sirt1 protein are known to fluctuate in tissues such as the liver, white adipose tissue, brown adipose tissue and muscle under different metabolic conditions (such as calorie restriction and starvation) [11,12]. In addi- tion to changes in its activity, as a result of fluctua- tions in NAD levels [13–15], the modulation of Sirt1 protein levels is also important for its functions [11]. In the pancreatic islets, insulin secretion is linked to glucose availability and is controlled by several factors, including changes in ADP ⁄ ATP, mitochondrial mem- brane potential and expression of proteins involved in processes such as exocytosis [16]. Importantly, Sirt1 activity-dependent down-regulation of UCP2 levels has been shown to affect insulin secretion [13]. Transgenic mice that over-express Sirt1, specifically in the b-islets (BESTO mice), have been used to show that Sirt1 is a crucial player in GSIS [17]. Although, in these trans- genic mice, there was no change in insulin secretion under fed and starved conditions, there was a dramatic effect on insulin secretion in response to glucose chal- lenge (GSIS). These reports strongly suggest a role for Sirt1 in the regulation of insulin secretion. In spite of this, it is not known whether, in insulin-secreting cells, Sirt1 protein levels are regulated in a manner similar to that in other metabolically relevant tissues [11,12]. In this study, we have further investigated the role of mirs in the control of insulin secretion. We report that the 3¢UTR of Sirt1 mRNA is targeted by mir-9 and leads to a down-regulation of its protein levels. Interestingly, we observe that this control mechanism is relevant in insulin-secreting b-cells. In order to gain more insight into the physiological role of mir-9 in insulin secretion, we have specifically addressed the link between GSIS and the regulation of mir-9 expres- sion. We report, for the first time, that mir-9 levels are regulated during GSIS in vivo in pancreatic b-islets. We also show that this involves an increase in the transcript levels of mir-9 derived from different chro- mosomal loci. More importantly, Sirt1 protein levels are modulated in the b-islets during GSIS, consistent with mir-9 levels. In conclusion, our results indicate that, in insulin-secreting cells, Sirt1 protein levels are altered in response to changes in glucose availability, and this is brought about by mir-9. Results Glucose-dependent changes in mir-9 expression affect its levels in pancreatic b-islets Mir-9 has been implicated in insulin secretion and has been proposed to be regulated by glucose levels [18]. We therefore wished to determine whether mir-9 expression was regulated in vivo in pancreatic b-islets. To this end, we isolated pancreatic b-islets from mice that had been starved for 24 h and administered glu- cose, intraperitoneally, to stimulate insulin secretion (GSIS). Following glucose injections, sera and b-islets were isolated at different time intervals. We assayed for serum insulin levels in these mice (Fig. 1A). Mir-9 levels during GSIS were quantified from total RNA isolated from b-islets. It was very interesting to see that mir-9 levels showed no change at 30 min, but increased significantly by 60 min post-glucose injection (Fig. 1B). Importantly, this increase in mir-9 coincided with the time point at which insulin levels started to decline (Fig. 1A, B). Further, we also observed that mir-9 levels remained high until 4 h post-glucose injec- tion, when insulin secretion is expected to be low or decreasing (Fig. 1A). To our knowledge, this is the first report to clearly map the kinetics of mir-9 induc- tion in vivo in b-islets during GSIS. Given the implications of mir-9 function in the pan- creas, it is important to understand the roles of both cis- and trans-acting factors that control its transcrip- tion. Mir-9 is encoded from three chromosomal loci, in both humans and mice, and the molecular mecha- nisms that regulate its expression from these loci are not well understood. In mice, mir-9 is expressed from chromosomes 3, 13 and 7, and its precursors are denoted as pre-mir-9-1, pre-mir-9-2 and pre-mir-9-3, respectively, to indicate the loci from which they origi- nate. It should be noted that, although the mir-9 sequence is the same, those of the primary and precur- sor mirs (pri-mir and pre-mir) derived from these loci are different. Addressing the relative contributions of these loci to mir-9 levels is an important aspect in understanding the mechanisms and molecular factors involved in its transcriptional induction. Therefore, in order to determine whether mir-9 expression was dif- ferentially regulated during GSIS in vivo, we examined the levels of pri- ⁄ pre-mir-9 using primers designed to distinguish the three pri- ⁄ pre-mirs (Table S1). It was clear from our results that the levels of mir-9 tran- scripts from chromosomes 3 and 13 started to increase by 30 min post-glucose injection and peaked at 60 min (Fig. 1C). Together, our data clearly demonstrated that mir-9 levels were altered in vivo and this involved Mir-9-dependent regulation of Sirt1 in b-cells D. Ramachandran et al. 1168 FEBS Journal 278 (2011) 1167–1174 ª 2011 The Authors Journal compilation ª 2011 FEBS a differential contribution from chromosomes 3, 7 and 13. Further studies should help us to identify the mechanism involved in this differential expression from these loci, including the identification of transcription factors. Sequence analyses of mir-9 upstream regions indi- cated that there were CpG islands at these loci in humans and mice (Fig. S1). Indeed, mir-9 expression is known to be altered by hypermethylation in cancers [19,20]. Our results showed that pri- ⁄ pre-mir-9-3 expression was very low and barely detectable (Fig. 1C). The analysis of CpG methylation at the mir- 9-3 locus using methylation-sensitive enzymes, followed by PCR, showed that a low level of pri- ⁄ pre-mir-9-3 in the islets was probably not caused by hypermethyla- tion of this locus (Doc. S2 and Fig. S2). Mir-9 negatively regulates Sirt1 protein Mir-9 has been shown to target OC-2 in INS-1E cells and to regulate the exocytosis of over-expressed human growth hormone in these cells [5]. To further elucidate the role of mir-9 in insulin secretion, we looked for possible mir-9 targets using the online prediction tools Pictar and Targetscan. We found Sirt1 mRNA to be one of the candidate mRNAs for mir-9 targeting, among several others, based on seed complementarity and evolutionary conservation (Fig. 2A, B). Taking into consideration the importance of both Sirt1 and mir-9 in insulin secretion, we wished to determine whether this targeting was true. In order to assess this, Sirt1 3¢UTR from mouse cDNA was cloned into pmir-Report plasmid that encodes a firefly luciferase (Fig. S3). The pre-mir-9 sequence cloned into pSuper vector (pS9) was used for the over-expres- sion of mir-9, as described earlier by Plaisance et al. [5] (Doc. S3 and S4). Empty pmir-Report vector that lacked the 3¢UTR of Sirt1 did not show any changes in luciferase activity in the presence or absence of mir-9. Luciferase activity from pmir-Report carrying the 3¢UTR sequence of Sirt1 decreased only in the presence of mir-9 and in a dose-dependent manner. To confirm this targeting, the mir-9 binding site in Sirt1 3¢UTR was mutated and used in the luciferase assay. Unlike the wild-type 3¢UTR, there was no decrease in luciferase activity when mutant 3¢UTR was transfected together with pS9 (Fig. 2C). These results clearly show that mir-9 specifically targets the 3¢UTR of Sirt1. Further, to determine whether mir-9 reduced the endogenous Sirt1 protein levels, NIH3T3 cells were transfected with control and mir-9 precursor (pre-mir- 9). The results indicated that exogenously added mir-9 was able to down-regulate endogenous Sirt1 protein (Fig. 2D). Pre-mir-34a was used as a positive control [21]. Hence, our data show that mir-9 is able to target Fig. 1. Glucose-stimulated insulin secretion is accompanied by changes in mir-9 levels in pancreatic b-islet cells. (A) Insulin levels from the serum of mice were measured at 0, 15, 30, 60 and 240 min post-intraperitoneal glucose injections. (B) Mir-9 levels determined by RT-qPCR analysis from total RNA isolated from pan- creatic b-islets of the mice from (A). U6-snRNA levels were used for normalization. (C) Real-time qRT-PCR analysis of total RNA from pan- creatic b-islets of mice subjected to intraperitoneal glucose injec- tions at the indicated time points to determine the levels of transcripts pri ⁄ pre-mir-9-1, pri ⁄ pre-mir-9-2 and pri ⁄ pre-mir-9-3. ARBP was used as the normalization control. (A–C) One-way ANOVA was used for statistical analysis (n =3; *P < 0.05, **P < 0.01). In (B), * and # indicate significance with respect to the 0- and 30-min time points, respectively. In (C), # and * indicate significance with respect to the 0-min time point for 9-1 and 9-2, respectively. D. Ramachandran et al. Mir-9-dependent regulation of Sirt1 in b-cells FEBS Journal 278 (2011) 1167–1174 ª 2011 The Authors Journal compilation ª 2011 FEBS 1169 Sirt1 mRNA and post-transcriptionally regulate its expression. Sirt1 is regulated in vivo in response to GSIS Sirt1 is known to affect GSIS in b-islets [17]. However, it is unclear whether its expression is regulated in vivo. We found that mir-9 levels were regulated in response to GSIS in vivo and that it targeted Sirt1 and down- regulated its expression in cells in vitro. Hence, we wished to determine whether Sirt1 levels were regu- lated in vivo in pancreatic b-islets. If mir-9 targets Sirt1 in the b-islets, we would expect to see a decrease in Sirt1 levels under conditions in which mir-9 levels are high. Therefore, Sirt1 protein was assayed and, inter- estingly, we found that Sirt1 levels were modulated in pancreatic b-islets during GSIS (Fig. 3A). We observed that there was a significant decrease in Sirt1 expression 240 min post-glucose injection. Importantly, the reduc- tion in Sirt1 protein correlated well with increased mir-9 levels in the b-islets when serum insulin secretion Fig. 2. Mir-9 targets the 3¢UTR of Sirt1 and down-regulates its expression. (A) Alignment of mature mir-9 with the target sequence on the 3¢UTR of mouse Sirt1 (using Target Scan). (B) Conservation of the target site of mir-9 on the 3¢UTR of Sirt1 across vertebrates (using Target Scan). (C) pmir-Report, pmir-Report Sirt1 3¢UTR (wild- type or mutant) luciferase construct, pSuper-mir-9 and b-galactosi- dase vector were co-transfected into HEK293T cells for 24 h, as indicated. Luciferase activities were normalized to b-galactosidase activities. Student’s t-test was used for statistical analysis (n =3; *P < 0.05). (D) Western blot analysis of Sirt1 in NIH3T3 cells trans- fected with 20 and 60 n M of precursors of mir-control, mir-9 and mir-34a. b-Actin was used as a loading control. The relative protein levels were quantified using Adobe Photoshop and are indicated. Fig. 3. Glucose-stimulated insulin secretion is accompanied by changes in Sirt1 protein, but not mRNA levels, in pancreatic b-islet cells. (A) Western blot analysis of endogenous Sirt1 protein levels in pancreatic b-islets isolated at the indicated time points from mice subjected to intraperitoneal glucose injections. b-Actin was used as a loading control. (B) Relative Sirt1 protein levels in (A) were quanti- fied using Adobe Photoshop and are plotted. One-way ANOVA was used for statistical analysis (n =3;**P < 0.01). **indicates signifi- cance with respect to the 0-min time point. (C) Sirt1 mRNA expres- sion quantified by RT-qPCR from pancreatic b-islets isolated at the indicated time points from mice subjected to intraperitoneal glu- cose injections. ARBP was used as the normalization control. Mir-9-dependent regulation of Sirt1 in b-cells D. Ramachandran et al. 1170 FEBS Journal 278 (2011) 1167–1174 ª 2011 The Authors Journal compilation ª 2011 FEBS was decreasing (Fig. 1A, B). RT-qPCR analysis of Sirt1 mRNA showed that, although the protein was down-regulated (4 h post-glucose injection), this was not a result of decreased mRNA levels (Fig. 3C). To our knowledge, this is the first report to show that the amount of Sirt1 protein is regulated (post-transcrip- tionally) during GSIS in insulin-secreting b-islets in vivo. Sirt1 is down-regulated in insulin-secreting cells in a mir-9-dependent manner Although we have described the targeting in NIH3T3 cells, we wished to ascertain whether the post-tran- scriptional regulation of Sirt1 expression in b-cells was brought about by mir-9. We therefore used insulin- secreting b-TC-6 cells to address this. b-TC-6 cells were transfected with pre-mir-control ⁄ -9 and pS ⁄ pS9. From Fig. 4A, B, it can be seen that Sirt1 protein is reduced in cells transfected with pre-mir-9 and pS9, respectively. Increased expression of mir-9 in pS9 transfected cells is shown in Fig. 4C. These findings clearly demonstrate that mir-9 is indeed able to target Sirt1 in b-TC-6 cells. In order to confirm this, b-TC-6 cells were transfected with control and anti-mir-9 locked nucleic acid (LNA). To mimic a declining insu- lin secretion phase (Fig. 4D), the cells were subjected to 8 h of glucose withdrawal, 16 h post-transfection. The reduction in mir-9 after LNA transfection was quantified by RT-qPCR (Fig. 4E). It was very interest- ing to see that Sirt1 protein levels were significantly higher in cells that had been transfected with anti-mir- 9 LNA (Fig. 4F). This result is consistent with reduced Sirt1 expression in the pancreatic b-islets in vivo at 240 min post-glucose injection (when insulin secretion is decreasing) (Fig. 3A, B). Importantly, this provides mechanistic insight into the regulation of Sirt1 protein in insulin-secreting b-cells brought about by mir-9. Discussion In this study, we have provided in vivo evidence for the regulation of mir-9 expression in pancreatic b-islets. The study by Plaisance et al. [5] clearly elucidated the link between mir-9 and exocytosis in INS-1E cells. However, whether mir-9 actually participated in GSIS or whether it was itself regulated during this process was not evident until now. Our in vivo data from b-islets show, that mir-9 expression is temporally regu- lated during GSIS. Specifically, we show that mir-9 levels increase during the fall phase of insulin secretion (Fig. 1). We also show that, in b-islets, the increase in mir-9 levels is not a result of an equal contribution from the three chromosomal loci. Our results suggest that mir-9 obtained from chromosomes 3 and 13 con- tributes to an increase in its level in b-islets (Fig. 1C). Further studies are required to dissect out molecular players, such as transcription factors, involved in the control of mir-9 expression. Our results suggest that the increase in mir-9 levels in b-islets is an active Fig. 4. Mir-9 targets and reduces Sirt1 expression in insulin-secreting cells. b-TC-6 cells were transfected with 60 n M of precur- sors of mir-control and mir-9 (A) and 1.5 lg of pSuper and pSuper-mir-9 plasmids (B, C) for 72 and 24 h, respectively. (D) b-TC-6 cells were cultured in high-glucose-contain- ing medium and subjected to 8 h of glucose withdrawal. Insulin levels in cell culture supernatants were measured. (E, F) b-TC-6 cells were transfected with 100 n M LNA anti-mir-control and anti-mir-9 for 16 h and subjected to glucose withdrawal for 8 h as detailed in Experimental procedures. (C, E) Mir-9 expression was quantified by RT-qPCR normalized to U6 levels. (C–E) Student’s t-test was used for statistical anal- ysis (n =3;*P < 0.05, **P < 0.01). (A, B, F) Western blot analysis for Sirt1 protein with b-actin as the loading control. D. Ramachandran et al. Mir-9-dependent regulation of Sirt1 in b-cells FEBS Journal 278 (2011) 1167–1174 ª 2011 The Authors Journal compilation ª 2011 FEBS 1171 process. On the basis of our finding of the temporal regulation of its expression and the earlier report on exocytosis [5], we implicate mir-9 as a crucial factor in the control of insulin secretion in response to glucose stimulation in vivo. Furthermore, we have also identified mir-9 to be a key factor in the modulation of Sirt1 expression in vivo. Recently, Saunders et al. [22] have shown that mir-9 targets Sirt1 in mouse embryonic stem cells. However, our results show that mir-9 targeting of Sirt1 is physiologically significant in insulin-secreting cells (Figs 3 and 4). Earlier studies have linked Sirt1 to GSIS. In Sirt1 transgenic (BESTO) mice, Sirt1-mediated control of insulin secretion was found to be a result of the differ- ential expression of genes, including those involved in insulin secretion [17]. Banks et al. [23] used BAC trans- genic mice over-expressing Sirt1 to show that it is a crucial factor in insulin secretion under excess calorie conditions. However, until now, it was unclear whether Sirt1 itself was regulated in b-islets during insulin secretion. Our results show, for the first time, that Sirt1 expression is altered in insulin-secreting b-islets during GSIS in vivo. Using b-TC-6 cells and conditions that mimic the declining phase of insulin secretion, we clearly demonstrate that mir-9 is involved in the regu- lation of Sirt1 protein in these cells (Fig. 4). Our results on the expression profile of Sirt1 during GSIS fit well with the earlier findings on the ability of Sirt1 to positively regulate insulin secretion. Recent reports have also examined the role of Sirt1 in cytokine-dependent b-cell cytotoxicity [24]. Interest- ingly, the data show that interferon-c and interleukin-1b treatment of insulin-secreting cells leads to a down- regulation of Sirt1. In another study by Chen et al. [25], altered oxygen tension-mediated proliferation of insu- lin-secreting cells was linked to changes in Sirt1 expression in INS-1E cells. These reports suggest that, in addition to its significant role in insulin secretion, Sirt1 is a crucial factor in b-cell proliferation and survival. On the basis of our results that identify mir-9 as a negative regulator of Sirt1 in insulin-secreting cells, it would be interesting to determine whether a similar control is operating during cytokine-mediated changes in Sirt1 levels in b-islets. Given the known functions of Sirt1 in regulating insulin secretion, our study adds a new facet by show- ing, that Sirt1 protein levels are altered in insulin-secret- ing cells during GSIS. To conclude, we have discovered a functional interplay between glucose-dependent insu- lin secretion, mir-9 levels and Sirt1 protein in b-cells. In addition, we provide some evidence for the dynamic (and differential) nature of mir-9 expression in pancre- atic b-islets. Further insights into these mechanisms may help in the understanding and tackling of diseases such as diabetes. Experimental procedures Animal experiments Adult Swiss male mice were maintained at the Tata Insti- tute of Fundamental Research animal facility in accordance with the institute’s animal ethics regulations. These mice were used for GSIS. Briefly, the mice were starved over- night and injected with glucose (3 gÆkg )1 body weight) intraperitoneally. Serum and tissue samples were collected at 0, 15, 30, 60 and 240 min post-glucose injection. Three animals per group were used and the experiment was repeated at least twice. Cell culture NIH3T3 and HEK293T cells were grown in DMEM (Sigma, St. Louis, MO, USA cat. no. D7777) supplemented with 10% newborn calf serum (Gibco, USA cat. no. 16010- 159) and 10% fetal bovine serum (Gibco cat. no. 16000), respectively. b-TC-6 cells were grown in DMEM supple- mented with 15% heat-inactivated fetal bovine serum. For glucose withdrawal, cells were washed with NaCl ⁄ P i and then DMEM without glucose (Sigma cat. no. D5030), sup- plemented with 5% heat-inactivated fetal bovine serum, was added, as indicated in the figure legends. Primers and constructs The sequences of the primers used in this study are listed in Table S1. Cloning of Sirt1 3¢UTR (wild-type and mutant) into pmir-Report (Fig. S3) and the generation of pSuper- mir-9 construct [5] are detailed in Doc. S3 and S4. Transfections Cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA cat. no. 11668-019) according to the manufacturer’s instructions. Control pre-mir and pre-mir-9 were procured from Ambion (Austin, TX, USA). LNA con- structs for control and anti-mir-9 were obtained from Exiqon (Vedbaek, Denmark). Pre-mirs and anti-mirs were transfect- ed into cells at concentrations of 20 ⁄ 60 and 100 nm, respec- tively, and harvested after 24–48 h. Mir-9 was expressed in cells by transfecting 1.5 lg of pSuper-mir-9. RNA isolation and cDNA synthesis Total RNA was isolated using Trizol (Invitrogen cat. no. 15596-026). SuperScript-III (Invitrogen cat. no. 18080-044) Mir-9-dependent regulation of Sirt1 in b-cells D. Ramachandran et al. 1172 FEBS Journal 278 (2011) 1167–1174 ª 2011 The Authors Journal compilation ª 2011 FEBS was used for cDNA synthesis with 1 lg of total RNA employing random hexamers or with mir-specific RT-6 primers (Table S1). Real-time PCR qPCRs were performed in triplicate using SYBR green (Qiagen, USA cat. no. 204056) according to the manufac- turer’s instructions. qPCR for mir was performed as described in ref. [26]. Briefly, short-mir-9 and MP- fwd ⁄ MP-rev primers (Table S1) were used at concentra- tions of 4 and 100 nm, respectively. U6 and ARBP were used for the normalization of mir and mRNA expression, respectively. Pancreatic b-islet isolation b-Islets were harvested by perfusing the pancreas as described by Szot et al. [27] (Doc. S1). Luciferase and b-galactosidase assays HEK293T cells were transfected with pmir-Report-Sirt1 3¢UTR (wild-type or mutant), b-galactosidase vector and pSuper or pSuper-mir-9 (pS ⁄ pS9) in 24-well plates. Cells were harvested 24 h later and luciferase assay was carried out using the Stratagene Luciferase Assay kit (Agilent Technologies, Santa Clara, CA, USA cat. no. 219020) according to the manufacturer’s instructions. Luciferase activities were normalized to the b-galactosidase activity in each case. Western blots Equal amounts of protein (estimated using the BCA kit, Sigma-Aldrich, USA) were run on SDS ⁄ PAGE and trans- ferred to poly(vinylidene difluoride) membranes (Roche, Basel, Schweiz cat. no. 3 010 040 ⁄ ThermoFisher cat. no. 88518). Anti-Sirt1 (Millipore-Upsatate, MA, USA cat. no. 07-131) and anti-b-actin (Sigma cat. no. A1978) antibodies were used for immunoblotting. Horseradish peroxidase- conjugated secondary antibodies (Sigma cat. nos. A0545 and A2554) and Lumi-Light Western Blotting Substrate (Roche cat. no. 12 015 196 001) were used to visualize the bands. Insulin ELISA Sera and culture supernatants from b-TC-6 cells subjected to control and glucose withdrawal conditions were assayed for insulin. Insulin was quantified according to the manu- facturer’s protocol using the Rat ⁄ Mouse Insulin 96-Well Plate Assay Kit (Millipore, MA, USA cat. no. EZRMI- 13K). Statistical analysis All statistical analyses were carried out using GraphPad InStat3 or SigmaPlot. 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Exp Cell Res 314, 3075– 3080. 26 Sharbati-Tehrani S, Kutz-Lohroff B, Bergbauer R, Scholven J & Einspanier R (2008) miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample. BMC Mol Biol 9, 34. 27 Szot GL, Koudria P & Bluestone JA (2007) Murine pancreatic islet isolation. J Vis Exp 7, 255. Supporting information The following supplementary material is available: Fig. S1. Genomic loci encoding Mus. musculus (mmu)- mir-9-1 and 9-3. Fig. S2. Analysis of the methylation status of the mir- 9-3 promoter region. Fig. S3. Generation of Sirt1 3¢UTR luciferase con- struct. Doc. S1. Pancreatic b-islet isolation [27]. Doc. S2. Analysis of the methylation status of the mir- 9-3 promoter region. Doc. S3. pSuper-mir-9 construct. Doc. S4. Sirt1 3¢UTR wild-type and mutant luciferase construct. Table S1. Primer sequences. This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Mir-9-dependent regulation of Sirt1 in b-cells D. Ramachandran et al. 1174 FEBS Journal 278 (2011) 1167–1174 ª 2011 The Authors Journal compilation ª 2011 FEBS . was regulated in b-islets during insulin secretion. Our results show, for the first time, that Sirt1 expression is altered in insulin- secreting b-islets during. Sirt1 and mir-9 expression is regulated during glucose-stimulated insulin secretion in pancreatic b-islets Deepti Ramachandran*, Upasana