SIRT6 deacetylase transcriptionally regulates glucose metabolism in heart
Authors: Nowrin Fathma
Faiz Ahamed
Nagalingam R Sundaresan
Babukrishna Maniyadath
Sneha Mishra
Ullas Kolthur-Seetharam
Subhajit Dasgupta
Anwit S Pandit
Abhinav Dubey
Hanudatta S Atreya
Mohsen Sarikhani
Danish Khan
Journal: Journal of Cellular Physiology (2018)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: FoxO1; PDC; PDK4; SIRT6; cardiovascular diseases.
Affiliations: Cardiovascular and Muscle Research Laboratory, Department of Microbiology and Cell BiologyIndian Institute of ScienceBengaluruIndia, Tata Institute of Fundamental ResearchColabaMumbaiIndia, NMR Research CentreIndian Institute of ScienceBengaluruIndia, Tata Institute of Fundamental Research; Colaba Mumbai India

Abstract:  Sirtuins are a family of enzymes, which govern a number of cellular processes essential for maintaining physiological balance. SIRT6, a nuclear sirtuin, is implicated in the development of metabolic disorders. The role of SIRT6 in regulation of cardiac metabolism is unexplored. Although glucose is not the primary energy source of heart, defects in glucose oxidation have been linked to heart failure. SIRT6+/- mice hearts exhibit increased inhibitory phosphorylation of PDH subunit E1Œ±. SIRT6 deficiency enhances FoxO1 nuclear localization that results in increased expression of PDK4. We show that SIRT6 transcriptionally regulates the expression of PDK4 by binding to its promoter. SIRT6+/- hearts show accumulation of lactate, indicating compromised mitochondrial oxidation. SIRT6 deficiency results in decreased oxygen consumption rate and concomitantly lesser ATP production. Mechanistically, SIRT6 deficiency leads to increased FoxO1-mediated transcription of PDK4. Our findings establish a novel link between SIRT6 and cardiac metabolism, suggesting a protective role of SIRT6 in maintaining cardiac homeostasis. 

Retracted: No

PubPeer comments:
 The article by Khan et al., u201cSIRT6 deacetylase transcriptionally regulates glucose metabolism in heartu201d is highly interesting. While I have noticed some scientific issue in this article as follows: (i) This article is mainly focused on SIRT6 genetic modifications like SIRT6 knockdown and overexpression experiments. (a) The SIRT6 stable knockdown was generated using only 21 nucleotides u201c5 aaggaaacatgtttgtagagg 3 and explained as shRNA cloned, generated lentivirus for the stable knockdown. I am totally wondering about the shRNA without a short hairpin structure for the stable gene knockdown. (b) Importantly, the so-called 21 nucleotides of shRNA sequence [5 aaggaaacatgtttgtagagg 3 ] is a positive sense sequence of SIRT6 mRNA. It makes me surprise, about the stable knockdown using positive sense small RNA interference (RNAi). (c) They confirmed the insert using sequencing. Please provide the sequencing result. This positive sense sequence of SIRT6 mRNA [5 aaggaaacatgtttgtagagg 3 ] in the lentivirus, might have inserted randomly in any of the genes in the cell which leads to the shutdown of the inserted gene expression, but not SIRT6 knockdown. (d) Similarly, the details about the SIRT6 overexpression adenovirus were not provided in this article Collectively, the SIRT6 stable knockdown and overexpression may not properly be carried out (ii) Second important experiment in this article is ChIP (a) The FoxO1 binding DNA sequence is not present in the primer amplification range in the promoter of SOD2 [F:CCTCTTGTTTATTACATCAAGATTG, R: CTGGTTGTCAACTTGACTATATC] (b) Similarly, FoxO1 binding DNA sequence is present more than 1000bp away from the primer amplification range in the promoter of PDK4 [F GCGAACACCAATGCACTCTC, R: ATTGGCACCCTTGGGATAGG] (c) Moreover, the direct binding of SIRT6 in the intact (without damage) DNA (promoter) is unsettled. However, the SIRT6 binding DNA sequence is not established in the SOD2 and PDK4 primer amplification range. Collectively, the ChiP data generated in this article may not scientifically sound to confirm the SIRT6 mediated transcriptional activity of FoxO1 (iii) Third most focused one is SIRT6 and FoxO1 interaction (a) SIRT6+ - mice itself increased the FoxO1 at mRNA and protein levels, and SIRT6 overexpression reduced the FoxO1 at mRNA and protein levels. So, there is no much need to study the SIRT6 and FoxO1 interactions. (b) In figure 4a, FoxO1 IP condition, the FoxO1 band seems to be non-specific smearing. IP experiment may not work  (c) In figure 4d, SIRT6-KD cells shows fewer FoxO1 levels in the confocal  (iv) Fourth, a rescue experiment (a) FoxO1 DN adenovirus experiment was performed without Null-adenovirus control (figure 5c)  (b) Figure 3g and Figure 5c are identical  Finally, this study may not scientifically prove the SIRT6 deacetylase transcriptionally regulates glucose metabolism in the heart Thanks , 
 Dear colleague, Thank you for your interest in the manuscript. Please find below our point-by-point response for the concerns raised here. **1. Comment** (i) This article is mainly focused on SIRT6 genetic modifications like SIRT6 knockdown and overexpression experiments. (a) The SIRT6 stable knockdown was generated using only 21 nucleotides u201c5 aaggaaacatgtttgtagagg 3 and explained as shRNA cloned, generated lentivirus for the stable knockdown. I am totally wondering about the shRNA without a short hairpin structure for the stable gene knockdown. (b) Importantly, the so-called 21 nucleotides of shRNA sequence [5 aaggaaacatgtttgtagagg 3 ] is a positive sense sequence of SIRT6 mRNA. It makes me surprise, about the stable knockdown using positive sense small RNA interference (RNAi). (c) They confirmed the insert using sequencing. Please provide the sequencing result. This positive sense sequence of SIRT6 mRNA [5 aaggaaacatgtttgtagagg 3 ] in the lentivirus, might have inserted randomly in any of the genes in the cell which leads to the shutdown of the inserted gene expression, but not SIRT6 knockdown. (d) Similarly, the details about the SIRT6 overexpression adenovirus were not provided in this article Collectively, the SIRT6 stable knockdown and overexpression may not properly be carried out **Response** The commenter correctly points out that the shRNA sequence mentioned in the manuscript is the positive strand sequence of SIRT6. We obtained this sequence from a database (RNAicodex) which we have mentioned in the manuscript, rather than make our own. Since then, the RNAiCodex database is NO LONGER hosted on the CSHL servers, so, unfortunately, we cannot go back and link it here. As anyone in the field would know, the hairpin shRNA would have BOTH sense and antisense sequences. Our cloning method was EXACTLY as outlined in https: www.addgene.org protocols plko #B . For cloning, the actual forward oligo used was 5u2019 CCGG aaggaaacatgtttgtagagg CTCGAG cctctacaaacatgtttcctt TTTTTG 3u2019 and the reverse oligo was 5u2019 AATTCAAAAA aaggaaacatgtttgtagagg CTCGAG cctctacaaacatgtttcctt 3u2019 However, we do acknowledge that we should have mentioned the sequence presented in the manuscript as shRNA target sequence or given the complete sequence with the hairpin sequences. The likely reason for this oversight in the methods section of our manuscript was that the lentiviral constructs and the stable knockdown cells were GENERATED during the study, but NOT USED for any of the experiments presented in the manuscript. For all the SIRT6 depletion experiments in cells (1c, 2a, 2b, 3e, 3g, 4d, 4e, 5a, 5b, 5c, S3a) presented in the manuscript, we have used only siRNA which clearly reduces SIRT6 levels (shown in Figure 2a). The siRNA does not integrate into the genome and therefore rules out any concerns of random insertions or shutdown of unintended genes. We have used SIRT6 stable knockdown cells only for some validation experiments which were not added in the manuscript as they were redundant with the experiments performed using siRNA. We will now contact the journal to remove the details the stable knockdown generation from the methods section, to avoid any confusion. As for the SIRT6 adenovirus, we acknowledge that we had missed to mention the details of the adenovirus and thank the commenter for pointing it out. The SIRT6 adenovirus was obtained from Vector Biolabs (1556). The same adenovirus was also used in several of our other studies on SIRT6 (Maity et al., 2020; Ravi et al., 2019; Sundaresan et al., 2012). In addition, please note that the adenovirus infected cells displayed robust overexpression of SIRT6 which was confirmed by western blotting and shown in this manuscript (Figure 2e, 3i). We hence do not doubt the adenoviral overexpression of SIRT6 and the conclusions drawn from the experiments. We will contact the journal to update the catalog details of adenovirus in the methods section. In summary, we thank the commenter for pointing out the missing details in the methods section, but strongly disagree with any conclusion that SIRT6 knockdown and overexpression experiments may not properly be carried out. We have clearly shown the successful knockdown and overexpression using western blotting in Figures 2a, 2e and 3i. Therefore, the presented results are very much valid and DOES NOT affect any of the findings in the paper. **2. Comment** (ii) Second important experiment in this article is ChIP (a) The FoxO1 binding DNA sequence is not present in the primer amplification range in the promoter of SOD2 [F:CCTCTTGTTTATTACATCAAGATTG, R: CTGGTTGTCAACTTGACTATATC] (b) Similarly, FoxO1 binding DNA sequence is present more than 1000bp away from the primer amplification range in the promoter of PDK4 [F GCGAACACCAATGCACTCTC, R: ATTGGCACCCTTGGGATAGG] (c) Moreover, the direct binding of SIRT6 in the intact (without damage) DNA (promoter) is unsettled. However, the SIRT6 binding DNA sequence is not established in the SOD2 and PDK4 primer amplification range. Collectively, the ChIP data generated in this article may not scientifically sound to confirm the SIRT6 mediated transcriptional activity of FoxO1 **Response** **a) The FoxO1 binding DNA sequence is not present in the primer amplification range in the promoter of SOD2 [F:CCTCTTGTTTATTACATCAAGATTG, R: CTGGTTGTCAACTTGACTATATC]** The commenteru2019s assumption is a little faulty here. The consensus binding sequence of FoxO1 does not occur just once but at multiple places in the promoter (Obsil Obsilova, 2011). Our primers was experimentally validated in the H9C2 cell line by Junichi Sadoshimau2019s lab in 2014 ( u201cA functional interaction between Hippo-YAP signalling and FoxO1 mediates the oxidative stress responseu201d ; https: www.nature.com articles ncomms4315) in figure 2a and 2b of their main paper (Shao et al., 2014). We have independently verified the primer sequences to work in our hands as well.  **b) Similarly, FoxO1 binding DNA sequence is present more than 1000bp away from the primer amplification range in the promoter of PDK4 [F GCGAACACCAATGCACTCTC, R: ATTGGCACCCTTGGGATAGG]** Once again, this is not true. The site is -347bp from TSS and very much inside the amplicon. This amplicon was designed after bioinformatic analysis (as rat promoter binding site for FoxO1 on PDK4 was not published at that time). The PWM consensus sequence of FoxO1 was used from JASPAR 2018 (MA0480.1), and the site specificity to the PWM consensus was set at p 0.0001. We have attached the sequence directly, where the TSS of PDK4 (NM_053551) is marked (coding region is in bold capitals), along with forward primer (cyan), reverse primer (yellow) and putative FoxO1 binding site (green). Screenshots from the Eukaryotic promoter database and JASPAR are also added. Further, a study that was published around the same time as our study also validated a FoxO1 binding element within the amplification range of our primer pairs (Gopal et al., 2017).     https: epd.epfl.ch cgi-bin get_doc?db=rnEpdNew format=genome entry=Pdk4_1 **(c) Moreover, the direct binding of SIRT6 in the intact (without damage) DNA (promoter) is unsettled. However, the SIRT6 binding DNA sequence is not established in the SOD2 and PDK4 primer amplification range.** SIRT6 is a chromatin binding protein and has been to shown to the enriched in the promoter region of numerous genes. There is cornucopia of evidence in the literature in this regard (Etchegaray et al., 2019; Kawahara et al., 2009; Liu et al., 2020; Mostoslavsky et al., 2006; Zhong et al., 2010). Please note that the ChIP experiments show binding in the context of chromatin (hence **Ch**romatin **I**mmuno **P**recipitation) which is what we show in the manuscript as well. Collectively, many of the assumptions made here on the ChIP experiments seem to be faulty and renders the doubts raised untenable. **3. Comment** (iii) Third most focused one is SIRT6 and FoxO1 interaction (a) SIRT6+ - mice itself increased the FoxO1 at mRNA and protein levels, and SIRT6 overexpression reduced the FoxO1 at mRNA and protein levels. So, there is no much need to study the SIRT6 and FoxO1 interactions. (b) In figure 4a, FoxO1 IP condition, the FoxO1 band seems to be non-specific smearing. IP experiment may not work (c) In figure 4d, SIRT6-KD cells shows fewer FoxO1 levels in the confocal **Response** **(a) SIRT6+ - mice itself increased the FoxO1 at mRNA and protein levels, and SIRT6 overexpression reduced the FoxO1 at mRNA and protein levels. So, there is no much need to study the SIRT6 and FoxO1 interactions.** This comment seems to ignore the true complexities of transcriptional regulation. There are often multiple causal reasons for the same observed increase in the transcription of the target genes by transcription factors. While SIRT6 depletion does increase FoxO1 levels, that itself may not be sufficient for its recruitment to promoters. We therefore evaluated whether SIRT6 interacts with FoxO1 (Figure 4a), affects the sub-cellular localisation of FoxO1 (Figure 4b) and also whether SIRT6 affects FoxO1 recruitment to its target promoters (Figure 4e). **(b) In figure 4a, FoxO1 IP condition, the FoxO1 band seems to be non-specific smearing. IP experiment may not work** In addition to the blot in the manuscript. we checked lower exposures of the blot and confirmed a specific band at a molecular weight between 75 and 80 kDa. Please note that FoxO transcription factors undergo multiple modifications and often result in multiple closely located bands in this molecular weight range. Besides our IP experiment, we also documented the colocalization of SIRT6 with FoxO1 using confocal microscopy (Figure 4B). Further, the interaction between SIRT6 and FoxO1 has also been observed by several other research groups and we have also quoted one such reference in the manuscript itself (Jung et al., 2019; Song, Wang, Ka, Bae, Park, 2016; Zhang et al., 2014). **(c) In figure 4d, SIRT6-KD cells shows fewer FoxO1 levels in the confocal** The cells in the lower half of Figure 4d (please refer to full image pasted here with proper labelling as in manuscript) which corresponds to SIRT6 KD clearly shows markedly lower levels of SIRT6 and more FoxO1 levels compared to controls in the upper half. If the commenter was trying to point out that few odd cells in the whole field do not show expected high staining for FoxO1, this might be due to inherent heterogeneity of cells. Also, the increased FoxO levels under SIRT6 deficient conditions were confirmed by qPCR as well as western blotting in both in vitro and in vivo conditions (Figures 3c, 3d, 3e, 5a). On the other hand, SIRT6 overexpression reduces FoxO1 levels both at mRNA and protein level (Figures 3f, 3i). Indeed, the commenter itself has alluded to these data in his first comment in this section (Comment iii (a)).  **4. Comment** (iv) Fourth, a rescue experiment (a) FoxO1 DN adenovirus experiment was performed without Null-adenovirus control (figure 5c) (b) Figure 3g and Figure 5c are identical **Response** **(a) FoxO1 DN adenovirus experiment was performed without Null-adenovirus control (figure 5c)** The experiment did include Null-adenovirus control and is clearly mentioned in the figure legends. Here is the legend from the manuscript for reference, u201c(c) Representative confocal images of PDK4 and pu2010PDH in control or SIRT6u2010depleted (SIRT6u2010KD) cells infected with either Adu2010Null or Adu2010DNu2010FoxO adenovirus.u201d **(b) Figure 3g and Figure 5c are identical** We thank the commenter for pointing this out. There is indeed an unintended duplication which occurred while assembling figures. The oversight occurred possibly because the experiments in figure 3g and 5c involved similar conditions and labels and were carried out parallelly. We regret this inadvertent error and we are in the process of contacting the journal to get it corrected. However, this error does not affect the validity of our findings as we have also shown additional evidences that SIRT6 regulates phosphorylation of PDH complex in in vivo condition (Figure 3c) as well as in vitro (Figure 3i, 5a) conditions using western blotting. **Comment** Finally, this study may not scientifically prove the SIRT6 deacetylase transcriptionally regulates glucose metabolism in the heart **Response** This does not follow from the comments when it is read along with the response posted here. We would like to emphasize that we have employed multiple experimental approaches including a combination of in vitro and animal-based studies. Besides, we have clearly validated knockdown and overexpression of SIRT6 using western blotting. Therefore, concerns such as u201cThis article is mainly focused on SIRT6 genetic modifications like SIRT6 knockdown and overexpression experimentsu201d and u201cSIRT6 stable knockdown and overexpression may not properly be carried outu201d are unfounded. The concerns regarding ChIP experiments such as u201cFoxO1 binding DNA sequence is not present in the primer amplification range in the promoter of SOD2u201d and u201cFoxO1 binding DNA sequence is present more than 1000bp away from the primer amplification range in the promoter of PDK4u201d are not true as explained above. The binding sites have been arrived by systematic bioinformatic approaches and experimentally validated by ChIP experiments. Moreover, the regions within the range of our primers have been shown to be bound by FoxO1 by other studies as well (Please see the response). Next, the comments such as u201cthere is no much need to study the SIRT6 and FoxO1 interactionsu201d seem to ignore the true complexities of transcriptional regulation as explained in our response. Also, the statement u201cFoxO1 DN adenovirus experiment was performed without Null-adenovirus controlu201d seemed to have missed the information in the figure legends. Collectively, the findings in the study are backed by multiple evidences contained within the manuscript and elsewhere in the literature and hence we do not agree with the statement made here. If any further clarifications needed, please contact the corresponding author N. RAVI SUNDARESAN @ rsundaresan@iisc.ac.in. We are happy to clarify any doubts, share the detailed experimental protocols and original data if required. Thank you for carefully reading the manuscript and critically commenting on the contents and findings. Sincerely, Danish Khan **References** Etchegaray, J.-P., Zhong, L., Li, C., Henriques, T., Ablondi, E., Nakadai, T., . . . Mostoslavsky, R. (2019). The Histone Deacetylase SIRT6 Restrains Transcription Elongation via Promoter-Proximal Pausing. Molecular Cell, 75(4), 683-699.e687. doi:https: doi.org 10.1016 j.molcel.2019.06.034 Gopal, K., Saleme, B., Al Batran, R., Aburasayn, H., Eshreif, A., Ho, K. L., . . . Ussher, J. R. (2017). FoxO1 regulates myocardial glucose oxidation rates via transcriptional control of pyruvate dehydrogenase kinase 4 expression. Am J Physiol Heart Circ Physiol, 313(3), H479-h490. doi:10.1152 ajpheart.00191.2017 Jung, S. M., Hung, C.-M., Hildebrand, S. R., Sanchez-Gurmaches, J., Martinez-Pastor, B., Gengatharan, J. M., . . . Guertin, D. A. (2019). Non-canonical mTORC2 Signaling Regulates Brown Adipocyte Lipid Catabolism through SIRT6-FoxO1. Molecular Cell, 75(4), 807-822.e808. doi:https: doi.org 10.1016 j.molcel.2019.07.023 Kawahara, T. L. A., Michishita, E., Adler, A. S., Damian, M., Berber, E., Lin, M., . . . Chua, K. F. (2009). SIRT6 links histone H3 lysine 9 deacetylation to control of NF-u03baB dependent gene expression and organismal lifespan. Cell, 136(1), 62-74. doi:10.1016 j.cell.2008.10.052 Liu, W. H., Zheng, J., Feldman, J. L., Klein, M. A., Kuznetsov, V. I., Peterson, C. L., . . . Denu, J. M. (2020). Multivalent interactions drive nucleosome binding and efficient chromatin deacetylation by SIRT6. Nature Communications, 11(1), 5244. doi:10.1038 s41467-020-19018-y Maity, S., Muhamed, J., Sarikhani, M., Kumar, S., Ahamed, F., Spurthi, K. M., . . . Sundaresan, N. R. (2020). Sirtuin 6 deficiency transcriptionally up-regulates TGF-u03b2 signaling and induces fibrosis in mice. Journal of Biological Chemistry, 295(2), 415-434. doi:10.1074 jbc.RA118.007212 Mostoslavsky, R., Chua, K. F., Lombard, D. B., Pang, W. W., Fischer, M. R., Gellon, L., . . . Alt, F. W. (2006). Genomic Instability and Aging-like Phenotype in the Absence of Mammalian SIRT6. Cell, 124(2), 315-329. doi:10.1016 j.cell.2005.11.044 Obsil, T., Obsilova, V. (2011). Structural basis for DNA recognition by FOXO proteins. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1813(11), 1946-1953. doi:https: doi.org 10.1016 j.bbamcr.2010.11.025 Ravi, V., Jain, A., Khan, D., Ahamed, F., Mishra, S., Giri, M., . . . Sundaresan, N. R. (2019). SIRT6 transcriptionally regulates global protein synthesis through transcription factor Sp1 independent of its deacetylase activity. Nucleic Acids Research, 47(17), 9115-9131. doi:10.1093 nar gkz648 Shao, D., Zhai, P., Del Re, D. P., Sciarretta, S., Yabuta, N., Nojima, H., . . . Sadoshima, J. (2014). A functional interaction between Hippo-YAP signalling and FoxO1 mediates the oxidative stress response. Nature Communications, 5(1), 3315. doi:10.1038 ncomms4315 Song, M.-Y., Wang, J., Ka, S.-O., Bae, E. J., Park, B.-H. (2016). Insulin secretion impairment in Sirt6 knockout pancreatic u03b2 cells is mediated by suppression of the FoxO1-Pdx1-Glut2 pathway. Scientific Reports, 6, 30321-30321. doi:10.1038 srep30321 Sundaresan, N. R., Vasudevan, P., Zhong, L., Kim, G., Samant, S., Parekh, V., . . . Gupta, M. P. (2012). The sirtuin SIRT6 blocks IGF-Akt signaling and development of cardiac hypertrophy by targeting c-Jun. Nature Medicine, 18, 1643. doi:10.1038 nm.2961 Zhang, P., Tu, B., Wang, H., Cao, Z., Tang, M., Zhang, C., . . . Zhu, W.-G. (2014). Tumor suppressor p53 cooperates with SIRT6 to regulate gluconeogenesis by promoting FoxO1 nuclear exclusion. Proceedings of the National Academy of Sciences, 111(29), 10684-10689. Zhong, L., D Urso, A., Toiber, D., Sebastian, C., Henry, R. E., Vadysirisack, D. D., . . . Mostoslavsky, R. (2010). The Histone Deacetylase Sirt6 Regulates Glucose Homeostasis via Hif1u03b1. Cell, 140(2), 280-293. doi:https: doi.org 10.1016 j.cell.2009.12.041 **** ,