Beneficial Role of HO-1-SIRT1 Axis in Attenuating Angiotensin II-Induced Adipocyte Dysfunction
Authors: Nitin Puri
Hari Vishal Lakhani
Sneha S Pillai
Komal Sodhi
Joseph I Shapiro
Nader G Abraham
Mishghan Zehra
Journal: International Journal of Molecular Sciences (2019)
Discipline: CHEMISTRY
Keywords: adipocytes; angiotensin II; heme oxygenase 1; mineralocorticoid receptor; oxidative stress
Affiliations: Department of Surgery, Internal Medicine, and Biomedical Sciences, Marshall University Joan C. Edwards School of Medicine, Huntington, WV 25701, USA., Department of Pharmacology, New York Medical College, Valhalla, NY 10595, USA., Department of Surgery, Internal Medicine, and Biomedical Sciences, Marshall University Joan C. Edwards School of Medicine, Huntington, WV 25701, USA. .

Abstract:  Background: Angiotensin II (Ang II), released by the renin-angiotensin-aldosterone system (RAAS), contributes to the modulatory role of the RAAS in adipose tissue dysfunction. Investigators have shown that inhibition of AngII improved adipose tissue function and insulin resistance in mice with metabolic syndrome. Heme Oxygenase-1 (HO-1), a potent antioxidant, has been demonstrated to improve oxidative stress and adipocyte phenotype. Molecular effects of high oxidative stress include suppression of sirtuin-1 (SIRT1), which is amenable to redox manipulations. The mechanisms involved, however, in these metabolic effects of the RAAS remain incompletely understood. Hypothesis: We hypothesize that AngII-induced oxidative stress has the potential to suppress adipocyte SIRT1 via down regulation of HO-1. This effect of AngII will, in turn, upregulate mineralocorticoid receptor (MR). The induction of HO-1 will rescue SIRT1, hence improving oxidative stress and adipocyte phenotype. Methods and results: We examined the effect of AngII on lipid accumulation, oxidative stress, and inflammatory cytokines in mouse pre-adipocytes in the presence and absence of cobalt protoporphyrin (CoPP), HO-1 inducer, tin mesoporphyrin (SnMP), and HO-1 inhibitor. Our results show that treatment of mouse pre-adipocytes with AngII increased lipid accumulation, superoxide levels, inflammatory cytokine levels, interleukin-6 (IL-6) and tumor necrosis factor α (TNFα), and adiponectin levels. This effect was attenuated by HO-1 induction, which was further reversed by SnMP, suggesting HO-1 mediated improvement in adipocyte phenotype. AngII-treated pre-adipocytes also showed upregulated levels of MR and suppressed SIRT1 that was rescued by HO-1. Subsequent treatment with CoPP and SIRT1 siRNA in mouse pre-adipocytes increased lipid accumulation and fatty acid synthase (FAS) levels, suggesting that beneficial effects of HO-1 are mediated via SIRT1. Conclusion: Our study demonstrates for the first time that HO-1 has the ability to restore cellular redox, rescue SIRT1, and prevent AngII-induced impaired effects on adipocytes and the systemic metabolic profile. 

Retracted: Yes

PubPeer comments:
 Looks like the vector is integrating in the genome - the TALEN, the beta-lactamase, etc ... 8 diff studies show this (CRISPR, and ZFN too) https: sanchakblog.wordpress.com 2019 03 08 adeno-associated-virus-vector-delivery-method-in-gene-editing-is-not-safe-it-inserts-the-vector-including-the-bacterial-nuclease-in-the-genome-5-studies-in-mice ![file] These are too low to be an sequencing artifact. Contamination? Also, if the parental line were not edited (?), why is a vector containing the TALEN used??? Here is an example GNL.SRA.SRR3290535.194392907.2 194392907 CTAATGAATGGTGGAAAGTCTATCCATCTTCAGTAACAGAATT TAAGTTCTTGTTTGTGAGTGGTCATTTCAAAGGAAACTACAAA GCTCAGCTTACAAGATTGAATCATATCACTAATTGTAAT encodes a 44 aa from the TALEN protein in the vector NEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCN , 
 Here is another better example - a pair read encoding TALENs. A paired read helps gives more confidence, since it reads from [both ends ] ![file] Here is a paired read that encodes TALENS SRR3290535.3624337.1 3624337 length=125 AAGATTGCAAAACGTGGCGGCGTGACCGCAATGGAGGCAGTGCATGCATCGCGCA ATGCACTGACGGGTGCCCCCCTGGAGACGGGCGCCGCTACAGGGCGCGTCCCATT CGCCATTCAGGCTGC encoded a KIAKRGGVTAMEAVHASRNALTGAPLETGAATGRV 35 aa while the other read SRR3290535.3624337.2 3624337 length=125 CAATACGGCGATTGACTCTTCTGATCAATTCCGGCGCGTGCGGCAATCCCTTTTTCACTGCA TCCATGGCAGGACGTCCGCCGAGGCAGGCCAAGGCGACGAGGTGGTCGTTGGAGACGACCGGT encodes the 41 aa PVVSNDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRI This spans from 517 to 1190 in the plasmid pCS2TAL3-DD - showing this has been integrated in the genome. , 
 This is now a pre-print (not from me)... https: www.biorxiv.org content 10.1101 715482v1.article-info , 
 I could not figure the TALEN sequence used. In fact, if I had the exact DNA sequence they used, maybe the integration will be more apparent. Also, how did they deliver the TALEN - they say  3 TALENs mRNA (2 u03bcg) with the following RVD codes (Left monomer-NGNGNGHDNGNGNNNNNGNINNNNHDNGNN) and (Right monomer- NNNININININNNINNNINNNGNGNGNGNNNING) and 1 u03bcg of the Pc HDR template (see below) was co-transfected into 500,000 early passage fibroblasts from 2120. But, how does just mRNA get transcribed into protein without the supporting mechanisms (promoter, etc). They need plasmid (DNA), right? Also, is not mRNA very unstable? If its mRNA, and not DNA, then I am wrong - i.e. there is no TALEN integration. I just did not think thats possible (and it is very good news that this works). , 
 #4 Promoters are needed for transcription, not translation...  Also, is not mRNA very unstable? That s kind of the point of using RNA... so that you only get transient expression. , 
 Yes, thanks (that was a slip of the tongue - the promoter thing if mRNA is being sent directly). But, that must be really an arduous task for the mRNA - 1) avoid being degraded 2) get translated 3) find the correct DNA site to bind (and its soulmate - ie left and right) 4) have the template around to correct also somewhere nearby. But, great if it works. At least the worry of [nuclease integration ] is taken care of. Also, they should have done a better job with the contamination. Even the parental lines have TALEN reads. Contamination can drive a wrong story a long way. https: www.nature.com articles s41586-019-1451-5 , 
 Similarly, this [data from Massachusetts General Hospital] shows a very high level of AAV integration... The data also shows Cas9 integration, [as did this DMD study] . However, that is not mentioned in their abstract. ![file] , 
 No mention of TALEN in the genomic sequences in the FDA-preprint - if Recombinetics used mRNA (as they said they did), there should have been no TALEN in the genomic sequence. And yet, here they are: [They just cant come around to saying that the nuclease integrates.] ![file] SRR3290535.3624337.1 3624337 AAGATTGCAAAACGTGGCGGCGTGACCGCAATGGAGGCAGTGCATGCATCGCGCAATGCACTGACGGGTGCCCCCCTGGAGACGGGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGC SRR3290615.170159348.1 170159348 CCGGGTGTTGGCTGAGCGCAACGATGTGCGCGTGTGTAAACCCATGGCCCACCAGTGCCTCGTGGTGCTGCGCCACTGTCGAACGCACCTTCGGTTTGATCTTCTCTTGCTGCTGCTGACTGTAG SRR3290615.3766163.2 3766163 GAACCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCCACGATGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGG SRR3290615.3766163.1 3766163 GAGCGCTTGCTTGCCGCCATCGTGGCTGGCGATAGCCACCACTTGGTCCGGAGTCAGGCCATGGTCCTGGCACAGCACCGGCAACAGCCGCTGCACCGTTTCGAGCGCTTGCTTGCCGCCATTGT SRR3290615.229638680.1 229638680 TGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACAATGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTG SRR3290615.129716599.1 129716599 CTATCGCCAGCAACGGTGGCGGCAAGCAAGCGCTCGAAACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGACCATGGCCTGACCCCGGACCAAGTGGTGGCTATCGCCAGCAACAATGGCGGC SRR3290615.229638680.2 229638680 AGCTGGGCCACAATGCTTTCGAGCGCTTGCTTGCCGCCAATGTTGCTGGCGATAGCCACCACTTGGTCCGGGGTCAGGCCATGGTCCTGGCACAGCACCGGCAACAGCCGCTGCACCGTTTCGAG SRR3290615.129716599.2 129716599 CAGGCCGGCTCAGCTGGGCCACAATGCTTTCGAGCGCTTGCTTGCCGCCAATGTTGCTGGCGATAGCCACCACTTGGTCCGCGGTCAGGCCATGGTCCTGGCACAGCACCGGCAACAGCCGCTGC , 
 This is from their Supplemental Methods:  RCI-001 was the only animal from progenitor 2122 and a coin-flip was used to randomize between RCI-002 and RCI-004. Why not just sequence both? , 
 Yes the supplemental methods clearly state that TALEN mRNA was introduced into the cells:  Briefly, for HP-24.8, btHP1.3 TALENs mRNA (2 u03bcg) with the following RVD codes (Left monomer-NGNGNGHDNGNGNNNNNGNINNNNHDNGNN) and (Right monomer- NNNININININNNINNNINNNGNGNGNGNNNING) and1 u03bcg of the Pc HDR template (see below) was co-transfected into 500,000 early passage fibroblasts from 2120. For transfection, cells and nucleic acids were resuspended in buffer u201cRu201d and electroporated using the 100u03bcl tips by the following parameters: input Voltage; 1800V; Pulse Width; 20ms; and Pulse Number; 1, using the NEON electroporation system (Life Technologies). Individual colonies were propagated and evaluated for introgression of the Pc as previously reported1. So theoretically there should be no way this DNA is present in the cow genome unless somehow it got reverse transcribed in the cells. Sandeep Chakraborty, do you find any evidence for non-specific integration of the HDR template??? I can imagine a few possibilities though. I think the less likely possibility is that there was contaminating dsDNA in the mRNA prep. I m assuming the mRNA was made using an in vitro transcription kit that uses dsDNA as template. Typically dsDNA is removed by DNase treatment but I suppose there might still be some around. I think this is unlikely simply because the amount would be really really low. I think the real explanation is simpler and it s just plasmid contamination in the lab. They probably had a bunch of plasmid preps for the TALEN constructs and some of the DNA got spread around the lab. We do lots of NGS experiments in the lab and this has occurred several times. It s very obvious when DNA seq experiments get contaminated with plasmids encoding cDNA expression clones. I expect that s probably what happened here. Sandeep Chakraborty, are these TALEN sequences linked to parts of the cow genome? Or are the reads only covering the TALEN? , 
 #9 Also coverage is very low - they should have done amplicon sequencing of the target site, that would also have given integration events (or even translocations) Like [this ] , 
 #10 No, there is no exact integration of these TALEN reads - the paired reads are fully from the TALEN. I doubt this is plasmid contamination - then the numbers would be more. Remember, the coverage is very low - the N for targetted sequence reads are themselves very low. So, its easy to miss the integration site. The integration for other plasmid elements are there (as the pre-print also says) - but they should have mentioned this, whatever be the final explanation. Here are the TALEN fingers...![file] , 
 #12 Since the TALEN DNA can t be mapped to an integration site, I would wager that this is low level plasmid contamination in the sample. The other possibilities just seem really unlikely (reverse transcription of TALEN mRNA or DNA contaminating the TALEN mRNA prep). The simplest explanation is that a small amount of the TALEN plasmid got into the samples at some stage in the DNA sequencing process (must have been prior to NGS library construction since it would need to have illumina adapters added). , 
 A guess or wager is not good enough. Whatever be the explanation, the pre-print should have mentioned that - and its upto the Recombinetics authors to provide an explanation. Not you or me. Also, they have not provided the the mRNA used for the TALENs, and thus the target site (which they used for an off-target analysis). Finally, they should have done a targeted amplicon sequencing - that would have easily shown this integration. Like [here] or [here] .. , 
 And there is one [off-target integration] in the calves - WGS is not really a good way to find off-targets, one hardly gets coverage. They should just have done amplicon sequencing of the [edited region] . ,