Irisin Mediates Effects on Bone and Fat via αV Integrin Receptors
Authors: Daniel J Brooks
Bruce M Spiegelman
Alexander M Roche
Kaitlyn K Gerber
Mark Jedrychowski
Hyeonwoo Kim
Timothy S Strutzenberg
Hua Tu
Wenjing Chen
Sara Vidoni
Virginia-Jeni A Parkman
Christiane D Wrann
Kenichi Nagano
Patrick R Griffin
Joshua Chou
Lynda F Bonewald
Roland Baron
Phuong T Le
Yukiko Kitase
Bruce D Pascal
Chenhe Zhou
Mary L Bouxsein
Scott J Novick
Clifford J Rosen
Laura Mattheis
Journal: Cell (2018)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Irisin receptor; bone resorption; integrin αV; irisin; osteocyte
Affiliations: Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA; Department of Cell Biology, Harvard University Medical School, Boston, MA 02115, USA., Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA; Department of Cell Biology, Harvard University Medical School, Boston, MA 02115, USA; Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02219, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA., Department of Anatomy and Cell Biology, School of Medicine, Indiana University, Indianapolis, IN 46202, USA., Department of Oral Medicine, Infection, and Immunity, Harvard School of Dental Medicine, Boston, MA 02115, USA., Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL 33458, USA., Maine Medical Center Research Institute, Scarborough, ME 04074, USA., Center for Advanced Orthopedic Studies, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA., LakePharma, Inc., San Carlos, CA 94070, USA., Center for Advanced Orthopedic Studies, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA; Department of Orthopedic Surgery, Harvard Medical School, Boston, MA 02215, USA., Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Department of Oral Medicine, Infection, and Immunity, Harvard School of Dental Medicine, Boston, MA 02115, USA., Department of Anatomy and Cell Biology, School of Medicine, Indiana University, Indianapolis, IN 46202, USA; Department of Orthopedic Surgery, School of Medicine, Indiana University, Indianapolis, IN 46202, USA., Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA; Department of Cell Biology, Harvard University Medical School, Boston, MA 02115, USA. Electronic address: .

Abstract:  Irisin is secreted by muscle, increases with exercise, and mediates certain favorable effects of physical activity. In particular, irisin has been shown to have beneficial effects in adipose tissues, brain, and bone. However, the skeletal response to exercise is less clear, and the receptor for irisin has not been identified. Here we show that irisin binds to proteins of the αV class of integrins, and biophysical studies identify interacting surfaces between irisin and αV/β5 integrin. Chemical inhibition of the αV integrins blocks signaling and function by irisin in osteocytes and fat cells. Irisin increases both osteocytic survival and production of sclerostin, a local modulator of bone remodeling. Genetic ablation of FNDC5 (or irisin) completely blocks osteocytic osteolysis induced by ovariectomy, preventing bone loss and supporting an important role of irisin in skeletal remodeling. Identification of the irisin receptor should greatly facilitate our understanding of irisin's function in exercise and human health. 

Retracted: No

PubPeer comments:
 Identical blots different conditions: ![file] , 
 Loading control panel has been re-used in two western blots showing completely different experiments. Changes to brightness and contrast. https: i.imgur.com tk4wAzt.jpg , 
 We enjoyed reading the paper about possibility of using FNDC5 in osteoporosis. Bone related parameters reported only for vertebrae and osteocyte lacunae for tibiae. Did the authors evaluate what happens to bone parameters in long bones under the same experimental conditions?, any changes that are significant between the groups? , 
 #3 Thank you for your comments. We did not do histomorphometry of long bones but microCT was done and showed a higher BV TV in the KO both at baseline and after OVX, with significance on several parameters. , 
 #1 We unintentionally duplicated the beta-actin western blot image from Figure S6A in Figure S4C when we prepared the figures. The corrected figure S4C appears below. We have sent a correction to the journal. ![file] , 
 Thanks Hyeonwoo Kim. It is surprising to see the run pattern of B-Actin blot is totally different from the run pattern other experimental protein blots(for eg: CREB around 50 KDa) in your corrected blot image. This shows that the beta actin blot was not obtained by probing the same gel-blot (See below for cell journal instructions and recommendations) at the same time. Could you upload the raw western full length blots for comparison. ------------ 2. Loading controls More questions get raised about duplication or misuse of loading controls in figures with western blot or RT-PCR data than almost any other issue. Just to make sure we are all on the same page, the point of a loading control is to be able to see how consistently samples were loaded onto and run transferred through the same gel. ****It is against this control that changes in levels of the band of interest are calibrated, so it s important that the loading control be run through the gel from the same loading well as the experimental sample you want to probe.**** That is to say, running two separate gels won t yield a good loading control even if using the same pool lysate of nucleic acids proteins and the same pipetman setting for loading. -------------- , 
 #4 Thanks. Is there a reason the authors used 36 week old OVX animals for analysis of bone parameters instead of well reported time points of 6-8 weeks. How important is the age of the mice in bone phenotypes seen in FNDC5 KO animals? , 
 THanks for the note; we wanted to use adult animals that were older; a decision agreed upon by all the authors; generally 4 months is peak bone mass in trabecular compartments in female B6 (see several publications with Dr. Bouxsein) so we wanted to at least reach that age one important aspect that informed our decision (and will be presented in a second paper on overexpressing Mck Fndc5Tg moouse) was our finding that low trabecular bone mass in the Tg at 4.5 months vs + + controls suggesting this would be an optimal time for studying OVX in the KO. Of course age is a major determinant of bone mass in controls and mutants so there is always the chance that different phenotypes would be present. Six to 8 weeks however we feel are too young for OVX experiments. , 
 Thanks Clifford. If the feeling is  Six to 8 weeks however we feel are too young for OVX experiments , then what is the rationale behind estimating and showing increased levels of plasma irisin levels from OVX mice at 8 weeks in Figure S2G? Is this data even relevant for overall conclusions of the paper. We could not find any data in the paper about irisin levels at the preferred age(36-39 months) when there are changes in bone mass shown between control and knockouts. Is there a reason why plasma irisin levels were not described for experiments done at 9 months of age. Is plasma irisin even detected at this time point and increased upon OVX to be attributable to the bone phenotypes? , 
 ![file] Another figure from the same paper where beta actin blot was not obtained by probing the same gel-blot. Arrows show differences between beta actin run pattern vs CREB run pattern that clearly indicate these data were not obtained by same gel-blot. This makes beta actin an imperfect loading control for the experimental data shown in this figure. , 
 This paper was published on 13th of December 2018. So fishing out the original full length blots should not be difficult. , 
 #6, #11 These experiments monitor phosphorylated species, and in each case the phospho-protein blot is individually controlled with a total-protein blot. Because of that, the actin blots are essentially superfluous. If you think loading has not been suitably controlled, perhaps you could explicitly explain for which of the results or interpretations you think the authors were misled as a result of unequal loading. And explain your reasoning. Otherwise, your criticisms of imperfect loading controls strike me as nitpicking. Separately, what exactly do you imagine that  original full length blots would clarify that is not already evident in the portions that are shown? Do you have something in mind? I m having trouble imagining why this would be helpful, but perhaps that isn t your goal. , 
 #10 Why is b-actin even needed? The blot already shows FAK, Zyxin, and CREB to serve as controls for p-FAK, p-Zyxin, and p-Creb. , 
 Leucanella Acutissima your statement that  These experiments monitor phosphorylated species, and in each case the phospho-protein blot is individually controlled with a total-protein blot is based on what evidence. For example, If you see the blot in comment #10 by Microtus Oaxacensis, your reasoning about loading control being taken care by phospho-protein blot individually controlled with a total-protein blot(blots depicting FAK and FAKpY397 and CREB and CREBpS133) would be wrong as those panels are definitely not obtained by probing the same blot? or do you want phospho-protein blot and total protein blot should be probed on individual blots? Please explain your reasoning in support of your arguments with examples. Multiple figures in this paper, the authors seem have obtained apart from beta actin blot, phosphorylated and total protein panels also by probing separate blots. Also I don t agree with your argument that a separate beta actin blot is unnecessary in these figures? Lets follow cell press guidelines and stop creating case by case rules that will suit our arguments. As far as full length blots are concerned, they will give us more information for example, about molecular weight of individual proteins depicted in the figures and also tell us whether same blots were stripped and re-probed to obtain phospho-protein and total protein. If the authors indeed obtained the each panel of the figures from bands obtained from probing separate blots, they can comment by offering more insights into the same as they already responded for obvious duplication of beta actin blots earlier. , 
 There are enough differences in band sizes and run patterns between FAK and FAKpY397 blots in the images mentioned in this discussion to support comment #12 of phospho-protein blot is individually controlled with a total-protein blot. On the contrary, these differences between phosho-protein and total protein panels in these figures differences essentially strengthen #6, #11. , 
 #12 The point of seeing the entire blots is the following. First, the authors showed the same actin blot for two different experiments. When this is pointed out, they claim this was a mistake, and here is the correct blot. The shape of the new  correct blot makes it (very?) unlikely to be from the same gel. This is pointed out and the easiest way to resolve this would be to show the entire gel. If indeed, the  corrected actin bands are not from the same gel, than this means the authors made the same mistake again, whilst correcting a first mistake, or deliberately chose to show a second incorrect actin blot. Which means the authors would be either sloppy or worse. If the  corrected actin bands are indeed correct and the full gel image confirms this, then it is clear this was just an honest mistake the first time round. It does not really matter if you need the actin loading control or not. If as an author, you choose to show an actin loading control, it should be the correct one! This is data integrity 101... , 
 Same sample but separate blot is acceptable practice especially when there are too many proteins to blot in the same molecular weight range. There is nothing untoward in this manuscript amendement. , 
 Leucanella Acutissima, your statement  These experiments monitor phosphorylated species, and in each case the phospho-protein blot is individually controlled with a total-protein blot is simply wrong because in this paper, a number of western Blots for phosphorylated protein panels seem not to fit the corresponding total protein blot panels controls provided in the figures which is evident if you just look at the blots included in this discussion so far. It is not a small superfluous issue, because the purpose of such gel analysis is to compare the levels of a certain phosphorylated protein among different samples, say control versus various treatment or a the effect of a treatment at different time points. This is why it is important to ascertain that exactly same amounts of protein-containing cell lysates are present in each gel lane. Here, one does not rely solely on prior protein concentration measurements, but also performs loading controls of the same gel, for a protein which levels are not expected to change in the course of treatments. In the case of phosphorylation analysis, one usually probes with specific antibodies for the same total, non-phosphorylated protein. Only when the equal loading is ascertained can one compare the changing levels of the phosphorylation of your protein of interest, or in fact of the overall expression of any other protein you wish to analyze, using corresponding antibodies. Key point here is it must always be the same gel. It is of no use to detect amazing differences in a protein expression or phosphorylation, while not having a proof that the gel was indeed equally loaded. To probe a different gel where same samples were re-run for an allegedly corresponding loading control does not really make much sense: no two gels are ever exactly same, and a lot of incidental and not so incidental mistakes can happen during the sample loading or the gel run or transfer. This is also why one can spot if the two images really belong to the same physical gel: the lane widths and band sizes and shapes are a give-away if they do not match. Original scans of western blots will inform if any tricks were employed with loading controls, antibody specificity and stealthy gel splicing or even band duplications. It is now a required practice to include these in full length scans in all the accepted papers in many journals including nature cell biology and nature communications. Uploading them in this forum only prove that authors are meticulous in using the internal controls and reported the same. , 
 A lot of responses, but not a single one rose to the challenge: * ...explicitly explain for which of the results or interpretations you think the authors were misled as a result of unequal loading * Apparently those in the zero-tolerance loading-control crowd experience a lot of trouble with pipetting accurately. They seem to imagine that the authors here have excellent loading technique for all of the total protein blots (FAK, zyxin, CREB, Integrins, actin), but for some reason get sloppy and have drastically unequal loading for the phospho-protein blots. It really makes no sense. So, the concerns are theoretical, and they conflict with the empirical evidence. There is no objective basis for thinking that the results are misleading due to unequal loading, there is only criticism that the results don t meet some standard of perfection. I recognize those critics can never be swayed. , 
 #19 It seems like the key difference is in the assumption of good faith, something that is a little hard to justify on this site. You may be right that the likelihood of a careful and precise scientist messing up the pipetting is low. But the possibility and temptation to create a result by varying loading exists in the absence of the loading control. And the possibility of a screw up does still exist, although performing and showing replicates would essentially eliminate that concern. , 
 #19 #19 If you believe CELL journal editors are wrong about loading controls you can enlighten them directly by writing an editorial. I believe that the anonymous reviewers as well as the readers were indeed mislead by not stating clearly in the STAR methods section that separate gel-blots were used for some or all of their western blots while probing for loading controls(B-Actin, phospho-protein, total protein etc) contrary to the standard practices and journal instructions(see below). ** Loading controls** More questions get raised about duplication or misuse of loading controls in figures with western blot or RT-PCR data than almost any other issue. Just to make sure we are all on the same page, the point of a loading control is to be able to see how consistently samples were loaded onto and run transferred through the same gel. It is against this control that changes in levels of the band of interest are calibrated, so it s important that the loading control be run through the gel from the same loading well as the experimental sample you want to probe. *That is to say, running two separate gels won t yield a good loading control even if using the same pool lysate of nucleic acids proteins and the same pipetman setting for loading.* , 
 Loading control blot shown in the erratum in in figure S4C seem to be obtained through running a new gel as its run pattern is totally different from all the other blots shown in that figure. Per cell press recommended guidelines  it s important that the loading control be run through the gel from the same loading well as the experimental sample that needs to be probed . Could authors provide more clarity on how they obtained the blots shown in the original and corrected data and whether cell press has changed their guidelines for this paper. ,