Effects of GFP on C2C12 metabolism

I wanted to share some results we recently generated on the Seahorse Bioanalyzer, looking at the effect of GFP on cell metabolism. I was really surprised at the size of the response, and how reproducible it was. Hopefully others will find this data useful/interesting when interpreting their own results. Please let me know in the comments section below if you have found something similar.

This year my BSc(Hons) student (the very talented Ms Alex Webster) has been working on a C2C12 cell based project involving transient overexpression of a GFP tagged protein. Alex has completed several experiments so far looking at the effect of her protein on rates of proliferation and differentiation. Because of my ongoing interest in cell metabolism, Alex also used the Seahorse XF24 Bioanalyzer to examine the effect of her protein on OxPhos and glycolysis. She compared metabolism across three groups, control, GFP alone and her protein tagged with GFP, the results of the first two groups are presented below (we are planning on publishing a study with the tagged protein, so it isn’t included).

GFP_bioenergeticsMETHODS: The two graphs are from a single mitochondrial stress-test, run on a Seahorse XF24(3) machine, measuring oxygen consumption rate (OCR, top graph), and extracellular acidification rate (ECAR, bottom graph). In each graph, the green line/points indicate the GFP group, and black the control. Cells were seeded at a density of 25,000 cells/well and transfected with a commercially available GFP plasmid from Origene (pCMV6-AC-GFP) using Lipofectamine3000. The assay was run the next day as previously described by Dr David Nicholls (I will provide a full outline of our methodology in the ‘Muscle Methods’ section at a later date).

RESULTS: As you can see, overexpression of GFP in C2C12 cells results in a significant 3-fold increase in basal glycolysis (bottom graph) and a minor decrease in maximal oxidative capacity (peak observed between points B and C in top graph). Alex has repeated this experiment several times, and in every case experiment she has observed a 2-3 fold increase in glycolysis.

These results have important implications for the use of fluorescent tagging in cell culture studies. Altered cellular metabolism can lead to altered metabolite levels which can directly influence the transcriptional profile of the cell. Importantly, these results also indicate the need to confirm the metabolism of cells isolated in lineage tracing models (such as the often used ROSA26eYFP mouse).

As we have only used a single plasmid at this stage, I would be really interested to hear what experiences others have had when using fluorescent tags and the Seahorse.


Reflections on metabolism and skeletal muscle stem cells

The last few months have been a bit of a whirlwind at work, with plenty of successes to celebrate – the biggest of which has been the acceptance and publication of my postdoctoral research in Cell Stem Cell  (Ryall et al. 2015) investigating the process of metabolic reprogramming in skeletal muscle stem cells. This project began in 2008 in Dr Vittorio Sartorelli’s Laboratory of Muscle Stem Cells & Gene Regulation, when we noticed that there was a distinct metabolic gene profile in quiescent compared to proliferating muscle stem cells. What was particularly clear was the increased expression of glycolytic genes in the proliferating population (nearly every glycolytic enzyme was increased 2-20 fold). A similar upregulation can be observed in other previously published datasets, such as those from Margaret Buckingham and Tom Rando. This shift towards glycolysis is not unexpected in a proliferating cell population, and similar changes in metabolism have been observed in other stem cell and cancer cell populations. One of the reasons for this change is the requirement for new biomass (nucleotides, proteins and phospholipids for the generation of new cells, see here and here for recent reviews). However, what was particularly exciting was the link between this change in metabolism, gene transcription, and cell identity. We observed that the increase in glycolysis led to a reduction in the availability of NAD (a substrate required for the deactylase activity of SIRT1) and, as a result, an increase in the acetylation of one of the histone targets of SIRT1, histone H4 lysine 16 (H4K16). Using the Pax7cre X SIRT1fl/fl mouse model we could demonstrate elevated global H4K16ac in skeletal muscle stem cells, while culturing cells in galactose based growth media (instead of glucose) could prevent/delay the decline in NAD and H4K16ac. These results suggest that skeletal muscle stem cells can undergo a process of metabolic reprogramming during activation, and clearly link metabolism to cell identity (see our graphical abstract below).

While the last two-decades have focused extensively on the molecular revolution, and defining the transcriptional networks that regulate skeletal muscle stem cell identity and the processes of activation and proliferation, I expect the next decade to reveal the underlying cellular signals that initiate and regulate these networks. I’m excited about the role metabolism may play in regulating these processes, particularly the importance of metabolite availability (NAD for SIRT mediated deacetylation, acetyl-coA for acetylation, methionine for DNA/histone etc).

Refers to Ryall JG, Dell’Orso S, Derfoul A, Juan A, Zare H, Feng X, Clermont D, Koulnis M, Gutierrez-Cruz G, Fulco M, Sartorelli V (2015). The NAD+-Dependent SIRT1 Deacetylase Translates a Metabolic Switch into Regulatory Epigenetics in Skeletal Muscle Stem Cells. Cell Stem Cell Feb 5;16(2):171-183.


Just wanted to quickly post a big congratulations to all of the students in the Department of Physiology who handed in their Honours theses on Friday. A huge amount of work goes in to an Honours year (by both student and supervisor!), and such an effort should be recognised. In particular, I want to acknowledge the efforts of my own student, an awesome effort over the whole year…… now the real work begins!

On a related note, for students interested in doing Honours or Masters in the Department of Physiology at The University of Melbourne starting in 2014 please see the link here 

Musings on the future of academic publishing

I have read a number of really great articles recently, regarding the future of academic publishing, and I have long been excited by the potential of altmetrics as a mechanism to judge the true impact of a research paper. But having spent the last week chatting with my partner (who just happens to be a rising star at one of the largest publishing houses in NYC) about the future of publishing in general, I am convinced that we need to completely reinvent the way we approach presenting and sharing data. The current system of peer-review and publishing in academic journals was perfectly suited to the environment 20 years ago, it was appropriate for the volume of publications and the size of the data sets presented. This is no longer the case.

The sheer size of some data-sets (think next-gen sequencing, or next-gen imaging), and type (3D modelling, video) cannot be presented in print editions of journals. The current solution is to present these files as supplemental data which are not available in print editions, and are (generally) separate to the main document online. Similarly, there are problems related to journal paywalls (for publicly funded research), lack of detailed methods sections (when did we arrive at the point of a single sentence being acceptable for particular methods?), impact factors (see some great blog posts from Stephen Curry and Michael Eisen), author lists and author credit, time between submission and eventual publication, the single-blind peer-review system, and many more. The open-access revolution has begun to address many of these problems (I am a massive fan of the article-level metrics and the comments section – even if this is currently underused, in the PLOS journals), but these journals are still limited to the standard article format (abstract, introduction, methods, results, discussion).

At the risk of oversimplifying and generalizing, labs tend to have a particular research focus (a disease, a protein/transcription factor, a model organism/cell etc), and that lab will build on that research focus over an extended period of time. A lab may publish several smaller papers (or one large paper) that build around a central issue, and then branch out in a new direction. The several smaller papers may be published in different journals with different first authors. The single large paper may take years from inception to publication. As each lab builds upon each publication, wouldn’t it be great to be able to see a story develop from inception to conclusion (and not the “future studies will investigate….” conclusion,  but rather “this has led us to a completely new area….” or “we are sufficiently satisfied with our conclusions and have shifted our attention to…”). Wouldn’t it be great to be able to access the entire story from a single destination, and to be able to comment on the work prior to publication? Wouldn’t it be great to see some of the raw data?

What I’m leading to with this discussion is a proposal to change the way science is presented, shared and judged. What if we had a Facebook of science? What if we took something like ResearchGate a step further and uploaded results directly on a pre-publication weekly/monthly/quarterly basis? Each researcher could have their own page which would include a short summary of their research interest, an introduction that puts their research in context, a detailed methods section (this would include the type of protocols used in the lab, step-by-step protocols to allow for reproducibility), and then (well organised!) figures and tables that are regularly updated with both raw data files and condensed figures. Finally, a discussion of the results obtained and what the next step/s will be. Importantly, all of these sections would be open for comments (from registered users). Researchers would have the option of ‘following’ pages/projects that were of interest to them and could receive notifications of updates. The relative impact of the science being conducted could be judged via page views, links to pages, number of followers, this could be judged on both an immediate level (a researcher uploads an incredibly exciting result once), and across the course of their career (a researcher who consistently uploads interesting results).

A system such as this would increase peer-review from 2-3 reviewers, to however many people find the topic interesting. It would allow for the generation of a complex story over a number of years, will still providing clear evidence of progress (especially important for early- and mid-career researchers). It should encourage the publication of both positive and negative results (imagine how many experiments have been repeated by different labs but never published? Think of how much money is wasted because we don’t publish negative results). It should reduce the likelihood of getting ‘scooped’ after spending several years on a single project (or at least reducing the pain to getting scooped on months rather than years work).  It should encourage more open collaboration between research groups, and it should encourage uploading of raw data, thus reducing the likelihood of data manipulation.

I think some of the open-access journals have started this revolution, but I also believe that the revolution is only just beginning.

New Review on Metabolic Reprogramming of Stem Cells Published

My new review describing the metabolic reprogramming of stem-cells has been published in FEBS Journal (Click here). It is currently available in open-access format.

I’m really excited about the potential of metabolic reprogramming as a mechanism for controlling cell-fate, and while my focus remains on the resident population of skeletal muscle stem-cells, I think that work related to this topic has wide reaching applications for other stem-cell populations.

Science Policies: Liberal Party of Australia

When I first decided to write an article about science policy in Australia, I really had no idea how complex it would be, with science and research spread over a range of portfolio’s including: Innovation, industry and science; Health and ageing; Climate action, environment and heritage; and, Agriculture and food security. In fact, one could argue that the blanket term “science” could cover items in almost every single portfolio. With this in mind I will try and cover the leader, and individual ministers views towards science (based on speeches and current policy positions), as well as try and cover any specific science agenda as described in official party statements. Throughout, I have tried to provide as many links to further information as possible for those who might be interested, consider this a short introduction to Liberal Party Science Policies.

Current Policy: In a recent #asktony Twitter Q&A, I asked Tony Abbott where I could find the Liberal Parties science policy, the reply I got linked to a Dec 2011 interview transcript (see here) discussing the Coalition’s support for a science education fund and a promise that there was more to come. I’m still waiting…….

The current Liberal party policy for Innovation, Industry and Science can be found here. This policy document was written in 2010, and I can only assume that it represents the current views of the party as it lists costings running into the 2012/13 financial year. There are 15 key points laid out in this document and they give some insight into the agenda of the Liberal party in relation to science. One of the most interesting points listed in this document is number 9 “Enhance Australia’s venture capital market”. I really believe that Australia has enormous potential when it comes to the biotech sector, which is heavily under-represented in total venture capital investment. Our country does extremely well in the biotech industry, despite a real lack of investment. In fact, in a recent article in Scientific American (click here) Australia was ranked as the tenth most innovative country with regards to biotechnology. So, if the Liberal party is serious about enhancing the VC market, then I applaud them. However, at the moment it appears that any enhancement will be limited to a review of the Innovation Investment Fund.

A second key point from the Innovation, Industry and Science Policy document is point 14 “expand the Research Training Scheme”. This tends to be something both of the major parties focus on, and increasing the number of PhD/masters research students is obviously an important goal. Just as important is providing the funds, infrastructure and career paths for all of these graduating students. It is this next step that requires a lot more thought, and action.

Leader: The Hon Tony Abbott

Tony Abbott has a number of very strong (and very clear) opinions about some of the most widely discussed scientific issues facing both Australia and the world, namely climate change and embryonic stem-cell research. Mr. Abbott has repeatedly stated that he does not believe that human behaviour is responsible for climate change, and he is against embryonic stem-cell research, believing that it is a “slippery slope towards human cloning”. Mr. Abbott’s religious upbringing and beliefs have been discussed in great detail, and I don’t want to harp on them here. Instead, I want to know whether a Tony Abbott lead Coalition government would be good for science, and that really comes down to who he takes advice from.

Shadow Minister for Innovation, Industry and Science: Mrs Sophie Mirabella

Sophie Mirabella has received a lot of news recently regarding her reaction to Simon Sheikh’s collapse on Q&A, and the news generated this event far outweighs any news coming from her office regarding science policy. A quick scan of both her website (here) and twitter feeds (here) shows that she has a good handle on industry and manufacturing, but also indicates a clear absence of any science related news. In fact, the best I could find were a number of articles criticising the carbon-tax and ETS (which, despite her criticisms, Mirabella was unable to vote against after being removed from the house for 24hrs, see here). I would really like to see what the opposition MP for science would like to propose to tackle climate change, possibly the single most important factor regarding the future of industry and manufacturing. I would also like to hear more about the Coalition’s proposed R&D tax credit program, how the Coalition will promote science and research (is expanding the RTS still a priority?), are there any other plans to promote private-public research interactions. In short, instead of just hearing about what the government has done wrong, I want to know how the Coalition plans to do things differently, and what the benefit for science is likely to be.

Shadow Minister for Health and Ageing: The Hon Peter Dutton

I think that it is worth noting that in April of last year, in response to the rumour of cuts to medical research funding, Peter Dutton was quoted as saying “If they [ALP] took away a dollar, it would be a terrible outcome for medical research … for the health of our nation as well.” Dutton and the Liberal party also proposed a $212 million dollar increase for medical research (see here and here). After the way Dutton (and in fact the whole Liberal party) went after the government on this matter, it would be very hard for them to rationalize any cuts to medical research. However, all this said, both Dutton and the Coalition were very quiet on medical research following the 2012 budget. I did try and avoid focusing on science budget policy, but it is really slim pickings in regards to Coalition science policy statements.


I found it frustrating that I had to dig through a number of different Liberal party websites before I could really get much in the way of details in regards to science and research policies. I like that the Liberal party has proposed to enhance the VC market and public-private interactions, but I am disappointed that these ideas are in a document that is almost three years old, and have not been referred to once by the current minister for science. To me, this does not bode well, and indicates that science may either just continue along as it currently is, or will in fact go backwards. This is worrying as now (more than ever) is the time to invest in science.

OVERALL RATING – C+ Some good ideas, but (as yet) no follow-up.