This post is a bit late, but I wanted to record the story behind our recent eLife paper on ALKBH7 somewhere less ethereal than Twitter.

The story for us started as a collaboration with Dragony Fu in UofR’s Department of Biology. Dragony has worked on ALKBH7 for a number of years, and had shown that it plays a critical role in programmed necrosis in response to DNA alkylation. In addition there was some earlier work showing that the Alkbh7-/- mouse was obese and had some fat oxidation problems.

ALKBH7 is a mitochondrially-localized member of the alpha-ketoglutarate (aKG) dependent dioxygenase family. This includes enzymes you’ve probably heard of such as the TETs, the Jumonji domain demethylases, and the EGLN family of prolyl hydroxylases that regulate HIF. All these proteins add -OH onto something, for varied reasons. For example the DNA demethylases perform a “demethylation via hydroxylation” fundtion – they add -OH to the methyl group, which then spontanously decomposes, giving back the original non-methylated DNA base and formaldehyde. ALKBH7 is a homolog of the E. coli DNA repair enzyme ALKB, and pretty much all the other members are involved in repairing DNA alkylation damage.

The problem is, nobody has ever found a substrate for ALKBH7! It lacks the usual DNA binding pocket that other ALKB proteins contain. In-fact, the only thing it’s ever been shown to do is hydroxylate itself on a leucine. So, we hypothesized that ALKBH7 might be a mitochondrial prolyl-hydroxylase. We sent a bunch of WT and KO heart samples off to ‘Tish Murphy’s lab, where Leslie Kennedy performed a proteomic analysis – the idea was that if ALKBH7 is a prolyl-hydroxylase, we should see less P-OH in its substrate proteins, in the knockout.

Nothing! No differences. Well, there was one protein that showed lower hydroxylation… hydroxyacyl-CoA dehydrogenase. This was interesting at first, because remember the knockouts are obese. What if prolyl-hydroxylation was a novel mechanism of regulating fatty acid beta-oxidation? Well we did a bunch of enzymology on the dehydrogenase and nothing panned out, so that was a dead end.

As part of the proteomic analysis, we also had an abundance data set, giving us the levels of 3700 proteins in the WT vs. KO hearts, and here’s where things got interesting… only 2 proteins were up, and one of them was glyoxylase I (GLO-1). We confirmed this both by western blot and by doing GLO-1 activity assays, and the effect was real.

So, what’s GLO-1? (it’s also worth noting we ignored this finding for several months because the protein was listed as “lactoylglutathione lyase” in the data set, and we didn’t know what the hell that was all about, so…) Anyway, GLO-1 is a key enzyme involved in the detoxification of methylglyoxal (MGO), which is a toxic byproduct of glycolysis. Excess glucose metabolism such as occurs in hyperglycemia and diabetes, leads to more MGO, which can react with various biomolecules to form “advanced glycation end products” (AGEs). These post-translational modifications essentially gum up protein fucntion, and this is what’s believed to drive a lot of the pathology of diabetes. When a diabetic patient gets tested for glycated hemoglobin (aka “HbA1C” or simply A1C), that’s the MGO adducted form of hemoglobin, used to indicate long term trends in blood glucose. We also did a bunch of metabolomics analysis, to show that glycolysis is up in the ALKBH7 knockouts.

So, what does any of this have to do with cardiac ischemia-reperfusion (IR) injury, the main thing that we study? Well, the main mode of cell death in IR injury is necrosis, and remember ALKBH7 is required for necrosis. So, sure enough, we were able to show that the Alkbh7-/- mouse is protected against cardiac IR injury. We also made an inhibitor for ALKBH7 and showed that it is protective too. AND we showed that blocking the GLO-1 enzyme prevents the protection in the Alkbh7-/- mice. So, GLO-1 is required for the cardioprotection.

For most who study mitochondria and cardiac IR injury or protection, the mitochondrial permeability transition (PT) pore is the go-to target when we find an effect. But, we measured PT pore opening in Alkbh7-/- and it just wasn’t very impressive. Sure, there was a small change, but nowhere near enough to protect the heart from IR injury. So we struck out again. We also had recently shown that the induction of the mitochondrial unfolded protein response (UPRmt) was capable of inducing protection, but again no differences seen in the UPRmt in Alkbh7-/- mice. More negative data!

So, the upshot of all this is that we STILL don’t really understand how ALKBH7 is required for necrosis in heart attack. When you knock out ALKBH7, there’s an upregulation of GLO-1 and a rewiring of all the bits of metabolism that make MGO, and that appears to be required for the protection. But exactly how this protein in the mitochondrion signals to MGO production in the cytosol (where glycolysis is), is still not well understood.

Of course, the final writing and revision of the paper took place during the whole #Covid19 lock down and gradual reopening process, which essentially killed our ability to take a deeper dive and really close out the story. At the end of the day, it’s hard to study an enzyme for which there is no known activity and no known substrate! We’re still working in this area, and hope to be able to address some of these unknowns soon (for example by developing a screening assay for potential ALKBH7 substrates).

In sum, this started out as a collaboration with a biology colleague, took in a multi-omics approach (proteomics, metabolomics, PTM proteomics) and a bunch of other methods (the paper has >60 panels of data), and frankly most of what we found was negative. It’s frustrating, but that’s sometimes how science works. The good thing is we learned a bunch of interesting stuff along the way, and that brings us closer to understanding cardiac metabolism and how it can be manipulated for therapeutic benefit in situations such as IR.

As is usual for us now, the paper was posted as a preprint on @BioRxiv, and we also posted full data sets (humongous proteomics files) on FigShare.

So, like most of the world, the lab has been in lock-down since early March (we ramped everything down, including culling our mouse colonies to 1/3 of their original size, and turned off the lights on March 12th). Nevertheless, things have been moving along…

(1) Work with our collaborator Sabzali Javadov at University of Puerto Rico was published. along with an editorial for AJP Lung written with Mike O”Reilly, all about Scott Ballinger’s trans-mitochondrial mouse study.

(2) Lots of grants submitted and reviewed… In February we submitted an NIDDK R01 on the potential role of mitochondrial K+ channels as anti-obesity drug targets, as well as a metabolomics collaboration with Heiko Bugger in Austria. Paul served on an NIH special emphasis panel, and will also be ad-hocking (sp?) for NIH in June.

(3) Our mass spec’ went kaput! Apparently either the turbo pump or its controller are dead, so we’re awaiting a service call to fix it, but of course with the virus shut-down it’s not clear when that will be accomplished.

(4) Thankfully, when the lock-down occurred, we were at a place in the research cycle where we’re sitting on a LOT of data, and so now we’re writing up several papers for submission in a few weeks. While (my guess is) the rest of the world is going to be churning out review articles during this time, I’m hopeful that we can get some actual science published this year!

(5) We were intrigued by the report last fall from Yingming Zhao’s group regarding the potential for modification of histone lysine residues by “lactylation” (addition of lactate)… especially since, at the same time, Jim Galligan’s group in Arizona reported a possible mechanism. However, there were some problems in the Zhao paper related to the anti-lactyl-lysine antibody, and we currently have in revision at Nature a “Brief Matters Arising” paper, outlining some important caveats. We may post a pre-print on BioRxiv in the coming weeks, depending on how fast things move through the editorial process.

As seems to be the case with so much scientific communication recently, the blog is going the way of the dodo, and Twitter seems to be where it’s at, for more timely updates on happenings of both a scientific and non-scientific nature.

Some papers that have been in the pipeline for quite some time are now finally in the wild –

(1) Our paper showing how cardio-protection by the mitochondrial unfolded protein response (UPRmt) depends on the transcription factor ATF5, is now out in AJP Heart.

(2) From our long-running collaboration with Keith Nehrke, some work from his post-doc’ Yunki Im showing that the post-fertilization elimination of paternal mitochondria employs the FNDC1 mitophagy pathway. Published in Dev. Biol.

(3) From a collaboration with Paige Lawrence‘s lab, a paper in Scientific Reports on how early-life exposure to aryl-hydrocarbon receptor agonists has long-term impacts on mitochondrial function in T-Cells.

Other news –

Paige was also instrumental in the recent acquisition of a new Seahorse XFe96 analyzer for the URMC Shared Resource Laboratories. This came not a moment too soon, as our own ancient (serial # 003) XF24 machine died, and our XF96 will no longer be supported after next year.

PSBLAB grad’ student Alexander Milliken is about to do his qualifying exam in a couple of weeks, closely followed by former rotation student Jessica Ciesla (in the lab of Josh Munger).

As reported on Twitter, Alan Cash (the CEO of various corporations selling oxaloacetate as a supplement) called me up and threatened to sue over things written in this article. No letter yet.

We had a great time at the AHA BCVS meeting in Boston, catching up with old science friends and making new ones. Next travel is NHLBI mitochondria meeting in DC at the end of September, and then NIH MIM study section in DC in October.

Congratulations to our colleague George Porter, who got his R01 funded (on cyclophilin D and the PT pore in early cardiac development), and is now searching for a mitochondriac post-doctoral fellow.

I was at a meeting yesterday discussing cancer treatments, and the use of oxaloacetate (OAA) as an anti-cancer drug came up. This prompted some more reading, and my conclusion is there’s a lot of craaaazy stuff out there, with very little actual evidence for pushing this molecule into the clinic.

Background on OAA

First, for the uninitiated, OAA is one of the metabolites in the Krebs’ tricarboxylic acid (TCA) cycle in mitochondria. OAA is made by malate dehydrogenase (MDH)…

The MDH reaction is energetically unfavorable (ΔG +29.7 kJ/mol), but in reality that’s overcome by the next reaction in the Krebs’ cycle (citrate synthase, CS) being very favorable (ΔG -31.5 kJ/mol) so OAA gets removed as soon as it is made and that pulls the cycle forward.

OAA as a Drug?

The development of OAA from metabolite to drug appears to be pushed mostly by a company called “Terra Biological“. They’re selling OAA as a nutraceutical dietary supplement (only $49 for a month’s supply!) with all of the usual snake-oil claims that accompany such operations (“boosts energy”, “enhances mental focus”, blah-di-blah). Oh, and it allegedly mimics caloric restriction (CR) so will help you live longer, so there’s that. Here‘s a slide deck by the CEO of the company, Alan B. Cash, pushing all kinds of uses of OAA, from cancer to Parkinson’s and Alzhemier’s disease and everything in between (and here‘s another link in case that one goes down). The company received a warning letter from the FDA in 2017, for making drug-like claims about OAA supplements.

Despite this craziness, the company is now pushing OAA into the disease area of Glioma, by promoting “Cronoxal” (aka OAA) as a “medical food” for glioma patients. This DropBox link contains a bunch of hokey information from the company on the alleged benefits. For a time, Cronoxal was also known as “Gliaxal”. It’s also marketed under the name Benagene a “genomic aging supplement” (whatever that means) and as Jubilance for PMS relief. Cash also runs MetVital, yet another company developing OAA in a bunch of disease areas.

So where’s the evidence?

As is often the case with the murky waters of poorly-regulated dietary supplements are a number of shortcomings with the actual underlying scientific evidence for OAA’s effects. Most of the hype appears to hinge on this 2009 paper in Aging Cell, claiming that addition of 8 mM (!) OAA to growth media enhances lifespan in the nematode C. elegans. A notable feature of this paper is that it was published in September 2009 and contains no conflict of interest statement.

Compare and contrast with an October 2009 review article (Submitted April that year) in the OA journal Open Longevity Science (which no longer exists), in which author Alan B. Cash lists a conflict of interest – namely being an officer of a company that sells OAA supplements (that would be Terra Biological, founded in 2006).

So, Dr. Cash (and the lead author on the Aging Cell paper David Williams) appear to have seen fit to disclose this rather massive COI in an OA review article, but not in an Aging Cell primary research paper. Another notable find is that Cash submitted a patent application on OAA as a CR mimetic in 2005 – a full 4 years before the paper was published. You might think such an event would require disclosure?

Back to the science, Cash’s glorious PowerPoint deck from 2014 includes claims that studies in mice were underway in 2011, in collaboration with Steve Spindler at UC Riverside. A quick PubMed search indicates that nothing was ever published on this. Similarly, claims are made about lifespan extension in flies, but again there’s nothing published.

For brain cancer, there are a couple of papers showing alleged effects of OAA in mice, but the data are just not very believable, and the dose used – 2 grams per kg (that’s not a typo) equating to 150 grams in a human – is ridiculous!

So, that leaves just about the entirety of the medical claims about OAA based on a single published paper about worms, a shady pay-to-play OA review article by the CEO of the company, and a bunch of unsubstantiated and unpublished claims in PowerPoint slides. Despite this, some people saw fit to convince the FDA to allow clinical trials – one for Parkinson’s, which failed, and another for Alzheimer’s which is ongoing (wanna bet how it’ll turn out?)

OAA cannot work the way they say it does

The claimed mechanism-of-action for OAA is that it drives the MDH enzyme reaction backwards, and this enhances levels of NAD+, which boosts the activity of the Sirtuins, which are allegedly involved in the beneficial effects of CR. There’s another whole can of worms about SIRTs and NAD+ (covered briefly here), but that’s for another day. But anyway, driving MDH backwards to make NAD+ has a number of problems…

First, by using this redox reaction as a source of NAD+, the ultimate source is of course NADH. In cells, NAD+ is normally present at about 700-fold greater levels than NADH. As such, NADH cannot possibly serve as an adequate “reservoir” to provide more NAD+. Even if you could convert all the NADH in your cells to NAD+, the effective level of NAD+ would only go up by 0.14% (and you’d probably die because – duh – no NADH left!)

Second, OAA (above) is a dicarboxylic acid. As such, it is negatively charged at physiologic pH. Negatively charged species are generally excluded from cells. Any mitochondrial biologist can tell you the way to get dicarboxylates into cells is to block the carboxylate moieties with alkyl groups… You make methyl or ethyl esters and these get transported into cells, then the alkyl groups are cleaved off by cytosolic esterases, yielding the free dicarboxylate trapped inside the cell. This is done all the time for Krebs’ cycle metabolites, and you can buy methylated versions of succinate, alpha-ketoglutarate, etc. These molecules are proven bio-available, and the cancer drug dimethyl fumarate exploits this exact same biology to get the active agent (fumarate) into cells.

A third potential confounder is the achievable concentration in people. Remember, the worm study used a level of 8 millimolar in the media – that’s about a gram per liter (molecular weight of OAA = 132). To obtain a similar level in human plasma, assuming a typical human blood volume of 5 liters that would be 5 grams of OAA, or 50 times more than the 100 mg found in the dietary supplement being sold. It’s unlikely that 100 mg of oral OAA would appreciably increase the level of this metabolite in tissues.

Essentially, non-esterified OAA at a low external concentration will not get into cells, and unfortunately that’s where the MDH enzyme (required for the claimed mechanism-of-action) is located. It’s notable that the only evidence Alan Cash shows for an effect of OAA on NAD+/NADH levels comes from isolated mitochondrial studies in the 1960s, where of course the cell membrane is not present.

Another claimed mechanism for OAA is the lowering of blood glutamate levels. Glutamate at high levels in the brain causes excitotoxicity, and OAA is claimed to remove glutamate by converting it to a-ketglutarate, using the enzyme GOT (glutamate/oxaloacetate transaminase). Here’s the reaction:

Glutamate + OAA <–> Aspartate + a-ketoglutarate.

Except there are problems with this mechanism too. First, the enzyme GOT (there are 2 isoforms, GOT1 and GOT2) is not usually found in the plasma. In-fact, you probably know it by another name… aspartate aminotransferase, or AST. That would be the very same AST that’s used (along with ALT) as a biomarker for liver injury. AST is not supposed to be in the bloodstream – if it’s there you have liver failure!

In addition, there are several reasons to believe that the activity of the GOT enzyme is actually pro-cancer. Specifically, cancer cells often hijack the Krebs’ cycle in mitochondria to leech out carbon skeletons for building other biomolecules such as lipids and proteins. This creates a deficit in the cycle, so it’s necessary to top up the cycle – a process known as “anaplerosis”. Cancer cells use glutamate (and glutamine) as a carbon source to replenish the Krebs’ cycle; and how do they get that carbon into the cycle? Via the GOT enzyme!

Furthermore, the very paper that is used to make these claims contains data that contradicts this mechanism of action. Specifically, Figure 7 shows that glutamate levels went UP slightly with OAA treatment (not down as desired). Here’s a snapshot from the brochure for Cronaxal (from that DropBox treasure trove), explaining the GOT mechanism. It makes my brain hurt…

For starters, there’s the ridiculous claim that OAA is ketone. This seems disingenuously pitched to jump on the bandwagon of the ketogenic diet fad. Yes, chemically speaking OAA contains a ketone group, but it’s not a ketone body in the classical medical terms intended here. There are only 2 ketone bodies: beta-hydroxybutyrate and acetoacetate. Nothing else is a “ketone” if what you mean is something involved in ketosis, ketogenenesis, diet effects, etc.

Then there’s the claim that D-2HG is inhibited by OAA. WTF? 2HG is an “oncometabolite” made by rogue enzymes in certain forms of cancer. It is not an enzyme. Enzymes get inhibited. Metabolites are processed by enzymes. As far as I know, metabolites cannot get “inhibited” by other metabolites, but whatever, I’m just a lowly biochemist. Like I said, this makes my brain hurt.

Someone needs to go read a biochemistry text book.

The biochemistry dumb-fuckery gets even worse in this awesome slide from the Cronaxal promotional materials…

To be clear… NAD+ availability is a key determinant of the rate of glycolysis in cells. Cancer cells LOVE glycolysis (the Warburg effect). More NAD+ means more glycolysis. NAD+ will thus promote the Warburg effect. Anyone suggesting the opposite (note the lack of a reference for the statement at the bottom of the slide) is being silly.

So how might OAA be working (if it is working at all)?

In my (not so professional) opinion, there’s no freakin’ way that OAA can be having its biological effects via an MDH >> NAD+ mechanism. The biochemistry just doesn’t add up. There’s also very little chance it’s working via a GOT-dependent lowering of glutamate (and if it is, this might actually be beneficial toward cancer).

So, how could it be working? There are a number of known G-protein coupled receptors for Krebs’ cycle metabolites expressed on the surface of cells – most notably the succinate receptor GPR91. Whether such receptors can respond to OAA in the plasma is not known. Another interesting property of OAA (and indeed all alpha-keto acids) is that it reacts directly with hydrogen peroxide. Mixing OAA with H2O2 gets you malonate, another metabolite with biological effects. It has been claimed that OAA is an antioxidant, but that’sa bit of a stretch. A third interesting property of OAA… it’s a potent inhibitor of mitochondrial respiratory complex II (succinate dehydrogenase), but of course such an effect would require it to get across cell membranes, oops!

Conclusion

So there you have it, a molecule that doesn’t get into cells, with no published primary scientific data in mammals indicating an effect at an achievable dose, and has failed in at least one clinical trial, hyped to hell by a zealous CEO who doesn’t disclose COIs when publishing, mostly based on a single paper in worms, now being pushed on desperate glioma patients, with a complete lack of understanding of the fundamentals of biochemistry, all wrapped up in a bunch of slick brochures and at least 5 different supplement companies all run by one person. What a shit-show!

Yes, you read that correctly. This revelation (to me at least) comes from a recent interaction with the Federal Office of Research Integrity (ORI), namely this reporting of several image duplications across papers from the same lab. Importantly, in most cases*** the images were re-used within a similar context and to describe the same experiments.  Here is the response I received from ORI…

Dear Dr. Brookes:
Thank you for your thorough email regarding the re-use of images and data across nine (9) different publications, over the span of fourteen (14) years. The 2012-2018 publications and after, are within the six year period of limitations, thus are under ORI’s jurisdiction.  In addition, the Current Drug Targets 2008 and Biocatal Biotransformation 2010 publications would also be under ORI’s definition, per 42 C.F.R. Part 93.105 (b)(1) Subsequent use exception.

However, re-use of images and data does not meet the definition of research misconduct.  Per 42 C.F.R. Part 93.103: Research misconduct means fabrication, falsification, or plagiarism in proposing, performing, or reviewing research, or in reporting research results. Falsification is manipulating research materials, equipment or processes such that the research is not accurately represented in the research record.

In each instance of re-use, the research is accurately represented in the research record; thus there is no falsification.  The re-use is consistent with self-plagiarism, which also does not meet ORI’s definition of plagiarism.  Thus ORI does not have jurisdiction.  This also may be consistent with a copyright violation, depending on the specific journal’s policies.

As ORI does not have jurisdiction, ORI considers this a closed matter.

Thank you,
XXXXXXXXXX, Scientist Investigator,
Division of Investigative Oversight,
Office of Research Integrity

This is news to me! It’s the polar opposite of what we teach in our mandatory research ethics course (i.e., self-plagiarism is bad). Even if the experiments are the same, simply republishing the same data and images more than once raises two important ethical issues:

(1) Double-Dipping. When you publish the same data twice you get to <i>game</i>  the metrics system by getting more publications for the same or less work (vs. others who do new experiments each time). Most would agree this is “not fair”, as important events in academia such as promotions and tenure depend on such metrics.

(2) Copyright. When you publish a figure in one journal, typically that journal owns the copyright to the image. Even if you’re a CC-BY hawk and do everything open access, publishing the same figure again elsewhere without acknowledgement is a breach of someone’s copyright.

This issue clearly raises a dilemma from the journal editorial perspective…. Say for example something is published first in Journal A and then Journal B.  If A sues B for copyright infringement and it results in retraction of the paper in B, all good.  But, if B acts alone and retracts the paper, they have to be VERY careful. If they implies any kind of misconduct occurred, and the authors are savvy about the above-mentioned ORI policy, this could open the door for a defamation lawsuit from disgruntled authors.  Caveat editor!

_____________________

***In some cases the images were not reporting the same experimental conditions. In other papers there were clear examples of image manipulaton such as splicing together unrelated western blots. But, all those papers were >6yrs old and so fell outside the ORI statute of limitations. Thus, overall there was nothing that both met the definition of misconduct AND was within the S.O.L.  Oh well.