An embarrassment of cress

Happy Solstice all.

I have to admit to having done something embarrassing. I grew some cress. Two lots in fact. One was on the dining room windowsill as a control, the other was sandwiched between a wireless router and an Apple base station, about 30 feet from the first tray.

Both grew. The dining room window plate:

And here is the plate growing between the router and the Apple base station:



















I know there are two wifi routers there in the picture as well as the base station but the second one was only turned on for the last two days of the six day growth period shown here.

Tasted as good as cress ever does. Not something I rave about personally.

Unfortunately I set the experimental seeds to sprout before we had eaten all the cress which Daniel has growing on the kitchen windowsill as a routine:

Well duh, as my daughter (frequently) says. There is a limit to how much cress a given household can consume, even with Daniel liking vegetables. I'm also embarrassed that I even contemplated that the cress might fail to germinate next to the router(s).

More fool me.

Peter

A little more on PCSK9 inhibitors

The author of the following open letter to Medscape (I think you have to register to read, so I've copy/pasted it to make access easy), a Dr Mandrola, is not a THINCS member that I'm aware of. Not a Dr Ravenskov or Dr Kendrick.

He appears to be a mainstream cardiologist who is aware of how essentially all lipid lowering trials have bombed and that statins, whatever small benefit they show in drug company funded secondary prevention trials, probably don't work by LDLc lowering.

He is urging caution on PCSK9s. This is good. Never mind the known cognitive decline. My guess he is worrying (but not talking) about what the cancer death rate will have risen to by five or six years down the road. He has read J-LIT.

I know this is not unprecedented. It just feels like it. There really are medics in the cardiological wall who do think and who possibly care. That's nice to see.

Peter

Here is the letter:


www.medscape.com June 16, 2015

Dear FDA: Resist the Urge on PCSK9 Drugs

Last week, an FDA advisory committee recommended approval of two proprotein convertase subtilisin kexin 9 (PCSK9)–inhibitor drugs. A formal decision is expected later this summer. The FDA usually follows the advice of its advisory committees, but not always.

This is a big moment in cardiology. It is also a huge gamble for the FDA.

I believe the FDA should break with its advisory committee and say no.

Not yet. It's too early to unleash these drugs on American patients and doctors.

Here are four reasons.

Target Confusion

The first reason the FDA should say no (not yet) is the target. PCSK9 drugs lower LDL cholesterol. That fact is clear. But our target is not a lab value; it's heart disease.

Any doctor who sees patients knows heart disease comes from many things. These factors, which affect individuals in genetically varied ways, accumulate over years, not months. LDL-C may be important, or very important, but it is just one risk factor. Even in patients with familial hypercholesterolemia, LDL-C may be one of many risk factors.

The stunning LDL-C lowering from PCSK9 drugs might prevent future heart attacks, strokes, and deaths. The key word in that sentence is might. We don't know. The biology of these drugs is beautiful, but that beauty should not obscure the current state of knowledge.

What we know now is that PCSK9 drugs are effective at LDL-C lowering. That is it. The OSLER[1] and ODYSSEY[2] trials were not powered to look at outcomes. Those data are forthcoming in the FOURIER trial, which has completed enrollment of 27,000 subjects, and results are expected in 2017. Why not wait?

Do No Harm

The second reason the FDA should hold off is the risk. The mission statement of the FDA says its charge is to "protect the public health by [ensuring] the safety, efficacy, and security of human and veterinary drugs, biological products, medical devices, our nation's food supply, cosmetics, and products that emit radiation."

What I read into that is harm avoidance. In this way, I see the FDA's role as similar to a physician's. Yes, we want to benefit our patients, but the guiding principle must be to avoid harm. This is especially critical when treating people for something (an MI, stroke, or death) that has yet to happen. Having a high risk for a disease is not the same as having a disease.

It is true; in both the OSLER and ODYSSEY studies, evolocumab (Repatha, Amgen) and alirocumab (Praluent, Sanofi/Regeneron) looked reasonably safe. But follow-up was only 11 months in OSLER and 78 weeks in ODYSSEY. That's too short. Heart-disease prevention is not a 2-year endeavor.

In both studies, more patients on the PCSK9 inhibitor reported neurocognitive effects. That may be significant. Is it implausible to think cholesterol might be important for brain cells? Here, let's also be mindful of euphemism. "Neurocognitive function" is a fancy way to say "think." Thinking is what makes us human. So if we think of this issue from a patient-centered standpoint, how many humans would trade a dull mind for a possible benefit 2 to 10 years in the future?


The third reason the FDA should say no is historical. The use of surrogate markers for cardiac drugs has proven to be a bad gamble. We can point to niacin[3], fibrates[4] and cholesteryl ester transfer protein (CETP) inhibitors[5] as evidence of that failure. Although statin drugs are potent LDL-C lowering agents and proven effective in reducing cardiac events in high-risk patients, no one argues these drugs don't have important pleiotropic effects. The case of ezetimibe is hardly supportive of the LDL-C hypothesis. In the IMPROVE-IT[6] trial, the tiny absolute benefit of ezetimibe (composite end point) was achieved against simvastatin—a straw-man comparator if there ever was one.

Distractions—Benefits Missed

The fourth reason the drugs are not ready is the potentially harmful effects of distraction and benefits missed. Many have argued, including patients with familial hypercholesterolemia, that PCSK9 drugs should be approved now because of the benefits missed while waiting for the FOURIER data. Of course, this assumes positive results are forthcoming.

Another way to look at benefits missed is to imagine the devastating impact of all that will not occur if these expensive drugs are approved. If we spend billions of dollars on these drugs—and make no mistake, if the FDA approves them, we will spend billions, then that takes money (and attention) away from many other facets of heart disease prevention.

Look at the case of ezetimibe, the last drug approved without outcomes data. Billions were spent on a drug with minimal to no effect on outcomes. What were the benefits missed of those billions? Healthcare budgets are limited. If we spend money on unproven drugs, we aren't spending it on cardiac rehab programs, parks, bike lanes, school nutrition programs, and many other useful heart-healthy public-health projects.

2017 is just around the corner. I say the FDA should resist the urge. Protect the public. Do no harm.

JMM

References

1. Sabatine MS, Giugliano RP, Wiviott SD, et al. Efficacy and safety of evolocumab in reducing lipids and cardiovascular events. N Engl J Med 2015; 372:1500-1509. Article
2. Robinson JG, Farnier M, Krempf M, et al. Efficacy and safety of alirocumab in reducing lipids and
cardiovascular events. N Engl J Med 2015; 372:1489-1499. Article
3. The AIM-HIGH Investigators. Niacin in patients with low HDL cholesterol levels receiving intensive statin therapy. N Engl J Med 2011; 365:2255-2267. Article
4. Ginsberg HN, Elam MB, Lovato LC, et al. Effects of combination lipid therapy in type 2 diabetes mellitus. N Engl J Med 2010; 362:1563-1574. Article
5. Nissen SE, Tardif JC, Nicholls SJ, et al. Effect of torcetrapib on the progression of coronary atherosclerosis. N Engl J Med 2007; 356:1304-1316. Article
6. Cannon CP, Blazing MA, Giugliano RP, et al. Ezetimibe added to statin therapy after acute coronary syndromes. N Engl J Med 2015; DOI:10.1056/NEJMoa1410489. Article © 2015 WebMD, LLC

PCSK9 inhibitors and upper limb amputations

Another one liner, this time from Heartwire:

The two new drugs, both of which are proprotein convertase subtilisin kexin 9 (PCSK9) inhibitors, effectively lower LDL-cholesterol levels.

"If you're a cardiologist, you must not have a pulse if you're not excited" about the potential availability of the two new agents, Dr Amit Khera (University of Texas Southwestern Medical Center, Dallas) told heartwire from Medscape.

"For those of us who take care of patients with familial hypercholesterolemia [FH], this is just an amazing option," he said. "I know this seems like a small group of patients—one in 500—but it's really not all that small. There's also the advanced coronary artery disease patient who still has high lipids. It almost seems arcane in this modern era that we're just watching patients who have recurrent events and our hands are tied. I think this offers an incredible option for those people."

The hands tied option which caught my eye. I’m not sure if it was Pat Wall or Ronald Melzack, writing about chronic pain, who came up with the gem that “The best surgeon to consult for a chronic pain problem was one who had no arms”.

Nothing changes. We’ll have to wait to see the body counts.

Peter

Gluten: Not your average plant toxin?

Time is in short supply at the moment so no time for more coeliac stuff, however interesting. I was forwarded a link from Keir Watson to a paper from Simon Murch's group at Warwick:

Matrix Expansion and Syncytial Aggregation of Syndecan-1+ Cells Underpin Villous Atrophy in Coeliac Disease

He's filled out the core findings (with lovely histo pictures) and posted about it on his wife's Rosemary Cottage Clinic blog. The paper is rather neat and I have a great deal of time for Prof Murch.

None of it is tempting me to do a gluten relapse.........

Peter

ATP Synthase

It's a very interesting time for origin of life speculation. You can look around at the work coming from Lane's group as well as the excellent work by Koonin's group and consider what fits together logically from the various scenarios on offer. None are completely satisfying alone but a mixture of the ideas gives a very pleasant brew. Let's give Koonin some time now:

How do you build an ATP synthase complex? On the surface it looks to be utterly incomprehensible that a lipid embedded, ion gradient driven rotor should turn a stalk to a set of molecular forceps each of which forces a phosphate on to an ADP to give ATP as the rotor turns. It's particularly striking that Nick Lane considers this to be one of the core molecular machines present in LUCA before the separation in to the populations which gave rise to the archaeal and bacterial lineages. Running on a proton gradient. It would need to have been a very early development.

If we buy in to the alkaline hydrothermal vent scenario (Koonin doesn't) for the origin of life we have microscopic "pockets" of metabolism in iron (+/- nickel) sulphide walled chambers at the boundary of alkaline vent fluid with acidic ocean fluid. In the progress towards something akin to life there has to be the development of both protein and RNA. In a vent system, where conditions at the microscopic scale vary from time to time, the ability for a given replicator (RNA based) to spread itself over the maximum number of protocells would provide a survival benefit. The development of any sort of cell membrane is clearly a hindrance to this process, potentially terminal. The formation of simple pores of protein derivation are not inconceivable and would allow the continued spread of successful RNA to a maximum number of protocells. Pores do not have to be complex. RNA spread can be concentration based.

RNA will form double helices. Not with the solidity of those derived from DNA, but double helices never the less. There is a large family of proteins which unwind helices to allow replication, be that of DNA or RNA. Many of them use ATP to facilitate the process. They are very basic machines by evolutionary standards and there is no reason why they were not as available early in evolution as simple membrane pores.

Bear in mind that one of the core products of protometabolism is an acetyl thioester capable, energetically speaking, of producing ATP (was that as long ago as February?).

If we have these basics in mind we are in a position to go to this rather nice speculative opinion piece by Koonin's group. Here is figure 2:

Across the top we have the possibility of an ATP driven RNA helicase sticking itself to a membrane pore and using ATP to power RNA export through a proto membrane bound protocell. The helicase, if it sticks itself to the membrane pore, will extrude an RNA strand out of the protocell. As any helicase passes down an RNA strand, it rotates. If the helicase is fixed to the pore, the RNA will rotate as it passes through the pore. The right hand diagram shows the next development with an RNA strand replaced by a protein strand, not an impossible transformation and still potentially of benefit to a proto replicator.

Working along the middle row from right to left we have a protein translocator using ATP to export a protein using the same rotary machinery inherited from the RNA helicase/translocase. The + and - within the circles represent electrostatic charges which normally hold the pore and helicase stationary relative to each other. The middle diagram has a red protein which is physically stuck in the pore. The rotary component, powered by ATP, pushes/twists against the membrane bound section while the electrostatic charges resist rotation. Something has to give and in the process an ion or more is squeezed outwards as the pore derived section moves against the stator section.

The machine extrudes ions by consuming ATP. Ion gradients have many uses to protocells so the development may well have been advantageous at the time. The machine also works perfectly well in reverse so, given an appropriate ion gradient, it will generate ATP as ions enter the protocell. The ion gradient itself is a whole different ball game. Just for now recall that in those archaea and bacteria which lack cytochromes, the gradient is of Na+ ions, not H+ ions. Another post there.

This is a plausible derivation for the bacterial/mitochondrial F type ATP synthase.

The archaeal V type ATP synthase has very similar ion pump and ATP synthase sections to the F type, but the central stalk is quite different. In fact it doesn't look much like a stalk at all, see the bottom left diagram in the above figure 2. It looks more like a protein used to stabilise the pore and helicase sections against each other. It does the same job as the central stalk of the F type synthase but its origins are clearly different.

The ATP synthase complex was developed twice.

It looks very much as if the pore/helicase combination was ubiquitous in LUCA but the system of locking them together is different in the archaeal vs bacterial lineages. That seems very profound to me when thinking about LUCA.

The scenario does not have the ATP synthase as primordial. The system evolved as an ATP consuming machine, not an ATP generating machine. It has no hallmarks of the core power producing system in LUCA. Where did the ATP come from to power the engine which was eventually to become the dynamo?

I commented in my post on the reduction of CO2 to CO that the acetyl thioester produced was quite capable of substrate level phosphorylation. It is able to produce molecules in the energetic range of ATP and undoubtedly did so, because we are here today we are and running on ATP.

ATP is undoubtedly what worked best for the RNA helicase as it appears to have carried this preference forwards to the protein translocase and hence the ion pump which reversed role to become a dynamo. If the helicase had been powered by acetyl phosphate rather than ATP I can see no reason why metabolism might not be based around such a molecule. We use ATP because that's what worked best in the motor which, once it reversed to become a dynamo, unleashed a ubiquitous supply of energetic substrate based on an ion gradient.

Just to recapitulate: A proton gradient is essential to power protometabolism because without it you cannot reduce CO2 to CO to develop acetyl thioester and its assorted phosphorylated derivatives. This substrate level phosphorylation is utterly proton gradient driven but does not require proton translocation per se, although proton translocation may have been used to produce a localised environment which reduced ferredoxin. A source of direct ATP synthesis was required to power an immediately useful translocase which allows development of an ion pumping motor which could subsequently be reversed to provide an ATP generating dynamo as the membrane energetics changed.

The change in membrane energetics was probably related to a biologically generated Na+ gradient in addition to the geochemical H+ gradient. If the sodium ion gradient was supported by anti porting on a geothermal H+ gradient, it was both free and nearly unlimited in availability. So we have to think about antiporters.

Peter

Polyprenyl derivatives at the origin of life

I just wanted to put something down about the possible early membranes used by LUCA. There is a good case to be made for leaky membranes (certainly to protons) composed of polyprenyl phosphates. Here's the logic:

Start with the modern mevalonate pathway, from wiki.

Now, let's butcher it down to its utter basics:

Isopentenyl pyrophosphate is the central cornerstone of polyprenyl phosphate generation by this reaction:

You can make the polyprenyl phosphate lipid as long as you like, just add more isopentenyl derived units. This gives us the entry point, top centre, for this diagram:

Down the left hand side we have the simple pathway to the archeal plasma membranes, as you might expect. Down the centre is the route to cholesterol, essential to animal cell membranes. The hopanoids, essential to bacteria but absent from archea, are left of centre.

Archea stayed with polyprenyls for their membrane, bacteria went for the fatty acids while retaining polyprenyl derived components. Derivatives of polyprenyls are ubiquitous. Metabolic cornerstones common to both archea and bacteria may well have been present in LUCA. Seems a reasonable argument to me.

There are a set of core enzymes present in both archea and bacteria which appear to have evolved before their divergence from LUCA. Perhaps the most significant is the rotator/stator ATP synthase, a piece of molecular nanotechnology of breathtaking complexity, considering that it appears to have evolved as one of the core early molecular machines of life. It's association with membranes suggest that membranes were there quite early on in evolution.

The presence of membranes allows us to think about some interesting questions. Such as why are cells powered by ATP? Because that is what ATP synthase produces. There is nothing intrinsically special about ATP. Reduced ferredoxin is a common power source in bacteria and archaea. Acetyl phosphate can be used too. But ATP is what is made by the ATP synthase machine and it can be produced in bulk, given a membrane potential. The hows and whys are interesting.

Peter

The Wall is coming down

Well, I guess it's going to become officially OK (as if I could care less) for me to fry my egg yolks in salted butter and add a little extra salt at serving time. And to skip any sugar in my tea. At the risk of glucose deficiency I suppose but, what the hell, live dangerously...

Malcolm Kendrick and Steve Cooksey have two nice accounts (amongst many) out there so there's not much for me to add. It just reminds me of listening to the news as the Berlin Wall came down, even if it's not headlining at the BBC yet. Amazing. Happy.

You still have to feel sorry for the people so badly injured by the consequences of the lipid hypothesis but at least the possibility of using real food to produce normoglycaemia is no longer specifically decried by official dietary advice. The floodgates are open. The information is out there.

Excellent.

Peter

BTW, the Japanese appear to be about to start the destruction of statination too, certainly for the elderly... Thanks to Liz for the full text link.

Prokaryotic Microbes with Eukaryote-like Genes Found

Jack Kruse sent me this link:

Archaea with eukaryotic genes

For those working through Nick Lanes latest book will realise (if you hadn't already!) that there is an energetic cost to everything. The ability to change shape at will, with a view to phagocytosis, is both an essential to the development of the eukaryotes and is energetically very costly.

In the absence of mitochondria the only place this appears to have been discovered is the deep sea alkaline hydrothermal vents, in this case at Loki's Castle. This is the environment where geothermal sources generate the electrochemical disequilibrium which probably got life started using the "free lunch" of CO2/CO plus R-SH giving acetyl-S-R, gateway to metabolism. It is still an energy rich environment.

Phagocytosis is costly.

The alpha proteobacterial precursor of our mitochondria had to get inside the archaeal host. This is an interesting discovery relating to how that might have happened. The phenomenon of archea performing ingestion may well have remained confined to the vents if it was too energetically costly in the open ocean. Unless that archaea was carrying little mitochondrial powerhouses, in which case it was pretty well already eukaryotic. Far from equilibrium environments make for interesting developments.

Looks like you can develop eating only if you already have a free lunch!

Peter

Ketogenic Diet: Eat Food

From page 148 of the third edition (2000) of Freeman, Freeman and Kelly’s “The Ketogenic Diet”.

Daily intake for a 13 month old child:

Commercial formula providing 28.3 grams of fat composed of equal parts high oleic safflower oil, soy oil and coconut oil, plus an extra 38.1 grams of almost pure omega six from from a standard safflower oil emulsion, a little soy protein 'n' glucose polymer. Vitamins and minerals. A maximum of 980ml of water in total.

This paediatric ketogenic diet is a child of its time, steeped in the benefits of massive PUFA ingestion and often combined with phenobarbitone. The child in the case study above could barely swallow as a result of the high dose of phenobarbitone she was taking. If you were to suggest hepatopathy, dyslipdaemia and potentially fatal pancreatitis as sequelae of the diet, I would not be surprised.

Please do not feed this to your child, we are no longer in the 1970s.

On the other hand, if you order a steak in a restaurant and it comes to you with salad and chips on the side, you do not HAVE to eat the chips to stop yourself developing fulminating pancreatitis.

If you are post-obese, via low carb eating, there is every likelihood that repeatedly consuming the chips (to avoid the pancreatitis, don’tchano) will cure you of the post-ness of your obesity. Enjoy the chips by all means.

Peter

HbA1c and Familial Hypercholesterolaemia

Ivor, over at thefatemperor, recently mentioned the lovely observational study, the Norfolk section of EPIC. That's where I live and it's HbA1c which correlates with CVD here. Cholesterol does not. At all. Tha' be 'ere in Norf'k, b'o'r. With apologies for the lapse in to the vernacular. My children are becoming experts. Computer is com-poo-er...

That nudged me to put this very brief observational post up on glucose dysregulation and CVD in the land where cholesterol is king, for people with heterozygous familial hypercholesterolaemia. There are many, many problems you could point out in this study, but those are intrinsic to a retrospective observational study. Take a group of hFH people who have survived a premature heart attack. Match a similar group of hFH people who haven't had a heart attack. How do you tell the difference between the groups? As a lipidologist perhaps you might suspect the LDL cholesterol level? That is exactly the problem in hFH after all... But:

"There was no difference in total and LDL cholesterol between the two groups. Patients with previous myocardial infarction had significantly higher levels of insulin, insulin resistance [and several other things I'm not interested in, which they would like to treat]..."

Insulin resistance is the problem, on a mixed diet. Do you think this might show in the HbA1c, just as it does here in Norfolk for us non hFH folks?

It probably does. In a similar observational study on hFH, using HbA1c itself rather than insulin/resistance parameters:

"Of special note is that HbA1c showed a significant correlation with average ATT [Achilles Tendon Thickness], independent of other parameters..."

Achilles Tendon Thickness is a marker of Badness in hFH. As your tendon thickens, so too does your carotid intima thicken.

In general, patients with hFH tend to have rather good glucose control compared to the general population. That might just be why they live as long as they do under the correct circumstances. But with hFH I suspect that, should you manage it, developing metabolic syndrome may be a very unforgiving problem. You have to wonder what side stepping the syndrome by low carb eating might do, giving chronic normoglycaemia without elevated post prandial insulin. Not holding my breath waiting for that one.

BTW, as statins worsen glycaemic control, could they actually make the CVD problems worse for hFH patients? Surely not. Surely we have stopped making booboos of this type. Of course we have. Of course.

Peter

Ketogenic vs moderate carbohydrate diets

I thought I might put up this graph:

It's from

Comparison of the Atkins, Zone, Ornish, and LEARN Diets for Change in Weight and Related Risk Factors Among Overweight Premenopausal Women: The A TO Z Weight Loss Study: A Randomized Trial

The lead author does not appear to be a LCer. In general his publications are rather pro plants and quite mainstream. To his credit he has published some negative studies amongst the pro plant stuff. Harvard, ultimately, is no hotbed of pro Atkins zealotry.

If we want to look at the macros we can check here:

A little arithmetic allows us to look at the carbohydrate intake on the first graph at differing time points for Atkins and Zone diets:

Of course the Atkins diet was an unrestricted calories diet, Zone has a caloric restriction applied.

I have the impression from the data of the A-Z study that low carb is good, slightly higher carb is acceptable, adding more carbs back in is a booboo and that the Zone is crap. Just an impression. From the graph.

As an aside, of course the unanswered question is what, exactly, would a sustained 54g carb intake have produced in terms of weight loss over 12 months? Or 20g/d over 12m?

It is very clear that carbohydrate restriction only works WHEN YOU RESTRICT CARBOHYDRATE. A low carb diet does not appear to be as effective as a low carb diet when it has morphed in to an ex low carb diet through added carbs. A similar pattern might apply to ultra low fat diets if anyone wants to go down that dark alley. They don't work when you add fat. Assuming you don't mind the biochemistry while you eliminate fat.

It is also very clear that when comparing an almost-ketogenic diet to a modestly restricted carbohydrate diet of around 133g/d carbs, something like the Zone diet, the modest carbohydrate diet is just as good, if not a little better, than a ketogenic diet. You know the graph:

My problem is trying to square the circle between these two studies. Obviously, no study is free of bias. I struggle somewhat with Dr Sears, of the Zone diet, being the group leader of the study which shows a diet with 133g/d of carbohydrate out performs a ketogenic diet. That is very strange and doesn't happen in Stanford.

People must make up their own minds.

Peter

Insulin detemir (3)

I think it is quite clear how I view insulin detemir. Kindke was unable to resist finding the link to the abstract with the diametrically opposing view. I'll just stick both links in here to keep them together, so people can look at both research findings and draw their own conclusions.

Insulin detemir is not transported across the blood-brain barrier.

versus

Insulin detemir is transported from blood to cerebrospinal fluid and has prolonged central anorectic action relative to NPH insulin.

I think it is reasonable to assume that at least one of these two papers is factually incorrect.

If you search on Begg and Woods as co-authors you will find papers redolent with words like "reward", "hedonic" and "dopamine".  That's Begg and Woods, if anyone can stomach it.

I was, in my normal confirmation biased way, much more interested in the sort of work produced by Banks, Morley and/or Mooradian. These folks appear to be scientists rather than psychiatrists and they have some great publications. They include major work on the blood brain barrier, leptin transport, insulin transport, leptin resistance, gerontology, diabetes, antioxidants, the list goes on and on.

Here are a few little gems I particularly enjoyed in abstract form which might be worth a mention.

I dislike antioxidants. This is quite interesting from Banks and Morley:

Effect of alpha-lipoic acid on memory, oxidation, and lifespan in SAMP8 mice.

Alpha lipoic acid is a mitochondrial component present in normal cells and is available in mega doses as a supplement. It's a serious and deeply mitochondrial penetrative antioxidant. It helps a lot with diabetic neuropathic pain. SAMP8 mice are oddities which have been bred for early onset senility and memory loss. They are used (probably totally inappropriately) for Alzheimers Disease research. Treating them with antioxidants improves their memory performance. You might think this is a good idea. The cost is measured by a shortening of their life as elderly SAMP8 mice from 34 weeks to 20 weeks after start of treatment (started at 11 months of age). This may or may not be a good thing if you are an SAMP8 mouse (death might be a release). How it applies to a person managing their diabetic neuropathy or trying to delay the progression of their Alzheimers Disease is fascinating and slightly worrisome. I'll stick to a life based around beta oxidation, normglycaemia and a little superoxide signalling, stuff the antioxidants. Last sentence of the abstract "These findings are similar to studies using other types of antioxidants". Sweet, provided you avoid sugar. And antioxidants.

The next snippet includes Morley and Mooradian as authors and relates to making your blood sweet,  literally this time, using intravenous glucose:

Mechanism of pain in diabetic peripheral neuropathy. Effect of glucose on pain perception in humans.

Simple hyperglycaemia in a normal person reduces the threshold for feeling pain. It reduces the severity of pain you can tolerate. This applies to a normal human being on a glucose infusion or a diabetic person on a diet designed by a diabetologist, no glucose infusion needed. If hyperglycaemia makes a tolerable stimulus in to a painful experience and makes just bearable pain become unbearable, how many chronic pain syndromes would go in to remission with sustained normoglycaemia? Fat phobia makes this question currently un-answerable. The paper was published in 1984. Does anyone fancy having a gangrenous foot amputated for diabetic complications and waking up on a dextrose saline infusion in recovery? And then being offered the "diabetes diet" on the post op ward?

Banks and Morley were also instrumental in the generation of data for the concept that trigycerides in plasma induce leptin resistance at the blood brain barrier, a few years old now but still quite a useful concept:

Triglycerides induce leptin resistance at the blood-brain barrier.

I find the cream bashing in this last paper a little distasteful and I have to admit that Banks appears to be unaware that high saturated fat low carbohydrate diets are THE way to reduce fasting trigycerides in real people. Can't have everything I suppose. But even if the cream effect applies to people, who cares if I am leptin resistant with a full stomach provided leptin will work perfectly well in the post absorptive (low triglyceride) period? I am a human, not a mouse. I ate a high fat meal without sugar last night, ergo I'm not hungry today. Low trigs equal leptin sensitivity...

I'll call a halt there. Life is full of interesting snippets which make sense. They usually come from the sort of people who say insulin detemir does not cross the blood brain barrier.

Peter

Insulin detemir (2)

Morphine is a rather odd opioid analgesic. It has a complex multi-ring structure with two rather prominent hydroxyl groups which render it rather more hydrophilic and significantly less lipid soluble than many of its relatives. If you bolus a patient with IV morphine there is a delay in its passage across the blood-brain barrier due to this relatively poor lipid solubility. Time to peak effect is significantly delayed to somewhere around 15 minutes because the brain concentration lags way behind the rapidly changing plasma concentration. The brain never "sees" the peak plasma concentration due to this delay.

Now, if you boil some morphine up with acetic acid you can form ester linkages joining acetate on to those two hydroxyl radicals to give you di-acetyl morphine, better known as diamorphine or heroin. Masking the hydroxyl radicals markedly increases the lipid solubility of the drug and so the brain concentration rapidly follows the plasma concentration. In general lipid soluble agents cross the blood brain barrier rather faster than more water soluble agents. Peak plasma concentration will give a rapid onset peak brain concentration, which appears to be associated with effects rarely seen with morphine itself. Giving the enhanced recreational potential. This is all basic anaesthesia pharmacology with excerpts from Trainspotting thrown in.

Insulin detemir was developed to give an insulin with a very flat glycaemia controlling effect for use as a basal or background insulin. The clever people at Novo Nordisk deleted the terminal threonine from the B chain and attached a medium chain fatty acid to the now terminal lysine at position B29. The rather nice 14 carbon saturated fat, myristic acid, sticks out from the insulin molecule and neatly binds to the fatty acid binding site of albumin. It does this very rapidly and keeps the insulin bound and ineffective. Over the hours which follow there is a slow dissociation of the insulin from albumin which allows a very shallow dose response rate for glucose control. Ideal for a basal insulin.

There is a suggestion that this tagging of insulin might facilitate its transport in to the brain, a sort of heroin-insulin tweak. The idea is that myristic acid might facilitate the transport of insulin in to the brain and lead to a massive suppression of eating and subsequent weight loss. Assuming you are a true believer in the central anorectic effect of insulin. Which, sadly, I'm not.

Years ago, when insulin determir was first paraded as the living proof of the central anorectic effect of insulin, I looked up its structure and thought, as you do, that FFAs in general have very limited access to the brain. Insulin is not morphine and the myristic acid is not acetic acid. That big, long side chain of detemir is directly related to the sorts of free fatty acids which are specifically excluded from the brain. My own prediction would be that insulin detemir would have a significantly REDUCED effect within the brain.

It turns out that, at least in some labs, that my idea was slightly correct. But my idea was limited compared to the actual effect. Insulin detemir not only fails to cross the blood brain barrier itself but it also blocks the ability of ordinary human insulin to pass from plasma in to the brain. There is probably a specific insulin transporter which is nicely blockaded by an insulin molecule with the fatty acid tail of detemir sticking out. This paper says it all:

Insulin Detemir is Not Transported Across the Blood-Brain Barrier

Not a lot of mincing of words there.

If we go to labs with an outlook on life which I find comprehensible we can clearly see that physiological doses of insulin, within the brain, augment lipid uptake in to adipocytes, enhance adipocyte sensitivity to insulin, increase lipogenesis and augment fat gain. Largely through the sympathetic nervous system. I can't see how anyone would be surprised by this. Quite why anyone would expect central insulin to do the opposite of what peripheral insulin does at a comparable concentration is beyond me. I enjoyed this paper:

Central insulin action regulates peripheral glucose and fat metabolism in mice

"Moreover, chronic intracerebroventricular insulin treatment of control mice increased fat mass, fat cell size, and adipose tissue lipoprotein lipase expression, indicating that CNS insulin action promotes lipogenesis. These studies demonstrate that central insulin action plays an important role in regulating WAT mass and glucose metabolism via hepatic Stat3 activation".

How clearly does it need to be spelled out? This one is fun too:

Brain insulin controls adipose tissue lipolysis and lipogenesis.

"Here, we show that insulin infused into the mediobasal hypothalamus (MBH) of Sprague-Dawley rats increases WAT lipogenic protein expression, inactivates hormone-sensitive lipase (Hsl), and suppresses lipolysis. Conversely, mice that lack the neuronal insulin receptor exhibit unrestrained lipolysis and decreased de novo lipogenesis in WAT".


If you go looking you can find papers from Oz and Cincinatti which show that insulin detemir DOES cross the blood brain barrier and DOES suppress food intake, far better than neutral insulin does. In their own labs of course.

But I cannot forget that if you transport a researcher out of a Cincinatti psychiatry department and put her in to an industrial insulin lab she cannot get any effect of centrally infused insulin detemir or neutral insulin for that matter. Novo Nordisk cannot demonstrate this marvellous effect of insulin, even their own special insulin, in their own lab. We all know that much of the mindset of obesity research is not particularly effective at producing results which work. How they get the results derived from their ideas in their labs is what fascinates me! You couldn't make stuff up this counter intuitive. Maybe in another post.

Back in the real world we have this:

Insulin detemir results in less weight gain than NPH insulin when used in basal-bolus therapy for type 2 diabetes mellitus, and this advantage increases with baseline body mass index

Insulin detemir causes a small weight loss in morbidly obese patients, those with BMI >35kg/m2. Why? Because it blocks the brain entry of the chronically (and markedly) elevated levels of insulin so common in the morbidly obese. It has limited or zero effect within the brain in its own right. The brain simply loses awareness of the systemic pathologically elevated insulin. If plasma insulin is high enough this sudden loss of insulin's access to the brain can result in a decrease in brain driven, neurologically mediated, forced lipid storage in adipocytes, i.e. a little weight loss.

In the absence of marked hyperinsulinamia, i.e. in less obese type 2 diabetics, insulin detemir causes weight gain because there is less tonically elevated plasma insulin for the central uptake blockade to neutralise. There is no weight loss effect, although gain is undoubtedly blunted.

Insulin detemir is the best indicator I have seen that the central role of physiological concentrations of insulin within the brain is to augment fat storage. This makes sense to me.

I wouldn't ask a psychiatrist to develop an anaesthetic protocol. Or a weight loss protocol!

Peter

Insulin detemir (1)

There is a certain belief structure within obesity research which maintains that the central action of insulin is to limit appetite. Obviously, not everyone agrees with this. I would like to do some wild speculating (any resemblance to real life events is purely accidental) about this paper:

Evaluation of the lack of anorectic effect of intracerebroventricular insulin in rats

The paper is very interesting, partly for what they failed to reproduce but mostly for the affiliations of the authors.

The first thing to say is that, if you work in the pharmaceutical industry, you want drugs which work. Hardcore. It’s no good fudging your results when working in pharmaceutical R&D because you’re going to get caught out as soon as anyone tries to actually use your drug. Which is guaranteed to happen. The drug has GOT to work. Industry has no fudge factor. You might have to lie, evade, obfuscate, misplace computer files and massage data to hide the serious adverse effects of your functional patented drug, but you wouldn’t want to have to do this for a molecule which is ineffective in the first place. Statins are very, very effective. At lowering cholesterol. The fudge factor comes from whether this does any good for any person and what multiple adverse effects the drugs might generate.

So I have respect for the integrity, within certain defined limits, of a drug company R&D team. The managers and PR crowd are another matter altogether. Think Dilbert.

Let’s look at the authors of this paper:

There are three. Jessen is the first author, so probably did the bulk of the work and wrote much of the paper. She works in the department of insulin pharmacology at Novo Nordisk, the company which makes insulin detemir. Bouman is last author so is possibly Jessen's line manager and also works for Novo Nordisk. Insulin detemir is interesting because it is the only insulin ever to have been shown to cause weight loss in any patient group. OK, this is limited to morbidly obese (BMI>35) type two diabetics and the weight loss is very small. But it does happen. Quite amazing really and quite different to any other insulin formulation on the market, all of which reliably cause weight gain. Hence I suspect the project at Novo Nordisk was to find out the hows and whys of this strange effect.

Jessen and Bouman will have started with generous (by academic standards) funding, because the drug industry will work at a potentially rewarding idea in a rather more motivated manner than an academic department. Neither author has any track record of publishing on the central anoretic effect of exogenous insulin. Their job is to get reliable and repeatable results about how insulin detemir is special. In this project they failed to achieve any sort of anorectic effect of insulin detemir, or of any other sort of insulin, within the brain. Mucho problemo.

Clegg is middle author and works in the Department of Psychiatry, University of Cincinnati. She has a vast number of publications, several of which feature the successful anorectic effect of insulin when administered directly in to the brain. In at least one such study she is the lead author.

Jessen has co published with Clegg back in 2001 on a non insulin related subject, presumably before Jessen moved to work for Novo Nordisk. They know each other and have worked together before.


I have this image of two industrial pharmacologists setting out to investigate the CNS effects of their rather promising systemic drug, insulin detemir, comparing it to routine and more obesogenic neutral insulin. They fully expect central insulin to be anorectic because they've read all of the papers. That's their job. They expect insulin detemir to be extra effective. In the first run of experiments using intra cerebral administration they failed to get any effect, of any type of insulin, on food intake. None.

This is big. And bad. EVERYONE in obesity research KNOWS that insulin, within the brain, suppresses appetite (excepting the few people who think this idea is bollocks of course, there are always a few people who think logically).

Jessen and Bouman probably think they have made a mistake somewhere along the line. They know that Clegg can, in academia at least, deliver results that show a suppression of appetite in rats following centrally administered insulin. They call her over from of Cincinatti to trouble shoot their problems.

In a hard nosed, financially driven situation, she can't do it. From the abstract of the study:

“Although we varied rat strain, stereotactic coordinates, formulations of insulin and vehicle, dose, volume, and time of injection, the anorectic effect of intracerebroventricular insulin could not be replicated”.

It seems to me that there are differences between academia and industry. It’s the difference between holding a religious belief in the central anorectic effect of insulin and looking for an effect which might suggest a marketable drug which will actually work to assist weight loss. I would call the latter "The Real World".

Time to discard the idea that centrally acting insulin is an anorectic agent? Kudos to the researchers for publishing.

Peter

Random musings on carbon monoxide

There are occasional days at work when I get a lunch break. Sometimes it is long enough to get home and back, often it’s not. Given an hour of free time in the basement, what might you do?

Well, usually I go and have a look whether Nick Lane's group has any new publications up. Pondering the origins of life is a great relief from the problem solving with limited information that makes up clinical work.

In recent months I’ve usually had three browser windows open to try and get an accurate understanding of what he and his coworkers are specifically saying.

As a preamble to discussing those methanogens and acetogens which do not use cytochromes I would just like to lay it on with a trowel that these strict anaerobic organisms are not “living fossils” or in anyway primitive. They appear to be using a method of carbon fixation which is developed from that of the first prebiotic synthesis. That does not make them simple. If this is the first technique for carbon fixation it has been developing for well over 3 billion years. It’s still used nowadays because it works.

So lets go to Figure 1 sections a and b of An Origin-of-Life Reactor to Simulate Alkaline Hydrothermal Vents to have a look at the energetics of fixing organic carbon:

This shows that hydrogen is unable to reduce CO2 to formaldehyde at pH 7. However, if you introduce a pH difference across a thin FeS barrier, it appears to be possible to put an electron or two on to FeS clusters which can then drive the reaction. At the risk of spoiling the paper, it works. Yield is low, but it's there.

Of much greater interest (to me anyway) is the ability to reduce CO2 to CO. I've added these potentials to Nick Lane's diagram like this:

There is a lower barrier to be overcome and, given the suggested pH gradient, greater likelihood of the reaction proceeding. I think the group are interested in formate and formaldehyde because they have the potential to do many things other than generate acetate/methane plus the reactions from there onwards are exergonic.

I'm more interested in CO formation because the carbon monoxide dehydrogenase (CODH) enzyme appears to be core to carbon fixation and is conserved between archaea and eubacteria, certainly in the methanogens and acetogens which lack cytochromes and are strictly anaerobic in their metabolism. So too is acetyl CoA synthase, which is directly linked to CODH. This suggests that this enzyme complex, or the abiotic reaction it preserves, is very ancient. We need to look at Early bioenergetic evolution, Fig 2 section b for a suggestion of how prebiotic acetate synthesis might have occurred. This is the top section:

If we work down from the CO2 at the top of the dashed line rectangle we can see that CO2 is being reduced by electrons from a very low potential FeS cluster (just where it says +2[H]), generated as in my modification of the blue bar chart above, using the pH gradient across a thin FeS barrier. The FeS cluster on the right is the actual catalytic cluster and the green sphere is a Ni atom crucial to its function.

The CO diffuses to a second FeS catalytic cluster which is doped with two more Ni atoms, shown as two green spheres. This reaction does not need a low potential FeS group, so I'm not sure why the black/yellow cluster is shown to the left of the "Ni" in the diagram, excepting that modern ACS does have an FeS cluster covalently attached to the FeNiS catalytic cluster. Doesn't make the diagram particularly easy to interpret. Anyhow, the CO binds to one of the two Ni atoms and waits for a methyl donor.

The methyl source is on the far left of the diagram, shown as CH3-X. This has to be of abiotic origin in an origin-of-life scenario and the simplest is undoubtedly CH3-SH. This methyl group attaches to the CO on the Ni atom and we then have CH3-CO- attached to the catalytic site. Thiolysis using a source of sulphydryl (shown as HS-R in the diagram, below the CH3CO-Ni, still within the dotted rectangle) generates a thioester of acetate, shown (outside the rectangle) as CH3CO-SR.

We've already speculated the availability of CH3-SH in vent fluids to supply methyl groups, so we could simply replace HS-R with HS-CH3 i.e. CH3-SH can provide both the methyl group and the sulphydryl group for the above reactions. The only step requiring any energy input is the initial reduction of CO2 to CO using a low potential FeS cluster.

So when you supply CO and CH3-SH to a slurry of FeS and NiS you get CH3CO-S-CH3 of abiotic origin. This was done back in 1997 by Huber and Wächtershäuser. The trick of reducing CO2 to CO is the hard knot to pick and which needs FeS and a proton gradient, not available in the slurry.

The thioester can hydrolyse to heat and acetate but more interestingly it carries enough energy to form a phosphate derivative which retains slightly more available energy than ATP.

After translating this in to a system which has developed genes and proteins, we still the have remnants visible.

Let's now look at the lower section of Figure 2 section b. The complex series of reactions down the left is how modern acetogens generate their methyl donor source, down the right is the system from methanogens. These are both replacing the speculated abiotic CH3-SH. These pathways are not particularly relevant to any origin of life speculation but clearly are interesting in their own right as they also support CO2 reduction by H2. There are some interesting speculations as to why tungsten or molybdenum are required cofactors... Today it's still the central rectangle we are interested in:

This is the modern version of what we have been looking at previously, with the two Ni doped FeS clusters on the right hand side. The reduction of CO2 to CO is no longer accomplished using a proton gradient, in fact these archea and eubacteria use a sodium gradient rather than a proton gradient. Instead, a low potential ferrodoxin protein with two FeS clusters is generated in the cytoplasm using electron bifurcation from molecular hydrogen (not shown, discussed here, that's the third window in my browser!). The systems used differ between methanogens and acetogens but the core energy currency for both is still reduced ferredoxin. This ferredoxin looks, to me, like a fossil of the FeS on a proton gradient which provided reducing power in the initial scenario.

The transport of CO to the second FeSNi cluster is down a molecular tunnel in the modern enzyme but the binding to a Ni atom at the end of that tunnel persists. Source of the methyl group is quite different between archaea (methanogens) and eubacteria (acetogens). This differing arrangement suggests development after the divergence of the two lineages.

The use of CH3-SH to form a thioester has been replaced by the use of coenzyme A, giving us the familiar acetyl-CoA (shown as CH3CO-SCoA) below the rectangular box. This can be used for substrate level phosphorylation of ADP to ATP or can be used as a source of cell carbon for metabolism. There is a lot of interest along these lines in this paper: The stepwise evolution of early life driven by energy conservation.

There are some interesting ideas which stem from this believable scenario.

Substrate level phosphorylation is clearly very ancient. There are suggestions from the speculated origins of ATP synthase that ATP usage may have been common before this enzyme complex was developed.

The possibility of generating an ATP-like moiety was present when all that was available was a proton gradient derived reduced FeS cluster and some CH3-SH.

Carbon fixation does not appear, initially, to have involved proton translocation, though the gradient was essential.

Carbon monoxide is utterly intrinsic to the formation of life. Strange, that.

Peter

Unclean

People may have seen this quote on Facebook. Lovely to see Steven Rentaquote Nissen publicly acknowledging that the death toll from cardiovascular medicine's lethal low fat diet has finally been halted by a couple of investigative journalists. Thank you Mr Taubes and Ms Teicholz. Oh, he missed that bit...... Here's the quote:



Steven Nissen, chairman of cardiovascular medicine at the famed Cleveland Clinic: For years, "we got the dietary guidelines wrong. They've been wrong for decades."

Advice to avoid foods high in fat and cholesterol led many Americans to switch to foods high in sugar and carbohydrates, which often had more calories. "We got fatter and fatter," Nissen says. "We got more and more diabetes."

Recent studies even suggest that longtime advice on saturated fat and salt may be wrong, Nissen says.




Personally I feel a little contaminated, unclean, by Nissen's falling in to line with what any sensible person with a laptop and net access realised fourteen years ago. Yeugh. Anyway: I thought I would help out by sketching out his next press release:



Steven Nissen, chairman of cardiovascular medicine at the famed Cleveland Clinic: For years, "we got the cholesterol guidelines wrong. They've been wrong for decades."

Advice to take drugs to lower cholesterol led many Americans to pay for statins which made them diabetic and increased their cancer risk. "We got sicker and sicker,” Nissen says. "We got more and more dementia.”

Recent studies even suggest that longtime advice in favour of statins was a bad as that against saturated fat and salt, Nissen says.


Y'all know it's coming! You saw it here first.

Peter

Ketosis and Protein

I just wanted to throw out a few comments about the inhibition of ketogenesis by protein. The obvious effect, that of stimulating gluconeogenesis, appears to be at best a partial explanation of what happens.

I've long been interested in how amino acids feed into (and are derived from) the citric acid cycle and related pathways. Clearly any amino acid which metabolises to oxaloacetate within the liver is simply going to remove that void in the citric acid cycle (oxaloacetate deficiency) which results in acetyl-CoA being diverted to ketogenesis. Aspartate is one such. Those metabolising to pyruvate are also going to do essentially the same thing. There is no need for increased gluconeogenesis in this scenario. Gluconeogenesis may happen at a increased rate. It may not. Providing a source of oxaloacetate in the liver mitochondria will stop ketogenesis, whatever gluconeogenesis does, whatever insulin does.

Personally I am very ketoadapted. I've drifted in and out of ketosis since just after that start of the current century, probably around the summer of 2001 if I recall correctly. I find carbohydrate restriction effortless. Limiting to 30-40 grams per day is easy. Protein limitation is much more difficult. With about 20grams of protein in each breakfast and a few grams derived from cream, chocolate or macadamias at lunch time this does not leave a huge allowance for meat intake at suppertime. At around 65kg bodyweight nowadays keeping to 1g/kg is not the easiest target. A decent steak and I miss it. Life is too short to stress about this, but I certainly don't eat steak every day. Urinary ketones are always there at the + or ++ level. Exercise (distance walking) usually gives +++ as does the evening meal post prandial period, unless there have been excess chips with supper or I've gone significantly over my protein limit.

So I've limited protein, mildly, for years. My degree of keto adaptation still allows free generation of ketone bodies, certainly to a level where I can detect acetoacetate in my urine.

For some reason the concept of amino acids being derivatives of (and inputs towards) the TCA pulls me back to Nick Lane's ideas, the origin of life and the throwing together of metabolism. I'm willing to buy the reduction of CO2 by H2 to give formate as the starting point of metabolism. There is an energetic cost to this initial step but once going it's all down hill, energetically, to pyruvate. Many amino acids are formed from pyruvate and close derivatives. This makes sense. Evolution doesn't plan but does progress within the framework of what is available.

In modern biology DNA doesn't do very much other than replicate (I simplify). RNA is much more active, it carries the message out, assembles itself in to ribosomes and does all of the picking and choosing of amino acids etc to make a protein. I like to think of DNA as a rather stable "hard copy" of the information which was originally carried by the less stable RNA. As such DNA is the fossilisation of the amino acid preferences of primordial RNA. Written in to DNA are the remnants of what was probably a chemical associations of RNA with specific amino acids. If DNA specifies a cytosine at the start of a triplet then the amino acid chosen via transport RNA will be derived from alpha-ketoglutarate, if an adenine the coded amino acid will be oxaloacetate derived, if thymine it comes from pyruvate and if guanine the amino acid will be derived from any one of several possible small molecules. The second base specifies how hydrophobic/hydrophilic the chosen amino acid might be and these two cover a high proportion of the biological amino acids. The third base is degenerate, i.e. it doesn’t carry any specific information but does allow a wider pool of amino acids to be selected.

I'm afraid this is all rather cool to me.

I love these glimpses in to the early mish-mash of chemicals and how they might have interacted before life became seriously organised. What you can and cannot say about LUCA, the last universal common ancestor, and the first steps away from prebiotic chemistry, is largely determined by such biochemical fossils.

All of this random musing came from wondering whether amino acids might supply oxaloacetate and so suppress ketosis. Some do.

Well. Eating a steak is not very ketogenic. It’s hard to separate this from the origins of metabolism and of life, for me anyway,

Peter

Shewanella and electrons

Have we found alien life? This interview did the rounds via Facebook recently. There is only one answer.

No.

We have found bacteria that can run their electron transport chain using an electrical connection to an external electron acceptor. In more ordinary bacteria electrons travel down the ETC to complex IV where, under oxidative metabolism conditions, they are passed to oxygen as the terminal acceptor, still within the cell.

Bacteria are the most sophisticated metabolists on Earth. To stick a series of cytochromes together to form a wire, from the end of an electron transport chain to an extracellular acceptor of electrons, is no big deal. Once a bacterium is using such a wire (which will, undoubtedly, be using quantum tunnelling effects much as the FeS clusters of complex I do) to access extra cellular terminal acceptors it is absolutely no problem to make that terminal acceptor anything with an appropriate redox potential.

Under these conditions a cathode electrode will allow the metabolism of NADH from the cytosol, via complex I and the CoQ couple down the ETC and out along the cytochrome wire. A cathode electrode, whatever it is made of, is an electron acceptor. That's the definition.

So growing bugs on a cathode is utterly unremarkable. NADH comes from "food". Electrons from NADH drive the ETC to make ATP and are dumped out-of-cell on to the cathode.

Where things get slightly interesting is that you can also apply a negative voltage to the end of the wire, making it an anode, a net supplier of electrons. Certain types of bacteria can function under these conditions without any carbon input. From the article:

“A lot of organisms that can put electrons onto an electrode can also do the opposite and take electrons from one”—though not at the same time—Rowe says. That ability to reverse course surprises me, and Rowe, too. “I’d think it would be really hard on the organisms. You’re basically stealing energy from them. But they do okay.”

This is incorrect, it lacks perception.

It is perfectly possible to run the ETC in reverse. Bacteria do this on occasions, for reasons best known to themselves, especially if their normal metabolic substrates cannot generate NADH directly. Usually ATP is used to drive ATP synthase in reverse to maintain the membrane potential. Electrons flow in reverse to reduce NAD+ to NADH which can then be used for anabolism. All that Shewanella spp need to do is to use the negative voltage of an external electrode to facilitate reverse electron flow and they can be generating NADH for anabolism without any carbon source. About a third of a volt does the trick, from the interview.

Whether this process would allow the fixation of atmospheric carbon dioxide to actually allow growth is irrelevant. What matters is that there is absolutely nothing about these bacteria living on "pure electricity" which suggests anything other than a clever piece of wiring added on to the end of a  fairly normal ETC.

This is what Shewanella oneidensis looks like with its complex I and CoQ couple:

A clever bacterium? Yes, with very busy periplasm. But anyone who thinks that any bacteria are simple is stupid. What if you think that these particular ones are alien life forms?

"Kenneth Nealson is looking awfully sane for a man who’s basically just told me that he has a colony of aliens incubating in his laboratory".

It is only an alien life form if aliens developed the same electron transport chain as any Earthly organism which uses oxidative phosphorylation to chemical acceptors.

How much does Nealson know?

Peter

GSD IIIa, ketones, MAD and Veech again

I mentioned the high protein/exogenous ketone approach to Glycogen Storage Disease IIIa in a recent post. It's very nice that an effective treatment can actually be achieved through a Modified Atkins Diet (MAD at <10g of carbs per day) involving food, w/o faking ketosis though those exogenous synthetic ketones.

Robert gave me the heads up on the latest paper on GSD IIIa using MAD, available as a free text through Pubmed. It's rather good as, again, it shows that there are medics out there who think matters through and occasionally come to correct conclusions. I love the clinical details of compliance/non compliance too. And that the early hypoglycaemia, treated with corn starch (bleugh), was asymptomatic under ketosis.

That's nice.

He also sent me the full text of Veech's

The Determination of the Redox States and Phosphorylation Potential in Living Tissues and Their Relationship to Metabolic Control of Disease Phenotypes

which is a fascinating personal insight in to what it was like to work in Hans Krebs' lab, combined with the sort of hard core math which implies rather more understanding of biochemistry than simply adding MitoSOX red to some cells and looking for colour changes to show oxidative stress. That's some complex reading to work through when clinical and home life combine to give me a chance!

Peter

Palmitic acid and hyperglycaemia in diabetic heart failure (1)

I've been gifted a paper of great interest. The authors of the paper are very good in their explicit discussions of the limitations of their models, the reasons for the choices they made and the limitations of all current models and probes. This is good and, needless to say, there are an awful lot of "ifs, buts and maybes" to the data! On the down side the paper is an epub of the crudest type, retaining numbered lines, the figures at the very end of the whole paper and all of the captions in a lump between the references and the figures. So not easy reading for a very complex paper, with very complex figures and very complex captions. I spent a large amount of time on this paper over the Christmas vacation but never hit post on any of it. Here is an introduction.

Over the years we have seen, largely via Veech, the ability of ketone bodies alone to rescue myocardial function to give mechanical work performance comparable to the combination of insulin with a relatively normal level of glucose in the perfusion of isolated rat hearts. Equally there is the concept that ketones essentially bypass insulin resistance to rescue metabolism.

The hearts in Veech's study were perfused with oxygenated buffer which was devoid of the free fatty acids which are a normal metabolic substrate for heart muscle.

As a lipophile I have long wondered whether, outside of the neurons, palmitic acid might be a reasonable substitute for ketones in rescuing muscle metabolism damaged by hyperglycaemia.

From the start of the discussion section of Bhatt's paper:

"We investigated the role of substrate-driven redox status on the contractile/energetic performance of heart trabeculae from the T2DM ZDF rat. The main findings are: i) HG exerts a detrimental action on contractility of T2DM heart trabeculae that Palm is able to rescue; ii) Palm prevents oxidative stress exacerbated by HG, an effect independent from insulin action; iii) insulin appears to worsen the negative effect of HG through higher oxidative stress and lower GSH; iv) ZDF heart mitochondria emit less ROS and display higher ROS scavenging capacity of the GSH/Trx antioxidant systems; v) cardiac redox balance in HG appears to play a causal rather than correlative role in the preservation of contractile performance in ZDF trabeculae"

Palmitic acid will rescue hyperglycaemia induced myocardial contractility failure. OK, I'm happy.

This was the core finding in the paper. There are a whole slew of spin off concepts which grow from it but if I wait until I have them all sorted out it might be a very long time before I get another post out!

But here we have it. Hyperglycaemia is bad. Palmitic acid rescues hyperglycaemia induced dysfunction. A paradigm shift in glucose vs FFAs!

Peter

ACCORD and musings on insulin

There are a couple of things I would just like to mention in passing. Jenny Ruhl has just posted a nice entry about ACCORD. This is very important. Lowering your blood glucose is significantly protective against CVD events. This is the exact opposite of the initial analysis of the results where a flawed interpretation of the data led to vociferous suggestions that lowering the HbA1c of diabetics might be actually dangerous. Hopefully this reanalysis will put an end to such stupid ideas which are still dangerously prevalent today.

Before Jenny posted the above I had been thinking about both metformin and insulin for the management of diabetes. I have posted on insulin, which is probably the ideal drug for diabetes management provided it is combined with low carbohydrate eating, in the past but it bears reiterating.

This is my opinion. If you can control your diabetes with metformin and wish to eat lots of carbohydrate, by all means get on with it, that's your choice. Not checking or not worrying about your blood glucose excursions might be a mistake. What you mean by good control might not involve HbA1c values in the 5% region or below.

If you need insulin to control your blood glucose, you have no choice. It's low carb. Live with it.

Why?

Some researchers (who write third rate papers using a rather inappropriate pancreatectomised dog model) are now starting to wake up to the fact that the pancreas secretes insulin in to the portal vein, not the subcutaneous tissues. In normal individuals insulin/glucose arrives at the liver via the portal vein and insulin facilitates transport of the glucose in to the liver, being metabolised in the process. Relatively little post pranial insulin or glucose penetrates to the peripheral circulation in a normal individual.

EDIT: Gretchen has kindly pointed out that the liver uptakes glucose through GLUT2, not GLUT4. The function of insulin, acting on its receptor (facilitating its degradation), is to suppress gluconeogenesis and hepatic glucose output. This is the correct function of the elevated portal insulin level. It makes no difference to the issues with peripheral vs portal insulin, but the correction is welcome. END EDIT

Once a person needs insulin to control their blood sugar levels they inject it subcutaneously. This will invariably elevate the systemic concentration of insulin. It will only modestly elevate the portal vein level. This is very important.

In the cited paper the dogs get a meal with 50% of calories from carbohydrate, 30% from fat and 20% from protein. In control dogs, instrumented but not pancreatectomised, the portal vein insulin after the meal is, at certain time points, ten times the peripheral systemic concentration. This is what is needed to allow the liver deal with the glucose load from the meal while simultaneously protecting the body from both hyperglycaemia and hyperinsulinaemia.

Subcutaneous insulin will make the rest of the body do the liver's job of clearing post prandial glucose, the liver can't manage because it never sees the requisite ten times the normal peripheral insulin concentration needed to deal effectively with the portal glucose load.

What happens when you use the rest of the body as a glucose sump? From the paper:

"Peripheral hyperinsulinemia is associated not only with increased risk of hypoglycemia, but also an increase in catechol and cortisol secretion and lipolysis [14], deleterious effects on vessel walls [6], ischemic heart disease, hypertension and hyperlipidemia [8], and abnormalities in hemostasis [10]"

As always, I would add that it's the obese, blind, legless person in the queue for dialysis who pays the bill for eating the carbohydrate.

This is the situation for all type I diabetics and the more advanced type 2 diabetics. The only route round it is to use intra peritoneal insulin which is, in part, absorbed through the mesenteric veins so is partially portal vein selective. There are, needless to say, a stack of complications to intra peritoneal insulin infusion. Tight control of glucose using subcutaneous insulin from a blood glucose controlled pump is no solution. Though glycaemia is better controlled it is still at the cost of too little insulin in the portal vein and too much in the periphery, using the body as a glucose sump. Over the years I have never been quite able to decide whether hyperinsulinaemia or hyperglycaemia is the primary factor which kills nerves and kidneys. It's a difficult call. And a fascinating discussion in its own right.

What happens if you eat a diet very low in starch?

Very little insulin is ever secreted by the pancreas, especially as glucokinase down regulates. Very little glucose ever needs to be taken up by the liver. Very little insulin will be metabolised by the liver. The insulin gradient between the portal vein and the systemic circulation will be as low as you can practically get it. If someone still needs to inject insulin alongside a very low carbohydrate diet, and many might not, injecting a very small amount subcutaneously will deliver an arterial concentration to the gut, pancreas and eventually to the portal vein and liver which is still quite close to what the portal vein might have supplied. If the body is not using insulin the tissues will not extract it, so portal and systemic concentrations will converge. Everything pans out at some near basal level.

A very low carbohydrate diet is not perfect for insulin dependent diabetics but it is streets ahead of anything else. What people do or do not consider a "normal" human diet will not get around this. Need exogenous insulin? You are not in a position to eat ancestral starch. It's a simple matter of anatomy, physiology and biochemistry.

Peter

The P479L gene for CPT-1a and fatty acid oxidation

In order to work out what is happening with a given child having an episode of hypoglycaemia as a result of having the P479L version of CPT-1a, we need some information.

My thanks to Mike Eades for the full text of the paper on the Canadian Inuit, which does include a certain amount of useful clinical data.

Here is the snippet about a young girl having a hypoglycaemic episode while hospitalised:

“Plasma free fatty acid was 3.8 mmol/L and plasma 3-hydroxybutyrate was 0.5 mmol/L”

Blood glucose was 1.9 mmol/l at the time. An FFA level of 3,800 micromol/l is impressively high. She was generating a small amount of ketones.

No one would argue with intravenous glucose at this point, the question is about how she got here.

So. The problem here does not (as I'd initially thought) appear to insulin induced suppression of FFAs to a level at which beta oxidation fails to support metabolism. FFAs are very high, even for an P479L person after a short fast. With ketones starting to be produced (and low blood glucose) I feel it is reasonable to assume that her liver glycogen is depleted and, while some fatty acids are entering the hepatocytes, not enough of them are being oxidised to support ketogenesis. Glycogen is being depleted to keep liver cells functional. Gluconeogenesis from protein is unable to meet the hepatic (and whole body) demand for glucose calories in the situation of limited access to FFA calories.

However much glycogen derived glucose you consider that the ancestral diet contained I feel it is very, very unlikely to be greater than the glucose and fructose of a modern diet. I feel that getting enough glycogen in to the liver to fully fuel its metabolism in the absence of adequate fatty acid oxidation is a non starter. The P479L mutation was not "permitted" by high oral carb loading, it was permitted by conditions which facilitated fatty acid oxidation. You don't have to agree.


What starts to look much more interesting is what controls CPT-1a activity and how this might vary from the ancestral diet to the modern diet.

The paper makes the point that omega 3 fatty acids appear to up regulate fatty acid oxidation (in rats at least) by the liver. If this is true in humans then a high level of omega 3 fatty acids from marine fats might up regulate fatty acid oxidation to a level which no longer necessitates the depletion of hepatic glycogen derived form oral glucose intake or protein catabolism.

In support of this is that the distribution of P479L within Alaska is not uniform, it's significantly commoner in the coastal regions compared to the inland areas.

"The allele frequency and rate of homozygosity for the CPT-1a P479L variant were high in Inuit and Inuvialuit who reside in northern coastal regions. The variant is present at a low frequency in First Nations populations, who reside in areas less coastal than the Inuit or Inuvialuit in the two western territories"

I'm open to other explanations, there are papers suggesting that the mutation helps to preferentially dispose of omega 6 PUFA, with omega 3 fatty acids as the facilitator.

In summary: Maintaining adequate FFA oxidation to avoid glycogen depletion looks to be the core need in P479L. A high fat diet with a large proportion of omega 3 fats might be a plausible way of maintaining adequate hepatic fatty acid oxidation. Hyperglycaemia (via Crabtree effect) looks to be anathema. Glycogen loading with a normal starch/sugar based modern diet is clearly ineffective to prevent hypoglycaemia for some individuals. Resistant starch as a reliable nightly adjunct to infant feeding seems very unlikely in the ancestral diet. Repeated periods of fasting were probably routine when hunting was poor and does not appear to have selected against P479L in weaned children. Unweaned children are unlikely to be exposed to fasting, provided milk was available from lactation.

Well, there are some more thoughts on the biochemistry.

People clearly have very differing ideas of what the Inuit did or did not eat as an ancestral diet. The P479L gene eliminates the need for source of dietary glucose to explain very limited levels of ketosis recorded in the Inuit. While it is perfectly possible to invoke a high protein diet to explain a lack of ketosis in the fed state this goes nowhere towards explaining the limited ketosis of fasting. P479L fits perfectly well as an explanation.

I have some level of discomfort with using the Inuit as poster people for a ketogenic diet. That's fine. They may well have eaten what would be a ketogenic diet for many of us, but they certainly did not develop high levels of ketones when they carried the P479L gene.

However. Over the months Wooo and I seem to have come to some sort of conclusion that, while systemic ketones are a useful adjunct, a ketogenic diet is essentially a fatty acid based diet with minimal glucose excursions and maximal beta oxidation. Exactly how important the ketones themselves are is not quite so clear cut. From the Hyperlipid and Protons perspective I would be looking to maximise input to the electron transport chain as FADH2 at electron-transferring-flavoprotein dehydrogenase and minimise NADH input at complex I. Ketones do not do this. Ketones input at complex II, much as beta oxidation inputs at ETFdh, but ketones also generate large amounts of NADH in the process of turning the TCA from acetyl-CoA to get to complex II, which ETFdh does not. I'm not a great lover of increasing the ratio of NADH to NAD+. These are my biases.

Confirming that the Inuit are not poster boys for ketosis is a "so what?" moment for me. Using their P479L mutation to argue against ketogenic diets is more of a problem. It's a massive dis-service to any one of the many, many people out there who are eating their way in to metabolic syndrome to suggest that a ketogenic diet is a Bad Thing because no one has lived in ketosis before. Even the Inuit didn't! My own feeling is that everyone comes from stock who occasionally practiced and survived intermittent fasting so we are should be adapted to this. I'd guess that if you are of Siberian, Inuit or First Nations extraction you might benefit from Jay Wortman's oolichan oil as part of a ketogenic diet.

I'm always amazed by the concept that a ketogenic diet might be temporarily therapeutic but must be discontinued because it eventually becomes Bad For You. It reminds me so much of the converse concept that low fat diets, which might worsen every marker of health which people may care to look at, will deliver major benefits at some mythical future date.

Ultimately, point scoring on the internet about what the Inuit did or didn't eat shouldn't destroy people's chances of health. Destroying a circular argument about Inuit diets may may the destructor feel good. Destroying the feet, eyes and kidneys of a person with type 2 diabetes, who need a ketogenic diet, as a spin off from that victory must be difficult to live with. I don't know how anyone can do this.

I think that's probably all I have to say for now.

Peter