Tuesday, August 30, 2016

Sometimes, you get thumped on the head with a scientific paper...

Now and then, a member of some "low carbing" communities tries to "educate" me. In fact, I am not fundamentally opposed to a lower carb diet, or even no carb periods, but I probably not "faithful" enough to avoid a virtual Sunday morning "Jehova Witness style" call. Here's a paragraph from a scientific paper I have already been sent three times.

A claim so dramatic and peremptory that I should either see the light or crawl back into my hole. This is the type of statement that closes all debates and can easily be summarized for the choir. (it comes from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2129159/#B21


Let's dig a bit into it, in details.
"Contrary to popular belief supported by the leading physiology and biochemistry textbooks, there is sufficient population of glucose transporters in all cell membranes at all times to ensure enough glucose uptake to satisfy the cell's respiration, even in the absence of insulin"


Popular belief, maybe... But textbooks are fully aware of the existence of the different transporters (some of them insulin dependent, some of them gradient dependent, etc...), their respective distribution and roles in different tissues and of the absence of barrier. So, this is basically creating a giant out of a windmill. The "giant" is extraordinary and as such deserves an extraordinary proof. The proof comes from (21), the only article cited multiple times in the paragraph, which you can find here http://joe.endocrinology-journals.org/content/170/1/13.long This is, by the way, an article devoted to doping in sports which summarizes, sometimes without references, some information about insulin. The article hasn't been cited too often as a fundamental work on Insulin metabolism... but I digress..
"Insulin can and does increase the number of these transporters in some cells but glucose uptake is never truly insulin dependent."


What does "truly" actually mean? Is it dependent or not, partially, mostly, not at all, falsely dependent, somewhat falsely dependent? I don't know... "not only dependent" would have been acceptable though.

"Even under conditions of extreme ketoacidosis there is no significant membrane barrier to glucose uptake – the block occurs "lower down" in the metabolic pathway where the excess of ketones competitively blocks the metabolites of glucose entering the citric acid cycle."

This is an almost verbatim quote from (21) - even in ketoacidosis, glucose can enter the cell. But it is blocked "lower down" (which means at the Krebs Cycle level).

So, let's rephrase that: nothing prevents glucose from entering the cell, but it will be under utilized. Fair enough. It is a bit like saying "Falls are never dangerous to humans. The danger occurs "lower down" when the human hits some hard surface.

"Thus, insulin is not needed for glucose uptake and utilization in man"

Aha... Did he just prove that in a flash? Yes, some uptake does occur without insulin. That is the windmill the author had changed into a giant. But utilization? The author just stated that there is some kind of block "lower down" (which happen to be ketones that, in short, insulin would prevent)... I am somewhat confused here.

Anyway, let's go for another verbatim quote of 21, blah blah blah...

In fact, the process appears to be general for all polar (water-soluble) substrates, as transporters are the mechanism by which they are transported across the highly non-polar (lipid) cell membranes. 

and follow that by another interesting statement.

When insulin is administered to people with diabetes who are fasting, blood glucose concentrations falls. It is generally assumed that this is because insulin increases glucose uptake into tissues. However, this is not the case and is just another metabolic legend arising from in vitro rat data.

The claim that insulin lowers BG in diabetics because it increases uptake into tissues is now a "legend". Not bad. We'll get to that...

It has been shown that insulin at concentrations that are within the normal physiological range lowers blood glucose through inhibiting hepatic glucose production [].

Ah, finally, some truth. Yes, insulin does suppress endogenous glucose production. Among many other things. And, indeed, in some ranges, fasting and at rest, uptake does not increase and may even decrease. No demand from the cells. Except when you move your muscles a bit...

Now, let's go back to article 21. An article that deals mostly with doping and ends up on the usual customary note... If it was quoted three times, it must contain additional supporting evidence...


"Both methods need further validation before implementation. Research work carried out as part of the fight against doping in sport has opened up a new and exciting area of endocrinology."

That article also states, the very conventional... (the language is a bit dated, see below)

"The truth is that insulin acts exactly as Schafer had predicted – it acts as both an autacoid and a chalone. Through stimulating the translocation of ‘Glut 4’ glucose transporters from the cytoplasm of muscle and adipose tissue to the cell membrane it increases the rate of glucose uptake to values greater than the uptake that takes place in the basal state without insulin."

Ah, OK, then uptake does increase after all... Why skip that?

Finally, it completes the "lower down" paragraph quoted verbatim in the first paper

"... the block occurs ‘lower down’ in the metabolic pathway where the excess of ketones competitively blocks the metabolites of glucose entering the Krebs cycle. Under these conditions, glucose is freely transported into the cell but the pathway of metabolism is effectively blocked at the entry point to the Krebs cycle by the excess of metabolites arising from fat and protein breakdown. As a result of this competitive block at the entry point to the Krebs cycle, intracellular glucose metabolites increase ‘damming back’ throughout the glycolytic pathway, leading to accumulation of free intracellular glucose and inhibiting initial glucose phosphorylation. As a result, Figure 1 Insulin exhibits both inhibitory (chalonic) and excitatory (autacoid) actions via the same receptor. In these experiments carried out on rat adipose tissue, in vitro insulin simultaneously inhibits lipolysis (the release of glycerol from stored triglyceride) and stimulates lipogenesis (formation of stored triglyceride from glucose). Thus its anabolic action is due to two mechanisms working synergistically. much of the ‘free’ intracellular glucose transported into the cell is transported back out of the cell into the extracellular fluid. Thus under conditions of ketoacidosis, glucose metabolism (but not glucose uptake) is impaired as a direct consequence of the metabolism of fat – the ‘glucose–fatty acid’ cycle (Randle et al. 1965)"

which is a bit old, is widely understood to be generally correct and has been fine tuned since but not drastically. On the whole, when the paragraph is quoted completely, the meaning changes a bit though...

This is followed by a quite longish discussion about other things we also now know to be mostly true and are, in many ways, in complete opposition with the revelations of the original paper...

1. Through facilitating glucose entry into cells in amounts greater than needed for cellular respiration it will stimulate glycogen formation. Thus hyperinsulinaemic clamps will both increase muscle glycogen concentrations prior to events and in the recovery phase after events. Since performance in many events is known to be a function of muscle glycogen stores, Figure 3 The data used in this illustration were obtained from healthy normal subjects using a series of euglycaemic and hyperglycaemic clamps at basal or increased insulin concentrations. Rd1: insulin independent glucose uptake. The data have been fitted to the generic model shown in Fig. 2 (see text for details). Insulin, GH and sport · P H SONKSEN 17 www.endocrinology.org Journal of Endocrinology (2001) 170, 13–25 ‘bulking up’ these stores will most probably enhance performance. There is no documental proof that this technique is being used but informed ‘street talk’ indicates that it is not uncommon. 2. Through use of similar hyperinsulinaemic clamps post-event and during training, it is likely that recovery and stamina will be improved. 3. ‘Street talk’ indicates that insulin is also being used in a more haphazard way, particularly to increase muscle bulk in body builders, weight lifters and power lifters. This use is allegedly by regular injections of shortacting insulin together with high carbohydrate diets. Through this therapeutic regime it is almost certainly possible to increase muscle bulk and performance not only through increasing muscle glycogen stores on a ‘chronic’ basis but also by increasing muscle bulk through inhibition of muscle protein breakdown. Just as insulin has a chalonic action in inhibiting glucose breakdown in muscle glycogen, it also has an equally important chalonic action in inhibiting protein breakdown. Indeed, the evidence now indicates that insulin does NOT stimulate protein synthesis directly (this process is under the control of GH and insulin-like growth factor-I (IGF-I)). It has long been known that insulin-treated patients with diabetes have an increase in lean body mass when compared with matched controls (Sinha et al. 1996). 


in some specific insulin and BG ranges to end up with the conclusion that

Taken together, all these points support the concern shown by the Russian medical officer in Nagano and the immediate response of the IOC to ban the use of insulin in those without diabetes.

To summarize...

The paper I have been mailed several times so I could "educate" myself
  • makes an outstanding sounding claim.
  • that it falsely presents as a revelation that contradicts fundamental textbooks.
  • attempts to justify its claim by cherry picking partial paragraphs
  • out of a single paper that attempts to address a completely different issue
  • and comes from an era where we had concerns that insulin and GH could be used for doping.
  • finally, the original article manages to cram a couple of non sequitur and to contradict itself.
That's a bit light to convince me...

Conclusion
But, of course, the fact that the article is.... hmmmm.... what it is, does not invalidate its premise. In other words, it is not because people e-mail you crap on a semi-regular basis that the pillar of their faith is wrong. It could very well be correct. One could make a completely stupid argument that the Earth is approximately spherical and still be correct.

My goal here is not to attack the idea of low carb in itself, but it would be nice if the people who mail me papers could at least attempt a bit of homework...

Monday, August 8, 2016

PTPN22 gene and type 1 diabetes: a short post about why the genetics of diabetes are so complex.

Apparently, this post has been sleeping as a draft for a while. Wrote it at the beginning of the year, never posted it...

The case of PTPN22

Outside of the major histocompatibility complex (yes, that HLA-DR... stuff), the PTPN22 gene that encodes a protein call Lyp (lymphoid protein tyrosine phosphatase) is one of the genes most associated with auto-immune diseases such as Type 1 Diabetes, RA, JIA, Addison, Psoriasis, etc... A single mutation in the coding part of that gene leads to an amino acid (the building blocks of proteins) substitution. We will call that "bad" mutation R620W.

Here's a short description of PTPN22, we'll talk more about the roles of the phosphatase it encodes a bit later.



You will note the phosphatase is a member of a sub-family (an indication that there are lots of them). You will also note the use of "may". Not necessarily the word you want to read when you are looking at a strong suspect... We'll also get to the reasons behind the uncertainty.


How do we know it is associated with auto-immune diseases?
There are a few "simple" cases where one single mutation in a single gene creates a well defined disease. Phenylketoneuria is the archetypal single gene disease. Certain forms of Type 1 Diabetes have a direct genetic cause. When the Human Genome Project started, we lived for a while under the illusion that we would soon explain hundreds of "genetic diseases". We were totally wrong! In most cases, when a genetic predisposition is present, the causal links are weak and many. Most of today's gene-disease association research is done through Genome Wide Association Studies. How do those GWAS work? Here is a simple analogy. Let's say that 50% of the citizens of the USA are republicans. Therefore, we know that 20%  of the US citizens (40% of 50%) vote for Donald Trump. We also know that only very few Muslims, a small percentage of Blacks and possibly a slightly bigger percentage of Latinos vote for our dear Donald. Crunch the numbers, and you will obtain the odds for each population category. In that simple case, you can safely say if you are Caucasian your risk of contracting Trumpism is higher than the general population. The danger is even worse if you are a Caucasian Republicans! Of course, Caucasian Democrats are less likely to contract the disease but some of them could catch it at some point. Calculate all the odds and predict the results of the elections. Not that simple, right?

But in fact, biology is even more complex than politics and the odds aren't always that clear. Consider that recent Chinese study for example.

The tiny genetic code modification in PTPN22 is more frequent in the T1DM patients population. But, and that is extremely important, approximately 80% of the T1DM patients and 92.5% of the healthy control do not carry that variation! In a nutshell, while more T1DM patients than healthy subjects carry that variation, the vast majority of the population does not carry it...

The risk factor is clear, but the big picture is extremely fuzzy.

The huge weakness of GWAS studies is that while they find a lot of strong or weak correlations, in most cases they do not help us much in terms of causal link. They are pointers that can help directing further research.

Another interesting link between the bad mutation R620W and auto immune diseases is that its geographic distribution mirrors the distribution of diseases (Finland for example has the highest T1D frequency in Europe and also the highest R620W frequency). 

What do we know about PTPN22?

We know an awful lot about PTPN22. In fact, we know almost everything there is to know about it. Here is a simpler description of the gene


It has been extensively studied. 437 citations in Pubmed. 309 Gene RIFs (roughly established connections to functions)

Note: the green rsxxxxxx numbers are the SNPs (single nucleotide polymorphisms) tested by the 23andMe version 3 chip. Our SNPs are constantly loaded as a browser extension and automatically highlighted on pages that I visit.

In fact, you could probably devote your life to the study of PTPN22, its product, the role of its product, its interactions and associations.

Now, lets interest ourselves with the product of PTPN22, the phosphatase itself. You'll find a good summary of what we knew in 2011 in the excellent article Why is PTPN22 a good candidate susceptibility gene for autoimmune disease? I will just summarize a few salient points below.

Lyp

Lyp, the product of PTPN22, is a protein tyrosin phosphatase (PTP). We know more than 110 variations of which at least 57 of them are present in Lymphocytes ("white" cells, in general cells involved in the immune system). Phosphatase (PTP) and Kinase (PTK) are enzymes that control the activity of signalling transmitters, the messengers that will inform the cell of external changes and change the cell behaviour (they are the control switches inside of cells). In general PTP control the amplification of the signal, PTK control its tempo and duration. Again, you could spend a full life studying kinases and phosphatases without running of material...

Reaction to outside signals is obviously extremely important for immune system cells. It is no surprise that several of those phosphatases are associated with auto-immune diseases (PTPN2, PTPRC, UBASH3A, PTPN11, PTPRT and our PTPN22). PTPN22 was linked to Type 1 diabetes in 2004 (N. Bottini, et al., A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes, Nat. Genet., 36 (4) (2004), pp. 337–338)

Lyp, the member of a family of 100+ enzymes, a sub family of 57+ enzymes has three forms, independently of its eventual mutation.

The expression of Lyp varies, depending on the type of lymphocyte and cells.


Lyp inactivates some of the kinases of the Src and Syk families.

Lyp interacts with many other molecules. Here's a rough overview

Source: http://www.sciencedirect.com/science/article/pii/S0014579311002791

and a closer view of one of the process. 

Source: http://www.sciencedirect.com/science/article/pii/S0014579311002791

At this point, I can't resist a few direct (unlinked) quotes.

 The precise stoichiometry of the interactions that promote Lyp phosphorylation by Lck is complex and remains to be determined, but Csk micro clusters may facilitate this interaction through SH2 binding sites that allows the docking of Lck and subsequent phosphorylation and inactivation of Csk associated Lyp. Clarification of the sequence of events that govern inhibition of Lck by Csk and Lyp is also required, since Csk preferentially targets activated Lck that is phosphorylated at Y394, as demonstrated by in vitro kinase assays and phosphopeptide mapping [72]. The regulation of Lck has also been brought into question in recent years since it is becoming evident that acute dephosphorylation of Y505 may not be necessary for Lck activation and TCR signalling

 Studies have yet to address the contribution of Lyp in regulating Vav function.

Because B cells share much of their signalling machinery with T cells it is likely that Lyp may regulate proximal B cell receptor signalling in a similar fashion, but substrate trapping experiments have not been undertaken in B cells. There is some evidence that there may be impairment of signalling in human individuals carrying the R620W variant  

Despite these two molecules having opposing kinase/phosphatase activity, they both serve to inhibit T cell function, possibly synergistically

Together these studies flag up two important questions. First, how does a mutation outside the catalytic domain lead to increased enzyme activity? Second, how could an apparent attenuation of TCR signalling lead to autoimmune disease?

There is still no clear consensus as to whether R620W is a gain- or loss-of-function variant and there is a possibility that in the future there may be newly discovered functions of the mutant phosphatase. It is not inconceivable that R620W may exert both gain- and loss-of-function activity in different pathways but within the same cell. Another possibility is that the mutant Lyp may have different (or opposing) effects in cells of different lineages, or that the mutant has opposing activities in different pathways in the same cell.

Note: I stopped my draft at this point - probably out of despair.

Addendum: interesting papers

Staging Presymptomatic Type 1 Diabetes: A Scientific Statement of JDRF, the Endocrine Society, and the American Diabetes Association (http://care.diabetesjournals.org/content/38/10/1964.full)

A convenient list of T1D SNPs and their weighted risk score

http://care.diabetesjournals.org/content/suppl/2015/11/02/dc15-1111.DC1/DC151111SupplementaryData.pdf

Genetics of Type 1 Diabetes (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3253030/)