Monday, August 29, 2011

Ask Dr. Andro: The Pharmacokinetics of Creatine (Part II/II) - How Is Creatine Transported into the Muscle?

Illustration 1: There is a bunch of things that could potentially go wrong with creatine uptake: The creatine from dietary sources could be mal-absorbed (1) in the small intestine, (2) not make it into the cell, or (3) be excreted too readily either before or immediately after it was transported into the muscle.
Question from Learner (via comments): Do Creatine Transporters behave the same as glucose transporters? (I.e., serum insulin binds to cellular insulin receptors, which causes Transporters to migrate from inside the cell to the plasma membrane - and the Transporters then pull in the external glucose.)

Answer Dr. Andro: As you may have noticed, I took the freedom to set Learner's question into a broader context. A context I broached in my dissertations on Athletic Edge Nutrition's new creatine product Creatine RT on Tuesday, Aug 16, 2011. Thus, the questions I will be trying to answer (unfortunately, I have to rely on existing studies and do not have my own lab, here ;-) are the following ones:

  1. How does creatine get into the blood? (cf. Part I)
  2. How does creatine get into the muscle?
  3. What can influence these processes?
Those of you who have already read part I of this installment of "Ask Dr. Andro", will know that, in view of the fact that this is quite an extensive topic, I decided to tackle it in a two part series, where in part 1 (yesterday) I focused on the issue of creatine absorption into the bloodstream, from where I will now go on to explain how the creatine eventually gets stored in the cells of your muscle or cleared by your kidneys (steps 2 and 3 in illustration 1).

How does creatine get into the muscle?

Now that the creatine molecules have successfully passed your digestive tract they are floating largely unbound (binding affinity of creatine to plasma proteins is less than 10%) in your bloodstream. Whatever happens from now on, is called "clearance" in pharmacological terms - this is counterintuitive at first, but it stands in line with what I have already stressed in my blogpost on Creatine RT, Athletic Edge's creatine monohydrate + Russian tarragon formula. You may remember that Jäger et al. assumed that the smaller increase in plasma creatine they observed upon co-administration of Russian tarragon indicated greater "creatine clearance", which would equal greater muscular creatine uptake. Now, it is true that upon supplementation, the main pathway by which your body "disposes" of the increasing level of serum creatinine is skeletal muscle, but firstly, the tarragon extract could have interfered with the absorption of creatine, for example by modifiying gastric pH levels or intestinal permeability (this is not completely unlikely, since this herb has traditionally been used to cure upset stomachs, cf. Tarragon Central), and secondly, muscular creatine uptake is obviously one way the creatine could have been "cleared" from the bloodstream, the kidneys are yet another.
Image 1: Caffeine + Creatine = Increased renal clearance? Yes! Increased renal clearance = lower performance? No!
Did you know that the longstanding myth that caffeine would counter the beneficial effects of creatine on exercise performance and lean mass gains is bunk despite the fact that caffeine does in fact increase urinary creatine clearance? In a recently published paper on the effect of co-adminsistration of caffeine + creatine to rats (Franco. 2011), the scientists observed statistically significant increases in urinary creatine clearance (+38% after the loading phase with 0.43g/kg creatine and +29% in week 6 of the maintenance phase) over creatine alone when the latter (0.143 g/kg creatine) was administered with 15mg/kg caffeine (human equivalent 2.4mg/kg; ~200mg or 2 small cups of coffee for an 80kg human). When it comes to the real-world results you are looking for, this is yet not likely to be significant.

While the increase in urinary loss may increase the time it will take until your muscle creatine stores are saturated, a study by Lee et al. which compared the effects of creatine alone and creatine + caffeine at a much higher dose equivalent to 480mg or 5 cups of coffee found that "caffeine ingestion after creatine supplements augmented intermittent high-intensity sprint performance" (Lee. 2011) - any fears that drinking coffee or even taking stims could completely negate the beneficial effects of creatine are thus unwarranted.
While researchers initially believed that renal creatine clearance would be equivalent to the glomerular filtration rate (GFR) of roughly 7.0L/h, Poortmans et al. found that, under unsupplemented conditions, creatine clearance is 0.3-0.8L/h, which clearly supports a previously forumlated hypothesis that creatine is reabsorbed and thus "recycled" by the kidneys. Evidence from supplementation studies, where the renal clearance rate increased to 9-22L/h supports the idea that (McCall. 2008)
[a]s blood concentrations increase and more creatine is filtered, less reabsorption occurs and a greater percent age of creatine will be lost in the urine [...] as skeletal muscle approaches its capacity to store creatine, the kidney and possibly other tissues are responsible for the removal of creatine from the blood.
If we follow Mc Call's line of thought and assume that renal creatine clearance is essentially determined by the filling level of muscular creatine stores, it becomes obvious that supplementation with agents that increase creatine transport into the cell would be most beneficial in the "loading phase" (max. 7), when there is actually enough "room" for the creatine to be "stored" within the cell.

Creatine storage - how does that work after all?

A pros pos storage, it's actually quite telling that we know much more about what happens to the creatine molecules within the cell, than about how they actually get there. If you are interested in how scientists initially believed that phosphocreatine (PCr) "would represent the long sought-for 'immediate' source of energy for muscle contraction" I suggest you read Chapter one of the aforementioned compendium Creatine and Creatine Kinase in Health and Disease (ed. Salomons. 2008). For our purposes here it is most important to know that the capacity of our organs (skeletal muscle, kidney and possibly other tissues) is limited and creatine clearance (remember, this includes both the uptake by muscle tissue, as well the urinary clearance by the kidneys) decreases when muscular creatine stores increase (cf. figure 1).
Figure 1: Serum creatine levels (in µM) upon administration of identical doses of creatine at the beginning (first dose) and in the course (steady state) of creatine supplementation (based on McCall. 2008)
This is taken into account with the standard dosing regime, which - after an initial loading phase - uses smaller doses over time. McCall and Persky, explain this as follows:
[...] during early doses (i.e., doses within the first one to three days) when clearance is high, doses of 10 to 15 g per day will give blood concentrations greater than the Km [this is the creatine level in the blood, where creatine transport into the cell maxes out] for the creatine transporter. As the muscle becomes saturated and clearance decreases, it may be necessary to ingest 3 to 5 g of creatine a day to maintain similar blood concentrations.
The higher serum creatine levels upon steady state supplementation you can see in the data in figure 1 clearly substantiate this assumption. Together with the previously mentioned inverse relation of serum creatine to urinary creatine loss, it should also be obvious that taking "loading doses" of more than 10g per day for an extended period of time will at best fill the muscular creatine stores of the rats and cockroaches in the sewer (in case they happen live right next to your sewer pipe ;-)

What controls the muscular creatine transporter?

In order to understand the fundamental biochemical underpinnings of this interplay of dietary, serum and intra-muscular creatine, we do yet still have to identify the pathway by which the creatine molecules eventually get into the muscle. According to the most fundamental (and essentially oversimplified) cell model, a cell is a three-dimensional entity that is surrounded by a protective wall - the cell wall. This wall, of which most of you will have heard that it consists of phospholipids (note the word "lipid" indicates that fats! not proteins are the fundamental building blocks of the cell membrane), has the fascinating characteristic of being selectively permeable. Under physiological conditions transporter proteins function as "gate-keepers" and "taxi-drivers". They select and pick up specific molecules from the bloodstream and carry them across the "border" and into the cell (cf. illustration 2).
Illustration 2: A transporter like the creatine transporter is an active gatekeeper within the cell membrane.
One of the best-known and most-studied group of these transporters is the solute carrier family 6, which play an important role in neurotransmitter regulation in the brain. In the early and late 1990s the gamma-aminobutryic acid (GABA) and norepinephrine transporters were among the first of these Na+/Cl- dependent neurotransmitter transporters to be discovered. It is due to their dependence on the electrical potential between negative Cl- and positive Na+ molecules that they have also become known as neurotransmitter:sodium symporters (NSS, Saier. 1999). They are functionally identical to the likewise Na+-dependent amino acid carriers for taurine, betaine and creatine.

Image 2: β-Guanidinopropionate
competes with creatine for transpor-
tation across the cell membrane
Contrary to many other carriers, the creatine transporter (CT) is yet highly specific for creatine. Among the few exceptions which compete with creatine transport across the cell membrane is β-Guanidinopropionate. Those of you who follow my advice and scrutinize the nutritional information on the labels of their supplements, may be rubbing their eyes in disbelief, now, because Guanidino Propionic Acid or β-GPA is one of the standard ingredients in many pre-workout products (cf. Supplement Shootout, NO-Xplode). The reason for that probably (I would have to ask the producers, though ;-) is its hypoglycemic effect (Meglasson. 1993), which will probably remind you of Athletic Edge's Russian tarragon (see above) or of a 2009 study Rocic et al. which found remarkably similar effects for creatine, itself (Rocic. 2009).

So after all creatine and β-GPA share the same transporter and artemisia dracunculus (Russian tarragon, RT), creatine and β-GPA share the same beneficial effect on muscular insulin sensitivity. Now, Jäger et al. suggest that by increasing insulin sensitivity their RT extract would increase muscular creatine uptake. While this does seem to make sense, the results of Rocic et al. who found creatine to be equally effective as metformin in reducing blood glucose levels would suggest that creatine administration alone should increase it's own uptake ;-) This formally logical, but not very realistic conclusion is yet undermined by the established effect of guanidino propionic acid, which despite identical effects on insulin sensitivity, decreased creatine uptake by muscle cells by 82% (Willot. 1999) in vitro!
Image 3: Of sugar and salt, the "worst enemies" of many dieting body builders and figure athletes, salt and not sugar (or insulin) turns out to be creatine's most eager supporter on its way across the cell membrane (img. squidoo.com)
In the context of insulin sensitivity, it is interesting to note that Willot also tested the hypothesis that insulin would increase creatine uptake into the cell and found that "insulin had no effect on 14C-labeled creatine uptake at concentrations and under conditions in which effects are seen on glucose uptake glycogen synthesis and glycolysis." This finding does not essentialy contradict previous (Green. 1996), as well as very recent findings (Pittas. 2010), which support the idea of increased creatine retention upon coadministration of insulinogenic nutrients such as carbohydrates and/or protein , because "those may be owing to the expression of the creatine transporter, as opposed to acute effects on the transporter" (Willot. 1999). While it should be mentioned that a previous study by Oodom et al. found a 2x increase in creatine accumulation (again, not uptake! Oodom. 1996) after incubation with 3nM/ml insulin for 48h. The latter lacks real world significance, since even after high-carb meals blood insulin levels do hardly get up to 0.3-0.4pM/ml!.

In view of the fact that a -82% decrease in the Na+ concentration of the incubation medium reduced the creatine uptake by 77%, the addition of sodium to your creatine drink may be of greater importance than the fattening loads of simple carbs, anyway.
A 2003 study by Brault et al. confirms Willot's findings on the effect of guanidino propionic acid on creatine influx and retention into skeletal muscle. In the course of seven weeks on a β-GPA-enriched chow the muscular creatine levels of Brault's laboratory animals dropped by -85% (Brault. 2003). Notwithstanding, the flip side of this apparently undesirable effect of β-GPA are increased insulin sensitivity and, more importantly, at least in this context, profoundly augmented creatine uptake.
Figure 2: Effect of 7 weeks of β-GPA supplementation followed by 3 weeks of creatine supplementation on creatine and β-GPA content of the white gastrocnemius muscle in rats; data expressed relative to maximal concentrations (40µmol/g) of the two molecules (data calculated based on Brault. 2003).
Yet while the β-GPA induced creatine depletion increased creatine uptake in the subsequent supplementation phase (week 7+) by +24% and +33% in the soleus and the red gastrocnemius, respectively, creatine uptake in the glycolytic white muscle fibers of the gastrocnemius stayed constant. On the other hand, the white fibers of the gastrocnemius showed the expected decrease (-45%) in creatine uptake, when creatine was supplemented for 7 weeks at 0.85g/kg/day (~11g for 80kg human being) without prior β-GPA-induced creatine depletion (Brault. 2003a).
Figure 3: Creatine uptake (y-axis, in nmol/h/g) as a function of intramuscular creatine content (x-axis, in µmol/g) as measured by Brault. 2003.
These observation go challenge the previously formulated hypothesis that muscular creatine uptake via creatine transporter would always be linearly dependent on intra-muscular creatine stores. While this seems to be the case for the red, oxidative muscle fibers (violet regression in figure 3), the fast-twitch white glycolytic fibers appear to react assimilate creatine at a constant rate (green regression in figure 3) up to a certain threshold (in Brault's rat study that was ~17µmol/g, which is about +30% more than the maximal creatine content measured in red fibers in the same study), at the creatine uptake suddenly drops (cf. figure 3). What is even more confusing, though is that the insignificant changes in the creatine transporter protein expression measured by the scientists reflect neither the linear decrease nor the constant uptake rates. As Brault et al. point out "it is presently unclear what process may modulate Cr uptake" with high / low intra-muscular creatine levels. Possible mechanisms, according to Brault are...
  • with increasing intracellular creatine levels the Na+ gradient, which is necessary to drive the creatine into the cell, could become insufficient (unlikely)
  • with more creatine in the cell the release process that takes place once the creatine transporter enters the cell may slow down, as if the "taxi driver" would not find a parking lot 
  • the number of creatine transporters in the sarcolemmal membrane could be modulated according to intracellular creatine content in a similar manner as the expression of GLUT-4 is modulated by exercise (Goodyear. 1998)
  • high intramuscular creatine levels could lead to posttranslational modification of the creatine transporter, similar to what we see in its "relatives", the GABA/taurine transporters, whose activity
    is modified by protein phosphorylation
In fact, a 2002 study by Wang et al. (Wang. 2002) found an increase in creatine transporter phosphorylation that correlated with a reduction in creatine uptake and Zhao et al. observed a 38% increase in creatine uptake in response to a 30% reduction in serine phosphorylation of the CrT (Zhao. 2002). While it is thus most likely that posttranslational modification, something you probably have encountered in one of my blogpost related to the Akt/mTOR cascade, before is the underlying mechanism that controls how effective our "creatine shuttle" works, the unfortunate truth is that this does not go to tell us how we could possibly influence this process.

Conclusion - little do we know about the actual process of creatine uptake

If you look back at what you may or may not have learned from the second part of this write-up, you may notice that I have artistically evaded a direct response to Learner's question whether "creatine transporters behave the same as glucose transporters". Nevertheless, you should have been able to read between the lines that ...
  • despite studies showing increased creatine retention (not celullar uptake or creatine transporter protein expression) upon co-administration of insulinogenic nutrients (carbohydrates in Green. 1996 and carbohydrates + protein in Pittas. 2010), in-vitro studies have shown that insulin has no direct effect on muscular creatine uptake (Willot. 1999) - unless supraphysiological doses are used
  • at least in red oxidative muscle fibers, there is an inverse linear relationship between intra-muscular creatine levels and creatine uptake (Brault. 2003)
  • increases and decreases in creatine uptake are not mediated by respective increases in creatine transporter protein expression (Brault. 2003)
  • the most likely hypothesis explaining how intra-cellular creatine levels control the "effectivity" of the creatine transporter is via posttranslational modification, of which we do not yet know for sure how to influence it (the fact that tarragon and other insulin-sensitizers appear to increase creatine uptake could as well be related to changes in the phosphorylation of the creatine transporter as their insulin-sensitizing effects could be related to dephosphorylation of )
It would yet be unfair to leave you with all those additional gaps in your under understanding of the pharmacokinetics of creatine without a few words on the most important aspect of creatine supplementation, i.e. what works in practice.
Image 4: If its not the insulin, then
maybe a steadier influx of creatine
into the blood which can explain the
increased creatine retention upon co-
administration of carbohydrates.
A final note on the issue of carbohydrates: In view of what I have stated in the first installment of this series, i.e. the increase in gastric emptying time due to carbohydrate (and other foodstuff), an alternative explanation for the increase in creatine retention (again, not uptake ;-) upon co-administration of carbohydrates or carbohydrates + protein could be the steadier incline in plasma creatine levels. While the 1996 study by Green lacks the respective data, the figures in Pittas (2010) clearly show that creatine clearance in the creatine-only group increases dramatically after the initial spike in serum creatine levels 30min after the administration of 5g creatine. In view of the negative results of Willot's in-vitro studies on the effects of physiological levels of insulin on creatine uptake and the fact that renal creatine clearance increases with serum creatine levels, while the muscular uptake is maxes out at a relatively low threshold (10-100µM) is surpassed, it is at least possible that it is the steady influx of creatine into the bloodstream and not the insulinogenic effects of carbohydrates that facilitates creatine retention (I hope you remember from the first part of this series that the reduction in creatine influx into the blood due to degradation in the stomach is probably negligible, as long as the dose is large enough to reach blood levels beyond the Km value of 10-100µm)
As I have already hinted at in part I of this installment of "Ask Dr. Andro", for most of us, it does not really matter whether it takes 3, 5 or 10 days until the creatine stores in our muscles are saturated. Moreover, even high quality creatine monohydrate is so "dirt cheap" that you do not really have to care about potential losses (in the 0.3-0.6mg/day range) due to caffeine supplementation or potentially sub-optimal creatine retention in the absence of large boluses of fattening carbohydrates. Personally, I would just stick to what has been working for generations of trainees, now: plain creatine monohydrate taken at a dose of 10-15g/day for 3-5 days followed by a maintenance dose of 3-5g/day.