Alternative Methodology
For the reason that the determined insulin threshold is low enough to be reached via non-pharmacological methods, oral ingestion of glucose may be used.
For the reason that the carnitine turnover rate in skeletal muscle is low (190 +/- 20 hours) daily carnitine increases will result in a continued build up of total muscle carnitine.
This allows the use of low bioavailable methods such as daily ingestion of oral carnitine w/ glucose load to increase carnitine in muscle at an incremental rate such that within 100 days carnitine content will have increased by 10%.
blunt PDC activity (carbohydrate oxidation)
reduce muscle lactate content
increase glycogen content in muscle (i.e. reduce oxidation of glucose in favor of storage)
reduce muscle glycolysis
increase fat oxidation
Results: This amount of carnitine is sufficient to:
How do you increase carnitine in muscle?
Studies have consistently failed to increase skeletal muscle carnitine content either through oral supplementation or intravenous L-carnitine administration. Watcher et al (2002) fed 2 grams of L-carnitine twice a day for 3 months to normal people and failed. Similar studies by Barnet et al. (1994) and Vulkovich et al. (1994) demonstrated similar failures with oral feedings of l-carnitine for 3 months.
Intravenous infusion of L-carnitine for up to 5 hours similarly failed to have any effect on muscular carnitine content (Brass et al. (1994); (Stephens Group, Insulin stimulates… (2006)).
How carnitine is normally transported into the cell
The reason for these failures is very simple. Normal people have no deficiency in circulating plasma levels of carnitine. What they have is a fully saturated transport mechanism. No amount of carnitine load is sufficient without a concurrent increase in the ability of the transport mechanism to transport carnitine across the cellular membrane.
The cellular membrane is a lipid bilayer easily permeable to water molecules and a few other small, uncharged, molecules such as oxygen and carbon dioxide but little else. The cellular membrane is not permeable to ions such as K+, Na+.
In the normal course of things molecules and ions move about spontaneously down what is known as their concentration gradient (i.e., from a region of higher to a region of lower concentration) by diffusion.
Molecules and ions are capable of moving against their concentration gradient, but this process requires a process known as active transport.
It is the active transport that is lacking in regard to carnitine movement and unless this is changed additional carnitine will not be allowed to enter the cell.
Active transport is the pumping of molecules or ions through a membrane against their concentration gradient. It requires: a transmembrane protein (usually a complex of them) called a transporter and energy. The source of this energy is ATP.
The transmembrane protein responsible for carnitine transport into skeletal muscle is OCTN2. The half-saturation concentration of L-carnitine uptake by OCTN2 is 4.34 umols (Tamai et al. (1998)). In the normal state skeletal muscle carnitine uptake is saturated since plasma total carnitine concentration is 50 umols.
OCTN2 has a high affinity for carnitine and sodium ions (Na+) and readily binds to both and so carnitine is transported into skeletal muscle against a substantial concentration gradient via a transport process involving sodium Na+ flow. In essence carnitine hitches a ride on OCTN2 which hitches a ride on Na+.
A detailed description of this process is beyond the scope of this article so a general reduction will suffice. One method of direct active transport across the cellular membrane is the Na+/K+ ATPase pump.
The concentration of potassium ions (K+) is as much as 20 times higher inside the cell then outside. Conversely, the fluid outside the cell contains a concentration of sodium ions (Na+) as much as 10 times greater than that within the cell. Because of this difference a concentration gradient amenable to flow exists and the Na+/K+ ATPase pump effects the transfer of these two ions pushing out 3 Na+ ions for every 2 K+ ions pumped back into the cell. This activity establishes a net charge across the plasma membrane with the interior of the cell being negatively charged with respect to the exterior.
So with this basic understanding that OCTN2 is a cotransporter of sodium & carnitine and that under normal conditions it is fully saturated and thus unable to benefit further carnitine inflow via Na+/K+ ATPase pump activity, lets examine how insulin overcomes this equilibrium and brings about an increased inflow of carnitine into skeletal muscle.
How insulin increases the flow of carnitine into muscle
The Na+ dependent, active transport of carnitine into human skeletal muscle is mediated via a high-affinity, transporter OCTN2.
The Stephens Group found that the combination of increased carnitine and increased insulin (above a threshold) increased skeletal muscle OCTN2 mRNA expression by 2.3 fold (Stephens Group, Insulin stimulates… (2006)) in addition to increasing the activity of Na+/K+ ATPase pump. This results in an increased availability of transporter which potentially increases the amount of carnitine that may be carried into the muscle cell.
The action of insulin however is most important in changing the membrane permeability in favor of carnitine inflow.
It has been demonstrated that the Na+ dependent uptake of other nutrients into skeletal muscle is increased by insulin, for example amino acids (Zorzano et al. 2000) and creatine (Green et al. 1996; Steenge et al. 1998).
Insulin is able to increase the flow of carnitine into skeletal muscle as follows.
Insulin increases Na+/K+ ATPase pump activity by increasing translocation (or movement) of alpha2 and beta1 pump subunits from an intracellular storage site to the plasma membrane (Sweeney & Klip, 1998), and through an increase in the sensitivity of the Na+/K+ ATPase pump to intracellular Na+ (Clausen, 1986, 2003; Ewart & Klip, 1995).
OCTN2 has equal affinity for sodium ion (Na+) and carnitine and bonds to both. With an increased Na+/K+ ATPase pump activity brought about by an increase in circulating insulin concentration intracellular Na+ concentration is lowered which increases the electrochemical gradient for Na+ and therefore increases Na+/carnitine cotransport.
This results in an increase of carnitine inside the muscle cell.
Methodology and results
The Stephens Group undertook a series of experiments building on each to create an overall understanding. In Insulin stimulates L-carnitine accumulation in human skeletal muscle, they were able to increase muscle total carnitine content by 13%. They achieved this by using what I will call an “overkill amount of L-carnitine” administered by infusion. They administered a 15mg/kg bolus w/in the 1st 10 minutes rapidly achieving a supraphysiological plasma concentration of about 500 umol/L. This was followed by 10mg/kg infused over the next 290 minutes to maintain hypercarnitinemia.
In addition they infused insulin at a dose I will call an “overkill amount”. The aim of study was to determine whether insulin could increase Na+/dependent skeletal muscle carnitine up-take in healthy human subjects as a result of increasing Na+/K+ ATPase pump activity. They were very much successful. The positive results of the study are incorporated in the previous section.
Using an identical protocol and intravenous L-carnitine & insulin amounts they undertook another study reported in An Acute Increase in Skeletal Muscle Carnitine Content Alters Fuel Metabolism in Resting Human Skeletal Muscle with the broader aim of determining the effect that an increase in skeletal muscle carnitine content would have on the integration of muscle fat and carbohydrate oxidation during and after hyper-insulinemia.
As in the previous study total carnitine content increased in skeletal muscle, this time by 15%.
This resulted in a 30% decrease in muscle PDC activity (carbohydrate metabolism) and a 40% decrease in muscle lactate content. After an overnight fast, muscle glycogen and LCA-CoA (long-chain acyl-CoA) content had increased by 30% and 40% respectively, in the carnitine group compared with control. The difference between the control and carnitine visits was not attributable to a difference in the amount of carbohydrate administered.
“Taken together, these findings lead us to conclude that the increase in muscle carnitine content observed in the present study inhibited glycolytic flux (decrease in lactate) and carbohydrate oxidation at the level of the PDC, thereby diverting muscle glucose uptake toward glycogen storage (nonoxidative glucose disposal).”
“The reciprocal relationship between carbohydrate and fat oxidation in skeletal muscle would suggest that the apparent decrease in carbohydrate flux observed was the result of, or resulted in, an increase in fat oxidation. Thus, these findings could be of major importance in the treatment of insulin-resistant states, such as obesity and type 2 diabetes, because both conditions are associated with an impaired ability of skeletal muscle to oxidize fatty acids, both at rest and during exercise. Furthermore, reducing or preventing intramuscular lipid accumulation increases insulin sensitivity.”
The implications of these results should be clear and having read the previous portions of this article and examined the figures, self-explanatory. To reiterate the decrease in PDC activity indicates a substantial drop in carbohydrate metabolism, while the decrease in muscle lactate indicates a decrease in glycolysis activity. The increase in muscle glycogen storage given the constants of the study indicate that glucose was preferentially stored not metabolized. This also indicates that existing muscle glycogen stores where enhanced rather then drawn upon.
The reciprocal relationship between carbohydrate oxidation and fat oxidation indicates that fuel sources utilized for energy where fats.
The meaning of the one item that may not be readily apparent is that of LCA-CoA (long-chain acyl-CoA) increasing overnight. Remember from the early discussion in this article that carnitine transports long-chain acyl groups from fatty acids into the mitochondria where they are broken down through beta-oxidation. The fact that these groups had increased strongly indicated that carnitine is increasing its activity as a transporter in fatty acid oxidation and that fatty acid oxidation is increased.
In the words of the Stephens Group in an overall review of their work:
“…the apparent reduction in glycolytic flux and carbohydrate oxidation… (decreased PDC activity and lactate content, and increased glycogen accumulation), in the face of high carbohydrate availability, could have been caused by a carnitine-mediated increase in skeletal muscle long-chain fatty acid oxidation, i.e. an increase in long-chain acyl-CoA translocation into the mitochondrial matrix via CPT1, resulting in an increase in beta-oxidation.
According to Randle’s glucose–fatty acid cycle (Randle et al. 1963, 1964; Garland et al. 1963; Garland & Randle, 1963), a concept proposed in the 1960s from experiments involving rat heart and diaphragm muscle, an increase in beta-oxidation would result in an elevation of muscle acetyl-CoA concentration and, consequently, an increase in muscle citrate and glucose-6-phosphate content. This, in turn, would result in the down-regulation of carbohydrate flux, due to product inhibition of PDC….
Indeed, the decrease in PDC activity observed in our study was paralleled by a reduction in muscle lactate content and resulted in an accumulation of muscle glycogen overnight, conditions which are both consistent with the premise that glycolytic flux, and therefore carbohydrate oxidation, was inhibited. In support of this… muscle long-chain acyl-CoA content returned to basal overnight during the l-carnitine infusion visit (whereas it remained suppressed during the control visit), which suggests that beta-oxidation was indeed increased.”
Alternative Methodology
Obtaining a lower insulin threshold
In an attempt to discover the lowest amount of insulin needed to drive carnitine into muscle and activate the switch from carbohydrate oxidation to fatty acid oxidation, the Stephens Group undertook a study the reports of which are discussed in A threshold exists for the stimulatory effect of insulin on plasma L-carnitine clearance in humans.
They reasoned that while their previous studies with insulin infusion in an amount in the upper physiological range were successful, it would be difficult to achieve by dietary means alone.
They discovered that administered insulin will not stimulate muscle carnitine retention unless a serum insulin concentration greater than 90 mU/l is achieved during hypercarnitinemia. This level was substantially lower (and obtainable via dietary means) then the previous high concentrations used and stimulated muscle carnitine transport to a similar degree.
Extrapolating from data, skeletal muscle total carnitine content in this study with this threshold insulin amount would have been increased by about 10%.
Orally ingesting lower bioavailable L-carnitine together with high glycemic index carbohydrates
The Stephens Group in a study the results of which are reported in Carbohydrate ingestion augments L-carnitine retention in humans, investigated whether physiologically significant increases in skeletal muscle carnitine content can be achieved through the use of L-carnitine feeding in conjunction with a dietary-induced elevation in circulating insulin.
They examined serum insulin levels achieved from glucose ingestion, the plasma total carnitine level and the urinary total carnitine excretion levels in order to determine the amount of carnitine taken up in muscle by performing both a one day study and a 14 day study.
Both studies used oral ingestion of:
4.5 g L-carnitine L-tartrate (3 g L-carnitine) dissolved in 200 ml of water
followed by
94 g of simple sugars (CHO) either ingested twice at 1 hour & 4 hours after L-carnitine ingestion as in the 14 day study or as in the one day study four time across a 5 hour period.
Serum insulin concentrations during the period when simple sugars were ingested are graphed below. Surprisingly peak serum insulin concentrations of about 70mU/l proved to be sufficient.
The graph below indicates that the rise in insulin eliminated carnitine from plasma. The control subjects had more carnitine in plasma then those on the protocol. See below.
If the carnitine is not in plasma is it excreted? The graph below indicates that urinary excretion rates were lower over the measured 14 days in those following the protocol. See below.
“We suggest, therefore, that the lowering of plasma total carnitine (TC) concentration occurring immediately following CHO ingestion, and the lower urinary TC excretion during the CHO visit, collectively indicate that an increase in whole body carnitine retention occurred when L-carnitine feeding was accompanied by CHO ingestion. Given that skeletal muscle is the major site of carnitine storage within the body, and that maintaining hypercarnitinemia for 5h in the presence of hyperinsulinemia increases skeletal muscle TC accumulation (other Stephens Group studies), it is not unreasonable to suggest that this greater retention occurred mainly in this tissue.”
Extrapolating an accumulation strategy
Given that the increase in muscle carnitine content following a single dose, or 2 weeks, of L-carnitine feeding in the presence of elevated circulating insulin is likely to be small due to the poor bioavailability of orally administered L-carnitine (less then 20%), muscle carnitine accumulation was estimated indirectly from measurements of plasma and urinary carnitine concentration.
In this study 3 grams of carnitine results in at most 560 mg of absorbable plasma carnitine.
“Assuming all absorbed carnitine was either taken up into skeletal muscle tissue or excreted in the urine, it can be calculated that L-carnitine feeding in conjunction with CHO ingestion would have increased skeletal muscle total carnitine concentration by a further 0.1% (i.e., 60 mg) compared with L-carnitine ingestion alone.”
In fact “urinary total carnitine excretion was on average 70 mg/day lower in the CHO group over the 14 days of study. Consequently, if maintaining a daily L-carnitine feeding regime with CHO has an additive effect on muscle carnitine content, L-carnitine feeding for 100 days could increase muscle carnitine content by an additional 10%, which we believe could have a significant metabolic impact in contracting skeletal muscle.”
In the other comprehensive Stephens Group study they found that muscle total carnitine content was not reduced 24 h after a 15% increase, suggesting that a daily increase in muscle carnitine content can be maintained. In addition release of carnitine from skeletal muscle is a slow process, with skeletal muscle carnitine turnover time of 190 +/- 20 hours (Rebouche (1984)).
“Taken together with the maintained effect on whole body total carnitine retention observed in the 14 day study, these findings would suggest that daily L-carnitine and carbohydrate administration could well have an additive effect on skeletal muscle total carnitine accumulation. Importantly, if L-carnitine supplementation is to be used as a tool to modify skeletal muscle energy metabolism, the findings in the 14 day study also suggest that, at most, only two 500-ml CHO drinks (2 x 94 g CHO) are required to achieve the effect on L-carnitine retention.”
In conclusion:
“…muscle free carnitine availability becomes limiting to carnitine palmitoyltransferase I (CPT1) at a concentration of about 6 mmol/kg dry muscle….
Thus, assuming the average 70 mg/day retention in the present studies resided within skeletal muscle and that daily L-carnitine/carbohydrate feeding for 100 days would have an additive effect, then muscle carnitine content would increase by about 2 mmol/kg dry muscle, which could alleviate the decline in fat oxidation rates routinely observed at exercise intensities above 70% VO2 max, which could be of major relevance to exercise performance due to the sparing of muscle glycogen.
In line with this theory, increasing skeletal muscle carnitine availability has been reported to delay fatigue development by 25% in rat soleus muscle strips in vitro (Brass (1993)).”
Further more it is worth reiterating that the Stephens Group has demonstrated in the study involving intravenous L-carnitine administration that a 15% increase in skeletal muscle carnitine content, achieved during hyperinsulinemia, resulted in a 30% decrease in muscle PDC activity and 40% decrease in muscle lactate content compared with control. Furthermore, following an overnight fast, muscle glycogen and long-chain acyl-CoA content was 30% and 40% greater than control, respectively, despite carbohydrate administration over the previous 24 hours being exactly the same.
This is the first study to demonstrate that the retention of orally supplemented L-carnitine can be increased if accompanied by carbohydrate ingestion and that this retention is likely to reside in skeletal muscle, because insulin is known to stimulate muscle total carnitine accumulation. “These findings could have a significant effect on the integration of fat and carbohydrate oxidation in contracting skeletal muscle.”
Final Note
An immediate threshold amount of increase in muscle carnitine concentration can be had with administration of highly bioavailable Synthetine™ (sterile L-Carnitine) with insulin or oral ingestion of two high glycemic index drinks such as SyntheDEXTRIN™ (Maltodextrin Pure Carbohydrate).
An accumulation strategy of daily oral ingestion of low bioavailable l-carnitine with oral ingestion of two high glycemic index drinks such as SyntheDEXTRIN™ (Maltodextrin Pure Carbohydrate) will lead to a threshold amount of muscle carnitine concentration within 100 days.
These strategies should enable reversing the switch – Turning up the rate of fat oxidation & turning down the rate of carbohydrate oxidation.