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Researched
and Composed by
Jacob Wilson, BSc. (Hons), MSc. CSCS
Address correspondence to:
jwilson@abcbodybuilding.com
Journal of HYPERPLASIA
Research 6(3):
Published August 3, 2006
Abstract
Leucine is known for its capacity to stimulate insulin release. In turn a
number of investigators have sought to analyze how much of leucines effects are
mediated by insulin. The purpose of the following paper is to analyze the role
that insulin has in modulating leucine’s effects on protein synthesis and most
importantly how these effects can be manipulated for optimizing protein
balance.
Introduction
In part
one of this series, leucine’s general role in protein balance was discussed. In
particular it was found that this Branched Chain Amino Acid (BCAA) appeared to
be the primary regulator of protein synthesis. It has long been known that
leucine has insulinogenic properties (stimulates insulin secretion). Currently
the three major components of protein balance studied include an analysis of
leucine, insulin, and exercise. The following paper will discuss the role that
insulin has in modulating leucine’s effects on protein synthesis and most
importantly how these effects can be manipulated for optimizing protein
balance.
Leucine and Insulin
Secretion
Studies
from Anthony and colleagues (2000a, 2000b) have found that leucine administered
alone to fasting rats can stimulate protein synthesis and the major machinery
responsible for protein synthesis (e.g. initiation factors and ribosomal protein
6, see article 1 for an explanation) absent of other macronutrients (e.g.
carbohydrates & fats). Further Anthony et al. found that the administration of
leucine in both studies did not elicit an increase in insulin secretion. While
these findings suggest that leucine enhances protein synthesis independent of
insulin, the situation is a bit more complicated than it appears. This is due
to the fact that other studies have reported that leucine administration can
stimulate insulin secretion (Greiwe et al., 2001; Koopman et al., 2005).
Anthony et al (2002) therefore suggested that leucine may enhance insulin
secretion early and only transiently after administration , which leads to
enhanced protein synthesis. An alternative role is that lower levels of insulin
may play a permissive role in leucine’s effects.
To
further analyze the role of insulin in modulating leucine’s effects, Anthony et
al. (2002) administered leucine to food deprived rats along with somatostatin,
which inhibits the secretion of insulin from the pancreas. Without somatostatin
results indicated that leucine transiently increased insulin secretion between
15 and 45 min, followed by a return to basal levels by 60 minutes. During this
process all of the markers of enhanced protein synthesis were increased (e.g
initiation factors, and S6). When somatostatin was administered leucine’s
effects on ribosomal protein S6 (the protein responsible for enhancing the
protein synthetic capacity of the cell) was completely inhibited. Further
leucine’s effects on enhancing initiation factor assembly was partly, but not
fully inhibited. A number of studies have further investigated the role of
insulin needed to maximally facilitate leucine’s effects on protein synthesis.
Administration of insulin antibodies, which cause a rapid fall in plasma insulin
either partly or fully inhibits leucine’s effects on protein synthesis (Preedy,
1986; Svanberg et al., 1996), while Diazoxide administration, which blocks
insulin can lower protein synthesis to values below fasting despite being fed a
complete meal (Balage, 2001). These results suggest that leucine has both
insulin dependent and independent effects on protein synthesis.
The amount of Insulin
Needed to Maximally Stimulate Protein Synthesis
Perhaps
the most relevant studies for the amount of insulin needed to maximally
stimulate protein synthesis are found in studies analyzing leucine
administration in diabetics. Studies indicate that in mildly diabetic
conditions, when insulin is only slightly lower then controls that leucine and
other anabolic stimuli are still able to maximally stimulate protein synthesis
(Kimball et al., 2002). However, in severely diabetic rats, when insulin is
extremely low protein synthesis is attenuated, and even lowered (Kimball et al.,
2002).
An interesting and classic study was performed by Fedele et al. (2000) to
investigate the amount of insulin needed to promote the full benefits of
exercise’s stimulatory effects on protein synthesis. While this is slightly
different than leucine administration, both appear to operate by up regulating
the mTOR system. The results are displayed in figure 1.
Figure 1. Plasma insulin levels and their effect on protein synthesi.
As can
be seen protein synthesis is maximally stimulated at low plasma insulin levels
which correspond to fasting conditions.
In
summary results from diabetic models suggest that insulin plays either a direct
or passive role in maximizing the effects of both leucine and exercise on
protein synthesis. However this effect is maximally stimulated at very low
physiological concentrations corresponding to fasting levels of insulin.
Insulin’s capacity to
stimulate protein synthesis independently of Leucine and Other Amino Acids
Insulin’s capacity to stimulate protein synthesis has been a confusing problem
for some time. Wolfe (2000) summarizes by stating that
“In vitro studies document a stimulatory effect of insulin
on muscle protein synthesis, but in vivo results are conflicting. Everything
from decreased muscle protein synthesis to increased muscle protein synthesis in
response to insulin has been reported. A recent publication suggests that the
response of muscle protein synthesis to insulin is dose dependent, and that only
supraphysiological dose of insulin stimulate muscle protein synthesis. On the
other hand, some studies show a stimulatory effect of insulin in low doses.”
However
the situation may be able to be resolved when amino acid availability is
considered (Wolfe, 2000). Evidence suggests that when amino acid levels are
maintained that insulin infusion can increase protein synthesis. An efficient
techniques to measure this is by infusing insulin systemically (e.g. to the
whole body) or locally in the arm region for example. Systemic increases in
insulin without amino acids lower plasma amino acid concentrations, while local
infusion does not (amino acid concentrations are maintained).
In an intriguing study Bell and colleagues (2005) infused glucose, lipids, and
insulin in a high energy (162 kcals per hour) condition systemically to a group
of 6 participants. They then had a low energy condition (35 kcals per hour), in
which insulin was infused locally to the thigh region (specifically to the
femoral artery). Results found that the high energy condition lowered protein
breakdown, but did not affect protein synthesis. In contrast the low energy
condition had no change in protein breakdown, but increased protein synthesis.
Overall the high energy condition had a net negative protein balance, while the
low energy condition had a net positive protein balance (figure 2). This
suggests that the maintenance of amino acids is critical for insulin to be able
to exert any effects on protein synthesis, but it is not necessary for insulin
to decrease protein degradation (discussed further on in the article).
Figure
2. Protein synthesis in low and high energy conditions.
Insulin’s Mechanisms of
Action and Synergistic Effects with Leucine
Like
leucine, insulin’s actions appear to be mTOR dependent (see article 1).
Specifically it is thought that insulin binds to its receptor, which leads to
downstream events that activate Protein Kinase B. Protein Kinase B then
activates mTOR, leading to up regulation of initiation factors and ribosomal
protein S6 (see article 1).
In an interesting study Greiwe et al. (2001) infused insulin alone, leucine
alone, or insulin + leucine to human participants. The investigators then
measured 70-kDa ribosomal protein S6 kinase (p70S6k)
p70S6k, is the protein kinase that mTOR stimulates, which
activates ribosomal protein S6. So as not to complicate the situation however,
I should again stress that measures of either p70S6k or ribosomal
protein S6 are really measures of the cells capacity to enhance protein
synthesis. Results can be found in figure 3.
Figure
3. Level of phosphprylated p70S6k
As can
be seen, leucine alone increased the phosphorylation (a measure of activation)
of the protein kinase 4 fold, while insulin increased it 8 fold. If their
effects were additive then we would expect a 12 fold increase in the combined
condition. However, instead an 18 fold increase was found. This means that
their combined effects were greater than their effects alone.
There are a number of explanations which can be derived
from these results. First the investigators analyzed to see if insulin and
leucine operated through the activation of protein kinase B. Results found that
only insulin stimulated protein kinase B, suggesting that they operate through
independent mechanisms. Secondly plasma insulin concentrations were measured in
each condition. Here results indicated that leucine alone did not stimulate
insulin, while leucine combined with insulin raised insulin in the plasma by 20
%. However it was found that the increase in plasma insulin did not further
increase activation of protein kinase B, meaning that the rise in insulin may
not explain the synergistic effects seen.
However
the synergistic effects seen may be related to insulin’s capacity to increase
blood flow and amino acid availability to the musculature. This was supported
by a 2006 study by Volpi and colleagues. These investigators used the local
infusion technique of insulin. They postulated that insulin could not stimulate
protein synthesis unless it increased amino acid delivery and thus availability
to muscle tissue. They had low, intermediate, and high insulin conditions.
They found no increases in amino acid delivery in muscle tissue in the low
condition. However blood flow and amino acid delivery increased in the moderate
condition as well as protein synthesis. In the high condition amino acid
concentration began to lower and protein synthesis was not as pronounced.
Finally correlations between changes in blood flow (r = 0.79) and amino acid
delivery (r = 0.80) and changes in protein synthesis were both high, supporting
the authors hypothesis.
Returning to the Greiwe et al. (2001) study, it is important to emphasize that
leucine administration alone did not increase plasma insulin levels, yet when
combined with insulin leucine did increase plasma insulin concentrations. One
possible explanation is that leucine may have inhibited the degradation of
insulin. Because degradation (specifically receptor mediated degradation) is
the primary mechanism to clear insulin, leucine’s anticatabolic effects may have
enhanced insulin availability. It would be interesting to research the
possibility of HMBs capacity to enhance insulin levels.
Insulin decreases
protein degradation
While leucine appears to be the major dietary regulator of protein synthesis,
insulin appears to be the major regulator of protein degradation. Studies
indicate that insulins effect are dependent on blood flow to muscle tissue.
Briefly, muscle tissue receives blood slowly during rest relative to the liver
and kidneys, but during exercise this can increase up to 30 %. This increase in
blood flow has been demonstrated to be significantly correlated to enhancing
positive protein balance (Wilson & Wilson, 2006).
However recently the effects of insulin and other hormones have been analyzed in
their capacity to enhance blood flow to skeletal muscle tissue. Liu et al
(2006) found that insulin, IGF-1, and GH infused locally all significantly
increased blood flow to skeletal muscle tissue (figure 4). Further, along with
an increase in blood flow insulin and IGF-1 increased protein balance. What was
interesting is that they appeared to do so through different mechanisms.
Insulin did not increase protein synthesis, but greatly lowered protein
breakdown, while IGF-1 had opposite effects.
The
mechanism is that insulin is able to hinder proteolytic pathways through protein
kinase B (specifically the Ubiquitin pathway discussed in article 1).
The
investigators also found that administering insulin at increasing concentrations
(low, moderate, and high) increased protein balance from low to moderate, but
not from moderate to high.
Figure
4. Protein balance at low, moderate, and high levels of insulin and IGF-1.
The low,
moderate and hi conditions corresponded to increased insulin levels at ~20, 60,
and 110 µU/mL above fasting levels.
It is
interesting as a recent study conducted may further shed light on the insulin
protein balance relationship. The study was conducted by Koopman et al. (2005),
and they had participants consume three different beverages after exercise and
analyzed their effects on protein synthesis, degradation, and net protein
balance. The first beverage was comprised of a 0.3 grams of dextrose and
maltodextrin combination per kg of bodyweight (CHO condition). The second
beverage was the CHO combo plus 0.2 grams of whey protein per kg of bodyweight.
Finally in condition three participants consumed the CHO-PRO drink with 0.1
grams of leucine added per kg of bodyweight. For a 91 kg bodybuilder (200
pounds), this would correspond to a beverage of 27 grams of carbohydrate, 18.2
grams of whey, and 9.1 grams of leucine per hour.for approximately 330 minutes
following exercise. Results found that protein balance was negative in the CHO
condition, became positive in the CHO-PRO condition, and was greatest in the
CHO-PRO-LEU condition.
Figure
5.0 Net Protein Balance, Adapted from Koopman et al. (2005)
What was
interesting however is that these results paralleled increases in both leucine
and insulin concentrations. It was found that leucine was significantly
correlated to protein synthesis, while insulin was not. Further insulin was
inversely correlated to protein breakdown (more insulin less breakdown), while
leucine was not. Finally, when analyzing the insulin response in figure 6, it
can be seen that the greatest protein balance was found when insulin levels
peaked at around 60-80 µU/mL.
Figure
6. Protein balance as a function of insulin concentration. Adapted from
Koopman et al. (2005)
Further discussion as to the optimal dosage of insulin to enhance protein
balance is beyond the scope of this paper, but will be addressed in a future
series on insulin, and body composition (currently a work in progress for the
journal).
What it
does demonstrate however is the relationship between positive protein balance
and increasing insulin levels, along with the role that leucine plays in
regulating this response.
Summary and Conclusions
Leucine
appears to affect the capacity of a cell to increase protein synthesis in both
insulin independent and dependent mechanisms. However, in order for these
effects to be manifested via significant increases in protein synthesis, both
dependent and independent mechanisms must accompany each other.
Leucine and insulin’s effects appear to be independent of each other, and when
both insulin and leucine levels increase they have a synergistic effect on pKS6,
a marker of the cells capacity for protein synthesis. The mechanism appears to
be related to insulin’s capacity to increase blood flow and amino acid deliver
to skeletal muscle tissue.
While insulin’s effects on protein synthesis can occur at low levels, its
effects on protein degradation appear to increase in hyperinsulemic conditions
as indicated by post workout nutrition studies, which show a continual decrease
in protein degradation to increasing levels of insulin. Studies also indicate
that adding leucine or having a large dose of leucine in a protein containing
meal enhances the insulinogenic response to carbohydrates (for an in depth
review see Knowlden, 2003). This suggests that higher glycemic index
carbohydrates combined with leucine rich protein sources, or protein combined
with EAAs for caloric efficiency may be optimal for protein balance post
exercise.
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