Insulin Action and Its Disturbances in Disease - part 3 pptx

108 THE EFFECT OF INSULIN ON PROTEIN METABOLISM
mechanisms whereby insulin selectively affects mRNA stability have not been
well defined.
Initiation of mRNA translation into protein begins with formation of the 7-
methyl guanine cap at the 5-prime end of the RNA. A number of cap-associated
proteins including eukaryotic initiation factor 4E (eIF-4E), eIF-4G and phospho-
rylated heat–acid-stable protein (PHAS-1) are influenced by insulin. PHAS-1
binds to the eIF-4E cap binding protein, insulin enhances phosphorylation of
PHAS-1 and favours dissociation of eIF-4E and PHAS-1.
14
This allows for
binding of eIF-4E to eIF-4G and hence favours association with the 40S ribo-
somal subunit and translation initiation.
15, 16
Also important in the binding of
the 40S ribosomal subunit is eIF-2, and the binding of this initiation factor
is dependent on its association with GTP. Controlling the recycling of the
GTP/GDP-bound state of eIF-2 is eIF-2B. Insulin increases the activity of eIF-
2B and favours the GTP-bound (active) state of eIF-2, which in turn enhances
translation initiation.
16, 17
Protein elongation depends on the action of multiple elongation factors.
Among these are elongation factor 2 (eEF-2). This factor is important for move-
ment of the ribosomal complex along the mRNA and for the migration of the
amino acyl-tRNA from the acceptor site to the peptidyl site of the ribosome.
18
Insulin enhances eEF-2 activity by reducing its phosphorylation via inhibition
of its kinase.
19
A comprehensive description of the molecular mechanisms of
insulin’s effect on translation is available in review form.

20
The abundance of ribosomes and RNA content in part determines the cellu-
lar capacity to synthesize protein.
21
Ribosomes are made up of approximately
80 proteins and 4 ribosomal RNA (rRNA) species. Production and assembly
of ribosomes takes place in the nuclei. In chick embryo fibroblasts insulin
has been shown to induce a fourfold increase in the synthesis of ribosomal
proteins.
22
Similar findings have been made in mouse myoblasts.
23
This appears
to in part be due to post-transcriptional events. Messenger RNAs that encode
ribosomal proteins appear to be preferentially associated with polysomes in
mouse myoblasts treated with insulin.
23
The synthesis of rRNAs has been
shown to increase after insulin treatment in a variety of cell types including
fibroblasts,
22, 24
myoblasts
23
and hepatocytes.
25
Finally, insulin may also reduce
the rate of ribosome degradation.
25–27
Effect of insulin on intracellular events controlling protein breakdown
Cellular protein breakdown is a tightly controlled and highly specific process. In

catabolic states such as starvation, sepsis or insulin deprivation, protein break-
down can markedly increase. At the intracellular level, proteins can be degraded
through several pathways including the lysosomal pathway, the calcium-depen-
dent protease pathway or the ubiquitin–proteosome path.
28
The majority of
proteins in mammalian cells are degraded through the ubiquitin–proteosome
MOLECULAR MECHANISMS OF INSULIN’S EFFECT ON PROTEIN TURNOVER 109
pathway. Proteins are targeted for breakdown by covalent conjugation to ubiq-
uitin. This is an ATP-dependent process, and multiple ubiquitin molecules are
added such that a ubiquitin chain is formed.
29
Proteins with a ubiquitin chain
attached are degraded by the ATP-dependent 26S proteosome complex. The
rate-limiting step in this process is ubiquitin conjugation. Indirect evidence from
animal studies suggests that ubiquitin-dependent protein degradation is important
in states of insulin deprivation. Protein breakdown rates increase markedly in
rats that are made insulinopenic by treatment with streptozotocin. Treatment with
selective inhibitors of the lysosomal or calcium-dependent protease pathways
did not affect protein breakdown. When ATP synthesis was blocked, how-
ever, protein breakdown declined.
30
This suggests that ATP-dependent ubiqui-
tin–proteosome-mediated protein breakdown is important in insulin deficiency.
Others have shown that mRNAs for ubiquitin–proteosome proteins are increased
in the insulin-deficient state.
31
If diabetic rats are treated with insulin, pro-
tein breakdown is reduced, and ubiquitin–proteosome mRNAs are reduced to
control levels.

32
Acidosis and increased cortisol levels, which occur following
insulin deprivation, stimulate protein degradation in the ubiquitin–proteosome
pathway.
32, 30
In summary, insulin deficiency in a diabetic animal model shows
coordinate time-dependent changes in different proteolytic pathways in muscle,
resulting in increased overall proteolysis. Only the capacity of non-lysosomal
processes seems to be altered in muscle in response to insulin deficiency. The
many intracellular mechanisms of insulin action to affect protein turnover are
summarized in Figure 4.2.
Insulin effect on protein synthesis
ribosomes
mRNA transcription
-IRE promoter
elements
Insulin effect on protein breakdown
Ubiquinone–proteosome path
activity
mRNA
stability
Translation initiation
-PHAS-1
phosphorylation
eIF-2B activity
Elongation
eEF-2
phosphorylation
-
-

mRNA
-
-
Figure 4.2 Effect of insulin on protein turnover
110 THE EFFECT OF INSULIN ON PROTEIN METABOLISM
Insulin as a regulator of protein turnover in vitro and in situ
Studying the effect of insulin on protein turnover in humans is complicated. The
body is an intricate system with many hormonal mechanisms that interact with
one another. Therefore, altering a single hormone such as insulin can lead to
changes in other hormones, including growth hormone, glucagon, cortisol and
epinephrine to name a few. These in turn can lead to changes in concentrations
of substrates, such as amino acids, glucose and fatty acids, in heart rate and
in blood flow or have more direct effects on protein synthesis and breakdown.
Because of these complexities several in vitro and in situ systems have been
used to study insulin’s effect on protein turnover. By using an in vitro or in situ
model one can simplify the experiment by removing confounding factors like
other hormones and alterations in other parameters such as blood flow.
The simplest model is an in vitro cell culture system. In this system a homoge-
neous population of cells can be studied under very controlled conditions. Using
a specific cell line such as L6, a rat skeletal muscle myoblast line, allows one
to determine insulin’s effect on protein turnover within a single cell type. The
components of the cell medium and the insulin concentration can be well defined.
Using this model, it has been shown that insulin stimulates protein synthesis in L6
myoblasts.
33, 34
This type of model is ideal for studies of signal transduction path-
ways stimulated by insulin.
35, 36
The biggest disadvantage of a cell culture model
is that it may not be representative of the whole body system. Cell lines are gener-

ally transformed in some manner and even when differentiated the cells lack some
characteristics of cells in vivo. For example, even when L6 myoblasts are differen-
tiated into myotubes, they do not express the same myosin heavy chain isoforms as
adult skeletal muscle.
37
In addition, within a tissue such as skeletal muscle, there
are many different cell types such as fibroblasts, vascular muscle cells, vascular
epithelium etc. These may be important modulators of skeletal muscle cells and
the effect would be missed in a simple cell culture system.
In order to account for these parameters but to still maintain a very con-
trolled system, several investigators have used in situ methods to study the
effect of insulin on protein turnover. These models have utilized perfused animal
diaphragm, heart, skeletal muscle, or whole limbs.
38–40, 16
Consistently, insulin
reduces tissue protein breakdown. Although in situ studies provide a simpli-
fied system that may be optimal for understanding mechanisms behind insulin’s
effect on protein metabolism, they too may not fully represent the in vivo situa-
tion. Ultimately, to fully understand insulin’s regulation of protein metabolism
in humans, one must study an in vivo system. A number of methods have been
used to measure protein turnover in animal models and in human subjects.
Animal studies
Rodent models have been extensively used to study insulin effect on protein
metabolism. In studies performed in growing rodents indicated that insulin
MEASUREMENT OF PROTEIN METABOLISM 111
deficiency was associated with reduced synthesis rates of muscle proteins, where-
as in fully grown rodents insulin failed to stimulate muscle protein synthesis.
41
Similarly, in piglets insulin stimulates muscle protein synthesis rates
42

and with
increasing age the magnitude of synthesis rates decreases.
43, 44
These mea-
surements were performed on mixed tissue proteins, representing the average
fractional synthesis rates of many proteins. Recent studies in sexually matured
miniature pigs demonstrated that when the insulin effect was determined on
different subfractions of muscle proteins a specific stimulatory effect on mus-
cle mitochondrial protein synthesis was observed, with no significant effect on
synthetic rates of sarcoplasmic and myosin heavy chain proteins.
45
In contrast,
the insulin effect on liver proteins in mini-pigs is variable, showing no effect on
liver tissue protein synthesis whereas synthesis rate of fibrinogen was inhibited.
46
Since human adult life is much longer than that of rodents and pigs it is impor-
tant to study the insulin effect on adult humans to understand the regulation of
protein turnover in humans after the genetic potential for growth is passed.
4.3 Measurement of protein metabolism (synthesis and
breakdown or turnover) in human subjects
Measurement of protein turnover
Net protein turnover, a result of both synthesis and breakdown, can be quanti-
fied using a number of different methods. Some of the more global techniques
include whole body nitrogen balance, 3-methylhistidine excretion (specifically
for myofibrillar protein breakdown), regional amino-acid balance and systemic
amino-acid tracer incorporation. By using biopsies or separation techniques,
protein synthesis can also be measured within a specific tissue or for a specific
protein. Ultimately, the regulation of protein concentrations may be a result of
many factors including changes in gene expression, mRNA stability and trans-
lation efficiency. Assessment of changes in protein turnover induced by insulin

can take place at many levels: (1) the cellular level, where one may observe
the mRNA changes and changes in translation efficiency; (2) the tissue level,
where one can study the effect of insulin on a specific tissue or set of proteins
(such as skeletal muscle on myofibrillar proteins); (3) the regional or whole
body level, where one can more globally assess insulin’s effects (Figure 4.3). In
this section, the various methods of studying protein turnover in human subjects
will be discussed. Following the description of each method, we shall review
the use of the method to assess the effect of insulin on protein turnover.
Whole body nitrogen balance
When protein is broken down, free amino acids and their metabolites are released
into the circulation. All amino acids contain at least one nitrogen molecule.
Transamination is a critical process necessary to transfer nitrogen for synthesis
112 THE EFFECT OF INSULIN ON PROTEIN METABOLISM
Whole body
Regional
Tissue
Amino-acid
availability
Specific
proteins
Transcriptional and
translational
regulation
DNA and
mRNA
Figure 4.3 Sites to assess insulin effect on protein metabolism
of non-essential amino acids. Amino acids that are oxidized or transaminated
can give rise to ammonia. Most of this circulating ammonia is converted to urea
in the liver via the ornithine cycle and can be excreted in the urine. Urinary
nitrogen is composed of 80–85 per cent urea and ammonia. Another 5–10 per

cent of urinary nitrogen is accounted for by creatine, creatinine, uric acid and
free amino acids.
47
By collecting urine and stool for 24 hours, one can quantify
total body nitrogen loss. To determine net nitrogen loss daily nitrogen intake also
has to be measured. This reflects the summation of multiple processes including
changes in protein breakdown, protein synthesis, dietary protein intake, and
alterations in the recycling of amino acids.
Although this method seems straightforward in concept, there are several
problems with it. First, results can be affected by changes in renal function,
hydration status, certain medications and the amount of protein that is ingested.
Generally, subjects are asked to maintain a specific diet (normalized for protein
intake) for several days before a study. This reduces the variability in nitro-
gen generated by dietary protein intake. In diabetic patients with reduced renal
function, proteinuria or renal tubular acidosis, the results of whole body nitrogen
balance can be unreliable.
Insulin effect as measured by nitrogen balance and free amino-acid
concentrations
Early studies on diabetic patients used whole body nitrogen balance to assess
the effect of insulin on whole body protein metabolism. Withdrawal of insulin
MEASUREMENT OF PROTEIN METABOLISM 113
treatment has been shown to increase urinary nitrogen losses and to increase
the concentrations of several essential amino acids, especially branched chain
amino acids.
48, 1, 49
Insulin treatment normalizes the increased urinary nitrogen
loss and the increased circulating amino-acid concentrations.
5, 49, 50
In 1976,
Walsh and colleagues

51
studied 18 uncontrolled diabetic patients before and
after 6–8 weeks of treatment. This group was a mixture of type 1 and type 2
diabetic patients. In subjects who were given insulin to control blood sugars,
there was an average weight gain of 8.7 per cent and average nitrogen balance
of +13 per cent. In the diabetic patients treated with diet alone or with diet and
an oral agent there was no change in weight, and only a +3.8 per cent nitrogen
balance.
51
This increase in body mass and a positive nitrogen balance shows
that in patients who are relatively insulin deficient (diabetic patients) treatment
with insulin has an anabolic effect.
3-methylhistidine quantification
Skeletal muscle actin and myosin contain 3-methylhistidine (3-MH). This mod-
ified amino acid is not further metabolized or reutilized after release from actin
or myosin. The only fate is urinary excretion. These properties make 3-MH a
potential surrogate for muscle protein breakdown. 3-MH measurements com-
paring arterial versus venous concentrations have been made across local tissue
beds (the forearm or leg). In this type of study, the increase in venous concentra-
tion of 3-MH can provide good estimates of muscle protein breakdown. Whole
body studies quantifying urinary excretion of 3-MH are difficult to interpret
and do not necessarily reflect only skeletal muscle protein breakdown because
smooth muscle (particularly intestinal) can give rise to as much as 10 per cent of
urinary 3-MH.
47
Moreover, myofibrillar proteins have slow turnover (approxi-
mately 1–2 per cent/day), which makes it difficult to perform short term studies
on the effect of insulin on myofibrillar protein breakdown.
Insulin effect as measured by 3-MH
In healthy volunteers, insulin infusion does not change the flux of 3-MH across

the leg or forearm.
52, 53
In contrast, in a study of poorly controlled diabetic
patients there was a substantially greater excretion of urinary 3-MH as com-
pared with healthy volunteers. When the same diabetic patients were restudied
after achieving satisfactory glycemic control, urinary 3-MH excretion was not
different from that of healthy volunteers.
54
This suggests that insulin deficiency
results in increased muscle (we cannot differentiate between skeletal and smooth)
protein breakdown and that replacement of insulin inhibits this breakdown. The
available techniques to measure 3-MH have widely varying coefficients of vari-
ation, which makes these measurements insensitive to small differences.
114 THE EFFECT OF INSULIN ON PROTEIN METABOLISM
4.4 Whole body and regional protein turnover
The effect of amino-acid availability
Amino acids are the building blocks of proteins. The availability of these build-
ing blocks can determine whether protein synthesis can take place. Based on
the k
m
value of amino-acyl tRNA ligase it was argued that normal physiologi-
cal changes in free amino acids have little effect on protein synthesis. However,
recent studies have clearly demonstrated that amino acids by themselves enhance
translational efficiency of gene transcripts.
55, 56
Amino acids can be provided
by reuse of amino acids provided by protein breakdown or they can be provided
in the form of a meal or infusion. Amino-acid availability is of great impor-
tance when considering insulin’s effect on protein turnover, because amino-acid
availability has been shown to be a major factor controlling muscle protein

synthesis.
57–59
Systemic or regional infusion of insulin has been shown to
reduce blood concentrations of amino acids (hypoaminoacidemia).
60, 48, 61, 62
A reduced rate of protein breakdown by insulin is the likely cause of this
insulin-induced hypoamino acidaemia. Another potential site of the insulin effect
is on transmembrane transport of amino acids. Transmembrane transport of
neutral amino acids in skeletal muscle is mediated by at least four different sys-
tems (A, ASC, L and N
m
). Regional studies of forearm skeletal muscle using
methylaminoisobutyric acid (MeAIB), a non-metabolizable amino-acid analogue
specific for system A amino-acid transport, showed that physiologic hyperinsuli-
naemia stimulates the activity of system A amino-acid transport.
63
This effect
may play a role in determining the response of muscle amino-acid transport
and protein metabolism in response to insulin. When trying to reconcile the
results of whole body and regional studies in humans, it is important to note
whether blood amino-acid concentrations were monitored and/or clamped during
the study. When discussing results below, we shall note this.
Amino-acid tracer techniques
Use of a labelled amino-acid tracer allows simultaneous determination of pro-
tein synthesis and breakdown rates at the whole body level and across tissue
beds. Quantifying incorporation of the tracer into a specific protein or protein
fraction or mixed proteins can yield the synthesis rate. Measuring the dilution
of the tracer (provided it is a labelled essential amino acid) in the free tracee
(amino-acid) pool in the steady state is extensively used for calculation of pro-
tein breakdown rates. During a steady state condition the rate of appearance

of an essential amino acid such as leucine is the same as its disappearance
rate. Therefore, in a fasted state, rate of appearance is equivalent to protein
breakdown because essential amino acids only appear from protein breakdown,
and rate of disappearance (sum of catabolism and incorporation into protein)
can be estimated. Once the catabolic rate (e.g. leucine oxidation, phenylalanine
WHOLE BODY AND REGIONAL PROTEIN TURNOVER 115
hydroxylation to tyrosine etc.) and flux (appearance or disappearance rate) are
measured, rate of incorporation of amino acid into protein (protein synthesis)
can be calculated by subtracting the catabolic rate of the amino acid from its
flux
7
(Figure 4.3).
In addition, from tracer and tracee measurements in artery and vein (e.g.
femoral vein for leg or hepatic vein for splanchnic bed) as well as blood flow
measurements (usually based on indicator dye dilution) the kinetics of protein
(breakdown and synthesis) and net balances can be estimated in the respective
tissue beds.
49
In addition, serial needle biopsy of skeletal muscle and infusion
of an isotopic tracer and measurements of isotopic abundance of the tracer
in muscle protein or proteins will allow the estimation of fractional synthesis
rates of mixed proteins or specific proteins.
64
Similar approaches can be applied
to measure fractional synthesis rates of circulating plasma proteins.
65, 66
The
tracer technique, therefore, can be used to determine whole body, regional and
specific protein (such as myosin heavy chain) synthesis rates. In most cases, if
the appropriate samples are taken (including blood, breath samples and tissue

biopsies), a single experiment can determine all of these parameters. Two tracer
methods are widely used for determination of tissue protein synthesis rates in
humans – flooding dose and continuous infusion.
The flooding dose technique
With the flooding dose a large amount of unlabelled amino acid (tracee) is
injected as a bolus along with the labelled amino acid (tracer).
67
The goal of
infusing this large dose is to quickly achieve an equilibrium of tracer concentra-
tion between the plasma and the intracellular ‘precursor pool’. The obligatory
‘precursor pool’ is the amino acid acylated to its transfer RNA (amino-acyl
tRNA). This is the step just prior to incorporation of the amino acid into a
protein. To accurately calculate synthesis rates based on extracellular tracer
enrichment, the extracellular tracer enrichment and intracellular ‘precursor pool’
enrichment must be in equilibrium.
The primary advantage of this technique is that protein synthesis rates can be
determined in a short period of time (10–30 minutes). Since a large amount of
tracer is infused, it will make up a greater percentage of the amino acids incor-
porated into protein. This is particularly useful in studies of acute interventions
such as short term infusion of a compound.
The main disadvantage of this technique is that a number of assumptions
need to be made. First, the large bolus of amino acid must be assumed to
have no effect on protein dynamics. Second, in order for rates of synthesis and
breakdown to be calculated, one must assume that enrichment is at steady state
during the study period, which may not be the case during a declining phase of
both tracer and tracee. These assumptions can be incorrect if certain requirements
of the flooding dose condition are not met, particularly if the concentration of
116 THE EFFECT OF INSULIN ON PROTEIN METABOLISM
the ‘flooding dose’ is too low or the study period is too long. Either of these
can cause non-equilibrium conditions in tracer enrichment between extracellular

and intracellular compartments. The tracer in this approach is not truly in the
‘tracer amount’ and the high concentration of ‘tracer’ may affect the protein
synthesis measurements.
68
The advantages and disadvantages of this technique
have been described in detail elsewhere.
69–74
The continuous infusion technique
With the continuous infusion technique, a continuous lower level infusion of
tracer is given. In order to reach a steady state more quickly, the continuous
infusion is typically preceded by a priming bolus of tracer.
75
The continuous
infusion technique allows study over a long period of time (several hours).
Hence, this technique is better suited to the study of proteins that have a
slow rate of turnover. Most skeletal muscle proteins fall into this category.
On the other hand, this technique is not ideal for quick turn over proteins
because of the amino-acid recycling that can occur over a prolonged time period.
Another disadvantage is that in most cases a surrogate measure of the oblig-
atory precursor (amino-acyl tRNA) has to be used for calculation of protein
synthesis. This results in underestimation of protein synthesis calculation.
76
For whole body measurements surrogate measures of intracellular pool, such
as ketoisocaprioate in the case of leucine tracer, have been used with some
strong theoretical reasons.
77
However, this approach is not practical with every
amino-acid tracer.
Amino-acid tracers
In the past, radiolabelled amino acids were used as tracers. More recently, sta-

ble isotope amino-acid tracers have been more widely used, which has many
theoretical advantages and is more acceptable for volunteers for studies and
institutional ethical committees. The incorporation of the tracer into protein can
then be quantified by mass spectrometry. The amount of incorporated tracer is
a reflection of the amount of newly synthesized protein over the time of the
infusion.
7
The amino acid chosen for the tracer varies from study to study, and
it is not uncommon to use more than one tracer within a single study.
7
For whole body studies tracers such as L[1-
13
C] leucine and labelled pheny-
lalanine (e.g. L[
15
N] phenylalanine L[
2
H
5
] phenylalanine) are extensively used.
For regional studies involving skeletal muscle bed phenylalanine has many
advantages, which include its small intracellular pool and thus the shorter period
needed to equilibrate with the free amino-acid pool. Within skeletal muscle
and the other tissues of the forearm or leg, phenylalanine is not metabolized.
Protein synthesis rates can be determined by measuring the rate of disappear-
ance of phenylalanine. However, if one is studying the splanchnic bed (liver
and intestine), it is important to account for the conversion of phenylalanine
WHOLE BODY AND REGIONAL PROTEIN TURNOVER 117
to tyrosine within the liver using an independent tracer of tyrosine.
7

Recent
studies have also demonstrated that phenylalanine is converted to tyrosine in
the kidney
78
besides in the liver. Therefore, for regional studies involving kid-
ney as well, phenylalanine and tyrosine tracers have to be used to measure
protein turnover.
Leucine is an essential amino acid that composes 6–8 per cent of protein.
Because leucine concentrations are high within most proteins, it provides large
enrichment when used as a tracer. This is particularly useful when studying
synthesis rates of proteins that have slow rates of turnover. Use of leucine in
regional studies, however, can make calculations more complicated because it
can be either directly incorporated into protein (non-oxidative metabolism) or
reversibly transaminated to form ketoisocaproic acid (KIC). KIC can then be
further oxidized to carbon dioxide and isovaleryl CoA (Figure 4.3) or reami-
nated back into leucine. If leucine is labelled at the carboxyl carbon (e.g.
13
C)
and the amino group with
15
N, it is possible to quantify leucine transamination
rates. Leucine tracers with both labels have been used to measure transami-
nation rates at the whole body
79
and regional levels.
80, 49
In order to account
for the metabolic products one must collect breath samples for measurement of
label within expired carbon dioxide or
13

CO
2
production across tissue beds in
regional studies.
80
Measurement of
13
C-KIC enrichment can be a useful sur-
rogate of the precursor pool leucyl-tRNA enrichment. Measurement of this
compound requires far less muscle tissue and labour than does direct mea-
surement of leucyl tRNA. It has been demonstrated in human studies to be a
good surrogate
81
although muscle tissue fluid is closer to tRNA enrichment.
81
For studies involving liver proteins (plasma proteins such as albumin, fibrino-
gen, APOB
100
etc.) plasma [
13
C] KIC is an excellent surrogate measure of liver
leucyl-tRNA enrichment. For skeletal muscle, muscle tissue fluid leucine enrich-
ment is a better indicator of leucyl tRNA. Amino-acid tracers can thus be used
to study protein kinetics of the whole body, of a region, of a certain tissue or
of specific proteins.
Insulin and protein turnover in type 1 diabetic patients using whole
body leucine flux
Type 1 diabetic patients are deficient in insulin, so perhaps the most dramatic
effects of insulin can be observed in these subjects. One can study these patients
in the insulin deficient state and compare these results to the insulin replete

state. Using the whole body leucine flux technique, several groups have con-
firmed that insulin deprivation in type 1 diabetic patients results in increased
protein breakdown as demonstrated by increased leucine flux, phenylalanine and
tyrosine flux.
82–85, 62, 49, 86–89
In the majority of studies, insulin infusion nor-
malized leucine flux, providing strong evidence that insulin suppresses protein
breakdown. Somewhat surprisingly, whole body protein synthesis also increased
118 THE EFFECT OF INSULIN ON PROTEIN METABOLISM
with insulin deprivation and was suppressed with insulin treatment. The mag-
nitude of synthesis suppression is less than breakdown suppression. In whole
body leucine flux studies that also included an amino-acid infusion to maintain
levels, there was a further decrease in protein breakdown and leucine oxida-
tion in response to insulin treatment.
82, 90, 84
Results are mixed regarding whole
body protein synthesis when amino acids were infused and leucine flux was
measured. Some studies showed an increase in whole body protein synthesis
(as measured by non-oxidative leucine flux)
90, 84
and others did not.
83
During
insulin deprivation there is also a marked increase in amino-acid oxidation,
which is largely due to a substantial increase in the transamination process in
the case of leucine.
7
Not all of the changes in type 1 diabetic patients during
insulin deficiency are directly related to insulin deficiency per se. There are
many secondary events that occur following insulin withdrawal in type 1 dia-

betic patients. Such secondary changes include increase in levels of circulating
amino acids (especially branched chain amino acids), glucagon, non-esterified
fatty acids and β hydroxybutyrate levels. Increased amino-acid levels (due to
increased protein breakdown) contribute substantially to the large increase in
leucine transamination and leucine oxidation. Increased amino acid levels also
cause increased protein synthesis in splanchnic bed.
59
Increased glucagon levels
also cause increased oxidation of leucine, which has been shown in patients
with type 1 diabetes.
91
Beta-hydroxybutyrate has been shown to stimulate syn-
thesis of muscle protein synthesis.
7
This effect of β-hydroxybutyrate and the
inhibitory effect of fatty acids on protein breakdown may limit the catabolic
effect of insulin deficiency.
Regional protein turnover
Within a local area – across the forearm, across the leg or across the splanch-
nic bed – protein turnover can be assessed by amino-acid balance and tracer
measurements. Amino-acid balance and tracer enrichment are determined by
infusing, systemically, a labelled amino-acid tracer to achieve a steady state in
the plasma and precursor pools. The amount of tracer enrichment and amino-
acid concentration present in the venous and arterial sides are then determined.
Amino-acid balance is the arterio-venous difference in amino acid multiplied
by blood flow. Rate of appearance (protein breakdown) and rate of disappear-
ance (synthesis and catabolism) can be estimated by mathematical equations.
92, 7
The estimation of catabolic rate and synthesis rates are possible using multiple
amino-acid tracers. The details of the models used for these measurements are

given elsewhere.
93, 49, 7
Measurement of protein turnover in a region accounts
for turnover in all of the tissues of that region. In the leg or forearm this would
include skin, connective tissue, adipose tissue and skeletal muscle although
skeletal muscle accounts for the major portion when deep veins such as femoral
vein are used to sample. In the splanchnic bed, tissues of the abdomen including
liver and intestine are the main contributors to protein turnover.
WHOLE BODY AND REGIONAL PROTEIN TURNOVER 119
Insulin and regional protein metabolism in type 1 diabetic patients
and healthy controls
The results of regional studies across the leg or forearm using either phenylala-
nine or leucine as tracers in type 1 diabetic patients are mixed. Some show a
reduction in protein breakdown after insulin infusion
83, 49, 94
while others show
no effect.
95
None of these studies showed any effect on protein synthesis. How-
ever, the relative fraction of muscle protein synthesis to whole body protein
synthesis increases after insulin replacement.
92
Results from the splanchnic bed are interesting. In the insulin deprived state,
splanchnic bed protein synthesis exceeds breakdown
49
(Figure 4.4). Figure 4.4
shows that insulin deprivation I(−) results in increased protein breakdown par-
ticularly in skeletal muscle (sk. muscle). I(−) also increases protein synthesis
(only in the splanchnic bed) but to a lesser degree. Insulin treatment I(+) results
in suppressed protein breakdown, particularly in skeletal muscle and protein syn-

thesis (only in splanchnic bed) Insulin levels are the lowest in the fasting state
between meals. It has been suggested that since there is a net breakdown in
skeletal muscle protein during insulin deficient states muscle may serve as a
reservoir of amino acids. Figure 4.5 shows that between meals, when insulin
levels are low, there is a preservation of splanchnic bed protein synthesis while
there is a net degradation of muscle protein. After a meal, when insulin levels
are high, there is a net gain in muscle protein and a decline in splanchnic bed
protein. The regulatory sites of insulin and amino acids are indicated in the
figure. During the fasting state, expendable muscle proteins could be broken
down in order to provide the necessary amino-acid supply to the splanchnic
Breakdown Synthesis
other
splanchnic
sk. muscle
3.5
2.5
mmol/h
2
1.5
1
0.5
0
3
I(−)I(+)I(−)I(+)
Figure 4.4 Effect of insulin on protein breakdown and synthesis in type 1 diabetic patients
120 THE EFFECT OF INSULIN ON PROTEIN METABOLISM
Circulating proteins
AA efflux
AA efflux
Insulin PB

Circulating proteins
AA uptake
AA efflux
Insulin PB
AA PB PS
AA P
B PS
PB
PS
PB
PS
(a)
(b)
Figure 4.5 Sites of protein accretion between and after meals – based on studies with
insulin and AA infusion
59
bed for synthesis of crucial proteins such as clotting factors. Studies performed
in non-diabetic people demonstrated that protein synthesis in splanchnic bed is
higher than protein breakdown in the fasted state.
96
Muscle releases amino acids
in the fasted state because muscle protein breakdown is higher than muscle pro-
tein synthesis. Therefore muscle is a provider of amino acids to the splanchnic
bed to maintain synthesis of proteins. Recent studies have shown the kidney is
a net producer of tyrosine and contributes to the systemic circulation.
78
When
insulin levels increase muscle protein breakdown decreases and the output of
amino acids decreases. This occurs in association with a decrease in splanchnic
protein synthesis. However, if amino acids are infused along with insulin, there

is a further reduction in muscle protein breakdown and there is an increase in
muscle protein synthesis.
59
Amino acids have an independent effect on splanch-
nic protein synthesis and increase in splanchnic protein synthesis. While insulin
is the major regulator of muscle protein turnover with minimal effect on splanch-
nic protein turnover, amino acids have major effects on both these two tissue
beds. Based on these studies, it is proposed that reduced insulin levels during
WHOLE BODY AND REGIONAL PROTEIN TURNOVER 121
the fasted state result in an increased output of amino acids from muscle bed
and these amino acids are vital for synthesis of essential proteins in the liver.
Following a mixed meal the amino acids from meal enhance protein accretion
in both splanchnic and muscle beds. Insulin plays a key facilitative role in all
these processes.
In type 1 diabetic patients insulin deficiency causes a substantial increase
in muscle and splanchnic protein breakdown (Figure 4.4). While muscle pro-
tein synthesis is not significantly affected by short term insulin deficiency in
type 1 diabetic patients, splanchnic protein synthesis increases. The net effect
is that both protein breakdown and protein synthesis increase at the whole
body level. However the greater increase in protein breakdown results in net
protein catabolism. The increased splanchnic protein synthesis and prevention
of decline in muscle protein synthesis during short term insulin deficiency is
thought to be related to increased circulating amino acids, based on studies in
non-diabetic people.
59
Tissue-specific protein synthesis
Biopsy of a specific tissue during an infusion or flooding dose of a tracer is par-
ticularly useful when one wishes to study a tissue with a slow rate of turnover
such as skeletal muscle. A biopsy is taken at baseline and then at some point
after an intervention. In these biopsy samples one can measure the synthesis

rates of particular groups of proteins by determining the amount of tracer incor-
porated over the intervention time from a defined precursor pool. In skeletal
muscle samples our laboratory typically determines the tracer incorporation into
mixed muscle protein (a mixture of the proteins present in the muscle), into
sarcoplasmic protein (protein present within the cytosol) and into myosin heavy
chain (a key structural and contractile protein). In addition, mitochondria are
isolated and the amount of tracer incorporated into mitochondrial proteins is
determined. The purification of these fractions and measurement techniques is
described elsewhere.
97
Similarly, fractional synthesis rates of circulating proteins
can be measured after purifying specific proteins.
65, 98, 66, 99
Insulin and mixed muscle protein in type 1 diabetic patients
Several groups have looked at the effect of insulin on the synthesis of mixed
muscle protein in type 1 diabetic patients after insulin treatment. In none of these
was there a change in the mixed muscle protein synthesis,
82, 91, 85, 86
even with
amino-acid infusion.
82
Fractional synthesis rate of a specific protein
If a specific protein can be purified from a biopsy specimen it is possible to
determine the fractional synthesis rate of the protein.
100, 97
In our laboratory
122 THE EFFECT OF INSULIN ON PROTEIN METABOLISM
we use SDS PAGE protein electrophoresis to purify myosin heavy chain from
muscle myofibrillar protein fraction separated by ultracentrifugation.
101

Mass
spectrometry is used in measuring the isotopic abundance of a protein such
as myosin heavy chain
102
that has a very slow turnover rate. After protein or
proteins are purified one has to hydrolyse the protein into amino acids and
use gas chromatography/combustion/isotope ratio mass spectrometry to deter-
mine the change of isotopic enrichment between two biopsy periods. Knowing
the precursor pool isotopic enrichment (amino-acyl tRNA or its surrogate mea-
sures), fractional synthesis rates of specific protein or protein subfractions (e.g.
mitochondrial proteins or sarcoplasmic proteins) can be measured.
7
Insulin’s effect on specific proteins
When the synthesis rate of myosin heavy chain was studied in type 1 diabetic
patients before and following insulin treatment, there was no change observed.
91
Insulin, however, has a specific effect on muscle mitochondrial protein syn-
thesis. When insulin was infused at high physiological levels while replacing
glucose and amino acids, muscle mitochondrial protein synthesis was stimulated
(Figure 4.3).
10
This increase in muscle mitochondrial protein synthesis occurred
in association with an increase in muscle mitochondrial enzyme activity and
ATP production. This important finding demonstrated a pivotal role of insulin
and amino acids in the regulation of mitochondrial oxidative phosphorylation
in skeletal muscle. Certain liver proteins appear to be responsive to changes
in insulin concentration. De Feo and colleges
65, 98
have studied the fractional
synthesis rates of albumin, antithrombin III, fibrinogen and apoB-100 in healthy

volunteers and in type 1 diabetic patients. In type 1 diabetic patients deprived
of insulin, the synthesis of albumin was reduced but the synthesis of fibrinogen
was increased.
98
The authors proposed that the increase in fibrinogen repre-
sented an acute phase response, because in healthy subjects insulin infusion
stimulated the synthesis of albumin but reduced the synthesis of fibrinogen and
antithrombin III.
Effect of insulin in healthy volunteers
Some of the insulin effect on protein metabolism has already been discussed
in comparison with type 1 diabetes. Healthy volunteers are typically studied in
the post-absorptive (fasting) state. Baseline insulin levels are low in this state
and calculations of amino-acid flux are simplified because there is no dilution of
tracer due to dietary intake of amino acid. With only a few exceptions,
52, 103–105
there seems to be good agreement that insulin inhibits protein breakdown in
normal subjects. Using a variety of tracers, many regional and whole body
studies have reached the same conclusion.
82, 106–112, 53, 113
However, there is
not good agreement regarding the effect of insulin on protein synthesis in healthy
volunteers – about half of the studies show no effect of insulin on protein
WHOLE BODY AND REGIONAL PROTEIN TURNOVER 123
synthesis.
52, 82, 106, 108, 110, 112, 114, 96, 104
Several others show an increase in
regional or whole body synthesis with insulin.
103, 109, 115, 105
These differences
may be related to insulin dose, methodologies, duration of insulin infusion and

levels of circulating amino acids and other substrates. On the whole, there is
quite convincing data to suggest that insulin can inhibit protein breakdown in
healthy volunteers. If protein synthesis rates are unchanged or increased, the
overall effect of insulin would be a whole body accretion of protein. Based on
recent studies it is clear that plasma amino acids have a critical role in stim-
ulating muscle protein synthesis and insulin alone reduces circulating amino
acids in vivo, which may explain some of the discrepancies between in vivo and
in vitro studies. Insulin also has a specific effect on synthesis of certain mus-
cle protein fractions such as mitochondrial proteins and plasma proteins such
as albumin.
Effect of insulin in type 2 diabetics patients
Type 2 diabetic patients are insulin resistant and as a result, at least early in the
course of the disease, they have chronically high insulin levels. The effect of
this insulin resistance to glucose metabolism and hyperinsulinaemia on protein
turnover is not well defined. Oral hypoglycemic agents (glyburide) have been
shown to reduce endogenous glucose production in type 2 diabetic patients
but have no effect on protein turnover.
116
When type 2 diabetic patients have
been infused with relatively high dose insulin over a short three hour clamp
period, there is a suppression of protein breakdown similar to that seen in
matched control subjects.
107, 117
However, intensive insulin treatment had no
effect on protein turnover in comparison with less stringent glycemic control
with insulin.
118
Interestingly, after a longer term infusion (overnight) after 10
days of subcutaneous insulin there appears to be a resistance to the insulin effect
on protein breakdown. Under these conditions, insulin does not significantly sup-

press protein breakdown.
119
This resistance to the effect of insulin to suppress
protein breakdown is important. Since type 2 diabetic patients have chronically
high insulin levels, normal tissue turnover could not take place if protein break-
down continued to be suppressed. A resistance to the action of insulin on glucose
disposal is not necessarily coupled with resistance to insulin’s suppression of
protein breakdown. This allows for normal protein turnover to take place even
in the face of high insulin levels. At this point, it is unclear whether the intra-
cellular mechanisms behind the resistance to insulin’s effect on glucose disposal
and protein metabolism are similar or whether they are independent.
As in control subjects, the fractional synthesis rates of mixed muscle pro-
tein, myosin heavy chain and mitochondrial protein were unchanged in diabetic
patients infused with insulin.
120
It is interesting that intensive insulin treatment
in type 2 diabetic patients did not stimulate mitochondrial protein synthesis,
which is consistent with the recent report that increasing insulin levels to the
124 THE EFFECT OF INSULIN ON PROTEIN METABOLISM
same extent failed to increase muscle mitochondrial ATP production in type 2
diabetic patients, in contrast with non-diabetic control subjects.
10
Summary
Considering all of the data, including those from intracellular studies, in situ
studies and human subject studies, there is good evidence that insulin can reg-
ulate protein turnover in many ways. Insulin can (1) selectively enhance the
transcription of certain mRNAs through insulin response elements in promoters,
(2) selectively enhance the stability of certain RNAs, (3) enhance translation
initiation and elongation and (4) enhance ribosomal abundance. Insulin can
also selectively inhibit the degradation of some proteins through the ubiqui-

tin–proteosome system. Studies in the whole organism help us to understand
the relative influence of changes in protein synthesis and breakdown in the over-
all protein balance. They also help us understand the differential effects within
certain tissue beds.
Studies in type 1 diabetic patients and non-diabetic people indicate that insulin
has differential effects on skeletal muscle protein turnover from those on the
splanchnic bed protein turnover. This is particularly important when consider-
ing the fluctuation in insulin levels in relation to meals. The effects observed
in type 1 diabetic patients are more dramatic than those in healthy controls
because they can be studied in the insulin deficient state. It may be that this
same paradigm is true in healthy subjects but that it is more difficult to detect
the differences. Insulin stimulates muscle mitochondrial protein synthesis and
mitochondrial biogenesis when amino acids are provided. Insulin-induced fall
in circulating amino acids blunts the stimulatory effect of insulin on synthesis
of muscle proteins. Insulin’s primary effect on muscle appears to be an inhibi-
tion of protein breakdown. While amino acids have a key role in modulating
insulin effect on muscle protein synthesis, amino acids are the main regulators
of splanchnic protein synthesis. Insulin, however, has highly specific effects on
certain liver proteins and muscle mitochondrial proteins and more research in
the area is warranted.
In type 2 diabetic patients, there is a resistance to insulin’s effect on glucose
disposal, and there is also a resistance to insulin-induced suppression of protein
breakdown. The mechanism is unclear; however, this resistance is important in
maintenance of normal protein turnover in type 2 diabetic patients. Insulin has no
stimulatory effect on muscle mito-protein synthesis in type 2 diabetic patients.
The question of how insulin might regulate protein turnover, and hence tissue
mass, is complicated. Currently, the limiting factor in more thoroughly under-
standing this process is one of technology. We have been limited to studying,
for the most part, groups of proteins. For example, the measurement of mixed
muscle protein or mitochondrial protein fractional synthesis rates may be the

summation of results from hundreds of different proteins. The currently available
REFERENCES 125
techniques make it difficult to analyse the effect of insulin on a large number
of specific proteins. As techniques become more refined for the purification and
profiling of many proteins simultaneously (proteomics), we will gain a detailed
understanding of the regulation of the entire network of proteins within specific
cell types in response to insulin.
Acknowledgements
This work was supported by NIH grants RO1 DK41973 and MO1RR00585, the
David Murdock-Dole Professorship (K. S. Nair) and the Mayo Clinic Clinician
Investigator Training Program (L. J. S. Greenlund).
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