Improvement of protein synthesis in muscle will greatly enhance the production of lean pork. This improvement can be traced to changes at the cellular level. The object of this study was to identify the effects of insulin and the branched chain amino acid, leucine on the extent and rate that messenger RNA (mRNA) is translated into protein. Porcine satellite cells were isolated from a 30 lb pig and cultured. The cultured cells were treated with varying levels of insulin and leucine. Increasing levels of insulin and leucine caused an increase in ribosomes, the organelles responsible for synthesis, only after leucine was present in the media in adequate concentrations. With increasing levels of insulin there was an increase in the recruitment of ribosomes into polyribosomes for mRNA translation. However, increasing leucine levels had no effect on polyribosome percentage. In conclusion, insulin stimulates translation of mRNA by increasing both ability and rate. However, adequate levels of amino acid must be available for the stimulation to occur. Increased levels of branched chain amino acid do not create a synergistic effect with insulin to increase polyribosomes for protein synthesis

Creamer, Brad

Jones, Steven J.

Scheffler, Jason

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Nebraska Swine Reports Animal Science Department January 2005 Does Insulin and Leucine Stimulate Muscle Protein Synthesis? Brad Creamer University of Nebraska-Lincoln Jason Scheffler University of Nebraska-Lincoln Steven J. Jones University of Nebraska-Lincoln, sjones1@unl.edu Follow this and additional works at: https://digitalcommons.unl.edu/coopext_swine  Part of the Animal Sciences Commons Creamer, Brad; Scheffler, Jason; and Jones, Steven J., "Does Insulin and Leucine Stimulate Muscle Protein Synthesis?" (2005). Nebraska Swine Reports. 26. https://digitalcommons.unl.edu/coopext_swine/26 This Article is brought to you for free and open access by the Animal Science Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Nebraska Swine Reports by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 2005 Nebraska Swine Report — Page 40Does Insulin and Leucine Stimulate MuscleProtein Synthesis?IntroductionPork products are a valuableproduct of American agricultureand consumers demand lean porkproducts. Therefore, producersmust continue to improve theefficiency of lean meat produc-tion. Improved efficiency of leanpork production can be directlyrelated to the rate and efficiencyof skeletal muscle protein accre-tion. Understanding the cellularmechanisms regulating proteinaccretion in skeletal muscle is animportant factor for improving theefficiency of lean-meat produc-tion.Skeletal muscle is the singlemost valuable component in thepig and is the largest contributorto protein accretion within theanimal. Postnatal muscle growthin meat animals is highly depen-dent on protein accretion. Skel-etal muscle growth via proteinaccretion is highly dependent onthe rate and efficiency of both tran-scription of DNA into messengerRNA (mRNA) and its subsequenttranslation by ribosomal RNA(rRNA) into protein. This studyfocused on the translation ofmRNA into protein. This wasaccomplished by measuring thetotal number of ribosomes andtheir activity in translating mRNAinto protein. The activity wasdetermined by measuring the per-cent of ribosomes in the polyribo-some form.Insulin and branched chainamino acids — for example, leu-cine — have been shown to havesignificant roles in stimulation ofmuscle protein synthesis. Thepurpose of this study was todetermine if insulin or leucinealtered the quantity of ribosomesor their rate of recruitment forprotein synthesis. Satellite cellswere used in this study becausethey could be grown in cell cul-ture and more closely representwhat would be observed in vivo.MethodsPrimary satellite cells werecollected from the semimembrano-sus and semitendinosus muscles ofa 6-week-old female pig weigh-ing 30 lb. After the gilt was sacri-ficed, muscles were removed fromboth the left and right side of theanimal. Muscles were cut into1-cm3 cubes and satellite cells wereliberated from the tissue using an(enzymatic protease) solution.After cells were released fromconnective tissue, they were sepa-rated from the cellular debris bycentrifugation. Cells were platedon 75-mm culture flasks MinimumEssential Media-alpha (α-MEM)with 10% FBS and allowed to pro-liferate. Once they reached 80percent surface density, they wereremoved from the plate and fro-zen (-80o C).Cells were removed from thefreezer, allowed to thaw and platedBrad CreamerJason SchefflerSteven J. Jones1Summary and ImplicationsImprovement of protein synthe-sis in muscle will greatly enhancethe production of lean pork. Thisimprovement can be traced to changesat the cellular level. The object of thisstudy was to identify the effects ofinsulin and the branched chain aminoacid, leucine on the extent and ratethat messenger RNA (mRNA) istranslated into protein. Porcine sat-ellite cells were isolated from a 30 lbpig and cultured. The cultured cellswere treated with varying levels ofinsulin and leucine. Increasinglevels of insulin and leucine causedan increase in ribosomes, the organellesresponsible for synthesis, only afterleucine was present in the media inadequate concentrations. Withincreasing levels of insulin there wasan increase in the recruitment of ribo-somes into polyribosomes for mRNAtranslation. However, increasing leu-cine levels had no effect on polyribo-some percentage. In conclusion,insulin stimulates translation ofmRNA by increasing both ability andrate. However, adequate levels ofamino acid must be available for thestimulation to occur. Increased levelsof branched chain amino acid do notcreate a synergistic effect with insu-lin to increase polyribosomes for pro-tein synthesis.2005 Nebraska Swine Report — Page 41on 6-well plates with 15 ml ofculture media and allowed to pro-liferate until they reached a 80%confluence. Cells at this stage ofdevelopment were identified asproliferating satellite cells (PSC)and used as one of the cell types.Additionally, replicate plates con-taining PSC media were replacedwith a fusion media consisting ofα-MEM plus two percent horseserum to stimulate cell fusion toform myotubes. Previous researchindicated that 48 hours aftermedia replacement was the opti-mum time for treatment ofmyotubes. This cell type will bereferred to as fused satellite cells(FSC).Both porcine derived PSC andFSC were used in the study usinga serum free media. The studywas designed using five concen-trations of insulin (0 ,0.0025, 0.045,0.09, and 0.18 µg/ml) with 0.09µg/ml representing post feedingphysiological concentrations andfour concentrations of leucine, 0,0.029, 0.058, 0.116 mg/ml inserum-free media. Each treatmentcombination was representedwith three wells per treatment andthe entire experiment was repli-cated twice.Cells were removed from theplate using a buffer to preventRNA degradation and protein,DNA and RNA were determined.The polyribosomes were isolatedand separated using an ultracen-trifuge then quantified using agradient fractionator. The percent-age of ribosomes in the polyribo-some form was determined andused as a measure of the rate oftranslation. Total protein, DNAand RNA were quantified.The statistical design was arandomized complete blockdesign with each plate serving asblocking criteria. The experimentwas replicated and analysis wasdone using Statistical AnalysisSystems (SAS). Mean separationwas accomplished using the leastsquare means statement. All dif-Figure 1. RNA/DNA in proliferating porcine satellite cells treated with serum-free mediacontaining combined levels of insulin and leucine. (n= 6: SEM = .006)0.250.20.150.10.050RNA:DNA0 0.029 0.058 0.116 0.145Leucine Level (mg/mL)Insulinµg/ml00.0450.090.18(Continued on next page)Insulinµg/ml0 0.029 0.058 0.116 0.145Leucine Level (mg/mL) Figure 2. RNA/DNA in fused porcine satellite cells 48 hours after addition of fusion media, andtreated with serum-free media containing combined levels of insulin and leucine. (n=6: SEM= .002))0.090.080.070.060.050.040.030.020.010RNA:DNA 00.0450.090.182005 Nebraska Swine Report — Page 42ferences are reported at P < .05probably.ResultsConcentrations of RNA andprotein were adjusted by cell den-sity by presenting data as a ratioto DNA. Differences in total DNA,and therefore cell densities, werecorrected for RNA and protein bypresenting the data as a ratio toDNA. No differences wereobserved in protein and DNAwithin cell types. In both cell types,RNA concentration increased asthe level of leucine increased(Figures 1 and 2). Differences(P<.05) in RNA due to insulin werenot observed until the media con-tained levels of leucine compa-rable to that normally found inthe α-MEM media (0.058 mg/ml).Once the leucine requirement wassatisfied, RNA concentrations in-creased (P < .05) with increasinglevels of insulin. In the PSC, thehigh level of insulin respondedwith a greater increase at lowerleucine levels compared to FSC.The increase in polyribosome per-centage with high levels ofinsulin occurred with leucine con-centrations at 0.058 mg/ml in PSCcompared to 0.116 mg/ml in FSC.This difference may be due to theleucine requirement being higherfor FSC versus PSC or it may berelated to the responsiveness ofthe FSC to insulin. Other studiesperformed in our lab have shownthat the response of FSC to insu-lin, as well as other hormones, isless than PSC. Another observa-tion in FSC was a decline in RNAlevels at the highest insulin andleucine levels. This drop-off inRNA levels may be an aberrationof data; it was expected for RNAlevels to plateau as they did inPSC.There was no interactionobserved between insulin andFigure 3. Polyribosome percentages in proliferating satellite cells exposed to serum-free mediacontaining combined levels of insulin and leucine. (n= 6: SEM = 3.2)00.0450.090.18706050403020Percent of Total Ribosomes as Polyribosomes0 0.029 0.058 0.116 0.145Leucine Level (mg/mL)Insulinµg/ml00.0450.090.18Figure 4. Polyribosome percentages in porcine satellite cells 48 hours after the addition offusion media, and exposed to serum-free media containing combined levels ofinsulin and leucine. (n= 6: SEM= 2.29)706050403020Percent of Total Ribosomes as Polyribosomes0 0.029 0.058 0.116 0.145Leucine Level (mg/mL)Insulinµg/ml2005 Nebraska Swine Report — Page 43leucine with regard to polyribo-some percentage within PSC orFSC (Figure 3 and 4). Regardlessof leucine levels, polyribosomepercentages were higher withincreasing levels of insulin for PSC,with main effect estimates of 42.5,47.4, 50.6, and 52.5 percent forinsulin levels 0, 0.045, 0.09, and0.18 µg/mL treatments, respec-tively. Similar results wereobserved in FSC, with insulineffects estimates, regardless ofleucine levels, being 42.6, 47.4,50.9, and 55.7 percent for insulinlevels 0, 0.045, 0.09, and 0.18 µg/mL treatments, respectively. Inboth PSC and FSC increasinglevels of leucine had no effect onribosome recruitment to poly-ribosomes.DiscussionOther researchers haveobserved that both insulin andamino acids have been shown tohave significant roles in the stimu-lation of protein synthesis byincreasing the total amount ofrRNA; (the machinery responsiblefor synthesis and the rate ofrecruitment for protein synthesisin muscle). One group of research-ers demonstrated in neonatal pigsthat insulin and amino acidsincrease protein synthesis, butthese effects are not additive. Ourstudy demonstrated insulin’spowerful effect on protein syn-thesis by increasing both thenumber of ribosomes and ratepolyribosome formation whichfunctions to synthesize proteinsin muscle. Leucine concentrationsonly impacted the rate of proteinsynthesis by increasing the amountof rRNA. This response is greaterin myoblasts when compared tomyotubes. Other studies in ourlab have shown fractional pro-tein synthesis rates are higher inthe myoblasts compared tomyotubes. Only when the aminoacids requirements are met canthe full anabolic effect of insulinbe realized. If essential aminoacids are restricted, increased con-centrations of insulin will haveno anabolic impact on muscle.The results of this study dem-onstrate insulin plays a large rolein increasing RNA synthesis aswell as its activity in the forma-tion of polyribosomes, which areactive in both PSC and FSC invitro.  This response can beincreased if leucine or possiblyother branched chain aminoacids are supplemented. It wasalso observed if leucine concen-trations decrease below normalmedia concentrations, the produc-tion of RNA, as well as its activ-ity, was significantly decreased.While insulin caused an increasein RNA and its activity, it doesnot appear to work synergisticallywith leucine to increase RNA pro-duction in either cell type. Leu-cine only appeared to impactsynthesis rates when it was re-duced to below baseline levels.1Brad Creamer and Jason Schefflerare graduate students and Steven Jones isa professor in the Animal Science Depart-ment at the University of Nebraska-Lincoln.

Does Insulin and Leucine Stimulate Muscle Protein Synthesis?

UNL | Libraries

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Nebraska Swine Reports Animal Science Department 
January 2005 
Does Insulin and Leucine Stimulate Muscle Protein Synthesis? 
Brad Creamer 
University of Nebraska-Lincoln 
Jason Scheffler 
University of Nebraska-Lincoln 
Steven J. Jones 
University of Nebraska-Lincoln, sjones1@unl.edu 
Follow this and additional works at: https://digitalcommons.unl.edu/coopext_swine 
 Part of the Animal Sciences Commons 
Creamer, Brad; Scheffler, Jason; and Jones, Steven J., "Does Insulin and Leucine Stimulate Muscle Protein 
Synthesis?" (2005). Nebraska Swine Reports. 26. 
https://digitalcommons.unl.edu/coopext_swine/26 
This Article is brought to you for free and open access by the Animal Science Department at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Nebraska Swine Reports by 
an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
2005 Nebraska Swine Report — Page 40
Does Insulin and Leucine Stimulate Muscle
Protein Synthesis?
Introduction
Pork products are a valuable
product of American agriculture
and consumers demand lean pork
products. Therefore, producers
must continue to improve the
efficiency of lean meat produc-
tion. Improved efficiency of lean
pork production can be directly
related to the rate and efficiency
of skeletal muscle protein accre-
tion. Understanding the cellular
mechanisms regulating protein
accretion in skeletal muscle is an
important factor for improving the
efficiency of lean-meat produc-
tion.
Skeletal muscle is the single
most valuable component in the
pig and is the largest contributor
to protein accretion within the
animal. Postnatal muscle growth
in meat animals is highly depen-
dent on protein accretion. Skel-
etal muscle growth via protein
accretion is highly dependent on
the rate and efficiency of both tran-
scription of DNA into messenger
RNA (mRNA) and its subsequent
translation by ribosomal RNA
(rRNA) into protein. This study
focused on the translation of
mRNA into protein. This was
accomplished by measuring the
total number of ribosomes and
their activity in translating mRNA
into protein. The activity was
determined by measuring the per-
cent of ribosomes in the polyribo-
some form.
Insulin and branched chain
amino acids — for example, leu-
cine — have been shown to have
significant roles in stimulation of
muscle protein synthesis. The
purpose of this study was to
determine if insulin or leucine
altered the quantity of ribosomes
or their rate of recruitment for
protein synthesis. Satellite cells
were used in this study because
they could be grown in cell cul-
ture and more closely represent
what would be observed in vivo.
Methods
Primary satellite cells were
collected from the semimembrano-
sus and semitendinosus muscles of
a 6-week-old female pig weigh-
ing 30 lb. After the gilt was sacri-
ficed, muscles were removed from
both the left and right side of the
animal. Muscles were cut into
1-cm3 cubes and satellite cells were
liberated from the tissue using an
(enzymatic protease) solution.
After cells were released from
connective tissue, they were sepa-
rated from the cellular debris by
centrifugation. Cells were plated
on 75-mm culture flasks Minimum
Essential Media-alpha (α-MEM)
with 10% FBS and allowed to pro-
liferate. Once they reached 80
percent surface density, they were
removed from the plate and fro-
zen (-80o C).
Cells were removed from the
freezer, allowed to thaw and plated
Brad Creamer
Jason Scheffler
Steven J. Jones1
Summary and Implications
Improvement of protein synthe-
sis in muscle will greatly enhance
the production of lean pork. This
improvement can be traced to changes
at the cellular level. The object of this
study was to identify the effects of
insulin and the branched chain amino
acid, leucine on the extent and rate
that messenger RNA (mRNA) is
translated into protein. Porcine sat-
ellite cells were isolated from a 30 lb
pig and cultured. The cultured cells
were treated with varying levels of
insulin and leucine. Increasing
levels of insulin and leucine caused
an increase in ribosomes, the organelles
responsible for synthesis, only after
leucine was present in the media in
adequate concentrations. With
increasing levels of insulin there was
an increase in the recruitment of ribo-
somes into polyribosomes for mRNA
translation. However, increasing leu-
cine levels had no effect on polyribo-
some percentage. In conclusion,
insulin stimulates translation of
mRNA by increasing both ability and
rate. However, adequate levels of
amino acid must be available for the
stimulation to occur. Increased levels
of branched chain amino acid do not
create a synergistic effect with insu-
lin to increase polyribosomes for pro-
tein synthesis.
2005 Nebraska Swine Report — Page 41
on 6-well plates with 15 ml of
culture media and allowed to pro-
liferate until they reached a 80%
confluence. Cells at this stage of
development were identified as
proliferating satellite cells (PSC)
and used as one of the cell types.
Additionally, replicate plates con-
taining PSC media were replaced
with a fusion media consisting of
α-MEM plus two percent horse
serum to stimulate cell fusion to
form myotubes. Previous research
indicated that 48 hours after
media replacement was the opti-
mum time for treatment of
myotubes. This cell type will be
referred to as fused satellite cells
(FSC).
Both porcine derived PSC and
FSC were used in the study using
a serum free media. The study
was designed using five concen-
trations of insulin (0 ,0.0025, 0.045,
0.09, and 0.18 µg/ml) with 0.09
µg/ml representing post feeding
physiological concentrations and
four concentrations of leucine, 0,
0.029, 0.058, 0.116 mg/ml in
serum-free media. Each treatment
combination was represented
with three wells per treatment and
the entire experiment was repli-
cated twice.
Cells were removed from the
plate using a buffer to prevent
RNA degradation and protein,
DNA and RNA were determined.
The polyribosomes were isolated
and separated using an ultracen-
trifuge then quantified using a
gradient fractionator. The percent-
age of ribosomes in the polyribo-
some form was determined and
used as a measure of the rate of
translation. Total protein, DNA
and RNA were quantified.
The statistical design was a
randomized complete block
design with each plate serving as
blocking criteria. The experiment
was replicated and analysis was
done using Statistical Analysis
Systems (SAS). Mean separation
was accomplished using the least
square means statement. All dif-
Figure 1. RNA/DNA in proliferating porcine satellite cells treated with serum-free media
containing combined levels of insulin and leucine. (n= 6: SEM = .006)
0.25
0.2
0.15
0.1
0.05
0
R
N
A
:D
N
A
0 0.029 0.058 0.116 0.145
Leucine Level (mg/mL)
Insulin
µg/ml
0
0.045
0.09
0.18
(Continued on next page)
Insulin
µg/ml
0 0.029 0.058 0.116 0.145
Leucine Level (mg/mL)
 Figure 2. RNA/DNA in fused porcine satellite cells 48 hours after addition of fusion media, and
treated with serum-free media containing combined levels of insulin and leucine. (n=
6: SEM= .002))
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
R
N
A
:D
N
A 0
0.045
0.09
0.18
2005 Nebraska Swine Report — Page 42
ferences are reported at P < .05
probably.
Results
Concentrations of RNA and
protein were adjusted by cell den-
sity by presenting data as a ratio
to DNA. Differences in total DNA,
and therefore cell densities, were
corrected for RNA and protein by
presenting the data as a ratio to
DNA. No differences were
observed in protein and DNA
within cell types. In both cell types,
RNA concentration increased as
the level of leucine increased
(Figures 1 and 2). Differences
(P<.05) in RNA due to insulin were
not observed until the media con-
tained levels of leucine compa-
rable to that normally found in
the α-MEM media (0.058 mg/ml).
Once the leucine requirement was
satisfied, RNA concentrations in-
creased (P < .05) with increasing
levels of insulin. In the PSC, the
high level of insulin responded
with a greater increase at lower
leucine levels compared to FSC.
The increase in polyribosome per-
centage with high levels of
insulin occurred with leucine con-
centrations at 0.058 mg/ml in PSC
compared to 0.116 mg/ml in FSC.
This difference may be due to the
leucine requirement being higher
for FSC versus PSC or it may be
related to the responsiveness of
the FSC to insulin. Other studies
performed in our lab have shown
that the response of FSC to insu-
lin, as well as other hormones, is
less than PSC. Another observa-
tion in FSC was a decline in RNA
levels at the highest insulin and
leucine levels. This drop-off in
RNA levels may be an aberration
of data; it was expected for RNA
levels to plateau as they did in
PSC.
There was no interaction
observed between insulin and
Figure 3. Polyribosome percentages in proliferating satellite cells exposed to serum-free media
containing combined levels of insulin and leucine. (n= 6: SEM = 3.2)
0
0.045
0.09
0.18
70
60
50
40
30
20
Pe
rc
en
t o
f T
ot
al
 R
ib
os
om
es
 a
s 
Po
ly
ri
bo
so
m
es
0 0.029 0.058 0.116 0.145
Leucine Level (mg/mL)
Insulin
µg/ml
0
0.045
0.09
0.18
Figure 4. Polyribosome percentages in porcine satellite cells 48 hours after the addition of
fusion media, and exposed to serum-free media containing combined levels of
insulin and leucine. (n= 6: SEM= 2.29)
70
60
50
40
30
20
Pe
rc
en
t o
f T
ot
al
 R
ib
os
om
es
 a
s 
Po
ly
ri
bo
so
m
es
0 0.029 0.058 0.116 0.145
Leucine Level (mg/mL)
Insulin
µg/ml
2005 Nebraska Swine Report — Page 43
leucine with regard to polyribo-
some percentage within PSC or
FSC (Figure 3 and 4). Regardless
of leucine levels, polyribosome
percentages were higher with
increasing levels of insulin for PSC,
with main effect estimates of 42.5,
47.4, 50.6, and 52.5 percent for
insulin levels 0, 0.045, 0.09, and
0.18 µg/mL treatments, respec-
tively. Similar results were
observed in FSC, with insulin
effects estimates, regardless of
leucine levels, being 42.6, 47.4,
50.9, and 55.7 percent for insulin
levels 0, 0.045, 0.09, and 0.18 µg/
mL treatments, respectively. In
both PSC and FSC increasing
levels of leucine had no effect on
ribosome recruitment to poly-
ribosomes.
Discussion
Other researchers have
observed that both insulin and
amino acids have been shown to
have significant roles in the stimu-
lation of protein synthesis by
increasing the total amount of
rRNA; (the machinery responsible
for synthesis and the rate of
recruitment for protein synthesis
in muscle). One group of research-
ers demonstrated in neonatal pigs
that insulin and amino acids
increase protein synthesis, but
these effects are not additive. Our
study demonstrated insulin’s
powerful effect on protein syn-
thesis by increasing both the
number of ribosomes and rate
polyribosome formation which
functions to synthesize proteins
in muscle. Leucine concentrations
only impacted the rate of protein
synthesis by increasing the amount
of rRNA. This response is greater
in myoblasts when compared to
myotubes. Other studies in our
lab have shown fractional pro-
tein synthesis rates are higher in
the myoblasts compared to
myotubes. Only when the amino
acids requirements are met can
the full anabolic effect of insulin
be realized. If essential amino
acids are restricted, increased con-
centrations of insulin will have
no anabolic impact on muscle.
The results of this study dem-
onstrate insulin plays a large role
in increasing RNA synthesis as
well as its activity in the forma-
tion of polyribosomes, which are
active in both PSC and FSC in
vitro.  This response can be
increased if leucine or possibly
other branched chain amino
acids are supplemented. It was
also observed if leucine concen-
trations decrease below normal
media concentrations, the produc-
tion of RNA, as well as its activ-
ity, was significantly decreased.
While insulin caused an increase
in RNA and its activity, it does
not appear to work synergistically
with leucine to increase RNA pro-
duction in either cell type. Leu-
cine only appeared to impact
synthesis rates when it was re-
duced to below baseline levels.
1Brad Creamer and Jason Scheffler
are graduate students and Steven Jones is
a professor in the Animal Science Depart-
ment at the University of Nebraska-
Lincoln.


https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1025&context=coopext_swine

Does Insulin and Leucine Stimulate Muscle Protein Synthesis?

Abstract

Similar works

Full text

Available Versions

DigitalCommons@University of Nebraska

UNL | Libraries