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From the Clinical Research Centers |
Departments of Internal Medicine (E.V.), Surgery (B.M., B.B.R., R.R.W.), and Anesthesiology (R.R.W.), University of Texas Medical Branch, and Shriners Hospital (E.V., B.M., B.B.R., R.R.W.), Galveston, Texas 77550
Address all correspondence and requests for reprints to: Elena Volpi, M.D., Ph.D., Department of Medicine, Division of Endocrinology, University of Southern California, 1333 San Pablo Street, BMT-B11, Los Angeles, California 90033.
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Abstract |
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We measured muscle protein synthesis and breakdown, and amino acid transport in healthy young (30 ± 3 yr) and elderly (72 ± 1 yr) volunteers in the basal postabsorptive state and during the administration of an amino acid-glucose mixture, using L-[ring-2H5]phenylalanine infusion, femoral artery and vein catheterization, and muscle biopsies. Basal muscle amino acid turnover was similar in young and elderly subjects. The mixture increased phenylalanine leg delivery and transport into the muscle in both groups. Phenylalanine net balance increased in both groups (young, -27 ± 8 to 64 ± 17; elderly, -16 ± 4 to 29 ± 7 nmol/(min·100 mL); P < 0.0001, basal vs. mixture), but the increase was significantly blunted in the elderly (P = 0.030 vs. young). Muscle protein synthesis increased in the young, but remained unchanged in the elderly [young, 61 ± 17 to 133 ± 30 (P = 0.005); elderly, 62 ± 9 to 70 ± 14 nmol/(min·100 mL) (P = NS)]. In both groups, protein breakdown decreased (P = 0.012) and leg glucose uptake increased (P = 0.0258) with the mixture.
We conclude that the response of muscle protein anabolism to hyperaminoacidemia with endogenous hyperinsulinemia is impaired in healthy elderly due to the unresponsiveness of protein synthesis.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The failure of nutritional supplementation in the treatment of sarcopenia of the elderly may be due to age-related alterations of muscle protein metabolism in response to feeding. This hypothesis is indirectly supported by recent data in humans suggesting that protein requirements increase with age (9). Increased protein requirements in aging suggest a decline in the ability of old muscle to use and maintain dietary amino acids. Recent studies in old rats have shown that muscle protein synthesis is blunted during balanced feeding (10). In humans, muscle protein synthesis has been found to be slower in elderly compared with young controls during a 5-h meal (11). However, when these data were compared with those obtained in different subjects in the fasting state the stimulation of muscle protein synthesis from the basal level appeared to be similar in elderly and young (11). On the other hand, another study from the same group showed that high protein meals do not stimulate muscle protein synthesis in elderly volunteers undergoing exercise training (12). Nevertheless, we recently reported that muscle protein turnover is similar in healthy elderly to that in younger counterparts in the fasting state and in response to an amino acid load (13, 14). Our results appear to be in conflict with those of other investigators (7, 8, 10, 12), although this is not necessarily the case, as different combinations of nutrients were used. We used a balanced mixture of amino acids alone (13, 14), whereas all other studies (7, 8, 10, 12) used a mixture of protein, fat, and carbohydrate, which stimulated the endogenous insulin response.
Like amino acids, insulin is a very potent anabolic stimulus for muscle protein metabolism. Different researchers have shown that in young adults insulin stimulates muscle protein anabolism when given alone or in combination with amino acids, and that the combination of insulin and amino acids appears to be more beneficial than either stimulus alone in young individuals (reviewed in Ref. 15). Although the mechanism(s) is still under debate (16), aging is associated with increased prevalence of insulin resistance that results in glucose intolerance and diabetes (17). However, no data are available on the physiologic interaction between amino acids and insulin on muscle protein metabolism in the elderly.
Based on prior studies, we hypothesized that the failure of mixed nutritional supplementation to increase muscle mass in the elderly depends on specific alterations in the response of muscle to the combined effects of exogenous amino acids and endogenous insulin. Consequently, we compared in healthy young and elderly subjects the response of muscle protein synthesis, breakdown and net balance to ingestion of a mixture of amino acids and glucose that stimulates endogenous hyperinsulinemia.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We recruited five healthy young (one woman and four men; age, 30 ± 3 yr; body mass index, 24.7 ± 0.4 kg/m2) and five healthy elderly (one woman and four men; age, 72 ± 1 yr; body mass index, 25.9 ± 0.9 kg/m2) volunteers through the Center on Aging Volunteers Registry of the University of Texas Medical Branch. All subjects gave informed written consent before participating in the study, which was approved by the Institutional Review Board of the University of Texas Medical Branch (Galveston, TX).
Volunteer eligibility was assessed by physical examination and laboratory tests, including electrocardiogram, blood count, plasma electrolytes, liver and renal function tests, blood glucose, and oral glucose tolerance test. Exclusion criteria were heart disease, coagulation disorders, artery or vein diseases, hypertension, obesity, cancer, acute or chronic pulmonary diseases, infectious diseases, and diabetes mellitus or glucose intolerance, as defined by the report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus (18).
Both young and elderly volunteers were physically active, i.e. living on their own with no limitation in ambulation or history of falls, although not exercise trained. We specifically excluded subjects of any age who were engaged in exercise training programs.
Study design
Each subject was studied after an overnight fast. The volunteers were instructed to eat their usual diet during the week preceding the study to avoid metabolic changes due to recent dietary modifications. The evening before the study the subjects were admitted to the General Clinical Research Center of the University of Texas Medical Branch. They ate a light dinner, after which they were allowed only water ad libitum. The morning of the study, at approximately 0600 h, polyethylene catheters were inserted into a forearm vein for tracer infusion, in the wrist vein of the opposite hand for arterialized blood sampling, and in the femoral artery and vein of one leg for blood sampling. The arterial catheter was also used for the infusion of indocyanine green (ICG; Cardio-Green, Becton Dickinson and Co., Cockeysville, MD).
After a blood sample was drawn for the measurement of background
phenylalanine and glucose enrichments and ICG concentration, a primed
continuous infusion of
L-[ring-2H5]phenylalanine
(priming dose, 2 µmol/kg; infusion rate, 0.05 µmol/(kg·min);
Cambridge Isotope Laboratories, Andover, MA) and
[6,6-2H2]glucose (priming
dose, 19 µmol/kg; infusion rate, 0.22 µmol/(kg·min); Cambridge
Isotope Laboratories) were started and maintained at constant rate for
480 min until the end of the experiment (Fig. 1
). At 120 min a muscle biopsy was taken
from the lateral portion of the muscle vastus lateralis of the leg with
the femoral catheters, about 20 cm above the knee, using a 5-mm
Bergström biopsy needle. The tissue was immediately frozen in
liquid nitrogen and stored at -80 C until analysis. At 230 min a
continuous infusion of ICG dye (0.5 mg/min) was started in the femoral
artery and was maintained until 270 min to measure leg blood flow. To
measure the plasma ICG concentration, four blood samples were taken
every 10 min between 240–270 min from the femoral and wrist veins.
Subsequently, between 270–300 min, four blood samples were taken every
10 min from the femoral artery and vein to measure plasma phenylalanine
concentrations and enrichments, and glucose concentration. At 240 and
300 min additional blood samples were drawn from the femoral artery to
measure insulin, GH, and insulin-like growth factor I (IGF-I)
concentrations. At 300 min a second muscle biopsy was taken as
previously described. Immediately after the biopsy was taken, oral
administration of a liquid amino acid-glucose mixture was started and
continued for 3 h until the end of the study. The mixture
contained a total of 40 g glucose and 40 g crystalline amino
acids (Sigma, St. Louis, MO) in the same proportion as
that found in beef proteins (19). The mixture was
dissolved in 540 mL water containing a sugar-free flavoring (Crystal
Light, Kraft Foods, Inc., White Plains, NY). The mixture was given as
small boluses (30 mL) every 10 min to achieve steady state plasma
phenylalanine enrichments and concentrations, and steady state blood
glucose concentrations. The amount of glucose (young, 17.83 ±
0.15; elderly, 18.04 ± 0.33 mg/(kg·min); P =
0.57), phenylalanine (young, 0.75 ± 0.03; elderly, 0.73 ±
0.05 µmol/(kg·min); P = 0.70), and total amino
acids (young, 3.08 ± 0.12; elderly, 2.95 ± 0.22
mg/(kg·min); P = 0.64) given were similar in the two
groups. To avoid an excessive reduction of blood glucose enrichment
during oral glucose intake, the infusion rate of
[6,6-2H2]glucose was
increased to 0.85 µmol/(kg·min). We chose to increase the iv
infusion of labeled glucose rather than add the tracer to the oral
mixture because the oral glucose was given at a constant rate, and
therefore the problem of isotopic nonsteady state was not an issue.
Between 420 and 480 min the measurement of leg blood flow was repeated,
and blood samples were taken as described for the fasting period. At
480 min, before stopping the tracer and amino acid infusion, a third
muscle biopsy was taken. All of the subjects but one young volunteer,
who was lost at follow-up, underwent leg magnetic resonance imaging
(MRI) to measure muscle leg volume approximately 1–2 weeks after the
study.
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The concentration of plasma insulin was measured with a
commercial RIA kit (Diagnostic Products, Los Angeles, CA).
Plasma human (h) GH was measured using a commercial immunoassay
(Nichols Institute Diagnostics, San Juan Capistrano, CA),
and IGF-I was measured using a commercial immunoradiometric assay
(Nichols Institute Diagnostics, San Juan Capistrano, CA).
Plasma glucose concentrations were measured with an enzymatic method
(YSI, Inc., Yellow Springs, OH). The serum ICG
concentration was measured with a spectrophotometer set at 
=
805 nm.
The arterial and venous samples were processed as previously described (20), and phenylalanine and glucose enrichments and phenylalanine concentrations were measured using gas chromatography-mass spectrometry (GCMS; GC HP 5890, MSD HP 5989, Hewlett-Packard Co., Palo Alto, CA).
Muscle samples were processed as previously described (20), and free tissue phenylalanine enrichments and concentrations were determined by GCMS. The intracellular concentration of phenylalanine was then calculated from the tissue value, accounting for the ratio of intracellular to extracellular water (21). Mixed muscle protein-bound phenylalanine enrichment was measured by GCMS (GC 8000 series, MD 800, Fisons Instruments, Manchester, UK) using the standard curve approach previously described (22).
Leg skeletal muscle volume was determined by analysis of images collected by MRI (GE Signa 1.5 Tesla whole body imager: General Electric, Milwaukee, WI). Initial images were taken of the frontal plane to establish the anatomical landmarks used to demarcate upper (greater trochanter to patella) and lower (patella to ankle) leg series. Consecutive transaxial images, 10 mm thick, were collected between these landmarks. The differences between fat (200–300 ms) and muscle (800–900 ms) relaxation times in T1 enabled analysis using an intensity threshold method. The images were subsequently processed using NIH Image software (NIH public domain analysis package).
Calculations
We calculated skeletal muscle amino acid kinetics from the enrichments and concentrations of phenylalanine in the femoral artery and vein and from the enrichment of tissue-free phenylalanine, using a three-pool model previously described and validated (21). Phenylalanine was used because it is an essential amino acid, i.e. it is not produced in the body and is not oxidized in the muscle tissue. Thus, it allows calculation of muscle protein synthesis and breakdown in addition to the measurement of amino acid delivery to the leg, release from the leg, transport into and from the muscle tissue, and shunting from the artery to the vein without entering the tissue space. The three-pool model parameters were calculated as follows: delivery to the leg, Fin = CA x BF (Eq I); release from the leg, Fout = CV x BF (Eq II); net balance across the leg, NB = (CA - CV) x BF (Eq III); transport into muscle, FM,A = {[(EM - EV)/(EA - EM) x CV] + CA} x BF (Eq IV); transport from muscle, FV,M = {[(EM - EV)/(EA - EM) x CV] + CV} x BF (Eq V); artery-vein shunting, FV,A = Fin - FM,A (Eq VI); muscle protein breakdown, FM,O = FM,A x [(EA/EM) - 1] (Eq VII); and muscle protein synthesis, FO,M = FM,O + NB (Eq VIII). CA and CV are plasma phenylalanine concentrations in the femoral artery and vein, respectively; EA, EV, and EM are phenylalanine enrichments (tracer/tracee ratio) in femoral arterial and venous plasma and in muscle, respectively; BF is leg blood flow. Data are presented per 100 mL leg volume (21).
Leg plasma flow was calculated from the steady state dye concentration values in the femoral and wrist vein as previously described (23, 24). Leg blood flow was calculated by correcting the plasma flow by the hematocrit.
Additionally, we determined the fractional synthetic rate (FSR) of
mixed muscle proteins by measuring the incorporation rate of
L-[ring-2H5]phenylalanine
into the proteins using the precursor-product model (25)
as follows: FSR =
{[(
EP/t)/](EM(1)
+ EM(2))/2]} x 60 x 100 (Eq
IX). EP is the increment in
protein-bound phenylalanine enrichment between two sequential biopsies,
t is the time interval between the two sequential biopsies,
and EM(1) and EM(2) are the
phenylalanine enrichments (tracer/tracee ratio) in the free muscle pool
in the two subsequent biopsies. The results are presented as the
percentage per hour.
Glucose delivery to the leg, release from the leg, and uptake across the leg were calculated as follows: glucose delivery = GA x BF (Eq X), glucose release = GV x BF (Eq XI), and glucose uptake = (GA - GV) x BF (Eq XII). GA and GV are the blood glucose concentrations in the femoral artery and vein, respectively.
The whole body glucose rate of appearance (Ra) was calculated using the single pool model as follows (26): glucose Ra = I/EA (Eq XIII). EA the arterial glucose enrichment, and I is the tracer infusion rate. During the intake of the oral amino acid-glucose mixture, the above formula represented the total glucose Ra, i.e. endogenous plus exogenous glucose Ra. Therefore, during oral glucose intake endogenous glucose Ra was calculated by subtracting the oral glucose administration rate from the total Ra, assuming that all of the oral glucose was absorbed: endogenous glucose Ra = total glucose Ra - oral glucose intake (Eq IV).
Statistical analysis
The effect of age on the response variables in the fasting state and during infusion of the amino acid-glucose mixture was analyzed using two-way ANOVA with repeated measures. Post-hoc pairwise multiple comparisons were carried out when appropriate using the t test modified with Bonferroni’s correction. Differences were considered significant at P < 0.05. Data are presented as the mean ± SE.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Total leg volume was slightly, but not significantly, lower in the elderly (young, 10.17 ± 0.45; elderly, 9.16 ± 0.58 L; P = 0.21). Similarly, leg muscle volume was slightly, but not significantly, lower in the elderly (n = 5) compared with the young (n = 4, one volunteer did not undergo the measurement) using total volume (young, 5.6 ± 0.9; elderly, 4.5 ± 0.5 L; P = 0.34), percent leg volume (young, 48 ± 5%; elderly, 45 ± 4%; P = 0.60), or muscle volume corrected by the squared height to obtain a leg muscle index (young, 1.86 ± 0.25; elderly, 1.52 ± 0.1 L/m2; P = 0.29).
Blood flow
Leg blood flow was similar in young and elderly in the basal state [young, 3.38 ± 0.49; elderly, 3.82 ± 0.58 mL/(min·100 mL leg)]. During amino acid-glucose intake, leg blood flow slightly increased in the young, whereas it decreased in the elderly [young, 4.31 ± 0.79; elderly, 2.99 ± 0.54 mL/(min·100 mL leg)], with a significant treatment by group interaction (P = 0.0178).
Hormones
Arterial insulin concentrations were similar in young and elderly
in the basal state, and increased significantly during amino
acid-glucose intake (P < 0.0001, basal vs.
amino acid-glucose mixture) with no differences between the groups
(Table 1). Arterial hGH concentrations
were slightly, but not significantly, lower in the elderly compared
with those in the young during the basal period, and did not change
significantly during administration of oral amino acids with glucose,
although there was a trend in the young for a reduction in hGH
concentrations with amino acid-glucose intake (P =
0.076) (Table 1). IGF-I concentrations were higher (P =
0.003) in the young than in the elderly both in the basal state and
during administration of oral amino acids with glucose (Table 1).
However, amino acid-glucose intake did not exert any effect on plasma
IGF-I in either age group.
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Arterial essential amino acid concentrations were similar in the
basal state in young and elderly and increased significantly and
similarly in both groups during amino acid-glucose intake with the
exception of tryptophan, which increased significantly only in the
elderly, not in the young (Table 2).
Nonessential amino acid concentrations were similar in the elderly and
the young in the basal state with the exception of serine, which was
lower in the elderly. The administration of oral amino acids with
glucose significantly increased the concentrations of all nonessential
amino acids except tyrosine, which did not change in either group. The
increase in the concentrations of asparagine, histidine, and serine
were blunted in the elderly (Table 2). Free phenylalanine
concentrations in the femoral artery and vein were at steady state
during the last hour of each study period (data not shown).
Phenylalanine concentrations in the femoral artery and vein and in the
muscle tissue fluid were similar in the basal postabsorptive state in
young and elderly (Table 3). During amino
acid-glucose intake, the concentrations of free phenylalanine in the
femoral artery and vein and in the muscle intracellular fluid
significantly increased from the fasting values, with no differences
between young and elderly. Plasma phenylalanine enrichments in
the femoral artery and vein were at steady state during the last hour
of the basal and amino acid-glucose periods in both groups (Fig. 2). In the basal state, phenylalanine
enrichments in the femoral artery and vein were similar in the young
and the elderly (Table 3). During amino acid-glucose intake,
phenylalanine enrichments decreased significantly in the femoral artery
and vein. Tissue fluid enrichment (Table 3) was higher in the elderly
in the basal state and decreased significantly during amino
acid-glucose intake only in the elderly, with a significant treatment
by group interaction (P = 0.0373).
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Mixed muscle protein FSR was similar in the basal state in young
and elderly (young, 0.0598 ± 0.0087%/h; elderly, 0.0601 ±
0.0110%/h). The amino acid-glucose mixture caused a significant
increase in mixed muscle protein FSR only in the young
(P = 0.029, basal vs. amino acid-glucose
mixture), whereas there was no change in the elderly (young,
0.0857 ± 0.0119%/h; elderly, 0.0476 ± 0.0153%/h;
P = 0.0241, treatment by group interaction; Fig. 3).
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The model-derived parameters of leg muscle free phenylalanine
kinetics in the young and the elderly in the basal period (240–300
min) and during amino acid-glucose intake (420–480 min) are reported
in Table 4. In the basal state, all of
the model-derived parameters of free phenylalanine kinetics were
similar in young and elderly. Amino acid-glucose intake significantly
increased phenylalanine delivery to the leg and its release from the
leg as well as phenylalanine transport into and from the muscle cells,
with no differences between young and elderly. The rate at which
phenylalanine was shunted from the artery to the vein did not change
significantly in either young or elderly subjects. Muscle protein
breakdown decreased significantly during amino acid-glucose intake in
both groups. The tissue phenylalanine rate of appearance, calculated as
the sum of inward transport and protein breakdown, was similar in the
basal state [young, 172 ± 24; elderly, 209 ± 17
nmol/(min·100 mL/leg)] and increased (P = 0.019)
similarly in the elderly and the young [young, 306 ± 52;
elderly, 278 ± 58 nmol/(min·100 mL/leg)]. The rate of
utilization of phenylalanine for protein synthesis increased
significantly during amino acid-glucose intake only in the young,
whereas there was no change in the elderly. This result was consistent
with the FSR data. Phenylalanine net balance across the leg, which was
negative in the fasting state, became positive during the intake of the
amino acid-glucose mixture in both groups, indicating a shift from net
protein breakdown to net protein synthesis. However, such an increase
was blunted in the elderly.
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Blood glucose enrichments (Fig. 2
) and concentrations (data not
shown) were at steady state during the sampling hour of the basal and
the amino acid-glucose study periods. The results of glucose kinetics
are reported in Table 5. Whole body
glucose Ra, glucose delivery to the leg and release from the leg, and
leg glucose uptake were similar in the basal state in the young and the
elderly and increased significantly and similarly during amino
acid-glucose intake. Endogenous glucose production decreased
significantly and similarly in both young and elderly.
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Discussion |
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In the young, we observed an increase in muscle protein anabolism in response to amino acid-glucose intake as indicated by the shift of phenylalanine net balance across the leg from a negative value, indicating net catabolism, to a positive value. The improvement in net balance was due to both increased protein synthesis and decreased protein breakdown. The results of protein synthesis are consistent with previous data from young subjects showing that hyperinsulinemia during hyperaminoacidemia stimulates muscle protein synthesis (27). As recent studies have shown that muscle protein breakdown is not decreased by the administration of amino acids alone in either the young or the elderly (13, 14, 28), the reduction of muscle protein breakdown observed in the present study was probably caused by the glucose-induced hyperinsulinemia. The effect of insulin on muscle protein breakdown is still controversial; some of the studies show a reduction and others show no effect (reviewed in Ref. 15). It is possible that amino acid availability and/or the physiological conditions during the experiments play an important role in determining the response to insulin. Further investigations are required to clarify this issue.
In the elderly, the shift of net balance from negative to positive in response to amino acids with glucose was significantly blunted compared with that in the young controls. We considered the possibility that the apparent difference in net balance between elderly and young was due to the presentation of the data per unit of total leg volume. If the elderly had a reduced muscle volume relative to the total leg volume, the net balance per unit of total leg volume would be underestimated. Although we could not present our data per unit of muscle volume due to the fact that one volunteer could not undergo the leg MRI, we are convinced that this was not the case. In fact, the older volunteers in our study not only had a smaller muscle volume, but also had a smaller total leg volume, so that the muscle volume relative to the total leg volume was not different between elderly and young (45% vs. 48%). In addition, changes in leg volume would linearly affect the values for both basal and amino acid-glucose periods, so that the magnitude of the response to the amino acid-glucose stimulus (i.e. the change over basal) would not be influenced.
The difference in muscle protein anabolism between elderly and young
subjects was due to a lack of responsiveness of muscle protein
synthesis (FSR and three-pool model) to the amino acid-glucose stimulus
in the elderly. Thus, the net balance shift to a positive value
during amino acid-glucose intake in the elderly was entirely due to a
reduction in protein breakdown. These findings are surprising if we
consider that we have shown in previous experiments that amino acids
alone stimulate net muscle protein anabolism in the elderly, as in the
young, by increasing protein synthesis in the absence of effects on
breakdown (13, 14). One of the previous studies was
performed by administering the same dose of amino acids given in the
present experiment to comparable groups of young and elderly volunteers
(14). This gave us the opportunity to compare the general
effects of amino acids with glucose vs. amino acids alone on
net muscle protein anabolism in young and elderly (Fig. 4). Whereas glucose increased the
positive effect of amino acids in the young, phenylalanine net balance
was slightly, but not significantly, higher during amino acid-glucose
treatment compared with amino acid treatment alone in the elderly.
Overall, these results suggest that whereas in young adults the
addition of glucose to an amino acid mixture augments the effect of
amino acids alone in stimulating muscle protein anabolism, such a
combination in the elderly does not add a real benefit compared with
that of amino acids alone. In fact, the addition of carbohydrate may
even be detrimental, as the added carbohydrate reduces protein turnover
by inhibiting protein breakdown and preventing the stimulatory effect
of amino acids on protein synthesis, thereby reducing muscle tissue
remodeling.
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Glucose per se is an unlikely candidate for the observed negative effect of amino acid-glucose on muscle protein synthesis in the elderly. If anything, the extra calories provided by glucose should have improved, not limited, the response of muscle protein synthesis.
As a direct effect of the administered substrates or differences in substrate availability could be safely excluded, we analyzed the possible role of the endogenous hormonal response on the surprisingly different response of muscle protein synthesis in the elderly.
Previous studies have shown that acute GH administration may (30, 31) or may not (32, 33) stimulate muscle protein synthesis. GH secretion is physiologically increased by amino acids and decreased by glucose administration, and it is generally reduced in older people (34). Assuming that GH directly stimulates muscle protein synthesis, a bigger suppression of GH secretion in the elderly compared with the young could have explained the differences in muscle protein synthesis between the two groups. However, we found that there was a trend for GH concentration to decrease during amino acid-glucose intake only in the young, whereas GH concentrations did not change from basal in the elderly.
On the other hand, consistent with previous data (35), we found that the IGF-I concentration was lower in the elderly than in the young. However, the IGF-I concentration did not change during amino acid-glucose intake in either the elderly or the young. If IGF-I had a role in the alteration of muscle protein synthesis during amino acid-glucose intake, we should have also observed differences in muscle protein synthesis both in the basal state and during the administration of amino acids alone (13, 14). This was not the case.
Thus, we hypothesize that the defect responsible for the age-related impairment of muscle protein synthesis during the intake of amino acids with glucose is due to an alteration in the response of muscle protein synthesis to the glucose-induced endogenous hyperinsulinemia. The finding that leg blood flow was reduced in the elderly during amino acid-glucose treatment is suggestive of an impaired response to insulin in the elderly, because normally insulin increases muscle blood flow (36, 37, 38). In fact, the altered response of blood flow observed in the present study is consistent with previous studies that found a paradoxical reduction of peripheral blood flow in the elderly during insulin infusion (39), as opposed to the vasodilatation observed in young adults (36, 37, 38). This may reflect a general response of insulin resistance, because previous studies have reported a similar alteration in the response of blood flow to insulin in type 2 diabetes patients (40, 41). Nonetheless, the impairment of the response of muscle protein synthesis to the intake of amino acids with glucose with aging is surprising, because the elderly volunteers in our study had normal glucose tolerance and insulin concentrations. In addition, whole body glucose turnover was similar in elderly and young subjects both in the basal state and during amino acid-glucose treatment, and muscle glucose uptake during the intake of amino acid-glucose was not different when comparing the elderly and the young subjects. This raises the possibility that a defect in the anabolic effect of insulin on muscle protein metabolism can develop in elderly individuals who retain the normal or quasi-normal responsiveness of muscle to the stimulatory effect of insulin on glucose uptake. This dichotomy is most likely due to the fact that glucose and protein metabolism are controlled by insulin through distinct intracellular signaling pathways, which can be independently affected by insulin resistance. This hypothesis is indirectly supported by a recent study showing that in conditions characterized by impaired glucose metabolism (obesity and type 2 diabetes) the signaling pathway affected by insulin resistance is the one controlling glucose metabolism (phosphatidylinositol 3-kinase), whereas the mitogen-activated protein kinase pathway, which is linked to cell growth and gene expression, appears to be normally responsive to insulin (42). As both insulin and amino acids stimulate protein synthesis using in part the same intracellular signaling pathways, specifically those that lead to the phosphorylation of the eukaryotic initiation factor 4E-binding protein-1 (43, 44, 45, 46, 47), it is possible that an age-related alteration in the responsiveness to insulin of one or more of these factors or enzymes may lead to the inhibition of the positive effects of amino acids on muscle protein synthesis. This speculation needs to be addressed in future studies.
Consistent with our previous data (14), basal protein turnover was similar in the elderly and the young. Several researchers have reported an age-related reduction in total muscle (48), myofibrillar (11, 49, 50), or myosin heavy chain (51) protein synthesis rates, whereas others (52) and our laboratory (Ref. 14 and the present study) did not find any difference. As previously mentioned (14), the amount of daily physical activity performed by the volunteers might have played a role in the apparent discrepancy between our results and those previously reported. However, our elderly subjects were neither remarkably fit nor exercising regularly, and they had a slightly reduced muscle mass. Nonetheless, it is possible that undetectable differences in the activities of daily living might have been responsible for the discrepancies between our results and those in the literature. Modeling issues can be ruled out, because all of the studies used the same methodology, i.e. the tracer incorporation technique (FSR). In addition, in our studies the lack of a difference between young and elderly subjects in muscle protein synthesis rate was observed using either the three-pool model or the traditional labeled amino acid incorporation technique.
The lack of a difference in basal muscle protein synthesis between elderly and young subjects makes the results of the present study even more striking, suggesting that an altered response of muscle proteins to the combination of amino acids and glucose, with associated hyperinsulinemia, may be one of the mechanisms by which muscle mass is lost with aging, regardless of physical activity. Our results may also provide an explanation for the recent observation that protein requirements may increase with age (8, 9). Essential amino acids are physiologically replenished during the ingestion of proteins, usually occurring in the context of the ingestion of a meal containing carbohydrates. It is reasonable to assume that a diminished response of muscle amino acid utilization similar to that observed in this study occurs during a regular meal. If so, the lack of stimulation of muscle protein in response to a meal will probably contribute over the long term to a progressive loss of muscle. Consequently, due to the diminished effectiveness of the amino acids absorbed after the ingestion of proteins eaten in the context of a mixed meal, additional protein intake is needed to achieve a protein balance. This explanation raises the question of the fate of the absorbed amino acids that are not incorporated into proteins. It is possible that these amino acids are oxidized, and the carbons excreted as CO2. In addition, the carbons from these amino acids could be routed through the lipogenic and/or gluconeogenic pathways. Indirect evidence supporting the utilization of dietary amino acids for fat synthesis when given in combination with carbohydrate comes from the study by Fiatarone et al. (7). They found that whereas mixed multinutritional supplements failed to increase muscle mass when given alone or in combination with exercise training (7), body weight nonetheless increased, suggesting that fat mass increased with the multinutrient supplement.
In conclusion, the results of this study show that muscle protein anabolism, but not glucose turnover, is impaired in healthy elderly volunteers during combined hyperaminoacidemia and endogenous hyperinsulinemia after the ingestion of amino acids with glucose due to the unresponsiveness of protein synthesis. This alteration is likely to contribute to the development, over the long term, of sarcopenia in the elderly.
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Acknowledgments |
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Footnotes |
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2 Recipient of the Brookdale National Fellowship.
Received March 24, 2000.
Revised June 27, 2000.
Revised July 13, 2000.
Accepted August 26, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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76 yr old. Am J Physiol.
277:E118–E125.
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S. Fujita, H. C. Dreyer, M. J. Drummond, E. L. Glynn, E. Volpi, and B. B. Rasmussen Essential amino acid and carbohydrate ingestion before resistance exercise does not enhance postexercise muscle protein synthesis J Appl Physiol, May 1, 2009; 106(5): 1730 - 1739. [Abstract] [Full Text] [PDF]
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N. A. Burd, J. E. Tang, D. R. Moore, and S. M. Phillips Exercise training and protein metabolism: influences of contraction, protein intake, and sex-based differences J Appl Physiol, May 1, 2009; 106(5): 1692 - 1701. [Abstract] [Full Text] [PDF]
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 E. L. Dillon, M. Sheffield-Moore, D. Paddon-Jones, C. Gilkison, A. P. Sanford, S. L. Casperson, J. Jiang, D. L. Chinkes, and R. J. Urban Amino Acid Supplementation Increases Lean Body Mass, Basal Muscle Protein Synthesis, and Insulin-Like Growth Factor-I Expression in Older Women J. Clin. Endocrinol. Metab., May 1, 2009; 94(5): 1630 - 1637. [Abstract] [Full Text] [PDF]
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 S. Verhoeven, K. Vanschoonbeek, L. B Verdijk, R. Koopman, W. K. Wodzig, P. Dendale, and L. J. van Loon Long-term leucine supplementation does not increase muscle mass or strength in healthy elderly men Am. J. Clinical Nutrition, May 1, 2009; 89(5): 1468 - 1475. [Abstract] [Full Text] [PDF]
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 D. K. Layman, E. M. Evans, D. Erickson, J. Seyler, J. Weber, D. Bagshaw, A. Griel, T. Psota, and P. Kris-Etherton A Moderate-Protein Diet Produces Sustained Weight Loss and Long-Term Changes in Body Composition and Blood Lipids in Obese Adults J. Nutr., March 1, 2009; 139(3): 514 - 521. [Abstract] [Full Text] [PDF]
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M. C. Thivierge, J. A. Bush, A. Suryawan, H. V. Nguyen, R. A. Orellana, D. G. Burrin, F. Jahoor, and T. A. Davis Positive net movements of amino acids in the hindlimb after overnight food deprivation contribute to sustaining the elevated anabolism of neonatal pigs J Appl Physiol, December 1, 2008; 105(6): 1959 - 1966. [Abstract] [Full Text] [PDF]
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A. L. D. Riis, J. O. L. Jorgensen, P. Ivarsen, J. Frystyk, J. Weeke, and N. Moller Increased Protein Turnover and Proteolysis Is an Early and Primary Feature of Short-Term Experimental Hyperthyroidism in Healthy Women J. Clin. Endocrinol. Metab., October 1, 2008; 93(10): 3999 - 4005. [Abstract] [Full Text] [PDF]
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 A. Raynaud-Simon, M. Kuhn, J. Moulis, J. Marc, L. Cynober, and C. Loi Tolerance and Efficacy of a New Enteral Formula Specifically Designed for Elderly Persons: An Experimental Study in the Aged Rat J. Gerontol. A Biol. Sci. Med. Sci., July 1, 2008; 63(7): 669 - 677. [Abstract] [Full Text] [PDF]
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R. J. Manders, R. Koopman, M. Beelen, A. P. Gijsen, W. K. Wodzig, W. H. Saris, and L. J. van Loon The Muscle Protein Synthetic Response to Carbohydrate and Protein Ingestion Is Not Impaired in Men with Longstanding Type 2 Diabetes J. Nutr., June 1, 2008; 138(6): 1079 - 1085. [Abstract] [Full Text] [PDF]
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D. Paddon-Jones, K. R Short, W. W Campbell, E. Volpi, and R. R Wolfe Role of dietary protein in the sarcopenia of aging Am. J. Clinical Nutrition, May 1, 2008; 87(5): 1562S - 1566S. [Abstract] [Full Text] [PDF]
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D J. Millward, D. K Layman, D. Tome, and G. Schaafsma Protein quality assessment: impact of expanding understanding of protein and amino acid needs for optimal health Am. J. Clinical Nutrition, May 1, 2008; 87(5): 1576S - 1581S. [Abstract] [Full Text] [PDF]
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Z. Cao, Y. Wu, K. Curry, Z. Wu, Y. Christen, and Y. Luo Ginkgo biloba Extract EGb 761 and Wisconsin Ginseng Delay Sarcopenia in Caenorhabditis elegans J. Gerontol. A Biol. Sci. Med. Sci., December 1, 2007; 62(12): 1337 - 1345. [Abstract] [Full Text] [PDF]
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 G. I. Smith, D. T. Villareal, and B. Mittendorfer Measurement of human mixed muscle protein fractional synthesis rate depends on the choice of amino acid tracer Am J Physiol Endocrinol Metab, September 1, 2007; 293(3): E666 - E671. [Abstract] [Full Text] [PDF]
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A. M. W. Petersen, F. Magkos, P. Atherton, A. Selby, K. Smith, M. J. Rennie, B. K. Pedersen, and B. Mittendorfer Smoking impairs muscle protein synthesis and increases the expression of myostatin and MAFbx in muscle Am J Physiol Endocrinol Metab, September 1, 2007; 293(3): E843 - E848. [Abstract] [Full Text] [PDF]
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 S. Fujita, H. C. Dreyer, M. J. Drummond, E. L. Glynn, J. G. Cadenas, F. Yoshizawa, E. Volpi, and B. B. Rasmussen Nutrient signalling in the regulation of human muscle protein synthesis J. Physiol., July 15, 2007; 582(2): 813 - 823. [Abstract] [Full Text] [PDF]
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 S. Fujita, B. B. Rasmussen, J. G. Cadenas, M. J. Drummond, E. L. Glynn, F. R. Sattler, and E. Volpi Aerobic Exercise Overcomes the Age-Related Insulin Resistance of Muscle Protein Metabolism by Improving Endothelial Function and Akt/Mammalian Target of Rapamycin Signaling Diabetes, June 1, 2007; 56(6): 1615 - 1622. [Abstract] [Full Text] [PDF]
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D. Remond, M. Machebeuf, C. Yven, C. Buffiere, L. Mioche, L. Mosoni, and P. P. Mirand Postprandial whole-body protein metabolism after a meat meal is influenced by chewing efficiency in elderly subjects Am. J. Clinical Nutrition, May 1, 2007; 85(5): 1286 - 1292. [Abstract] [Full Text] [PDF]
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 J. I. Barzilay, C. Blaum, T. Moore, Q. Li Xue, C. H. Hirsch, J. D. Walston, and L. P. Fried Insulin Resistance and Inflammation as Precursors of Frailty: The Cardiovascular Health Study Arch Intern Med, April 9, 2007; 167(7): 635 - 641. [Abstract] [Full Text] [PDF]
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S. Fujita, B. B. Rasmussen, J. A. Bell, J. G. Cadenas, and E. Volpi Basal muscle intracellular amino acid kinetics in women and men Am J Physiol Endocrinol Metab, January 1, 2007; 292(1): E77 - E83. [Abstract] [Full Text] [PDF]
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S. Fujita, B. B. Rasmussen, J. G. Cadenas, J. J. Grady, and E. Volpi Effect of insulin on human skeletal muscle protein synthesis is modulated by insulin-induced changes in muscle blood flow and amino acid availability Am J Physiol Endocrinol Metab, October 1, 2006; 291(4): E745 - E754. [Abstract] [Full Text] [PDF]
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R. Koopman, L. Verdijk, R. J. Manders, A. P Gijsen, M. Gorselink, E. Pijpers, A. J. Wagenmakers, and L. J. van Loon Co-ingestion of protein and leucine stimulates muscle protein synthesis rates to the same extent in young and elderly lean men. Am. J. Clinical Nutrition, September 1, 2006; 84(3): 623 - 632. [Abstract] [Full Text] [PDF]
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I. Rieu, M. Balage, C. Sornet, C. Giraudet, E. Pujos, J. Grizard, L. Mosoni, and D. Dardevet Leucine supplementation improves muscle protein synthesis in elderly men independently of hyperaminoacidaemia J. Physiol., August 15, 2006; 575(1): 305 - 315. [Abstract] [Full Text] [PDF]
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J. J. G. C. van den Borne, M. W. A. Verstegen, S. J. J. Alferink, F. H. M. van Ass, and W. J. J. Gerrits Synchronizing the Availability of Amino Acids and Glucose Decreases Fat Retention in Heavy Preruminant Calves J. Nutr., August 1, 2006; 136(8): 2181 - 2187. [Abstract] [Full Text] [PDF]
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G. Caso, P. J. Garlick, L. M. Ballou, J. A. Vosswinkel, M. C. Gelato, and M. A. McNurlan The Increase in Human Muscle Protein Synthesis Induced by Food Intake Is Similar When Assessed with the Constant Infusion and Flooding Techniques J. Nutr., June 1, 2006; 136(6): 1504 - 1510. [Abstract] [Full Text] [PDF]
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 R. Siddiqui, D. Pandya, K. Harvey, and G. P. Zaloga Nutrition Modulation of Cachexia/Proteolysis Nutr Clin Pract, April 1, 2006; 21(2): 155 - 167. [Abstract] [Full Text] [PDF]
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J. J. Kaczor, W. Ziolkowski, J. Antosiewicz, S. Hac, M. A. Tarnopolsky, and J. Popinigis The effect of aging on anaerobic and aerobic enzyme activities in human skeletal muscle. J. Gerontol. A Biol. Sci. Med. Sci., April 1, 2006; 61(4): 339 - 344. [Abstract] [Full Text] [PDF]
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S. Chevalier, R. Gougeon, N. Choong, M. Lamarche, and J. A. Morais Influence of adiposity in the blunted whole-body protein anabolic response to insulin with aging. J. Gerontol. A Biol. Sci. Med. Sci., February 1, 2006; 61(2): 156 - 164. [Abstract] [Full Text] [PDF]
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R. R. Wolfe Skeletal Muscle Protein Metabolism and Resistance Exercise J. Nutr., February 1, 2006; 136(2): 525S - 528S. [Abstract] [Full Text] [PDF]
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S. Fujita and E. Volpi Amino Acids and Muscle Loss with Aging J. Nutr., January 1, 2006; 136(1): 277S - 280S. [Abstract] [Full Text] [PDF]
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J. A. Bell, S. Fujita, E. Volpi, J. G. Cadenas, and B. B. Rasmussen Short-term insulin and nutritional energy provision do not stimulate muscle protein synthesis if blood amino acid availability decreases Am J Physiol Endocrinol Metab, December 1, 2005; 289(6): E999 - E1006. [Abstract] [Full Text] [PDF]
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C. S Katsanos, H. Kobayashi, M. Sheffield-Moore, A. Aarsland, and R. R Wolfe Aging is associated with diminished accretion of muscle proteins after the ingestion of a small bolus of essential amino acids Am. J. Clinical Nutrition, November 1, 2005; 82(5): 1065 - 1073. [Abstract] [Full Text] [PDF]
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L. Holm, B. Esmarck, C. Suetta, K. Matsumoto, T. Doi, M. Mizuno, B. F. Miller, and M. Kjaer Postexercise Nutrient Intake Enhances Leg Protein Balance in Early Postmenopausal Women J. Gerontol. A Biol. Sci. Med. Sci., September 1, 2005; 60(9): 1212 - 1218. [Abstract] [Full Text] [PDF]
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R. G. Highstead, K. D. Tipton, D. L. Creson, R. R. Wolfe, and A. A. Ferrando Incidence of associated events during the performance of invasive procedures in healthy human volunteers J Appl Physiol, April 1, 2005; 98(4): 1202 - 1206. [Abstract] [Full Text] [PDF]
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 H. C. Dreyer and E. Volpi Role of Protein and Amino Acids in the Pathophysiology and Treatment of Sarcopenia J. Am. Coll. Nutr., April 1, 2005; 24(2): 140S - 145S. [Abstract] [Full Text] [PDF]
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D. Paddon-Jones, M. Sheffield-Moore, A. Aarsland, R. R. Wolfe, and A. A. Ferrando Exogenous amino acids stimulate human muscle anabolism without interfering with the response to mixed meal ingestion Am J Physiol Endocrinol Metab, April 1, 2005; 288(4): E761 - E767. [Abstract] [Full Text] [PDF]
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M. Prod'homme, M. Balage, E. Debras, M.-C. Farges, S. Kimball, L. Jefferson, and J. Grizard Differential effects of insulin and dietary amino acids on muscle protein synthesis in adult and old rats J. Physiol., February 15, 2005; 563(1): 235 - 248. [Abstract] [Full Text] [PDF]
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C. Guillet, A. Zangarelli, P. Gachon, B. Morio, C. Giraudet, P. Rousset, and Y. Boirie Whole Body Protein Breakdown Is Less Inhibited by Insulin, But Still Responsive to Amino Acid, in Nondiabetic Elderly Subjects J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6017 - 6024. [Abstract] [Full Text] [PDF]
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W. J. Evans Protein Nutrition, Exercise and Aging J. Am. Coll. Nutr., December 1, 2004; 23(suppl_6): 601S - 609S. [Abstract] [Full Text] [PDF]
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D. K. Layman Protein Quantity and Quality at Levels above the RDA Improves Adult Weight Loss J. Am. Coll. Nutr., December 1, 2004; 23(suppl_6): 631S - 636S. [Abstract] [Full Text] [PDF]
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D. Paddon-Jones, M. Sheffield-Moore, R. J. Urban, A. P. Sanford, A. Aarsland, R. R. Wolfe, and A. A. Ferrando Essential Amino Acid and Carbohydrate Supplementation Ameliorates Muscle Protein Loss in Humans during 28 Days Bedrest J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4351 - 4358. [Abstract] [Full Text] [PDF]
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M. G. Cree, B. R. Newcomer, C. S. Katsanos, M. Sheffield-Moore, D. Chinkes, A. Aarsland, R. Urban, and R. R. Wolfe Intramuscular and Liver Triglycerides Are Increased in the Elderly J. Clin. Endocrinol. Metab., August 1, 2004; 89(8): 3864 - 3871. [Abstract] [Full Text] [PDF]
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J. D. Parkington, N. K. LeBrasseur, A. P. Siebert, and R. A. Fielding Contraction-mediated mTOR, p70S6k, and ERK1/2 phosphorylation in aged skeletal muscle J Appl Physiol, July 1, 2004; 97(1): 243 - 248. [Abstract] [Full Text] [PDF]
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D. Paddon-Jones, M. Sheffield-Moore, X.-J. Zhang, E. Volpi, S. E. Wolf, A. Aarsland, A. A. Ferrando, and R. R. Wolfe Amino acid ingestion improves muscle protein synthesis in the young and elderly Am J Physiol Endocrinol Metab, March 1, 2004; 286(3): E321 - E328. [Abstract] [Full Text]
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T. Trappe, R. Williams, J. Carrithers, U. Raue, B. Esmarck, M. Kjaer, and R. Hickner Influence of age and resistance exercise on human skeletal muscle proteolysis: a microdialysis approach J. Physiol., February 1, 2004; 554(3): 803 - 813. [Abstract] [Full Text] [PDF]
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E. Volpi, H. Kobayashi, M. Sheffield-Moore, B. Mittendorfer, and R. R Wolfe Essential amino acids are primarily responsible for the amino acid stimulation of muscle protein anabolism in healthy elderly adults Am. J. Clinical Nutrition, August 1, 2003; 78(2): 250 - 258. [Abstract] [Full Text] [PDF]
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M. Louis, J. R. Poortmans, M. Francaux, E. Hultman, J. Berre, N. Boisseau, V. R. Young, K. Smith, W. Meier-Augenstein, J. A. Babraj, et al. Creatine supplementation has no effect on human muscle protein turnover at rest in the postabsorptive or fed states Am J Physiol Endocrinol Metab, April 1, 2003; 284(4): E764 - E770. [Abstract] [Full Text] [PDF]
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R. R. Wolfe Regulation of Muscle Protein by Amino Acids J. Nutr., October 1, 2002; 132(10): 3219S - 3224. [Abstract] [Full Text] [PDF]
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M.-A. Arnal, L. Mosoni, D. Dardevet, M.-C. Ribeyre, G. Bayle, J. Prugnaud, and P. Patureau Mirand Pulse Protein Feeding Pattern Restores Stimulation of Muscle Protein Synthesis during the Feeding Period in Old Rats J. Nutr., May 1, 2002; 132(5): 1002 - 1008. [Abstract] [Full Text] [PDF]
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C. Simon and G. Brandenberger Ultradian Oscillations of Insulin Secretion in Humans Diabetes, February 1, 2002; 51(90001): S258 - 261. [Abstract] [Full Text] [PDF]
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 E. Volpi, M. Sheffield-Moore, B. B. Rasmussen, and R. R. Wolfe Basal Muscle Amino Acid Kinetics and Protein Synthesis in Healthy Young and Older Men JAMA, September 12, 2001; 286(10): 1206 - 1212. [Abstract] [Full Text] [PDF]
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R. Roubenoff and C. Castaneda Sarcopenia--Understanding the Dynamics of Aging Muscle JAMA, September 12, 2001; 286(10): 1230 - 1231. [Full Text] [PDF]
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M. J Rennie Grandad, it ain't what you eat, it depends when you eat it - that's how muscles grow! J. Physiol., August 15, 2001; 535(1): 2 - 2. [Full Text] [PDF]
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