Influence of age on insulin stimulation of amino acid uptake in rat diaphragm.

Abstract 1. The transport of α-amino[1-C14]isobutyric acid by intact rat diaphragm was studied in vitro over a 60-min course of incubation, after a 180-min prior incubation period. Insulin stimulated α-aminoisobutyric acid accumulation by muscle from 10-, 25-, 37-, 50-, and 100-day-old animals by 41, 147, 109, 54, and 23%, respectively. 2. Insulin stimulated uptake by increasing the maximum velocity of active transport (Vmax) in muscle from 10-day-old animals. In contrast, insulin increased the apparent affinity (Km) of the carrier mechanism for α-aminoisobutyric acid in muscle from 25- and 50-day-old animals. 3. Puromycin dihydrochloride inhibited uptake by resting, nonstimulated muscle from 10-, 25-, and 37-day-old animals when the antibiotic was added 180 min before α-aminoisobutyric acid. This inhibitory effect decreased with increasing age. Puromycin dihydrochloride did not inhibit α-aminoisobutyric acid uptake by muscle from 50- and 100-day-old animals. 4. Puromycin had no effect on insulin stimulation of α-aminoisobutyric acid transport in muscle from 10-day-old; reduced uptake in muscle from 25-day-old; but completely abolished subsequent insulin stimulation in muscle from 50-day-old animals. 5. These studies show that insulin stimulates α-aminoisobutyric acid uptake by different mechanisms in skeletal muscle from rats of different ages. The results suggest that insulin affects its stimulatory response in two distinct ways: directly by interacting with membrane carrier mechanisms; and indirectly by initiating the synthesis of a specific protein(s) which enhance transport. Muscle from 10-day-old rats responds only to the direct stimulatory mechanism. Muscle from the 25-day-old rats responds to both direct and indirect stimulation. Muscle from 50-day-old rats has lost responsivity to direct stimulation and relies completely on indirect stimulation through new protein synthesis.


The transport
of cr-amino[l-C4]isobutyric acid by intact rat diaphragm was studied in vitro over a 60-min course of incubation, after a HO-min prior incubation period. Insulin stimulated ol-aminoisobutyric acid accumulation by muscle from lo-, 25-, 37-, 50-, and loo-day-old animals by 41, 147, 109, 54, and 23%, respectively. 2. Insulin stimulated uptake by increasing the maximum velocity of active transport (V,,,) in muscle from lo-day-old animals.
In contrast, insulin increased the apparent affinity (K,) of the carrier mechanism for ar-aminoisobutyric acid in muscle from 25-and 50-day-old animals. 3. Puromycin dihydrochloride inhibited uptake by resting, nonstimulated muscle from lo-, 25-, and 37-day-old animals when the antibiotic was added 180 min before a-aminoisobutyric acid. This inhibitory effect decreased with increasing age. Puromycin dihydrochloride did not inhibit a-aminoisobutyric acid uptake by muscle from 50and loo-day-old animals. 4. Puromycin had no effect on insulin stimulation of cu-aminoisobutyric acid transport in muscle from lo-day-old; reduced uptake in muscle from 25-day-old; but completely abolished subsequent insulin stimulation in muscle from 50day-old animals.
5. These studies show that insulin stimulates cr-aminoisobutyric acid uptake by different mechanisms in skeletal muscle from rats of different ages. Several groups of investigators have demonstrated that insulin stimulates the accumulation of the nonutilizable amino acid, a-aminoisobutyric acid, by intact rat diaphragm muscle (l-3). Insulin also stimulates the incorporation of naturally occurring amino acids into new protein (4). After 180 or 120 min of exposure to inhibitors of protein synthesis before initiating transport studies, an association of these dual stimulatory effects was seen (5, 6). However, a different relationship was found between stimulation of amino acid transport and protein synthesis by insulin when diaphragm muscles from 25-day-old (60 to 90 g) and 50.day-old (220 to 250 g) rats were compared. In the younger animals, prolonged inhibition of protein synthesis inhibited both resting cr-aminoisobutyric acid uptake and the stimulatory effect of insulin.
In the older animals, exposure to inhibitors of protein synthesis did not affect resting transport, but abolished insulin stimulation of subsequent transport. It was postulated that insulin stimulated cY-aminoisobutyric acid transport by skeletal muscle in two ways: directly by affecting a plasma membrane carrier protein; and indirectly by stimulating the synthesis of specific protein(s), which then enhanced transmembrane transport (7). The present studies explore further the relationship of age to the mechanisms by which insulin stimulates amino acid transport. The lo-day-old pups were received as sucklings with their mother.
The older rats were fed Purina rat chow ad lib&m until they were killed by stunning and decapitation.
Diaphragm muscle from suckling lo-day-old animals accumulated cr-aminoisobutyric acid against a concentration gradient to 6.67 ll~ 0.50 times the medium during the 60-min incubation period.
The mean resting control distribution ratio fell to 1.47 A 0.14 in 25-day-old animals, 0.85 f 0.06 in 37-day-old animals, and 0.76 A 0.06 in loo-day-old animals.
The response to insulin stimulation rose and then fell with increasing age. Insulin stimulation was 147% in the 25.day-old group, whereas transport by the younger lo-day-old animals was increased by only 45%.
Note that the lower response to insulin was associated in the youngest group with the highest control distribution ratios.
Peak insulin stimulation occurred in the 25-day-old animals. Further increase in age was associated with decreasing insulin responsiveness.
The 37-and 50-day-old animals responded with 109 and 54% stimulation, respectively, and the oldest animals (loo-day-old) responded least with 23% stimulation.
Doubling the insulin concentration to 0.8 unit per ml failed to cause further stimulation in 50-or loo-day-old animals. In$uence of Age on Inhibition of a-Aminoisobutyric Acid Transport by Puromycin-In previous studies with 25-day-old animals, 180 or 120 min of previous exposure to puromycin inhibited subsequent amino acid transport.
In 50-day-old animals, however, puromycin failed to inhibit ar-aminoisobutyric acid uptake (7). The data in Table II describe the influence of age on the inhibition of resting, non-insulin-stimulated cr-aminoisobutyric acid transport.
The inhibitory response to puromycin decreased with increasing age. In diaphragms from the youngest age group (lo-day-old) the per cent inhibition was greatest (78% inhibition), whereas in muscle from the older 50-and loo-day-old animals, puromycin failed to inhibit a-aminoisobutyric acid uptake.
In separate experiments not presented here puromycin did not impair transport by diaphragm muscle from 50-day-old animals even after 6 hours of prior incubation, or when present in twice the concentration (1.1 MM). Despite the absence of an inhibitory effect of puromycin on a-aminoisobutyric acid uptake in the older animals, inhibition of lysine incorporation into protein was as rapid and as complete as in younger animals (Table  III). This antibiotic inhibited lysine incorporation into protein by 99% in the lo-day-old,  1.46 f 0.09 (9) 4.60 i 0.47 (9) 0.86 f 0.07 (6) 1.70 f 0.08 (6) 0.60 St 0.04 (11) 1.12 f 0.06 (9) 0.94 f 0.12 (9) 1.01 f 0.08 (9) 0.63 f 0.09 (9) 0.71 f 0.06 (9 Table  conditions identical  with those described in Tables I, II, and IV. IV. KD was calculated as described in a and was presented in Each point represents the mean of at least triplicate observations.  Table IV. Despite 3 hours of exposure to puromycin, insulin stimulated cr-aminoisobutyric acid uptake into lo-day-old rat diaphragm by 215%.
Insulin stimulation in the presence of puromycin decreased to 97 and 86% in 25. and 35.day-old animals. By 50 and 100 days of age, no significant insulin stimulation was seen after previous exposure to puromycin.
In the lo-day-old group, insulin stimulated transport more in the presence of puromycin than in its absence (cf. Table I).
The influence of age on insulin stimulation of new protein synthesis was then investigated. As seen in Table V, insulin unexpectedly  failed to stimulate  lysine  incorporation into muscle protein from lo-day-old rats, but effected an expected stimulatory response in muscle from 25day-old animals.
In the younger animals a higher control rate of incorporation (1769 dpm per mg per hour) was seen which remained unchanged in the presence of insulin. In the older animals insulin increased the rate of lysine incorporation from 481 to 703 dpm per mg per hour, a 46% stimulatory effect. Zn$uence of Age on Transport Kinetics-The age-related differences in puromycin inhibition and insulin stimulation of a-aminoisobutyric acid transport were subjected to kinetic analysis.

Akedo and Christensen
(10) previously demonstrated two dis-quadruplicate observations. The results observed at the five highest concentrations were indicated on the expanded scale above. Values for Y were expressed in millimoles per liter per hour.
Values for A, were expressed in millimoles per liter.
tinct processes in diaphragm from 25-to 37-day-old rats: (a) a saturable process was subject to insulin stimulation; (6) a nonsaturable process was unaltered by insulin. Using their mathematical formalism, we defined the diffusion constant (K.) as a parameter of the second, nonsaturable uptake process (Table VI).
Under control conditions the K, was 0.69, 0.29, and 0.36 per hour in diaphragm muscle from lo-, 25., and 50-day-old rats, respectively. These values were unaltered by puromycin and insulin in the older animals, but puromycin reduced the K, in lo-day-old muscle to 0.40 per hour (Fig. la). Representative data for the calculations of KD in 25-and 50-dayold animals are presented in Fig. lb. Neither puromycin alone nor puromycin and insulin changed either the distribution ratio or the K D in 50-day-old animals.
The absent effect of these conditions on the KD for a-aminoisobutyric acid uptake in diaphragms from 25-day-old animals has been reported previously (7). With the observed values for K D the apparent active velocity of transport (Y) was calculated and plotted to obtain the maximum transport velocity (V,,,) and the apparent' affinity (K,).
In the absence of insulin, puromycin reduced the V,,, from 18.2 to 2.6 mmoles per liter per hour in lo-day-old and from 10.0 to 5.0 mmoles per liter per hour in 25-day-old animals. Puromycin had no effect on the V,,, in 50-day-old animals or on the K, in either 25. or 50.day-old animals.
Control values for K, rose with increasing age from 2.2 to 6.7 to 20.0 mM at 10, 25, and 50 days.
Insulin affected transport kinetics differently in the different age groups.
In the 25-and 50-day-old animals insulin stimulated a-aminoisobutyric acid transport by lowering the apparent K, ( lowered the resting K, of 20.0 to 6.7 mM. No change in transport kinetics was seen when puromycin was added 180 min before the initiation of uptake studies, but as seen in Fig. 2, puromycin abolished the expected reduction in K, by insulin.
Insulin could still lower the apparent affinity in diaphragm muscle from 25-day-old animals, although this stimulatory response was partially impaired.
In this age group, despite 3 hours exposure to puromycin, insulin lowered the K, from 6.7 to 4.0 mmoles per liter per hour, but not to 3.0 mmoles per liter per hour noted in the absence of puromycin.
In the lo-day-old animal, insulin evoked a quite different kinetic response (Fig. 3).
Insulin raised the resting Vm,, from 18.2 to 22.2 mmoles per liter per hour.
In the presence of puromycin, insulin increased the V ma* from 2.6 to 6.7 mmoles per liter per hour.
In contrast to the 25-and 50-day-old animals in this age group, insulin did not affect the K, in either the presence or absence of puromycin. That the V,,, was observed at saturation and was significantly raised by insulin is shown in the legend to Fig. 3. Y values of 16.8, 14.9, and 16.7 mmoles per liter per hour were observed at 5,10, and 20 mM cr-aminoisobutyric acid in the absence of insulin. Insulin elevated these values to 28.3, 26.3, and 24.2 mmoles per liter per hour.
This paper describes the developmental changes with aging for a well defined mechanism of insulin action, the acceleration of oc-aminoisobutyric acid transport into rat diaphragm muscle. Previous investigators using 25-to 40-day-old animals defined this saturable, stereospecific, and energy-dependent concentrative process.
In this age range insulin stimulated uptake by raising the apparent affinity of a saturable transport site for a-aminoisobutyric acid (10). These characteristics of a-aminoisobutyric acid transport suggested enzyme mediation across the plasma membrane.
Their existence as a component of the diaphragm muscle transport system remains speculative.
Indirect evidence from our previous studies added evidence for the existence of such "protein carriers" in this system. When prolonged prior Influence of Age and Insulin on Amino Acid Transport Vol. 246, No. 21 incubation with protein synthesis inhibitors preceded t,he t,ransport experiment, the entry process was impaired. Since the antibiotics shown to inhibit or-aminoisobutyric acid transport had no effect on tissue water spaces, cation concentration, oxygen consumption, or the concentration of a-amino nitrogen in the incubation medium, their use in a development'al study would presumably clarify the relationship between stimulation of a-aminoisobutyric acid transport and protein synthesis by insulin.
It is presumed, but not proven, that insulin accelerates a-aminoisobutyric acid transport across muscle membranes by int,eraction with the plasma membrane. Indirect support for this hypothesis comes from the observations of Cuatrecasas (14) that insulin bound to large polymers of sepharose increase glucose utilization and suppress lipolysis by intact fat cells. That insulin stimulates the cell membrane directly was also shown by Martin and Carter (15). These investigators demonstrated that insulin catalyzed glucose transport into isolat'ed microsomal membrane vacuoles.
From the results of prolonged exposure to puromycin and kinetic analysis of insulin stimulat,ion, we have formulated a model for the changes in insulin membrane interacbion which occur with aging (Fig. 4).
Several unexpected observations were made in the lo-day-old animal.
First, puromycin failed to inhibit insulin stimulation of transport despite maximal inhibition of protein synthesis and control transport.
The large inhibitory response to puromycin by non-insulin-stimulated muscle presumably reflected the rapid turnover of carrier proteins, catabolic rates of which were fastest in this age group.
Second, insulin failed to stimulate the incorporation of lysine into new protein, but accelerated ol-aminoisobutyric acid uptake. Finally, insulin stimulated the capacity rather than the apparent affinity of the carrier for its substrate. Similar relationships between insulin kinetics and protein synthesis have been reported in chick heart muscle. Guidotti et al. (16) found that insulin stimulated a-aminoisobutyric acid uptake into 5-day-old chick embryo heart by increasing the maximal velocity of influx. At this developmental stage, insulin did not stimulate new protein synthesis (17). Since insulin did not stimulate new protein in either chick heart or rat diaphragm muscle, the increased capacity cannot be ascribed to new "carrier" protein synthesis. These kinetic data suggest that insulin either uncovers or increases the rate of substrate binding and release by existing mobile carrier units.
Furthermore, these observations indicate that insulin stimulates a-aminoisobutyric acid membrane transport in this age group by a direct effect, independent of new protein synthesis. In contrast to the lo-day-old group, several observations support a dual role for insulin's stimulatory effect in the 25-day-old animals.
First, insulin stimulation was greater in this age group, a finding which suggested additional stimulatory mechanisms. Second, puromycin partially inhibited insulin stimulation, suggesting two mechanisms: one protein dependent, and anot.her independent of new protein synthesis. In both the presence and absence of puromycin, insulin stimulated a-aminoisobutyric acid uptake by increasing the apparent affinity of the transport site for ar-aminoisobutyric acid. However, this observed reduction in K,,, was lessened by puromycin, indicating partial dependence on new protein synthesis. Thus, our model (Fig. 4) suggests that in the 25-day-old group, insulin enhances the affinity of a carrier protein directly through interacting with the membrane taansport site and indirectly by initiating the synthesis of new proteins which stimulate transport.
The experiments in older animals provided strong evidence for two effects of insulin on transport. Puromycin failed to decrease resting non-insulin-stimulated uptake after 2-fold increases in exposure time and 4-fold excesses of inhibitor, implying a slower rate of turnover of postulated carrier proteins. Despite this resistance to puromycin with increasing age, the transport mechanism was absolutely dependent on new protein for insulin stimulation.
In the 50-day-old group, puromycin ablated subsequent stimulatory effects on a-aminoisobutyric acid transport.
This observation suggests that with increasing age, muscle membranes lose their direct responsivity to insulin and rely instead on the initiation or activation of "specific" proteins which then accelerate membrane transport. Our kinetic analysis indicates that these specific proteins act, as in the 25.day-old group, by enhancing the affinity of the transport mechanism for cu-aminoisobutyric acid.
We are unaware of other observations indicating absolute dependence of hormonal stimulation of transport on protein synthesis. However, Garren,Ney,and Davis (18)  Our studies imply that the resistance to insulin stimulation which occurs with increasing age is associated with a progressive decrease in the turnover rate and loss of the direct effect of insulin on oc-aminoisobutyric acid transport proteins. Although the rat diaphragm muscle cannot be directly related to man, as a laboratory model for one mechanism of insulin action, it offers some pertinent points.
In man, resistance to the effects of insulin develops with increasing age (20). Nonspecific effects of obesity, inhibitors, and vascular impermeability to insulin are some of the mechanisms invoked (21). Obviously, in the rat diaphragm model, neither plasma antagonists nor vascular abnormalities can be implicated since neither are present.
Perhaps the increased resistance to insulin expressed by aging muscle reflects a nonspecific effect of increased cell size and obesity.
DiGirolamo and Rudman (22) found that an altered pattern of glucose utilization and resistance to insulin stimulation seen in epididymal adipose tissue from obese rats virtually disappeared with fasting. Reduction of muscle cell size through fasting is impossible without killing the animal, but several points mitigate against obesity as a cause for the observed differences in insulin stimulation of muscle transport at different ages. First, although that portion of uptake mediated through a nonsaturable, non-insulin-responsive diffusion process accounted for a greater portion of the distribution ratio with increasing age, this factor was eliminated when calculating the active velocity of uptake (Y).
With the use of these corrected Y values, the apparent affinity of the carrier mechanism for its substrate decreased with increasing age. Second, the stimulatory effect of insulin exhibited different kinetic changes at different ages: in lo-day-old animals, insulin enhanced the capacity for a-aminoisobutyric acid uptake whereas in 25. and 50.dagold animals, the affinity was increased.
Finally, insulin responsivity did not change proportional to body weight. Ten-dayold animals demonstrate less insulin response than larger 25-or 37-day-old animals.
Our findings in muscle suggest developmental mechanisms independent of obesity. In this regard, Stauffacher and Renold (23) found that medium glucose incor-by guest on March 23, 2020 http://www.jbc.org/ Downloaded from pot&ion into glycogen by diaphragm muscle responded normally to insulin from mice the obesity of which was induced by gold thioglucose.
In contrast, these investigators observed that muscle from genetically obese mice (ObOb and NZO) was resistant to insulin stimulation (23). In our results, normal development is associated with a iise and fall in the inherent sensitivity and a change in the mechanism by which insulin stimulates muscle membrane transport. Perhaps a similar change in end organ sensitivity to insulin develops as man ages resulting in the syndrome(s) now called "maturity onset diabetes."