Contribution of Amino Acids and Insulin to Protein Anabolism During

Contribution of Amino Acids and Insulin toProtein Anabolism During Meal AbsorptionElena Volpi, Paola Lucidi, Guido Cruciani, Francesca Monacchia, Gianpaolo Reboldi, Paolo Brunetti,Geremia B. Bolli, and Pierpaolo De Feo

The contribution of dietary amino acids and endogenoushyperinsulinemia to prandial protein anabolism still hasnot been established. To this end, leucine estimates ([1-14C]leucine infusion, plasma a-ketoisocaproic acid [KIC]specific activity [SA] as precursor pool SA) of whole-bodyprotein kinetics and fractional secretory rates (FSRs) ofalbumin, fibrinogen, antithrombin III, and immunoglobu-lin G (IgG) were measured in three groups of healthyvolunteers during intragastric infusion of water (con-trols, n = 5), liquid glucose—lipid—amino acid (AA) meal(meal+AA, n = 7), or isocaloric glucose—lipid meal(meal-AA, n = 7) that induced the same insulin responseas the meal+AA. The results of this study demonstratethat i ) by increasing (P < 0.01) whole-body proteinsynthesis and decreasing (P < 0.01) proteolysis, dietaryamino acids account for the largest part (—90%) ofpostprandial protein anabolism; £) the ingestion of anisocaloric meal deprived of amino acids exerts a modestprotein anabolic effect (10% of postprandial proteinanabolism) by decreasing amino acid oxidation and in-creasing (P < 0.01) albumin synthesis; #) albumin FSR isincreased (—20%) by postprandial hyperinsulinemia(meal—AA) and additionally increased (—50%) by aminoacid intake (meal+AA); 4) IgG FSR is stimulated (-40% )by amino acids, not by insulin; and 5) fibrinogen andantithrombin III FSR are not regulated by amino acids orinsulin. Diabetes 45:1245-1252, 1996

The effects of meal ingestion on protein kinetics arethe overall result of the individual effects of anumber of nutrients and hormones in whichplasma concentrations are increased by the ab-

sorptive state. Several studies suggest that among nutrientsand hormones, amino acids and insulin play a primary role inpromoting postprandial protein anabolism (1-4). Differentgroups of investigators have indirectly evaluated the poten-tial contribution of amino acids and insulin by reproducingtheir postprandial plasma concentrations (plasma insulin,—400-500 pmol/1) with isolated or combined intravenousinfusions (1-4). In this regard, Castellino et al. (2) published

From the Department of Internal Medicine, Endocrine and Metabolic Sciences,University of Perugia, Perugia, Italy.

Address correspondence and reprint requests to Dr. Pierpaolo De Feo, DI.M.I.S.E.M., Via E. Dal Pozzo, 06126 Perugia, Italy. E-mail: [email protected].

Received for publication 21 November 1995 and accepted in revised form 2 May1996.

AA, amino acid; apo, apolipoprotein; FSR, fractional secretoiy rate; KIC,a-ketoisocaproic acid; meal+AA, liquid glucose-lipid-amino acid meal;meal-AA, isocaloric glucose-lipid meal; NEFA, nonesterified free fatty acid; SA,specific activity.

the most exhaustive study and reported that amino acidspromoted protein anabolism through the concomitant sup-pression of proteolysis and the stimulation of protein syn-thesis, whereas isolated hyperinsulinemia had only a modestbeneficial effect on protein balance because it induced aparallel decrease of both rates of protein breakdown andsynthesis. Extrapolation of these results to the absorptivestate suggests that amino acids promote postprandial proteinsynthesis, whereas the suppression of whole-body proteoly-sis is mediated by both amino acids and insulin (2). However,any conclusion on the real contribution of amino acids andinsulin to prandial protein anabolism is weakened by thelimits intrinsic to the intravenous infusion approach. Theintravenous administration of amino acids and insulin can-not reproduce the physiology of nutrient absorption, includ-ing the venous portal/peripheral gradient of substrates andhormones. For instance, the intravenous model might under-estimate the effects of amino acids and insulin on hepaticprotein metabolism and overestimate those on extrahepatictissues. At present, the role played by amino acids andinsulin in the regulation of postprandial protein anabolismhas not been established, and, also, the mechanism(s) re-sponsible for whole-body protein accretion are still debated.The results of some studies indicate that the positive proteinbalance induced by the enteral absorption of a mixed meal isexclusively achieved through the suppression of whole-bodyproteolysis (5-7), whereas the results of other studies alsosupport a role for an increase in whole-body protein synthe-sis (8-13).

Recent studies demonstrating that the fractional secretoryrates (FSRs) of albumin and fibrinogen are differentiallyaffected by nutrient intake suggest that meal-inducedchanges in protein synthesis are not in the same directionand that the regulation of protein metabolism in the absorp-tive state is more complex than originally believed (12,14).Individual proteins in which synthesis is affected by mealingestion and the respective contribution of amino acids andinsulin to this regulation are still to be identified.

The aim of this study was to assess the role played byamino acids and insulin in the regulation of whole-bodyprotein kinetics and the FSR of some representative plasmaproteins of hepatic (albumin, fibrinogen, antithrombin III)and extrahepatic (IgG) synthesis during meal absorption inhealthy humans. An original experimental approach basedon the selective intragastric infusion of nutrients was used tosimulate the physiology of meal absorption. The combinedeffect of meal ingestion and prandial hyperinsulinemia wasassessed by comparing protein kinetics during the intragas-tric infusion of water (control study) or of a mixed glucose-lipid-amino acid meal. The isolated contribution of prandial

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Er VOLPI AND ASSOCIATES

hyperinsulinemia was determined by the intragastric infu-sion of an isocaloric meal deprived of amino acids thatresulted in plasma glucose, nonesterified free fatty acids(NEFAs), C-peptide, insulin, and glucagon concentrationssimilar to the complete mixed meal.

RESEARCH DESIGN AND METHODSMaterials. Purity, sterility, and radio purity of L-[l-14C]leucine (specificactivity [SA] 54 mCi/mmol, Amersham International, Buckinghamshire,U.K.) were determined before use (15).Protocol. After receiving Institutional Review Board approval, informedconsent was obtained from 19 healthy volunteers (5 women, 14 men).The subjects were randomly divided into a water (control, n - 5), aliquid glucose-lipid-amino acid (AA) meal (meal+AA) (n = 7), or anisocaloric glucose-lipid meal (meal-AA) (n = 7) group and matchedfor sex, age (control 25 ± 6, meal+AA 23 ± 1, meal-AA 24 ± 1 years,means ± SE), weight (water 64 ± 4, meal+AA 62 ± 2, meal-AA 66 ±4 kg), and BMI (water 22.0 ± 3, meal+AA 22 ± 1, meal-AA 23 ± 1kg/m2). All subjects were studied 3 days after consuming a weight-maintenance diet of 35 kcal-kg"1 -day"1 containing 55, 30, and 15%carbohydrate, fat, and protein, respectively. After fasting overnight, thevolunteers were admitted to the Clinical Research Unit of the Diparti-mento di Medicina Interna e Scienze Endocrine e Metaboliche of theUniversity of Perugia (I) at -0730. At -0800, an 18-gauge plasticcatheter needle was placed in an antecubital vein for the infusion of[l-^CJleucine (Harvard syringe pump, Harvard Apparatus, Ealing, SouthNatick, MA) and saline (0.5 ml/min, Vial Medical pump, Grenoble,France). A contralateral hand vein was cannulated in a retrogradefashion with a 19-gauge butterfly needle, and the hand was maintained at65°C in a thenuoregulated Plexiglas box to permit intermittent samplingof arterialized venous blood (16), and a feeding nasogastric tube wasinserted for intragastric meal or water infusion (Vial Medical pump). At—0900 (0 min), a primed constant intravenous infusion of L-[l-MC]leucine (9 jxCi prime, 0.3 |xCi/min) was started and continued for 8 h.At 240 min, after blood and breath sampling, the intragastric infusion ofwater (water group) or of a liquid mixed meal (meal+AA, meal—AAgroups) was started at a rate of 1.75 ml/min and continued until the endof the study (480 min). The meal+AA provided 17% of total calories fromamino acids, 33% from lipids, and 50% from carbohydrates, for a totalamount of 632 kcal. It was prepared by mixing a complete formula(Isopuramin Plus 10%, Bieffe Medical, Modena, Italy) of nonessential andessential (leucine infusion rate 1.25 ± 0.02 (xmol-kg"1 -min"1) aminoacids with 84 g of glucose and a mixed oil solution (Lipofundin S, B.Braun, Melsungen, Germany). The meal-AA provided the same amountof calories (632 kcal) in the form of glucose (110 g, 56% of total kcal) andlipids (44%).

There were 16 ml of blood and 2-min breath samples collected at -15,0, 60, 120, 180, 200, 220, 240, 300, 360, 420, 440, 460, and 480 min tomeasure the plasma concentrations of albumin, fibrinogen, antithrombinIII, IgG, glucose, NEFA, insulin, C-peptide, glucagon, isoleucine, leucine,a-ketoisocaproic acid (KIC), the plasma SA of leucine and KIC, the SA ofleucine bound to the above plasma proteins and to the lipoprotein VLDLApoB-100 (apolipoprotein [apo]B-100), and the rates of expired total'"COa and '"CO2 SA.Analytical methods. The plasma concentrations of glucose (Beckmanglucose analyzer; Beckman, Palo Alto, CA), NEFA (17), insulin (18),C-peptide (19), glucagon (20), albumin (21), fibrinogen (22), and IgG(radial immunodiffusion, Boehringer Mannheim, Germany) (23) weredetermined, as previously described.

The plasma concentrations of isoleucine, leucine, and KIC; theleucine concentration into the infused mixed meal and the SA of leucine;and KIC were determined by high-performance liquid chromatography(24). Precisely measured volumes of the tracer leucine infusate weremixed with known amounts of unlabeled leucine and analyzed simulta-neously by the same means to determine radioactivity and to calculatethe infusion rates of the tracers (25).

Expired rates of 14CO2 were measured on breath samples collected bydirecting 2 min of expired air via a two-way valve to a 50-liter gas bag.The collected air was slowly aspirated through 200 ml of 1 N ethanol-amine solution to trap the CO2. Triplicate 1-ml aliquots were counted byliquid scintillation. The SA of breath 14CO2 was determined at eachbreath-sampling time by aspirating expired air through hyamine hydrox-ide. The leucine SA in plasma albumin, fibrinogen, antithrombin III (25),IgG, apoB-100, and the 14C radioactivity in KIC, leucine, and CO2 wasdetermined, as previously described (12). During meals, to avoid

apoB-48 contamination, chylomicrons were removed by ultracentrifuga-tion using a density gradient <1.0. SDS-PAGE confirmed the purity ofapoB-100 in the VLDL fraction.Calculations. Rates of radiolabeled isotope administration were deter-mined multiplying the disintegrations per minute (dpm) per milliliter ofinfusate by the infusion rate (ml/min). Estimates of whole-body leucinemetabolism were made at substrate and isotopic steady state between180-240 min (postabsorptive state) and 420-480 min (absorptive state).The rate of total leucine appearance (jjimol • kg"1 • min"1) was calcu-lated by dividing the infusion rate of [14C]leucine (dpm • kg"1 • min"1)by the plasma [14C]KIC SA (dpm/(xmol) (15). During meal+AA infusion,the rate of endogenous leucine appearance (|xmol • kg"1 • min"1) wascalculated by the intragastric leucine infusion rate from the rate of totalleucine appearance, assuming that intragastric delivered leucine iscompletely absorbed and reaches the systemic pool in the form ofleucine or KIC (26). The rate of leucine oxidation (fxmol • kg"1 • min"1)was calculated by dividing the expired rate of 14CO2 (dpm • kg"1

• min"1) by the plasma [14C]KIC SA (dpm/^mol) (15) and assuming a 70and 82% CO2 recovery in the postabsorptive and absorptive state (27),respectively. The rate of nonoxidative leucine disposal (iJimol-kg"1

•min"1) was calculated by subtracting the rate of leucine oxidationfrom the total leucine flux. Net leucine balance (fjimol • kg"1 • min"1)was calculated by subtracting the endogenous leucine flux from thenonoxidative leucine disposal.

The FSR (percent per hour) of plasma albumin, fibrinogen, antithrom-bin III, and IgG was calculated by dividing the increase (slope) in the SAof leucine derived from hydrolyzed proteins (dpm • nmol"1 • min"1)from 180 to 240 min (basal state) and from 420 to 480 min (absorptivestate) by the mean plasma KIC SA (dpm/nmol) over the same timeperiods and corrected to an hourly FSR. The abbreviation FSR is used toindicate the fractional secretory rate of the plasma protein, i.e., thefraction of the total pool of the plasma protein that is secreted by anorgan (liver, plasma cells) per unit of time. We preferred this expressionto that more currently used, fractional synthesis rate, because it betterdefines what we can really measure. In fact, it is possible that a fractionof synthesized protein undergoes degradation before being secreted.Statistical analysis. Statistical analysis was performed by SAS/STATsoftware (rel. 6.08). The effect of treatment on response variables in thepostabsorptive and absorptive state was analyzed using analysis ofvariance for repeated measures (28). Data are expressed as means ± SE.Linearity of label incorporation into plasma proteins was tested accord-ing to the method suggested by Snedecor and Cochran (29).

RESULTSPlasma concentrations of glucose, NEFA, insulin, C-peptide, and glucagon. In the basal state (0-240 min), theplasma concentrations of glucose, NEFA, insulin, C-peptide,and glucagon were not different among the three groups (Fig.1). When compared with water (control group), meal absorp-tion increased (P < 0.01) the plasma concentrations ofglucose, insulin, and C-peptide and decreased those ofNEFA, without differences between the meal+AA or themeal-AA groups. The plasma glucagon concentration wasnot significantly affected by meal intake.Plasma concentrations of leucine, isoleucine, and KIC.During the basal period, the plasma concentrations ofleucine, isoleucine, and KIC did not differ among the threegroups (Fig. 2). The plasma concentrations of leucine andisoleucine were increased (P < 0.05) by meal+AA adminis-tration in comparison to that of water or of the meal-AA.When compared with water, the plasma KIC concentrationswere decreased (P = 0.0007) by meal+AA or meal-AAintake. In each study, the plasma concentrations of leucineand KIC were nearly at steady state over the last hour of thebasal (180-240 min) and absorptive (420-480 min) periods(Fig. 2).Leucine kinetics. The infusion rates of radioactive leucineare reported in Table 1. In each study, 14CO2, [14C]KIC, andleucine SA were nearly at steady state over the last hour ofthe basal (180-240 min) and absorptive (420-480 min)

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mM

mM 0.5

0500

pM 250

nM

ng/L

2

1

0150

100

50

GlucosemM

mM

0 120 240 360 480minutes

FIG. 1. Plasma concentrations of glucose, NEFA, insulin, C-peptide, andglucagon in three groups of healthy subjects during the overnightpostabsorptive state (0-240 min) and during the intragastric infusion ofwater (O), a 632-kcal glucose-lipid-amino acid meal (•, meal+AA), oran isocaloric glucose-lipid meal (D, meal-AA).

periods (Fig. 2). In the basal period, leucine rates of appear-ance, oxidative, and nonoxidative disposal were not differentamong the three groups (Table 1). When compared withwater or meal-AA, meal+AA administration decreased (P <0.01) the rate of endogenous leucine appearance, increased(P < 0.01) the rate of nonoxidative leucine disappearance,doubled (P < 0.01) the rate of leucine oxidation, and resultedin a positive net leucine balance. When compared withwater, meal-AA intake resulted in a marginally significant(P = 0.060) decrement in the rate of leucine oxidation and ina 36%, yet not statistically significant, improvement in therate of net leucine balance.

POSTPRANDIAL PROTEIN METABOLISM

Concentrations and FSR of plasma proteins. The con-centrations of plasma proteins were not different among thethree groups during either the basal or the absorptiveperiods (data not shown). The SA of leucine derived from thehydrolysis of apoB-100 reached a plateau after ~6 h (Fig. 3),and it was not statistically different from the plasma KIC SAover the last hour (420-480 min) independently on water,meal+AA, or meal-AA administration.

The SA of leucine derived from hydrolyzed albumin,fibrinogen, antithrombin III, and IgG increased linearly (P <0.01) from 180 to 240 min and from 420 to 480 min. A straightline fit was adequate to describe the time course of labelincorporation for the data from each subject during the threestudies. The FSR of albumin, fibrinogen, antithrombin III,and IgG are reported in Table 2 and Fig. 4. During thepostabsorptive period, the FSRs of these plasma proteinswere not significantly different among the three groups.When compared with water, albumin FSR was increased(P = 0.0338) by meal-AA administration and additionallyincreased (P = 0.0002) by meal+AA intake (P = 0.0171 vs.meal-AA), whereas fibrinogen and antithrombin III FSRwere not affected by either the deprived or the aminoacid-enriched meals. When compared with water, IgG FSRwas significantly increased (P = 0.0038) by meal+AA, not bymeal-AA administration (P = NS vs. water, P = 0.0007 vs.meal+AA). If the FSR of IgG were calculated using plasmaleucine SA instead of plasma KIC SA as a precursor pool forprotein synthesis, similar statistical results were obtained(data not shown).

DISCUSSIONThe relative contribution of amino acids and insulin toprandial protein anabolism was assessed using a selectiveintragastric infusion of nutrients that evoked the sameendogenous insulin response. When compared with theprevious model based on the intravenous infusion of aminoacids and/or insulin (1-4), our experimental approach bettermimics the physiology of nutrient absorption, since it doesnot bypass splanchnic tissues and reproduces the venousportal/peripheral gradient of substrates and hormones. Thefollowing conclusions can be drawn from the present results:i ) protein anabolism induced by the absorption of a mixed

TABLE 1Tracer infusion rates, plasma leucine, and KIC SAs and whole-body leucine kinetics

Intravenous [l-14C]leucine infusion idpm • kg"1 • min"1)

Leucine SA (dpm/nmol)KIC SA (dpm/nmol)

rate (103

CO2 excretion rate (dpm • kg"1 • min"1)Total leucine Ra (ixmol • kg"1 • minNasogastric leucine infusion rate

(jjunol • kg"1 • min"1)Endogenous leucine Ra

((jtmol • kg"1 • min"1)Oxidative leucine disposal

(|jimol • kg"1 • min"1)Nonoxidative leucine disposal

(ixmol • kg"1 • min"1)Leucine balance (ixmol -kg"1 • min"

"')

Control group

Basal state

8.16 d6.05 ± 0.283.76 ± 0.20803 ± 1112.18 ± 0.12

—

2.18 ± 0.12

0.30 ± 0.04

1.87 ± 0.12-0.30 ± 0.04

Water

:0.596.57 ± 0.244.05 ± 0.24848 ± 1302.02 ± 0.12

—

2.02 ± 0.12

0.30 ± 0.03

1.73 ± 0.14-0.30 ± 0.03

Meal+AA group

Basal state Meal

7.74 ± 0.435.00 ± 0.413.14 ± 0.19599 ± 612.48 ± 0.07

—

2.48 ± 0.07

0.28 ± 0.04

2.19 ± 0.07-0.28 ± 0.04

4.12 ± 0.362.61 ± 0.201234 ± 1033.01 ± 0.12

1.41 ± 0.13

1.60 ± 0.18*

0.58 ± 0.03*

2.43 ± 0.12*0.83 ± 0.13*

Meal-AA group

Basal state Meal

7.87 ± 0.485.89 ± 0.463.43 ± 0.16546 ±442.31 ± 0.15

—

2.31 ± 0.15

0.23 ± 0.02

2.08 ± 0.13-0.23 ± 0.02

7.07 ±3.86 ±605 ±2.05 ±

—

2.05 ±

0.19 ±

1.86 ±-0.19 ±

0.520.14780.12

0.12

0.03

0.100.03

Data are means ± SE. *P < 0.01 vs. water and meal—AA.


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200

150

100

50

130

90

50

1060

uM 40

20

uM

Leucine h+ju CO2SA

Leucine SA

0 120 240 360 480 180minutes

240 420 480

21000

14000

7000

o1 8

6

4

2

5

3

2

n m o 1

dpmnmol

dpmnmol

FIG. 2. Plasma concentrations of leucine, isoleucine,ketoisocaproate, and SAs of expired CO2, plasmaleucine, and ketoisocaproate in three groups of healthysubjects during the overnight postabsorptive state(0-240 min) and during the intragastric infusion ofwater (O), a 632-kcal glucose-lipid-amino acid meal(•, meal+AA), or an isocaloric glucose-lipid meal (D,meal-AA). Leucine kinetics were calculated using theSA of the last hour (180-240 and 420-480 min) of thetwo study periods.

meal is largely (—90%) sustained by dietary amino acids thatinduce a concomitant increase in whole-body protein syn-thesis and decrease in whole-body proteolysis; 2) the inges-tion of an isocaloric glucose-lipid meal slightly reducesprotein catabolism by decreasing amino acid oxidation andpromoting the synthesis of selected proteins (albumin); and3) the absorptive state stimulates the FSR of a number oftarget proteins through the individual or the combined actionof amino acids and insulin: the FSR of albumin is increasedby isolated postprandial hyperinsulinemia and is additionallystimulated by amino acid intake; the FSR of IgG is stimulatedby amino acids, yet it is not under insulin control; in contrast,the FSR of two proteins of the coagulative system (fibrinogenand antithrombin III) is not affected by increased amino acidand/or insulin availability.

The results of this study, in agreement with other recentstudies (9-13), demonstrate that the absorption of a mixedmeal significantly increases the estimates of whole-bodyprotein synthesis. When compared with the control group,the increase in nonoxidative leucine disposal induced by thecomplete mixed meal (0.70 ixmol • kg • min ) accountedfor ~60% of the net leucine balance (1.13 ixmol-kg"1

•min"1), whereas the decrease in the rate of endogenousleucine appearance (0.42 ixmol • kg"1 • min"1) accounted forthe remaining 40%. The fact that the increase in proteinsynthesis is responsible for postprandial protein anabolismshould not be surprising if we consider that this is the onlyway to spare essential dietary amino acids from irreversibleoxidation. Furthermore, it would be difficult to accept thattissue and whole-body protein synthesis are increased whenamino acids are given by the parenteral (2,30-32) and not theenteral route (6,7). In this study, first-pass splanchnic re-moval of dietary leucine was not measured, and we assumedthat intragastric delivered leucine was completely absorbedreaching the systemic pool in the form of leucine or KIC (26).This might not be completely true because Horber et al. (9),who administered to normal subjects a liquid meal with acomposition similar to ours, calculated that —90% of enter-ally delivered leucine reached the systemic circulation. Thus,it is possible that our estimates of the suppressive effect of

the complete mixed meal on the rate of endogenous leucineappearance have been overestimated by ~ 10%.

The administration of the glucose-lipid meal reduced by0.26 ± 0.05 |xmol • kg"1 • min"1 the rate of leucine appear-ance versus the 0.15 ± 0.07 ixmol • kg"1 • min"1 decrementobserved in the control study. Because whole-body proteinsynthesis can only be indirectly calculated by subtracting therate of leucine oxidation from the rate of leucine appearance(whole-body proteolysis), this reduction of leucine appear-ance, albeit not significant, was sufficient to offset the sparingeffect of the glucose-lipid meal on leucine oxidation and toprevent the increase of whole-body protein synthesis. Ourresults suggest that in the absence of dietary amino acids, theenergy delivered as carbohydrates or lipids is used to pro-mote glycogen and fat synthesis but not to increase proteinsynthesis at the expense of endogenous amino acids. In thisregard, postprandial hyperinsulinemia is candidated to play akey regulatory role, considering the well-known stimulatoryeffects of insulin on glycogen (33) and triglyceride synthesis(34) and inhibitory effects on protein breakdown (1,2). Theresults of this study, obtained by inducing a physiologicalinsulin response, substantially confirm the conclusions onthe anabolic effects of insulin drawn by the previous intra-venous infusion studies (1-4). Isolated postprandial hyper-insulinemia promotes protein anabolism by reducing therates of amino acid oxidation and endogenous proteolysisbut does not increase protein synthesis at a whole-bodylevel. It must be stressed that the insulin response induced inthis study by the ingestion of a glucose-lipid meal appears tobe less effective in comparison to the direct intravenousinfusion of insulin (1-4,25) in reducing the rate of whole-body proteolysis, suggesting that the meal-induced reductionof protein breakdown is mainly due to hyperaminoacidemia(10,35) more than to hyperinsulinemia. The rate of netleucine balance improved by —0.1 (xmol • kg"1 • min"1 afterthe glucose-lipid meal and by —1 ixmol • kg"1 • min"1 afterthe isocaloric amino acid—enriched meal (water —0.30 ±0.07, meal-AA -0.19 ± 0.03, meal+AA 0.83 ± 0.15ixmol-kg"1 -min"1) in the presence of similar plasmaconcentrations of glucose, NEFA, insulin, C-peptide, and


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* 2

I 1

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§. 3

| 2CO

£ 1

60 120 180 240 300 360 420 480

60 120 180 240 300 360 420 480

120 180 240 300 360 420 480

minutesFIG. 3. Comparison of plasma ketoisocaproate SA (•, plasma KIC SA;420-480 min) and SA of leucine derived from the hydrolysis of plasmaVLDL apoB-100 (O, leucine SA from ApoB-100; apoB 60-480 min) inthree groups of healthy subjects during the intravenous infusion of[l-l4C]leucine in the postabsorptive state (0-240 min) followed by theintragastric infusion of water (A), a 632-kcal glucose-lipid-amino acidmeal (H), or an isocaloric glucose-lipid meal (C). No statisticaldifferences were observed under these three experimental conditions.

glucagon (Fig. 1). Thus, together, endogenous hyperinsulin-emia, glucose, lipid, and energy meal content accounted for~10% of whole-body prandial protein anabolism, primarilyby reducing the rate of amino acid oxidation, whereas aminoacids alone contributed to —90% of postprandial protein

anabolism by inducing a concomitant stimulation of proteinsynthesis and reduction of protein breakdown. Because inthis study, amino acids alone were not given, further studiesare required to establish whether the ingestion of othernutrients and the associated insulin response play a syner-gistic role by augmenting the anabolic effects of dietaryamino acids on the rates of protein breakdown and synthe-sis.

The differential effects of meal ingestion on the FSR ofindividual plasma proteins indicate that protein synthesis inthe absorptive state is under a complex hormonal andsubstrate regulation. Among liver proteins, the FSR of fibrin-ogen and antithrombin III was unchanged, whereas that ofalbumin was increased by the amino acid-enriched anddeprived meals. The former result suggests that the synthesisof the two proteins of the coagulative system is primarilyregulated by factors different from increased substrate orinsulin availability. This is also supported by in vitro studiesshowing that fibrinogen synthesis is not stimulated by insulin(36), but like that of other acute phase reactant proteins, isenhanced by interleukin-6 (37). The fact that fibrinogen FSRwas decreased by —20% after the intravenous infusion ofphysiological amounts of insulin (25) yet was unchanged inthis study by meal-induced endogenous hyperinsulinemiasuggests that some substrates (possibly glucose or lipids) orhormones/enterohormones stimulated by meal ingestionmust have counterbalanced the negative effects of insulin onfibrinogen synthesis.

In regard to albumin, the results of this study and those ofprevious in vitro and in vivo studies clearly support theconclusion that insulin plays a key role in modulating thesynthetic rate of the protein. In cultured rat hepatocytes,hyperinsulinemia increases (38), and in diabetic rats, insulindeficiency (39) decreases albumin synthesis by directly reg-ulating the transcription of albumin mRNA (40). In healthyhumans, the intravenous infusion of exogenous insulin (25)or the endogenous postprandial insulin response (this study)increase albumin FSR by —20%, while the association ofamino acid ingestion and postprandial hyperinsulinemia in-creases it by —50% (this study). These data, combined withthe demonstration that in insulin-deficient type I diabeticpatients, albumin FSR is decreased in spite of elevatedplasma amino acid concentrations, prove that, in contrast towhole-body protein (41-43), hyperaminoacidemia is not ableto stimulate albumin synthesis in the absence of insulinaction. Very likely, the tool of the insulin's modulatory effectof the albumin synthetic rate should be located at the level ofalbumin mRNA transcription (40). As far as we know, theeffect of insulin on albumin synthesis is at present the onlyavailable demonstration in humans that systemic hyperinsu-linemia can increase protein synthesis in the absence ofexogenous amino acid administration, i.e., despite a reducedintracellular amino acid availability (44). The peculiarity ofthis insulin effect raises the question of what is the essentialmeaning of a direct control of albumin synthesis by insulin.Because insulin increases both the synthesis and the trans-capillary escape rate (45) of albumin and albumin is the mostwidely represented plasma protein (14), we postulate thatinsulin, by promoting the postprandial increase in albuminsynthesis, exerts an important anabolic role because itfacilitates the incorporation into the protein of part of dietaryamino acids and their delivery to peripheral tissues wherethe protein undergoes degradation.


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TABLE 2Increase in SA of leucine derived from the hydrolysis of plasma proteins and hourly FSR of albumin, fibrinogen, antithrombin III, andIgG

Control group Meal+AA group Meal—AA group

Basal state Water Basal state Meal Basal state Meal

AlbuminSA slope (1(T3 dpm • nmol"1 • min"1) 0.212 ± 0.021 0.230 ± 0.018 0.178 ± 0.013FSR (%/h)

FibrinogenSA slope (10~3 dpm • nmol"1

FSR (%/h)Antithrombin III

SA slope (10~3 dpm • nmol"1

FSR (%/h)IgG

SA slope (10 3 dpm • nmol 1

FSR (%/h)

0.338 ± 0.057 0.341 ± 0.038 0.342 ± 0.018

min"1) 0.574 ± 0.045 0.641 ± 0.067 0.554 ± 0.0440.917 ±0.056 0.949 ±0.049 1.058 ± 0.082

min"1) 0.816 ± 0.096 0.931 ± 0.091 1.058 ± 0.1891.302 ± 0.114 1.378 ± 0.102 2.022 ± 0.276

0.219 ± 0.021 0.189 ± 0.021 0.264 ± 0.0320.504 ± 0.039*t 0.330 ± 0.026 0.411 ± 0.040$

min 0.098 ± 0.008 0.113 ± 0.011 0.096 ± 0.0100.155 ± 0.006 0.167 ± 0.008 0.183 ± 0.017

0.489 ± 0.0511.124 ± 0.064

0.871 ± 0.1592.004 ± 0.252

0.109 ± 0.0090.252 ± 0.008§

0.514 ± 0.062 0.643 ± 0.0740.899 ± 0.081 0.999 ± 0.102

1.057 ± 0.130 1.120 ± 0.1081.848 ± 0.206 1.742 ± 0.170

0.112 ± 0.0150.194 ± 0.021

0.127 ± 0.0140.197 ± 0.018

Data are means ± SE. *P < 0.01 vs. water, tP < 0.02 vs. meal-AA, +.P < 0.04 vs. water, §P < 0.01 vs. water and meal-AA.

IgG FSR, like albumin, was increased by the ingestion ofthe complete mixed meal. This result confirms, also from aquantitative point of view (40% increment), our previousobservation in a different group of healthy volunteers (12). Incontrast to albumin, IgG synthesis remained unchangedduring the absorption of the amino acid-deprived meal.Because the only evident variable between the two studieswas the presence or the lack of dietary amino acids, thisresult demonstrates that the postprandial increase in IgGsynthesis (12) is selectively promoted by amino acid intake.Such a direct relationship between immune humoral func-tion and protein nutrition might explain the reduced serumIgG concentrations (46,47) and responses to infectiousagents (48), reported in experimental (47) or clinical (46,48)protein malnutrition and outlines the importance of anadequate amino acid nourishment to all patients with infec-tious diseases and/or reduced immune defenses.

A methodological point that requires detailed discussionconcerns the validity of our estimates of hepatic protein FSR

Albumin Fibrinogen- 0.6

0.5

0.4

0.3

Basalstate

e.

Meal+AA(p<0.01)

Meal-AA(p<0.04)Water

1.2

1.0

0.8

0.6

Meal+AAMeal-AAWater

Basalstate

Antithrombin Immunoglobulins G

r 2.5

o•c:

I 1.5

0.5

Meal+AA ^Meal-AA •?

Basalstate

0.30

0.24

0.18

0.12

Basalstate

o

A Meal+AAy " (p<0.005)

----A Meal-AA—-0 Water

FIG. 4. FSRs of plasma hepatic (albumin, fibrinogen, antithrombin III)and extrahepatic (IgG) proteins in three groups of healthy subjectsduring the overnight postabsorptive state (basal) and during theintragastric infusion of water (O), a 632-kcal glucose-lipid-amino acidmeal (•, meal+AA), or an isocaloric glucose-lipid meal (D, meal+AA).The FSR of all plasma proteins were not different in the basal state;albumin FSR was increased (P = 0.0338) by meal-AA administrationand additionally increased (F = 0.0002) by meal+AA intake (P = 0.0171vs. meal-AA); IgG FSR was increased (P = 0.0038) only by meal+AAadministration; fibrinogen and antithrombin HI FSRs were unaffected bymeal administration.

under the different experimental conditions of this study.Because technical difficulties, i.e., the rapid turnover oftRNAs and the need of large tissue samples, prevent theroutine determination of leucyl-tRNA SA/enrichment in hu-mans, the plasma SA/enrichment of the intracellular leucinemetabolite KIC is currently used as a surrogate for thedetermination of hepatic or muscle protein FSR (49,50). Thevalidity of this assumption can be tested in humans bymeasuring the plateau isotopic enrichment of a fast-turning-over plasma protein secreted by the liver. In fact, the isotopicenrichment/SA of plasma proteins reaches a steady statewhen the precursor (leucyl-tRNA enrichment) and producthave the same value (50). In this regard, the use of apoB-100offers several methodological advantages: 1) apoB-100 isalmost exclusively synthesized by the liver and secreted asVLDL (51), 2) the time required to completely change theplasma protein pool (12,52-54) is relatively short (5-6 h)(Fig. 3), and 3) after secretion, VLDLs are continuouslyconverted to LDLs (51); consequently, at plateau, the enrich-ment/SA of leucine bound to apoB-100 is representative ofthe most recently secreted protein, i.e., reflects eventualchanges in the enrichment/SA of its tRNA (12). The determi-nation of the FSR of different hepatic proteins based onapoB-100 assumes that the labeling of the leucyl-tRNA ofapoB-100 does not differ from that of the leucyl-tRNAs of theother secreted liver proteins. In theory, it is not possible torule out that for the synthesis of specific proteins, anintracellular compartmentalization or an interorgan zonationmight occur, resulting in a differential labeling of the precur-sor pools (55,56). However, it is very likely that the bulk ofleucyl-tRNAs used for the synthesis of exported hepaticproteins is similarly labeled because these proteins aremainly synthesized on membrane-bound ribosomes (57), andtheir tRNAs have too rapid turnover rates (58) to postulate adifferential labeling in the same intracellular region. Re-cently, different groups of investigators (53,54) reported thatplasma KIC enrichment closely approximates the plateauenrichment of apoB-100 in postabsorptive healthy humans.To the best of our knowledge, this is the first study compar-ing plasma KIC SA with the plateau leucine SA of apoB-100under different conditions, such as fasting (basal aminoaci-demia and insulinemia) and the absorption of a mixed meal(hyperaminoacidemia, hyperinsulinemia) or of an aminoacid-deprived meal (hypoaminoacidemia, hyperinsulin-


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emia). The results, summarized in Fig. 3, show that plasmaKIC SA can be used as a reliable surrogate of intrahepaticprecursor pool SA. In fact, independent of the fasted or fedstate, it was not different from the plateau SA of leucinederived from apoB-100. However, we would like to empha-size that during feeding, the use of plasma KIC SA/enrich-ment as a reliable index of the hepatic leucyl-tRNA SA/enrichment cannot be generalized but must be validatedunder the particular experimental circumstances of eachstudy. For instance, Reeds et al. (59), who fed normalsubjects with hourly boluses of a liquid meal containingproteins, carbohydrates, and lipids, observed that plasmaKIC enrichment overestimated by ~30% the enrichment ofleucine bound to apoB-100. The different results between thetwo studies can be explained by the different study designsand by the different plasma KIC/leu enrichment/SA ratiosobtained by Reeds et al. (0.90) and in our study (0.63). Thatour estimates of albumin FSR, based on plasma KIC SA, arecorrect is indirectly suggested by the fact that Hunter et al.(60), using a completely different technique (flooding dose oflabeled phenylalanine), reported albumin FSR values in thefasted and fed (ingestion of a regular meal containingone-third of daily energy requirements) state similar to ours.

In conclusion, the results of this study indicate thatpostprandial protein anabolism in humans is the mean resultof the differential effects of substrates and hormones onprotein kinetics. Amino acids play a primary role, since theyare alone responsible for the postprandial increase in whole-body protein synthesis and they are the major determinantsof the postprandial decrease in whole-body endogenousproteolysis. The ingestion of other substrates (glucose andlipids) and the associated endogenous insulin response con-tribute to protein anabolism primarily by reducing aminoacid oxidation and by increasing the synthesis of albumin.Despite the fact that estimates of whole-body protein syn-thesis are significantly increased, there is a differentialregulation of the synthetic rates of individual proteins bysubstrates and hormones. Albumin synthesis is selectivelypromoted by insulin and IgG synthesis is enhanced by aminoacids, whereas fibrinogen and antithrombin III synthesis isnot affected by meal ingestion.

ACKNOWLEDGMENTSThis study was supported by a research grant of the CNR(Consiglio Nazionale delle Ricerche, Italy), Project #94024112.CTO4.

The authors are indebted to Vania Cesarini for skillfultechnical assistance.

REFERENCES1. Tessari P, Inchiostro S, Biolo G, Trevisan R, Fantin G, Marescotti MC, Iori

E, Tiengo A, Crepaldi G: Differentia] effects of hyperinsulinemia andhyperaniinoacidemia on leucine-carbon metabolism in vivo: evidence fordistinct mechanisms in regulation of net amino acid deposition. J ClinInvest 79:1062-1069, 1987

2. Castellino P, Luzi L, Simonson DC, Haymond M, DeFronzo RA: Effect ofinsulin and plasma amino acid concentrations on leucine metabolism inman: role of substrate availability on estimates of whole body proteinsynthesis. J Clin Invest 80:1784-1793, 1987

3. Luzi L, Castellino P, Simonson DC, Petrides AS, DeFronzo RA: Leucinemetabolism in IDDM: role of insulin and substrate availability. Diabetes39:38-48, 1990

4. Inchiostro S, Biolo G, Bruttomesso D, Fongher C, Sabadin L, Carlini M,Duner E, Tiengo A, Tessari P: Effects of insulin and amino acid infusionon leucine and phenylalanine kinetics in type 1 diabetes. Am J Physiol262:E203-E210, 1992

5. Hoffer LJ, Yang RD, Matthews DE, Bistrian BR, Bier DM, Young VR:

Effects of meal consumption on whole body leucine and alanine kineticsin young adult man. Br J Nutr 53:31-38, 1985

6. Melville S, McNurlan MA, McHardy KC, Broom J, Milne E, Calder AG,Garlick PJ: The role of degradation in the acute control of proteinbalance in adult man: failure of feeding to stimulate protein synthesis asassessed by L-[l-13C]leucine infusion. Metabolism 38:248-255, 1989

7. McNurlan MA, Garlick PJ: Influence of nutrient intake on proteinturnover. Diabetes Metab Rev 5:165-189, 1989

8. Rennie MJ, Edwards RHT, Halliday D, Matthews DE, Wolman SL,Millward DJ: Muscle protein synthesis measured by stable isotopetechniques in man: the effects of feeding and fasting. Clin Sci 63:519-523,1982

9. Horber FF, Haymond MW: Human growth hormone prevents the proteincatabolic side effects of prednisone in humans. J Clin Invest 86:265-272,1990

10. Collin Vidal C, Cayol M, Obled C, Ziegler F, Bommelaer G, Beaufrere B:Leucine kinetics are different during feeding with whole protein oroligopeptides. Am J Physiol 267:E907-E914, 1994

11. Pacy PJ, Price GM, Halliday D, Quevedo MR, Millward DJ: Nitrogenhomeostasis in man: the diurnal responses of protein synthesis anddegradation and amino acid oxidation to diets with increasing proteinintakes. Clin Sci 86:103-118, 1994

12. De Feo P, Volpi E, Lucidi P, Cruciani C, Monacchia F, Reboldi G,Santeusanio F, Bolli GB, Branetti P: Ethanol impairs post-prandialhepatic protein metabolism. J Clin Invest, 95:1472-1479, 1995

13. El-Khoury AE, Sanchez M, Fukagawa NK, Young VR: Whole body proteinsynthesis in healthy adult humans: 13CO2 technique vs. plasma precursorapproach. Am J Physiol 268:E174-E184, 1995

14. De Feo P, Horber FF, Haymond MW: Meal stimulation of albuminsynthesis: a significant contributor to whole body protein synthesis inhumans. Am J Physiol 263:E794-E799, 1992

15. Schwenk WF, Beaufrere B, Haymond MW: Use of reciprocal pool specificactivities to model leucine metabolism in humans. Am J Physiol 249:E646-E650, 1985

16. McGuire E, Helderman J, Tobin J, Andres R, Berman M: Effects of arterialvenous sampling on analysis of glucose kinetics in man. J Appl Physiol41:565-573, 1976

17. Dole V, Meinertz H: Microdetermination of long-chain fatty acids inplasma and tissues. J Biol Chem 235:2595-2599, 1960

18. Herbert V, Lau K, Gottlieb C, Bleicher S: Coated charcoal immunoassayof insulin. J Clin Endocrinol Metab 25:1375-1384, 1965

19. Faber O, Binder C, Markiissen J, Heding L, Naithani V, Kuzuya H, Blix P,Horwitz D, Rubenstein A: Characterization of seven C-peptide antisera.Diabetes 27 (Suppl. l):170-177, 1978

20. Faloona G, Unger R: Glucagon. In Methods of Hormone Radioimmuno-assay. Jaffe B, Behrman H, Eds. New York, Academic Press, 1974, p.317-330

21. Doumas BT, Watson W, Biggs HG: Albumin standards and the measure-ment of serum albumin with bromcresol green. Clin ChimActa 31:87-96,1971

22. Jacobsoon K: Studies on determination of fibrinogen in human bloodplasma. Scand J Clin Lab Invest 7:14-19, 1955

23. Whicher JT, Warren C, Chambers RE: Immunochemical assays forimmunoglobulins. Ann Clin Biochem 21:78-91, 1984

24. Horber FF, Kahl J, Lecavalier L, Krom B, Haymond MW: Determination ofleucine and a-ketoisocaproic acid concentrations and specific activity inplasma and leucine specific activities in proteins using high-performanceliquid chromatography. J Chromatogr 495:81-94, 1989

25. De Feo P, Volpi E, Lucidi P, Cruciani G, Reboldi G, Siepi D, Mannarino E,Santeusanio F, Branetti P, Bolli GB: Physiological increments in plasmainsulin concentrations have selective and different effects on synthesis ofhepatic proteins in normal humans. Diabetes 42:995-1002, 1993

26. Hoerr RA, Matthews DE, Bier DM, Young VR: Leucine kinetics from [2H.,]-and [13C]leucine infused simultaneously by gut and vein. Am J Physiol26O:E111-E117, 1991

27. Hoerr RA, Yu YM, Wagner DA, Burke JF, Young VR: Recovery of 13C inbreath from NaH13CO., infused by gut and vein: effect of feeding. Am JPhysiol 257:E426-E438, 1989

28. Winer BJ: Statistical Principles in Experimental Design. 2nd Ed. NewYork, McGraw-Hill, 1972, p. 261-308

29. Snedecor GW, Cochran WG: Statistical Methods. 7th Ed. Ames, IA, TheIowa State University Press, 1980

30. Bennet WM, Connacher AA, Scrimgeour CM, Rennie MJ: The effect ofamino acid infusion on leg protein turnover assessed by L-[l5N]phenyl-alanine and L-[l-13C]leucine exchange. EurJ Clin Invest 20:41-50, 1990

31. Wolfe RR, Goodenough RD, Burke JF, Wolfe MH: Response of proteinand urea kinetics in burn patients to different levels of protein intake.Ann Stirg 197:163-171, 1983

32. Goulet O, DePotter S, Salas J, Robert JJ, Rongier M, Ben Hariz M, KozietJ, Desjeux JF, Ricour C, Darmaun D: Leucine metabolism at gradedamino acid intakes in children receiving parenteral nutrition. Am JPhysiol 265:E540-E546, 1993

33. Stalmans W, Bollen M, Mvumbi L: Control of glycogen synthesis in healthand disease. Diabetes Metab Rev 3:127-161, 1987

34. Denton R, Brownsey RW, Belsham GJ: A partial view of the mechanismof insulin action. Diabetologia 21:347-362, 1981


Dow



35. Biolo G, Inchiostro S, Tiengo A, Tessari P: Regulation of postprandialwhole-body proteolysis in insulin-deprived IDDM. Diabetes 44:203-209,1995

36. Grieninger G, Plant PW, Liang J, Kalb RG, Amrani D, Mosesson MW,Hertzbergh KM, Pindyck J: Hormonal regulation of fibrinogen synthesisin cultured hepatocytes. Ann NY Acad Sci 408:469-489, 1983

37. Heinrich PC, Castell JV, Andus T: Interleukin-6 and the acute phaseresponse. Biochem J 265:621-636, 1990

38. Lloyd CE, Kalinyak JE, Hutson SM, Jefferson LS: Stimulation of albumingene transcription by insulin in primary cultures of rat hepatocytes. Am JPhysiol 252:C205-C214, 1987

39. Peavy DE, Taylor JM, Jefferson LS: Time course of changes in albuminsynthesis and mRNA in diabetic and insulin-treated diabetic rats. Am JPhysiol 248:E656-E663, 1985

40. Kimball SR, Horetsky RL, Jefferson LS: Hormonal regulation of albumingene expression in primary cultures of rat hepatocytes. Am J Physiol268:E6-14, 1995

41. Nair KS, Garrow JS, Ford C, Mahler RF, Halliday D: Effect of poordiabetic control and obesity on whole body protein metabolism in man.Diabetologia 25:400-403, 1983

42. Nair KS, Ford GC, Halliday D: Effect of intravenous insulin treatment onin vivo whole body leucine kinetics and oxygen consumption in insulin-deprived type 1 diabetic patients. Metabolism 36:491-495, 1987

43. Pacy PJ, Nair KS, Ford C, Halliday D: Failure of insulin infusion tostimulate fractional muscle protein synthesis in type 1 diabetic patients:anabolic effect of insulin and decreased proteolysis. Diabetes 38:618-624, 1989

44. Alvestrand A, DeFronzo RA, Smith D, Wahren J: Influence of hyperinsu-linaemia on intracellular amino acid levels and amino acid exchangeacross splanchnic and leg tissues in uraemia. Clin Sci 74:155-163, 1988

45. Hilsted J, Christensen NJ: Dual effect of insulin on plasma volume andtranscapillary albumin transport. Diabetologia 35:99-103, 1992

46. Aref GH, El-Din MK, Hassan AI, Araby II: Immunoglobulin in kwashior-kor. J Trop Med Hyg 73:186-191, 1970

47. Dionigi R, Zonta A, Dominioni L, Gnes F, Ballabio A: The effects of totalparenteral nutrition on immunodepression due to malnutrition. Ann Surg185:467-474, 1977

48. Reddy V, Srikantia SG: Antibody response in kwashiorkor. Indian J Med

Res 52:1154-1158, 196449. Bier DM: Intrinsically difficult problems: the kinetics of body proteins and

amino acids in man. Diabetes Metab Rev 5:111-132, 198950. De Feo P, Haymond MW: Principles and calculations of the labelled

leucine methodology to estimate protein kinetics in humans. DiabetesNutr Metab 7:165-184, 1994

51. Ginsberg HN: Lipoprotein physiology and its relationship to atherogene-sis. Endocrinol Metab Clin North Am 19:211-228, 1990

52. Venkatesan S, Pacy PJ, Wenham D, Halliday D: Very-low-density lipopro-tein-apolipoprotein B turnover studies in normal subjects: a stableisotope study. Biochem Soc Trans 18:1192-1193, 1990

53. Venkatesan S, Cullen P, Halliday D, Scott J: Stable isotopes show a directrelation between VLDL apoB overproduction and serum triglyceridelevels and indicate a metabolically and bichemically coherent basis forfamilial combined hyperlipidaemia. Arterioscler Thromb 13:1110-1118,1993

54. Cummings MH, Watts GF, Umpleby AM, Hennessy TR, Naoumova R,Slavin BM, Thompson GR, Sonksen PH: Increased hepatic secretion ofvery-low-density lipoprotein apolipoprotein B-100 in NIDDM. Diabetolo-gia 38:959-967, 1995

55. Bennet WM, Haymond MW: Plasma pool source for fibrinogen synthesisin postabsorptive conscious dogs. Am J Physiol 260:E581-E587, 1991

56. Nair KS, Adey D, Chartlon M, Ljungqvist O: Protein metabolism indiabetes mellitus. Diabetes Nutr Metab 8:113-122, 1995

57. Rolleston FS: Membrane-bound and free ribosomes. Subcell Biochem3:91-117, 1974

58. Scornik OA: Faster protein degradation in response to decreased steadystate levels of amino acylation of tRNA"is in Chinese hamster ovary cells.JBiol Chem 258:882-886, 1983

59. Reeds PJ, Hachey DL, Patterson BW, Motil KJ, Klein PD: VLDL apoli-poprotein B-100, a potential indicator of the isotopic labeling of thehepatic protein synthetic precursor pool in humans: studies with multiplestable isotopically labeled amino acids. J Nutr 122:457-466, 1992

60. Himter KA, Ballmer PE, Anderson SE, Broom J, Garlick PJ, McNurlanMA: Acute stimulation of albumin synthesis rate with oral meal feeding inhealthy subjects measured with [ring-2H5]phenylalanine. Clin Sci 88:235-242, 1995


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Contribution of Amino Acids and Insulin to Protein Anabolism During

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