help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rochon, C.
Right arrow Articles by Grizard, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rochon, C.
Right arrow Articles by Grizard, J.
The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 2 697-706
Copyright © 2000 by The Endocrine Society

Original Studies

Response of Leucine Metabolism to Hyperinsulinemia in Hypothyroid Patients before and after Thyroxine Replacement1

Cecile Rochon, Igor Tauveron, Catherine Dejax, Patrick Benoit, Pierre Capitan, Gerard Bayle, Jacques Prugnaud, Andrea Fabricio2, Christiane Berry, Claude Champredon, Philippe Thieblot and Jean Grizard

Centre de Recherche en Nutrition Humaine d’Auvergne: Unité d’Etude du Métabolisme Azoté, Institut National de la Recherche Agronomique, Centre de Clermont-Ferrand (C.R., P.C., G.B., J.P., C.C., J.G.), 63122 Saint-Genès Champanelle; Service d’Endocrinologie et Maladies Métaboliques CHU de Clermont-Ferrand (C.R., I.T., A.F., C.B., P.T.), B.P. 69, 63003 Clermont-Ferrand cedex; Service de Médecine nucléaire in vivo (C.D.), centre Jean Perrin 63003 Clermont-Ferrand; and Service de Médecine Interne (P.B.), Centre Hospitalier 03000 Moulins, France


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
We have investigated the effect of hypothyroidism and insulin on protein metabolism in humans. Six hypothyroid patients were studied in a postabsorptive state before and after 5 months of regular treatment for hypothyroidism (153 ± 17 µg/day of L-T4). The effect of insulin was assessed under hyperinsulinemic euglycemic and eukalemic conditions. Insulin was infused for 140 min at 0.0063 ± 0.0002 nmol/kg·min. An amino acid infusion was used to blunt insulin-induced hypoaminoacidemia. Whole body protein turnover was measured using L-[1-13C] leucine. When compared to L-T4-induced subclinical thyrotoxic state, hypothyroidism induced a significant decrease (P < 0.05) in leucine endogenous appearance rate (a reflection of proteolysis; 0.89 ± 0.09 vs. 1.33 ± 0.05 µmol/kg·min), oxidation (0.19 ± 0.02 vs. 0.25 ± 0.03 µmol/kg·min), and nonoxidative disposal (a reflection of protein synthesis; 0.87 ± 0.11 vs. 1.30 ± 0.05 µmol/kg·min). Insulin lowered proteolysis during both the subclinical thyrotoxic and hypothyroid states. Hypothyroidism impaired the antiproteolytic effects of insulin. Thyroid hormones are, therefore, essential for the normal antiproteolytic action of insulin.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
HYPOTHYROIDISM is associated with numerous alterations in metabolisms, such as hypercholesterolemia (mainly due to an increase in low-density lipoprotein concentration; Refs. 1, 2), decreased lipolysis (3), and decreased plasma nonesterified fatty acids. Glucose production has been shown to be either decreased (4, 5) or unchanged (6, 7), whereas the glucose-alanine cycle is generally thought to be decreased (8). These metabolic changes may be responsible for the pathophysiological events that affect the general prognosis of hypothyroidism, atherosclerosis, and coronary heart disease. However, although hypothyroidism is known to be associated with muscular weakness, there are few studies examining the state of protein metabolism in these patients (9, 10). Studies that have been performed show that during hypothyroidism whole body leucine flux and protein synthesis are decreased, whereas leg tyrosine efflux is unchanged.

Thyroid hormone treatment can normalize T3, T4, and TSH levels; motor activity and strength; and perhaps whole body protein breakdown and synthesis (9, 10). Studies examining protein metabolism in the hypothyroid state and during thyroid hormone replacement would provide a better understanding of the effect of thyroid hormone treatment on protein metabolism. Additionally, data on the role played by thyroid hormones in the physiological regulation of protein metabolism in healthy humans are very limited, and studies performed in humans with hypothyroidism provide indirect information on the potential role of thyroid hormones in normal individuals.

We have previously demonstrated an interaction between thyroid hormones and insulin on proteolysis during hyperthyroidism (11) (i.e. the ability of exogenous insulin to inhibit whole body proteolysis was increased). The present study was performed to examine whether the lack of thyroid hormones in hypothyroidism lead to an alteration of the antiproteolytic activity of insulin.

We have, therefore, studied the effect of insulin on in vivo protein metabolism in hypothyroid patients before and after regular thyroid hormone treatment. We used an hyperinsulinemic euglycemic potassium clamp with isotopic measurements of leucine turnover. Because amino acid supply is crucial for the effects of insulin on protein synthesis (12), the insulin-induced hypoaminoacidemia was blunted by concomitant amino acid infusion (11).


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patients

The study protocol was approved by the local Ethics Committee (Comité Consultatif pour la Protection des Personnes en Recherche Biomédicale d’Auvergne), and each subject gave written informed consent for the study.

Six hypothyroid patients (mean age, 48.2 ± 4.0 yr; body weight, 74.8 ± 5.5 kg; body mass index, 28.0 ± 2.1 kg/m2, Table 1Go) were studied before and after receiving regular treatment for hypothyroidism. The hypothyroid state was assessed either before the thyroid hormone treatment for primary hypothyroidism (two subjects) or after a 6-week period of thyroid hormone withdrawal to perform postoperative scinti-scans (four subjects). The thyroid treatment included 153 ± 17 µg L-T4 (Levothyrox1; Merck-Lipha, Lyon, France) taken orally each morning (mean, 2.1 ± 0.2 µg/kg; range, 1.3 and 2.5 µg/kg). Based on hormone assays, the patients displayed frank hypothyroidism before treatment (Table 2Go). After 5 months of hormone treatment, plasma free T4 and T3 concentrations increased substantially. The T4-treated patients had a lower TSH level compared to the normal range. A consequence of the regular treatment of hypothyroidism after thyroidectomy for TSH-dependent cancer is always a slightly decreased level of TSH. This is the consequence of negative feedback from T4 treatment. Patients were, therefore, in subclinical thyrotoxic state after treatment. Treatment resulted in a significant decrease in body weight (-2.5 ± 0.2 kg, P < 0.001). Resting heart rate increased from 62.0 ± 4.1 to 76.3 ± 2.8 with treatment (P < 0.05), and clinical symptoms of hypothyroidism were resolved.


View this table:
[in this window]
[in a new window]
  		Table 1. Age, weight, body mass index (BMI), and resting heart rate in subclinical thyrotoxic and hypothyroid state

 

View this table:
[in this window]
[in a new window]
  		Table 2. Plasma thyroid hormones and thyreostimuline concentrations in subclinical thyrotoxic and hypothyroid state

 
Materials

L-[1-13C] leucine and [13C] NaHCO3 (all 99% pure; Mass Trace, Woburn MA) were prepared aseptically in water (final concentration 75.6 and 11.7 µmol/mL, respectively). A beet-derived glucose solution (100 g/L, with a low natural abundance of 13C) was obtained from Braun (St Gallen, Germany). The commercially available amino acid solution (Primène 51; Baxter, Maurepas, France) contained amino acids in the following concentrations (in µmol/mL): L-leucine, 38.17; L-isoleucine, 25.57; L-valine, 32.48; L-lysine, 37.67; L-methionine, 8.05; L-phenylalanine, 12.73; L-threonine, 15.55; L-tryptophan, 4.90; L-alanine, 44.94; L-arginine, 24.14; L-aspartic acid, 22.56; L-cysteine, 10.16 (chlorydrate); L-glutamic acid, 34.01; glycine, 26.67; taurine, 2.40; L-histidine, 12.26; L-proline, 13.05; L-serine, 19.05; L-tyrosine, 2.49; L-ornithine, 8.56.

Experimental procedure (Fig. 1Go)

The same patients were studied in both a hypothyroid (h) and subclinical thyrotoxic (t) state. The subclinical thyrotoxic state was achieved by regular treatment with T4 for 5 months (see above). All studies were performed in a postabsorptive state at 0800 h after a 12-h overnight fast. A sampling catheter (Venflon 21, 20G; Viggo, Helsingborg, Sweden) was inserted in a dorsal hand vein in a retrograde fashion. Another catheter was placed in a contralateral forearm vein for infusions.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 1. Study protocol. Subjects underwent a 320-min period of L-[1-13C]leucine infusion, which included, first a 180-min control period (leucine infusion alone) and then a 140-min period of hyperinsulinemic, euglycemic clamp. Amino acids and potassium were concomitantly infused to blunt insulin-induced hypoaminoacidemia and hypokalemia, respectively. The bicarbonate pool was primed with an injection of 13C-bicarbonate at time zero. At the same time, a primed constant infusion of (L-1-13C)-leucine was begun and continued throughout the whole experiment to determine leucine kinetics during both the basal and the hyperinsulinemic periods. Blood sampling and expired air collections are indicated by the arrows.

 
Each experiment began with a 320-min period of L-[1-13C]leucine infusion (Fig. 1Go), which included first a 180-min control period (leucine infusion alone) and then a 140-min hyperinsulinemic euglycemic clamp. Hyperinsulinemia was produced by a primed (0.031 ± 0.001 nmol/kg in 15 mL saline) continuous infusion (0.0063 ± 0.0002 nmol/kg·min) of human insulin (Actrapid HM1; Novo, Copenhagen, Denmark) using a syringe pump (Precidor; Infors AG, Basel, Switzerland). Plasma glucose concentrations were maintained at basal levels by an iv infusion of glucose using a peristaltic pump (Vip 2; Becton Dickinson, St. Etienne de St. Geoirs, France). The infusion rate was adjusted every 3–5 min for the first 45 min and every 7–10 min thereafter based on rapid blood glucose concentration measurements (within 1 min after blood sampling).

To blunt the insulin-induced hypoaminoacidemia, amino acids were infused at the constant rate of 1.00 ± 0.01 mL/min using another Vip 2 peristaltic pump. This infusion delivered leucine at a rate of 0.529 ± 0.007 µmol/kg·min. Potassium chloride was administered with the amino acid mixture (67 µmol/min) to avoid insulin-induced hypokalemia. Normal levels of kalemia were maintained during the hyperinsulinemic period in both the hypothyroid and subclinical thyrotoxic states (3.69 ± 0.02 and 3.36 ± 0.02 mEq/L, respectively; each individual value was the mean of nine determinations). In separate experiments, the natural abundance of 13C in the glucose, amino acid, and insulin solutions was determined. From these determinations it was shown that the compounds infused did not artefactually modify 13C enrichments during L-[1-13C] leucine infusion.

To determine leucine kinetics, L-[1-13C] leucine was injected iv as a priming dose (7.01 ± 0.29 µmol/kg in 7 mL fluid) at the beginning of the control period and then was continuously infused at a constant rate (0.16 ± 0.01 µmol/kg·min) for 320 min. The bicarbonate pool was primed with an injection of 13C-bicarbonate (1.47 ± 0.07 µmol/kg in 9 mL).

Sampling

Frequent capillary blood samples were taken for rapid measurement of blood glucose concentrations (Glucotide1 strip; Ames, Bayer Diagnostics, Puteaux, France). Arterialized venous blood was taken from the dorsal hand catheter, during the control and hyperinsulinemic periods (Fig. 1Go), by placing the forearm and hand in a heating box (60C). Blood samples were collected in heparinized tubes, centrifuged at 4C, and the resulting plasma stored at -20C for subsequent analyses. Expired air was analyzed in a custom-made apparatus (11). Expired-air was collected for seven 4-min periods (i.e. at the last three sampling points in the control period and the last four sampling points during the hyperinsulinemic euglycemic clamp). The expired-air samples were dried on CaCl2 and passed through a gasometer (Slonic, Schlumberger, France) for determination of the volume. A fraction of the expired-air sample was drawn into a bottle filled with a soda lime-sodium hydroxide mixture (1:4 wt/wt) to measure the CO2 content. Another fraction was collected into evacuated glass tubes (Vacutainer1; Becton Dickinson, Grenoble, France) for storage before 13C enrichment analysis. The rate of expired CO2 was significantly lower in the hypothyroid vs. the subclinical thyrotoxic state (7.5 ± 0.8 vs. 9.5 ± 0.3 mmol/min during the control period; 8.4 ± 0.9 vs. 10.3 ± 0.2 mmol/min during the hyperinsulinemic period; P < 0.05).

Analysis of 13C enrichment

Plasma L-[1-13C] leucine enrichments and {alpha}-ketoisokaproic acid ({alpha}-KIC) were determined by GC-MS. Leucine was extracted with chloroform-acetone and {alpha}-KIC with dichloromethane acid (13). Ter-butyldimethylsilyl derivatives of leucine and {alpha}-KIC were prepared from plasma extracts as described previously (13) and analyzed by gas chromatography-mass spectrometry (mass selective detector 5972, coupled with a gas chromatograph 5890 series II; Hewlett Packard, Les Ullis, France). 13CO2 enrichments were measured directly by isotopic ratio mass spectrometry using a V6-Isocrom II GC (Micromass HK, Manchester, UK).

Metabolite assays

Rapid whole blood glucose measurements were performed during the clamp with a glucose analyzer using the glucose oxidase procedure (Glucometer 4; Bayer Diagnostics, Puteaux, France). These glucose determinations were verified on plasma samples using a more accurate colorimetric enzymatic (GOD/PAP) procedure, (Cobas Mira diagnostic systems, Neuilly sur Seine, France). Plasma potassium was assayed using flame emission (PHF 90 apparatus with K filter; ISA Biologie). Plasma tryptophan was determined using a fluorometric method (using a LS30; Perkin-Elmer, Buckinghamshire, England; Refs. 14, 15).

The concentration of the other amino acids in plasma were measured by ion-exchange chromatography. A special protein precipitation protocol has been developed to avoid sample dilution. Two hundred fifty microliters of a sulfosalicylic acid solution (0.916 mol/L dissolved in ethanol with 0.533 mol/L thiodiglycol and 1.040 mmol/L norleucine) were added to tubes and evaporated to dryness. One milliliter of plasma was then added to tubes and then incubated on ice for 1 h. The tubes were then centrifuged for 1 h (3500 g, +4C), and 500 µL supernatant were added to 250 µL of 0.1 M lithium acetate buffer (pH 2.2). Amino acid concentrations were determined on these extracts using an automated amino acid analyzer (Biotronic LC 3000; Roucaire, Velizy, France; with BTC 2410 resin).

Hormone assays

Plasma insulin was determined by homologous RIA with a commercial kit (INSI-PR; Cis Bio International, Gif sur Yvette, France). The intra- and interassay coefficients of variation were approximately 5% and 10%, respectively. The results for each of the samples taken during the control period (n = 3) and during the hyperinsulinemic period (n = 5) were averaged to give representative results for these three periods. Plasma free T3, free T4 (FT3 Amerlex M and FT4 Amerlex MAB; Kodak, Clinical Diagnostics, Amersham, UK) and TSH levels (Gnost-hTSH; OCPL, Behringwerke AG, Marburg, Germany) were also measured by RIA.

Calculations

Leucine kinetics were calculated using samples taken during the last hour of the control period (at times 130, 150, and 170 min) and the hyperinsulinemic euglycemic clamp (at times 260, 280, 300, and 320 min). Both the primary and reciprocal pool models were used. In the latter, the labeled {alpha}-KIC, the transaminated product of leucine is supposed to better reflect the intracellular-labeled leucine than the plasma-labeled leucine.

Total leucine turnover rate (Q) (µmol/kg·min) was determined using the equation (11, 16, 17, 18, 19):

where F is the L-[1-13C] leucine infusion rate (µmol/kg·min), IEinf is the isotopic enrichment of the infusate (i.e. 99 mol% excess), and IEa (also in mol% excess) is either the plasma L-[1-13C] leucine enrichment (when using the primary pool model) or the plasma [13C] {alpha}-KIC enrichment (when using the reciprocal pool model). Note that Q includes the tracer infusion.

The whole body leucine oxidation rate (Ox) (µmol/kg·min) was calculated from the following equation:

where IECO2 (mol% excess) is the expired 13CO2 enrichment; VCO2 is the amount of expired CO2 (µmol of CO2/kg·min); and 0.8 is a factor correcting for incomplete recovery of labeled bicarbonate.

According to the model, the following equation applies:

where F is the L-[1-13C] leucine infusion rate (µmol/kg·min), I is the rate of iv unlabeled leucine infusion, Ra is the endogenous leucine appearance, and Rd the nonoxidative leucine disposal rate from plasma (all in µmol/kg·min). Knowing Q, F, I, and Ox, Ra (an index of protein breakdown), and Rd (an index of protein synthesis) can be determined. Net leucine balance, an index of protein deposition, is calculated as Rd - Ra. Due to the fact that expired CO2 was not available in one patient, Ox, Rd, and leucine balance were obtained from only five subjects. Moreover, [13C] {alpha}-KIC enrichment was not available in another subject. Value from this subject was calculated from the L-[1-13C] leucine enrichment according to the following equation: [13C] {alpha}-KIC enrichment = 0.761L-[1-13C] leucine enrichment. This relationship was already found in another study (20). It was verified that it did not change with thyroid status or insulin infusion in the present experiment.

Statistical analysis

All data are expressed as means ± SE. Paired t tests were used to compare data obtained during the control and the hyperinsulinemic period. Paired t tests and two-way ANOVA were also used to compare the results obtained during the hypothyroid state with those during the subclinical thyrotoxic state. P <= 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Insulin

Plasma insulin concentrations during the control period (i.e. before insulin infusion) were similar in the hypothyroid and subclinical thyrotoxic states (0.056 ± 0.006 and 0.062 ± 0.011 nmol/L, respectively). Combined insulin, glucose, amino acid, and potassium infusion, which started at 180 min, resulted in a stable hyperinsulinemic state from 230–320 min; the plateau level of plasma insulin was higher in the hypothyroid vs. the subclinical thyrotoxic state (0.652 ± 0.114 vs. 0.417 ± 0.029 nmol/L, respectively, P < 0.05).

Glucose

Plasma glucose concentrations remained constant during the control period and were not different for the hypothyroid and subclinical thyrotoxic states (4.86 ± 0.15 vs. 5.13 ± 0.11 mmol/L, respectively). Plasma glucose was maintained during hyperinsulinemia at a level similar to the control levels (5.36 ± 0.40 and 4.87 ± 0.21 in hypothyroid and subclinical thyrotoxic states, respectively). The mean coefficients of variation for plasma glucose over the whole hyperinsulinemic period were 6.0 and 7.2% in hypothyroid and subclinical thyrotoxic states, respectively.

To maintain glycemia during hyperinsulinemia, the rate of exogenous glucose infusion was started at 5.52 ± 0.53 µmol/kg·min. It was increased gradually during the 180–250-min period in the hypothyroid state and during the 180–280-min period in the subclinical thyrotoxic state and remained constant thereafter. The mean coefficients of variation of the exogenous rate of glucose infusion during the plateau period were 6.1% and 2.8% in hypothyroid and subclinical thyrotoxic state, respectively. The mean rates of exogenous glucose infusion at plateau were lower in the hypothyroid than the subclinical thyrotoxic state (22.41 µmol/kg·min vs. 34.28 µmol/kg·min, P < 0.001).

Plasma free amino acids (Table 3Go)

Leucine concentrations were similar in the hypothyroid and subclinical thyrotoxic state during the control period (130 min, 108.9 ± 6.7 and 129.8 ± 6.9 µmol/L·min, respectively; 170 min, 108.9 ± 7.6 and 130.3 ± 7.2). This was also the case during insulin infusion (min, 138.4 ± 1.5 and 142.1 ± 5.3; min, 128.4 ± 5.9 and 137.7 ± 6.5; min, 127.5 ± 2.4 and 127.5 ± 6.6; min, 123.0 ± 8.4 and 127.0 ± 7.8). Leucine concentration significantly increased during the insulin period compared to the control period in both states. Importantly, plasma leucine concentrations were at steady state, during the time period used for estimation of 13C leucine enrichments (the coefficients of variation were 2.9% and 4.4% in hypothyroid and subclinical thyrotoxic state, respectively, during the 120–180-min period). The corresponding values during the insulin infusion period (260–320 min) were 5.5% and 5.4%.


View this table:
[in this window]
[in a new window]
  		Table 3. Plasma-free essential amino acid concentrations and serine (in µmol/L) in the subclinical thyrotoxic and hypothyroid states during the control period and the euglycemic euaminoacidemic hyperinsulinemic clamp

 
During the control period, there were similar concentrations of the other free amino acids in the hypothyroid and subclinical thyrotoxic states, with the exception of serine whose concentration was increased in hypothyroid state. The amino acid concentrations were at steady state during the entire 130–170-min control period (data not shown). The mean coefficient of variation of plasma free amino acids was between 2% and 10%.

To maintain aminoacidemia under hyperinsulinemic conditions, a constant rate of exogenous amino acids was infused (1.00 ± 0.01 mL/min). After 40 min of combined insulin, glucose, potassium and amino acids infusion (230-min time point), a moderate but significant (P < 0.05) increase in most free amino acids (leucine, isoleucine, phenylalanine, lysine, arginine, tryptophan and histidine) was observed in both the hypothyroid and subclinical thyrotoxic state; this was also the case for alanine and valine in the hypothyroid state. In contrast, tyrosine and citrulline concentrations decreased in both states, and threonine, proline, and aspartic acid + asparagine decreased in the subclinical thyrotoxic state (P < 0.05). There was no significant difference in plasma free amino acid concentrations between 50 and 250 min of the hyperinsulinemic clamp (250–330-min period): the mean coefficients of variation for the amino acids were between 4% and 10%. There was no significant change during the control and hyperinsulinemic periods for serine concentration for both groups.

Plasma leucine 13C enrichment, {alpha}KIC 13C enrichment, and expired 13CO2 enrichment

In both states, the 13C leucine, 13C {alpha}KIC, and expired 13CO2 enrichments were measured at 130, 150, and 170 min during control period and at 260, 280, 300, and 320 min after the start of 13C leucine infusion (i.e. during insulin infusion, Tables 4Go and 5Go). Steady-state leucine, {alpha}-KIC, and expired CO2 enrichments were achieved after 130 min of 13C leucine infusion. The mean coefficients of variation for individuals were 4.2%, 2.5%, and 6.1% during the control period and 5.2%, 3.3%, and 5.0% during the hyperinsulinemic period in the hypothyroid state for leucine, {alpha}-KIC, and expired CO2 enrichments, respectively. In the subclinical thyrotoxic state, the mean coefficients of variation were 4.9%, 6.7%, and 4.6% during the control period and 6.7%, 7.0%, and 4.6% in the hyperinsulinemic period, for leucine, {alpha}-KIC and expired CO2 enrichments, respectively. Enrichments of all compounds during the control period were significantly higher in the hypothyroid state compared to the subclinical thyrotoxic state (except at time 130 min for expired 13CO2 enrichments where the increase did not reach significance).


View this table:
[in this window]
[in a new window]
  		Table 4. 13C enrichment of plasma leucine and KIC as a function of time in subclinical thyrotoxic and hypothyroid states

 

View this table:
[in this window]
[in a new window]
  		Table 5. 13C enrichment of expired CO2 as a function of time in subclinical thyrotoxic and hypothyroid states

 
During insulin infusion, 13C-leucine enrichments decreased only at the 260-min time point in both states (P < 0.05). Insulin infusion failed to modify mean 13C-leucine enrichment regardless of the thyroid status. Therefore, the absolute difference between the insulin and control periods is not different from zero. Enrichments of leucine during hyperinsulinemia were always higher in the hypothyroid state compared to the subclinical thyrotoxic state.

13C-{alpha}KIC enrichments decreased in both groups during hyperinsulinemia (P < 0.05). The mean decrease in 13C-{alpha}KIC enrichments from the control to the insulin period was not significantly different for the hypothyroid and subclinical thyrotoxic states. During hyperinsulinemia, 13C-{alpha}KIC enrichments were always higher in the hypothyroid state compared to the subclinical thyrotoxic state (P < 0.05).

Expired 13CO2 enrichments increased in both groups compared to the control period (Table 5Go). Combined insulin/amino acid/glucose infusion induced a significant increase in 13CO2 enrichments in hypothyroid and subclinical thyrotoxic states. Therefore, we have evaluated whether the magnitude of this effect differed between the hypothyroid and subclinical thyrotoxic state. Thyroid status had no effect on the insulin-induced incremental increase in 13CO2 enrichment. Enrichments of CO2 during hyperinsulinemia were always higher in the hypothyroid compared to the subclinical thyrotoxic state.

Whole body leucine kinetics

Parameters of leucine kinetics are presented in Table 6Go. Based on the "primary pool model," the total leucine flux (including tracer) was 33% lower in the hypothyroid state compared to the subclinical thyrotoxic state during the control period, (P < 0.05). The total leucine flux did not change with insulin infusion. Therefore, there was a similar insulin-induced decrease in total flux in the hypothyroid and subclinical thyrotoxic states (-26%, P < 0.05).


View this table:
[in this window]
[in a new window]
  		Table 6. Effect of insulin on tracer kinetics in the subclinical thyrotoxic and hypothyroid states

 
The rate of appearance of endogenous leucine, an index of protein breakdown, was less in the hypothyroid state compared to the subclinical thyrotoxic state during the control (-35%) and hyperinsulinemic periods (-48%, P < 0.05). Protein breakdown decreased during insulin infusion in both the hypothyroid and subclinical thyrotoxic states (P < 0.05), but the absolute decrease was less in the hypothyroid state (-0.37 ± 0.08 vs. -0.48 ± 0.07 µmol/kg·min, respectively, P < 0.05, Fig. 2aGo). However, when proteolysis is expressed as a percentage of basal, insulin-induced suppression was similar in both groups (-43.9 ± 10.2 and -36.2 ± 5.4 in hypothyroid and subclinical thyrotoxic states, respectively, Fig. 2cGo). The sensitivity of protein breakdown to insulin is presented in Fig. 2bGo as a mean of each individual’s slope of a dose-response curve. As can be seen in Fig. 2bGo, the slope is less steep in the hypothyroid state than in the subclinical thyrotoxic state (0.78 ± 0.19 vs. 1.38 ± 0.19 µmol Leu/kg·min·nmol insulin·L-1, P < 0.003), indicating that in the hypothyroid state there is a reduction in sensitivity to the effect of insulin.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 2. Effect of insulin on endogenous leucine appearance rate (an index of proteolysis) in hypothyroid (h) and subclinical thyrotoxic (t) state. Values (from the primary pool model) are presented as the absolute decrease in leucine appearance between the hyperinsulinemia and the control states (a, in µmol Leu/kg·min), the slope of the dose-response curve of insulin affects on proteolysis (b, in µmol Leu/kg·min/nmol insulin·L-1), and the percentage decrease in proteolysis with insulin infusion (c, in % from basal values). Values are means ± SE for six subjects. *, P < 0.05 subclinical thyrotoxic state compared with hypothyroid state.

 
The nonoxidative leucine disposal (an index of protein synthesis) was significantly decreased in the hypothyroid state when compared to subclinical thyrotoxic state during the control period (-34%, P < 0.05). Insulin infusion had no effect on protein synthesis in the hypothyroid state but decreased protein synthesis moderately (-19%, P < 0.05) in the subclinical thyrotoxic state (as absolute or relative terms).

Oxidative leucine disposal was less in the hypothyroid state compared to the subclinical thyrotoxic state (-36 and -33% during the control and hyperinsulinemic periods, respectively). Leucine oxidation was stimulated by insulin infusion to a similar extent in both states when expressed in absolute (+0.23 ± 0.03 and +0.28 ± 0.03 µmol Leu/kg·min in hypothyroid and subclinical thyrotoxic states, respectively) or relative terms (+127 ± 16% and +120 ± 14%), or as a sensitivity index (0.16 ± 0.02 and 0.20 ± 0.03 µmol Leu/kg·min·nmol insulin·L-1, respectively).

In the control state, the net leucine balance was not significantly affected by hypothyroidism. Insulin infusion increased leucine balance to similar positive values in both groups (as expressed as absolute or relative values).

The reciprocal pool analysis of the data is presented in Table 6Go. As has been observed by others, all flux values were higher when using the reciprocal pool compared to the primary pool model (total flux (+30%), endogenous appearance (+48%), oxidative disposal (+33%), nonoxidative disposal (+33%)). In contrast, the balance seemed to be lower when using the reciprocal pool method (-115%). Total leucine flux was increased by insulin infusion in the subclinical thyrotoxic and hypothyroid states when using the reciprocal pool, whereas it did not when using the primary pool. The reciprocal pool model did not reveal a decrease in leucine oxidation in the hypothyroid state as the primary pool model did. The two analyses yielded the same results with all the other leucine kinetic parameters.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In agreement with previous experiments in humans (9, 10), whole body protein synthesis was lower in the hypothyroid than the subclinical thyrotoxic state. Animal studies have demonstrated that this is a result of a marked decrease in protein synthesis rates in many tissues (e.g. liver, brain, small intestine, and skeletal muscles (21, 22, 23, 24). Furthermore, it has been shown that thyroid hormones are able to stimulate gene expression and transcriptional synthesis of proteins (25). In the present study, whole body proteolysis was also decreased by hypothyroidism. Morisson et al. (10) suggested that thyroid hormone deficiency in man mainly decreased proteolysis in liver and viscera. However, studies in rats clearly show that skeletal muscles are also affected (23, 26).

The origin of the decrease in protein turnover in hypothyroidism is not completely understood. It could be an indirect effect. It is noteworthy that hypothyroid patients have slower metabolic rate. Because their protein mass was approximately constant (see below), it suggests that they were eating less food. A reduction in food intake, especially dietary protein is known to decrease whole body protein turnover in man (27, 28). However, there is some evidence that the decrease in protein turnover in hypothyroid patients could also result of the direct effect of thyroid hormones depletion.

Conversely, we have previously shown that in hyperthyroidism, protein synthesis and degradation are increased (11). Combining the results of the present experiment with the previous study on hyperthyroidism, a positive correlation between plasma thyroid hormone levels and protein synthesis and degradation (Fig. 3Go) is obtained. Thus, it is clear that thyroid hormone is a major regulator of protein metabolism in humans. Considering protein metabolism, the subclinical thyrotoxic patients, therefore, represent an intermediate state between hypothyroidism and euthyroidism. In other words, the treatment did not completely normalize protein metabolism.



View larger version (11K):
[in this window]
[in a new window]
  		Figure 3. Relationship between plasma-free T3 and rates of whole body protein degradation and synthesis. Data are from the present study ({square}, hypothyroid state; {blacksquare}, subclinical thyrotoxic state) and from Tauveron et al., 1995 ({circ}, euthyroid state; •, hyperthyroid state) and were calculated from the primary pool model. Each individual is represented by the same number before and after treatment: numbers 1–6 before and after hypothyroid treatment; numbers 7–12 before and after experimental hyperthyroidism.

 
The fall in muscle protein breakdown in hypothyroid animals seems to be primarily a result of a suppression of the ATP-dependent process (29, 30, 31). This occurs along with a marked reduction in conjugation of ubiquitin to soluble proteins (32). Thyroid hormones not only stimulate ATP-ubiquitin dependent proteolysis but they also promote intralysosomal proteolysis and increase the content of lysosomal enzymes in muscle (33, 34, 35).

Using continuous infusion of labeled leucine or phenylalanine, many investigators have shown that insulin administration reduces endogenous leucine appearance rate, an index of proteolysis (11, 12, 36, 37, 38, 39, 40, 41). Whole body proteolysis is very sensitive to insulin with consistent effects occurring at low doses of insulin. Skeletal muscle has been recognized as a target for the antiproteolytic action of insulin (39, 40, 42, 43, 44). The mechanism for insulin’s effect on muscle remains unclear, although it is likely that it has a direct role in regulating the ATP-ubiquitin proteasome-dependent pathway. It has been shown in vivo that insulin depresses ubiquitin messenger RNA (mRNA) levels in muscle (45). In cultured myoblasts, insulin down-regulates the levels of ubiquitin-conjugating enzyme mRNA (via a decrease in mRNA stability) (46). A direct effect of insulin on muscle proteolysis is also supported by studies in incubated muscles (47, 48). In addition, Tessari et al. (41) have demonstrated that hyperinsulinemia acutely decreases endogenous amino acid appearance by acting primarily at sites other than skeletal muscle. In liver, insulin’s major effect is through suppression of macroautophagy and cathepsin enzyme activities (see Ref. 49 for a review).

In the present study, hypothyroidism impaired the antiproteolytic effect of insulin. The higher insulin levels in hypothyroid patients were associated with a lower reduction in proteolysis. Conversely, we previously observed that during experimental hyperthyroidism in healthy volunteers the antiproteolytic effects of insulin are increased (11). The inhibition of proteolysis by insulin is, therefore, a direct function of the thyroid hormone state. Perhaps proteolysis is depressed during the hypothyroid state to an extent that it is unable to respond normally to insulin infusion. There may be an interaction between thyroid hormones and insulin in the regulation of the ATP-ubiquitin-proteasome proteolytic pathway in skeletal muscle. Indeed, as has been mentioned above, both these hormones affect the ubiquitin-conjugation pathway. A final mechanism may involve thyroid hormones influencing events within the insulin signaling cascade. In accordance with this hypothesis is the demonstration of increased activity of the insulin receptor tyrosine kinase in skeletal muscle with thyroid hormone treatment. However, it is unlikely that alterations in insulin binding to liver and skeletal muscle receptors is a mechanism because it does not seem to be affected by thyroid status (see Ref. 50 for a review).

In the present experiment, coinfusion of leucine and amino acids produced a large increase in leucine oxidation. The results of previous studies are inconsistent: some studies showed an increase in oxidation with amino acid and insulin infusion (36, 37, 40), whereas others showed no effect. Consistent with an increase in whole body oxidation are studies that have shown that insulin stimulates the activity of the mitochondrial branched-chain ketoacid dehydrogenase enzyme complex in adipose tissue by promoting its dephosphorylation. The infusion of insulin and high amounts of branched-chain amino acids in the present study may have produced a synergistic effect, leading to a large increase in oxidation.

Serine was the only amino acid whose plasma concentration was affected by the hypothyroid state. This may reflect the marked effect of thyroid hormones on liver gluconeogenesis. Several sites of action of thyroid hormones have been identified: at the level of amino acid transport, of the enzyme {alpha}-glycerophosphate deshydrogenase and the gluconeogenic enzymes, such as pyruvate carboxylase and PEPCK (see Ref. 51 for a review).

In this study, the effect of insulin was assessed during a combined glucose, potassium, amino acid clamp. Most essential free amino acids levels in plasma were increased during insulin infusion. Thus, it seems unlikely that these amino acids were limiting for protein synthesis. Amino acids at normal plasma levels are also needed to maintain the sensitivity of muscle protein synthesis to insulin (52) and insulin responsiveness to proteolysis (12).

Because whole body total leucine flux was unchanged by insulin infusion, the stimulation of leucine oxidation revealed a reduction of nonoxidative leucine disposal (an estimate of whole body protein synthesis). As for most studies in man, these results consider labeled leucine and KIC in plasma as a reflection of leucine label in the precursor pool for protein synthesis. In fact, the immediate precursor pool is amino acyl-tRNA. The label in the latter compartment is difficult to assess since tRNA pool is small, tRNA half-life is very short and amino acyl-tRNA are very rapidly renewed (within 1 min). Amino acyl-tRNA originate both from extra and intracellular free amino acid pools. Recent experiments, using infusions of labeled leucine in rat, pig, and man showed that leucyl-tRNA label is intermediate between plasma-free leucine label (a reflection of extracellular leucine label) and muscle-free leucine label (a reflection on intracellular leucine label) (53). Thus, when estimated from plasma 13C leucine enrichment, the total leucine flux is underestimated. In contrast, plasma KIC label was very close to (or slightly higher than) leucyl-tRNA label (53). In other words, the transaminated product reciprocal to the infused tracer may more accurately reflect intracellular events than the primary pool plasma enrichments [e.g. L-[1-13C] leucine (54, 18)]. Other methodologies are needed to correctly analyze the effect of insulin on in vivo protein synthesis, especially at the tissue level (11, 20, 47, 55, 56, 57). Note that contrasting with the primary pool model, the reciprocal pool model did not show any difference in leucine oxidation between the hypothyroid and subclinical thyrotoxic states. This is consistent with the lack of significant differences in leucine concentration between the two states. Indeed, the reciprocal pool model is known to give accurate values for leucine oxidation unlike the primary pool model (58).

There have been several studies examining body composition in hypo- and hyperthyroidism. The most important feature of hypothyroidism is the increase in body fat mass, which occurs even during mild hypothyroidism (59, 60). An increase in extracellular water has also been reported (61). Body cell mass is unchanged or decreased in hypothyroid patients (59, 60). In our study, the weight loss with thyroid hormone replacement may be due partly to a decrease in fat mass. The increase in the ratio, total leucine flux to body weight in subclinical thyrotoxic state, may be a reflection of the decrease in the body weight after thyroid treatment of hypothyroid patients. This increase would be artifactual if body protein did not change. However, when expressed in absolute terms, the increase in total flux with thyroid treatment is very significant (78.27 ± 11.75 vs. 105.70 ± 7.63 µmol/min in hypothyroid and subclinical thyrotoxic state, respectively, P < 0.002).

Changes in the physicochemical properties of the islet membranes induced by hypothyroidism in rats, especially with respect to lipid composition and microviscosity results in a decrease in glucose-stimulated insulin release (62). A similar effect is also seen in vivo after a glucose challenge (63). Surprisingly, basal plasma insulin in the current experiment was normal during the hypothyroid state. Moreover, the steady-state plasma insulin concentration during insulin infusion was significantly higher in the hypothyroid state. A possible explanation for this finding is that the inhibition of insulin secretion by exogenous insulin infusion is reduced. However, this seems unlikely because it has been shown that this phenomenon is not sensitive to the thyroid states in man. Alternatively, there may be a decrease in the metabolic clearance rate of exogenous insulin in hypothyroidism. This would be consistent with the increased metabolic clearance rate and accelerated half-life of insulin that is often observed during hyperthyroidism (64, 65).

In conclusion, our study extends to the hypothyroid state the concepts derived from studies in experimentally induced hyperthyroidism (11). In particular, we have shown that the ability of insulin to inhibit whole body proteolysis in vivo is increased as a function of thyroid hormone levels. Additional studies are needed to determine the origin of the interaction between insulin and thyroid hormones at the level of proteolytic pathways in various tissues.


   Acknowledgments
 
We thank D. Bonin and H. Lafarge for literature requisitions; S. Corny and A. Arvouet for preparing and testing stable isotopes; Dr. S. Brionnet, Baxter France, and G. Dutot for providing the amino acid mixture; and A. Kee and M. Balage for critical comments.


   Footnotes
 
Address for all correspondence and requests for reprints to: J. Grizard, Laboratoire d’Etude du Métabolisme Azoté, INRA Centre de Clermont-Ferrand, 63122 Saint-Genès Champanelle, France.

1 This study was supported by grants from the French Ministère de l’Education Nationale (Programme Hospitalier de Recherche Clinique), Région Auvergne, and Lipha Santé Lyon. Back

2 Present address: Serviço de Endocrinologia, Hospital Universitario Evangelico de Curitiba, Curitiba/pr-CEP 80730-150, Brazil. Back

Received February 12, 1999.

Revised July 22, 1999.

Accepted October 19, 1999.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Thompson GR, Soutar AK, Spengel FA, Jadhav A, Gavigan SJ, Myant NB. 1981 Defects of receptor-mediated low density lipoprotein catabolism in homozygous familial hypercholesterolemia and hypothyroidism in vivo. Proc Natl Acad Sci USA. 78:2591–2595.[Abstract/Free Full Text]
  2. Scarabottolo L, Trezzi E, Roma P, Catapano AL. 1986 Experimental hypothyroidism modulates the expression of the low density lipoprotein receptor by the liver. Atherosclerosis. 59:329–333.[CrossRef][Medline]
  3. Armstrong, KJ, Stouffer JE, Van Inwegen RG, Thompson WJ, Robison GA. 1974 Effects of thyroid hormone deficiency on cyclic adenosine 3':5'-monophosphate and control of lipolysis in fat cells. J Biol Chem. 249:4226–4231.[Abstract/Free Full Text]
  4. Okajima F, Ui M. 1979 Metabolism of glucose in hyper- and hypo-thyroid rats in vivo. Glucose-turnover values and futile-cycle activities obtained with 14C- and 3H-labeled glucose. Biochem J. 182:565–575.[Medline]
  5. Müller MJ, Seitz HJ. 1981 Starvation-induced changes of hepatic glucose metabolism in hypo- and hyperthyroid rats in vivo. J Nutr. 111:1370–1379.
  6. Müller MJ, Paschen U, Seitz HJ. 1983 Thyroid hormone regulation of glucose homeostasis in the miniature pig. Endocrinology. 112:2025–2031.[Abstract/Free Full Text]
  7. Müller MJ, Mitchinson PE, Paschen U, Seitz HJ. 1988 Glucoregulatory function of glucagon in hypo-, eu- and hyperthyroid miniature pigs. Diabetologia. 31:368–374.[CrossRef][Medline]
  8. Huang MT, Lardy HA. 1981 Effects of thyroid states on the Cori cycle, glucose–alanine cycle, and futile cycling of glucose metabolism in rats. Arch Biochem Biophys. 209:41–51.[CrossRef][Medline]
  9. Crispell KR, Parson W, Hollifield G. 1956 A study of the rate of protein synthesis before and during the administration of L-triiodothyronine to patients with myxedema and healthy volunteers using N-15 glycine. J Clin Invest. 35:164–169.
  10. Morrison WL, Gibson JN, Jung RT, Rennie MJ. 1988 Skeletal muscle and whole body protein turnover in thyroid disease. Eur J Clin Invest. 18:62–68.[Medline]
  11. Tauveron I, Charrier S, Champredon C, et al. 1995 Response of leucine metabolism to hyperinsulinemia under amino acid replacement in experimental hyperthyroidism. Am J Physiol. 269:E499–E507.
  12. Flakoll PJ, Kulaylat M, Frexes-Steed M, et al. 1989 Amino acids augment insulin’s suppression of whole body proteolysis. Am J Physiol. 257:E839–E847.
  13. Collin-Vidal C, Cayol M, Obled C, Ziegler F, Bommelaer G, Beaufrère B. 1994 Leucine kinetics are different during feeding with whole protein or oligopeptides. Am J Physiol. 267:E907–E914.
  14. Denckla WD, Dewey HK. 1967 The determination of tryptophan in plasma, liver, and urine. J Lab Clin Med. 69:160–169.[Medline]
  15. Bloxam DL, Warren WH. 1974 Error in the determination of tryptophan by the method of Denkla and Dewey. A revised procedure. Anal Biochem. 60:621–625.[CrossRef][Medline]
  16. Matthews DE, Motil KJ, Rohrbaugh DK, et al. 1980 Measurement of leucine metabolism in man from a primed, continuous infusion of L-[1–3C]leucine. Am J Physiol. 238:E473–E479.
  17. Reeds PJ. 1991 Isotopic estimation of protein synthesis and proteolysis in vivo. In: Niessen S, ed. Modern methods in protein nutrition and metabolism. London: Academic; 249–273.
  18. Schwenk WF, Beaufrère B, Haymond MW. 1985 Use of reciprocal pool specific activities to model leucine metabolism in humans. Am J Physiol. 249:E649–E650.
  19. Tesseraud S, Grizard J, Debras E, et al. 1993 Leucine metabolism in lacting and dry goats: effect of insulin and substrate availability. Am J Physiol. 265:E402–E413.
  20. Cayol M, Tauveron I, Rambourdin F, et al. 1995 Whole-body protein turnover and hepatic protein synthesis are increased by vaccination in man. Clin Sci. 89:389–396.[Medline]
  21. Hayase K, Naganuma Y, Moriyama M, Yoshida A, Yokogoshi H. 1997 Effect of a thyroid hormone treatment on brain protein synthesis in rats. Biosci Biotechnol Biochem. 61:1536–1540.[Medline]
  22. Brown JG, Bates PC, Holliday MA, Millward DJ. 1981 Thyroid hormones and muscle protein turnover. The effect of thyroid-hormone deficiency and replacement in thyroidectomized and hypophysectomized rats. Biochem J. 194:771–782.[Medline]
  23. Flaim KE, Li JB, Jefferson LS. 1978 Effects of thyroxine on protein turnover in rat skeletal muscle. Am J Physiol. 235:E231–E236.
  24. Hasselgren PO, Chen IW, James JH, Sperling M, Warner BW, Fischer JE. 1987 Studies on the possible role of thyroid hormone in altered muscle protein turnover during sepsis. Ann Surg. 206:18–24.[Medline]
  25. Müller MJ, Seitz HJ. 1984 Thyroid hormone action on intermediary metabolism. Klin Wochenschr. 62:11–18.[CrossRef][Medline]
  26. Carter WJ, van der Weijden Benjamin WS, Faas FH. 1981 Effect of experimental hyperthyroidism on skeletal-muscle proteolysis. Biochem J. 194:685–690.[Medline]
  27. Soares MJ, Piers LS, Shetty PS, Jackson AA, Waterlow JC. 1994 Whole body protein turnover in chronically undernourished individuals. Clin Sci (Colch). 86:441–446.
  28. Stein TP, Rumpler WV, Leskiw MJ, Schluter MD, Staples R, Bodwell CE. 1991 Effect of reduced dietary intake on energy expenditure, protein turnover, and glucose cycling in man. Metabolism. 40:478–483.[CrossRef][Medline]
  29. Tawa Jr NE, Odessey R, Goldberg AL. 1997 Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles. J Clin Invest. 100:197–203.[Medline]
  30. Tawa NE, Goldberg AL. 1991 Prolonged fasting dietary protein restriction or lack of thyroid hormones suppress both lysosomal and ATP-dependent proteolytic system in muscle. Surg Forum. 42:25–28.
  31. Kettelhut IC, Wing SS, Goldberg AL. 1988 Endocrine regulation of protein breakdown in skeletal muscle. Diabetes Metab Rev. 4:751–772.[Medline]
  32. Solomon V, Lecker SH, Goldberg AL. 1998 The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle. J Biol Chem. 273:25216–25222.[Abstract/Free Full Text]
  33. Tawa NE, Goldberg AL. 1994 Protein and amino acid metabolism in muscle. In: Engel AG, Franzini-Armstrong C, eds. Myology, 2nd ed. New York: McGraw-Hill Book Co.; 683–707.
  34. Mitch WE, Goldberg AL. 1996 Mechanisms of disease: mechanisms of muscle wasting the role of the ubiquitin-proteasome pathway. N Engl J Med. 335:1897–1905.[Free Full Text]
  35. DeMartino GN, Goldberg AL. 1978 Thyroid hormones control lysosomal enzyme activities in liver and skeletal muscle. Proc Natl Acad Sci USA. 75:1369–1373.[Abstract/Free Full Text]
  36. Bennet WM, Connacher AA, Scrimgeour CM, Jung RT, Rennie MJ. 1990 Euglycemic hyperinsulinemia augments amino acid uptake by human leg tissues during hyperaminoacidemia. Am J Physiol. 259:E185–E194.
  37. Castellino P, Luzi L, Simonson DC, Haymond M, DeFronzo RA. 1987 Effect of insulin and plasma amino acid concentrations on leucine metabolism in man. Role of substrate availability on estimates of whole body protein synthesis. J Clin Invest. 80:1784–1793.
  38. Fukagawa NK, Minaker KL, Young VR, Matthews DE, Bier DM, Rowe JW. 1989 Leucine metabolism in aging humans: effect of insulin and substrate availability. Am J Physiol. 256:E288–E294.
  39. Gelfand RA, Barrett EJ. 1987 Effect of physiologic hyperinsulinemia on skeletal muscle protein synthesis and breakdown in man. J Clin Invest. 80:1–6.
  40. Heslin MJ, Newman E, Wolf RF, Pisters PWT, Brennan MF. 1992 Effect of hyperinsulinemia on whole body and skeletal muscle leucine carbon kinetics in humans. Am J Physiol. 262:E911–E918.
  41. Tessari P, Barazzoni R, Zanetti M, et al. 1996 Protein degradation and synthesis measured with multiple amino acid tracers in vivo. Am J Physiol. 270:E733–E741.
  42. Tessari P, Inchiostro S, Biolo G, Vincenti E, Sabadin L. 1991 Effects of acute systemic hyperinsulinemia on forearm muscle proteolysis in healthy man. J Clin Invest. 88:27–33.
  43. Newman E, Heslin MJ, Wolf RF, Pisters PWT, Brennan MF. 1994 The effect of systemic hyperinsulinemia with concomitant amino acid infusion on skeletal muscle protein turnover in the human forearm. Metabolism. 43:70–78.[CrossRef][Medline]
  44. Fryburg DA, Jahn LA, Hill SA, Oliveras DM, Barrett EJ. 1995 Insulin and insulin-like growth factor-I enhance human skeletal muscle protein anabolism during hyperaminoacidemia by different mechanisms. J Clin Invest. 96:1722–1729.
  45. Larbaud D, Debras E, Taillandier D, et al. 1996 Euglycemic hyperinsulinemia and hyperaminoacidemia decreases skeletal muscle ubiquitin mRNA in goats. Am J Physiol. 271:E505–E512.
  46. Wing SS, Bedard N. 1996 Insulin-like growth factor I stimulates degradation of an mRNA transcript encoding the 14 kDa ubiquitinconjugating enzyme. Biochem J. 319:455–461.
  47. Sinaud S, Balage M, Bayle G, et al. 1999 Diazoxide-induced insulin deficiency greatly reduced muscle protein synthesis in rats. Involvement of eIF-4E. Am J Physiol. In press.
  48. Price SR, Bailey JL, Wang X, et al. 1996 Muscle wasting in insulinopenic rats results from activation of the ATP-dependent, ubiquitin-proteasome proteolytic pathway by a mechanism including gene transcription. J Clin Invest. 98:1703–1708.[Medline]
  49. Mortimore GE, Poso AR, Lardeux BR. 1989 Mechanism and regulation of protein degradation in liver. Diabetes Metab Rev. 5:49–70.[Medline]
  50. Tauveron I, Grizard J, Thiéblot PH, Bonin D. 1992 Adaptation métabolique en hyperthyroidie, implication de l’insuline. Diabete Metab. 18:131–136.[Medline]
  51. Beylot M, Laville M. 1994 Thyroid hormones and intermediary metabolism. In: Orgiazzi J, Leclère J, Hostalek U, eds. The thyroid and tissues. Stuttgart and New York: Schattauer; 47–59.
  52. Garlick PJ, Grant I. 1988 Amino acid infusion increases the sensitivity of muscle protein synthesis in vivo to insulin. Effect of branched-chain amino acids. Biochem J. 254:579–584.[Medline]
  53. Watt PW, Lindsay Y, Scrimgeour CM, et al. 1991 Isolation of aminoacyl-tRNA and its labeling with stable isotope tracers: Use in studies of human tissue protein synthesis. Proc Natl Acad Sci USA. 88:5892–5896.[Abstract/Free Full Text]
  54. Horber FF, Horber-Feyder CM, Krayer S, Schwenk WF, Haymond MW. 1989 Plasma reciprocal pool specific activity predicts that of intracellular free leucine for protein synthesis. Am J Physiol. 257 (Endocrinol Metab) 20:E385–E399.
  55. Davis TA, Burrin DG, Fiorotto ML, Reeds PJ, Jahoor F. 1998 Roles of insulin and amino acids in the regulation of protein synthesis in the neonate. J Nutr. 128:347S–350S.[Abstract/Free Full Text]
  56. Russell-Jones DL, Umpleby M. 1996 Protein anabolic action of insulin, growth hormone and insulin-like growth factor I. Eur J Endocrinol. 135:631–642.[Abstract/Free Full Text]
  57. De Féo P. 1996 Hormonal regulation of human protein metabolism. Eur J Endocrinol. 135:7–18.[Abstract/Free Full Text]
  58. Grizard J, Dardevet D, Balage M, et al. 1999 Insulin action on skeletal muscle protein metabolism during catabolic states. Reprod Nutr Dev. In press.
  59. Seppel T, Kosel A, Schlaghecke R. 1997 Bioelectrical impedance assessment of body composition in thyroid disease. Eur J Endocrinol. 136:493–498.[Abstract/Free Full Text]
  60. Wolf M, Weigert A, Kreymann G. 1996 Body composition and energy expenditure in thyroidectomized patients during short-term hypothyroidism and thyrotropin-suppressive thyroxine therapy. Eur J Endocrinol. 134:168–173.[Abstract/Free Full Text]
  61. Nazarov AN, Lobachik VI, Zhidkov VV, Borisov GI, Abrosimov SV. 1987 Characteristics of the hydration status of patients with hypothyroidism. Probl Endokrinol (Mosk). 33:18–21.
  62. Diaz GB, Paladini AA, Garcia ME, Gagliardino JJ. 1993 Changes induced by hypothyroidism in insulin secretion and in the properties of islet plasma membranes. Arch Int Physiol Biochim Biophys. 101:263–269.[Medline]
  63. Doong ML, Wang JW, Chung SC, et al. 1997 Regulation of thyroid hormones in the secretion of insulin and gastric inhibitory polypeptide in male rats. Metabolism. 46:154–158.[CrossRef][Medline]
  64. Dimitriadis G, Baker B, Marsh H, Mandarino L, Rizza R, Bergman R. 1985 Effect of thyroid hormone excess on action, secretion, and metabolism of insulin in humans. Am J Physiol. 248:E593–E601.
  65. Randin JP, Tappy L, Scazziga B, Jequier E, Felber JP. 1986 Insulin sensitivity and exogenous insulin clearance in Graves’ disease. Measurement by the glucose clamp technique and continuous indirect calorimetry. Diabetes. 35:178–181.[Abstract]



This article has been cited by other articles:


Home page
 J. Clin. Endocrinol. Metab.Home page
G. Dimitriadis, P. Mitrou, V. Lambadiari, E. Boutati, E. Maratou, D. B. Panagiotakos, E. Koukkou, M. Tzanela, N. Thalassinos, and S. A. Raptis
Insulin Action in Adipose Tissue and Muscle in Hypothyroidism
J. Clin. Endocrinol. Metab., December 1, 2006; 91(12): 4930 - 4937.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
A. L. D. Riis, J. O. L. Jorgensen, S. Gjedde, H. Norrelund, A. G. Jurik, K. S. Nair, P. Ivarsen, J. Weeke, and N. Moller
Whole body and forearm substrate metabolism in hyperthyroidism: evidence of increased basal muscle protein breakdown
Am J Physiol Endocrinol Metab, June 1, 2005; 288(6): E1067 - E1073.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
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]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rochon, C.
Right arrow Articles by Grizard, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rochon, C.
Right arrow Articles by Grizard, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals