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Insulin Does Not Stimulate Protein Synthesis Acutely in Prepubertal Children with Insulin-Dependent Diabetes Mellitus¹

Department of Pediatrics, Division of Pediatric Endocrinology, Baylor College of Medicine (M.G.V., P.R.B., K.C.C.), Houston, Texas 77030; and the Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: Kenneth C. Copeland, M.D., Department of Pediatrics, Baylor College of Medicine, 6621 Fannin, Suite 850, MC: 3–2351, Houston, Texas 77030. E-mail: Copeland{at}msmail.his.tch.tmc.edu

	Abstract

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Insulin treatment in adult type I diabetic patients decreases protein loss primarily by inhibiting protein breakdown without stimulating protein synthesis. In young growing rodents, insulin treatment has been reported to stimulate protein synthesis. We examined whether insulin stimulates protein synthesis in normally growing prepubertal children with insulin-dependent diabetes mellitus.

Five prepubertal children with insulin-dependent diabetes mellitus (aged 8.6–11.25 yr) were studied in the postabsorptive state on two occasions: once during insulin deprivation (I-; blood glucose, 325 ± 67.8 mg/dL; mean ± SD) and once during insulin administration for 4 h (I+; blood glucose, 96 ± 23.6 mg/dL). Leucine kinetics were measured using a 4-h primed continuous infusion of L-[1-¹³C]leucine.

Serum insulin concentrations were lower (I- vs. I+, 0.6 ± 0.3 vs. 7.5 ± 4.3 µU/mL; mean ± SD; P = 0.02), whereas serum ß-hydroxybutyrate (I- vs. I+, 3.4 ± 0.5 vs. 0.9 ± 0.5 mg/dL; P < 0.001) and free fatty acid concentrations (I- vs. I+, 2.9 ± 0.4 vs. 0.9 ± 0.4 mEq/L; P < 0.001) were higher in the insulin-deprived state than during insulin administration. Leucine Ra, an index of protein breakdown (I- vs. I+, 200.5 ± 23.4 vs. 167 ± 17 µmol/kg·h; P = 0.008), and leucine oxidation (I- vs. I+, 56.5 ± 20.7 vs. 29.6 ± 9.3 µmol/kg·h; P = 0.03) were reduced by insulin treatment. Nonoxidative leucine disposal, an index of protein synthesis, was not affected by insulin treatment (I- vs. I+, 144 ± 20.8 vs. 137.5 ± 13.5 µmol/kg·h; P = 0.4). We conclude that the acute decline in net protein loss during insulin treatment in growing prepubertal children, like that in adults, is due primarily to an inhibition of protein breakdown without stimulation of protein synthesis.

	Introduction

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THE PRIMARY action of insulin on protein metabolism in adults, both healthy subjects and patients with type 1 diabetes mellitus, is an inhibition of protein breakdown without stimulation of protein synthesis (1, 2, 3, 4, 5, 6, 7, 8). However, in vitro studies using rodent tissues have demonstrated that insulin directly stimulates protein synthesis (9, 10, 11). Animal studies indicate that insulin is capable of stimulating protein synthesis in young, rapidly growing rats, but not in adult animals (12, 13). Whether the effect of insulin on protein synthesis in the human is similarly age dependent has never been examined.

In this study we examined the mechanism of insulin’s anticatabolic action in prepubertal children with insulin-dependent diabetes mellitus (IDDM) by measuring whole body protein metabolism using stable isotope methodology in five children both during insulin deprivation (I-) and insulin replacement (I+). Although growth is faster during puberty, in this study only prepubertal children were studied to avoid potential interactions of insulin with the complex hormonal setting of puberty.

	Subjects and Methods

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Subjects

Five prepubertal children with IDDM, one girl and four boys, were studied. All were free of diabetic complications and other medical problems. Ages ranged between 8.6–11.25 yr (mean ± SD, 9.7 ± 1.6 yr), and the mean body mass index was 18 ± 1.5 kg/m². The duration of diabetes was 4 ± 2.8 yr, the daily insulin requirement was 0.8 ± 0.1 U/kg, and the total glycated hemoglobin level was 8.8 ± 0.9% (nondiabetic range, 4.0–6.8%) at the time of the study. The mean growth velocity the year before the study was 5.7 ± 1.4 cm. After disclosure of risks and benefits, written informed consent was obtained. This study was approved by the institutional review board for human subjects at Baylor College of Medicine (Houston, TX).

Study design

Subjects were studied on two occasions separated by at least a 2-month interval (mean ± SD, 3.4 ± 2 months; range, 2–7 months). For each study, subjects were admitted to the Clinical Research Center of Texas Children’s Hospital for a 1-day hospitalization. One study was performed in an insulin-deprived state (I-), and the other was conducted during insulin replacement (I+). Studies were performed in random order.

Insulin deprivation (I-). NPH or other long or intermediate acting insulin was discontinued 72 h before the study. This was done to ensure a uniform state of profound insulinopenia in all subjects after short term withdrawal of all forms of insulin. Glycemic control was achieved with multiple sc injections of short acting insulin (regular insulin) given before each meal, at bedtime, and at 0200 h. The last regular insulin injection was given 9 h before the metabolic study.

Insulin replacement (I+). Subjects remained on their usual home insulin regimen (usually split/mixed NPH and regular insulin) until the morning of the metabolic study. At that time, the usual morning insulin dose was omitted, and each subject received an iv bolus of regular insulin according to the following sliding scale: no insulin for blood glucose less than 120 mg/dL, 0.025 U/kg for blood glucose concentrations between 120–180 mg/dL, and 0.05 U/kg for blood glucose levels greater than 180 mg/dL, followed immediately by a continuous insulin infusion for 4 h (Fig. 1). Blood glucose concentrations were monitored every 15 min, and the insulin infusion was adjusted to maintain the blood glucose level between 80–120 mg/dL. Exogenous iv or oral glucose was not administered to any subject during the iv insulin infusion.

View larger version (32K): [in this window] [in a new window]   Figure 1. Study diagram.

The study was performed after an overnight fast of 12 h. At 0800 h, a priming bolus dose of NaH¹³CO₃ (0.2 mg/kg) and L-[1-¹³C]leucine (0.35 mg/kg) was administered through a peripheral iv line, followed immediately by a constant iv infusion of L-[1-¹³C]leucine (0.65 mg/kg·h) for 4 h. Blood samples were collected for -ketoisocaproate (KIC) from a contralateral heated dorsal hand vein at baseline and every 15 min during the last hour of the study. This method of blood sampling has been shown to serve as a suitable surrogate for direct arterial sampling of plasma isotopic enrichment in whole body studies (14). Expired air samples for determination of CO₂ isotopic enrichment were collected at baseline and every 15 min during the last hour of the study. Total CO₂ production was determined by indirect calorimetry for 30 min, using a ventilated hood (SensorMedics, Yorba Linda, CA). Blood for plasma hormone and substrate measurements was obtained at 180 and 240 min of the study (Fig. 1).

Analytical methods

NaH¹³CO₃ and L-[1-¹³C]leucine were purchased from CIL (Andover, MA). All samples were frozen and stored at -20 C until analysis in the author’s laboratory (K. S. Nair, Mayo Clinic, Rochester, MN) at the end of the project. Plasma KIC was measured as its quinoxalinol-trimethyl silyl derivative under positive ammonia chemical ionization conditions, as previously described (15), by gas chromatography-mass spectrometry. Isotopic enrichment was monitored using ions m/z 276/275. Expired air ¹³CO₂/¹²CO₂ ratios were measured by gas isotope ratio mass spectrometry. Plasma amino acid concentrations were determined by high pressure liquid chromatography with precolumn O-phthalaldehyde derivatization (16). Total insulin was measured by RIA, and glucose was determined using a One Touch II (Lifescan, Milpitas, CA) bedside meter. Plasma free fatty acids (FFAs) were measured using a nonesterified fatty acid-colorimetric kit (WACO Chemicals USA, Richmond, VA); ß-hydroxybutyrate was determined using a ß-hydroxybutyrate kit (Sigma Chemical Co., St. Louis, MO); serum epinephrine, norepinephrine, and dopamine were measured by reverse phase high pressure liquid chromatography with electrochemical detection after extraction on activated alumina (ESA, Inc., Chelmsford, MA); plasma glucagon concentrations and C peptide were determined by RIA (Linco Research, St. Charles, MO); serum GH was measured by RIA (ICN Pharmaceuticals, Costa Mesa, CA); and serum cortisol was determined by a competitive binding immunoenzymatic assay (Sanofic Diagnostics Pasteur, Chaska, MN). The inter- and intraassay variabilities for total insulin were 5.6% and 4.2%, those for C peptide were 13% and 5.7%, those for ß-hydroxybutyrate were 2.3% and 3.7%, those for cortisol were 16% and 8.4%, those for glucagon were 9% and 7.1%, those for GH were 7% and 5.7%, those for epinephrine were 6.2% and 3.6%, those for norepinephrine were 13.6% and 4.5%, and those for dopamine were 9% and 5.5%, respectively.

Calculations

Calculations of the leucine rate of appearance or flux (Ra) and leucine oxidation (Ox) were based on mean values of KIC isotopic enrichment in plasma and of ¹³CO₂ in expired air at the plateau during the last hour of the leucine infusion (17). KIC isotopic enrichment was used for these calculations because it is considered a better indicator of intracellular leucine enrichment than is plasma leucine enrichment (18). Ra was calculated using the formula: Ra = i[Ei/Ep) - 1], where i is the infusion rate of tracer in micromoles per kg/h, Ei is the isotopic enrichment of tracer in atoms percent excess (APE), and EP is the isotopic enrichment of plasma KIC in APE at the isotopic plateau.

The production of ¹³CO₂ (F¹³CO₂) in micromoles per kg/h was calculated using the formula F¹³CO₂ = (VCO₂ x 60 x ECO₂)/(0.0224 x 100 x Wt x 0.8), where VCO₂ is the rate of expired CO₂ (milliliters per min), 60 converts minutes to hours, ECO₂ is the isotopic enrichment of expired CO₂ (APE) at the isotopic plateau, 0.0224 is milliliters of CO₂ per µmol, 100 converts APE from a percentage to a fraction, Wt is the body weight (kilograms), and 0.8 is a parameter used for the recovery of ¹³CO₂ in breath. Leucine Ox in micromoles per kg/h was calculated according to the formula: Ox = F¹³CO₂ x [(1/Ep) - (1/Ei)] x 100. Nonoxidative leucine disposal (NOLD) was calculated as Ra - Ox.

Statistical analysis

The mean ± SD are presented. Ra, Ox, and NOLD as well as serum concentrations of measured hormones between the I+ and I- studies were compared using paired t test. The mean value of the two hormone/substrate determinations (obtained at 180 and 240 min) was used for calculations and analyses. Regression analysis was performed to compare changes in leucine kinetics and measured hormone concentrations. P < 0.05 was considered significant.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
C peptide concentrations were less than 0.1 ng/mL in all samples, documenting endogenous insulin deficiency in all subjects. In the 3 days before the I- study, daily insulin requirements remained constant (0.7 ± 0.1 U/kg the first day on multiple injections of regular insulin, 0.8 ± 0.1 U/kg the second day, and 0.8 ± 0.05 U/kg the third day; not different from the 0.8 ± 0.1 U/kg/day insulin requirement before conversion to multiple injections of regular insulin; P > 0.05). Daily insulin requirements remained the same before I+ (0.8 ± 0.1 U/kg). During I+, a mean (±SD) bolus dose of 0.035 ± 0.02 U/kg regular insulin was given, with mean (±SD) insulin infusion rates of 0.35 ± 0.16, 0.18 ± 0.16, 0.07 ± 0.13, and 0.1 ± 0.16 mU/kg·min at the end of the first, second, and third hours of the infusion and at the end of the study, respectively.

Insulin deprivation resulted in low insulin levels (I- vs. I+, 0.6 ± 0.3 vs. 7.5 ± 4.3 µU/mL; mean ± SD; P = 0.02) and increased serum ß-hydroxybutyrate and FFA concentrations compared to those after insulin administration (P < 0.001; Table 1). The mean blood glucose concentration was 325 ± 67.8 mg/dL (range, 247–418 mg/dL) during the last 2 h of the insulin deprivation study and 96 ± 23.6 mg/dL (range, 66–132 mg/dL) during the last 2 h of insulin administration. Steady state isotopic enrichment of [¹³C]KIC (APE) was achieved in all subjects during the last hour of the study (coefficients of variation of the last four isotopic determinations ranged from 2–9%; Fig. 2).

View larger version (14K): [in this window] [in a new window]   Figure 2. [13C]KIC enrichment at baseline and every 15 min during the last hour of the leucine infusion during insulin deprivation and replacement. Values are expressed as the mean ± sd.

Leucine Ra was lower during insulin administration (I- vs. I+, 200.5 ± 23.4 vs. 167 ± 17 µmol/kg·h; P = 0.008). Leucine oxidation was also lower during insulin replacement (56.5 ± 20.7 vs. 29.6 ± 9.3 µmol/kg·h; P = 0.03). NOLD was not different between groups (I- vs. I+; 144 ± 20.8 vs. 137.5 ± 13.5 µmol/kg·h; P = 0.4; Fig. 3).

View larger version (27K): [in this window] [in a new window]   Figure 3. Leucine Ra, Ox, and NOLD, in micromoles per kg/h, during insulin deprivation (I-) and insulin replacement (I+). Values are expressed as the mean ± SD.

Serum concentrations of the branched chain amino acids (BCAA), valine, leucine, and isoleucine, were increased significantly during insulin deprivation (P < 0.01). The concentrations of the rest of the amino acids remained unchanged, except for glycine, which was marginally lower during insulin deprivation (Table 2).

Serum epinephrine, norepinephrine, dopamine, and GH concentrations were not significantly different between the two studies (I- vs. I+; Table 1). Serum cortisol and glucagon concentrations were higher during insulin deprivation [I- vs. I+: serum cortisol, 60 ± 18 vs. 27.2 ± 17 µg/dL (P < 0.05); serum glucagon, 202 ± 74.8 vs. 80.3 ± 18.7 pg/mL (P < 0.05)]. Leucine Ra correlated with serum ß-hydroxybutyrate levels (r² = 0.54; P = 0.02) and inversely with serum insulin concentrations when insulin concentrations were transformed logarithmically (r² = 0.45; P = <0.05). No correlation between Ra and cortisol, glucagon, or FFA concentrations was found. There were no significant correlations among leucine oxidation, NOLD, and serum hormone or substrate concentrations.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
This study represents the first of its kind examining the effects of insulin on protein metabolism in children with IDDM. The data described here indicate that acute insulin administration in prepubertal children with IDDM in the postabsorptive state decreases leucine Ra (indicative of protein breakdown) and leucine oxidation without a concurrent increase in nonoxidative leucine disposal (indicative of protein synthesis). Previous reports pertaining to insulin administration in adults with type I diabetes mellitus describe similar effects, including decreased rates of protein breakdown with either decreased or no change in nonoxidative leucine disposal (2, 3, 4, 6, 7). This study was designed to test the hypothesis that insulin stimulates protein synthesis in young growing humans based on animal data indicating that insulin stimulates protein synthesis in young growing animals, but not in adult animals (12, 13). Contrary to the animal data, this study failed to demonstrate stimulation of protein synthesis by insulin in prepubertal children with IDDM. Although the explanation for these divergent observations is unknown, species differences in the response to insulin at different stages of physical maturity seem likely. It should be noted, however, that a potential effect of insulin on protein synthesis during puberty, when growth is even faster, cannot be excluded from the current study.

In this study the anticatabolic effects of insulin in diabetic patients were examined both during profound insulin deficiency and at concentrations similar to those in nondiabetic subjects after a short fast. During insulin deprivation, insulin concentrations were approximately 0.6 µU/mL, approximately an order of magnitude lower than normal nondiabetic basal insulin concentrations (7 µU/mL) and less than concentrations reported to stimulate protein synthesis in vitro (10).

In the current study protein breakdown was suppressed by insulin, similar to results reported previously in adult diabetic patients (2). In addition, we observed a significant inverse relationship between the rate of protein breakdown and the logarithmic concentrations of serum insulin, similar to that reported previously in healthy adults (19). We observed a 20% suppression of protein breakdown between insulin concentrations of 0.7–7 µU/mL, similar to the findings of Flakoll et al., who also observed a 20% suppression of protein breakdown from 10–100 and from 100-1000 µU/mL insulin (19). These results indicate that suppression of protein breakdown is sensitive to insulin over a wide range of concentrations, and that protein breakdown is markedly suppressed even at normal fasting insulin concentrations. Studies in adult diabetic patients indicate that most of these changes in protein metabolism occur in skeletal muscles (16).

Insulin administration in the current study reduced leucine oxidation by 47%, similar to the magnitude observed in adult diabetic patients (4). In adult diabetic patients, it has been demonstrated that increased leucine oxidation during insulin deprivation occurred due to increased leucine Ra and increased KIC production rates (16). Whether this change in leucine oxidation is a direct effect of insulin or is related to changes in counterregulatory hormones and/or substrate concentrations remains unclear. Both glucocorticoids and glucagon increase amino acid oxidation (20, 21, 22), and either or both may be related to the higher oxidation rates observed during I-. In normal adult subjects, insulin deficiency alone does not increase leucine oxidation, but hyperglucagonemia during insulin deficiency increases leucine oxidation by almost 100% (21). FFA and ß-hydroxybutyrate concentrations have been reported to correlate inversely with rates of amino acid oxidation (23, 24), but such correlations were not found in this study. Finally, GH reduces amino acid oxidation (25). Although random serum GH concentrations were the same in I- and I+ groups, a difference in GH concentrations between groups cannot be excluded given the pulsatile secretion of GH.

Studies in adults indicate that insulin infusion also reduces amino acid availability and thus may reduce the rate of protein synthesis. Conversely, hyperaminoacidemia per se has been shown to stimulate protein synthesis (26). The effect of insulin on protein synthesis when amino acids are supplied concurrently and the potential differences between children and adults were not addressed in this study. In the current study, the BCAAs were the only amino acids shown to be increased during insulin deprivation. Elevated circulating concentrations of BCAA relative to those of the other amino acids have been reported previously in IDDM (27). Insulin deprivation is known to reduce both the activation of the enzyme branched chain keto acid dehydrogenase (BCKAD) in muscle (28) and the specific activity (units of activity per g tissue) of hepatic BCKAD (29). Consequently, during insulin deprivation, protein breakdown increases, skeletal muscle BCKAD activity is less responsive to the resultant increase in leucine concentrations, and the hepatic capacity to oxidize BCAA is reduced. The result is a marked elevation in BCAA concentration, as observed in the current study.

In summary, the current study demonstrates that the acute protein metabolic effects of insulin in the postabsorptive state in prepubertal children are similar to those reported in adults; the principal protein metabolic effects of insulin are reductions in protein breakdown and amino acid oxidation, without stimulation of protein synthesis. As in the current study the induction of a state of marked insulin deficiency was associated with numerous secondary hormonal and substrate changes, it was not possible to determine whether insulin deficiency per se or secondary events brought about these changes. Nevertheless, the study demonstrates that the acute protein anabolic actions of insulin are similar in prepubertal children and adults studied under conditions of insulin deprivation and administration.

	Footnotes

 
¹ This work was supported in part by a grant from Serono Laboratories (Norwell, MA), USPHS Grants RO1-DK-41973 and RR-00585, and NIH Grant MO1-RR-00188.

Received March 5, 1997.

Revised August 6, 1997.

Accepted August 20, 1997.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vogiatzi, M. G. Articles by Copeland, K. C. Search for Related Content PubMed PubMed Citation Articles by Vogiatzi, M. G. Articles by Copeland, K. C.