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Departments of Endocrinology and Metabolism (P.H.B., H.P.S.), Biochemistry (A.J.M.), Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands; Department of Metabolic Diseases (M.G.M.d.S.-v.d.V.), University Medical Center Utrecht, 3500 AB Utrecht, The Netherlands; Center for Liver (F.K., F.S.), Digestive and Metabolic Diseases, Academic Hospital Groningen, 9713 GZ Groningen, The Netherlands; and Department of Endocrinology (P.H.B., J.A.R.), Leiden University Medical Center, 2333 AZ Leiden, The Netherlands
Address all correspondence and requests for reprints to: J. A. Romijn, M.D., Department of Endocrinology and Metabolism (C4Q), Leiden University Medical Center, Albinusdreef 2, 2333 AZ Leiden, The Netherlands. E-mail: J.A.Romijn{at}lumc.nl.
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Abstract |
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The low-carbohydrate/high-fat diet resulted in lower absorptive and postabsorptive plasma insulin concentrations, and higher rates of nitrogen excretion compared with the other two diets: 15.3 ± 0.9 vs. 12.1 ± 1.1 (P = 0.03) and 10.8 ± 0.5 g/24 h (P = 0.005), respectively. Postabsorptive rates of appearance of leucine and of leucine oxidation were not different among the three diets. In addition, dietary carbohydrate content did not affect the synthesis rates of fibrinogen and albumin.
In conclusion, eucaloric carbohydrate deprivation increases 24-h nitrogen loss but does not affect postabsorptive protein metabolism at the hepatic and whole body level. By deduction, dietary carbohydrate is required for an optimal regulation of absorptive, rather than postabsorptive, protein metabolism.
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Introduction |
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We hypothesized that alterations in dietary carbohydrate intake also change plasma insulin concentrations in humans and may thus lead to altered protein metabolism. Welle et al. (7) showed that carbohydrate overfeeding induced protein accretion, as indicated by decreased 24-h urinary nitrogen loss. Whether this effect was caused by overfeeding or by increased carbohydrate intake per se remains unclear. It is also not clear whether physiological modulation of insulin secretion by means of variation of carbohydrate intake affects the synthesis of fibrinogen and albumin. Therefore, the specific aim of our study was to establish the effects of long-term dietary modulation of insulin secretion on whole body and hepatic protein metabolism in healthy subjects by three isocaloric diets with different carbohydrate and fat content. The carbohydrate content of the diets varied from 2–85% of total energy, whereas protein content and composition were identical.
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Subjects and Methods |
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The study group consisted of six healthy, nonsmoking men aged 29–55 yr with a body mass index of 21–26 kg/m2. The subjects had no family history of diabetes and used no medications. All subjects were recruited among hospital employees and participated because of their special interest in this field of research. All subjects gave written, informed consent, and the study was approved by the Medical Ethical Committee of the Academic Medical Center. Subjects refrained from alcohol consumption during the experimental diets, and physical activity was limited to usual daily activities. In addition to the diets, the subjects were allowed to drink only water ad libitum. The postabsorptive state is defined as the period that starts when appearance of nutrients from the gut into plasma stops, which is after at least 8 h of fasting. The absorptive state is defined as the period between food intake and start of the postabsorptive state.
Diets
The three diets were custom-made (Nutricia, Zoetermeer, The Netherlands) and consisted of liquid formulas. Each diet was consumed for a period of 11 d. The order of the diets was determined by balanced assignment. After each diet, the subject entered an 8- to 10-wk wash-out period, followed by the next diet. During the wash-out period, subjects used their habitual diet. All three diets were isocaloric and contained identical amounts of protein (15% of energy) and identical protein composition. In healthy subjects with normal insulin sensitivity, carbohydrate intake is the main controlling factor of insulin secretion. To maximize potential effect of carbohydrate intake on protein metabolism diets with extremely low-, normal-, and extremely high-carbohydrate content were used. The low-carbohydrate/high-fat (LCHF) diet provided 83% of energy as fat and 2% of energy as carbohydrate; the intermediate-carbohydrate/intermediate-fat (ICIF) diet provided 41% of energy as fat and 44% of energy as carbohydrate, and the high-carbohydrate/low-fat (HCLF) diet provided 0% of energy as fat and 85% of energy as carbohydrate. The saturated:monounsaturated:polyunsaturated fat ratios were 2:2:1 for all diets containing fat. The ratio mono- and disacharides:polysacharides was 1:1. Energy requirements for each subject were assessed by a dietitian by means of a detailed 3 d dietary journal. The diets provided 138 (SE 6) kJ/kg body weight.
Liquid meals with predetermined amounts of energy were consumed at six fixed time points each day between 0800 and 2130 h for 11 d. Urine was collected in acid (6 N HCl 7.5 ml/liter urine) for 24 h on d 10 and 11 of each diet for determination of urinary nitrogen excretion.
Protocol
On d 7 of each diet, the subjects fasted from 1800 h. A catheter was inserted in an antecubital vein of each arm. One catheter was used to sample blood. The other catheter was used to infuse [1-13C]valine (>99% enriched, Cambridge Isotope Laboratories, Andover, MA). At 0200 h, a primed, continuous infusion of [1-13C]valine was started at a rate of 15 µmol·kg-1·h-1 (prime 15 µmol·kg-1). At 0200, 0230, 0300 h, and subsequently every hour until 1000 h, blood samples were taken to determine the tracer/tracee ratios of plasma KIV (ketoisovaleric acid) and valine in fibrinogen and albumin.
At 0800 h on d 10 of each diet, the subjects ingested 300 kcal of the respective diet, after an overnight fast of 10 h. Plasma glucose, insulin, and C-peptide concentrations were measured just before ingestion and every half-hour after ingestion for 3 h.
At d 11 of each diet, the subjects were studied again, after an overnight fast. At 0700 h, a catheter was inserted in an antecubital vein of each arm. One catheter was used to sample arterialized blood using a heated-hand box (60 C). The other catheter was used to infuse [1-13C]leucine (>99% enriched, Cambridge Isotope Laboratories). After collection of baseline blood and breath samples a bolus of 0.1 mg·kg-1 NaH13CO3 (>99% enriched, Cambridge Isotope Laboratories) was administered and a primed, continuous infusion of [1-13C]leucine was started at a rate of 5 µmol·kg-1·h-1 (prime 5 µmol·kg-1) for 4 h. At 0800, 1130, 1145, and 1200 h blood and breath samples were taken for tracer/tracee ratios of 13C-KIC (ketoisocaproic acid) and 13CO2, respectively. Breath sample collection was performed by breathing through a straw into a 10-ml gas testing vials (Labco Ltd., Buckinghamshire, UK). Carbon dioxide production (VCO2) was measured continuously between 1130 and 1200 h with the ventilated-hood technique (model 2900, Sensormedics, Anaheim, CA). The mean rates of VCO2 between 1140 and 1200 h were used to calculate leucine oxidation (Lox).
Analysis of [13C]KIV, albumin, and fibrinogen
KIV was isolated from 0.75-ml heparin plasma by cation exchange chromatography (8). The isolation of albumin from heparin plasma was based on differential solubility in absolute ethanol from trichloric acetic acid precipitated proteins as described in detail previously (9). Fibrinogen was isolated from citrate plasma as previously described (9). Both proteins were hydrolyzed with 6 N HCl for 24 h at 110 C. The hydrolysates were supplied to cation-exchange resin as described previously (9). Derivatization of isolated KIV and of the hydrolyzed proteins was performed according to the method of Hu
ek (10). The tracer molar ratios of [13C]KIV in plasma were measured by gas chromatography mass spectrometry on a Hewlett-Packard HP 5890 type II gas chromatograph interfaced to a HP 5989B mass spectrometer (Hewlett-Packard, Palo Alto, CA). The gas chromatograph was equipped with a CP Sil 19CB capillary column (Chrompack, Bergen op Zoom, The Netherlands). Tracer molar ratios of [13C]valine in both proteins were measured by gas chromatography combustion isotope ratio mass spectrometry as described previously (9). All isotopic enrichments were measured against standard calibration curves as described by Kulik et al. (11).
Analysis of [13C] plasma KIC and breath CO2
Calibration samples with a tracer molar ratio for [1-13C]KIC ranging from 0–20% were prepared. Fifty microliters of each prepared [1-13C]KIC calibration sample solution was then processed in one batch with the patient plasma samples. Five hundred microliters of plasma were deproteinized with sulfasalisilic acid, and the supernatant derivatized to the quinoxalinol-O-t-butyldimethylsilyl derivative (12). Isotopic enrichment of plasma KIC was measured by use of electron impact ionization mass spectrometry in the selected ion monitoring mode recording the fragments mass to charge ratio 259 and 260 of the quinoxalinol-O-t-butyldimethylsilyl derivative on a SSQ 7000 gas chromatograph quadrupole mass spectrometer (Finnigan MAT, San Jose, CA) by use of methane positive ion chemical ionization. The quinoxalinol-O-t-butyldimethylsilyl derivatives of amino acids were separated on an AT 1701 capillary column using helium as carrier gas, temperature programming, and splitless injection.
Measurement of [13C] enrichment in CO2 was performed directly in breath with a Finnigan TracerMat continuous flow isotope ratio mass spectrometer (Finnigan MAT).
Analytical procedures
Plasma insulin concentration was determined by RIA (insulin RIA 100, Pharmacia Diagnostic AB, Uppsala, Sweden), intraassay coefficient of variation (c.v.) 3–5%; interassay c.v., 6–9%; detection limit, 15 pmol/liter. C-peptide was determined by RIA (RIA-coat c-peptide, Byk-Sangtec Diagnostica GmbH & Co. KG, Dietzenbach, Germany), intraassay c.v., 4–6%; interassay c.v., 6–8%; detection limit, 50 pmol/liter. Ammonia and urinary nitrogen concentration were measured as follows. Ammonia was measured enzymatically with the Monotest Ammonia kit (Roche, Almere, The Netherlands). For measurement of urinary nitrogen excretion, the nitrogen from nitrogenous compounds was converted to ammonia by wet oxidation according to the Kjeldahl technique in a digestion mixture of sulfuric acid, sodium sulfate, and mercuric sulfate. The amount of ammonia generated was measured as described above. Plasma concentrations of albumin were measured with standard laboratory methods on a Ektachem 950 (Johnson & Johnson, Clinical Diagnostics, NY). Plasma fibrinogen concentration was measured using the Von Clauss method (13). Plasma free fatty acids were measured by an enzymatic method (NEFAC, Wako Chemicals GmbH, Neuss, Germany; intraassay c.v., 2–4%; interassay c.v., 3–6%; detection limit, 0.02 mmol/liter). Quantitative amino acid analysis (including hydroxyproline) was performed on a Beckman 7300 amino acid analyzer (Beckman, Palo Alto, CA). Plasma was deproteinized with sulfosalicylic acid. Separation was done on a cation-exchange column using lithium citrate buffers as eluents. The amino acids were reacted with ninhydrin and quantitated using the absorbance of the reaction products at 440 and 570 nm. Urinary 3-hydroxybutyrate was measured with an enzymatic/spectophotometric assay.
Calculations and statistics
Plasma leucine kinetics were determined using steady-state isotope dilution equations and the reciprocal model, which uses the enrichment of KIC, the transamination product of leucine, and the preferred indicator of intracellular leucine tracer/tracee ratio (14). The rate of appearance of leucine (LRa) was calculated as follows: LRa = I/Ep - I. I is the infusion rate of [1-13C]leucine and Ep is the plasma [13C]KIC enrichment at plateau. Lox was calculated as follows: Lox = (ECO2 x VCO2)/(K x Ep). ECO2 is the 13CO2 enrichment in expired air and K is a correction factor (0.8) (15). The factor K accounts for the fraction of 13CO2 released into the body’s bicarbonate pool from the oxidation of [1-13C]leucine but not recovered in the expired air. Nonoxidative leucine disposal (LNOD) was calculated by subtracting Lox from LRa. LNOD was used to estimate incorporation of leucine into protein.
The fractional synthesis rates (FSR) of albumin and fibrinogen were estimated using the SAAM II compartmental module (SAAM Institute, University of Washington). A model was constructed consisting of two compartments representing plasma [13C]KIV and plasma [13C]albumin or [13C]fibrinogen connected by a delay, which represents the time needed for the label to appear in either albumin or fibrinogen. A forcing function was associated with the [13C]KIV compartment. The delay time was set at 0.5 h and the catabolic rate was fixed 0.1%/h. The k value that resulted from fitting the model to the data represents the FSR. Absolute synthetic rates of fibrinogen and albumin were calculated by multiplying their FSR with the plasma pool size. To calculate the plasma pool size plasma volume was assessed at 40 ml/kg body weight.
The data were analyzed by ANOVA for randomized block design. A post hoc analysis was done when appropriate using Fisher’s least significant difference test. P < 0.05 was considered to indicate a significant difference. Data are presented as means ± SE. We used SPSS (SPSS Inc., Chicago, IL) for the statistical analysis.
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Results |
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Insulin, fatty acid, and amino acid concentrations
Postabsorptive plasma glucose and insulin concentrations were lower on the LCHF diet compared with the other diets (Table 1
). Postabsorptive plasma fatty acid concentrations were 0.78 ± 0.12, 0.36 ± 0.05, and 0.36 ± 0.04 mmol/liter, respectively (P < 0.01 low-carbohydrate diet vs. other diets). Postabsorptive plasma concentrations of amino acid concentrations are shown in Table 2. There were no differences in the total amino acid concentrations between the three diets, although there were slight changes in some of the individual amino acids.
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). The integrated plasma insulin and C-peptide response increased from 2.5 ± 0.4 to 15.6 ± 1.6 to 24.8 ± 3.5 nmol/liter·min (P < 0.001 among the three diets) and from 20 ± 5 to 160 ± 30 to 215 ± 28 nmol/liter·min (P < 0.001 among the three diets), respectively, with increasing dietary carbohydrate contents.
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The excretion of urinary nitrogen in 24-h urine collections was approximately 28% and approximately 44% higher on the LCHF than on the ICIF or HCLF diet, respectively (P < 0.05, Fig. 2). Urinary ammonia excretion was not different between the diets: 609 ± 124, 603 ± 78, and 608 ± 159 mg/24 h.
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Leucine kinetics and fibrinogen and albumin synthesis
The rates of appearance of leucine, Lox, and nonoxidative leucine disposal are presented in Table 3. The postabsorptive LRa and oxidation rates were not different between the three diets.
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). Dietary carbohydrate content affected neither synthesis rates nor catabolic rates of fibrinogen and albumin (Table 3). |
Discussion |
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Insulin secretion paralleled dietary carbohydrate content, reflected by the increase in plasma insulin and C-peptide concentrations after the different meals and by the difference in postabsorptive insulin concentrations. Insulin differentially affects fibrinogen and albumin synthesis. De Feo et al. (6) showed that acute hyperinsulinemia (insulin 175 pmol/liter), induced by a hyperinsulinemic euglycemic clamp, increased the fractional synthetic rate of albumin, but decreased the fractional synthetic rate of fibrinogen. These authors also showed that acute insulin withdrawal in patients with type 1 diabetes mellitus is associated with a decrease in the fractional synthetic rate of albumin and an increase in that of fibrinogen (16). The aim of our study was to manipulate insulin levels by extreme variations in dietary carbohydrate content to evaluate the physiological relevance of the effects of postabsorptive insulin concentrations on hepatic protein metabolism. Our data indicate that, in the presence of constant protein intake, the postabsorptive rates of fibrinogen and albumin synthesis are not affected by a maximal variation in dietary carbohydrate and fat content. Although there were no changes in postabsorptive whole body protein kinetics, the low-carbohydrate diet markedly increased 24-h urinary nitrogen loss, indicative of protein loss. This discrepancy is most likely caused by differences in absorptive protein metabolism. Absorptive alterations of protein metabolism would be reflected in 24-h nitrogen excretion, but not necessarily in postabsorptive protein metabolism. In the absorptive phase, plasma amino acid concentrations increase by absorption of amino acids from the intestine. Although plasma amino acids are known to stimulate insulin secretion, amino acids are quantitatively less important than glucose-mediated insulin secretion. This is reflected by the marked difference in insulin secretion in response to the LCHF diet compared with the other diets, despite identical dietary protein content. Fereday et al. (17) showed that insulin-mediated inhibition of endogenous proteolysis is required for efficient protein utilization, in addition to the stimulatory effects of insulin on protein synthesis (5). This may imply that the LCHF diet increased 24-h nitrogen excretion in our study, because it failed to stimulate insulin secretion to a degree that enabled efficient utilization of dietary protein in addition to the absent effects of meal-induced insulin secretion on endogenous protein metabolism. In other words, dietary carbohydrate is required for an optimal utilization of dietary proteins. In contrast to protein metabolism, postabsorptive glucose and fat metabolism have been shown to change in response to changes in dietary carbohydrate and fat intake (18, 19), but this response was at least in part caused by induction of insulin resistance (20).
Previous studies have shown that insulin differentially affects splanchnic and muscle amino acid and protein metabolism (21, 22). The data obtained by infusion of [1-13C]leucine do not permit a conclusion with respect to potential, tissue-specific differences in protein metabolism between the three diets. For this purpose, we assessed hepatic protein synthesis by measuring synthesis rates of fibrinogen and albumin, proteins known to be regulated by insulin. The results indicated that dietary carbohydrate content did not affect postabsorptive fibrinogen and albumin synthesis, but possible effects on other liver-derived proteins might exist.
Despite the absence of changes in protein kinetics, there were slight differences in amino acids among the three diets. For instance, leucine concentration increased during the LCHF diet. Because there was no change in the rate of leucine appearance, this indicates that leucine clearance was decreased. Although it is likely that the diets differentially affected the interorgan exchange of amino acids, this did not affect the parameters of postabsorptive protein metabolism, measured in the present study.
The low-carbohydrate diet in this study is likely to produce ketosis. As a result, several physiological and metabolic adaptations occur, which might affect our conclusions with respect to urinary nitrogen excretion. Ketoacidosis could lead to increased nitrogen excretion by stimulation of renal conversion of glutamine into ammonia. However, urinary excretion of ammonia was not different between the diets. Alternatively, ketosis might cause bone loss and collagen breakdown. However, urinary excretion of hydroxyproline and N-telopeptides, markers of bone loss and collagen breakdown, were not different between the diets. Thus, the HFLC diet did not increase the conversion of glutamine to ammonium ions in the kidney or increase bone and collagen breakdown. Therefore, these consequences of ketosis are unlikely explanations for the effects of dietary carbohydrate deprivation on urinary nitrogen excretion.
In conclusion, eucaloric carbohydrate deprivation results in increased 24-h nitrogen loss. However, postabsorptive parameters of protein metabolism at hepatic and whole body level are not affected by carbohydrate deprivation. By deduction, dietary carbohydrate content predominantly affects protein metabolism in the absorptive rather than in the postabsorptive phase.
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Acknowledgments |
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Footnotes |
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Abbreviations: c.v., Coefficient of variation; FSR, fractional synthesis rates; HCHF, high-carbohydrate, high-fat; ICIF, intermediate-carbohydrate/intermediate-fat; KIC, ketoisocaproic acid; KIV, ketoisovaleric acid; LCHF, low-carbohydrate, high-fat; Lox, leucine oxidation; LRa, rate of appearance of leucine; VCO2, carbon dioxide production.
Received July 12, 2002.
Accepted May 5, 2003.
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References |
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13C values of valine in protein hydrolysate by gas chromatography-combustion isotope ratio mass spectrometry. J Chromatogr B Biomed Sci Appl 710:37–47[CrossRef][Medline]
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