Effect of protein intake on plasma and erythrocyte free amino acids and serum IGF-I and IGFBP-1 levels in rats

J. C. Divino Filho1, S. J. Hazel2, B. Anderstam1, J. Bergström1, M. Lewitt3, and K. Hall2

1 Divisions of Renal Medicine and Baxter Novum, Department of Clinical Science and 2 The Endocrine and Diabetes Unit, Department of Molecular Medicine, Karolinska Hospital, Karolinska Institutet, S-141 86 Stockholm, Sweden; and 3 Department of Medicine (Endocrinology), Royal Prince Alfred Hospital and The University of Sydney, Sydney NSW 2006, Australia


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Amino acid (AA) levels in plasma and erythrocytes (RBC) were determined in rats (n = 29) fed diets with 6, 21, and 35% protein, and their association with insulin-like growth factor I (IGF-I), insulin, or IGF-binding protein (IGFBP)-1 levels was studied. Free AA in plasma and RBC were determined by reversed-phase high-pressure liquid chromotography, and IGF-I, IGFBP-1, and insulin plasma levels were determined by RIA. Rats fed the low-protein (6%) diet were growth-retarded and had lower serum IGF-I levels and higher serum IGFBP-1 levels than the other two groups (P < 0.0001). In rats fed the low-protein diet, most of the nonessential AA (NEAA) in both plasma and RBC increased, whereas the essential AA (EAA), with the exception of threonine, decreased. When the groups were combined, both RBC and plasma EAA-to-NEAA ratios were positively correlated to IGF-I (r = 0.76 and 0.80, respectively; P < 0.0001) and inversely correlated to IGFBP-1 levels (r = -0.67, P < 0.001 and r = -0.78, P < 0.0001, respectively). A significant inverse correlation was found between RBC glutamate and IGF-I (r = -0.85, P < 0.0001, n = 25) and insulin (r = -0.72, P < 0.001, n = 21), and a positive correlation was found for IGFBP-1 (r = 0.78, P < 0.0001, n = 24). In multiple regression analysis, only IGF-I remained as an independent variable. Threonine was the only EAA with a significant inverse correlation to insulin (r = -0.66, P < 0.001). We hypothesize that AA metabolism is associated to changes in IGF-I, insulin, and IGFBP-1 levels in rats on different protein intakes.

intra- and extracellular amino acids; glutamate; threonine; catabolism; insulin-like growth factor I; insulin; insulin-like growth factor-binding protein 1


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RESTRICTION OF DIETARY protein in young rats results in stunted growth, reduced serum insulin-like growth factor (IGF) I, elevated IGF-I-binding protein (IGFBP)-1 concentrations, and abnormalities in amino acid (AA) concentrations (23, 32, 36-38). IGF-I is a polypeptide growth factor expressed in multiple tissues with an important role in cell proliferation and differentiation throughout the life cycle. The effect of IGF-I is modulated by IGFBP (16). A family of six IGFBPs with high affinity for IGFs has been fully characterized and cloned (16). IGFBP-1 was the first to be purified (26), is produced primarily in the liver, and can inhibit the biological action of IGF-I in vitro (5) and in vivo (19). Circulating levels of IGFBP-1 are regulated acutely in response to changes in nutrient supplies and insulin concentrations. An increase in both the expression and serum levels of IGFBP-1 has been reported in rats fed low-protein diets, which could further reduce the IGF-I bioavailability by competing for IGF-I binding with the IGF-I receptor (32). Straus et al. (32) have also demonstrated that limitation of essential AA (EAA) may be a factor contributing to the induction of IGFBP-1 gene expression in the liver of protein-restricted animals.

Plasma AA are generally used as an index of the circulating AA levels, but several studies have implicated the erythrocytes (RBC) as important carriers in the net flux of various AA between peripheral tissues, gut, and liver in humans and rats (2, 7-9, 25, 30). The need to carry out simultaneous determinations of AA concentrations, both in RBC and plasma, to obtain a more complete picture of the interorgan AA relationship under different physiological situations has been suggested (15, 30). We have previously reported that, in hemodialysis patients with elevated RBC glutamate levels, the RBC glutamate (but not the plasma) levels were positively correlated to IGFBP-1 and inversely correlated to IGF-I levels (6). These findings raised the question of how the RBC AA levels are influenced by dietary protein intake.

The aim of this study was to investigate in a rodent model how the protein content in the diet changes the plasma and RBC AA levels and whether these changes are associated with changes in circulating IGF-I and IGFBP-1 levels.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Animals and diets. Female Sprague-Dawley young growing rats were purchased from BK Universal (Sollentuna, Sweden). A total of 29 rats were used in the experiments and were divided randomly into groups fed 6, 21, and 35% protein diets ad libitum (n = 9, 10, and 10, respectively). The body weights of the groups of animals at the beginning of the experiment were 159 ± 1, 162 ± 1, and 163 ± 1 (means ± SE) for the 6, 21, and 35% protein groups, respectively. The diets were calculated to contain the following: raw protein 6.1, 21.0, and 35.0% and nonfat energy 67.9, 53.0, and 39.0%, respectively, with 7.0% raw fat, 4.0% fiber, 5.0% ash, 10.0% water, and 13.3 MJ/kg in each diet (AnalyCen Nordic; Special Diets, Lidköping, Sweden). The raw materials were casein, corn starch, sucrose, dextrose, cellulose powder, mineral premix, and soybean oil. All rats were maintained on a 12:12-h day-night rhythm with free access to water. The rats were fed these diets for 47 days. The experimental design was approved by the Animal Ethics Committee of the Karolinska Hospital.

Sampling procedures. Forty-seven days after the start of the experiment, the rats received an intraperitoneal injection of fluanisonum and fentalynium (Hypnorm; 10 and 0.2 mg/ml, respectively) followed by exsanguination using cardiac puncture. Rats were killed in the period 1000 to 1730. For practical reasons, the rats could not be killed simultaneously, but the groups were uniformly distributed to ensure that there was no biased effect of the time of the day.

Analytical procedures. Blood and tissue samples were collected, and the following analyses were performed.

Serum biochemistry. Serum rat IGF-I was measured using an RIA. Samples were acid-ethanol extracted and cryoprecipitated before the assay, and des(1-3)IGF-I was used as a ligand to eliminate interaction of the IGFBPs (3). The intra- and interassay coefficients of variation were 4 and 11%, respectively. The detection limit was 8 µg/l. Immunoreactive insulin was measured by RIA using guinea pig antibodies raised against porcine insulin and 125I-labeled porcine insulin as a tracer. Rat insulin (Novo) was used as a standard. Dextran-coated charcoal was used to separate bound from free insulin. The sensitivity of the assay was ~0.2 µU/tube, and the intra-assay coefficient of variation was 5.1%. Serum IGFBP-1 was measured by RIA using rat IGFBP-1 standard (21). The sensitivity of the IGFBP-1 assay was 50 pg/tube, and the within-assay imprecision was 3.7% at 0.82 ng.

Serum biochemistry measurements for urea, creatinine, and glucose were evaluated by routine methods.

AA analyses. A heparinized blood sample was centrifuged for 10 min at 4°C to obtain plasma, which was then deproteinized with sulfosalicylic acid (SSA, 30 mg/ml plasma) and centrifuged. The supernatant was stored at -70°C until analysis of AA.

For measurement of RBC AA, white blood cells and platelets were carefully removed, and packed RBCs were rapidly hemolyzed by adding 1% saponin (Sigma, St. Louis, MO). The sample was then extracted with 50% SSA, mixed, and centrifuged at 1,700 g for 20 min at 4°C. The supernatant was filtered using a 0.45-µm HA filter (Millipore) and was frozen at -70°C until analyzed. For calculation of AA concentrations in RBC, the water was taken as 66% of RBC weight in all samples (10). In four rats (2 in the 6% group and 1 in each other group), insufficient blood was obtained to perform the separation, and they were therefore excluded.

Free AA concentrations in plasma and RBC were determined using an automated on-line high-pressure liquid chromatography system with precolumn derivatization (ortho-phthaldialdehyde/3-mercaptopropionic acid) and norvaline as the internal standard. AA were conventionally classified as EAA (those that can not be endogenously synthesized) or nonessential AA (NEAA; those that can be synthesized). Tyrosine, considered as an indispensable AA under special conditions as uremia and infancy, was listed as an EAA.

Statistical analysis. Data are reported as means ± SE if not stated otherwise. Values on IGF-I, insulin, and IGFBP-1 were log transformed before analyses because the transformed values more closely approximated the Gaussian distribution. For statistical analysis of the data, ANOVA followed by Student's t-test with the Bonferroni procedure was used. Pearson's coefficient of correlation (r) was used to calculate the correlation between different variables. Multiple regression analysis was performed to test the influence of IGF-I, insulin, and IGFBP-1 on AA concentrations. The value of acceptance for statistical significance was set at P < 0.01.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Serum biochemistry. The 6% protein intake group showed significantly lower serum levels of urea and glucose than the 21% group and the 35% group (Table 1). On the other hand, no significant differences in serum urea and glucose levels were seen between the 21 and 35% protein groups.

                              
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Table 1.   Weight gain and serum concentration of urea, creatinine, glucose, insulin, IGF-I, and IGFBP-1 in rats

AA concentration in plasma and RBC. The AA concentrations in plasma and RBC are shown in Tables 2 and 3, respectively. The plasma EAA levels, apart from threonine, were significantly lower in the 6% than in the 21% protein group (Table 2). Conversely, most of the plasma NEAA displayed an opposite pattern with higher values in the 6% group when compared with the 35% group. However, plasma glutamate and arginine showed no differences between the groups, and taurine followed the pattern of EAA with lower values at low-protein intake.

                              
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Table 2.   Plasma free amino acid concentrations in rats


                              
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Table 3.   RBC free amino acid concentrations in rats

Similar to plasma EAA, most of the RBC EAA levels were significantly lower in the 6% group than in the 21 and 35% groups, with the exception being threonine, which had higher values as protein intake decreased (Table 3). Methionine did not show any difference between the groups. Among the RBC NEAA, glutamate, glutamine, glycine, and serine levels were significantly higher in the 6% group than in both the 21 and 35% groups, whereas RBC ornithine was only higher when compared with the 35% group. Moreover, serine levels in the 21% group were also significantly higher than in the 35% group. In plasma, but not RBC, the EAA-to-NEAA ratio was lower in the 35% group than in the 21% group (P < 0.0001).

The EAA-to-NEAA ratios in both plasma and RBC were significantly lower in the 6% protein intake group when compared with the 21 and 35% groups (P < 0.0001; Tables 2 and 3).

The RBC/plasma gradients of most of the EAA (threonine, phenylalanine, tyrosine, valine, isoleucine, and leucine) and some NEAA (alanine, citrulline, and glutamine), with mean values ranging between 1.1 and 1.7, were independent of the protein intake (Table 4). In contrast, tryptophan and methionine had low RBC/plasma gradients and were, likewise, also independent of the protein intake (0.2-0.3 and 0.5-0.7, respectively). Glutamate was the only AA in which the RBC/plasma gradient increased significantly as the protein intake decreased (4.3-12.7, P < 0.0001).

                              
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Table 4.   Gradient RBC/plasma free amino acids in rats

RBC glutamate levels, which were weakly related to plasma glutamate (r = 0.44, P < 0.02), were significantly correlated to RBC and plasma glutamine (r = 0.70, P < 0.0001 for both). The branched-chain AA isoleucine, leucine, and valine displayed highly significant positive correlations within RBC (r = 0.95-0.99) or within plasma (r = 0.93-0.99), whereas the correlations between these two compartments were much lower (r = 0.41-0.45).

Growth and IGF-I, IGFBP-1, and insulin concentrations. The growth of rats fed 6% protein were significantly retarded compared with the other two groups (P < 0.0001; Table 1). The low-protein group lost ~5% of their initial weight during the experiment, whereas the normal and high-protein groups gained ~60% of their original body weight during the same period.

The mean IGF-I and insulin levels in the 6% group (83 µg/l and 13 µU/ml, respectively) were significantly lower (P < 0.0001) than the values of the 21% group (262 µg/l and 32 µU/ml, respectively) and the 35% group (281 µg/l and 37 µU/ml, respectively). The mean IGFBP-1 levels displayed an opposite pattern in relation to protein intake, with significantly higher values in the 6% group (238 µg/l) than in the 21% (20 µg/l) and 35% (38 µg/l) groups (P < 0.0001; Table 1). There was an inverse correlation between IGFBP-1 and insulin (r = -0.68, P < 0.001, n = 21) as well as between IGFBP-1 and IGF-I (r = -0.84, P < 0.0001, n = 24) when the three groups were combined.

Correlations between AA, urea, insulin, IGF-I, and IGFBP-1 levels. When the results from the three groups were combined, the RBC EAA-to-NEAA ratio showed a positive correlation to IGF-I and insulin (r = 0.76, P < 0.0001 and r = 0.58, P < 0.01, respectively), whereas an inverse correlation was found for IGFBP-1 (r = -0.67, P < 0.001; Fig. 1, A-C). The plasma EAA-to-NEAA ratio showed a positive correlation to IGF-I (r = 0.80, P < 0.0001) and insulin (r = 0.54, P < 0.01) and an inverse correlation to IGFBP-1 (r -0.78, P < 0.0001). In multiple regression analyses, with the EAA-to-NEAA ratio as dependent and IGF-I and IGFBP-1 levels as independent variables, the adjusted r2 for the ratio in plasma was 0.68 (P < 0.0001) and in RBC 0.60 (P < 0.0001). Only IGF-I remained as a significant independent variable, and addition of insulin did not increase the adjusted r2.


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Fig. 1.   Erythrocyte (RBC) essential amino acid (EAA)-to-nonessential amino acid (NEAA) ratios, RBC threonine, and RBC glutamate levels in relation to serum insulin-like growth factor I (IGF-I; A, D, and G), insulin (B, E, and H), or IGF-binding protein 1 (IGFBP-1; C, F, and I) levels in rats fed different protein diets: 35% (), 21% (open circle ), and 6% (black-triangle). ICW, intracellular water. Univariate correlation coefficients in linear regression analysis are shown in Table 5. Observe that RBC EAA/NEAA and RBC glutamate values in the 35% group displayed a tendency to apposition between the 6 and 21% groups, whereas the RBC threonine values followed the protein intake.

Several plasma AA correlated to IGF-I, insulin, and IGFBP-1 (Table 5). In general, plasma EAA were positively correlated to IGF-I and insulin and negatively to IGFBP-1. However, threonine displayed an opposite pattern similar to most of the plasma NEAA, which were inversely related to IGF-I and positively related to IGFBP-1, glutamate and taurine being exceptions. The highest univariate correlation between plasma AA and IGF-I was observed with tyrosine, glutamine, glycine, and serine as dependent variables. Addition of IGFBP-1 or insulin as an independent variable did not increase these correlation coefficients significantly. The relation between RBC AA and IGF-I, insulin, or IGFBP-1 followed the plasma pattern. In contrast to the other RBC EAA, threonine was inversely correlated to IGF-I and insulin. In addition, threonine was also the only RBC EAA that displayed a significant relation to insulin (r = -0.66, P < 0.001; Fig. 1E). In multiple regression with RBC and plasma threonine as dependent variables and insulin, IGF-I, and IGFBP-1 as independent variables, the adjusted r2 were 0.54 and 0.48 (P < 0.001), respectively.

                              
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Table 5.   Correlations between AA, IGF-I, insulin, and IGFBP-1 levels in rats

Among RBC AA, the most impressive correlations to IGF-I and IGFBP-1 were found with RBC glutamate and serine as dependent variables. A highly significant inverse correlation existed between RBC glutamate and IGF-I (r = -0.85, P < 0.0001, n = 25) and insulin (r = -0.72, P < 0.001, n = 21) levels, whereas a positive correlation was found between RBC glutamate levels and IGFBP-1 (r = 0.78, P < 0.0001, n = 24; Fig. 1, G-I).

In multiple regression analysis with RBC glutamate as dependent and IGF-I, IGFBP-1, and insulin as independent variables, the adjusted r2 was 0.72 (P < 0,0001) and did not differ from r2 (0.73) with IGF-I alone. Thus only IGF-I remained as a significant independent variable. When RBC glutamine was added to IGF-I as an independent variable, the adjusted r2 rose to 0.82 (P < 0.0001). No correlations were found between plasma glutamate and IGF-I, IGFBP-1, or insulin.

Serum urea was inversely correlated to RBC glutamate (r = -0,62, P < 0.001; Fig. 2), plasma glutamine (r = -0.57, P < 0.01), and IGFBP-1 (r = -0.46, P < 0.02) and positively correlated to the EAA-to-NEAA plasma ratio (r = 0.69, P < 0.0001) and IGF-I (r = 0.72, P < 0.0001; Fig. 2).


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Fig. 2.   Serum urea (S-Urea) levels in relation to serum IGF-I (A) or RBC glutamate (B) levels in rats fed different protein diets: 35% (), 21% (open circle ), and 6% (black-triangle). A: r = 0.72, P < 0.0001, n = 25; B: r = -0.62, P < 0.001, n = 25 (linear regression).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Significant changes in the patterns of AA concentrations were found in both the plasma and RBC of rats fed a low-protein diet (6%), which, as expected, displayed reduced weight gain, lower serum urea and IGF-I, and higher IGFBP-1 levels when compared with rats on normal or high-protein intake (36). In general, the changes in EAA and NEAA concentration with different protein content in the diet were in opposing directions. On a low-protein diet, most of the NEAA in both plasma and RBC increased, whereas the EAA decreased. The higher NEAA with decreased protein intake contributed to the significantly lower EAA-to-NEAA ratio observed in plasma and RBC in the 6% protein group. An increase in NEAA and a decrease in EAA are generally found during protein malnutrition and have been assumed to reflect the enhanced protein breakdown (12), which in vivo is reduced by both insulin and IGF-I (29).

The rise in NEAA with decreased protein intake contributed significantly to the ratio of EAA to NEAA. A high plasma EAA-to-NEAA ratio is considered to be an index of positive protein nutritional status (33). In this study, we could observe significantly higher positive correlations between the plasma or RBC EAA-to-NEAA ratio and IGF-I than insulin; conversely, there were inverse correlations between these ratios and the serum IGFBP-1 levels. Because high IGFBP-1 levels may reduce the bioavailability of IGFs, this may indicate that a relationship exists between the EAA-to-NEAA ratio and IGF bioavailability. It is well established that both malnutrition and starvation decrease the IGF-I levels in humans and rats. Both growth hormone (GH) and IGF-I resistance have been suggested during protein and caloric restriction in both species, since GH or IGF-I treatment fails to normally stimulate growth and anabolism (4, 38, 39). The IGF-I insensitivity has, at least partly, been attributed to reduced bioavailability of IGFs due to an increase of inhibitory IGFBPs. Hazel et al. (14) have shown that, during protein restriction in rats, the hepatic expression of IGFBP-1 and IGFBP-2 increased, whereas no changes were seen in hepatic IGFBP-4 mRNA. Previously, it has been shown that plasma levels of IGFBP-3 and IGFBP-2 declined concomitantly with IGF-I during protein restriction in rats (18). IGFBP-1 transgenic mice have impaired growth (27). Total IGF-I concentrations were not altered; however, measurements of free IGF-I were not made. IGFBP-1 in vitro (41) and in vivo (20) suppresses the IGF-induced glucose uptake in muscle. Therefore, high IGFBP-1 levels, which reduce the levels of free IGF-I (11), may contribute to reducing the AA uptake in muscle tissue and/or enhancing muscle protein breakdown and muscle protein synthesis.

Threonine was the only EAA to decrease in plasma and RBC as protein intake increased. High-protein diets consistently induce a fall in the concentration of the glucogenic AA threonine, glycine, and serine in plasma, liver, and muscle (22), but the RBC concentrations have not been determined previously. The decreased availability of an EAA such as threonine by excess dietary protein constitutes a dietary paradoxical response, probably representing an adaptation to high-protein diets (22). Threonine shares a common metabolic pathway [threonine-serine dehydratase (TSH)] with serine, and high-protein intake was shown to stimulate induction of hepatic TSH (24). Interestingly, plasma and RBC threonine levels, in the present study, were inversely correlated to insulin, implicating that insulin could play a role in liver TSH expression. Taurine was the only NEAA (as tyrosine is considered as an EAA in the present study) to increase in plasma and RBC as protein intake increased. Taurine is shown to be actively absorbed by the various tissues of the rat (31), and the reduction in plasma and RBC taurine levels in the low-protein diet group may reflect a mechanism of preventing the loss of taurine via urine.

The RBC/plasma gradients for most of the AA did not differ between rats on different diets. Only glutamate accounted for a striking change in the RBC-to-plasma AA ratio, with a threefold higher ratio in the 6% compared with the 22% protein group. This increase in RBC glutamate in relation to plasma glutamate may be attributed to a decreased capacity of the peripheral muscle tissue to extract glutamate from the circulating blood and/or an increased hepatic glutamate release. Aoki et al. have demonstrated that insulin selectively increases muscle glutamate uptake from whole blood in humans (1) and that changes occur in whole blood fluxes of glutamate with changing states of nutrition (2). IGF-I is expected to have a similar effect to insulin. Previous studies have demonstrated that the RBC are involved in AA transport between different tissues of the body (2, 7-9), playing an independent and opposing role to plasma in the exchange of several AA across the liver and gut. Plasma carries free AA from gut or peripheral tissues to liver, whereas AA are carried from liver to peripheral tissues as free AA in RBC or as protein in plasma (7). The positive relationship between RBC-glutamate versus both RBC and plasma glutamine but not plasma glutamate reinforces the importance of the RBC in AA transport and also suggests a role for the RBC in the interconvertibility of glutamine and glutamate. Glutamate and glutamine are known to be linked to gluconeogenesis, renal ammoniagenesis, and cycling of nitrogen among various organs of the body and are therefore two of the most important NEAA.

In catabolic states, nitrogen flows from muscle to the gut and liver largely in the form of glutamine (40), where it ultimately supports accelerated ureagenesis. In the low-protein diet group, the serum urea levels were significantly lower than in the other groups, indicating depressed ureagenesis. In addition, the serum urea levels were inversely related to RBC glutamate levels. Rémésy et al. (28) have recently reported that in rats fed a protein-deficient diet (11%), nitrogen is extensively recycled (64%) as a result of glutamate and glutamine release by the liver. This cycling is much lower (15%) when the dietary protein level is sufficient (22%) to fulfill the nitrogen requirements (28). Metabolic adaptation in the protein-deficient animal preserves AA and cell proteins, avoiding their use in energy production, gluconeogenesis, or lipogenesis (17, 34). The results of the present study, with high RBC glutamate and low urea levels, suggest that a low-protein diet may result in the shunting of nitrogen in the form of glutamine from urea to hepatic glutamate release.

The elevated IGFBP-1 levels on the low-protein diet can be attributed to an increased expression of IGFBP-1 mRNA (14, 35). Glucagon and cytokines have been considered as stimulators of IGFBP-1 expression during starvation, but their effect on glutamine and glutamate turnover in the liver is unknown. Whether the change in hepatic glutamine/glutamate metabolism shares a common regulator with increased hepatic IGFBP-1 expression and decreased IGF-I expression during reduction of protein intake has to be considered (32). Hazel et al. (14) also demonstrated that hepatocyte expression of IGF-I and IGFBP-1 mRNA displays a regional distribution according to the relative position of the hepatocyte within the liver acinus. In rats fed a low-protein diet, higher levels of IGF-I mRNA were found in the periportal versus the perivenous region. In contrast, expression of IGFBP-1 was higher in the perivenous versus the periportal region in rats fed a normal but not a low-protein diet. Because glutamate/glutamine metabolism in the liver acinus is also zonally distributed (13), it is possible that common regulators may exist. Deprivation of AA in the medium of primary rat hepatocytes decreased IGF-I mRNA and increased IGFBP-1 mRNA (35), and in rat hepatoma cells the IGFBP-1 expression increased by the limitation of single EAA, such as phenylalanine, methionine, leucine, and tryptophan (32).

In this study, associations between AA, IGF-I, insulin, and IGFBP-1 levels were found in rats on different protein intake. The increase in RBC glutamate levels with a low-protein diet may indicate alterations in glutamate flux and interorgan nitrogen transport, and it is possible that RBC glutamate could be used as a sensitive index of catabolism.


    ACKNOWLEDGEMENTS

We thank Inga-Lena Wivall, Monika Eriksson, and Kristina Rustas for technical assistance.


    FOOTNOTES

These studies were supported by grants from the Swedish Medical Research Council, Project nos. 1002 and 4224, Novo Nordic Foundation, and Swedish Diabetes Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. C. Divino Filho, Dept. of Renal Medicine, Huddinge University Hospital K56, S-141 86 Huddinge, Sweden (E-mail: jose.divino{at}klinvet.ki.se).

Received 26 August 1998; accepted in final form 21 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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