Parenteral amino acid intake alters the anabolic actions of insulin-like growth factor I in rats

Anthony J. Kee1, Robert C. Baxter2, Anthony R. Carlsson1, and Ross C. Smith1

1 Department of Surgery, University of Sydney, and 2 The Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney, New South Wales 2065, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The anabolic properties of insulin-like growth factor (IGF) I are attenuated by oral diets that are low in protein. However, it is not known whether parenteral nutrition (PN) providing a low amino acid (AA) input will influence IGF-I action. With the use of a rat model, this study examined the interaction between AA input (1.27 and 0.62 g N · kg body wt-1 · 24 h-1, AA and 1/2AA groups, respectively) and recombinant human IGF-I (rhIGF-I, 2.5 mg · kg body wt-1 · 24 h-1) infusion on the composition of the carcass and organs and on plasma insulin, IGF-I, IGF-binding protein 1 (IGFBP-1), and acid-labile subunit (ALS) concentrations. Carcass protein deposition only occurred in the AA groups (P < 0.003) and was not influenced by administration of rhIGF-I. However, visceral protein loss persisted in the AA group but was prevented by rhIGF-I infusion. The changes in water content of the carcass and the organs were generally in the expected proportion of normal lean tissue. The accumulation of lipid that follows the infusion of the AA-deficient PN was prevented by rhIGF-I infusion, which may indicate an improved energy utilization. Neither serum insulin nor ALS concentrations were influenced by the level of AA infusion but were reduced by rhIGF-I administration. However, plasma IGF-I levels were elevated by higher AA infusion and by IGF-I administration. Also, IGFBP-1 concentrations were reduced by the higher AA infusion and increased with rhIGF-I administration. Interestingly, there was a significant interaction effect between both of these influences. It is concluded that free IGF-I concentration, which may be regulated by IGFBP-1 through a direct effect of AAs on the liver, may have an important role in regulating anabolism in visceral and possibly skeletal tissue during PN.

parenteral nutrition; insulin-like growth factor I administration; body composition; organ composition; insulin-like growth factor-binding proteins


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ALTHOUGH PARENTERAL NUTRITION (PN) has become an important therapy for patients unable to take adequate oral nutrition, it has not proven to be the panacea for malnutrition. PN is plagued by a number of important problems that limit its wider use. These problems include atrophy of the intestinal mucosa, impaired immune function, and impaired renal function (12), and it is unable to prevent the loss of protein that occurs during stressful situations (47). The level of amino acid (AA) input is important for the maintenance of body composition in perioperative patients (33) and in preventing the net efflux of AAs from peripheral tissues in depleted nonstressed patients (22). However, lesser amounts of PN input are frequently used to avoid the intolerance seen with rapid infusions (7), and with such infusions sepsis is more common. It has been postulated that the deficiencies of PN may be improved by the use of growth factors.

Growth hormone (GH) was the first of these agents to be shown to increase the anabolic effects of PN (16, 27). It is suggested that the anabolic effects of GH may be such that a net protein anabolic state can be achieved with PN that contains less than the normal daily energy and N requirements (21, 27). However, severely malnourished and critically ill patients are resistant to the normal growth-promoting effects of GH (30, 31, 35, 41), possibly due to an uncoupling of the normal GH/insulin-like growth factor (IGF) I axis with decreased rather than increased IGF-I levels in response to GH (1, 27, 43). It is this suppression of IGF-I levels that is thought to attenuate the protein anabolic effects of GH in these conditions. Consequently, IGF-I may be more effective than GH in improving the efficacy of PN in hypermetabolic patients. This hypothesis is supported by recent studies in animals indicating that IGF-I administration can reduce the loss of weight and protein induced by starvation (15), endotoxemia (9), surgery (26), and dexamethasone treatment (44). There are now also studies in unstressed and stressed animals demonstrating an enhancement of the protein anabolic actions of PN with IGF-I administration (20, 21, 44).

In a previous study of PN in young male rats, an infusion rate of 1.24 MJ/kg body weight was determined the minimum to allow for limited growth. This was 30% less than that required for growth at the rate seen in the chow control group and therefore represents a minimal N input for growth at a rate that avoids hepatobiliary dysfunction (18). It is in this situation that IGF-I should have a most beneficial effect. If growth factors are influential at this lower infusion rate, they may be used to improve the efficacy of PN in the clinical setting.

Exogenous IGF-I may exert an influence in the protein-restricted state through a number of different mechanisms. The serum IGF-I concentration is expected to be increased above the reduced levels occurring in energy and/or protein restriction (14, 37, 38, 42), but endogenous production may be reduced (32). GH effect as measured by the serum acid-labile subunit (ALS) concentration may be reduced because of reduced GH production, decreased hepatic GH binding caused by a decrease in GH receptors (25), and postreceptor downregulation (8). Nutritional deprivation also influences the IGF-binding proteins (IGFBP), which are also potential mediators of the alterations in IGF-I activity (2). Furthermore, the increase in serum IGFBP-1 during fasting and diabetes has been shown to be at least partly due to the decreased insulin concentrations associated with these conditions (46). The level of protein and energy intake also affects the anabolic actions of exogenous IGF-I (23, 34, 38). Protein restriction (38) attenuates the anabolic effects of IGF-I even when serum IGF-I concentrations are increased. In contrast, energy restriction appears to enhance the anabolic effects of exogenous IGF-I (34, 45).

Under the condition of limited oral protein diet, IGF-I infusion appears to increase the weight of kidneys and spleen (38). Similar results were obtained by Ling et al. (20), in which IGF-I infusion in parenterally fed rats produced anabolic effects on the whole body but not on liver or muscle. Whether alterations in parenteral protein and/or energy intake produce similar resistance to the anabolic effects of exogenous IGF-I is unknown. Thus the present studies were undertaken to determine whether the level of parenteral AA input in disease-free relatively unstressed rats affects the anabolic actions of exogenous IGF-I.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Recombinant human IGF-I. Recombinant human IGF-I (rhIGF-I) was kindly supplied by Pharmacia & Upjohn (Stockholm, Sweden). rhIGF-I was added each day to the PN solution for continuous intravenous infusion with the PN solution. The daily dose of rhIGF-I was 2.5 mg/kg body wt, based on the weight of the rats at the start of rhIGF-I infusion. This dose of rhIGF-I is similar to the dose of IGF-I found to be anabolic by other investigators (40). To assess whether there was loss of rhIGF-I during coinfusion of rhIGF-I with PN, rhIGF-I was mixed with the PN solution and infused through the PN catheter system. Determination of IGF-I in the PN solution before and after 30 h of infusion showed that only small amounts (5-6%) of rhIGF-I were lost. This level of rhIGF-I loss during coinfusion with PN is similar to that reported by Yang et al. (44).

Experimental protocol. The Animal Care and Ethics Committee of Royal North Shore Hospital and University of Technology, Sydney, Australia, approved the study design. Male Sprague-Dawley rats were received (Laboratory Animal Services, University of Sydney, Australia) at 4 wk of age (90-110 g) and were placed individually in metabolic cages in a light (0700 lights on, 1900 lights off)- and temperature (23-25°C)-controlled environment. After a 7-day acclimatization period when the animals were given free access to deionized water and rat chow [20% (wt/wt) protein, 5.2% (wt/wt) fat, 3.8% (wt/wt) crude fiber; 16.7 MJ/kg total energy content or 12.0 MJ/kg digestible energy content; Doust and Rabbidge, Sydney, Australia], a catheter was implanted aseptically into the right internal jugular vein as described previously (18).

After surgery, there was a 3-day postoperative recovery period in which the animals received slow (0.5 ml/h) intravenous isotonic saline infusions and were given continued access to chow and water. PN was introduced in the morning (0800-0900) of the fourth postoperative day, at one-half the target rate for 1 day, and thereafter for 8 consecutive days at the target rate of PN infusion. Chow was withdrawn at the start of target-rate PN infusion. Body weight was measured daily. The syringes containing the PN solutions were weighed before and after each day of infusion, and the volume infused was calculated on the basis of a PN solution density of 1.070 g/ml.

The animals were randomized into the following six groups on the morning of the fourth postoperative day: maintenance AA infusion with IGF-I infusion (AA + IGF); maintenance AA infusion without IGF-I infusion (AA - IGF); half-maintenance AA infusion with IGF-I (1/2AA + IGF); half-maintenance AA infusion without IGF-I (1/2AA - IGF), saline, and baseline. The latter two groups received the same surgical procedures as the PN groups. The baseline group animals were killed in the morning of the fourth postoperative day and thus represent a baseline for estimation of changes in body and organ composition that occurred with treatment. The saline group received a continuous infusion of sterile isotonic saline to control for the stress of intravenous infusion. The rate of saline infusion was identical to the rate of PN infused in the PN groups. The saline group rats were fed chow ad libitum throughout the experiment.

Because the postmortem and tissue harvesting protocol was described in detail previously (18), only a brief outline of the procedures will be given. All animals were killed by intravenous pentobarbitone sodium overdose (20 mg Nembutal; Boehringer Ingelheim, Sydney, Australia). After death, catheters, silicone anchor plates, and jackets were removed from the catheterized animals, and the animals were weighed. Catheter tips from all animals were removed aseptically and were sent to the Institution's Microbiology Laboratory for measurement of microbial growth. Three to five milliliters of blood were taken by cardiac puncture and were placed immediately on ice. The time from anesthetic euthanasia until blood collection never exceeded 5 min. The liver, kidneys, lungs, heart, thymus, stomach, small intestine, duodenum, cecum, colon, spleen, testes, pancreas, left gastrocnemius muscle, and diaphragm were removed, and the volume of urine in the bladder was measured. Surface fat and connective tissue were removed from the organs, and the organs were then washed briefly with isotonic saline, blotted briefly on filter paper (Whatman), and weighed. The intestinal tissues were cut open longitudinally, the contents of the intestinal lumen were removed with tweezers, and the tissues were reweighed. The wet weight of the intestinal contents and the urine in the bladder were subtracted from the animal weight at death to calculate the whole body tissue weight. The wet weight of the eviscerated carcass was calculated by subtracting the whole body weight from the total weight of the visceral organs.

PN solutions. The PN solutions were prepared using aseptic techniques in a laminar flow hood (model HWS120; Gelman Science, Sydney, Australia). The maintenance (AA) PN solution consisted of a 1:1:1 mixture of Synthamin 17 (Baxter HealthCare, Sydney, Australia)-50% glucose-20% Intralipid (Kabi Pharmacia, Stockholm, Sweden) with the addition of essential vitamins, minerals, and trace elements (see Table 1). The half-maintenance (1/2AA) PN solution was identical to the maintenance PN solution except one-half of the Synthamin solution was replaced with an electrolyte solution (Baxter HealthCare). These solutions contained a 1:1 ratio of glucose to lipid nonprotein energy, which was the same for all PN protocols.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Composition of the parenteral nutrition solutions

The daily target rate of energy infusion for all animals receiving PN was 1.30 MJ/kg body weight, and the daily target rates of AA N infusion for the AA and 1/2AA groups were 1.30 and 0.65 g N/kg body wt, respectively. The target volumetric rate of PN infusion was 230 ml · kg body wt-1 · 24 h-1. The PN infusion rate was adjusted daily according to body weight of the animal on the day.

Organ and carcass analysis. The organs were minced with scissors and homogenized in deionized water using a Polytron Tissue Homogenizer (model PT10St "OD" S; Kinematica, Lucerne, Switzerland). The eviscerated carcass was minced using a domestic kitchen mincer and was homogenized in ~400 ml of distilled water using a blender (model BLE-37; Breville, Sydney, Australia).

Aliquots of organ (5-20 g) and eviscerated carcass (200 g) homogenates were dried in an oven at 70°C until a constant weight was reached. From the dry weight of the aliquots, the wet weight of the aliquots, and the total wet weight of the homogenates, the total dry material in the organs and eviscerated carcass was calculated. Water content was determined by subtracting the total dry weight from the wet weight of the tissue. Five 0.5-g samples of the dried eviscerated carcass homogenate and a single 50- to 250-mg sample of the dried organ homogenates were taken for N analysis by a macro-Kjeldahl procedure (10). Total protein content was calculated by multiplying total N content by 6.25. Total lipid content of the eviscerated carcass was measured using the chloroform/methanol extraction procedure of Folch et al. (11), modified as previously described (18).

Serum glucose was determined using a commercially available kit based on the glucose oxidase method (Peridochrom; Boehringer Mannheim, Sydney, Australia). Serum insulin was determined by RIA (Pharmacia, Stockholm, Sweden). The human insulin antibody has ~90% cross-reactivity with the rat insulin molecule (40). Rat serum IGFBP-1, ALS, and total IGF-I (endogenous rat IGF-I + rhIGF-I) concentrations were determined by RIA as previously described (3, 5, 19). The polyclonal antibody used in the assay for total IGF-I was shown to react equipotently with recombinant rat IGF-I (Gropep, Adelaide, Australia) and rhIGF-I (Genetech, San Francisco, CA; results not shown).

N balance. All PN syringes were weighed before and after each 24 h of infusion, the weight of chow was measured before and after each 24 h, and the differences were used to calculate the N input. All urine and other debris were collected in the metabolic cages for each 24 h. The aliquots were combined for each 2-day period. Measurement of N was made for N analysis by a macro-Kjeldahl procedure (10). The N balance for each 2-day period was calculated, and the accumulated N balance was calculated by adding the values for the period of days 2-10.

Statistical analysis. Results are presented as means ± SE. Initially, comparisons of results among all groups, including the baseline and saline groups, were made using ANOVA. In addition, the four groups receiving PN were compared by two-way ANOVA using IGF-I infusion and level of AA infusion as the two effects. If statistically significant differences were detected by ANOVA, individual comparisons between groups were made using Fisher's protected least significant difference (PLSD) test (29). A further Fisher's PLSD test was undertaken to determine the influence of IGF-I infusion at the different AA infusion rates. Results were considered significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Forty-one of 47 animals receiving venous catheters successfully reached the study end point (87% success rate). Of the six animals that failed to reach the end point, two died in the immediate postoperative period due to respiratory depression, one died of unknown causes (not septic), and three animals were killed before the end point because of leaking catheters (n = 2) or sepsis (n = 1; positive culture on catheter tip). Apart from the latter animal, there were no positive cultures on any catheter tip at the end point. Thus the septic complication rate in this study was 2%.

Nutrient intakes. The rate of energy infusion was similar for all PN animals (1.29 ± 0.02, 1.29 ± 0.02, 1.30 ± 0.01, and 1.30 ± 0.01 MJ/kg body wt-1 · 24 h-1 for 1/2AA - IGF, 1/2AA + IGF, AA - IGF, and AA + IGF groups, respectively), whereas the rate of N infusion of the 1/2AA groups was one-half that of the AA groups (0.64 ± 0.01, 0.64 ± 0.03, 1.30 ± 0.01, and 1.29 ± 0.02 g N · kg body wt-1 · 24 h-1 for 1/2AA - IGF, 1/2AA + IGF, AA - IGF, and AA + IGF groups, respectively). The chow-fed animals (saline and reference groups) consumed a similar amount of energy (1.2 ± 0.1 MJ · kg body wt-1 · 24 h-1) but a greater amount of N (2.3 ± 0.2 g N · kg body wt-1 · 24 h-1) compared with the PN-infused animals.

Body weight changes. The mean body weights of the animals in each group at the time of operation and randomization to treatment were not significantly different (P > 0.2, ANOVA). The cumulative weight changes during target-rate PN administration are shown in Fig. 1. The AA content of the PN infusion had a significant effect on the cumulative weight change for days 2-8 inclusive (P < 0.001; 2-way ANOVA). rhIGF-I did not have an independent effect on weight (P = 0.29; 2-way ANOVA), but there was an interaction effect (P < 0.028). This appears to be due to a greater effect on the 1/2AA group.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 1.   Cumulative body weight change during target-rate parenteral nutrition (PN) administration. Results are means ± SE for 7 animals in all groups except the amino acid (AA) - insulin-like growth factor (IGF) I group in which there were 6 animals. Day 0 is start of target-rate PN administration. There was a significant effect of the level of AA infusion (P < 0.001) but not IGF-I infusion (P = 0.518) on cumulative weight change from the start of target-rate PN infusion to days 2-8 inclusive (2-way ANOVA). There was no interaction between the level of AA infusion and IGF-I infusion (P = 0.058, 2-way ANOVA) over the same time period. However, there was a significant effect of IGF-I infusion and an interaction between the level of AA infusion and IGF-I infusion on the change in weight from day 5 to day 8 (P = 0.049 and 0.004, respectively; 2-way ANOVA). There was a significant difference in cumulative weight change of the 1/2AA + IGF and 1/2AA + IGF groups from the start of target-rate PN infusion to days 5-8 inclusive [P < 0.05, Fisher's protected least significant difference (PLSD) test].

N balance. The accumulated N balance from day 2 to day 10 was positive for all groups, but this does not take into consideration accumulated errors inherent in the methodology, which, of necessity, will be different between the chow-fed animals and the PN animals (Table 2). With two-way ANOVA with the PN groups, there was a significant effect of AA input (P < 0.0001), but the effect of rhIGF-I failed to reach significance (P = 0.21). There was no significant interaction effect, and Fisher's PLSD showed no significance for the effect of rhIGF-I on the AA subgroups.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Carcass composition of rats

Carcass composition. The composition of the eviscerated carcass is shown in Table 2, and the changes in composition during treatment are presented in Fig. 2. As was the case for body weight change (Fig. 1), the animals receiving the 1/2AA PN regimen lost carcass weight, whereas the animals in the AA groups gained significant amounts of carcass weight (Fig. 2). There was also a trend toward a greater, but not statistically significant, loss of carcass weight with the addition of rhIGF-I to the 1/2AA PN regimen (Table 2 and Fig. 2). The gain in carcass weight of the animals receiving the AA PN regimen was primarily due to a net gain in lipid and protein (Fig. 2). However, the loss of weight of the rats in the 1/2AA groups was mainly due to a loss of carcass water. These rats had no change in carcass protein, and only those in the 1/2AA - IGF-I group had a significant gain in lipid (Fig. 2). rhIGF-I administration had no significant effect on the changes in carcass water or protein during PN administration. However, rhIGF-I infusion greatly reduced the net gain in carcass lipid that occurred with 1/2AA PN infusion but had little effect on carcass lipid deposition during AA PN administration (Fig. 2).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   Changes in eviscerated carcass weight and water, lipid, and protein contents after 8 days of saline infusion + chow (Saline group) or complete PN (AA) or AA-deficient PN (1/2AA) with or without recombinant human IGF-I infusion. Results are means ± SE for 7 animals in all groups except the AA - IGF group in which there were 6 animals. There was a significant effect of the level of AA infusion (P < 0.001) but not IGF-I infusion (P > 0.5) on change in carcass weight or water and protein contents during 8 days of treatment (2-way ANOVA). There was also no interaction between the level of AA infusion and IGF-I infusion for any parameter (P > 0.5, 2-way ANOVA). * P < 0.05, significant differences between the saline group and the groups receiving PN (Fisher's PLSD test). dagger  P < 0.05, changes significantly different from 0 (t-test).

When the carcass composition is examined in relation to the saline/chow control group (Table 2), carcass weight was maintained by AA infusion but not by 1/2AA (P < 0.001), and although there was no direct effect of rhIGF-I there was an interaction effect of AA and rhIGF-I. Carcass protein results followed the weight results, with a positive effect of AA infusion rate and no direct effect of rhIGF-I. Carcass lipid deposition was different, with no direct influence of AA infusion on lipid weight but a direct reduction of lipid by rhIGF-I and also with a significant interaction effect of both these influences.

The ratio of water to protein in the lean carcass was similar in all groups (PN-infused and saline groups; see Table 2) and is similar to what is thought to be the ratio of water to protein in normal lean carcass tissue (3.7 g water/g protein; see Ref. 13).

Organ weights and composition. Fractional organ weights (organ weight as a percentage of body weight) of all organs in which there were significant effects of IGF-I or AA infusion are presented in Table 3. There was no effect of AA infusion or IGF-I treatment on fractional weight of the liver, lungs, heart, and diaphragm (results not shown). The fractional weight of the small intestine/duodenum, colon, cecum, pancreas, and thymus was significantly greater in the saline group compared with the AA - IGF and 1/2AA - IGF groups. IGF-I infusion increased the fractional weight of the kidneys, small intestine/duodenum, colon, cecum, stomach, pancreas, thymus, and spleen (2-way ANOVA). The level of AA infusion had a significant effect on the fractional weight of the colon, stomach, thymus, testis, and left gastrocnemius muscle (2-way ANOVA).

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Organ weights as a percentage of final body weight

The changes in absolute weight and water and protein contents of the kidneys, small intestine/duodenum, thymus, and spleen during 8 days of treatment are presented in Table 4. Both PN regimens resulted in a large and significant loss of kidney, small intestine/duodenum, thymus, and spleen weight. In all of these organs, the loss of weight during PN administration was accompanied by losses of water and protein. When these data are analyzed by two-way ANOVA, the catabolic effect of PN can be shown to be influenced by both AA infusion rate and by the infusion of IGF-I. However, there was only an interaction effect between these two influences for spleen weight loss and the water content of the kidney and spleen. It is of interest that there was a strong influence of IGF-I in the 1/2AA subgroup on the kidney and small intestine (P < 0.001) and a weaker influence on the spleen, but this was not significant for the thymus (Table 4). In the AA + IGF-I group, there was preservation of the weight of kidneys, small intestine, thymus, spleen, and of their water and protein contents (Table 4). In the 1/2AA + IGF group, the kidney and spleen were also strongly influenced by IGF-I, where, for protein in particular, the values reached were not different from the saline group. In all other groups, these measures of organ composition were significantly different from the saline group values (Table 4).

                              
View this table:
[in this window]
[in a new window]
 
Table 4.   Changes in organ composition

Serum hormones, IGFBPs, and glucose concentrations. The 1/2AA PN infusion reduced serum total (endogenous + exogenous) IGF-I concentrations, whereas there was no significant change in total IGF-I concentrations between the baseline and the AA - IGF groups (Table 5). rhIGF-I infusion increased serum total IGF-I concentrations during infusion of both PN regimens; however, the increase in serum total IGF-I concentrations was greater during AA PN infusion (Table 5). rhIGF-I infusion suppressed serum insulin concentrations independent of AA intake (Table 5). The mean serum insulin concentrations of the animals in the 1/2AA - IGF and AA - IGF groups were greater than the baseline and saline groups (P < 0.05, Fisher's PLSD test), and rhIGF-I infusion resulted in a reduction of insulin concentration in both subgroups studied. Serum glucose concentration increased to a similar extent during all treatments.

                              
View this table:
[in this window]
[in a new window]
 
Table 5.   Serum concentrations of total IGF-I, insulin, glucose, IGFBP-1, and ALS

There was an increase in serum IGFBP-1 concentration with IGF-I infusion, the increase being significantly greater during 1/2AA PN than AA PN (Table 5). In contrast to the AA PN regimen, the 1/2AA regimen produced an increase in serum IGFBP-1 concentration (compared with baseline values). ALS concentrations decreased during IGF-I infusion (Table 5). There was a greater suppression of ALS concentration by IGF-I infusion during 1/2AA PN (P < 0.01) but not during AA PN. Serum ALS concentrations were significantly lower in all PN groups than the saline group; however, only the animals receiving rhIGF-I had statistically lower ALS concentrations compared with the baseline group (Table 5).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This PN model has demonstrated that carcass protein growth (mainly skeletal muscle) is dependent on an adequate infusion of AAs and is not influenced by the additional infusion of IGF-I. However, visceral protein is lost with an infusion giving PN providing 1.3 g N · kg body wt-1 · 24 h-1, which was adequate for an increase in muscle mass, but this loss is prevented by the infusion of IGF-I. In the kidney and spleen, there was an interaction between the AA infusion and IGF-I infusion for water retention. The changes in water content of the carcass and the organs were generally in the expected proportion of normal lean tissue. The accumulation of "excess" lipid that follows the infusion of PN compared with the saline group was prevented by the infusion of IGF-I. In this study, the PN infusion rate was chosen to provide the minimal nutrition to allow growth of a young male rat but below the normal growth rate (18). Therefore the 1/2AA group represents significant protein restriction, which may limit the skeletal muscle response to IGF-I.

rhIGF-I and lipid balance. Lipid deposition frequently occurs with PN and is usually attributed to increased insulin secretion (24) when glucose-based solutions are used. The increased lipolysis that occurred with IGF-I infusion requires consideration of interactions with insulin, GH axis, and binding proteins.

A net increase in lipolysis with IGF-I infusion has been observed on a number of occasions and is thought to be mainly due to suppression of insulin secretion (6). Although in this study insulin concentrations were not significantly increased during the lipid-based PN infusion, they were significantly reduced by rhIGF-I infusion when tested by two-way ANOVA (Table 5). Although there was not a statistically significant interaction between AA and IGF-I for insulin concentration, there was a greater significance of the fall in the 1/2AA group. Furthermore, the decreases in serum insulin in response to rhIGF-I (~60% in 1/2AA group and ~40% in the AA group) are proportional to the decreases in lipid content. Therefore, the influence of rhIGF-I may have been through its effect on insulin. However, these changes need to be examined with other factors that are known to influence lipid deposition.

GH is another major effector of lipid balance; in general, it has a net lipolytic effect (6). Although serum GH concentrations were not measured in this study, the increase in lipid deposition during PN infusion, when there was no added rhIGF-I, was associated with a decrease in ALS. ALS may be used as a qualitative assessment of GH activity because GH, which is pulsatile and therefore difficult to measure, stimulates ALS production, which is constant (28). However, GH activity does not provide a simple explanation for all of the carcass lipid results because exogenous rhIGF-I given to the 1/2AA group further reduced ALS concentration, but lipid deposition was not increased further. Alternatively, there is a reduced accumulation of carcass lipid in animals receiving rhIGF-I, which is possibly related to an overriding effect of the lower plasma insulin concentration in this circumstance.

The IGFBPs are other potential regulators of IGF-I activity during protein restriction. Under normal conditions, the relatively high concentration of ALS ensures that most of the circulating IGFs and IGFBP-3 are complexed in a 150-kDa ternary complex (28). In the present study, the molar ratio of serum ALS to IGF-I was 8.3 and 7.3 in the 1/2AA - IGF-I and AA - IGF-I groups compared with 0.9 and 1.8 in the 1/2AA + IGF-I and AA + IGF-I groups. This result, taken together with the decrease in the serum ALS concentration and concomitant increase in IGFBP-1, implies that during 1/2AA + IGF-I there is a shift of IGF-I from the ternary complex to IGFBP-1. Any effect of IGF-I on lipid metabolism would therefore be inhibited, as previous cell culture studies have shown that IGFBP-1 tends to inhibit IGF-I activity (46). Thus the changes in ALS and IGFBP-1 concentrations seen with the 1/2AA PN and both rhIGF-I infusion groups would promote lipid deposition. However, the opposite was found, with a trend of reduction of carcass lipid concentration in these groups. Therefore, it is hypothesized that binding of free IGF-I to IGFBPs occurs more readily than with the ternary complex. Further studies are required to define a cause for the greater suppression of carcass lipid deposition during AA-restricted PN and rhIGF-I infusion.

rhIGF-I and carcass lean tissue. There are a number of potential reasons for the lack of effect of rhIGF-I on carcass lean tissue in the present study. Although the IGF-I dose used in the present study (2.5 mg · kg body wt-1 · 24 h-1) has been shown to stimulate body weight gain in orally fed rats (39), it is possible that the dose of IGF-I required to stimulate growth is greater in parenterally fed rats. This is supported by a recent study where rhIGF-I infused at a dose of 3.6 mg · kg body wt-1 · 24 h-1 stimulated growth of rats fed a PN regimen with a similar energy and AA content to that used in the present study (21).

It is also possible that the relatively high concentration of lipid in the PN solution in the present study was responsible for the lack of effect of IGF-I. Snyder et al. (34) found that the growth-promoting properties of exogenous GH were greater during a high-carbohydrate diet (72% of nonprotein energy was carbohydrate) than a high-lipid diet (80% lipid) in energy-restricted obese women. In the present study, the rats received a 1:1 ratio of glucose to lipid energy. Thus the relatively high concentration of lipid in the PN solutions in the present study may have led to an IGF-I-resistant state. Clearly, studies are required to investigate the effect of dietary lipid-to-glucose ratios on the anabolic actions of IGF-I.

Effects of rhIGF-I and AA intake on visceral tissues. PN led to a decrease in the weight of many visceral organs, and the decrease was not affected by the level of AA intake. rhIGF-I infusion reduced much of the PN-induced loss of visceral mass; however, this effect was more marked during AA PN, particularly for the kidney, thymus, and spleen. The ratios of change in water to protein in these organs were similar to the ratio of water to protein in normal lean tissue (3.7-4.5). Thus, although PN produced a loss of organ weight and organ lean tissue weight, the composition of the lean organ tissue was not altered. Similarly, these results suggest that rhIGF-I stimulated a gain in normal lean tissue in these organs.

Exogenous IGF-I has been shown to have anabolic effects on visceral tissues, but not carcass, when AA input is limiting. It is well known that IGF-I has greater trophic effects on the intestine, kidneys, thymus, and spleen than on many other visceral tissues (37, 39). However, there are only a few reports on the effects of dietary AA deficiency on the trophic effects of exogenous IGF-I in visceral tissues (37, 39). In this study, AA-deficient PN led to a relative resistance to the normal anabolic effects of IGF-I in the kidneys, spleen, and thymus (see Tables 4 and 5).

Regulation of IGFBP-1 concentrations. The hepatic production of IGFBP-1 is activated by nutrient deprivation and is potently inhibited by insulin. It is generally thought that the increase in IGFBP-1 production during nutrient deprivation is primarily due to a fall in insulin concentration (46). However, in the present study, the greater IGFBP-1 concentration of the 1/2AA - IGF-I group compared with the AA - IGF-I group was not associated with lower insulin concentrations. Furthermore, the rhIGF-I-induced increase in serum IGFBP-1 was much greater during 1/2AA PN compared with AA PN (158 vs. 67 g/l, respectively), even though the decreases in insulin concentrations were similar for the two groups. Changes in glucose concentrations do not account for the increased IGFBP-1 during 1/2AA PN as there were no differences in glucose concentration between any of the PN groups. Therefore, the increase in IGFBP-1 concentration during AA restriction in this rat model is independent of insulin and glucose. Baxter et al. (4) also observed that neither glucose nor insulin was the major mediator of the increase in IGFBP-1 concentration in fasted individuals with IGF-I infusion.

The present results suggest that AA input may influence the serum IGFBP-1 concentration. This is because IGFBP-1 and total IGF-I concentrations demonstrated strong associations with AA input and exogenous IGF-I, and there was an interaction effect between both of these influences. This is supported by a recent study that showed an increase in IGFBP-1 mRNA abundance in hepatocytes deprived of AAs (37). Jousse et al. (17) demonstrated that leucine deficiency exaggerated the production of IGFBP-1 in cultures of Hep G2 cells. It remains to be determined whether there are other specific AAs responsible for this effect. It is of interest to note that the spleen was the only organ that responded to AAs and IGF-I with a similar interaction effect to that seen with total IGF-I and IGFBP-1 concentrations. Further work is needed to explore these findings.

The loss of visceral tissue and impaired growth of skeletal tissue in the 1/2AA - IGF-I group were possibly modulated by a reduction in total endogenous IGF-I and an increase in IGFBP-1, the latter likely to further reduce the biological effects of IGF-I. Furthermore, there was a decrease in ALS concentrations, indicating a reduction in GH activity. The addition of IGF-I to this AA-deficient regimen had the interesting effect of increasing visceral tissue mass but had no effect on skeletal tissue. Although total IGF-I was increased to normal levels with this treatment, there was an increase in IGFBP-1 concentrations and a reduction in ALS levels, the latter indicating a further reduction in GH activity. This indicates that IGF-I has a powerful effect on visceral tissues even in the circumstance of a relative deficiency of AAs where there is a lack of growth of skeletal tissue and a suppression of GH. By increasing the AA infusion (from 1/2AA PN to AA PN), without exogenous IGF-I, skeletal tissue growth occurred, but visceral tissue growth did not. These changes occurred without marked changes in total IGF-I, insulin, or ALS levels, but the IGFBP-1 levels remained at baseline values, which may have made IGF-I more available. Thus the presence of adequate AA intake may influence the activity of IGF-I through an effect on the IGFBP-1, possibly through an influence on the liver. The addition of IGF-I to the AA PN regimen reduced visceral tissue loss that occurred during 1/2AA PN. This was associated with an increase in total IGF-I and IGFBP-1 levels. The suppression of ALS by IGF-I infusion was less during AA PN than 1/2AA PN. Therefore, it is likely that the changes in visceral tissues are due to a direct effect of IGF-I on these tissues.

This study confirms that exogenous IGF-I has greater anabolic effects in visceral tissues than carcass lean tissue. The anabolic effect of exogenous IGF-I in the kidneys, thymus, and spleen is influenced by AA-deficient PN. Conversely, AA deficiency appeared to enhance the IGF-I-induced suppression of net lipid deposition. Alterations in insulin or GH secretion do not seem to be mediators of these effects, which are more cogently explained by the nutrient-specific regulation of IGF-I activity by the IGFBPs. Because of the clinical importance of maintaining the viscera and the immune system and preventing excessive lipid accretion in patients requiring intravenous nutrition, further work should be undertaken to improve our knowledge of the mechanisms influencing these observations.


    ACKNOWLEDGEMENTS

This study was supported by the National Health and Medical Research Council, the Northern Sydney Area Health Service, and Pharmacia&Upjohn.


    FOOTNOTES

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: R. C. Smith, Dept. of Surgery, Royal North Shore Hospital, St. Leonards NSW 2065, Australia (E-mail: rsmith{at}med.usyd.edu.au).

Received 22 June 1998; accepted in final form 3 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abribat, T., P. Brazeau, I. Davignon, and D. R. Garrel. Insulin-like-growth factor-1 blood levels in severely burned patients: effects of time post injury, age of patient and severity of burn. Clin. Endocrinol. Metab. 39: 583-589, 1993.

2.   Baxter, R. C. Insulin-like growth factor binding proteins in the human circulation: a review. Horm. Res. 42: 140-144, 1994[Medline].

3.   Baster, R. C., and J. Dai. Purification and characterization of the acid-labile subunit of rat serum insulin-like growth factor binding protein complex. Endocrinology 134: 848-852, 1994[Abstract].

4.   Baxter, R. C., N. Hizuka, K. Takano, S. R. Holman, and K. Asakawa. Responses of insulin-like growth factor binding protein-1 (IGFBP-1) and the IGFBP-3 complex to administration of insulin-like growth factor-I. Acta Endocrinol. 128: 101-108, 1993[Medline].

5.   Baxter, R. C., Z. Zaltman, J. R. Oliver, and J. O. Willoughby. Pulsatility of immunoreactive somatomedin-C in chronically cannulated rats. Endocrinology 113: 729-734, 1983[Abstract].

6.   Berneis, K., and U. Keller. Metabolic actions of growth hormone: direct and indirect. Bailliere's Clin. Endocrinol. Metab. 10: 337-352, 1996[Medline].

7.   Brennan, M. F., P. W. Pisters, M. Posner, O. Quesada, and M. A. Shike. A prospective randomized trial of total parenteral nutrition after major pancreatic resection for malignancy. Ann. Surg. 220: 436-441, 1994[Medline].

8.   Clemmons, D. R., and L. E. Underwood. Nutritional regulation of IGF-I and IGF binding proteins. Annu. Rev. Nutr. 11: 393-412, 1991[Medline].

9.   Dickerson, R. N., C. B. Manzo, S. L. Charland, R. G. Settle, T. P. Stein, D. A. Kuhl, and J. J. Rajter. The effect of insulin-like growth factor-1 on protein metabolism and hepatic response to endotoxemia in parenterally fed rats. J. Surg. Res. 58: 260-266, 1995[Medline].

10.   Fleck, A., and H. N. Munro. The determination of organic nitrogen in biological materials. A review. Clin. Chim. Acta 11: 2-12, 1965.

11.   Folch, J., M. Lees, and G. H. Sloane-Stanley. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497-509, 1957[Free Full Text].

12.   Fong, Y., M. A. Marano, A. Barber, W. He, L. L. Moldawer, D. Bushman, S. M. Coyle, G. T. Shires, and S. F. Lowry. Total parenteral nutrition and bowel rest modify the metabolic response to endotoxins in humans. Ann. Surg. 210: 449-457, 1989[Medline].

13.   Forbes, R. M., H. M. Mitchell, and A. R. Cooper. Further studies on the gross composition and mineral elements of the adult human body. J. Biol. Chem. 223: 969-975, 1956[Free Full Text].

14.   Isley, W. L., L. E. Underwood, and D. R. Clemmons. Changes in plasma somatomedin-C in response to ingestion of diets with variable protein and energy content. J. Parenter. Enteral Nutr. 8: 407-411, 1984[Abstract].

15.   Jacob, R., E. Barrett, G. Plewe, K. D. Fagin, and R. S. Sherwin. Acute effects of insulin-like growth factor I on glucose and amino acid metabolism in the awake fasted rat. Comparison with insulin. J. Clin. Invest. 83: 1717-1723, 1989[Medline].

16.   Jiang, Z.-M., G.-Z. He, S.-Y. Zhang, X.-R. Wang, N.-F. Yang, Y. Zhu, and D. W. Wilmore. Low-dose growth hormone and hypocaloric nutrition attenuate the protein-catabolic response after major operation. Ann. Surg. 210: 513-525, 1989[Medline].

17.   Jousse, C., A. Bruhat, M. Ferrara, and P. Fafournoux. Physiological concentrations of amino acids regulate insulin-like-growth-factor-binding protein 1 expression. Biochem. J. 334: 147-153, 1998[Medline].

18.   Kee, A. J., and R. C. Smith. The effect of the rate and route of nutrient delivery on total body and organ composition in rats. Nutrition 12: 180-188, 1996[Medline].

19.   Lewitt, M. S., H. Saunders, J. L. Phuyal, and R. C. Baxter. Regulation of insulin-like growth factor-binding protein-1 in rat serum. Diabetes 43: 232-239, 1994[Abstract].

20.   Ling, P. R., C. Gollaher, E. Colon, N. Istfan, and B. R. Bistrian. IGF-I alters energy expenditure and protein metabolism during parenteral feeding in rats. Am. J. Clin. Nutr. 61: 116-112, 1995[Abstract].

21.   Lo, H.-C., P. S. Hinton, C. A. Peterson, and D. M. Ney. Simultaneous treatment with IGF-I and GH additively increases anabolism in parenterally fed rats. Am. J. Physiol. 269 (Endocrinol. Metab. 32): E368-E376, 1995[Abstract/Free Full Text].

22.   Loder, P. B., R. C. Smith, A. J. Kee, S. R. Kohlhardt, M. M. Fischer, M. Jones, and T. S. Reeve. What rate of infusion of intravenous nutrition is required to stimulate uptake of amino acids by peripheral tissues in depleted patients? Ann. Surg. 211: 360-368, 1990[Medline].

23.   Lowe, W. L., Jr., M. Adamo, H. Werner, C. T. Roberts, Jr., and D. Leroith. Regulation by fasting of rat insulin-like growth factor I and its receptor. Effects on gene expression and binding. J. Clin. Invest. 84: 619-626, 1989[Medline].

24.   MacFie, J., A. G. Yule, and G. L. Hill. Effect of added insulin on body composition of gastroenterological patients receiving intravenous nutrition---a controled clinical trial. Gastroenterology 81: 285-289, 1981[Medline].

25.   Maes, M., Y. Amand, L. E. Underwood, D. Maiter, and J. M. Ketelslegers. Decreased serum insulin-like growth factor I response to growth hormone in hypophysectomized rats fed a low protein diet: evidence for a postreceptor defect. Acta Endocrinol. 117: 320-326, 1988[Medline].

26.   Malmlöf, K., Z. Cortova, H. Saxerholt, E. Karlsson, V. Arrhenius-Nyberg, and A. Skottner. Effects of insulin-like growth factor-I and growth hormone on the net flux of amino acids across the hind limbs in the surgically traumatized pig. Clin. Sci. Mol. Med. 88: 285-292, 1995.

27.   Manson, J. M. K., and D. W. Wilmore. Positive nitrogen balance with human growth hormone and hypocaloric intravenous feeding. Surgery 100: 188-197, 1986[Medline].

28.   Martin, J. L., and R. C. Baxter. Insulin-like growth factor binding protein-3: biochemistry and physiology. Growth Regul. 2: 88-99, 1992[Medline].

29.   Milliken, G. A., and D. E. Johnson. Analysis of Messy Data. New York: Van Nostrand Reinhold, 1984, p. 33.

30.   Ross, R. J. M., J. P. Miell, J. M. P. Holly, H. Maheshwari, M. Norman, A. F. Abdulla, and C. R. Buchanan. Levels of GH binding activity, IGFBP-1, insulin, blood glucose and cortisol in intensive care patients. Clin. Endocrinol. Metab. 35: 361-367, 1991.

31.   Roth, E., L. Valentini, M. Semsroth, T. Holzenbein, S. Winkler, W. F. Blum, M. B. Ranke, M. Schemper, A. Hammerle, and J. Karner. Resistance of nitrogen metabolism to growth hormone treatment in the early phase after injury of patients with multiple injuries. J. Trauma 38: 136-141, 1995[Medline].

32.   Schalch, D. S., H. Yang, D. M. Ney, and R. D. Dimarchi. Infusion of human insulin-like growth factor-I (IGF-I) into malnourished rats reduces hepatic IGF-I mRNA abundance. Biochem. Biophys. Res. Commun. 160: 795-800, 1989[Medline].

33.   Smith, R. C., L. Burkinshaw, and G. L. Hill. Optimal energy and nitrogen intake for gastroenterological patients requiring intravenous nutrition. Gastroenterology 82: 425-452, 1982[Medline].

34.   Snyder, D. K., D. R. Clemmons, and L. E. Underwood. Dietary carbohydrate content determines responsiveness to growth hormone in energy-restricted humans. J. Clin. Endocrinol. Metab. 69: 745-752, 1989[Abstract].

35.   Tayek, J. A., and J. A. Brasel. Failure of anabolism in malnourished cancer patients receiving growth hormone: a clinical research center study. J. Clin. Endocrinol. Metab. 80: 2082-2087, 1995[Abstract].

36.   Thissen, J. P., J. B. Pucilowska, and L. E. Underwood. Differential regulation of insulin-like growth factor I (IGF-I) and IGF binding protein-1 messenger ribonucleic acids by amino acid availability and growth hormone in rat hepatocyte primary culture. Endocrinology 134: 1570-1576, 1994[Abstract].

37.   Thissen, J. P., L. E. Underwood, D. Maiter, M. Maes, D. R. Clemmons, and J.-M. Ketelslegers. Failure of insulin-like growth factor-I (IGF-I) infusion to promote growth in protein-restricted rats despite normalization of serum IGF-I concentrations. Endocrinology 128: 885-890, 1991[Abstract].

38.   Tirapegui, J., M. Baldi, and S. L. Ribeiro. Effect of protein deficiency on plasma insulin-like growth factor-I (IGF-I) and protein and proteoglycan synthesis rates in skeletal muscle and bone. Nutr. Res. 16: 869-879, 1996.

39.   Tomas, F. M., S. E. Knowles, P. C. Owens, L. C. Read, C. S. Chandler, S. E. Gargosky, and F. J. Ballard. Effects of full-length and truncated insulin-like growth factor-I on nitrogen balance and muscle protein metabolism in nitrogen-restricted rats. J. Endocrinol. 128: 97-105, 1991[Abstract].

40.   Tomas, F. M., A. B. Lemmey, L. C. Read, and F. J. Ballard. Superior potency of infused IGF-I analogues which bind poorly to IGF-binding proteins is maintained when administered by injection. J. Endocrinol. 150: 77-84, 1996[Abstract].

41.   Voerman, B. J., R. J. M. Strack Van Schijndel, A. B. J. Groeneveld, H. de Boer, J. P. Nauta, and L. G. Thijs. Effects of human growth hormone in critically ill nonseptic patients: results from a prospective randomized, placebo-controlled trial. Crit. Care Med. 23: 665-673, 1995[Medline].

42.   Weller, P. A., M. J. Dauncey, P. C. Bates, J. M. Brameld, P. J. Buttery, and R. S. Gilmour. Regulation of porcine insulin-like growth factor-I and growth hormone receptor mRNA expression by energy status. Am. J. Physiol. 266 (Endocrinol. Metab. 29): E776-E785, 1994[Abstract/Free Full Text].

43.   Wojnar, M. M., J. Fan, R. A. Frost, M. C. Gelato, and C. H. Lang. Alterations in the insulin-like growth factor system in trauma patients. Am. J. Physiol. 268 (Regulatory Integrative Comp. Physiol. 37): R970-R977, 1995[Abstract/Free Full Text].

44.   Yang, H., M. Grahn, D. S. Schlach, and D. M. Ney. Anabolic effects of IGF-I coinfused with total parenteral nutrition in dexamethasone-treated rats. Am. J. Physiol. 266 (Endocrinol. Metab. 29): E690-E698, 1994[Abstract/Free Full Text].

45.   Yang, H., D. S. Schalch, and D. M. Ney. Anabolic effects of insulin-like growth factor I in calorie restricted and ad libitum fed rats. Nutr. Res. 10: 1151-1160, 1990.

46.   Zapf, J. Physiological role of insulin-like growth factor binding proteins. Eur. J. Endocrinol. 132: 645-654, 1995[Medline].

47.   Ziegler, T. R., C. Gatzen, and D. W. Wilmore. Strategies for attenuating protein-catabolic responses in the critically ill. Annu. Rev. Med. 45: 459-480, 1994[Medline].


Am J Physiol Endocrinol Metab 277(1):E63-E72
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society