Endotoxin-induced changes in IGF-I differ in rats provided enteral vs. parenteral nutrition

Margaret M. Wojnar1, Jie Fan2, Yue Hua Li2, and Charles H. Lang2

1 Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Internal Medicine, and 2 Departments of Cellular and Molecular Physiology and of Surgery, Pennsylvania State College of Medicine, Hershey, Pennsylvania 17033


    ABSTRACT
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Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

The purpose of the present study was to determine whether acute changes in the insulin-like growth factor (IGF) system induced by mild surgical trauma/fasting or endotoxin [lipopolysaccharide (LPS)] are differentially modulated by total enteral nutrition (TEN) or total parenteral nutrition (TPN). Rats had vascular catheters and a gastrostomy tube surgically placed and were fasted overnight. The next morning animals randomly received an isocaloric, isonitrogenous (250 kcal · kg-1 · day-1, 1.6 g N · kg-1 · day-1) infusion of either TEN or TPN for 48 h. Then rats were injected intravenously with Escherichia coli LPS (1 mg/kg) while nutritional support was continued. Time-matched control animals were injected with saline. After mild surgical trauma and an 18-h fast, TEN was more effective at increasing plasma IGF-I levels than TPN. Subsequent injection of LPS decreased IGF-I in blood, liver, and muscle in both TEN- and TPN-fed rats compared with saline-injected control animals. However, this decrease was ~30% greater in rats fed TPN compared with those fed TEN. LPS-induced downregulation of IGF-I mRNA expression in liver and muscle was also more prominent in TPN-fed rats. The LPS-induced increase in plasma corticosterone and tumor necrosis factor-alpha was greater (2- and 1.6-fold, respectively) in TPN-fed rats, and these changes were consistent with the greater reduction in IGF-I seen in these animals. In similarly treated rats allowed to survive for 24 h after LPS injection, the LPS-induced increase in the urinary 3-methylhistidine-to-creatinine ratio was smaller in TEN-fed rats. In summary, LPS reduced systemic levels of IGF-I as well as IGF-I protein and mRNA in critical target organs. Enteral feeding greatly attenuated this response. Maintenance of higher IGF-I levels in TEN-fed rats was associated with a reduction in inflammatory cytokine levels and lower rates of myofibrillar degradation.

insulin-like growth factor-binding protein-1; insulin; corticosterone; 3-methylhistidine; tumor necrosis factor-alpha ; lipopolysaccharide


    INTRODUCTION
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Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

SURGICAL TRAUMA, infection, and thermal injury are known to produce rapid and sustained decreases in the circulating concentration of insulin-like growth factor (IGF) I (10, 22, 23, 25). In experimental models of these conditions, the reduction in plasma IGF-I levels is closely correlated to decreases in IGF-I protein content and mRNA expression in liver, the primary site of synthesis, and in skeletal muscle, a key target tissue (25). This reduction in plasma and tissue IGF-I content has been suggested to be responsible, at least in part, for the muscle wasting and insulin resistance produced by critical illness (13). Hence, efforts have been made to increase IGF-I levels during catabolic conditions in an attempt to reverse or prevent some of these injury-related alterations in muscle metabolism. Numerous studies have now demonstrated the ability of either IGF-I or growth hormone, the proximal mediator of hepatic IGF-I secretion, to improve nitrogen balance and modulate injury-induced derangements in muscle protein balance (5, 15, 20).

Endogenous IGF-I synthesis is exquisitely sensitive to the nutritional status of the host (as reviewed in Ref. 38). Fasting and prolonged protein-calorie malnutrition lead to a sustained decrease in the plasma IGF-I concentration (31, 39). This decrease appears to result from both a reduction in hepatic synthesis of the peptide and an increased rate of IGF-I clearance from the circulation (31, 37). The increase in IGF-I levels after refeeding is at least partially dependent on nutrient composition. That is, the recovery of IGF-I is slower in subjects fed a diet in which protein calories are reduced compared with individuals fed an isocaloric diet with adequate protein (19, 39). Increases in IGF-I also appear greater in diet-restricted individuals fed a high-carbohydrate diet (80% of nonprotein calories) compared with those provided a lipid-rich diet (72% of nonprotein calories) in which protein intake was constant (36).

However, no information is available concerning whether the route (i.e., parenteral vs. enteral) of nutritional support affects the various components of the IGF system. The route of nutritional support in catabolic patients and experimental animals modulates a number of critical aspects of host defense, including bacterial translocation, immune function, hormone secretion, and cytokine production (as reviewed in Ref. 30). Because the IGF system can also be regulated by classical hormones (e.g., insulin and glucocorticoids) (3, 4, 25) as well as by cytokines [interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)] (8, 11, 22), nutritionally induced changes in these systems may secondarily modulate IGF-I synthesis and secretion.

The purpose of the present study was to determine whether there is a differential effect of parenteral vs. enteral feeding in modulating 1) the recovery of IGF-I levels after mild surgical trauma and an overnight fast, and 2) the reduction in IGF-I levels after an acute septiclike insult imposed by endotoxin [lipopolysaccharide (LPS)]). We also investigated whether the normally occurring increase in IGF-binding protein (IGFBP)-1 induced by either fasting or LPS was influenced by total enteral nutrition (TEN) or total parenteral nutrition (TPN). Moreover, we measured plasma levels of insulin, corticosterone, and TNF-alpha to determine whether there was a correlation between changes in these known regulators and the fasting- and LPS-induced changes in either IGF-I or IGFBP-1.


    MATERIALS AND METHODS
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

Animals and surgical procedure. Male Sprague-Dawley rats (400 ± 12 g; ~14-15 wk of age; Taconic Farms, Germantown, NY) were housed at a constant temperature, exposed to a 12:12-h light-dark cycle, and maintained on standard rodent chow (Purina, St. Louis, MO) and water ad libitum for 1 wk before experiments were initiated. All experiments were approved by the Institutional Animal Care and Use Committee, and these studies adhered to the National Institutes of Health guidelines for the use of experimental animals.

Rats were anesthetized with an intramuscular injection of ketamine (9 mg/100 g) and xylazine (0.9 mg/100 g), and sterile surgery was performed. Rats in the TEN group underwent a midline laparotomy and placement of a silicon rubber gastrostomy tube. Animals in the TPN group had a silicone catheter inserted in the right jugular vein. A midline laparotomy was also performed on animals in this group as a sham operation. In addition, rats in both groups had a catheter (PE-50) inserted into the carotid artery (8). Catheters were exteriorized between the scapulae, passed through a tightly coiled stainless steel spring, and fixed to a freely rotating swivel (Instech, Plymouth Meeting, PA). After surgery, animals were housed in wire-bottom cages and fasted overnight but had free access to water.

Nutritional support. The next morning, a continuous infusion of either TEN or TPN was started and continued for the next 48 h. Nutritional support was prepared and infused (Harvard Apparatus, South Natick, MA) under sterile conditions. TEN contained (per liter) 43 g of amino acids (16% of total calories), 45 g of lipid (38%), and 123 g of carbohydrate (46%), in addition to various vitamins and minerals (Ultracal, Mead-Johnson Nutritionals, Evansville, IN). Animals received ~250 kcal · kg-1 · day-1 (1.06 kcal/ml × 3 ml/h) containing 1.6 g N · kg-1 · day-1 (total calorie-to-N ratio of 153:1). The parenteral formulation was composed of essential and nonessential amino acids (Freamine III 8.5%, McGaw, Irvine, CA), dextrose (Baxter Healthcare, Deerfield, IL), and fat (Intralipid 20%, Kabi Vitrum, Stockholm, Sweden) and yielded a mixture that was isocaloric and isonitrogenous with that of the enteral preparation. The TPN was also supplemented with trace elements and vitamins (Multiple Vitamine Infusate, USV Pharmaceuticals, Tuckahoe, NY) such that the concentration of these components was comparable in both feeding regimens. The individual components of both nutritional solutions are provided in Table 1.

                              
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Table 1.   Composition of nutrition solutions

Arterial blood samples were obtained immediately before the start of feeding (i.e., after an overnight fast) and 24 and 48 h later. After the 48-h sample, rats were injected intravenously with Escherichia coli LPS (1 mg/kg; 026:B6; Difco, Detroit, MI). Time-matched control animals were injected with an equal volume (0.5 ml/100 g body wt) of sterile saline (0.9%). Additional arterial blood samples were taken at 90 and 240 min thereafter. Blood samples were used to determine the plasma concentrations of IGF-I, various IGFBPs, insulin, corticosterone, glucose, and triglycerides. An equal volume of sterile saline was used as volume replacement after each blood sample. After the final sampling point, rats were rapidly anesthetized with pentobarbital and exsanguinated, and selected tissues were obtained for the determination of IGF-I protein and mRNA abundance. All animals injected with LPS survived until the 4-h sample point. Tissue samples were rinsed in ice-cold saline and blotted extensively to minimize blood contamination.

A second study was performed with similarly prepared animals. In this study, animals were housed in metabolic cages before and for 24 h after the injection of LPS. One group of animals was infused with TPN and the other with TEN. An additional group of time-matched fasted rats was also included in this study. All animals in this protocol received an intravenous injection of LPS, as described above. Blood (0.5 ml) was obtained at 90 and 240 min after LPS administration for the determination of plasma TNF-alpha levels. The concentration of 3-methylhistidine (3-MH) and creatinine in a 24-h urine sample was determined to provide an estimate of myofibrillar breakdown. Plasma IGF-I levels were also measured in these animals at the conclusion of the study.

Analytical methods. The IGF-I concentration in plasma and tissues was determined by RIA, as previously described (8, 11, 22). Before assay, plasma was extracted with a modified acid-ethanol (0.25 N HCl:87.5% ethanol) procedure with overnight cryoprecipitation, and tissues were extracted with acid homogenization with subsequent Sep-Pak C18 extraction (8, 11, 22). The tissue eluate was evaporated, and the dried sample was reconstituted with phosphate buffer. Previous studies have indicated a strong correlation between liver and muscle IGF-I content and mRNA expression (11, 25). Moreover, LPS-induced changes in IGF-I peptide content are similar between freeze-clamped tissue in vivo and after the perfusion of the organ with a blood-free perfusate (9). Hence, although some contamination of the tissue with IGF-I present in the extracellular fluid is expected, measurements of tissue IGF-I peptide content appear to adequately represent intracellular and membrane-bound IGF-I.

To determine IGFBP-1 levels, plasma samples were first separated on a 12.5% SDS-PAGE gel under nonreducing conditions. Separated proteins were electroblotted overnight onto nitrocellulose and blocked with Tris-buffered saline containing nonfat dry milk, as previously described (22). Membranes were then incubated with antiserum against rat IGFBP-1 (kindly provided by Dr. S. Shimisaki; The Scripps Research Institute, La Jolla, CA). Antigen-antibody complexes were identified with goat anti-rabbit immunoglobulin G tagged with horseradish peroxidase (Sigma, St. Louis, MO) and were exposed to the enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL) and X-ray film (Kodak X-Omat AR; Eastman Kodak, Rochester, NY). Band intensities were determined in triplicate with a laser densitometer (Hoeffer, San Francisco, CA). Representative samples from all experimental groups were electrophoresed on the same gel; data are expressed as a percentage of control value.

Total RNA from whole tissue was prepared by a single-step acid guanidinium thiocyanate method (6), slightly modified by our laboratory (25). Aliquots of total RNA were quantitated by measuring the absorbance at 260 nm, and the accuracy was confirmed by comparing the intensity of ethidium bromide staining of the 28S and 18S ribosomal bands after agarose gel electrophoresis. All aliquots of RNA were frozen in liquid nitrogen and stored at -70°C until they were used in subsequent nuclease protection assays.

RNA (10 µg for liver and 25 µg for muscle) was hybridized with ~200,000 counts/min of the [32P]UTP-labeled riboprobe at 45°C for 16 h. The preparation of the antisense RNA probe has been previously described (1, 24). After RNase digestion, protected hybrids were denatured and resolved on 6% polyacrylamide-8 M urea gels. The dried gel was apposed to X-ray film to generate an autoradiograph at -70°C. Exposure times were varied to generate multiple autoradiographic exposures. Abundance of IGF-I mRNA was semiquantified by measuring protected bands with a laser densitometer.

The arterial plasma concentrations of insulin and corticosterone were determined on each sample by RIA (Diagnostic Products, Los Angeles, CA). The assay characteristics have been previously described (22, 25). Plasma TNF-alpha was determined with an enzyme-linked immunosorbent assay (Biosource International, Camarillo, CA) specific for rat TNF-alpha . Plasma triglyceride levels were measured colorimetrically after extraction with Dole's reagent. Plasma glucose concentrations were determined with a rapid glucose analyzer (YSI, Yellow Springs, OH).

3-MH was determined by HPLC analysis on 24-h urine samples. Briefly, urine samples were precipitated with perchloric acid and centrifuged to remove particulate material. Samples were then derivatized with fluorescamine before injection. Urine samples were injected onto a Waters Novapak C18 column, and fluorescence was detected with a fluorometric detector (ABI Analytical, Ramsey, NJ) with an excitation wavelength of 375 nm and a long-pass filter >418 nm. The isocratic mobile phase consisted of 1 M phosphate buffer (pH 6.5) and acetonitrile and water; the flow rate was 1.0 ml/min. Urinary 3-MH was calculated on the basis of an external 3-MH standard (Sigma). Urinary creatinine concentration was determined with a standard colorimetric method (Sigma).

Experimental values are presented as means ± SE. The number of rats per group is indicated in Figs. 1, 2, and 3-7 and in Table 2. To determine statistical significance of time-matched values, data were analyzed by one-way ANOVA followed by a Student-Newman-Keuls test to determine treatment effect, unless otherwise noted. Statistical significance was set at P < 0.05. For some variables, a paired t-test was used to determine whether values were significantly altered by 48 h of nutritional support (i.e., refeeding) compared with 18-h fasted values from the same treatment group. For these comparisons, statistical significance was set at P < 0.01. For the LPS-induced changes in plasma IGF-I, the area under the curve (AUC) was calculated with the trapezoidal solution after first subtracting the mean time-matched value from saline-injected animals in the same treatment group.


    RESULTS
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Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

IGF-I concentrations. The first blood sample from each animal was obtained after an 18-h fast, and, as expected, the plasma IGF-I concentration was not different between the various groups (Fig. 1; entire group average 497 ± 8 ng/ml). Thereafter, animals were continuously infused with either TPN or TEN for the next 2 days. IGF-I concentrations increased gradually in both groups of rats receiving nutritional support, albeit more slowly in the TPN-fed group. After 48 h of nutritional support, IGF-I levels were 30% higher in TEN-fed than in TPN-fed rats.


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Fig. 1.   Plasma insulin-like growth factor (IGF) I concentrations in animals infused with total parenteral (TPN) or total enteral (TEN) nutrition before and after iv injection of endotoxin [lipopolysaccharide (LPS)]. The -48-h sample was taken after an ~18-h fast. Thereafter, an isocaloric, isonitrogenous infusion of either TPN or TEN was started and maintained for remainder of study. At time 0, rats were injected iv with either LPS or an equal volume of sterile saline (SAL). Values are means ± SE; n = 8-9 rats/group. * P < 0.05 vs. time-matched values from TPN-SAL group. + P < 0.05 vs. time-matched values from TEN-SAL group. § P < 0.05 vs. time-matched values from TEN-LPS group.

Both groups of animals responded to the injection of LPS with a marked decrease in plasma IGF-I (Fig. 1). However, the fall in IGF-I in the TEN-infused rats was smaller than that observed in animals infused with TPN. Because IGF-I levels differed in TEN- and TPN-fed rats before the injection of LPS, the magnitude of the LPS-induced decrease in IGF-I is better illustrated by examining the change in this variable from baseline. When the AUC was calculated, the LPS-induced decrease in IGF-I in animals fed enterally was 44% greater than that observed in parenterally fed rats (AUC for TPN = 778 ± 86 vs. AUC for TEN = 540 ± 69 ng · ml-1 · h; P < 0.05, t-test). Circulating IGF-I levels remained unchanged in both groups of time-matched animals injected with saline.

After the final blood sample was collected, animals were anesthetized, and samples of liver and gastrocnemius were freeze-clamped for the determination of IGF-I protein and mRNA abundance. In animals that did not receive LPS, the IGF-I content in liver from TEN-infused rats was 23% higher than that detected in liver from TPN-infused animals (Fig. 2A). Although the hepatic IGF-I content was decreased in both groups after LPS, the reduction observed in the TPN group (44%) was greater than that observed in the TEN group (29%). The IGF-I content in gastrocnemius was not different between TPN and TEN groups injected with saline (Fig. 2B). However, as seen with liver, the LPS-induced decrease in IGF-I in muscle was greater in TPN (43%) compared with TEN-infused rats (25%). There were no significant changes in the wet-to-dry weight ratio induced by either LPS or route of nutritional support in either liver or muscle at this time point (data not shown). Hence, comparable changes in tissue IGF-I content were detected when data were expressed per milligram of tissue protein or per gram of dry weight.


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Fig. 2.   Content of IGF-I in liver (A) and gastrocnemius (B) in TPN- and TEN-infused rats 4 h after injection of LPS. Values are means ± SE; n = 8-9 rats/group. Groups with different letters (a, b, c) are statistically different from each other (P < 0.05; 1-way ANOVA). Groups with same letter are not significantly different.

IGF-I mRNA. Figure 3A is a representative autoradiograph from a nuclease protection assay of total RNA of liver from animals in each of the four experimental groups. Several bands are distinguishable in liver from all four groups. The smallest band at ~176 bp represents exon 2-derived mRNA (i.e., class 2). The remaining four bands represent exon 1-derived mRNAs (i.e., class 1). The two bands at ~520 and 420 bp represent mRNAs initiated at the two major transcription initiation sites. The third band (~260 bp) is also a class 1 mRNA from which the 186-bp sequence in exon 1 has been deleted. The fourth band at ~210 bp represents an additional transcription initiation site in exon 1. In liver, refeeding- and LPS-induced changes in IGF-I mRNA abundance appear to be coordinately regulated. Collectively for all transcripts, basal IGF-I mRNA expression was 31% greater in the TEN-saline group than in the TPN-saline group. Consistent with the changes in IGF-I protein content of liver as already described, both TPN- and TEN-infused animals responded to LPS with a decrease in IGF-I mRNA. However, as seen in Fig. 3A, the decrease in IGF-I mRNA expression in the TPN-LPS group was 54% greater than that observed in the TEN-LPS group (P < 0.05).


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Fig. 3.   Representative autoradiograph of solution hybridization-RNase protection assay with rat IGF-I Sau3A-NI-alpha -IV antisense RNA probe and total RNA from either liver (A) or gastrocnemius (B). Lanes under each of 4 experimental groups are samples from different animals. Lines at right indicate protected probe bands corresponding to mRNAs resulting from use of indicated start sites. A 32P-labeled hae digest of phi X174 DNA was used to indicate approximate size (bp) of protected bands (lines at left). Intensity of ethidium bromide staining of 18S and 28S rRNA was analyzed by agarose gel electrophoresis to ensure equal loading of protein (data not shown). See IGF-I mRNA for details.

The same pattern of IGF-I mRNA expression was seen in gastrocnemius in response to refeeding and LPS (Fig. 3B). There was no significant difference in the IGF-I mRNA abundance in muscle from animals in the TEN-saline and TPN-saline groups. However, IGF-I mRNA expression was decreased to a greater extent in the TPN-LPS group than in the TEN-LPS group (61%; P < 0.05).

IGFBP-1. Figure 4 is a representative Western blot for IGFBP-1 in plasma (Fig. 4A) in which levels have been semiquantitated by densitometry (Fig. 4B). The infusion of either TPN or TEN for 48 h was equally effective at reducing the normal increase in plasma IGFBP-1 produced by the 18-h fast. Circulating IGFBP-1 returned to fasting levels in TPN-infused rats 4 h after injection of LPS. However, enterally fed animals exhibited more than a 250% greater increase in IGFBP-1 than in rats fed intravenously (Fig. 4B). No statistical differences could be detected in IGFBP-2 levels in the plasma for either TPN- or TEN-fed rats in response to LPS (data not shown).


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Fig. 4.   A: representative Western blot of IGF-binding protein (IGFBP)-1 in plasma obtained from rats infused for 48 h with TPN (P) or TEN (E) and then subsequently injected with LPS or SAL. Plasma samples were obtained 4 h after injection of LPS or SAL. A total of 8 samples for each group were analyzed. B: semiquantitated data for all Western blots. Data were obtained by scanning densitometry of Western blots and are in arbitrary units. Values are means ± SE; n = 8 rats/group. Groups with different letters (a, b, c) are statistically different from each other (P < 0.01; 1-way ANOVA). Groups with same letter are not significantly different.

Plasma hormone concentrations. Plasma insulin and glucocorticoid levels were assessed because these hormones are known regulators of the IGF system. Both parenteral and enteral feeding increased plasma insulin concentrations two- and threefold, respectively, compared with values detected in 18-h fasted rats (Fig. 5, top). However, the feeding-induced hyperinsulinemia was of greater magnitude in TEN-infused rats. Regression analysis of insulin and IGF-I across both TEN and TPN groups after 48 h of refeeding indicated a significant positive linear relationship (y = 14.7x + 297; r2 = 0.61; P < 0.05). The injection of LPS did not significantly alter plasma insulin levels at either 90 or 240 min in either group compared with saline-injected time-matched control values.


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Fig. 5.   Plasma insulin (top) and corticosterone (bottom) concentrations in rats fed parenterally or enterally and then injected with either LPS or SAL. Plasma was collected after a 18-h fast, after 48-h of refeeding (TPN or TEN), and then 90 and 240 min after injection of LPS or SAL. Values are means ± SE; n = 8-9 rats/group. * P < 0.01 vs. basal 48-h refed values from same treatment group (paired t-test). + P < 0.05 vs. time-matched values from either TPN-SAL or TEN-SAL group. § P < 0.05 vs. time-matched values from TPN-LPS group. For later 2 comparisons, 1-way ANOVA was performed separately at 90 and 240 min.

The 2-day infusion of either TPN or TEN decreased circulating levels of corticosterone by an average of 43% compared with values determined in 18-h fasted rats (Fig. 5, bottom). Although both experimental groups responded to LPS with a marked increase in corticosterone at 90 and 240 min, the elevation was greater in rats infused enterally.

Plasma TNF-alpha and urinary 3-MH-to-creatinine ratio. In a separate group of rats, the concentration of TNF-alpha in the plasma and 3-MH in the urine was determined after 48 h of nutritional support (or an equivalent period of fasting) and then in the same animals after injection of LPS. Time-matched animals injected with saline were not included in this series.

TNF-alpha levels were similarly elevated in fasted and TPN-infused animals 90 min after LPS (Fig. 6). Although TNF-alpha levels were also elevated in the TEN group after LPS, the increase was significantly smaller than that observed in the other two groups. At the 240-min time point, TNF-alpha levels in all groups had returned to near-control levels, and there were no significant differences between the groups. Previous studies in our laboratory have indicated that TNF-alpha levels are below the detection limit (~15 pg/ml) in control animals not injected with LPS (8).


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Fig. 6.   Modulation of LPS-induced increase in plasma tumor necrosis factor (TNF)-alpha concentrations in rats fed enterally or parenterally. Animals were infused with TPN or TEN for 48 h before injection with LPS. Control animals (fasted) were time matched and provided no nutritional support but were also injected with LPS. Values are means ± SE; n = 8-10 rats/group. ANOVA was performed at each time point, and groups with different letters (a,b) are statistically different from each other.

The urinary 3-MH-to-creatinine ratio was lower in both TEN- and TPN-infused rats compared with time-matched fasted animals under both basal conditions and at 24 h post-LPS (Fig. 7). When the change in the 3-MH-to-creatinine ratio was calculated for each group, it was evident that the LPS-induced increase in 3-MH excretion was smaller in the TEN-fed rats (0.16 ± 0.02) than in either TPN-fed or fasted animals (0.34 ± 0.06 and 0.41 ± 0.08, respectively; P < 0.05).


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Fig. 7.   Effect of TPN or TEN on urinary 3-methylhistidine (3-MH)-to-creatinine (Cr) molar ratio. Rats were fed parenterally (TPN) or enterally (TEN) for 48 h or were fasted for a comparable period of time. Urine was collected during final 24 h of this period (basal). Thereafter, all rats were injected with LPS (24 h post-LPS), and urine was collected 24 h later. Values are means ± SE; n = 8-10 rats/group. ANOVA was performed at each time point, and groups with different letters (a, b) are statistically different from each other.

Approximately 24 h after injection of LPS, plasma IGF-I levels in TPN-fed rats were still significantly lower than levels in TEN-infused rats (505 ± 68 vs. 755 ± 93 ng/ml; P < 0.05). The plasma IGF-I concentration in the fasted group of animals treated with LPS was lower than that of both groups receiving nutritional support (417 ± 22 ng/ml; P < 0.05).

Alterations in body weight and plasma glucose and triglyceride concentrations. Body weight was determined after an overnight fast before nutritional support was begun and then again after 2 days of either TEN or TPN. Animals in both groups demonstrated a significant increase in body weight compared with their own 18-h fasted values, but there was no difference in growth rate between groups (TEN = 3.7 ± 0.8 g/day; TPN = 3.1 ± 0.7 g/day). Body composition was not determined in the present study.

The infusion of either TPN or TEN elevated plasma glucose levels compared with fasted values, and there was no significant difference between rats fed enterally and those fed parenterally (Table 2). In contrast, the hyperglycemic response to LPS at 90 min was ~40% greater in TPN-infused rats compared with time-matched values from enterally infused animals that received LPS. Glucose levels were not different from control values for either treatment group at 240 min post-LPS.

                              
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Table 2.   Alterations in plasma glucose and triglyceride concentrations in rats infused with either TEN or TPN

Plasma triglyceride concentrations were also increased in response to TEN and TPN compared with basal values, and there was again no difference based on the route of nutrient administration (Table 2). In TEN-infused animals, LPS increased triglyceride levels by 130% at 240 min. Triglyceride levels also tended to increase in TPN-infused rats in response to LPS, but because of the high variability, this trend did not reach statistical significance.


    DISCUSSION
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

Alterations in IGF-I induced by uncomplicated surgical trauma and fasting differed in rats fed TPN or TEN. After mild surgical trauma and an 18-h fast, both parenteral and enteral feeding increased plasma IGF-I levels. However, the recovery of IGF-I in the TEN group was greater than that observed in TPN-fed rats. Two days of enteral nutrition achieved IGF-I levels comparable with those reported in unstressed fed rats (25). The different response between the two groups cannot be attributed to differences in nitrogen content or to the relative percentage of lipid and carbohydrate in the infusate. However, we cannot unequivocally conclude that the route of nutritional delivery is responsible for the differences in IGF-I levels between the TPN and TEN groups because the two formulations did not have identical compositions.

The concomitant elevation in insulin and reduction in corticosterone levels in plasma are likely mediators of this IGF-I response. The ability of refeeding to decrease fasting-induced increases in plasma corticosterone has been previously described (16) and appears to be regulated, in part, by an increased expression of hypothalamic neuropeptide Y, which is mediated by a reduction in circulating levels of leptin (2). Pretreating animals with the glucocorticoid receptor antagonist RU-486 attenuates the fasting-induced decrease in IGF-I (24). Conversely, increasing of glucocorticoid levels has been shown to decrease IGF-I (28). Hence, the reduction in corticosterone levels in both TPN- and TEN-infused rats may partially contribute to the increase in plasma IGF-I. However, such a mechanism would not account for the relative greater increase of IGF-I in enterally fed rats. The mechanism by which TEN or TPN modulates the LPS-induced increases in corticosterone is unknown. However, in general, it may involve the attenuation of either some circulating stimulatory factor (such as TNF-alpha in the present study) and/or alterations in the regulation of adrenal secretion via the central nervous system.

IGF-I can also be regulated by the prevailing insulin concentration. Severe hypoinsulinemia, as is seen in models of insulin-dependent diabetes mellitus, results in a dramatic reduction in IGF-I, whereas the treatment of diabetics with insulin leads to an increase in plasma IGF-I (3, 4). In the present study, regression analysis of insulin and IGF-I level values for TPN- and TEN-fed animals after 48 h of refeeding indicated a strong positive relationship between these two variables. These data suggest, but do not prove, that circulating insulin levels are a primary determinant of the increase in IGF-I observed in response to refeeding.

The primary purpose of this study was to determine whether TPN or TEN influences the host IGF-I response to LPS. In this regard, animals fed parenterally had a larger decrease in plasma IGF-I than animals fed enterally. This response was associated with a more pronounced reduction in the protein content and mRNA abundance of IGF-I in liver. These data are consistent with liver being the primary site of production for circulating IGF-I (9). In addition, the protein level and mRNA abundance of IGF-I in skeletal muscle were also lower in rats fed TPN compared with those fed TEN. The ability of enteral feeding to better maintain muscle IGF-I content may have an important impact on protein balance in this tissue. Exogenous administration of IGF-I has been shown to both increase protein synthesis and decrease protein degradation in muscle under specific experimental conditions (3, 7, 14). Moreover, we have previously demonstrated a strong positive correlation between the IGF-I content and rates of protein synthesis in gastrocnemius during hypermetabolic infection (22). Our present data, indicating a smaller LPS-induced elevation in urinary 3-MH excretion in enterally fed rats, are consistent with the known inhibitory effect of IGF-I on muscle proteolysis (33).

The exact mechanism by which TPN or TEN produces this differential IGF-I response to LPS was not elucidated. Our study suggests at least three possible mediators may be responsible for the relatively smaller decrease in IGF-I in rats fed enterally. First, plasma insulin levels were maintained at a higher level during the early portion of the insult. Second, the LPS-induced increase in glucocorticoids was significantly smaller through the study period in TEN-fed rats. LPS-induced increases in the plasma concentration of other stress hormones, such as glucagon and epinephrine, have also been reported to be attenuated by TEN (12). Third, the plasma TNF-alpha response produced by LPS was also smaller. The scientific rationale behind the first two mechanisms has been described above. The rationale for the third alternative is based on in vivo studies that demonstrate that the infusion of nonlethal doses of TNF-alpha produces a rapid and sustained decrease in circulating IGF-I (8). Although a portion of this action may be mediated indirectly via elevations in glucocorticoids (25), TNF-alpha also directly inhibits the ability of growth hormone to stimulate IGF-I synthesis in isolated hepatocytes (40). The relative importance of each of these factors (e.g., insulin, glucocorticoids, TNF-alpha ), however, could not be ascertained in the present study. The ability of TEN to attenuate the systemic TNF response has been previously reported under some experimental conditions (12, 27) but not all (35).

Attenuation of the LPS-induced decrease in IGF-I by enteral feeding most likely represents a beneficial host response. Numerous studies have demonstrated that endogenous stimulation of IGF-I with growth hormone and/or administration of exogenous IGF-I enhances nitrogen retention and ameliorates aspects of the catabolic response (5, 15, 20, 21). Hence, maintenance of IGF-I levels may prevent the erosion of lean body mass and minimize the increased morbidity and mortality associated with the sustained loss of muscle mass. The sepsis-induced mobilization of skeletal muscle protein, however, does serve to maintain the hepatic output of acute-phase proteins and glucose. Therefore, if IGF-I was increased without concomitant provision of adequate nutritional support, the impairment of this metabolic reprioritization might be deleterious. Moreover, although growth hormone has been shown to enhance LPS-induced organ injury in rats, this effect appears to be IGF independent (26). Finally, other reports suggest maintenance of IGF-I might be expected to improve renal and immune function after a septic challenge (17, 18, 29).

The large majority of IGF-I in the circulation is not free but is carried by one of at least six different high-affinity binding proteins (32). The majority of the IGF-I is bound to IGFBP-3, which, because of its large molecular weight, is restricted to the plasma compartment and probably acts as a reservoir for IGF-I. In the present study, we focused on the alterations in IGFBP-1. This particular binding protein has been examined extensively and is known to undergo rapid and dramatic fluctuations in response to nutritional variations (24) as well as inflammation and stress (8, 11, 22-25). Moreover, IL-1, IL-6, and TNF-alpha have all been demonstrated to directly stimulate hepatic IGFBP-1 production (34). Because of its small molecular weight, IGF-I bound to this binding protein can cross the vascular endothelium. Under most experimental paradigms, an elevation in IGFBP-1 has been demonstrated to inhibit the anabolic actions of IGF-I (24, 32). In the present study, the 48-h infusion of either TPN or TEN reduced IGFBP-1 levels by the same extent. Insulin is a known inhibitor of hepatic IGFBP-1 production, and the feeding-induced increase in this hormone is consistent with the reduction in circulating levels of IGFBP-1 observed in the present study. However, elevations in glucocorticoids are also known to stimulate IGFBP-1 production (3). Therefore, we cannot exclude the possibility that the TPN- and TEN-induced reduction in corticosterone was also at least partially responsible for the decrease in IGFBP-1 observed after 48 h of nutritional support. In contrast, there was a marked difference in the LPS-induced increase in IGFBP-1 in parenterally and enterally fed rats. Although both LPS-treated groups responded with the expected elevation in IGFBP-1 (10, 25), the increment in IGFBP-1 was more than twofold greater in the enterally fed animals. At this time, we have no explanation for the more dramatic increase in IGFBP-1 observed in TEN-fed rats. The higher insulin levels as well as the lower glucocorticoid and TNF-alpha levels in this group would be expected to minimize, not enhance, the LPS-induced increase in IGFBP-1 (24, 32).

In summary, enterally fed rats have a more rapid normalization of plasma IGF-I levels in response to mild surgical stress and fasting, as well a smaller decrement in IGF-I in plasma, liver, and muscle in response to LPS, than parenterally fed rats. In contrast, enteral feeding results in a more dramatic increase in IGFBP-1 in response to LPS, which cannot be explained by concomitant changes in either insulin, glucocorticoids, or TNF-alpha . Overall, enteral nutritional support preferentially maintains IGF-I in plasma and muscle after LPS and is present in animals with lower rates of myofibrillar degradation.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of General Medical Sciences Grant GM-38032.


    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: C. H. Lang, Dept. of Cellular and Molecular Physiology, Pennsylvania State College of Medicine, Hershey, PA 17033.

Received 6 July 1998; accepted in final form 28 October 1998.


    REFERENCES
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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