1 Division of Pulmonary, 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
insulin-like growth factor-binding protein-1; insulin; corticosterone; 3-methylhistidine; tumor necrosis factor- 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- 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
ABSTRACT
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References
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-
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.
; lipopolysaccharide
INTRODUCTION
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References
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
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.
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- 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- was determined with an enzyme-linked immunosorbent assay
(Biosource International, Camarillo, CA) specific for rat TNF-
.
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.
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RESULTS |
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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.
|
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 · ml1 · 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.
|
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).
|
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).
|
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.
|
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- and urinary 3-MH-to-creatinine
ratio. In a separate group of rats, the concentration
of TNF-
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- levels were similarly elevated in fasted and TPN-infused
animals 90 min after LPS (Fig. 6). Although
TNF-
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-
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-
levels are below the detection limit (~15
pg/ml) in control animals not injected with LPS (8).
|
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).
|
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.
|
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.
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DISCUSSION |
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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- 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- 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-
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-
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-
), 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- 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-
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-. 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.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of General Medical Sciences Grant GM-38032.
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FOOTNOTES |
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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.
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