1 North West Injury Research Collaboration, 2 Medical Research Council Trauma Group, and Departments of 3 Endocrinology and 4 Rheumatology, Hope Hospital, Salford, M6 8HD, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Type 2 diabetes is associated with
biochemical evidence of low-grade inflammation, and experimental
studies have suggested that both insulin and glucose affect
inflammatory responses. To determine the effect of in vivo changes in
glucose availability and plasma insulin concentrations in humans, we
administered 20 U/kg Escherichia coli lipopolysaccharide
(LPS) or saline (control) to 14 subjects during a euglycemic
hyperinsulinemic clamp (n = 6) or an infusion of
sterile saline (n = 8). Parallel in vitro studies on human
whole blood were undertaken to determine whether there was a direct
effect of glucose, insulin, and leptin on proinflammatory cytokine
production. Infusion of glucose and insulin significantly amplified
and/or prolonged the cardiovascular, plasma interleukin-6 (IL-6), tumor
necrosis factor- (TNF-
), and counterregulatory hormone responses
to LPS, whereas the effects on fever, plasma norepinephrine
concentrations, and oxygen consumption were unaffected. In vitro
studies showed no modulation of LPS-stimulated IL-6 or TNF-
production by glucose, insulin, or leptin at physiologically relevant
concentrations. Hyperinsulinemia indirectly enhances key components of
the systemic inflammatory and stress responses in this human model of infection.
diabetes; cytokines; lipopolysaccharide; inflammation; sepsis
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE EFFECT OF
INFLAMMATION and injury on insulin sensitivity has been
recognized for many years. Inflammation associated with infection and
connective tissue disease is accompanied by profound reductions in
insulin sensitivity. The fact that corticosteroid treatment, a potent
cause of insulin resistance in itself, may actually reduce insulin
resistance in patients with rheumatoid arthritis (36)
indicates that the negative influence of inflammation on insulin
sensitivity exceeds even that of pharmacological doses of steroids. It
has also recently been suggested that low-grade, chronic inflammation
may play a role in the pathogenesis of insulin resistance in obesity
and type 2 diabetes. This suggestion is based on the demonstration of
elevated plasma concentrations of proinflammatory cytokines, such as
interleukin-6 (IL-6) (9, 28) and tumor necrosis factor-
(TNF-
) (14, 22), as well as acute- phase reactants
(21) in diabetes, increased expression of TNF-
mRNA in
the adipose tissue (13) and skeletal muscles (32) of obese and diabetic patients, and protection from
obesity-induced insulin resistance in mice lacking TNF receptor
function (38). Raised plasma IL-6 concentrations,
indicative of low-grade inflammation, have been suggested to represent
the link between the insulin resistance and the vascular disease that
characterize the metabolic syndrome X (29).
An alternative explanation for the demonstration of increased levels of
proinflammatory cytokines in type 2 diabetes might be that increased
plasma glucose and/or insulin concentrations enhance the inflammatory
response. In this scenario, the hyperglycemia/hyperinsulinemia or other
endocrine changes associated with insulin resistance might accentuate
the inflammatory response, rather than inflammation leading directly to
insulin resistance. This suggestion is supported by the finding that
administration of glucose significantly augments the TNF- and
hemodynamic responses to endotoxin administration in rabbits
(18), although the converse has been demonstrated in mice
(33). Glucose (23, 27) and insulin-like
growth factors (30) have also been shown to significantly
augment secretion of IL-1, IL-6, and TNF-
from mononuclear
cells in vitro. It is unclear whether glucose availability and/or
hyperinsulinemia upregulates the inflammatory response in humans,
although this could provide an alternative explanation for the
demonstration of increased circulating and tissue levels of
proinflammatory cytokines in patients with type 2 diabetes, in whom
hyperglycemia and hyperinsulinemia are common.
The aim of this study was therefore to test the hypothesis that hyperinsulinemia and increased glucose availability would significantly augment the systemic response to an inflammatory stimulus in humans and, if so, to determine whether this might be attributable to a direct effect on cytokine-producing cells.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In Vivo Study
Subjects. Fourteen subjects (12 male, 2 female), of mean age 32.7 (range 24-38) yr and mean body surface area (BSA) 1.9 (range 1.7-2.1) m2, were admitted to the clinical investigation facility of Hope Hospital, Salford. Before study, all subjects were screened by history, physical examination, and electrocardiogram. No subject had a history of cardiorespiratory, metabolic, or inflammatory disease, and none was receiving any medication. The study was approved by the local research ethics committee of Salford and Trafford Health Authority, and informed consent was obtained from each subject before enrollment in the study.
Study protocol. Lipopolysaccharide (LPS) was administered intravenously to six (4 male, 2 female) subjects during conditions of euglycemic hyperinsulinemia and to eight male subjects during an infusion of sterile normal saline.
Each subject was studied on two occasions, 10-14 days apart. On the first occasion, subjects received either an intravenous bolus of LPS or an equivalent volume (6-8 ml) of sterile isotonic saline. On the second occasion, subjects received the alternative infusion, the order being determined randomly. There were thus four study arms, with each subject receiving either LPS or saline (control), during an infusion of insulin-glucose or saline. All subjects were studied at 0830 after an overnight fast and having refrained from smoking or beverages containing caffeine for 24 h before the study. After voiding, subjects were weighed to the nearest 0.1 kg with an Avery beam balance and measured to the nearest 1 cm with a calibrated scale (Seca, Birmingham, UK). BSA was calculated accordingly (8). Catheters (Venflon, Helsingborg, Sweden) were inserted into an antecubital fossa vein of the right arm (for infusion) and retrogradely into a vein on the dorsum of the left hand (for sampling). The sampling hand was then placed in a hot box (air temperature 60°C) to produce arterialization of venous blood (1). The subjects, lightly clothed, then rested quietly in a supine position for the remainder of the study. Hand skin temperature was maintained at 42°C and was monitored continuously with a thermistor taped to the dorsum (Vickers Medical Limited, Hampshire, UK). All catheters were kept patent with sterile heparinized saline (50 U of heparin in 150 mmol/l sodium chloride, CP Pharma, Wrexham, UK) when not in use. After a 30-min rest period, an 8-h infusion of insulin-glucose (see Euglycemic hyperinsulinemic clamp) or saline (150 mmol/l, 50 ml/h) was commenced. One hundred twenty minutes after the start of these infusions, subjects received either an intravenous infusion of 20 U/kg National Reference Bacterial Endotoxin (Lot EC-6, prepared from E. coli 0113, USPC, Rockville, MD) over a 5-min period or an equivalent volume of sterile saline (150 mmol/l). Repeated measurements of mean arterial pressure (MAP), heart rate, oxygen consumption, and plasma hormone, cytokine, and leptin concentrations were performed over the following 6 h, as detailed in the following sections.Indirect calorimetry. Oxygen consumption was measured for the last 30 min of each hour by use of open-circuit indirect calorimetry (Deltratrac, Datex, Helsinki, Finland). The calorimeter was calibrated before and after each measurement with the manufacturer's recommended gases, having previously been validated by alcohol combustion and shown to deliver values within 98% of those predicted. All measurements were standardized to BSA.
Euglycemic hyperinsulinemic clamp.
Insulin (Humulin S, Eli Lilly, Basingstoke, Hampshire, UK) was
administered by primed-continuous infusion at 80 mU · m2 · min
1 for 8 h, during which euglycemia (5 mmol/l) was maintained by a variable
infusion of sterile aqueous glucose (20 g/100 ml, Baxter, Norfolk, UK).
The concentration of glucose in arterialized venous plasma was
monitored at 5-min intervals throughout the 8-h period using a Beckman
glucose analyzer, (Beckman Instruments, Fullerton, CA).
Sample collection.
Arterialized venous blood samples were taken hourly for measurement of
analyte concentrations. Samples for IL-6 and TNF- assays were taken
into EDTA. All other samples were taken into lithium heparin (10 U/ml).
Samples were centrifuged, and the plasma was separated and stored
immediately at
20°C except for catecholamine, cytokine,
insulin-like growth factor (IGF), IGF-binding protein (IGFBP), and
leptin samples, which were stored at
80°C pending analysis.
Biochemical analysis. substrate and hormone concentrations . Plasma glucose concentrations were measured spectrophotometrically by use of a Cobas Bio Centrifugal analyzer (Roche Products, Welwyn, Garden City, Hertfordshire, UK). Commercially available radioimmunoassay (RIA) kits were used for measurement of plasma insulin (Pharmacia, Milton Keynes, UK) and plasma cortisol concentrations (Wallac Oy, Turku, Finland). Plasma growth hormone (GH) concentration was measured by fluoroimmunometric assay (Delfia, Wallac Oy). The maximum coefficient of variation (CV) for these assays was 5.5%. Plasma concentrations of norepinephrine were measured by reversed-phase HPLC and electrochemical detection, as described previously (20). Plasma IGF-I concentration was determined by RIA as reported previously (11). The detection limit of the assay was 28 ng/ml, and inter- and intra-assay CVs were 5.2-7.4 and 4.0-5.7%, respectively. IGFBP-1 levels were also determined by RIA (42), utilizing human recombinant IGFBP-1 (donated by Dr. L. Fryklund, Pharmacia, Sweden) for standard and radiolabel and monoclonal antibody (MAb 6303, provided by Medix Biochemica, Kauniainen, Finland). MAb 6303 recognizes all isoforms of IGFBP-1, including the phosphoform characteristic of normal plasma. Detection limits for the assay were 3 µg/l, and inter- and intra-assay CVs were <8 and <6.8%, respectively. IGFBP-3 was measured by a commercially available RIA kit (Bioclone Australia Pty, NSW, Australia). Quoted sensitivity was 3.5 ng/ml, with inter- and intra-assay CVs of <8.5 and <6%, respectively.
CYTOKINES AND LEPTIN . Plasma TNF-Temperature, pulse, and blood pressure measurement. Tympanic membrane temperature was measured at 30-min intervals using an infrared probe (Thermoscan, San Diego, CA). Pulse rate and MAP were measured automatically every 30 min with an electronic monitor (BCI International, Waukesha, WI). In each case, the mean of three measurements taken over a 5-min period was used.
In Vitro Studies
Media and reagents. L-Glutamine and medium 199 (M199) were purchased from Life Technologies (Paisley, UK). Sterile sodium heparin and glucose infusate (20 g/dl) were obtained from C. P. Pharmaceuticals (Wrexham, UK) and Baxter-Clintec (Norfolk, UK), respectively. LPS (from E. coli 0128:B12), recombinant human insulin, and phenyldiamine dihydrochloride (OPD) were obtained from Sigma-Aldrich (Dorset, UK). Recombinant human leptin was purchased from R&D Systems (Minneapolis, MN). Hormone and LPS dilutions were prepared in M199 (+10% fetal calf serum and 2 mM glutamine).
Anti-human IL-6 monoclonal antibody was purchased from Eurogenetics (Middlesex, UK). Purified recombinant human IL-6 was obtained from Serono (Norwell, MA). Peroxidase-conjugated donkey anti-rabbit IgG was obtained from Jackson Immunoresearch Laboratories (Luton, UK). Rabbit anti-human IL-6 serum was produced after immunization of rabbits with recombinant human IL-6 (generously donated by Dr. L. A. Aarden, CLB, The Netherlands). Mouse anti-human TNF-IL-6 and TNF- ELISA.
IL-6 concentration was measured in supernatants with a sandwich ELISA.
Briefly, 96-well microtiter plates (Nalge NUNC, Roskilde, Denmark) were
coated overnight at 4°C with either human anti-IL-6 or human
anti-TNF-
MAb, at a concentration of 1 µg/ml in phosphate buffer
(pH 7.2-7.4). After washing and blocking stages, freshly thawed
samples and standards (recombinant human IL-6 at 0-1,000 pg/ml or
human TNF-
at 0-1,600 pg/ml) were incubated in duplicate overnight at 4°C. Plates were washed before incubation at room temperature with rabbit anti-IL-6 polyclonal antibody for 2 h or
with biotinylated anti-TNF-
for 1 h. Plates were then incubated at room temperature with peroxidase-conjugated donkey anti-rabbit IgG
polyclonal antibody (1 in 2,500 dilution) for 1 h, or with 0.625 µg/ml streptavidin-HRP for 30 min. After washing, enzyme substrate
(OPD 0.4 mg/ml in the presence of hydrogen peroxide) was added to each
plate. IL-6 and TNF-
concentrations were measured using an automated
plate reader (EL340 Microplate reader, Bio-tek Instruments,
Winooski, Vermont) at 490 nm. The minimum threshold for
detection in the assay was 10.2 pg/ml for IL-6 and 25 pg/ml for
TNF-
. The intra-assay CVs for IL-6 and TNF-
were <10% at 125-500 pg/ml and 10-20% at 150-900 pg/ml, respectively.
Methods: effect of glucose, insulin, and leptin on cytokine production. Six healthy male volunteers (age range 28-45 yr, body mass index range 18.5-22.4), free of metabolic or inflammatory disease and not receiving any form of medication, were studied at 0800 after a 10-h fast. A 40-ml sample of venous blood was taken from each subject, and sterile sodium heparin was added to a final concentration of 10 U/ml.
Aliquots of heparinized venous blood (1 ml) were dispensed in 24-well microtiter plates (Nalge NUNC International, Forskilde, Denmark) in the presence or absence of supplementary glucose and/or insulin (to provide final well glucose and insulin concentrations, after supplementation, of 5, 12, or 30 mmol/l glucose and 1 nmol/l insulin). Samples were then incubated for 2 h at 37°C, after which LPS (100 ng/ml) or M199 (control) was added, and the samples were then incubated for a further 6 h. Cell-free supernatants were obtained by centrifugation and stored atStatistical Analysis
Analysis of variance for repeated measures (MANOVA) was used to assess time effects and treatment-time interactions (clamp vs. saline). When MANOVA revealed a significant treatment-time interaction, post hoc unpaired comparisons between the groups were made with the Bonferroni test, with correction for multiple comparisons (24). For in vitro studies, one-way ANOVA was performed with post hoc analysis by the Neuman-Keuls test for intergroup comparisons and Dunnett's test for intragroup comparison with control study arms. All calculations were performed using Graphpad Prism software (Graphpad Prism, San Diego, CA). All data are expressed as means ± SD unless otherwise stated, and P < 0.05 was taken as the level of statistical significance. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vivo studies
Subject characteristics. Six subjects (4 male and 2 female), mean age 33.7 ± 4.2 yr, mean weight 72.5 ± 12.6 kg, and BSA 1.8 ± 0.1 m2, received LPS or saline during a euglycemic hyperinsulimemic clamp. Eight subjects (all male), mean age 32.0 ± 4.4 yr, mean weight 74.1 ± 7.8 kg, and BSA 1.9 ± 0.1 m2, received LPS or saline during saline infusion. There were no significant differences between groups with respect to age, weight, or BSA. No variable changed significantly between the two study arms within each group.
Temperature, pulse, MAP, and oxygen consumption.
Administration of LPS led to a significant pyrexia, irrespective of the
presence of euglycemic hyperinsulinemia (P < 0.001, Table 1). Although the febrile response
was, overall, significantly greater when LPS was administered under
conditions of euglycemic hyperinsulinemia (F = 4.7, P < 0.05), there was no significant difference between
the groups at any given time point, and peak pyrexia was similar in the
two groups (38.1 vs. 37.8°C, Clamp/LPS vs. Saline/LPS, respectively).
In parallel with the pyrexia, administration of LPS led to a
significant tachycardia in both Clamp/LPS and Saline/LPS groups (Table
1). Administration of LPS during conditions of euglycemic
hyperinsulinemia led to a significant prolongation of the tachycardia,
however (F = 600, P < 0.001), with heart
rate remaining at a plateau until 360 min, whereas heart rate peaked at
180 min in the absence of euglycemic hyperinsulinemia and declined thereafter. MAP did not change significantly in response to euglycemic hyperinsulinemia, with or without LPS, in any group of subjects (Table
1). Oxygen consumption increased markedly in response to LPS in both
groups (Table 1). This response was unaffected by euglycemic
hyperinsulinemia. All of the subjects who received LPS during
euglycemic hyperinsulinemia had marked symptoms of malaise, nausea, and
vomiting, whereas this was not observed in subjects who received LPS
during saline infusion or during euglycemic hyperinsulinemia alone.
|
Hormone, metabolite, cytokine, and leptin concentrations.
Plasma glucose concentrations (Table 2)
remained stable in the presence of euglycemic hyperinsulinemia (CV
5.9 ± 3.3%), and during insulin-glucose infusion they were
unaffected by LPS administration (CV 6.7 ± 3.5%). In the absence
of glucose-insulin infusion, however, saline infusion alone led to a
progressive decline in plasma glucose concentration, whereas LPS
administration led to a significant fall in plasma glucose
concentration at 120 min (P < 0.01, Table 2), followed
by a later recovery (F = 15.2, P < 0.003).
|
|
|
|
In Vitro Studies
In preliminary experiments, an LPS concentration was chosen to produce a significant but submaximal cytokine response to increase the possibility of observing any modification of cytokine production. Neither altering the glucose concentration in the incubation medium nor increasing the insulin concentration altered in vitro production of IL-6 or TNF-
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present data indicate, for the first time in humans, that key
elements of the systemic inflammatory response to endotoxemia are
significantly augmented by glucose-insulin infusion. Specifically, euglycemic hyperinsulinemic clamping significantly augmented the IL-6,
GH, and cortisol responses to LPS administration, and these effects
were associated with enhancement of LPS-induced tachycardia. In
contrast, the fever, increase in oxygen consumption, and TNF- and
catecholamine responses to LPS were unaffected by euglycemic hyperinsulinemia. Although the increased subjective malaise associated with LPS administration during euglycemic hyperinsulinemia must be
interpreted with caution, given the practical and ethical constraints that prevented blinding of the subjects in the study arms, it nevertheless suggests (when taken with the other findings) that the
observed augmentation of the LPS response is an integrated pathophysiological effect and not simply a collection of unrelated biochemical changes.
The increased counterregulatory hormone response to LPS in the clamp group was a particularly surprising finding, because the plasma glucose concentration was maintained at ~5 mmol/l, whereas a brisk hypoglycemia developed in subjects given LPS alone, as described previously (10). Preventing the hypoglycemic response to LPS therefore appears to have been associated with an augmentation of the counterregulatory hormone response. One would have anticipated the opposite to have occurred on theoretical grounds, and these findings therefore indicate that, in humans, hypoglycemia does not play a significant role in the counterregulatory hormone response to LPS.
These human findings contrast with data from mice, in which it has been
shown that glucose-induced increases in plasma insulin concentration
significantly attenuate the TNF- response to LPS (33),
and with data from rabbits in which the response was significantly augmented by intraportal glucose infusion (18). In neither
of these studies were counterregulatory hormone or other cytokine responses to LPS studied, although the cardiovascular response to LPS
was also augmented in rabbits by glucose administration (18). Insulin has been shown to be essential for normal
cytokine production in bronchoalveolar macrophages after LPS inhalation in rodents (3), although it is unclear whether, as
suggested in the current study, a further increase in the plasma
insulin concentration would have led to amplification of the
inflammatory response.
The primary metabolic and endocrine changes induced by glucose-insulin
infusion in the present study concern glucose availability, plasma
insulin and leptin concentrations, and the IGF axis. Although fasting
plasma glucose concentrations were maintained during conditions of
euglycemic hyperinsulinemia, glucose availability at the tissue level
would undoubtedly have been substantially increased, despite the modest
insulin resistance induced by LPS (2). Increased glucose
availability has been shown to increase basal TNF- and IL-6
(23) and LPS-stimulated IL-1 production (27)
by human monocytes in vitro, suggesting increased glucose availability as a possible mechanism for potentiation of LPS-mediated effects in
vivo. The findings of the in vitro studies, however, do not support
this suggestion, because we were unable to demonstrate any potentiation
of LPS-induced cytokine production by increasing glucose availability
in vitro. It is unclear why we were unable to confirm these previous
findings, but it should be noted that, unlike the previous studies, our
experiments were conducted over shorter incubation periods and in whole
blood as opposed to isolated monocytes. These circumstances thus more
closely reflect the physiological environment of the in vivo studies.
In addition, the glucose solution employed in the present study was
designed for clinical use and was free of endotoxin (manufacturer's
data), whereas the earlier studies employed glucose solutions prepared
in the laboratory that could conceivably have been contaminated with
endotoxin. This possibility is supported by the findings of additional
studies in this laboratory (H. Duxbury, S. J. Hopkins, and G. L. Carlson, unpublished observations). Similarly, although
pharmacological insulin concentrations have been shown to increase
basal TNF-
production by murine monocytes in vitro
(30), the insulin concentrations employed were far higher
than those observed in vivo in the present study. The findings of our
in vitro studies are in keeping with previous reports, which have also
failed to demonstrate increased basal TNF-
and IL-6 production after
incubation with insulin at concentrations similar to those employed in
vivo (26) and, in fact, suggest modest reductions in
LPS-induced cytokine production.
The in vivo studies confirm earlier reports of increased plasma leptin concentrations after glucose-insulin infusion in humans (19). In view of the demonstration that leptin might upregulate proinflammatory cytokine production from macrophages (17), the in vitro studies were undertaken to test the hypothesis that the enhanced in vivo systemic inflammatory and counterregulatory hormone response to LPS after glucose-insulin infusion might have been mediated by a direct action of leptin on cytokine-producing cells. The increase in plasma leptin after glucose-insulin infusion was, however, only statistically significant beyond 120 min (i.e., after 4 h of glucose-insulin infusion) and not at the time point at which LPS was administered. Although a modest dose-related increase in basal IL-6 production was observed after incubation of whole blood with leptin, the effect was observed only at supraphysiological leptin concentrations, and there was no apparent influence of leptin on LPS-stimulated cytokine production. These data contrast with those previously reported (17), but it should be noted that, in our study, the in vitro protocol matched as closely as possible the conditions of the in vivo experiment, and the lower dose of leptin used replicated the plasma leptin concentrations in vivo, for a similar time period before and after LPS exposure. In contrast, the study reported by Loffreda et al. (17) involved leptin concentrations that are unlikely to have been achieved under any physiological conditions, and a 24-h incubation period, so the physiological relevance of these data is unclear.
Although the in vitro studies were designed to match as closely as possible the conditions and time course of the in vivo studies, caution should be exercised when comparisons are drawn. The endotoxin exposure is likely to have been substantially different, and the cytokine response observed in vivo may reflect stimulation of cells such as Kupffer cells, as opposed to blood monocytes (10). Although it is unclear whether cytokine production in these cell populations is equally sensitive to endotoxin and subjected to the same regulatory factors, the data presented in the present study cast doubt on a direct role for increased glucose availability, hyperinsulinemia, or leptin in the augmentation of the cytokine response observed after LPS administration in humans and suggest that other, related factors might be of importance.
In vitro studies indicate that IGF-I may directly augment both basal
and LPS-induced TNF- and IL-6 production in vitro (30, 37). In vitro incubations with IGF-I were not undertaken in the
present study, because the aim of the in vitro experiments was to
mimic, as far as possible, the in vivo conditions under which
mononuclear cells would be subjected to the hormone and substrate
environment induced by hyperinsulinemia. Because the biological
availability of IGF-I is influenced by the plasma concentration of
binding proteins (and especially IGFBP-1), the in vivo conditions of
this experiment would have been impossible to reproduce accurately in
vitro by the simple addition of IGF-I. Nevertheless, the demonstration that glucose-insulin infusion in vivo significantly increases the molar
ratio of IGF-I to IGFBP-1 and that IGF-I concentrations are unaffected
by LPS administration confirms the findings of previous studies
(15, 34). The biological actions of IGF-I are thought to
be modulated by IGFBPs (5); in particular, the IGF-I-to-IGFBP-1 (IGF-I/IGFBP-1) molar ratio has been suggested as a
surrogate marker of bioavailability (16). The absence of an increase in the IGF-I/IGFBP-3 molar ratio after euglycemic hyperinsulinemia implies that more IGF-I would have been available at
the time when LPS was administered. It is therefore possible that at
least some of the augmented IL-6 production observed in vivo is related
to the increased biological availability of IGF-I.
The augmentation of TNF- release in the present study was modest
compared with that reported in previous animal studies
(18), and it seems unlikely that the enhanced IL-6
response could be attributed to this alone. Although infusion of
TNF-
largely reproduces the systemic inflammatory response to LPS,
suggesting a pivotal role of TNF-
as a proximal mediator, blockade
of endogenous TNF-
has been shown to attenuate, but not to abolish,
the systemic inflammatory response to LPS in primates (41)
and humans (35, 40). In addition, parenteral nutrition and
hyperinsulinemia have been shown to modulate expression of
cell-associated TNF-
receptors, providing a potential mechanism for
upregulation of TNF-
sensitivity (4, 12). Although this
suggests that glucose-insulin infusion might act via increased
sensitivity to TNF-
-mediated events, other sites of action or other
cytokine-mediated pathways might be equally or more
important, and further studies will be required to clarify the
precise mechanisms of action of glucose and insulin.
The plasma insulin concentrations achieved during the present study are
generally higher than those observed in clinical disease. Nevertheless,
these findings raise important questions about the relationship between
hyperinsulinemia associated with type 2 diabetes and increased plasma
concentrations of biochemical markers of inflammation (21, 22,
28) and monocyte proinflammatory cytokine production after in
vitro stimulation with LPS (7) in such patients. It has
been suggested that chronic low-grade inflammation may induce the
insulin resistance, hyperinsulinemia, and atherogenesis characteristic
of type 2 diabetes mellitus (29), although exogenous insulin infusion has also recently been reported to cause an acute reduction in markers of inflammation in obese humans (6).
However, the inhibition of inflammation was related primarily to
changes in monocyte intranuclear nuclear factor B and circulating
plasma concentrations of intercellular adhesion molecule-1, rather than to plasma cytokines or counterregulatory hormones. Furthermore, these
changes occurred under basal (as opposed to endotoxin-stimulated) conditions, at circulating insulin concentrations significantly lower
than those employed in the present study. The suggestion that insulin
acutely downregulates some features of basal inflammatory activity is not, therefore, incompatible with our demonstration of
increased responsiveness to an inflammatory stimulus in healthy subjects.
The present findings may also be of relevance to nutritional support in sepsis. Glucose-based nutrition in this patient population may cause marked hyperinsulinemia due to peripheral insulin resistance (31). The findings of the present study suggest that hyperinsulinemia associated with glucose-based nutrition in sepsis might augment proinflammatory cytokine production and the stress response in septic patients. It is unclear whether these effects occur during the clinically relevant conditions of glucose-based total parenteral nutrition as opposed to euglycemic clamping and whether they are of clinical significance, but a recent randomized controlled trial has shown that administration of insulin to critically ill patients substantially reduces mortality and severe infection rates (39). These beneficial effects of insulin therapy were observed primarily in patients who remained in the intensive care unit for >5 days. Although no measurements of plasma cytokine or counterregulatory hormone concentrations were presented, prolonged duration of critical illness is associated with a state of immune anergy, in which anti-inflammatory cytokines predominate, predisposing the long-stay critically ill patient to infective morbidity (25). The findings of the present study suggest a potential mechanism for the beneficial effects of insulin therapy, attributable to modulation of the immune anergy associated with critical illness.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by a Medical Research Council program grant.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: G. L. Carlson, NWIRC, Clinical Sciences Bldg., Hope Hospital, Salford M6 8HD, UK (E-mail: gcarlson{at}fs1.ho.man.ac.uk).
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. Section 1734 solely to indicate this fact.
First published February 26, 2002;10.1152/ajpendo.00535.2001
Received 3 December 2001; accepted in final form 14 February 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abumrad, NN,
Rabin D,
Diamond MP,
and
Lacy WW.
Use of a heated superficial hand vein as an alternative site for the measurement of amino acid concentrations and for the study of glucose and alanine kinetics in man.
Metabolism
30:
936-940,
1981[ISI][Medline].
2.
Agwunobi, AO,
Reid C,
Maycock P,
Little RA,
and
Carlson GL.
Insulin resistance and substrate utilization in human endotoxemia.
J Clin Endocrinol Metab
85:
3770-3778,
2000
3.
Boichot, E,
Sannomiya P,
Escofier N,
Germain N,
Fortes ZB,
and
Lagente V.
Endotoxin-induced acute lung injury in rats. Role of insulin.
Pulm Pharmacol Ther
12:
285-290,
1999[ISI][Medline].
4.
Braxton, CC,
Coyle SM,
Montegut WJ,
van der Poll T,
Roth M,
Calvano SE,
and
Lowry SF.
Parenteral nutrition alters monocyte TNF receptor activity.
J Surg Res
59:
23-28,
1995[ISI][Medline].
5.
Clemmons, DR.
Insulin-like growth factor binding proteins and their role in controlling IGF actions.
Cytokine Growth Factor Rev
8:
45-62,
1997[Medline].
6.
Dandona, P,
Aljada A,
Mohanty P,
Ghanim H,
Hamouda W,
Assian E,
and
Ahmad S.
Insulin inhibits intranuclear factor kappaB and stimulates IkappaB in mononuclear cells in obese subjects: evidence for an anti-inflammatory effect?
J Clin Endocrinol Metab
86:
3257-3265,
2001
7.
Desfaits, AC,
Serri O,
and
Renier G.
Normalization of plasma lipid peroxides, monocyte adhesion, and tumor necrosis factor-alpha production in NIDDM patients after gliclazide treatment.
Diabetes Care
21:
487-493,
1998[Abstract].
8.
DeWeir, JB.
New methods for calculating metabolic rate with special reference to protein metabolism.
J Physiol
109:
1-9,
1949[ISI].
9.
Fernandez-Real, JM,
Vayreda M,
Richart C,
Gutierrez C,
Broch M,
Vendrell J,
and
Ricart W.
Circulating interleukin-6 levels, blood pressure and insulin resistance in apparently healthy men and women.
J Clin Endocrinol Metab
86:
1154-1159,
2001
10.
Fong, Y,
Marano MA,
Moldawer LL,
Wei H,
Calvano SE,
Kenney JS,
Allison AC,
cerami AC,
Shires GT,
and
Lowry SF.
The acute splanchnic and metabolic response to endotoxin in humans.
J Clin Invest
85:
1896-1904,
1990[ISI][Medline].
11.
Gill, MS,
Whatmore AJ,
Tillman V,
White A,
Addison GM,
Price DA,
and
Clayton PE.
Urinary IGF and IGF binding protein-3 in children with disordered growth.
Clin Endocrinol
46:
483-492,
1997[ISI][Medline].
12.
Hauner, H,
Bender M,
Haastert B,
and
Hube F.
Plasma concentrations of soluble TNF-alpha receptors in obese subjects.
Int J Obes Relat Metab Disord
22:
1239-1243,
1998[Medline].
13.
Hotamisligil, GS,
Arner P,
Caro JF,
Atkinson RL,
and
Spiegelman BM.
Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance.
J Clin Invest
95:
2409-2415,
1995[ISI][Medline].
14.
Katsuki, A,
Sumida Y,
Murashima S,
Murata K,
Takarada Y,
Ito K,
Fujii M,
Tsuchihashi K,
Goto H,
Nakatani K,
and
Yano Y.
Serum levels of tumor necrosis factor- are increased in obese patients with noninsulin-dependent diabetes mellitus.
J Clin Endocrinol Metab
83:
859-862,
1998
15.
Lang, CH,
Pollard V,
Fan J,
Traber LD,
Traber DL,
Frost RA,
Gelato MC,
and
Prough DS.
Acute alterations in growth hormone-insulin-like growth factor axis in humans injected with endotoxin.
Am J Physiol Regulatory Integrative Comp Physiol
273:
R371-R378,
1997
16.
Lee, PD,
Giudice LC,
Conover CA,
and
Powell DR.
Insulin-like growth factor binding protein-1: recent findings and new directions.
Proc Soc Exp Biol Med
216:
319-357,
1997[Abstract].
17.
Loffreda, S,
Yang SQ,
Lin HZ,
Karp CL,
Brengman ML,
Wang DJ,
Klein AS,
Bulkley GB,
Bao C,
Noble PW,
Lane MD,
and
Diehl AM.
Leptin regulates proinflammatory immune responses.
FASEB J
12:
57-65,
1998
18.
Losser, MR,
Bernard C,
Beaudeux JL,
Pison C,
and
Payen D.
Glucose modulates hemodynamic, metabolic, and inflammatory responses to lipopolysaccharide in rabbits.
J Appl Physiol
83:
1566-1574,
1997
19.
Malmstrom, R,
Taskinen MR,
Karonen SL,
and
Yki Jarvinen H.
Insulin increases plasma leptin concentrations in normal subjects and patients with NIDDM.
Diabetologia
39:
993-996,
1996[ISI][Medline].
20.
Maycock, PF,
and
Frayn KN.
Use of alumina columns to prepare plasma samples for liquid chromatographic determination of catecholamines.
Clin Chem
33:
286-287,
1987
21.
McMillan, DE.
Increased levels of acute-phase serum proteins in diabetes.
Metabolism
38:
1042-1046,
1989[ISI][Medline].
22.
Mishima, Y,
Kuyama A,
Tada A,
Takahashi K,
Ishioka T,
and
Kibata M.
Relationship between serum tumor necrosis factor- and insulin resistance in obese men with type 2 diabetes mellitus.
Diabet Res Clin Pract
52:
119-123,
2001[ISI][Medline].
23.
Morohoshi, M,
Fujisawa K,
Uchimura I,
and
Numano F.
Glucose-dependent interleukin 6 and tumor necrosis factor production by human peripheral blood monocytes in vitro.
Diabetes
45:
954-959,
1996[Abstract].
24.
Motulsky, H.
Analyzing Data with Graphpad Prism. San Diego, CA: Graphpad Software, 1999.
25.
Oberholzer, A,
Oberholzer C,
and
Moldawer LL.
Cytokine signallingregulation of the immune response in normal and critcally ill states.
Crit Care Med
28:
N3-N12,
2000[ISI][Medline].
26.
Ohno, Y,
Aoki N,
and
Nishimura A.
In vitro production of interleukin-1, interleukin-6 and tumor necrosis factor-alpha in insulin dependent diabetes mellitus.
J Clin Endocrinol Metab
77:
1072-1077,
1993[Abstract].
27.
Orlinska, U,
and
Newton RC.
Role of glucose in interleukin-1 beta production by lipopolysaccharide-activated human monocytes.
J Cell Physiol
157:
201-208,
1993[ISI][Medline].
28.
Pickup, JC,
and
Crook MA.
Is type II diabetes mellitus a disease of the innate immune system?
Diabetologia
41:
1241-1248,
1998[ISI][Medline].
29.
Pickup, JC,
Mattock MB,
Chusney GD,
and
Burt D.
NIDDM as a disease of the innate immune system: association of acute phase reactants and interleukin-6 with metabolic syndrome X.
Diabetologia
40:
1286-1292,
1997[ISI][Medline].
30.
Renier, G,
Clement I,
Desfaits AC,
and
Lambert A.
Direct stimulatory effect of insulin-like growth factor-1 on monocyte and macrophage tumor necrosis factora production.
Endocrinology
137:
4611-4618,
1996[Abstract].
31.
Saeed, M,
Carlson GL,
Little RA,
and
Irving MH.
Selective impairment of glucose storage in human sepsis.
Br J Surg
86:
813-821,
1999[ISI][Medline].
32.
Sagizadeh, M,
Ong JM,
Garvey WT,
Henry RR,
and
Kern PA.
The expression of TNF-alpha by human muscle: relationship to insulin resistance.
J Clin Invest
97:
1111-1116,
1996
33.
Satomi, N,
Sakurai A,
and
Haranaka K.
Relationship of hypoglycaemia to tumor necrosis factor production and antitumour activity: role of glucose, insulin and macrophages.
J Natl Cancer Inst
74:
1255-1260,
1985[ISI][Medline].
34.
Soop, M,
Nygren J,
Brismar K,
Thorell A,
and
Ljungqvist O.
The hyperinsulinaemic-euglycaemic glucose clamp: reproducibility and metabolic effects of prolonged insulin infusion in healthy subjects.
Clin Sci (Colch)
98:
367-374,
2000[ISI][Medline].
35.
Suffredini, AF,
Reda D,
Banks SM,
Tropea M,
Agosti JM,
and
Miller R.
Effects of recombinant dimeric TNF receptor on human inflammatory responses following intravenous endotoxin administration.
J Immunol
155:
5038-5045,
1995[Abstract].
36.
Svenson, KL,
Lundqvist G,
Wide L,
and
Hällgren R.
Impaired glucose handling in active rheumatoid arthritis: relationship to peripheral insulin resistance.
Metabolism
37:
125-130,
1988[ISI][Medline].
37.
Tu, W,
Cheung PT,
and
Lau YL.
IGF-1 increases interferon-gamma and IL-6 mRNA expression and protein production in neonatal mononuclear cells.
Pediatr Res
46:
748-754,
1999[Abstract].
38.
Uysal, KT,
Wiesbrock SM,
Marino MW,
and
Hotamisligil GS.
Protection from obesity-induced insulin resistance in mice lacking TNF-alpha function.
Nature
389:
610-614,
1997[ISI][Medline].
39.
Van den Berghe, G,
Wouters P,
Weekers F,
Verwaest C,
Bruyninckx F,
Schtez M,
Vlasselaers D,
Ferdinande P,
Lauwers P,
and
Bouillon R.
Intensive insulin therapy in critically ill patients.
N Engl J Med
345:
1359-1367,
2001
40.
Van der Poll, T,
Coyle SM,
Levi M,
Jansen PM,
Dentener M,
Barbosa K,
Buurman WA,
Hack CE,
ten Cate JW,
Agosti JM,
and
Lowry SF.
Effect of a recombinant dimeric tumor necrosis factor receptor on inflammatory responses to intravenous endotoxin in normal humans.
Blood
89:
3727-3734,
1997
41.
Van der Poll, T,
Levi M,
van Deventer SJ,
ten Cate H,
Haagmans BL,
Biemond BJ,
Buller HR,
Hack CE,
and
ten Cate JW.
Differential effects of anti-tumor necrosis factor monoclonal antibodies on systemic inflammatory responses in experimental endotoxemia in chimpanzees.
Blood
83:
446-451,
1994
42.
Westwood, M,
Gibson JM,
Davies AJ,
Young RJ,
and
White A.
The phosphorylation pattern of insulin-like growth factor binding protein-1 in normal plasma is different from that in amniotic fluid and changes during pregnancy.
J Clin Endocrinol Metab
79:
1735-1741,
1994[Abstract].