1Department of Biochemistry, University of Tasmania, Hobart 7001, Australia; and 2Health Sciences Center, University of Virginia, Charlottesville, Virginia 22908
Submitted 20 March 2003 ; accepted in final form 11 May 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
tumor necrosis factor-; diabetes mellitus; blood flow; muscles; inflammation; capillaries
A number of studies in vitro have provided evidence that TNF- can
directly cause loss of insulin sensitivity over both long and short periods.
Thus 3-5 days of exposure of 3T3-L1 or 3T3-F442A adipocytes to TNF-
causes reductions in insulin receptor and insulin receptor substrate (IRS-1)
tyrosine phosphorylation in response to a maximum dose of insulin
(8,
14). Others report that 3-4
days of exposure of 3T3-L1 adipocytes to TNF-
gives rise to
transcriptional changes including decreases in GLUT4, insulin receptor, and
IRS-1 mRNA and protein (25,
26). These reported effects
from chronic exposure to TNF-
in cell culture have downstream
consequences consistent with insulin resistance; for example, decreased
insulin-stimulated glucose transport has been noted in L6 myocytes
(1). Over a shorter period (1
h), C2C12 muscle cells exposed to TNF-
exhibited
impaired insulin-dependent phosphatidylinositol 3-kinase activation mediated
by IRS-1 and -2. This was accompanied by a decrease in 2-deoxyglucose uptake
(5). Despite such reports,
isolated incubated muscle appears to be completely unaffected by TNF-
.
Thus incubation of isolated soleus and epitrochlearis muscles with 6 nmol/l
for 45 min or 4 h, or 2 nmol/l for 8 h, had no effect on insulin signaling or
glucose uptake (22). In
contrast, we have recently shown
(29) that TNF-
infusion
evoked acute insulin resistance [euglycemic hyperinsulinemic clamp (10 mU
· kg-1 · min-1) in
vivo], and this was accompanied by the loss of insulin-mediated hemodynamic
responses, including capillary recruitment and increases in total limb blood
flow. Taken together, this raises the interesting possibility that, although
muscle cell lines respond acutely to TNF-
in vitro, the vasculature in
vivo may be an important target for TNF-
. The loss of the hemodynamic
responses may limit insulin and/or glucose access and account for inhibition
of
50% of the insulin-stimulated glucose uptake by muscle
(29). Such a loss would be
apparent only in vivo and thus be consistent with the negative outcomes of
using TNF-
in isolated incubated muscles, as found by Nolte et al.
(22).
The mechanism for capillary recruitment by insulin is an early
(27) and nitric oxide
(NO)-dependent (28) process,
likely to be mediated at the endothelial cells. Brief treatment by TNF-
inhibits the insulin signaling in endothelium that leads to NO production
(19). Because capillary
recruitment in muscle can also be stimulated by muscle contraction and there
is evidence that TNF-
and insulin oppose each other in a dose-dependent
manner (9), we now explore
whether TNF-
inhibitory effects on capillary recruitment are opposed by
high doses of insulin or by muscle contraction.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Surgery. Rats were anesthetized using Nembutal (50 mg/kg body wt)
and had polyethylene cannulas (PE-50; Intramedic) surgically implanted into
the carotid artery for arterial sampling and measurement of blood pressure
(pressure transducer Transpac IV; Abbott Critical Systems) and into both
jugular veins for continuous administration of anesthetic and other
intravenous infusions. A tracheotomy tube was inserted, and the animal was
allowed to spontaneously breathe room air throughout the course of the
experiment. Small incisions (1.5 cm) were made in the skin overlying the
femoral vessels of both legs, and the femoral artery was separated from the
femoral vein and saphenous nerve. Unless indicated otherwise, the epigastric
vessels were then ligated, and an ultrasonic flow probe (VB series, 0.5 mm;
Transonic Systems) was positioned around the femoral artery of the right leg
just distal to the rectus abdominis muscle. The cavity in the leg surrounding
the flow probe was filled with lubricating jelly (H-R; Mohawk Medical Supply,
Utica, NY) to provide acoustic coupling to the probe. The probe was then
connected to the flowmeter (model T106 ultrasonic volume flowmeter; Transonic
Systems). This was in turn interfaced with an IBM-compatible PC computer,
which acquired the data (at a sampling frequency of 100 Hz) for femoral blood
flow (FBF), heart rate, and blood pressure by use of WINDAQ data acquisition
software (DATAQ Instruments). The surgical procedure generally lasted 30
min, and then the animals were maintained under anesthesia for the duration of
the experiment by means of a continual infusion of Nembutal (0.6 mg ·
kg-1 · min-1) via the left
jugular cannula. For clamps, the femoral vein of the left leg was used for
venous sampling by use of an insulin syringe with an attached 29-gauge needle
(Becton Dickinson). A duplicate venous sample was taken only on completion of
the experiment (120 min) to prevent alteration of the blood flow from the
hindlimb due to sampling and to minimize the effects of blood loss. The body
temperature was maintained using a water-jacketed platform and a heating lamp
positioned above the rat.
For animals undergoing twitch contraction studies in vivo, one leg was prepared for field stimulation (2 Hz, 0.1 ms at 30 V) and blood sampling. A platinum electrode was placed under the skin at the proximal end of the upper surface of the thigh and another at the Achilles tendon. Femoral blood was sampled at the end of the contraction period (10 or 60 min) from a cannula positioned in the epigastric vein. Blood samples were assayed for glucose (60 min) and 1-methylxanthine (1-MX; 10 min).
Experimental procedures. Once the surgery was completed, a 45- to
60-min equilibration period was allowed so FBF and blood pressure could become
stable and constant. Rats were then allocated into either protocol A
(Fig. 1), where animals were
infused with saline or TNF- for 3 h and underwent euglycemic insulin
clamp (3, 10, or 30
mU·kg-1·min-1
Humulin R; Eli Lilly, Indianapolis, IN) or saline alone for the final 2 h, or
protocol B (Fig. 1),
where animals were infused with saline or TNF-
for 2 h and underwent
muscle contraction by electrical field stimulation (n = 6 in each
group) for the last 1 h (2-deoxyglucose and glucose uptake) or the last 10 min
(1-MX metabolism). Before the start of these experiments, a force-tension
curve was constructed to optimize stimulation parameters.
|
TNF- (mouse recombinant; Sigma Aldrich) was dissolved in saline and
0.1% bovine serum albumin. Because 1-MX (Sigma Aldrich) clearance was very
rapid, it was necessary to partially inhibit the activity of xanthine oxidase
(23). To do this, an injection
of a specific xanthine oxidase inhibitor, allopurinol
(6) (10 µmol/kg), was
administered as a bolus dose 5 min before commencing the 1-MX infusion (0.5 mg
· kg-1 · min-1 for 1
h; Fig. 1). This allowed
constant arterial concentrations of 1-MX to be maintained throughout the
experiment.
At 45 min before the completion of each experiment, a 50-µCi bolus of 2-deoxy-D-[2,6-3H]glucose (2-DG, specific activity 44.0 Ci/mmol; Amersham Life Science) in saline was administered. Plasma samples (25 µl) were collected at 5, 10, 15, 30, and 45 min to determine plasma clearance of the radioactivity. At the conclusion of the experiment, the soleus, plantaris, gastrocnemius white, gastrocnemius red, extensor digitorum longus, and tibialis muscles were removed, clamp frozen in liquid nitrogen, and stored at -20°C until assayed for 2-DG uptake.
The total blood volume withdrawn from the animals before the final arterial and venous samples did not exceed 1.5 ml and was easily compensated for by the volume of fluid infused.
Duplicate arterial and venous samples (300 µl) were taken at the end of
the experiment (total time 180 min) and placed on ice. These blood samples
were immediately centrifuged, and 100 µl of plasma were mixed with 20 µl
of 2 M perchloric acid. The perchloric acid-treated samples were then stored
at -20°C until assayed for 1-MX. The rest of the plasma was used for
plasma glucose, insulin, and TNF- analysis.
Analytical methods. A glucose analyzer (model 2300 Stat plus,
Yellow Springs Instruments) was used to determine whole blood glucose (by the
glucose oxidase method) during the insulin clamp. A blood sample of 25 µl
was required for each determination. Human insulin levels at the end of the
euglycemic insulin clamp were determined from arterial plasma samples by ELISA
(Dako Diagnostics, Ely, UK), using human insulin standards. Plasma TNF-
levels were also determined using an ELISA based on mouse TNF-
(Pierce
Endogen). Perchloric acid-treated plasma samples were centrifuged for 10 min,
and the supernatant was used to determine 1-MX, allopurinol, and oxypurinol
concentrations by reverse-phase HPLC as previously described
(23,
24).
2-DG uptake assay. Individual frozen muscles from the clamps were ground under liquid nitrogen and homogenized using an Ultra Turrax. Free and phosphorylated parts of 2-DG were separated by ion exchange chromatography using an anion exchange resin (AG1-X8) (17, 20). Biodegradable Counting Scintillant-BCA (Amersham) was added to each radioactive sample and radioactivity determined using a scintillation counter (Beckman LS3801). From this measurement and a knowledge of plasma glucose and the time course of plasma 2-DG disappearance, R'g, which reflects glucose uptake into the muscle, was calculated as previously described by others (17, 20).
Data analysis. All data are expressed as means ± SE. Mean
FBF, mean heart rate, and mean arterial blood pressure were calculated from
5-s subsamples of the data, representing 500 flow and pressure
measurements every 15 min. Vascular resistance in the hindleg was calculated
as mean arterial blood pressure in millimeters of mercury divided by femoral
blood flow in milliliters per minute and expressed as resistance units (RUs).
Glucose uptake in the hindlimb was calculated from the arterial-venous (a-v)
glucose difference and multiplied by FBF and expressed as micromoles per
minute. The 1-MX metabolism was calculated from the a-v plasma 1-MX difference
and multiplied by FBF (corrected for the volume accessible to 1-MX, 0.871,
determined from plasma concentrations obtained after additions of standard
1-MX to whole rat blood) and expressed as nanomoles per minute.
Statistical analysis. To ascertain differences between treatment groups at the end of the experiment (120 min), one-way analysis of variance was used. When a significant difference (P < 0.05) was found, Dunnett's test was used to determine which times were significantly different from saline control (for FBF, arterial blood pressure, femoral vascular resistance, arterial glucose, and 1-MX, hindleg glucose extraction and uptake, and hindleg 1-MX extraction and disappearance). Pairwise comparisons were made using the Student-Newman-Keuls method. An unpaired Student's t-test was used to determine whether there was a significant difference (P < 0.05) between the glucose infusion rates at the conclusion of the experiments. All tests were performed using the SigmaStat statistical program (Jandel Software).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Metabolic effects of insulin. There was no significant difference
in the arterial blood glucose concentration between any of the treatments
either at the beginning of the experiments (time = 0 min) or at the end (time
= 120 min) (data not shown). During the euglycemic insulin clamp experiments
(insulin alone or insulin + TNF-), arterial blood glucose was
maintained at basal values by infusion of glucose. Glucose infusion rates are
shown in Fig. 3. Steady-state
rates were 11.5 ± 0.4, 21.0 ± 0.8, and 25.5 ± 0.8 mg
· kg-1 · min-1 for
3, 10, and 30 mU of insulin, respectively. The constant infusion of
TNF-
inhibited the glucose infusion rates for the two lower insulin
doses of 3 and 10 mU by
50 and 29%, respectively. TNF-
had no
effect on the glucose infusion rate due to 30 mU of insulin
(Fig. 3). End-of-experiment
(120 min) arterial plasma insulin concentrations (pmol/l) were 298 ± 33
for saline and 479 ± 50, 1,704 ± 152, and 7,391 ± 152 for
the insulin infusion rates of 3, 10, and 30 mU ·
kg-1 · min-1, respectively.
None of these was affected by TNF-
infusion. End-of-experiment (120
min) arterial plasma TNF-
concentrations were 354 ± 65 pg/ml
(n = 8).
|
Figure 4 shows data for
hindleg glucose uptake for three doses of insulin with and without
TNF-. Hindleg glucose uptake (extraction x FBF) was stimulated by
insulin 2.5-fold at 3 mU and 3.5-fold at 10 and 30 mU of insulin. TNF-
fully inhibited the increase due to insulin at 3 and 10 mU but was without
effect at 30 mU of insulin.
|
2-DG uptake. 2-DG was administered for the final 45 min of each
experiment. Figure 4 shows
combined uptake values for six lower leg muscles removed at completion. The
response to insulin varied depending on the muscle (data not shown), but in
general 3 mU of insulin led to a twofold increase, and maximal stimulation was
reached at 10 mU, as reflected by the combined data
(Fig. 4). The highest dose of
insulin (30 mU) did not further increase R'g for individual muscles (not
shown) or for the combination (Fig.
4). TNF- alone, as reported previously
(29), had little or no effect
(data not shown). When combined with insulin, TNF-
fully inhibited the
stimulation due to 3 mU of insulin, partly blocked that of 10 mU, but was
without effect on 30 mU of insulin (Fig.
4).
1-MX metabolism. No significant difference was found between
experimental groups in arterial plasma concentrations of 1-MX or oxypurinol
(data not shown; P = 0.81 and P = 0.29, respectively).
Figure 4 shows 1-MX metabolism
(A-V extraction x FBF) for the three doses of insulin (3, 10, and 30 mU)
with and without TNF-. Neither insulin nor insulin plus TNF-
had
any significant effect on 1-MX extraction (data not shown). However, insulin
alone did significantly increase 1-MX metabolism at all three concentrations,
and the increase compared with saline (control) was the same at all three
doses of insulin (Fig. 4).
TNF-
completely blocked the insulin-mediated increase in 1-MX
metabolism at 3 and 10 mU of insulin but was without effect at the highest
dose of 30 mU (Fig. 4).
Hemodynamic effects of contraction.
Figure 5 shows FBF and
calculated vascular resistance for the three experimental conditions: saline
infusion alone for 2 h, saline infusion for 2 h with electrical stimulation (2
Hz, 0.1 ms at 30 V) in the last 60 min (protocol B,
Fig. 1), and TNF-
infusion for 2 h and electrical stimulation in the last 60 min (protocol
B, Fig. 1). Electrical
stimulation increased FBF approximately threefold and heart rate by
approximately 14% (data not shown) but did not affect mean arterial pressure
(not shown). Vascular resistance calculated from FBF and mean arterial
pressure decreased from 150 to 61 RU, and this decrease was not affected by
TNF-
. TNF-
concentrations were not affected by muscle
contractions of either 10 min (246 ± 20 pg/ml, n = 5) or 60
min (411 ± 33 pg/ml, n = 5) duration, compared with
noncontraction controls (354 ± 65 pg/ml, n = 8).
|
Metabolic effects of contraction. Blood glucose concentrations
were constant for all three experimental conditions (data not shown). Because
measurement of 2-DG uptake required 45 min, uptake was determined after 1 h of
contraction. Measurement of hindleg glucose uptake was also determined at this
time. Figure 6 shows that
60-min electrical stimulation increased R'g in plantaris, gastrocnemius
red and white, extensor digitorum longus, and tibialis muscles 8- to 27-fold.
Soleus was increased only approximately twofold. Combined uptake for the six
muscles was 23-fold. TNF- had no significant effect on individual or
combined R'g (Fig. 6).
Hindleg glucose uptake was increased approximately ninefold, and this was
uninhibited by TNF-
(Fig.
6).
|
Contraction effects on 1-MX metabolism.
Figure 6 shows 1-MX metabolism
for saline control, contraction, and contraction plus TNF-. Contraction
had no effect on 1-MX extraction (data not shown) but increased 1-MX
metabolism. TNF-
had no effect on extraction (not shown) or metabolism
due to muscle contractions (Fig.
6).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Current views on TNF- and insulin resistance in rodents suggest a
key role linking them with obesity
(13). Thus overexpression of
TNF-
by adipose tissue is frequently found in different rodent models
of obesity (10,
15). In addition, acutely
administered TNF-
during insulin clamps in vivo creates resistance to
insulin, particularly evident as reduced muscle glucose uptake
(29) and complete loss of
insulin-mediated hemodynamic responses
(29). However, the mechanism
by which TNF-
exerts its effect on insulin action is not yet
understood. Effects may be direct via TNF-
itself or via an
intermediary that is released in vivo by the cytokine. An indirect effect via
fatty acid-dependent activation of the serine kinase IKK
, which plays a
role in the pathogenesis of insulin resistance through tissue inflammation, is
one possibility. In at least one system, there is evidence that TNF-
and insulin oppose each other
(9), suggesting, as does the
present study, that insulin's action is most vulnerable to
TNF-
-mediated inhibition when insulin levels are low and least
vulnerable when levels are high. At the low insulin level, inhibition of
insulin action appears to have occurred at both hemodynamic and metabolic
sites. Insulin-mediated increases in FBF as well as 1-MX metabolism were
completely blocked. Because it is unlikely that the hemodynamic responses
account for more than 50% of the insulin-mediated glucose uptake by muscle in
vivo, complete inhibition of hindleg glucose uptake suggests that insulin's
action at the myocyte is likely to have also been affected by TNF-
.
Exercise is now considered to be an insulin-independent mechanism for
stimulating muscle glucose uptake. Exercise also has marked hemodynamic
effects in vivo, including increases in limb blood flow
(4) and capillary recruitment
(11). Mechanisms for
exercise-mediated glucose uptake by myocytes are currently under intense
investigation and are quite distinct from the mechanism used by insulin.
Possibilities include Ca2+, protein kinase C, AMP
kinase, a combination of these, nitric oxide, or adenosine
(18). Mechanisms to account
for the hemodynamic responses are less well understood, but it is thought that
a metabolic vasodilator released from the myocytes or a radiating membrane
depolarization is responsible. To our knowledge, TNF- has not been used
against exercise previously. We now report that infused TNF-
at the
same dose that markedly inhibited physiological insulin had no effect on
either the hemodynamic or metabolic responses to electrical stimulation of the
hindleg muscle in vivo. In particular, capillary recruitment, which was
increased as a result of the exercise, was unaffected by the cytokine.
Together, the present findings suggest that insulin signaling at sites in
the vasculature where capillary recruitment and total flow are controlled by
insulin is particularly sensitive to inhibition by TNF-. Furthermore,
the findings imply that, if muscle insulin resistance results from the effects
of TNF-
, these can be circumvented by exogenous insulin, possibly by
agents that enhance insulin action at endothelial cells or by exercise.
![]() |
DISCLOSURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
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. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|