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Fails to Affect Insulin-Stimulated Glucose Metabolism of Isolated Rat Soleus Muscle1
Department of Medicine III (C.F., S.N., M.R., M.B., W.W.), Division of Endocrinology & Metabolism, and Department of Medical & Chemical Laboratory Diagnostics (O.W.), University of Vienna, Vienna, Austria A-1090
Address all correspondence and requests for reprints to: Clemens Fürnsinn, Ph.D., Department of Medicine III, Division of Endocrinology and Metabolism, Währinger Gürtel 18–20, A-1090 Vienna, Austria. E-mail: clemens.fuernsinn{at}akh-wien.ac.at
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF)
on insulin sensitivity, direct interaction of the peptide with freshly
isolated rat soleus muscle strips was investigated. Muscles were
exposed to TNF at concentrations ranging from 0.01–5 nmol/liter.
Rates of insulin-stimulated (5 or 100 nmol/liter) glucose metabolism
were determined after periods of TNF preexposure of 30 min, 6
h, and 24 h. Independent of exposure time, TNF failed to exert
any significant effect on rates of 3H-2-deoxy-glucose
transport (stimulation by 100 nmol/liter insulin after preincubation
without vs. with 5 nmol/liter TNF, cpm/mg·h: 30
min, 779 ± 29 vs. 725 ± 29; 6 h,
652 ± 56 vs. 617 ± 60; 24 h, 911
± 47 vs. 936 ± 31) or glucose incorporation into
glycogen (µmol/g·h: 30 min, 5.19 ± 0.22 vs.
5.25 ± 0.41; 6 h, 2.08 ± 0.10 vs.
2.09 ± 0.17; 24 h, 2.51 ± 0.21 vs.
2.41 ± 0.26). In parallel, TNF neither affected
insulin-stimulated rates of glucose oxidation (CO2
production) and anaerobic glycolysis (lactate release), nor muscle
glycogen content. In conclusion, these findings do not support the
hypothesis of muscle insulin desensitization by TNF via autocrine or
paracrine mechanisms. The obtained data favor the concept that
TNF-dependent muscle insulin resistance in vivo
depends on indirect effects rather than direct interaction of the
peptide with skeletal muscle. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF) may
play a key role in the etiology of obesity-associated insulin
resistance (for review see Ref. 3). This hypothesis is based on the
observations that expression and release of TNF is increased in
adipose tissue from obese rodents and humans (4, 5, 6) and that TNF
carries a distinct potential to induce insulin resistance as indicated
by blunted insulin effects on whole body glucose uptake and hepatic
glucose output in TNF-infused rats in vivo (7, 8). It is
of note that during TNF infusion, glucose uptake by skeletal muscle
and skin revealed to be primarily responsible for decreased
insulin-mediated whole body glucose utilization (7, 8). Furthermore,
neutralization of endogenous TNF in genetically obese insulin
resistant Zucker rats for 3 days led to a 2- to 3-fold increase in
insulin-stimulated peripheral glucose utilization in vivo
without any change in the rate of hepatic glucose output (4). In
parallel, insulin-induced autophosphorylation of the insulin receptor
as well as phosphorylation of insulin receptor substrate-1 were
restored to near control values in muscle and fat, but not liver (9),
which suggests that endogenous TNF contributes considerably to
peripheral insulin resistance in obese Zucker rats. In contrast to its
effects in Zucker rats, TNF neutralization over a period of 4 weeks
did not affect insulin sensitivity in obese NIDDM patients (10).
Whether blunting of whole body insulin sensitivity by exogenous and
endogenous TNF in vivo is due to its direct interaction
with insulin target tissues or rather mediated via indirect mechanisms
is, however, not completely understood.
Because up to 80% of insulin-stimulated glucose uptake is into
muscle (2, 11), any major changes in whole body glucose clearance in
response to infusion or neutralization of TNF (4, 7, 8) have to
involve considerable changes in muscle insulin sensitivity (7).
Although a decrease in insulin action has been described in cultured L6
rat muscle cells preexposed to TNF for 10 min to 12 h (12), no
insulin resistance was observed in the same cell line after TNF
treatment for 4–8 days (13) or in cardiomyocytes after short-term
exposure to the peptide (14). Hence, it is still unclear, whether
TNF is to influence glucose handling by direct interaction with
native skeletal muscle.
This study, therefore, was designed to elucidate if TNF is to
directly affect insulin-stimulated glucose metabolism in freshly
isolated rat soleus muscle strips. Because time dependency of TNF
action in vitro has been described in other experimental
settings (15, 16, 17), insulin-stimulated rates of glucose metabolism were
determined after muscle preexposure to TNF for 30 min, 6 h, and
24 h.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TNF
Unless stated otherwise, human recombinant TNF was from Sigma
Chemical Co. (St. Louis, MO). Biological activity of the peptide was
validated by TNF-dependent accumulation of plasminogen activator
inhibitor-1 in the supernatant of cultured human umbilical vein
endothelial cells (ng/ml after 24 h: control, 56 ± 11,
vs. 1.4 nmol/liter TNF, 275 ± 23; P
< 0.01; n = 3 each).
Muscle incubation procedures
Phase 1 (30 min, 6 h, or 24 h). Medium 199 (Sigma)
supplemented with 5 mmol/liter HEPES, 25,000 U/liter penicillin G, 25
mg/liter streptomycin, and 0.25% (wt/vol) BSA was used as incubation
medium (M199; pH 7.35). In the presence or absence of TNF-, muscle
strips were incubated for 30 min, 6 h, and 24 h,
respectively. For short-term incubation (30 min), 25-ml Erlenmeyer
flasks coated with Blue Slick solution (Serva, Heidelberg, Germany) and
provided with 3 ml M199 were employed (1 strip/flask), whereas for
long-term incubation (6 h and 24 h, respectively) muscles were put
into coated 50-ml flasks provided with 20 ml M199 (6 strips/flask) so
as not to touch the inner surface of the flask. Flasks were placed into
a shaking water bath (37 C; 130 cycles/min) and an atmosphere of 95%
O2:5% CO2 was continuously provided within the
flasks. During 24-h incubation, M199 was renewed every 5–7 h.
Phase 2 (1 h). After phase 1, muscles were immediately
transferred into a set of 25-ml flasks provided with 3 ml of M199 (1
strip/flask). In phase 2, M199 contained identical concentrations of
TNF as used in phase 1 and trace amounts of
D-[U-14C] glucose or, alternatively,
2-deoxy-D-[2,6-3H] glucose plus
[U-14C] sucrose (all from Amersham, Amersham, UK) to
determine rates of glucose transport, glucose incorporation into
glycogen, and glucose oxidation in the absence or presence of insulin
(Actrapid, Novo, Bagsvaerd, Denmark). After 60-min muscle strips were
quickly removed, blotted, and frozen in liquid N2. Later,
muscle strips were lysed in 1 mol/liter KOH at 70 C and the lysate used
for further analytical procedures as described below.
Experimental design
Before the effects of TNF were investigated, a series of
experiments was performed to confirm preservation of the stimulatory
action of insulin on glucose metabolism in rat soleus muscle strips
after long-term preincubation. To this end, muscle strips were
preincubated for 30 min, 6 h, or 24 h in the absence of any
exogenous peptide (phase 1) and selected parameters of glucose
metabolism were then measured in the absence or presence of insulin (1,
10, and 100 nmol/liter; phase 2).
To examine the dose-dependent effects of short- and long-term exposure
of isolated muscle to TNF on insulin-stimulated glucose metabolism,
muscle strips were preincubated for 30 min, 6 h, or 24 h in
the absence of insulin and in the presence of 10, 100, or 1000
pmol/liter TNF (phase 1). Subsequently, selected parameters of
glucose metabolism were measured in the presence of a maximally
stimulating concentration of insulin (100 nmol/liter; phase 2).
To investigate the effects of a concentration of 5 nmol/liter TNF on
glucose metabolism in the presence of submaximally and maximally
insulin-stimulated glucose metabolism, muscle strips were preincubated
for 30 min, 6 h, or 24 h in the absence vs.
presence of 5 nmol/liter TNF and in the absence of insulin (phase
1). Selected parameters of glucose metabolism were then measured in the
presence of 5 nmol/liter and 100 nmol/liter insulin, respectively
(phase 2).
To control for potential influence of peptide source and solubility,
the short-term effects of TNF (5 nmol/liter) on insulin-stimulated
(100 nmol/liter) glucose metabolism were examined employing TNF from
another source (GIBCO, Gaitherburg, MD) or, alternatively, in the
presence of 1% (vol/vol) dimethyl sulfoxide.
Analytical procedures
Net uptake rate of 2-deoxy-D-[2,6-3H]
glucose, a glucose analogue that does not enter glycolysis and
accumulates within the cell, was determined employing
[14C] sucrose as an extracellular space marker by methods
described previously (19). Glycogen synthesis is given as the net rate
of conversion of [14C] glucose to [14C]
glycogen as determined by methods described previously (18). Glucose
oxidation, i.e. CO2 production, was calculated
from conversion of [14C] glucose into
14CO2. To this end, the flasks were sealed
during the last 45 min of muscle incubation, after which the muscle
strips were quickly removed and the flasks were immediately resealed
with a stopper carrying a hang-in container provided with 200 µl
CO2-trapping solution (phenethylamine:methanol, 1:1). Using
a syringe, 200 µl of 3 mmol/liter perchloric acid were injected into
the incubation buffer within the flasks to quantitatively release
CO2 from the medium. After incubation for at least 1 h
at room temperature, the trapping solution was brought into
scintillation fluid, which was vigorously shaken and counted for
14C-content. Anaerobic glycolysis, i.e. rate of
lactate release, was calculated from M199 lactate concentration
measured enzymatically by the lactate dehydrogenase method (20). For
determination of muscle glycogen content, glycogen in the muscle lysate
was completely degraded to glucose with amyloglucosidase (21). Glucose
was then measured enzymatically by a commercial kit from Human
(Taunusstein, Germany).
Statistics
All data are presented as means ± SEM and a
P < 0.05 was considered significant. For comparison of
two groups, P values were calculated by two-tailed paired
Student’s t test. Multiple comparisons with a control were
performed after logarithmic transformation of data according to the
method of Dunnett (22), where the effect of individual rat was
controlled.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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demonstrate that soleus
muscle strips remained viable and well-responsive to insulin
stimulation during incubation periods up to 24 h. Nevertheless,
increased rates of glycolysis and glucose oxidation as well as
decreased rates of glucose incorporation into glycogen were observed
after prolonged incubation and were associated with lower muscle
glycogen content.
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failed to affect
insulin-stimulated muscle glucose metabolism. Rates of
insulin-stimulated glucose transport, glycogen synthesis, glycolysis,
and glycogen accumulation were neither influenced by short-term nor by
long-term treatment with TNF (Fig. 2).
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demonstrates glucose metabolic rates prevailing
in soleus muscle strips, which were exposed to 5 nmol/liter TNF, a
peptide concentration employed previously to describe insulin
desensitization by TNF of isolated nonmuscle tissue (14, 15, 16, 23, 24). Both short- and long-term actions of TNF on muscle glucose
metabolism were not only tested with respect to insulin responsiveness
(i.e. under maximal stimulation with 100 nmol/liter
insulin), but also with respect to insulin sensitivity (i.e.
under partial stimulation with 5 nmol/liter insulin). Under these
experimental conditions, no influence on insulin sensitivity or insulin
responsiveness of isolated rat soleus muscle was revealed, although
muscles were exposed for up to 25 h to 5 nmol/liter TNF.
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exposure on
insulin-stimulated glucose metabolism were observed, when the effects
of TNF from an alternative source or in the presence of detergent
were determined (Table 2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in rats increases peripheral glucose uptake in
the basal state but elicits a distinct loss in hepatic and peripheral
insulin sensitivity in vivo, which includes decreased
insulin-stimulated glucose uptake in rectus abdominus, gastrocnemius,
and red as well as white quadriceps muscle (7, 8). Furthermore,
experimental evidence has been provided that endogenous TNF may be
instrumental in the development of obesity-associated insulin
resistance and derangement of glucose homeostasis in rats (3, 4).
Because the circulating plasma levels of TNF found in association
with obesity are regarded too low to allow for endocrine effects on
muscle tissue, it has been hypothesized that peptide release from fat
cells located in the vicinity of muscle fibers may mediate TNF
action via paracrine mechanisms (3, 4, 25). Speculations also include
potential autocrine mechanisms in spite of the observation that TNF
is expressed at much lower rates in muscle than in adipose tissue
(25).
In this study, TNF at concentrations ranging from 0.01–5 nmol/liter
neither affected insulin-stimulated rates of glucose transport,
glycogen synthesis, CO2 production, and lactate release,
nor did TNF exert any influence on glycogen content of isolated rat
soleus muscle strips in vitro. Such failure of TNF to
directly affect insulin-stimulated soleus muscle glucose metabolism
in vitro was substantiated under various experimental
circumstances including TNF pretreatment for 30 min, 6 h, and
24 h followed by both submaximal and maximal insulin stimulation.
Although TNF release from isolated muscle strips in vitro
can not be excluded, it seems unlikely that autocrine mechanisms may
have triggered maximal TNF-stimulation in the control experiments
and hence may have masked any effect of exogenous TNF added to the
incubation medium. Thus, in spite of such potential autocrine
stimulation, others have found that plasma concentrations around 100
pmol/l TNF were sufficient to induce distinct muscle insulin
resistance in vivo (7) and that TNF-induced insulin
desensitization was well-describable for various other isolated tissues
(12, 16, 23, 24, 26, 27, 28). Poor quality of employed TNF was excluded
as the cause of negative results by evaluation of its capability to
release plasminogen activator inhibitor-1 from cultured human ubilical
vein endothelial cells. The obtained data thus do not support the idea
of TNF to induce insulin resistance via direct interaction with
skeletal muscle, which is known to express receptors for TNF (29).
Such lack of direct interaction with skeletal muscle glucose metabolism
is in line with described effects on protein catabolism, which is
markedly stimulated by TNF in vivo (30, 31), whereas the
peptide fails to directly affect protein breakdown of isolated muscle
tissue in vitro (30, 32).
In agreement with a previous report (33), the applied method for
incubation of freshly isolated rat soleus muscle strips displayed
blunted rates of glycogen synthesis, increased glycolysis, and
decreased glycogen content upon prolonged incubation in the absence of
plasma, hormones, and innervation when compared with short-term
incubation. Such time-dependent increase in the rate of carbohydrate
catabolism may be caused by depletion of im lipid stores resulting in
an enhanced requirement for glucose as a fuel substrate, or may reflect
a general increase in the metabolic rate due to the artificial
environment. Nevertheless, muscle tissue remained viable even after
incubation for 24 h as indicated by its preserved ability to
respond to insulin, which dose-dependently triggered increases in the
rates of glucose transport, glycogen synthesis, and glycolysis. The
employed rat soleus muscle preparation therefore appears adequate for
the investigation of short- and long-term TNF action on
insulin-stimulated glucose metabolism.
Peptide concentrations and exposure periods applied in our experiments
are in the range of that used to document TNF-dependent insulin
desensitization in isolated tissues including adipocytes, hepatoma
cells, and the cultured rat skeletal muscle cell line L6 (12, 16, 23, 24, 26, 27, 28). Most studies describing TNF-dependent insulin
desensitization in vitro did, however, not determine
insulin-stimulated rates of glucose metabolism, but rather focused on
processes involved in intracellular insulin signal transduction (16, 17, 23, 24, 26, 27), whereby phosphorylation of insulin receptor
substrate-1 serine residues (23, 26) and activation of phosphotyrosine
phosphatases (17) have been suggested to mediate TNF-induced insulin
resistance.
In a limited number of in vitro-studies, the stimulatory
effect of insulin on glucose metabolism was determined and glucose
transport has been found blunted in isolated adipocytes exposed to
TNF (15, 27). In the cultured rat skeletal muscle cell line L6, no
insulin resistance was induced by 4–8 d of exposure to TNF (13),
whereas a distinct decrease in insulin-stimulated glucose transport was
observed after TNF-pretreatment for 1 h or 12 h (12). The
latter finding was associated with reduced glucose incorporation into
glycogen and glycogen synthase activity (12) suggesting that major
differences exist in the interaction of TNF with insulin in cultured
L6 muscle cells vs. freshly isolated soleus muscle. Such
discrepancies can not be explained by different source of TNF, since
peptide both from GIBCO (used for L6 cells; 12) and Sigma (used in this
study) did not affect soleus muscle glucose handling in
vitro. Difference in responses to TNF thus are likely to
reflect different type of tissue employed with freshly isolated native
muscle tissue relating closer to the physiological situation than a
cultured muscle cell line. Even using freshly isolated muscle, however,
direct conclusions to the physiological situation are subject to
limitations principally applying in vitro studies, which
include that full muscle sensitivity to the effects of insulin and/or
TNF may depend on innervation and normal blood perfusion. Although
muscles dominated by white glycolytic fast-twitch fibers as well as
muscles dominated by red oxidative slow-twitch fibers are affected by
TNF-dependent insulin desensitization in vivo (7, 8),
deviations in metabolic response to TNF in vitro may to
some extent depend on muscle fiber type. In the case of soleus muscle
fiber type is mainly slow-twitch, although presence of a small amount
fast-twitch fibers can not be excluded. In that context it is of note
that TNF has also been described to inhibit insulin-dependent
activation of phosphatidylinositol 3-kinase in adipocytes, but not in
cardiomyocytes (14), which carry a high oxidative capacity and hence
resemble red rather than white skeletal muscle fibers.
In conclusion, this study demonstrates failure of TNF to affect
insulin-stimulated glucose metabolism by direct interaction with rat
soleus muscle in vitro and hence does not provide evidence
for TNF-dependent muscle insulin desensitization via autocrine or
paracrine mechanisms as hypothesized by others (3, 4, 25). Our findings
rather favour the concept that TNF-dependent muscle insulin
resistance in vivo is mediated indirectly via interaction
with other tissues and may involve counterregulatory hormone release
(7, 34) or lipid metabolism (9, 35).
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Acknowledgments |
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Footnotes |
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Received December 30, 1996.
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