ORIGINAL ARTICLE
Denervation and High-Fat Diet Reduce Insulin Signaling
in T-Tubules in Skeletal Muscle of Living Mice
Hans P.M. Mortensen Lauritzen,1 Thorkil Ploug,2 Hua Ai,2 Morten Donsmark,2 Clara Prats,2 and
Henrik Galbo3
OBJECTIVE—Insulin stimulates muscle glucose transport by
translocation of GLUT4 to sarcolemma and T-tubules. Despite
muscle glucose uptake playing a major role in insulin resistance
and type 2 diabetes, the temporal and spatial changes in insulin
signaling and GLUT4 translocation during these conditions are
not well described.
RESEARCH DESIGN AND METHODS—We used time-lapse
confocal imaging of green fluorescent protein (GFP) ADP-ribosylation factor nucleotide-binding site opener (ARNO) (evaluation of phosphatidylinositide 3-kinase activation) and GLUT4GFP–transfected quadriceps muscle in living, anesthetized mice
either muscle denervated or high-fat fed. T-tubules were visualized with sulforhodamine B dye. In incubated muscle, glucose
transport was measured by 2-deoxy-D-[3H]-glucose uptake, and
functional detubulation was carried out by osmotic shock. Muscle fibers were immunostained for insulin receptors.
RESULTS—Denervation and high-fat diet reduced insulin-mediated glucose transport. In denervated muscle, insulin-stimulated
phosphatidylinositol 3,4,5 P3 (PIP3) production was abolished in
T-tubules, while PIP3 production at sarcolemma was increased
2.6-fold. Correspondingly, GLUT4-GFP translocation to T-tubules
was abolished, while translocation to sarcolemma was increased
2.3-fold. In high fat–fed mice, a ⬃65% reduction in both insulininduced T-tubular PIP3 production and GLUT4-GFP translocation was seen. Sarcolemma was less affected, with reductions of
⬃40% in PIP3 production and ⬃15% in GLUT4-GFP translocation.
Access to T-tubules was not compromised, and insulin receptor
distribution in sarcolemma and T-tubules was unaffected by
denervation or high-fat feeding. Detubulation of normal muscle
reduced basal and abolished insulin-induced glucose transport.
CONCLUSIONS—Our findings demonstrate, for the first time,
that impaired insulin signaling and GLUT4 translocation is compartmentalized in muscle and primarily localized to T-tubules
and not sarcolemma during insulin resistance. Diabetes 57:
13–23, 2008
From the 1Research Division, Joslin Diabetes Center and Harvard Medical
School, Boston, Massachusetts; the 2Copenhagen Muscle Research Centre,
Department of Medical Physiology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark; and the 3Department of Rheumatology, Bispebjerg Hospital, Copenhagen, Denmark.
Address correspondence and reprint requests to H.P.M. Mortensen Lauritzen, PhD, Research Division, Joslin Diabetes Center, Harvard Medical School,
Boston, MA 02215. E-mail: [email protected].
Received for publication 13 April 2007 and accepted in revised form 29
September 2007.
Published ahead of print at http://diabetes.diabetesjournals.org on 3 October 2007. DOI: 10.2337/db07-0516.
Additional information for this article can be found in an online appendix at
http://dx.doi.org/10.2337/db07-0516.
[3H]2-DG, 2-deoxy-D-[3H]-glucose; 2-DG, 2-deoxy-glucose; ARNO, ADP-ribosylation factor nucleotide-binding site opener; EDL, extensor digitorum
longus; GFP, green fluorescent protein; PI3-K, phosphatidylinositide 3-kinase;
PIP3, phosphatidylinositol 3,4,5 P3; ROI, region of interest.
© 2008 by the American Diabetes Association.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
DIABETES, VOL. 57, JANUARY 2008
S
keletal muscle plays an important role in glucose
homeostasis, and defects in glucose uptake in
muscle are involved in states of insulin resistance
(e.g., type 2 diabetes). Upon stimulation with
insulin, the GLUT4 glucose transporters in skeletal muscle
fibers are translocated from intracellular compartments to
the plasma membrane and T-tubules, and, in turn, glucose
uptake is increased (1,2). In type 2 diabetes, the number of
GLUT4 transporters in muscle is normal (3,4), and in other
states of insulin resistance as well, reductions in GLUT4
content cannot fully explain the diminished glucose transport in muscle (5–7). Accordingly, the insulin signaling
and/or the trafficking of GLUT4 must be impaired (4).
However, the nature of these cellular defects is poorly
understood. By imaging the dynamic changes in localization of green fluorescent protein (GFP)-tagged proteins in
skeletal muscle fibers in situ in normal living mice (8), we
have revealed previously unnoticed signaling functions of
the T-tubule system (9). In the present study, using similar
techniques we investigated the temporal and spatial dynamics of insulin signaling and GLUT4 translocation in
two conditions accompanied by insulin resistance, muscle
denervation (6,10), and high-fat dieting (5,11). We found
that insulin signaling at the level of phosphatidylinositide
3-kinase (PI3-K) activation and GLUT4 translocation are
markedly impaired in the T-tubules during both conditions. In contrast, in sarcolemma increased insulin-mediated PI3-K activity and GLUT4 translocation were seen
after denervation, while these processes were only little
impaired after high-fat dieting. Our study demonstrates,
for the first time, that insulin signaling and, in turn, GLUT4
translocation are primarily compromised in the T-tubules
and not the sarcolemma of skeletal muscle during insulinresistant states.
RESEARCH DESIGN AND METHODS
Animal procedures. All experiments were approved by the animal research
committee of the Danish Ministry of Justice. Mice were always provided with
water and studied in the fed state.
High-fat diet treatment. At 3 weeks of age, male Friend virus B-type (FVB)
mice (Charles River, Sulzfeld, Germany) were given either normal standard
diet or high-fat diet for 12 weeks. Standard diet consisted of 14% energy from
fat, 66% from carbohydrate, and 20% from protein (Altromin, Lage, Germany).
The high-fat diet was prepared by Harlan Teklad (TD93075), as previously
described (5), and consisted of 55% of energy from fat, 24% from carbohydrate,
and 21% from protein.
Denervation. To study the effects of denervation, the right femoral nerve of
7- to 8-week-old FVB male mice (Charles River) was transsected 1 mm
proximal to its branch, innervating the quadriceps muscle. In other experiments, 7- to 8-week-old FVB male mice had one extensor digitorum longus
(EDL) and one soleus muscle denervated by resection of a 5-mm-long segment
of the sciatic nerve in the thigh. After denervation, the skin was closed by
sutures and confocal imaging analysis or glucose transport measurement was
performed 10 days later.
13
DENERVATION, DIET, AND INSULIN SIGNALING
Plasmid and transfection procedures. The construction of GLUT4-GFP (8)
and GFP-ADP–ribosylation factor nucleotide-binding site opener (ARNO) (12)
has been described previously. The vastus lateralis part of the quadriceps
muscles of 5-days– denervated or high fat–fed and control FVB mice (Charles
River) were transfected with either two times 1 ␮g GLUT4-GFP or 1 ␮g
ARNO-GFP cDNA/0.5 mg gold, as previously described (8).
Time-lapse microscopy. Five days after transfection, mice were anesthetized subcutaneously with 0.8 –1 ml/100 g body wt Hypnorm-Dormicum (25%
Hypnorm [5 mg/ml] ⫹ 25% Dormicum [5 mg/ml] in water), and the skin
covering the superficial part of the quadriceps muscle (86% type IIb fibers [13])
was opened. The mice were mounted on the confocal microscope, transfected
muscle fibers with similar probe expression in the various groups were
imaged, and insulin was stimulated during maintenance of euglycemia, as
previously described (9). Sulforhodamine B was administrated and imaged, as
previously described (9).
Image analysis. The TIFF (tagged image file format) images obtained with
the Leica confocal software were imported into Metamorf Software (version
6.1; Universal Imaging). Image stacks were created and pixel values were
corrected for noncellular fluorescence. Brightness and contrast were adjusted. For quantifications and visualization of GLUT4-GFP images, a Fourier
noise filter was applied. The degree of GLUT4-GFP translocation to membrane
surfaces was quantified by drawing a region of interest (ROI) surrounding the
surface of interest and measuring its pixel values. For sarcolemma, measurements from both sarcolemma edges (excluding perinuclear areas) of the
confocal picture were averaged. For T-tubules four ROIs, excluding big
vesicular GLUT4-EGFP stores, were drawn and measurements from these
were averaged. For GFP-ARNO quantification, ROI pixel values were measured at both sarcolemma edges (excluding perinuclear areas) and averaged.
For GFP-ARNO at T-tubules, measurements were averaged from two 2-␮mwide ROIs along the length of the fiber at identical depths. Arbitrary units
shown are the actual gray value of ROI fluorescence divided by ROI fluorescence value at t ⫽ 0. Uncompressed Quicktime movies were produced from
the image stacks and subsequently compressed with Sorensen 3 codec in
Quicktime Pro 6.
Study of the distribution of insulin receptors in single muscle fibers.
Quadriceps muscles were fixed and single fibers from the superficial white
part of the muscle were teased to perform immunofluorescence staining, as
previously described (14). A rabbit antibody against insulin receptor (no.
ab10991-50; Abcam) was used and immunodetected with a secondary antibody conjugated to Alexa-488 (no. A11008; Invitrogen).
Formamide-induced detubulation. Seven- to 8-week-old FVB male mice
(Charles River) were anesthetized subcutaneously with Hypnorm-Dormicum
(0.8 –1 ml/100 g body wt) and decapitated. EDL muscles (47% type IIa, 50%
type IIb fibers [13]) were gently taken out and preincubated in test tubes at pH
7.4 and 29°C for 60 min in Krebs-Henseleit buffer gassed with 95% O2 and 5%
CO2 and containing 8 mmol/l glucose, 1 mmol/l pyruvate, and 0.2% BSA. After
preincubation, the muscles were incubated with 5 mol/l formamide (Sigma) in
Krebs-Henseleit buffer for 60 min. The detubulation was induced by transferring muscles to Krebs-Henseleit buffer for 20 min, and then the muscles were
washed twice in glucose-free Krebs-Henseleit buffer.
Measurement of 2-DG transport in incubated muscles. EDL and soleus
(87% type I fibers [13]) muscles were preincubated for at least 90 min in
Krebs-Henseleit buffer gassed with 95% O2 and 5% CO2 and containing 8
mmol/l glucose, 1 mmol/l pyruvic acid, and 0.2% BSA at pH 7.4 and 29°C.
For glucose transport measurement, muscles were transferred to glucosefree Krebs-Henseleit buffer containing 2 mmol/l pyruvic acid and 0.2% BSA
with or without insulin (100 mU/ml Actrapid; Novo Nordisk). Glucose
transport was measured as a 2-deoxy-D-[3H]-glucose ([3H]2-DG) uptake with
[14C]sucrose as the extracellular marker (15). Isotopes and unlabeled sugars
were added to the incubation medium to yield final concentrations of 0.43 ␮Ci
[3H]2-DG and 0.32 ␮Ci [14C]sucrose/ml and 1 mmol/l of both unlabeled 2-DG
and sucrose. After 10 min of exposure to isotopes, muscles were briefly
blotted on filter paper and immediately frozen in liquid nitrogen. Muscles were
stored at ⫺80°C until analyzed.
Glucose concentration in tail vein blood. Blood was sampled at noon from
the tail vein of anesthetized (subcutaneously with Hypnorm-Dormicum)
15-week-old FVB male mice that had either a high-fat diet or a standard diet
for 12 weeks. Blood glucose concentrations were measured directly by
EuroFlash glucose-monitoring system using the sensor strips of this system
(Johnson & Johnson).
Calculations and statistics. Data are presented as means ⫾ SE. Statistical
comparisons were carried out by two-way ANOVA and Student’s t test, paired
or unpaired as applicable. Post hoc analysis was done by the Tukey test. P ⬍
0.05 was considered significant in two-tailed testing.
14
RESULTS
Insulin resistance in muscle. To study insulin signaling
in insulin resistance, we looked at two conditions accompanied by insulin resistance: long-term muscle denervation and high-fat feeding. Long-term denervation of muscle
(7–10 days) is known to result in an up to 60% decrease in
insulin-induced, biochemically determined PI3-K activity
and glucose transport (10,16). In agreement with this, we
found a 40% decrease (P ⬍ 0.05) in insulin-mediated 2-DG
uptake after 10 days of denervation of EDL (online appendix Fig. 1A [available at http://dx.doi.org/10.2337/db070516]) and soleus (data not shown) muscle. High-fat
feeding of FVB mice has also been shown to induce insulin
resistance and type 2 diabetes (5,11). In agreement with
this, we found a 30% decrease (P ⬍ 0.05) in insulinmediated 2-DG uptake in EDL (online appendix Fig. 1B)
and soleus (data not shown) muscles after 12 weeks of
high-fat diet feeding. Furthermore, in accordance with the
development of whole-body insulin resistance, after 12
weeks of high-fat feeding the noon glucose concentration
in tail vein blood had increased compared with values in
control mice (8.9 ⫾ 0.5 vs. 10.5 ⫾ 0.5 mmol/l, n ⫽ 6 – 8; P ⬍
0.05) (online appendix Fig. 1C).
Dynamic visualization of insulin signaling in insulinresistant muscle
Denervation. We analyzed the spatial and temporal
changes in insulin signaling in quadriceps muscle of denervated and high fat–fed mice by time-lapse analysis of
the early insulin signaling step, the activation of PI3-K
(17,18). For this we used the ARNO (19) fused to GFP
(12,20). ARNO binds with exquisite specificity via its
pleckstrin homology domain to phosphatidylinositol 3,4,5
P3 (PIP3), the product of insulin-activated PI3-K (21). The
production of PIP3 has been shown to be a direct measure
of PI3-K activation, and GFP-ARNO has been shown to
track and indicate local concentrations of PIP3 (20,22). By
imaging GFP-ARNO in skeletal muscle fibers of living
mice, we could dynamically follow the local PI3-K activity
with time (Fig. 1A). Skeletal muscle of living mice were
denervated 10 days before imaging, and GFP-ARNO–transfected muscle fibers were imaged in their natural surroundings. In the basal state, GFP-ARNO was distributed
throughout the muscle cell in both control (Fig. 1A, t ⫽ 0
min) and denervated (Fig. 1B, t ⫽ 0 min) muscle as
expected for a cytosolic protein (12). Furthermore, within
⬃2 min after insulin injection, GFP-ARNO translocated to
the sarcolemma in both groups (Fig. 1A and B, t ⫽ 2 min,
arrows). However, the PIP3 production persisted for a
longer time and at a higher level at the sarcolemma in
denervated compared with control muscle (Fig. 1A and B,
t ⫽ 4 min). In fact, image quantification of ROIs, including
sarcolemma (Fig. 2 E and F), revealed a 2.6-fold higher
PIP3 production at the sarcolemma over the 20 min of
observation in denervated muscle compared with control
(Fig. 2A and G). In contrast to findings in sarcolemma, the
wave-like spreading of the PIP3 production into the Ttubules seen in normal muscle (Fig. 1A, t ⫽ 4 and 14-min
arrows; online appendix movie 1) was completely absent
in denervated muscle (Fig. 1B; online appendix movie 2).
Instead, in the latter muscle only a few fragmented spots
of PIP3 production emerged in the T-tubules (Fig. 1B, t ⫽
7 min, arrow), and overall PIP3 production was markedly
reduced in the T-tubules (Fig. 1B, t ⫽ 4 –20 min). Image
quantification of ROIs at various distances from the sarcolemma (Fig. 2E and F, t ⫽ 0 min) also showed that the
DIABETES, VOL. 57, JANUARY 2008
H.P.M.M. LAURITZEN AND ASSOCIATES
FIG. 1. Muscle denervation markedly reduces insulin-mediated PIP3 production in the T-tubules but not in the sarcolemma. A and B: t ⴝ 0 shows
a confocal image of a basal GFP-ARNO expressing quadriceps muscle fiber in situ in a mouse just before intravenous insulin injection. Images of
GFP-ARNO were obtained every 2 s after insulin injection in control (A) and denervated (B) quadriceps. Numbers denote time points (min).
Arrows indicate local PIP3 production at the selected time points at sarcolemma and T-tubule areas illustrating less intense PI3-K activation at
T-tubules in denervated compared with control muscle. C: Confocal images of a denervated muscle fiber in vivo showing that intravenously
injected sulforhodamine B diffuses into the T-tubules. Images were collected every 2 s from insulin and sulforhodamine B injection at t ⴝ ⴚ14
s and are shown at time of arrival at sarcolemma (t ⴝ 0) and t ⴝ 2 and 4 min. Bar ⴝ 20 ␮m. Images are representative of findings in four mice.
wave of PIP3 production in the T-tubules in response to
insulin was almost completely abolished (⌬AUC, P ⬍ 0.05)
(Fig. 2B–D, H). So, not only did denervation alter the
progression pattern of PIP3 production, but the overall
PIP3 production was also strongly reduced.
To see if these changes in PIP3 production in T-tubules
were secondary to the rearrangement of the T-tubule
system, which has been shown by electron microscopy to
take place during long-term denervation (23), sulforhodamine B (24 –26) was coinjected with insulin and glucose.
This dye is nontoxic, is not taken up by muscle cells, and
has previously been used to load T-tubules both in vitro
(24 –26) and in vivo (9). As shown in Fig. 1C, a striated
pattern emerged as previously seen in normal innervated
muscle (9). Thus, no T-tubular rearrangement was visible
at the level of light microscopy and no changes in accessibility to the inner T-tubules had taken place, a fact
excluding that such rearrangement explained the denervation-induced change in appearance of PIP3 production.
Similarly, immunofluorescence staining of insulin receptors delineated the sarcolemma and, in addition, showed a
striated pattern of uniform intensity indicating an even
distribution of insulin receptors throughout the T-tubule
system, as previously described in normal muscle (9) (Fig.
3A and B).
High-fat diet. After 12 weeks of high-fat diet, quadriceps
muscles were transfected with GFP-ARNO and subjected
to confocal image analysis in vivo. During basal conditions, GFP-ARNO was distributed similarly throughout the
DIABETES, VOL. 57, JANUARY 2008
muscle cell in both control (Fig. 4A, t ⫽ 0 min) and fat-fed
muscle (Fig. 4B, t ⫽ 0 min). Within ⬃2 min after insulin
injection, GFP-ARNO translocated to the sarcolemma in
both groups, but translocation was slightly reduced in the
high fat–fed mice (Fig. 4A and B, t ⫽ 2 min, arrows).
Quantification showed the increase in PIP3 production at
the sarcolemma to be reduced by 42% over the time of
PI3-K activation in the high fat–fed mice compared with
controls (Fig. 5 A and E). Moreover, while in muscle of
mice fed normal diet a strong and regular wave-like
spreading of the PIP3 production along the T-tubules was
seen (Fig. 4A, t ⫽ 9 min, arrows), in mice fed a high-fat diet
a less strong GFP-ARNO signal was found (Fig. 4B, t ⫽ 9
min, arrows), indicating reduced PIP3 production with
only a few strong “spikes” running into the T-tubules (Fig.
4A and B). However, overall, the wave-like pattern was
still present, although weaker and more irregular than
seen in normal mice (Fig. 4A and B). Quantification at
various distances from the sarcolemma revealed a marked
reduction of ⬃65% of T-tubular PIP3 production over the
period of observation (⌬AUC, P ⬍ 0.05) (Fig. 5B–D and F).
Sulforhodamine B staining of T-tubules (results not shown),
as well as immunofluorescence staining of insulin receptors in sarcolemma and T-tubules (Fig. 3C and D), appeared as in normal muscle.
Dynamic visualization of insulin-mediated GLUT4
translocation in insulin-resistant muscle. We investigated if the observed changes in PI3-K activity after
denervation and high-fat diet had any functional conse15
DENERVATION, DIET, AND INSULIN SIGNALING
FIG. 2. Quantification of in vivo confocal recordings (Fig. 1) showing that insulin-stimulated PIP3 production is increased at the sarcolemma and
strongly reduced in the T-tubules in denervated compared with normal muscle. Image quantification of GLUT4-GFP at the sarcolemma (A) and
in the T-tubules at 10-␮m (B), 20-␮m (C), and 30-␮m (D) distances from sarcolemma. Values are means ⴞ SE, n ⴝ 4. Incremental areas under
the curve (ŒAUC), calculated from early nadir values to end of curve or to values below initial values, differed significantly (P < 0.05, t test)
between denervated and control muscle (B–D). E and F: Confocal image of a basal GFP-ARNO expressing quadriceps muscle fiber from control
(E) and denervated (F) muscle at t ⴝ 0. ROIs are drawn, including the sarcolemma surfaces and in 10-, 20-, and 30-␮m distances from the
sarcolemma surfaces. The mean of ROI measurements at the same locations were calculated at each time point. G and H: Raw nonnormalized and
nonaveraged fluorescence for representative single ROIs at the sarcolemma (G) and in T-tubule area 20 ␮m from sarcolemma (H). A–D, G, and
H: 䡺, control; F, denervated.
16
DIABETES, VOL. 57, JANUARY 2008
H.P.M.M. LAURITZEN AND ASSOCIATES
FIG. 3. Insulin receptors are always uniformly distributed throughout the T-tubules, and receptor numbers here and at the sarcolemma are
identical in denervated muscle fibers (A) and muscle fibers from high fat–fed (C) or control (B and D) mice. Single quadriceps muscle fibers were
immunostained. Bar ⴝ 20 ␮m. Images are representative of findings in five mice.
quences at the level of GLUT4 translocation. In control
muscles, insulin induced a threefold increase in GLUT4GFP at the sarcolemma and a sixfold increase in the
T-tubules in agreement with previous findings (Fig. 6A, t ⫽
30 min, arrows; C and D) (8,9,14). In basal denervated
muscles, GLUT4 localization in the T-tubule region was
changed compared with control, now having a diffuse
rather than dotted distribution, as also reported earlier
(27). After insulin stimulation of denervated muscle, in
sarcolemma the increased PIP3 signal (Figs. 1 and 2) was
accompanied by an increased GLUT4-GFP translocation
compared with findings in control muscle (Fig. 6B, t ⫽ 30
min, arrows). Image quantification showed the increase in
GLUT4-GFP translocation in denervated muscle to be
2.3-fold above control (Fig. 6C). Furthermore, corresponding with the finding of an abolished PIP3 signal in the
T-tubules in denervated muscle, insulin stimulation did not
induce any visible GLUT4-GFP translocation, as no enDIABETES, VOL. 57, JANUARY 2008
hancement of the striated pattern in the T-tubule area
emerged (Fig. 6B). Image quantification also showed a
complete abolition of GLUT4-GFP translocation in the
T-tubules in denervated muscle compared with control
(Fig. 6D). Thus, in denervated insulin-resistant muscle
both insulin signaling and GLUT4 translocation are markedly impaired in the T-tubules while being increased at the
sarcolemma.
Image analysis in the high-fat fed mice showed that at
the sarcolemma the diminished PIP3 signal in response to
insulin was accompanied by a slightly weaker staining in
GLUT4-GFP transfected compared with normal muscle,
indicating reduced sarcolemmal translocation (Fig. 7B vs.
a). Quantification supported this observation showing a
reduction in GLUT4-GFP translocation to sarcolemma of
16% in high fat–fed compared with control mice (Fig. 7C).
Furthermore, corresponding with the decrease in PIP3
signal in the T-tubules (Fig. 4A and B), the insulin-induced
17
DENERVATION, DIET, AND INSULIN SIGNALING
FIG. 4. High-fat diet reduces insulin-mediated PIP3 production in the sarcolemma and T-tubules. A and B: t ⴝ 0 shows a confocal image of a basal
GFP-ARNO expressing quadriceps muscle fiber in situ in a living FVB mouse just before intravenous insulin injection. Images of GFP-ARNO were
obtained every 2 s after insulin injection in controls (A) and high fat–fed (B) mice. Numbers denote time points (min). Arrows indicate PIP3
production at sarcolemma (2 min) and locally in the T-tubule area (9 min). Bar ⴝ 20 ␮m. Images are representative of findings in four mice. For
quantification see Fig. 5.
GLUT4-GFP translocation was also reduced in fat-fed compared with control mice, as indicated by emergence of only
weak striations (Fig. 7A vs. B). Image quantification supported this observation showing a reduction in GLUT4GFP translocation to the T-tubules of 68% (Fig. 7D).
Glucose transport in detubulated muscle. The above
findings pointed at an essential role of impaired insulin
signaling and GLUT4 translocation in T-tubules for insulin
resistance. Therefore, in order to directly evaluate the
importance of the T-tubules in normal mouse muscle, we
measured the effect of detubulation on insulin-mediated
2-DG transport in incubated muscles. EDL muscles were
subjected to osmotic shock by which the T-tubules lose
their connection to the sarcolemma and, accordingly, have
no access to insulin and glucose from the extracellular
fluid (28 –30). Detubulation reduced basal glucose transport by 50% (0.04 ⫾ 0.006 vs. 0.08 ⫾ 0.02 ␮mol 䡠 g⫺1 䡠 5
min⫺1; n ⫽ 5– 6; P ⬍ 0.05) and completely abolished
the insulin-induced increase in glucose transport (to
0.04 ⫾ 0.06 vs. 0.18 ⫾ 0.03 ␮mol 䡠 g⫺1 䡠 5 min⫺1; n ⫽ 5– 6;
P ⬍ 0.05) (Fig. 8).
DISCUSSION
In the present study, the spatial and temporal distribution
of insulin signaling and GLUT4 translocation was, for the
first time, followed in insulin-resistant skeletal muscle in
18
situ in living animals. A major new finding is that, whether
elicited by muscle denervation or high-fat diet, insulin
resistance is associated with a marked reduction of insulin-mediated PIP3 production and GLUT4 translocation in
the T-tubules, while these variables are less diminished or
even increased at the sarcolemma. Emphasizing the role of
T-tubules relative to that of sarcolemma for glucose transport in muscle, insulin-mediated glucose transport was
completely abolished by osmotic disruption of T-tubules in
normal muscle (Fig. 8). We are the first to report changes
in the opposite direction of both PIP3 production and
GLUT4 translocation in T-tubules compared with sarcolemma, as seen in denervated muscle. The finding shows
that the defects in insulin action are markedly compartmentalized in insulin-resistant muscle and further supports that GLUT4 translocation is controlled by local
insulin signaling (9).
Electron microscopy studies have shown that the arrangement of T-tubules changes within 10 days of muscle
denervation (23). So, it might be speculated that reduced
accessibility of insulin to its receptors might explain the
reduced insulin signaling in T-tubules seen after denervation in the present study. However, the total T-tubule area
increases with denervation (23). Furthermore, in the
present study the dye sulforhodamine B, which has previously been used to locate T-tubules (9,24 –26), diffused
DIABETES, VOL. 57, JANUARY 2008
H.P.M.M. LAURITZEN AND ASSOCIATES
FIG. 5. Quantification of in vivo confocal recordings (Fig. 4) showing that insulin-stimulated PIP3 production is reduced primarily in the T-tubules
and, to a lesser extent, in sarcolemma in high fat–fed compared with control mice. Image quantification of GLUT4-GFP at the sarcolemma (A) and
in the T-tubules at 10-␮m (B), 20-␮m (C), and 30-␮m (D) distances from sarcolemma (see Fig. 2 legend). A–D: Means ⴞ SE, n ⴝ 4. B–D:
Incremental areas under the curve (ŒAUC), calculated from early nadir values to end of curve or to values below initial values, differed
significantly (P < 0.05, t test) between muscles from high fat–fed and control mice (B–D). E and F: Raw nonnormalized and nonaveraged
fluorescence for representative single ROIs at the sarcolemma (E) and in the T-tubule area 20 ␮m from sarcolemma (F). A–F: 䡺, control; F, 12-h
high-fat fed.
into the T-tubules of denervated muscle with no delay and
caused a striated pattern, as previously seen (9) in normal
muscle (Fig. 1). Reduced insulin signaling in the T-tubules
was also not due to reduced presence of insulin receptors
DIABETES, VOL. 57, JANUARY 2008
in the T-tubules (Fig. 3). Thus, we show, for the first time,
that plasma molecule access to T-tubules and insulin
receptor distribution are not affected in insulin-resistant
denervated muscle.
19
DENERVATION, DIET, AND INSULIN SIGNALING
FIG. 6. Denervation reduces insulin-stimulated GLUT4-GFP translocation in T-tubules. A and B: t ⴝ 0 shows confocal images of basal GLUT4-GFP
expressing quadriceps muscle fibers in situ in living mice just before intravenous insulin injection. Images of GLUT4-GFP were obtained every
2 s after insulin injection in control (A) and denervated (B) muscle and are shown for the time points indicated (min). Bar ⴝ 20 ␮m. ROIs drawn
along the sarcolemma surfaces and in four locations within the T-tubule area are shown at t ⴝ 10 min. Arrows indicate sarcolemma and T-tubule
areas illustrating emergence of a striated pattern of GLUT4-GFP localization with insulin stimulation in control but not in denervated muscle.
At 30 min of insulin stimulation, GLUT4-GFP staining of sarcolemma is higher in denervated compared with control muscle. Images are
representative of findings in four mice. C and D: Image quantification of GLUT4-GFP translocation to sarcolemma (C) and T-tubules (D) in
denervated and control muscle. Values are means ⴞSE, n ⴝ 4. *Significant difference (t test) between denervated and control muscle (P < 0.05).
E and F: Raw nonnormalized and nonaveraged fluorescence for representative single ROIs at the sarcolemma (E) and in the T-tubule (F) area.
C–F: 䡺 control; F, denervated.
In previous studies of the mechanism of denervationinduced insulin resistance, biochemical methods have
been applied on crude muscle homogenate. In line with
the present findings, it was shown that after 7–10 days of
20
denervation insulin receptor content was normal, whereas
insulin-stimulated tyrosin phosphorylation of the receptor
and of its downstream signaling molecule, insulin receptor
substrate-1, as well as the association of insulin receptor
DIABETES, VOL. 57, JANUARY 2008
H.P.M.M. LAURITZEN AND ASSOCIATES
FIG. 7. High-fat diet reduces insulin-stimulated GLUT4-GFP translocation in the T-tubules. A and B: t ⴝ 0 shows confocal images of basal
GLUT4-GFP expressing quadriceps muscle fibers in situ in living mice, just before intravenous insulin injection. Images of GLUT4-GFP were
obtained every 2 s after insulin injection in control (A) and high fat–fed (B) mice and are shown for the time points indicated (min). Bar ⴝ 20
␮m. Arrows indicate sarcolemma and T-tubule areas illustrating less intense striation upon insulin stimulation in high fat–fed compared with
control muscle. Images are representative of findings in four mice. C and D: Image quantification of GLUT4-GFP translocation to sarcolemma (C)
and T-tubules (D) in high fat–fed and control mice (see Fig. 6 legend). Values are means ⴞ SE, n ⴝ 4. *Significant difference between high fat–fed
and control mice (P < 0.05, t test). E and F: Raw nonnormalized and nonaveraged fluorescence for representative single ROIs at the sarcolemma
(E) and in the T-tubule (F) area. C–F: 䡺, control; F, high-fat fed.
substrate-1 with PI3-K, insulin receptor substrate-1–associated PI3-K activity, PI3-K activity, insulin-mediated protein kinase B/Akt activation, and total amount of GLUT4
mRNA and protein were diminished (6,10). However,
those studies were able to reveal neither the dynamics nor
the location of the changes. In contrast, the present study
has indicated that after a similar denervation protocol,
insulin signaling and GLUT4-GFP translocation are exclusively reduced in T-tubules and that in these, the wave-like
DIABETES, VOL. 57, JANUARY 2008
spreading of PIP3 production seen in normal muscle, as
well as the GLUT4-GFP translocation, are absent in their
entire length (Figs. 1, 2, and 6).
These findings indicate that after long-term denervation,
diminished insulin-mediated glucose transport in skeletal
muscle reflects impaired GLUT4 translocation to T-tubules
rather than to sarcolemma. However, because denervation
reduces the total endogenous GLUT4 content of muscle by
25–50% (6,31,32), at similar GLUT4-GFP expression the
21
DENERVATION, DIET, AND INSULIN SIGNALING
FIG. 8. Detubulation markedly reduces basal and insulin-mediated
2-DG uptake in incubated EDL muscles. Values are means ⴞSE, n ⴝ
5– 6. *Significant difference between detubulated and control muscle
(P < 0.05, two-way ANOVA). f, control; u, detubulated.
labeled fraction of GLUT4 (specific activity) will be higher
in denervated compared with control muscle. Accordingly,
in spite of enhanced translocation of GLUT4-GFP to
sarcolemma, after denervation, translocation of endogenous GLUT4 might nevertheless be reduced and so account for the diminished insulin-mediated glucose
transport. Still, this was hardly the case because the
increase in GLUT4-GFP translocation to sarcolemma in
denervated compared with control muscle was 2.3-fold,
while increases in specific activity calculated from reported decreases in endogenous GLU4 would be only 1.3to 2-fold, allowing for a 1.15- to 1.73-fold higher endogenous GLUT4 translocation to sarcolemma in denervated
muscle.
The redistributed amounts of both GFP-ARNO and
GLUT4-GFP in absolute terms also depend on the total
amount of the probes expressed in the studied cells.
However, this did not vary consistently between groups,
and, furthermore, any random variation was corrected for
by expressing fluorescence upon insulin stimulation relative to local basal values.
In unstimulated denervated muscle we found, in agreement with others (27), in the T-tubule region a diffuse
distribution of GLUT4-GFP rather than a dotted distribution as seen in control muscle (Fig. 6). At the level of light
microscopy resolution, it is not possible to distinguish
between GLUT4-GFP in vesicles in the juxtaposition of the
T-tubules and GLUT4-GFP within these membranes. So,
we cannot completely exclude that in denervated muscle
part of the diffusively located GLUT4-GFP is locally translocated to the T-tubule membranes. However, judged from
the insulin-induced PIP3 production, any GLUT4 translocation would be minimal. Because we and others find no
increased basal glucose uptake after long-term denervation, it is also not likely that GLUT4 within T-tubule
membranes was high in the basal state in denervated
muscle.
Our preparation is set up for imaging of muscle fibers in
the superficial layers of the quadriceps muscle. This part
of the quadriceps muscle consists of white type IIb fibers
(13). However, in accordance with previous findings (6,31–
33), we found no differences in changes in insulin sensitivity after denervation in red soleus muscle, consisting
predominantly of type 1 fibers, compared with EDL muscle, consisting predominantly of type 2 fibers (13). Accordingly, our findings in the superficial quadriceps would be
22
expected to apply, as well, to other types of muscle
(6,31–33).
Others have found that insulin-mediated glucose uptake
in muscle is diminished after only 24 h of denervation
(16,34). In the present study, PIP3 production and GLUT4
translocation were also analyzed after 24 h of denervation.
We found no changes compared with controls (data not
shown). This is in agreement with the fact that insulinstimulated receptor phosphorylation, biochemically determined PI3-K activity, and GLUT4 translocation determined
by subcellular fractionation have also been shown to be
preserved (16,34,35). So, apparently the insulin resistance
associated with short-term denervation must be attributed
to downregulation of the metabolic machinery (e.g., hexokinase).
In previous studies of the mechanism of high-fat diet–
induced insulin resistance, the insulin binding in muscle
was normal (36), a finding agreeing with the observation
that the number of insulin receptors was reduced neither
in sarcolemma nor in T-tubules in high-fat– compared with
standard diet–fed mice (Fig. 3). Based on biochemical
measurements, some have argued that defects in insulin
signaling in muscle are late events seen after 30 but not
after 8 weeks of high-fat diet (7). In contrast, other studies
have reported reduced insulin-stimulated PI3-K and Akt/
protein kinase B activities after only 4 weeks of high-fat
diet (5,37) and reduced Akt/protein kinase B signaling
after 7 weeks of high-fat diet (38). Our study, for the first
time, shows that 12 weeks of high-fat diet reduce PI3-K
activity in the entire length of the T-tubules and, to a lesser
extent, in sarcolemma (Figs. 4 and 5) and, furthermore,
that this compartmentalized reduction in insulin signaling
is closely coupled with reduced GLUT4 translocation (Fig.
7). Previous biochemical studies have found either normal
(4,5) or reduced (6,37,39) overall GLUT4 content in muscle
from high fat–fed animals, while GLUT4 translocation was
reduced (4,5,7). One study using subcellular fractionation
found completely abrogated insulin-stimulated GLUT4
translocation to both sarcolemma and T-tubules (38).
In studies with the isolated perfused rat hindquarter, 7
days of muscle denervation has been shown to diminish
insulin-stimulated glucose transport in the three types of
skeletal muscle more than streptozotocin-induced diabetes (6). Compatible with impairments of the same mechanisms in the two disease states, the effect of combined
denervation and diabetes was not more marked than that
of denervation alone (6). These findings are in accordance
with our findings on GLUT4 translocation and PI3-K
activity in quadriceps muscle from muscle-denervated or
high fat–fed mice. However, the effect of denervation on
insulin-mediated glucose transport in incubated EDL and
soleus muscles was not markedly higher than the effect of
high-fat feeding. EDL and soleus muscles were used
instead of quadriceps muscle because quadriceps muscle
cannot be easily isolated. It is possible that the more
moderate effect of denervation in the present experiments
on incubated muscle compared with the findings in the rat
hindquarter reflects that the effect of impaired T-tubule
function is blurred in incubated muscle because diffusion
of insulin and glucose into the T-tubules may be hindered
when muscle is not stretched.
In conclusion, the present study demonstrates new in
vivo evidence of a marked compartmentalization of insulin
signaling in skeletal muscle and that impaired insulin
signaling in T-tubules is an essential part of development
of insulin resistance. Moreover, the study has, for the first
DIABETES, VOL. 57, JANUARY 2008
H.P.M.M. LAURITZEN AND ASSOCIATES
time, shown that insulin signaling may be changed in
opposite directions in T-tubules compared with sarcolemma. Future studies must clarify the mechanism for the
latter finding and for the effects of denervation and high-fat
diet on insulin signaling in muscle. In this context, it may
be recalled that although the T-tubules extend from the
sarcolemma, the membrane composition of these two
cellular compartments vary markedly regarding cholesterol, phospholipids, proteins, receptors, and enzymes
(40,41).
ACKNOWLEDGMENTS
This work was supported by the Faculty of Health Sciences, University of Copenhagen; the Danish Diabetes
Association; the Weimann Foundation; the Novo Nordic
Research Foundation; the Beckett Foundation; and the
Danish National Research Foundation. H.A. was partly
supported by the National Natural Science Foundation of
China (grant no. 30270636).
We thank L. Kall for bioanalytical assistance and S.
Lohmann for engineering assistance.
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