Articles in PresS. Am J Physiol Endocrinol Metab (December 9, 2014). doi:10.1152/ajpendo.00466.2014
1
Fiber Type Effects on Contraction-stimulated Glucose Uptake and GLUT4 Abundance in
2
Single Fibers from Rat Skeletal Muscle
3 4
Carlos M. Castorena1, Edward B. Arias1, Naveen Sharma1, Jonathan S. Bogan2 and Gregory D.
5
Cartee1, 3, 4
6 7 8 9 10
1
Muscle Biology Laboratory, School of Kinesiology, University of Michigan, Ann Arbor, MI
2
Section of Endocrinology and Metabolism, Dept. of Internal Medicine, and Dept. of Cell
Biology, Yale University School of Medicine 3
Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI
4
Institute of Gerontology, University of Michigan, Ann Arbor, MI
11 12
Running title: Contraction-stimulated glucose uptake by single fibers
13 14
Correspondence:
15
Gregory D. Cartee, Ph.D.
16
University of Michigan, School of Kinesiology, Room 4745F
17
401 Washtenaw Avenue, Ann Arbor, MI 48109-2214
18
Phone: (734) 615-3458
19
Fax: (734) 936-1925
20
email: [email protected]
21
1 Copyright © 2014 by the American Physiological Society.
22 23
Abstract To fully understand skeletal muscle at the cellular level, it is essential to evaluate single
24
muscle fibers. Accordingly, the major goals of this study were to determine if there are fiber
25
type-related differences in single fibers from rat skeletal muscle for: 1) contraction-stimulated
26
glucose uptake and/or 2) the abundance of GLUT4 and other metabolically relevant proteins.
27
Paired epitrochlearis muscles isolated from Wistar rats were either electrically stimulated to
28
contract (E-Stim) or remained resting (No E-Stim). Single fibers isolated from muscles
29
incubated with [3H]-2-deoxy-d-glucose (2-DG) were used to determine fiber type (myosin heavy
30
chain, MHC, isoform protein expression), 2-DG uptake and abundance of metabolically relevant
31
proteins, including the GLUT4 glucose transporter. E-Stim, relative to No E-Stim, fibers had
32
greater (P < 0.05) 2-DG uptake for each of the isolated fiber types (MHC-IIa, MHC-IIax, MHC-
33
IIx, MHC-IIxb and MHC-IIb). However, 2-DG uptake for E-Stim fibers was not significantly
34
different among these five fiber types. GLUT4, TUG (tethering protein containing a UBX
35
domain for GLUT4), COX IV (cytochrome C oxidase IV) and filamin C protein levels were
36
significantly greater (P < 0.05) in MHC-IIa versus MHC-IIx, MHC-IIxb or MHC-IIb fibers.
37
TUG and COX IV in either MHC-IIax or MHC-IIx fibers exceeded values for MHC-IIxb or
38
MHC-IIb fibers. GLUT4 levels for MHC-IIax fibers exceeded MHC-IIxb fibers. GLUT4, COX
39
IV, filamin C and TUG abundance in single fibers were significantly (P < 0.05) correlated with
40
each other. Differences in GLUT4 abundance among the fiber types were not accompanied by
41
significant differences in contraction-stimulated glucose uptake.
42 43
Key Words: glucose transport; glucose transporter; exercise; myofiber; myosin heavy chain
2
44
Introduction
45
Skeletal muscle is a heterogeneous tissue, and the magnitude of the exercise-induced
46
increase in glucose uptake is not uniform across all muscles (29, 50). Although the diversity
47
among muscles in their glucose uptake during in vivo exercise is greatly influenced by the levels
48
of muscle recruitment and blood flow, the muscle’s intrinsic metabolic characteristics may also
49
modulate contraction-stimulated glucose uptake. Supporting this idea, Henriksen et al. (25)
50
evaluated four different isolated rat skeletal muscles with varying fiber type compositions and
51
found that the greatest contraction-stimulated glucose uptake was in the flexor digitorum brevis
52
[FDB; 92% type IIa fibers (1, 9)], and the lowest values were in the epitrochlearis [51% type IIb
53
fibers (12)]. These results suggest that the capacity for contraction-stimulated glucose uptake of
54
type IIa fibers exceeds that of type IIb fibers in rat muscles.
55
However, analysis of muscle tissue has limitations for elucidating differences among
56
fiber types at the cellular level because: 1) no rat muscles have been found to exclusively
57
express a single MHC isoform; 2) no rat muscle has been recognized to be predominantly
58
composed of MHC-IIx fibers; 3) whole tissue analysis cannot determine the glucose uptake of
59
hybrid fibers that express multiple MHC isoforms, and hybrid fibers are common in rat skeletal
60
muscle (7, 8, 42, 56); 4) multiple cell types (including vascular, neural, and adipose cells)
61
contribute to glucose uptake by skeletal muscle tissue; and 5) it cannot be assumed that every
62
functional difference between two muscles with disparate fiber type profiles is necessarily a
63
direct consequence of their fiber type differences.
64
We recently developed a novel method to determine both glucose uptake and fiber type in
65
a single rat skeletal muscle fiber, making it possible to compare glucose uptake by single fibers
66
of different fiber types from the same muscle. Insulin-stimulated glucose uptake was much
3
67
greater for single fibers expressing type IIa MHC versus fibers expressing IIb, IIx or IIxb MHC
68
(42). However, insulin and contractile activity use distinct mechanisms to increase glucose
69
uptake (10, 11, 15, 21, 23, 45, 46), and muscle fiber type may not have identical relationships
70
with glucose uptake stimulated by insulin versus contraction. Our overarching goal was to gain
71
insights at a cellular level on the relationships among muscle fiber type, contraction-stimulated
72
glucose uptake and the expression of key proteins implicated in the regulation of glucose uptake.
73
The first specific aim of this study was to measure contraction-stimulated glucose uptake in
74
single fibers of differing fiber types from epitrochlearis muscles that were electrically stimulated
75
to contract.
76
Rather than myosin heavy chain expression directly controlling glucose uptake capacity,
77
it seems more likely that fiber type composition is co-regulated with other proteins that regulate
78
glucose uptake. Contraction-stimulated glucose uptake is ultimately dependent on the GLUT4
79
glucose transporter, and earlier studies have demonstrated a relationship between fiber type
80
composition and muscle GLUT4 protein abundance in muscle tissue (12, 25, 31, 44, 48).
81
Furthermore, Henriksen et al. (25) and Brozinick et al. (6) reported that contraction-stimulated
82
glucose uptake by several muscles was positively related to their GLUT4 abundance. Our
83
second specific aim was to determine if GLUT4 abundance differed according to MHC isoform
84
expression in single fibers.
85
Based on analysis of multiple muscles with diverse fiber type profiles, we recently found
86
that the relative protein levels of the GLUT4 glucose transporter and TUG (tether, containing a
87
UBX domain, for GLUT4) were highly correlated to each other, and each was related to fiber
88
type composition in muscle tissue (12). Our final specific aim was to investigate protein co-
4
89
expression patterns in single fibers for GLUT4 and TUG, as well as several other proteins with
90
potential metabolic relevance.
91 92 93
Materials and Methods
94
Materials
95
The reagents and apparatus for sodium dodecyl sulfate polyacrylamide gel
96
electrophoresis (SDS-PAGE), non-fat dry milk (no. 170-6404XTU) and Coomassie Brilliant
97
Blue (no. 161-0436) were from Bio-Rad (Hercules, CA). Bicinchoninic acid protein assay
98
reagent (no. 23227) was from Pierce Biotechnology (Rockford, IL). Anti-cytochrome C oxidase
99
IV (COX IV; no. 4850) and anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH; no.
100
2118) were from Cell Signaling Technologies (Danvers, MA). LuminataTM Forte Western
101
Horseradish Peroxidase Substrate (no. WBLUF0100) was from Millipore (Billerica, MA). Anti-
102
filamin C (no. sc-48496) was from Santa Cruz Biotechnology (Dallas, TX). Anti-GLUT4 and
103
anti-TUG antibodies (provided by JSB), were raised in rabbits using peptide immunogens
104
corresponding to the C-termini of these proteins, as previously described (5, 57). Collagenase
105
Type 2 (305 units/mg) was purchased from Worthington Biochemical (no. LS004177,
106
Lakewood, NJ).
107 108 109
Animal Treatment Procedures for animal care were approved by the University of Michigan Committee on
110
Use and Care of Animals. Male Wistar rats (250-300g; Harlan, Indianapolis, IN) were provided
111
with standard rodent chow and water ad libitum. Rats were fasted the night before the 5
112
experiment at ~1900. On the morning after the overnight fast (between 0700 and 0900), rats
113
were given an intraperitoneal injection of sodium pentobarbital (50 mg/kg wt). While rats were
114
under deep anesthesia, both epitrochlearis muscles were dissected out, rapidly rinsed in warm
115
(35°C) Krebs-Henseleit buffer (KHB) and transferred to vials for subsequent ex vivo incubations.
116 117 118
Muscle Incubations and Electrical Stimulation Isolated muscles were incubated in vials containing the appropriate media that was
119
gassed from above (95% O2/5% CO2) in a water bath (35°C for Steps 1-3 and 5; and Step 4 was
120
on ice) throughout all of the incubation steps. For Step 1 (30 min), both epitrochlearis muscles
121
from each rat were placed in vials containing 2 mL of Media 1 [KHB supplemented with 8mM
122
glucose]. For Step 2 (10 min), one of the paired muscles was suspended in a 5-ml bath
123
containing two platinum electrodes with one end of the muscle attached to a glass rod and the
124
other end attached to a force transducer (Radnoti, Litchfield, CT). The mounted muscle was
125
incubated in Media 1 and electrically stimulated to contract [E-Stim: 0.1 ms twitch, 100 Hz train
126
for 10 s, 1 train/min for 10 min; Grass S48 Stimulator; Grass Instruments, Quincy, MA (19, 22)].
127
The contralateral muscle, serving as a rested/non-electrically stimulated control (No E-Stim),
128
was transferred to a vial containing Media 1. For Step 3 (30 min), each muscle was transferred
129
to a vial containing 2 ml of Step 3 Media [KHB supplemented with 0.1% BSA, 0.1 mM 2-
130
deoxyglucose (2-DG; final specific activity of 13.5 mCi/mmol 3H-2-DG) and 9.9 mM mannitol].
131
For Step 4 (15 min), muscles underwent 3 washes (5 min/wash with shaking at 115 revolutions
132
per min) in ice-cold Wash Media [Ca2+-free KHB supplemented with 0.1% BSA and 8mM
133
glucose, to clear the extracellular space of 3H-2-DG (55)]. For Step 5 (60-70 min), muscles were
134
incubated in vials containing Collagenase Media [Wash Media + 1.5% Type II collagenase] for
6
135
enzymatic digestion of muscle collagen (collagenase-treated muscles are hereafter referred to as
136
fiber bundles).
137 138 139
Isolation and Processing of Single Fibers for Glucose Uptake and Immunoblotting After incubation Step 5, fiber bundles were removed from Collagenase Media, washed
140
with Wash Media at room temperature, and placed in a petri-dish containing Isolation Media
141
(Wash Media supplemented with 0.25% Trypan Blue, TB) (42). Under a dissecting microscope
142
(Leica EZ4D; Buffalo Grove, IL), intact single fibers were gently teased away from the fiber
143
bundle using forceps. Only fibers that were not permeable to TB (TB-permeable fibers were
144
very rare) were isolated. Each fiber was imaged using a camera-enabled microscope with Leica
145
Application Suite EZ software after isolation. Fiber dimensions were measured using Image J
146
software (National Institutes of Health, NIH). Width (mean value for width measured at 3
147
locations on each fiber: near the fiber midpoint and approximately halfway between the
148
midpoint and each end of the fiber) and length of each fiber were be used to calculate an
149
estimated volume (V
150
length). After imaging, each fiber was transferred by pipette with 10µl of Isolation Media to a
151
micro-centrifuge tube containing 10µl of lysis buffer (1 ml/muscle; 20 mM Tris-HCL, 150 mM
152
NaCl, 1% Triton X-100, 1 mM Na3VO4, 1 mM EDTA, 1 mM EGTA, 2.5 mM NaPP, 1 mM β-
153
glycerophosphate, 1 µg/ml leupeptin, 1 mM PMSF), for a total volume of 20 µl. Lysis buffer
154
(30µl) and 2X Laemmli buffer (50µl) were added to each isolated fiber tube and also to each
155
fiber’s corresponding Background Media tube. The tubes were then vortexed and heated to 95-
156
100o C for 5 min. Samples were then cooled and stored at -20o C until 2-DG uptake and MHC
157
isoform expression were determined.
πr l; r = radius as determined by half of the width measurement, l =
7
158 159 160
Fiber Bundle Homogenization and 2-DG Uptake After isolation of single fibers (~20-40 fibers per fiber bundle), the remainder of each
161
fiber bundle was homogenized in 1ml ice-cold lysis buffer using a glass-on-glass pestle and
162
grinding tubes (Kontes, Vineland, NJ) chilled on ice. Aliquots of fiber bundle homogenates
163
were added to vials containing scintillation cocktail (Research Products International, Mount
164
Prospect, IL) for scintillation counting, and 2-DG accumulation was determined (42). A portion
165
of the homogenate was used to determine protein concentration according to the manufacturer’s
166
protocol (Pierce Biotechnology no. 23227), and 2-DG uptake was normalized to protein content
167
(µmol • µg-1 protein for fiber bundles) (42).
168 169
Single Fiber 2-DG Uptake
170
An aliquot of each lysed single fiber were pipetted into separate vials with each
171
containing 10 ml of scintillation cocktail. The single fiber counts were then used to determine
172
the single fiber 3H-2DG accumulation which was calculated and normalized to the calculated
173
fiber volume and expressed as nmol • µl-1 (42).
174 175 176
Myosin Heavy Chain (MHC) Isoform Expression MHC isoforms from aliquots of single fiber lysates were separated and identified by
177
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) essentially as
178
previously described (41, 42, 54), with the following minor modifications made to the resolving
179
gel (final reagent concentrations): 6.5% acrylamide-bis (50:1), 30% glycerol gel, 210 mM Tris-
180
HCl (pH 7.4), 105 mM glycine, 0.4% SDS, 19% H2O, 0.1% ammonium persulfate, 0.05%
8
181
TEMED. Samples and a MHC isoform standard (6 μg protein of a 3:2 mixture of homogenized
182
rat soleus and extensor digitorum longus muscles containing all four MHC isoforms: I, IIa, IIb,
183
IIx) were run at a constant voltage (40 V) for 1 h (during electrophoresis that was performed in a
184
refrigerator cooled to 4oC, the gel box was placed in a secondary container that was packed with
185
ice) with continuous mixing by a magnetic stir bar inside the electrophoresis apparatus (Mini-
186
PROTEAN® Tetra cell no. 165-8004, Hercules, CA). After 1 h, the power supply was switched
187
from constant voltage mode to constant current mode, with the current setting at the end of the
188
first hour of electrophoresis maintained for the subsequent 23-25 h (with the gel box packed on
189
ice in a refrigerator at 4oC). Gels were subsequently removed and stained with Coomassie
190
Brilliant Blue overnight at room temperature while gently rotating on an orbital shaker. The gels
191
were then destained in 20% methanol and 10% acetic acid solution for 4-6 h while rotating
192
(destaining solution was replaced with fresh solution every 45-60 min). MHC isoform
193
expression was determined by comparing the migration of MHC protein bands from each fiber to
194
a standard prepared from rat soleus and extensor digitorum longus that included all MHC
195
isoforms.
196 197
Immunoblotting
198
Aliquots of single fiber lysates were to heated to 90˚C for 5-10 min, separated via SDS-
199
PAGE using 4-20% TGXTM gradient gels (Bio-Rad no. 456-1095), and then electrophoretically
200
transferred to nitrocellulose membranes. After transfer, gels were stained in Coomassie Brilliant
201
Blue overnight and then destained the following morning as described above. The Coomassie-
202
stained MHC bands in the gels were quantified by densitometry with these post-transfer MHC
203
band densities served as the loading controls for the subsequently immunoblotted proteins
9
204
(Alpha Innotech, San Leandro, CA) (32, 41, 42). Membranes were then rinsed with Tris-
205
buffered saline (TBS: 140 mM NaCl and 20 mM Tris base, pH 7.6), blocked with 5% nonfat dry
206
milk in TBS plus tween (TBST: 0.1% Tween-20) for 1 h at room temperature, washed 3 x 5 min
207
at room temperature and incubated with the appropriate primary antibody diluted 1:1000 with
208
5% BSA-TBST (anti-TUG was the only exception; diluted 1:1000 with 5% nonfat dry milk-
209
TBST) and rotated on orbital shaker overnight at 4°C. On the following morning membranes
210
were washed 3 x 5 min with TBST, then incubated with the appropriate IgG horseradish
211
peroxidase (HRP) conjugated secondary antibody (1:20,000 in 5% milk-TBST) for 3 h at room
212
temperature. Membranes were then washed again 3 x 5 min with TBST, 3 x 5 min TBS and
213
subjected to enhanced chemiluminescence to visualize protein bands. Protein bands from the
214
membranes were quantified by densitometry (Alpha Innotech, San Leandro, CA). The
215
individual values for samples were normalized to the mean value for all of the samples on the
216
blot, and then divided by the loading control (post-transfer MHC density determined for the
217
respective sample on Coomassie stained gels) (32).
218 219 220
Statistics A two-tailed t-test was used to compare the No E-Stim and E-Stim groups for glucose
221
uptake by muscle bundles. One-way ANOVA was used to determine the effect of fiber type (as
222
determined by MHC expression) on protein abundance, and the Bonferroni correction was used
223
to identify the source of significant variance for data when appropriate. Two-way ANOVA with
224
Bonferroni post-hoc analysis was used to determine the effects of fiber type and contraction on
225
glucose uptake. Spearman correlation analysis was used to assess the relationships between
226
abundance of each pair of the proteins studied. Data that were not normally distributed were
10
227
mathematically transformed to achieve normality prior to running the appropriate statistical
228
analysis using Sigma Plot version 11.0 (San Rafael, CA). Data were expressed as means ± SE.
229
A P-value < 0.05 was considered statistically significant.
230 231
Results
232
MHC Isoforms
233
Single muscle fibers isolated from the epitrochlearis muscle expressed five distinct fiber
234
types: MHC-IIa, MHC-IIax, MHC-IIx, MHC-IIxb and MHC-IIb (Figure 1). The respective
235
percentages of total isolated single fibers from these fiber types were: MHC-IIxb (~44%),
236
MHC-IIb (~28%), MHC-IIx (~17%), MHC-IIa (~6%) and MHC-IIax (~5%). Consistent with
237
earlier research using the collagenase method to isolate single fibers from rat epitrochlearis
238
muscles (41, 42), no MHC-I fibers were found among any of the single fibers that were isolated
239
in the current study.
240 241 242
2-DG Uptake E-Stim fiber bundles versus No E-Stim fiber bundles had significantly greater (P < 0.01)
243
contraction-stimulated 2-DG uptake (Figure 2). For 2-DG uptake of single fibers (Figure 3),
244
there were significant main effects of E-stim (P < 0.01) and fiber type (P < 0.01). Within the No
245
E-Stim group, 2-DG uptake was significantly greater (P < 0.01) for MHC-IIa fibers versus
246
MHC-IIx and MHC-IIxb fibers. For each fiber type, 2-DG uptake was significantly greater (P
1
Fiber Type Effects on Contraction-stimulated Glucose Uptake and GLUT4 Abundance in
2
Single Fibers from Rat Skeletal Muscle
3 4
Carlos M. Castorena1, Edward B. Arias1, Naveen Sharma1, Jonathan S. Bogan2 and Gregory D.
5
Cartee1, 3, 4
6 7 8 9 10
1
Muscle Biology Laboratory, School of Kinesiology, University of Michigan, Ann Arbor, MI
2
Section of Endocrinology and Metabolism, Dept. of Internal Medicine, and Dept. of Cell
Biology, Yale University School of Medicine 3
Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI
4
Institute of Gerontology, University of Michigan, Ann Arbor, MI
11 12
Running title: Contraction-stimulated glucose uptake by single fibers
13 14
Correspondence:
15
Gregory D. Cartee, Ph.D.
16
University of Michigan, School of Kinesiology, Room 4745F
17
401 Washtenaw Avenue, Ann Arbor, MI 48109-2214
18
Phone: (734) 615-3458
19
Fax: (734) 936-1925
20
email: [email protected]
21
1 Copyright © 2014 by the American Physiological Society.
22 23
Abstract To fully understand skeletal muscle at the cellular level, it is essential to evaluate single
24
muscle fibers. Accordingly, the major goals of this study were to determine if there are fiber
25
type-related differences in single fibers from rat skeletal muscle for: 1) contraction-stimulated
26
glucose uptake and/or 2) the abundance of GLUT4 and other metabolically relevant proteins.
27
Paired epitrochlearis muscles isolated from Wistar rats were either electrically stimulated to
28
contract (E-Stim) or remained resting (No E-Stim). Single fibers isolated from muscles
29
incubated with [3H]-2-deoxy-d-glucose (2-DG) were used to determine fiber type (myosin heavy
30
chain, MHC, isoform protein expression), 2-DG uptake and abundance of metabolically relevant
31
proteins, including the GLUT4 glucose transporter. E-Stim, relative to No E-Stim, fibers had
32
greater (P < 0.05) 2-DG uptake for each of the isolated fiber types (MHC-IIa, MHC-IIax, MHC-
33
IIx, MHC-IIxb and MHC-IIb). However, 2-DG uptake for E-Stim fibers was not significantly
34
different among these five fiber types. GLUT4, TUG (tethering protein containing a UBX
35
domain for GLUT4), COX IV (cytochrome C oxidase IV) and filamin C protein levels were
36
significantly greater (P < 0.05) in MHC-IIa versus MHC-IIx, MHC-IIxb or MHC-IIb fibers.
37
TUG and COX IV in either MHC-IIax or MHC-IIx fibers exceeded values for MHC-IIxb or
38
MHC-IIb fibers. GLUT4 levels for MHC-IIax fibers exceeded MHC-IIxb fibers. GLUT4, COX
39
IV, filamin C and TUG abundance in single fibers were significantly (P < 0.05) correlated with
40
each other. Differences in GLUT4 abundance among the fiber types were not accompanied by
41
significant differences in contraction-stimulated glucose uptake.
42 43
Key Words: glucose transport; glucose transporter; exercise; myofiber; myosin heavy chain
2
44
Introduction
45
Skeletal muscle is a heterogeneous tissue, and the magnitude of the exercise-induced
46
increase in glucose uptake is not uniform across all muscles (29, 50). Although the diversity
47
among muscles in their glucose uptake during in vivo exercise is greatly influenced by the levels
48
of muscle recruitment and blood flow, the muscle’s intrinsic metabolic characteristics may also
49
modulate contraction-stimulated glucose uptake. Supporting this idea, Henriksen et al. (25)
50
evaluated four different isolated rat skeletal muscles with varying fiber type compositions and
51
found that the greatest contraction-stimulated glucose uptake was in the flexor digitorum brevis
52
[FDB; 92% type IIa fibers (1, 9)], and the lowest values were in the epitrochlearis [51% type IIb
53
fibers (12)]. These results suggest that the capacity for contraction-stimulated glucose uptake of
54
type IIa fibers exceeds that of type IIb fibers in rat muscles.
55
However, analysis of muscle tissue has limitations for elucidating differences among
56
fiber types at the cellular level because: 1) no rat muscles have been found to exclusively
57
express a single MHC isoform; 2) no rat muscle has been recognized to be predominantly
58
composed of MHC-IIx fibers; 3) whole tissue analysis cannot determine the glucose uptake of
59
hybrid fibers that express multiple MHC isoforms, and hybrid fibers are common in rat skeletal
60
muscle (7, 8, 42, 56); 4) multiple cell types (including vascular, neural, and adipose cells)
61
contribute to glucose uptake by skeletal muscle tissue; and 5) it cannot be assumed that every
62
functional difference between two muscles with disparate fiber type profiles is necessarily a
63
direct consequence of their fiber type differences.
64
We recently developed a novel method to determine both glucose uptake and fiber type in
65
a single rat skeletal muscle fiber, making it possible to compare glucose uptake by single fibers
66
of different fiber types from the same muscle. Insulin-stimulated glucose uptake was much
3
67
greater for single fibers expressing type IIa MHC versus fibers expressing IIb, IIx or IIxb MHC
68
(42). However, insulin and contractile activity use distinct mechanisms to increase glucose
69
uptake (10, 11, 15, 21, 23, 45, 46), and muscle fiber type may not have identical relationships
70
with glucose uptake stimulated by insulin versus contraction. Our overarching goal was to gain
71
insights at a cellular level on the relationships among muscle fiber type, contraction-stimulated
72
glucose uptake and the expression of key proteins implicated in the regulation of glucose uptake.
73
The first specific aim of this study was to measure contraction-stimulated glucose uptake in
74
single fibers of differing fiber types from epitrochlearis muscles that were electrically stimulated
75
to contract.
76
Rather than myosin heavy chain expression directly controlling glucose uptake capacity,
77
it seems more likely that fiber type composition is co-regulated with other proteins that regulate
78
glucose uptake. Contraction-stimulated glucose uptake is ultimately dependent on the GLUT4
79
glucose transporter, and earlier studies have demonstrated a relationship between fiber type
80
composition and muscle GLUT4 protein abundance in muscle tissue (12, 25, 31, 44, 48).
81
Furthermore, Henriksen et al. (25) and Brozinick et al. (6) reported that contraction-stimulated
82
glucose uptake by several muscles was positively related to their GLUT4 abundance. Our
83
second specific aim was to determine if GLUT4 abundance differed according to MHC isoform
84
expression in single fibers.
85
Based on analysis of multiple muscles with diverse fiber type profiles, we recently found
86
that the relative protein levels of the GLUT4 glucose transporter and TUG (tether, containing a
87
UBX domain, for GLUT4) were highly correlated to each other, and each was related to fiber
88
type composition in muscle tissue (12). Our final specific aim was to investigate protein co-
4
89
expression patterns in single fibers for GLUT4 and TUG, as well as several other proteins with
90
potential metabolic relevance.
91 92 93
Materials and Methods
94
Materials
95
The reagents and apparatus for sodium dodecyl sulfate polyacrylamide gel
96
electrophoresis (SDS-PAGE), non-fat dry milk (no. 170-6404XTU) and Coomassie Brilliant
97
Blue (no. 161-0436) were from Bio-Rad (Hercules, CA). Bicinchoninic acid protein assay
98
reagent (no. 23227) was from Pierce Biotechnology (Rockford, IL). Anti-cytochrome C oxidase
99
IV (COX IV; no. 4850) and anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH; no.
100
2118) were from Cell Signaling Technologies (Danvers, MA). LuminataTM Forte Western
101
Horseradish Peroxidase Substrate (no. WBLUF0100) was from Millipore (Billerica, MA). Anti-
102
filamin C (no. sc-48496) was from Santa Cruz Biotechnology (Dallas, TX). Anti-GLUT4 and
103
anti-TUG antibodies (provided by JSB), were raised in rabbits using peptide immunogens
104
corresponding to the C-termini of these proteins, as previously described (5, 57). Collagenase
105
Type 2 (305 units/mg) was purchased from Worthington Biochemical (no. LS004177,
106
Lakewood, NJ).
107 108 109
Animal Treatment Procedures for animal care were approved by the University of Michigan Committee on
110
Use and Care of Animals. Male Wistar rats (250-300g; Harlan, Indianapolis, IN) were provided
111
with standard rodent chow and water ad libitum. Rats were fasted the night before the 5
112
experiment at ~1900. On the morning after the overnight fast (between 0700 and 0900), rats
113
were given an intraperitoneal injection of sodium pentobarbital (50 mg/kg wt). While rats were
114
under deep anesthesia, both epitrochlearis muscles were dissected out, rapidly rinsed in warm
115
(35°C) Krebs-Henseleit buffer (KHB) and transferred to vials for subsequent ex vivo incubations.
116 117 118
Muscle Incubations and Electrical Stimulation Isolated muscles were incubated in vials containing the appropriate media that was
119
gassed from above (95% O2/5% CO2) in a water bath (35°C for Steps 1-3 and 5; and Step 4 was
120
on ice) throughout all of the incubation steps. For Step 1 (30 min), both epitrochlearis muscles
121
from each rat were placed in vials containing 2 mL of Media 1 [KHB supplemented with 8mM
122
glucose]. For Step 2 (10 min), one of the paired muscles was suspended in a 5-ml bath
123
containing two platinum electrodes with one end of the muscle attached to a glass rod and the
124
other end attached to a force transducer (Radnoti, Litchfield, CT). The mounted muscle was
125
incubated in Media 1 and electrically stimulated to contract [E-Stim: 0.1 ms twitch, 100 Hz train
126
for 10 s, 1 train/min for 10 min; Grass S48 Stimulator; Grass Instruments, Quincy, MA (19, 22)].
127
The contralateral muscle, serving as a rested/non-electrically stimulated control (No E-Stim),
128
was transferred to a vial containing Media 1. For Step 3 (30 min), each muscle was transferred
129
to a vial containing 2 ml of Step 3 Media [KHB supplemented with 0.1% BSA, 0.1 mM 2-
130
deoxyglucose (2-DG; final specific activity of 13.5 mCi/mmol 3H-2-DG) and 9.9 mM mannitol].
131
For Step 4 (15 min), muscles underwent 3 washes (5 min/wash with shaking at 115 revolutions
132
per min) in ice-cold Wash Media [Ca2+-free KHB supplemented with 0.1% BSA and 8mM
133
glucose, to clear the extracellular space of 3H-2-DG (55)]. For Step 5 (60-70 min), muscles were
134
incubated in vials containing Collagenase Media [Wash Media + 1.5% Type II collagenase] for
6
135
enzymatic digestion of muscle collagen (collagenase-treated muscles are hereafter referred to as
136
fiber bundles).
137 138 139
Isolation and Processing of Single Fibers for Glucose Uptake and Immunoblotting After incubation Step 5, fiber bundles were removed from Collagenase Media, washed
140
with Wash Media at room temperature, and placed in a petri-dish containing Isolation Media
141
(Wash Media supplemented with 0.25% Trypan Blue, TB) (42). Under a dissecting microscope
142
(Leica EZ4D; Buffalo Grove, IL), intact single fibers were gently teased away from the fiber
143
bundle using forceps. Only fibers that were not permeable to TB (TB-permeable fibers were
144
very rare) were isolated. Each fiber was imaged using a camera-enabled microscope with Leica
145
Application Suite EZ software after isolation. Fiber dimensions were measured using Image J
146
software (National Institutes of Health, NIH). Width (mean value for width measured at 3
147
locations on each fiber: near the fiber midpoint and approximately halfway between the
148
midpoint and each end of the fiber) and length of each fiber were be used to calculate an
149
estimated volume (V
150
length). After imaging, each fiber was transferred by pipette with 10µl of Isolation Media to a
151
micro-centrifuge tube containing 10µl of lysis buffer (1 ml/muscle; 20 mM Tris-HCL, 150 mM
152
NaCl, 1% Triton X-100, 1 mM Na3VO4, 1 mM EDTA, 1 mM EGTA, 2.5 mM NaPP, 1 mM β-
153
glycerophosphate, 1 µg/ml leupeptin, 1 mM PMSF), for a total volume of 20 µl. Lysis buffer
154
(30µl) and 2X Laemmli buffer (50µl) were added to each isolated fiber tube and also to each
155
fiber’s corresponding Background Media tube. The tubes were then vortexed and heated to 95-
156
100o C for 5 min. Samples were then cooled and stored at -20o C until 2-DG uptake and MHC
157
isoform expression were determined.
πr l; r = radius as determined by half of the width measurement, l =
7
158 159 160
Fiber Bundle Homogenization and 2-DG Uptake After isolation of single fibers (~20-40 fibers per fiber bundle), the remainder of each
161
fiber bundle was homogenized in 1ml ice-cold lysis buffer using a glass-on-glass pestle and
162
grinding tubes (Kontes, Vineland, NJ) chilled on ice. Aliquots of fiber bundle homogenates
163
were added to vials containing scintillation cocktail (Research Products International, Mount
164
Prospect, IL) for scintillation counting, and 2-DG accumulation was determined (42). A portion
165
of the homogenate was used to determine protein concentration according to the manufacturer’s
166
protocol (Pierce Biotechnology no. 23227), and 2-DG uptake was normalized to protein content
167
(µmol • µg-1 protein for fiber bundles) (42).
168 169
Single Fiber 2-DG Uptake
170
An aliquot of each lysed single fiber were pipetted into separate vials with each
171
containing 10 ml of scintillation cocktail. The single fiber counts were then used to determine
172
the single fiber 3H-2DG accumulation which was calculated and normalized to the calculated
173
fiber volume and expressed as nmol • µl-1 (42).
174 175 176
Myosin Heavy Chain (MHC) Isoform Expression MHC isoforms from aliquots of single fiber lysates were separated and identified by
177
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) essentially as
178
previously described (41, 42, 54), with the following minor modifications made to the resolving
179
gel (final reagent concentrations): 6.5% acrylamide-bis (50:1), 30% glycerol gel, 210 mM Tris-
180
HCl (pH 7.4), 105 mM glycine, 0.4% SDS, 19% H2O, 0.1% ammonium persulfate, 0.05%
8
181
TEMED. Samples and a MHC isoform standard (6 μg protein of a 3:2 mixture of homogenized
182
rat soleus and extensor digitorum longus muscles containing all four MHC isoforms: I, IIa, IIb,
183
IIx) were run at a constant voltage (40 V) for 1 h (during electrophoresis that was performed in a
184
refrigerator cooled to 4oC, the gel box was placed in a secondary container that was packed with
185
ice) with continuous mixing by a magnetic stir bar inside the electrophoresis apparatus (Mini-
186
PROTEAN® Tetra cell no. 165-8004, Hercules, CA). After 1 h, the power supply was switched
187
from constant voltage mode to constant current mode, with the current setting at the end of the
188
first hour of electrophoresis maintained for the subsequent 23-25 h (with the gel box packed on
189
ice in a refrigerator at 4oC). Gels were subsequently removed and stained with Coomassie
190
Brilliant Blue overnight at room temperature while gently rotating on an orbital shaker. The gels
191
were then destained in 20% methanol and 10% acetic acid solution for 4-6 h while rotating
192
(destaining solution was replaced with fresh solution every 45-60 min). MHC isoform
193
expression was determined by comparing the migration of MHC protein bands from each fiber to
194
a standard prepared from rat soleus and extensor digitorum longus that included all MHC
195
isoforms.
196 197
Immunoblotting
198
Aliquots of single fiber lysates were to heated to 90˚C for 5-10 min, separated via SDS-
199
PAGE using 4-20% TGXTM gradient gels (Bio-Rad no. 456-1095), and then electrophoretically
200
transferred to nitrocellulose membranes. After transfer, gels were stained in Coomassie Brilliant
201
Blue overnight and then destained the following morning as described above. The Coomassie-
202
stained MHC bands in the gels were quantified by densitometry with these post-transfer MHC
203
band densities served as the loading controls for the subsequently immunoblotted proteins
9
204
(Alpha Innotech, San Leandro, CA) (32, 41, 42). Membranes were then rinsed with Tris-
205
buffered saline (TBS: 140 mM NaCl and 20 mM Tris base, pH 7.6), blocked with 5% nonfat dry
206
milk in TBS plus tween (TBST: 0.1% Tween-20) for 1 h at room temperature, washed 3 x 5 min
207
at room temperature and incubated with the appropriate primary antibody diluted 1:1000 with
208
5% BSA-TBST (anti-TUG was the only exception; diluted 1:1000 with 5% nonfat dry milk-
209
TBST) and rotated on orbital shaker overnight at 4°C. On the following morning membranes
210
were washed 3 x 5 min with TBST, then incubated with the appropriate IgG horseradish
211
peroxidase (HRP) conjugated secondary antibody (1:20,000 in 5% milk-TBST) for 3 h at room
212
temperature. Membranes were then washed again 3 x 5 min with TBST, 3 x 5 min TBS and
213
subjected to enhanced chemiluminescence to visualize protein bands. Protein bands from the
214
membranes were quantified by densitometry (Alpha Innotech, San Leandro, CA). The
215
individual values for samples were normalized to the mean value for all of the samples on the
216
blot, and then divided by the loading control (post-transfer MHC density determined for the
217
respective sample on Coomassie stained gels) (32).
218 219 220
Statistics A two-tailed t-test was used to compare the No E-Stim and E-Stim groups for glucose
221
uptake by muscle bundles. One-way ANOVA was used to determine the effect of fiber type (as
222
determined by MHC expression) on protein abundance, and the Bonferroni correction was used
223
to identify the source of significant variance for data when appropriate. Two-way ANOVA with
224
Bonferroni post-hoc analysis was used to determine the effects of fiber type and contraction on
225
glucose uptake. Spearman correlation analysis was used to assess the relationships between
226
abundance of each pair of the proteins studied. Data that were not normally distributed were
10
227
mathematically transformed to achieve normality prior to running the appropriate statistical
228
analysis using Sigma Plot version 11.0 (San Rafael, CA). Data were expressed as means ± SE.
229
A P-value < 0.05 was considered statistically significant.
230 231
Results
232
MHC Isoforms
233
Single muscle fibers isolated from the epitrochlearis muscle expressed five distinct fiber
234
types: MHC-IIa, MHC-IIax, MHC-IIx, MHC-IIxb and MHC-IIb (Figure 1). The respective
235
percentages of total isolated single fibers from these fiber types were: MHC-IIxb (~44%),
236
MHC-IIb (~28%), MHC-IIx (~17%), MHC-IIa (~6%) and MHC-IIax (~5%). Consistent with
237
earlier research using the collagenase method to isolate single fibers from rat epitrochlearis
238
muscles (41, 42), no MHC-I fibers were found among any of the single fibers that were isolated
239
in the current study.
240 241 242
2-DG Uptake E-Stim fiber bundles versus No E-Stim fiber bundles had significantly greater (P < 0.01)
243
contraction-stimulated 2-DG uptake (Figure 2). For 2-DG uptake of single fibers (Figure 3),
244
there were significant main effects of E-stim (P < 0.01) and fiber type (P < 0.01). Within the No
245
E-Stim group, 2-DG uptake was significantly greater (P < 0.01) for MHC-IIa fibers versus
246
MHC-IIx and MHC-IIxb fibers. For each fiber type, 2-DG uptake was significantly greater (P
