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J. Biol. Chem., Vol. 275, Issue 29, 22381-22386, July 21, 2000
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From the Departments of
Received for publication, March 9, 2000, and in revised form, March 30, 2000
Glut1 transgenic mice were bred with transgenic
mice that overexpress hexokinase II in skeletal muscle in order to
determine whether whole-body glucose disposal could be further
augmented in mice overexpressing glucose transporters. Overexpression
of hexokinase alone in skeletal muscle had no effect on glucose
transport or metabolism in isolated muscles, nor did it alter blood
glucose levels or the rate of whole-body glucose disposal. Expression of the hexokinase transgene in the context of the Glut1 transgenic background did not alter glucose transport in isolated muscles but did
cause additional increases in steady-state glucose 6-phosphate (3.2-fold) and glycogen (7.5-fold) levels compared with muscles that
overexpress the Glut1 transporter alone. Surprisingly, however, these
increases were not accompanied by a change in basal or
insulin-stimulated whole-body glucose disposal in the doubly transgenic
mice compared with Glut1 transgenic mice, probably due to an inhibition
of de novo glycogen synthesis as a result of the high
levels of steady-state glycogen in the muscles of doubly transgenic
mice (430 µmol/g versus 10 µmol/g in wild-type mice).
We conclude that the hexokinase gene may not be a good target for
therapies designed to counteract insulin resistance or hyperglycemia.
Transgenic mice that overexpress the Glut1 glucose transporter in
skeletal muscle have been extensively characterized. Muscles isolated
from these mice exhibit a dramatic increase in basal glucose transport
and basal glucose metabolism (1, 2). The Glut1-overexpressing mice are
hypoglycemic relative to non-transgenic littermates and demonstrate an
elevation in whole-body glucose disposal under both basal and
euglycemic/hyperinsulinemic clamp conditions (1, 3). These observations
demonstrate that transport is rate-limiting for muscle glucose
metabolism and for whole-body glucose disposal in normal mice, a
finding that is consistent with a large body of additional experimental
data that has accumulated over the past several decades (for a review,
see Ref. 4).
Surprisingly, however, muscles from the Glut1-overexpressing mice do
not exhibit further increases in glucose transport after treatments
that augment transport in normal muscles (insulin, hypoxia,
electrically induced contractions) (5), despite the fact that Glut4
translocates to the sarcolemma and transverse tubules normally in the
transgenic muscles (6). These data suggest that the intrinsic activity
of Glut4 can be subject to regulation in the plasma membrane domains of
muscle fibers.
The dramatic elevation in basal glucose uptake in the muscles of
Glut1-overexpressing mice results in the near equilibration of free
glucose across the muscle fiber membranes, indicating that the
phosphorylation of glucose by hexokinase has become the rate-limiting
step in glucose uptake in these muscles (2). This observation
demonstrates that hexokinase catalyzes a secondary rate-limiting step
in skeletal muscle glucose disposal that can become predominant if the
transport step is artificially elevated. It therefore seemed reasonable
that a further increase in muscle glucose uptake and whole-body glucose
disposal might be induced in Glut1-overexpressing mice by increasing
the expression of hexokinase in the muscles of these mice. This
hypothesis has an important practical implication for therapies
designed to counteract the insulin resistance and glucose intolerance
associated with type 2 diabetes (4).
In order to test this hypothesis, Glut1-overexpressing mice were mated
with mice that overexpress hexokinase II in skeletal muscle.
Surprisingly, the overexpression of hexokinase II in the muscles of
Glut1-overexpressing mice dramatically increased steady-state levels of
glucose 6-phosphate and glycogen, but did not augment whole-body
glucose disposal. Additionally, mice overexpressing hexokinase II alone
exhibited no increase in muscle glucose uptake or whole-body glucose
disposal compared with wild-type control mice. We conclude that
increasing hexokinase activity in muscle is not likely to be an
effective means of counteracting insulin resistance or glucose intolerance.
Materials--
Purified porcine insulin (Iletin II) and human
insulin (Humulin R U-100) were purchased from Eli Lilly and Co.
Radioimmunoassay grade bovine serum albumin
(BSA),1 and the
nonradioactive forms of D-glucose,
2-deoxy-D-glucose (2-DG), and D-mannitol were
obtained from Sigma. 2-Deoxy-D-
[1,2-3H]glucose, [5-3H]glucose, and
3H2O were purchased from American Radiolabeled
Chemicals. [U-14C]Mannitol and high pressure liquid
chromatography-purified 3-[3H]glucose were obtained from
NEN Life Science Products.
Preparation of Transgenic Mice Overexpressing Glut1 and
Hexokinase II--
Transgenic mice overexpressing human hexokinase II
in striated muscle (a kind gift from Drs. D. H. Wasserman and D. K. Granner) were constructed as described previously, using a transgene
containing the rat muscle creatinine kinase promoter-enhancer coupled
to the human hexokinase II cDNA and an 850-base pair cassette
(SVPA) containing the polyadenylation and splice site sequences of SV40 (7). The construction of transgenic mice overexpressing the human Glut1
glucose transporter was also described previously (1). The minigene in
this construct contains a 2.47-kilobase cDNA fragment encoding the
human Glut1 glucose transporter under the regulation of the
1.2-kilobase rat myosin light chain-2 promoter. Expression of the
transgene is restricted to skeletal muscle and does not affect
expression of the Glut4 isoform. Male mice heterozygous for the Glut1
transgene were bred with female mice heterozygous for the hexokinase II
transgene to produce mice of four separate genotypes: those carrying
both transgenes, those carrying one of the two, and those carrying none
of the transgenes. The presence of the transgenes was determined by PCR
analysis of genomic DNA obtained from tail biopsy and prepared using
the QiAmp tissue isolation kit (Qiagen, Valencia, CA). Sex-matched
littermate mice between the ages of 2 and 4 months were used for all experiments.
Animal Care and Tissue Preparation--
Animals were housed in a
room maintained at 23 °C with a fixed 12-h light/dark cycle (lights
on 6 a.m. to 6 p.m.) and given free access to Purina chow and
water. On the morning of the experiment, animals were anesthetized with
pentobarbital sodium (5 mg/100 g body weight by intraperitoneal
injection). Extensor digitorum longus muscles were excised and
incubated for determination of 2-DG uptake or glucose utilization.
Measurement of 2-Deoxyglucose Uptake--
Extensor digitorum
longus muscles were incubated for 30 min at 35 °C in 2 ml of
oxygenated Krebs-Henseleit buffer (KHB) supplemented with 8 mM glucose, 32 mM mannitol, and 0.1% BSA in
the absence or presence of 2 milliunits/ml porcine insulin. Next,
muscles were transferred to KHB containing 40 mM mannitol,
0.1% BSA, and 2 milliunits/ml insulin for 10 min to remove glucose
from the extracellular space prior to measurement of 2-DG uptake as
described previously (8). Briefly, muscles were incubated for 20 min in
1.5 ml of KHB containing 1 mM
2-deoxy-D-[1,2-3H]glucose (1.5 µCi/mmol),
39 mM [U-14C]mannitol (8.5 µCi/mmol), and
0.1% BSA. Insulin was added if it was present in previous incubations.
The gas phase was 95% O2, 5% CO2, and the
temperature was maintained at 29 °C. Muscle extracellular space and
intracellular 2-DG concentration (µmol·ml intracellular
water Measurement of Muscle Glucose Metabolism Using
[3H]Glucose--
In vitro rates of glucose
utilization via glycolysis and glucose incorporation into glycogen were
determined in skeletal muscle isolated from control and
hexokinase-overexpressing mice according to a modification (8) of
previously described methods (9, 10). Extensor digitorum longus muscles
were incubated for 3 h at 35 °C in 1 ml of oxygenated KHB
containing 2.25 µCi of [5-3H]glucose under one of two
conditions: either 8 mM glucose and 20 microunits/ml
porcine insulin or 24 mM glucose and 2 milliunits/ml insulin. Mannitol was added to the medium at a concentration sufficient to bring the total osmolality of glucose plus mannitol to 40 milliosmolal. Using this technique, it has been demonstrated previously
that in isolated skeletal muscle, the sum of glucose utilization via glycolysis (measured as [3H]water formation from
[5-3H]glucose) plus the amount of tritium retained in
glycogen accounts for nearly all of the [5-3H]glucose
transported into the cell (11).
Measurement of Whole-body Glucose Disposal:
Euglycemic-Hyperinsulinemic Clamps--
Clamp experiments were carried
out as described previously (3, 12) with the following modifications.
After placement of the infusion catheter, an infusion of
3-[3H]glucose at 0.04 µCi/min was begun for measurement
of the rate of appearance of glucose, hepatic glucose production, and
total body glucose utilization. The infusion was continued during a 75-min control period, and 20 µl of blood was taken from the tail for
determination of glucose specific activity at 60, 67.5 and 75 min.
After 75 min, an infusion of insulin (regular human) was begun at a
rate of 80 milliunits/kg/min and continued for at least 90 min. An
infusion of dextrose (25%) was begun, and the infusion rate varied
during the clamp period to maintain the blood glucose at approximately
160 mg/dl.
In all the clamps, the continuous infusion of
3-[3H]glucose tracer was continued during the insulin
infusion periods and, in addition, the tracer was added to the 25%
dextrose infusion to approximate the glucose specific activity in the
blood at the end of the control period. This approximation was based
upon measurement of specific activity during identical conditions in
the same type of mice in previous experiments. Blood samples for
determination of specific activity were taken 20 and 10 min prior to
and at the end of the experimental period. The glucose infusion rate was not changed for at least 20 min prior to the first determination of
specific activity. Both the blood glucose and the glucose specific activity were in steady state during the control period and the clamp
sampling periods. Blood for insulin measurement was obtained by cardiac
puncture at the conclusion of the experiment.
Blood glucose was measured using 5 µl of whole blood in the Hemocue
blood glucose meter (Hemocue, Mission Viejo, CA). Plasma insulin was
measured by double-antibody radioimmunoassay using rat insulin
standards (Eli Lilly). Specific activity of glucose in whole blood was
determined by aqueous scintillation counting of 20 µl of blood
deproteinized with barium hydroxide (0.3 N) and zinc
sulfate (0.3 N). Aliquots of the resulting supernatant were
dried at 70 °C to remove tritiated water prior to resuspension and
liquid scintillation counting. The rate of appearance of glucose (Ra), which equals the rate of total body
glucose utilization (Rd) when the blood glucose
is in steady state, was calculated by dividing the infusion rate of
3-[3H]glucose by the specific activity at the same time.
Hepatic glucose production was calculated by subtracting the cold
glucose infusion rate from Ra.
Measurement of Hexokinase Activity and Glut1 Glucose Transporter
Content--
Total hexokinase activity and heat-stable hexokinase I
activity in quadriceps muscle homogenates (7) were measured
enzymatically (13). Hexokinase II activity was subsequently calculated
by subtracting hexokinase I from total hexokinase activity. Glucose transporter protein levels were determined in muscle homogenates by
immunoblotting, using rabbit polyclonal antibodies directed against the
C terminus of either Glut4 (Phe349) or Glut1
(Phe350) followed by horseradish peroxidase-conjugated
donkey anti-rabbit IgG. Antibody-bound proteins were detected using ECL.
Measurement of Muscle Metabolites--
Measurements of glycogen
and glucose 6-phosphate were performed using gastrocnemius muscles
clamp frozen in situ. Frozen muscles were homogenized in
ice-cold 0.3 M perchloric acid; one aliquot of this
homogenate was analyzed for glycogen (14), while the remaining
homogenate was centrifuged, and the resulting supernatant neutralized
with buffer containing 0.4 M KCl, 2 N KOH, and
0.4 M imidazole. Glucose and glucose 6-phosphate in this
fraction were measured fluorometrically (15).
Measurement of Plasma Metabolites--
Mice fed ad
libitum were bled (~0.3 ml) from the tail. Plasma insulin
concentrations were measured by double-antibody radioimmunoassays using
human standards (Linco Research, Inc.). Blood glucose levels were
measured using a Hemocue glucose analyzer.
Overexpression of Hexokinase--
Muscles were resected from
littermate mice expressing the Glut1 transgene, the hexokinase II
transgene, both transgenes, or control non-transgenic littermates and
analyzed for the expression of hexokinase I, hexokinase II, and total
hexokinase activities. Fig. 1 shows that
the two groups of mice expressing the hexokinase II transgene exhibited
similar (7.1- and 8.2-fold, respectively) increases in total hexokinase
activity, due mostly to a specific increase in hexokinase II activity.
The small increase in hexokinase I activity observed in these two
groups is probably due to the fact that the assays do not
quantitatively distinguish between the two isoforms. Glut1 transgenic
mice exhibited a small increase in total hexokinase activity in muscle
compared with their wild-type siblings. These data are consistent with
previously published data concerning Glut1 (2) and hexokinase II
(7)-overexpressing transgenic mice.
Characterization of Mice Overexpressing Hexokinase Alone--
In
order to determine whether overexpression of hexokinase II induced an
alteration in glucose transport activity, 2-deoxyglucose uptake
measurements were performed on isolated extensor digitorum longus
muscles. Fig. 2 shows that neither basal
nor insulin-stimulated glucose transport was significantly altered in
hexokinase II-overexpressing muscles. Similar results were observed in
isolated soleus muscles (data not shown). These results suggest that
overexpression of hexokinase II in muscle did not directly or
indirectly impact on glucose transporter expression, regulation, or
activity. Immunoblot analysis confirmed that neither Glut1 nor Glut4
protein levels were altered in the skeletal muscle of hexokinase
overexpressing mice relative to wild-type littermates, nor were Glut1
or Glut4 protein levels altered in Glut1 × Hex mice relative to
Glut1 mice (data not shown).
In order to determine whether hexokinase overexpression by itself can
augment muscle glucose metabolite levels, steady-state glucose
6-phosphate and glycogen levels were measured in muscles clamp frozen
in situ from the four groups of mice. Fig.
3 shows that neither metabolite was
altered in the muscles of mice overexpressing hexokinase II compared
with wild-type littermates. This is consistent with the observation
that transport, not phosphorylation, is rate-limiting for glucose
metabolism in normal mouse muscle.
A previous study with hexokinase-overexpressing mice suggested that
hexokinase may become rate-limiting or partially rate-limiting for
glucose metabolism in skeletal muscle under conditions of hyperinsulinemia and/or hyperglycemia (7), but direct evidence in the
form of glucose tracer analysis in isolated muscles was not provided.
In order to address this issue, the metabolism of [5-3H]glucose was examined in muscles isolated from
control and hexokinase II-overexpressing mice. Fig.
4 shows that under both basal (8 mM glucose, 20 microunits/ml insulin) and
hyperglycemic/hyperinsulinemic (24 mM glucose, 2 milliunits/ml insulin) conditions, neither glycolytic nor glycogenic
utilization of glucose was increased in muscles from hexokinase II
transgenic mice compared with wild-type littermates. These data are in
complete agreement with the data presented above on steady-state levels
of glucose metabolites and further confirm the rate-limiting nature of
the transport step in normal muscle.
In order to determine the effect of the hexokinase transgene on
whole-body glucose homeostasis, blood glucose and whole-body glucose
disposal measurements were conducted. Fig.
5 shows that expression of the hexokinase
II transgene alone had no significant effect on fed blood glucose
levels compared with wild-type controls. Fig.
6 shows that the hexokinase transgene
alone had no significant effect on whole-body glucose disposal
(Rd) in either the basal state or during a
hyperinsulinemic/euglycemic clamp.
Characterization of Mice Overexpressing Both Hexokinase and
Glut1--
Steady-state Glucose-6-phosphate levels were increased
2.4-fold and glycogen was increased 5.4-fold in muscles from Glut1 transgenic mice (Fig. 3). Increasing hexokinase activity in muscles that also overexpress Glut1 caused a further dramatic elevation in
glucose 6-phosphate (7.6-fold compared with wild-type and 3.2-fold compared with Glut1-overexpressing muscles) and glycogen (40.5-fold compared with wild-type and 7.5-fold compared with Glut1-overexpressing muscles). These observations are completely consistent with the observation that phosphorylation is rate-limiting for glucose uptake in
the muscles of the Glut1 transgenic mice (2).
The massive overexpression of glycogen in muscles of the doubly
transgenic animals resulted in a dramatic disruption in the normal
ultrastructure of striated muscle (see Fig.
7). Myofibrils in the doubly transgenic
mice exhibited greater electron density compared with mice
overexpressing hexokinase alone, which exhibited a normal muscle
architecture, and the myofibril bundles were disrupted by numerous
glycogen deposits, some of them very large in size. Despite their
grossly abnormal muscle architecture, the doubly transgenic mice did
not display any obvious change in mobility or behavior compared with
their non-transgenic siblings.
Consistent with previously published results (3, 12),
Glut1-overexpressing mice exhibited increases in basal whole-body glucose disposal and insulin-stimulated whole-body glucose disposal under euglycemic/hyperinsulinemic clamp conditions compared with wild-type littermates (Fig. 6). Because overexpression of hexokinase in
the context of Glut1 overexpression dramatically increased steady-state
levels of muscle metabolites compared with overexpression of Glut1
alone (see Fig. 3), it was expected that whole-body glucose disposal
would be markedly elevated in the Glut1 × Hex mice relative to
the Glut1 mice. Surprisingly, however, no significant difference was
observed in basal or insulin-stimulated whole-body glucose disposal in
Glut1 × Hex mice compared with Glut1 mice.
The data presented in this study are consistent with a large body
of previous experimental data indicating that, under most conditions
and in most skeletal muscle types, transport of glucose is
rate-limiting for glucose uptake and metabolism (4). However, it is
possible that in some skeletal muscle types, i.e. those expressing a relatively high Glut4/hexokinase II ratio, the
phosphorylation step may become partially rate-limiting for overall
glucose metabolism under certain conditions (16).
One unanticipated finding of this study is that the hexokinase
transgene did not further augment whole-body glucose disposal in mice
that also overexpress Glut1 in muscle. The observation that
intracellular free glucose is dramatically elevated in the muscle of
Glut1 transgenic mice (2) indicates that the hexokinase step is
rate-limiting for glucose metabolism in Glut1-overexpressing muscles.
Consistent with this interpretation is the finding that the muscles of
doubly transgenic mice exhibited elevated steady-state glucose
6-phosphate and glycogen levels relative to muscles overexpressing Glut1 alone (Fig. 3), implying an increased flux through the glycogenic pathway mediated by the hexokinase transgene. Why then are the elevated
steady-state metabolite levels in doubly transgenic muscles not
associated with a further increase in whole-body glucose disposal relative to mice that only carry the Glut1 transgene? There are several
possible explanations, one being that the grossly elevated steady-state
glycogen level caused an inhibition of de novo glycogen synthesis in muscles of the doubly transgenic mice (17). Support for
this interpretation is provided by the observation that muscles of
Glut1 transgenic mice with a 5-fold elevation in steady-state glycogen
levels relative to control muscles exhibit a 50% reduction in the
proportion of glycogen synthase in the active form (2). Inhibition of
flux through the glycogenic pathway due to the very high glycogen
levels probably contributed to the grossly elevated glucose 6-phosphate
levels in the muscles of mice carrying both transgenes. Because
glycogen synthesis is the major pathway for the disposal of glucose in
muscle under hyperinsulinemic conditions (18), this could explain why
the doubly transgenic mice do not exhibit a further increase in
whole-body glucose disposal during the euglycemic/hyperinsulinemic
clamp procedure.
Our data are in apparent disagreement with data published previously on
hexokinase II transgenic mice. Chang and colleagues (7) reported that
hexokinase II-overexpressing extensor digitorum longus and soleus
muscles exhibited increased 2-deoxyglucose uptake relative to control
muscles, especially in the presence of pharmacologic concentrations of
insulin. We observed no increase in 2-deoxyglucose uptake in either
muscle type in the presence or absence of insulin (Fig. 2). The data of
Chang and colleagues (7) imply that hexokinase overexpression augmented
the activity and/or expression of endogenous glucose transporters in
skeletal muscle, because 2-deoxyglucose uptake reflects transport
activity and not hexokinase activity under the conditions employed in
these experiments (19). This is also definitively demonstrated by our
uptake data where severalfold increases in hexokinase activity had no
effect on the rate of 2-deoxyglucose uptake (see Fig. 2). However,
since hexokinase activity appears to be rate-limiting for glucose
uptake in the muscles of Glut1-overexpressing mice, one might expect to
observe an increase in 2-deoxyglucose uptake after transgenic
augmentation of hexokinase activity in these muscles. The explanation
for the inability of hexokinase to increase 2-deoxyglucose uptake in
Glut1-overexpressing mice lies in the lack of feedback inhibition of
hexokinase activity by 2-deoxyglucose-6-P. Even at a concentration of
30 mM, 2-deoxyglucose-6-P fails to inhibit hexokinase
activity in muscle homogenates, whereas glucose-6-P inhibits hexokinase
activity by 80% at a concentration of 500 µM (19). Thus,
in situ, with an ambient blood glucose concentration of ~8
mM and an intracellular glucose-6-P concentration of ~0.8
mM (see Fig. 3), hexokinase activity was severely inhibited and was rate-limiting for glucose uptake in the muscles of
Glut1-overexpressing mice. However, when muscles were removed and then
incubated under conditions used to measure 2-deoxyglucose uptake (no
glucose and 1 mM 2-deoxyglucose), the lack of feedback
inhibition restored hexokinase activity to the point where transport
once again became rate-limiting for uptake. Thus, 2-deoxyglucose uptake
in isolated tissues continues to reflect only the rate of glucose
transport in the muscles of Glut1-overexpressing mice, even though
hexokinase is rate-limiting for glucose uptake in muscle in
situ.
If indeed the hexokinase-overexpressing mice studied by Chang and
colleagues (7) also exhibited an increase in the activity of an
endogenous glucose transporter, then that would explain other
differences between their data and ours. For example, Chang and
colleagues (7) reported a small but significant increase in glucose
6-phosphate levels in gastrocnemius muscles isolated from hexokinase
transgenic mice after a bolus injection of glucose and insulin, but we
observed no such increase under steady-state conditions. The increase
they observed may in fact have been due to an increase in glucose
transport activity in their mice. In a different collaborative study
(20), some of the same investigators reported that
hexokinase-overexpressing mice exhibited increases in the glucose
utilization index of gluteal and gastrocnemius muscles under
hyperglycemic/hyperinsulinemic clamp conditions. It is possible that
this was also due to an increase in the activity of an endogenous
glucose transporter isoform expressed in the skeletal muscles of the
hexokinase transgenic mice. In support of this suggestion, mice that
overexpressed both Glut4 and hexokinase II had lower rates of muscle
glucose utilization than mice overexpressing the Glut4 transgene alone
(20), and these differences in the in situ glucose
utilization rates paralleled the differences in muscle 2-deoxglucose
uptake data, i.e. muscles overexpressing both hexokinase and
Glut4 exhibited lower rates of 2-deoxyglucose uptake in the presence of
insulin than muscles overexpressing Glut4 alone. Why mice that
overexpress both Glut4 and hexokinase might have lower glucose uptake
rates than mice that overexpress hexokinase alone remains unclear, but
this observation is consistent with a dominant role for transport as
compared with phosphorylation in determining the rate of muscle glucose
disposal, and supports the hypothesis that increases in metabolism
observed in the hexokinase-overexpressing muscles may in fact have been
due to an increase in endogenous glucose transport activity rather than
the increase in hexokinase activity.
The hexokinase transgenic mice used in our study were derived from the
line originally constructed by Chang and colleagues (7). One difference
is that we cross-bred the original hexokinase line (in a FVB/NJ
background) with a different hybrid strain of mice that overexpress
Glut1 (C57BL/6 × SJL), so that all four mouse genotypes studied
were in a FVB/NJ × (C57BL/6 × SJL) background. This may
account for some of the differences observed by Chang and colleagues
(7) and by us. Regardless, our data on hexokinase-overexpressing mice
as well as the data described previously by others, when analyzed in
the present context, are consistent with a dominant if not exclusive
role for glucose transport in determining the rate of muscle glucose
metabolism and whole-body glucose disposal in mice. This conclusion is
in agreement with a recent elegant study employing NMR that directly
measured the concentration of free glucose and glucose 6-phosphate in
human skeletal muscle (21). This study demonstrated that glucose
transport, as opposed to phosphorylation, is rate-limiting for muscle
glucose disposal under hyperglycemic/hyperinsulinemic conditions in
normal subjects and in type 2 diabetics.
*
This work was supported in part by National Institutes of
Health Grants DK38495 (to M. M.) and DK18986 (to J. O. H.) and by the Diabetes Research and Training Center at Washington
University School of Medicine.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
These authors contributed equally to this work.
**
To whom correspondence should be addressed: Dept. of Cell Biology
and Physiology, Washington University School of Medicine, Box 8228, 660 S. Euclid Ave., St. Louis, MO 63110. E-mail: mike@ cellbio.wustl.edu.
Published, JBC Papers in Press, April 11, 2000, DOI 10.1074/jbc.M001946200
The abbreviations used are:
BSA, bovine serum
albumin;
2-DG, 2-deoxyglucose;
KHB, Krebs-Henseleit buffer.
Transgenic Overexpression of Hexokinase II in Skeletal Muscle
Does Not Increase Glucose Disposal in Wild-type or
Glut1-overexpressing Mice*
§,,, and
**
Medicine, ¶ Pediatrics,
and Cell Biology and Physiology, Washington University
School of Medicine, St. Louis, Missouri 63110
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1·20 min1)
were determined as described previously (8).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Hexokinase activity in quadriceps muscles
from control and transgenic mice. Total, type I, and type II
hexokinase activities were determined on muscle homogenates from
wild-type mice or mice overexpressing Glut1 (Glut1),
hexokinase II (Hex), or both transgenes (Glut1 × Hex) as described under "Experimental Procedures." The values
represent the mean ± S.E. for 4-6 individual muscles per group.
*, p < 0.001 for Hex or Glut1 × Hex
versus wild-type or Glut1.
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Fig. 2.
Glucose transport activity of muscles from
wild-type and transgenic mice. Extensor digitorum longus muscles
were excised from wild-type mice or mice overexpressing Glut1
(Glut1), hexokinase II (Hex), or both transgenes
(Glut1 × Hex). Muscles were subjected to
[3H]2-deoxyglucose uptake assays as described under
"Experimental Procedures." The values represent the mean ± S.E. for 8-10 individual muscles per group. *, p < 0.001 for Glut1 or Glut1 × Hex versus wild-type or
Hex.
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Fig. 3.
Steady-state metabolite levels in muscles
from wild-type and transgenic mice. Gastrocnemius muscles from
wild-type mice or mice overexpressing Glut1 (Glut1),
hexokinase II (Hex), or both transgenes
(Glut1 × Hex) were clamp frozen in
situ, resected, homogenized, and then used to measure steady-state
concentrations of glucose 6-phosphate (A) and glycogen
(B) as described under "Experimental Procedures." The
values given represent the mean ± S.E. for 8-10 individual
muscles per group. *, p < 0.001 for Glut1 × Hex
versus all other groups; **, p < 0.01 for
Glut1 versus wild-type or Hex.
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Fig. 4.
Rates of glycolytic and glycogenic glucose
utilization in muscles from control and hexokinase transgenic
mice. Extensor digitorum longus muscles from wild-type and
hexokinase (Hex) transgenic mice were resected, incubated in
the presence of [5-3H] glucose and either 8 mM cold glucose, 20 microunits/ml insulin (Low
Gluc/Ins) or 24 mM cold glucose, 2 milliunits/ml
insulin (High Gluc/Ins). The amounts of the isotope
incorporated into H2O (A) or glycogen
(B) were then determined as described under "Experimental
Procedures." Values represent the mean ± S.E. for 6-8
individual muscles per group.
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Fig. 5.
Blood glucose levels in control and
transgenic mice. Blood was obtained from the tail veins of freely
feeding wild-type mice or mice expressing the hexokinase transgene
(Hex), the Glut1 transgene (Glut1) or both
transgenes (Glut1 × Hex). Glucose concentrations were
determined as described under "Experimental Procedures." Values
given represent mean ± S.E. for 11-22 animals per group. *,
p < 0.001 for Glut1 or Glut1 × Hex
versus wild-type or Hex.
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Fig. 6.
Rates of whole-body glucose disposal in
control and transgenic mice. Wild-type mice or mice carrying the
hexokinase transgene (Hex), the Glut1 transgene
(Glut1), or both transgenes (Glut1 × Hex) were subjected to a euglycemic/hyperinsulinemic clamp
procedure along with the infusion of [3H]glucose to
ascertain the rate of whole-body glucose disposal under basal and
hyperinsulinemic conditions (see "Experimental Procedures"). Values
given represent the mean ± S.E. for 4 animals per group. *,
p < 0.01 for Glut1 or Glut1 × Hex
versus wild-type or Hex; **, p < 0.05 for
Glut1 versus wild-type or Hex.
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Fig. 7.
Ultrastructure of muscle from transgenic
mice. Extensor digitorum longus muscles were resected from mice
overexpressing hexokinase (Hex) or both Glut1 and hexokinase
(Glut1 × Hex) and then processed for
electron microscopy as described previously (22). Cross-sections
through the muscle fibers are shown. Muscles from Hex mice exhibit a
normal ultrastructure, whereas muscles from Glut1 × Hex mice
display an abnormal architecture due to the large increase in glycogen
content. Note the unusually high electron density of the myofibrils in
the Glut1 × Hex muscle and the large and small glycogen deposits
interrupting the bundles of myofibrils. Mi, mitochondria;
F, myofibrils; G, glycogen. The small
arrows point to small glycogen deposits within myofibril
bundles.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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