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Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, Martí i Franquès, 1, 08028 Barcelona, Spain; and
* Departments of Biochemistry and Internal Medicine and Gifford Laboratories for Diabetes Research, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA
1Correspondence: Departament de Bioquímica i Biologia Molecular, Facultat de Química, Universitat de Barcelona, Martí i Franquès, 1, 08028-Barcelona, Spain. E-mail: anamaria{at}sun.bq.ub.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: glucose phosphorylating activity • glucokinase expression • GLUT4 expression • non-insulin-dependent diabetes mellitus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Additional
support for this idea is provided by studies of transgenic mice with
muscle-specific overexpression of GLUT1, which in contrast to GLUT4
does not require insulin for localization in the plasma membrane
(3)
. GLUT1-transgenic mice showed accumulation of free
glucose but unaltered intracellular glucose 6-P levels. On the other
hand, overexpression of hexokinase II in skeletal muscle of transgenic
mice had little effect on glucose homeostasis in vivo
(4)
. One potential explanation for the limited metabolic
effect of hexokinase II overexpression could be that this enzyme is
known to be allosterically inhibited by its product,
glucose-6-phosphate.
We have recently addressed the control strength of the glucose
phosphorylation step for muscle glucose disposal by adenovirus-mediated
expression of liver glucokinase in human primary myocytes
(5)
. Glucokinase (GK), in contrast to hexokinase II, is
only weakly inhibited by glucose-6-P. We showed that cultured human
muscle cells expressing their endogenous complement of hexokinase II
exhibited a saturable capacity to accumulate glucose 6-P at
concentrations of glucose above 5 mM. In contrast, muscle cells
engineered for GK expression gained the capacity to respond to glucose
concentrations over the physiological range (5–25 mM) as indicated by
glucose concentration-dependent increases in glucose 6-P levels,
glycogen synthesis, and lactate production. These in vitro
data suggested that expression of GK in muscle in vivo could
constitute a mechanism for overriding the limitation of the glucose
phosphorylation step for glucose disposal. However, extrapolation of
the in vitro findings to the in vivo setting is
complicated by the fact that cultured myocytes express relatively high
levels of GLUT1, making it possible that GK expression in muscle of
intact animals would have little or no impact.
In the current study, we have directly tested the metabolic impact of GK expression by delivery of a recombinant adenovirus containing this gene (AdCMV-GKL) into muscle of newborn rats. We show that intramuscular (i.m.) injection of AdCMV-GKL results in effective expression of GK. Further, expression of the enzyme in muscle is associated with increased in vivo glucose phosphorylation capacity and enhanced glucose clearance in fed rats. These results demonstrate that GK expression in muscle may be an effective means of enhancing glucose clearance and that expression or activation of this gene may have therapeutic potential for overcoming insulin resistance and hyperglycemia in non-insulin-dependent diabetes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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or the Escherichia
coli ß-galactosidase nls-LacZ gene (AdCMV-lacZ) (7)
have been described previously. Viruses were amplified in 293 cells and
concentrated to 1011-1012
pfu/ml by centrifugation through a CsCl gradient as described
previously (8)
. Viruses were aliquoted and stored at
-70°C in phosphate-buffered saline (PBS). Viruses were titrated by a
serial dilution plaque-assay in noble agar overlaid 293 cells.
Approximately 5 x 108 pfu of AdCMV-GKL or
AdCMV-lacZ were delivered to newborn (2–3 days) Wistar-Hannover rats
(Harlan-Iberica) by i.m. injection in the right hind leg, and the same
volume of PBS was injected into muscle in the left leg. Litters of 12
animals were used and the virus was administered to 6 animals. All
experiments were performed in at least four separate batches of
littermates. Animals were maintained at 23°C with a 12 h light-dark cycle. They were weaned at 22 days and thereafter provided with a standard chow diet and water ad libitum. When stated, animals were fasted by 18 h deprivation of food. Animals were killed after anesthesia with pentobarbital. Liver, gastrocnemius, and soleus muscles were excised and immediately frozen in liquid N2, followed by freeze-drying in an Alpha 1–4/RVC drier (B. Braun Inc.) and storage at -20°C. Animals treated with AdCMV-GKL or AdCMV-lacZ were analyzed for transgene expression in muscle. Only animals with detectable transgene expression were used for further analysis.
Analysis of ß-galactosidase activity
For histochemical detection of ß-galactosidase activity,
gastrocnemius and soleus muscles were excised, rinsed with PBS, and
immediately fixed with 4% paraformaldehyde for 2 h at 4°C.
After further washing with PBS, intact muscles were incubated at 37°C
with a solution of 1 mg/ml X-Gal containing 5 mM
K4Fe(CN)6, 5 mM
K3Fe(CN)6, 2 mM
MgCl2, and 0.04% Igepal CA-630 for 12 h. To
estimate the fraction of tissue that expressed ß-galactosidase
activity, every X-gal stained muscle was dissected and sections that
appeared blue under the a microscope were separated from those that
appeared unstained. Blue-stained and unstained sections of every muscle
were pooled and weighed for percentage calculation. Alternatively,
ß-galactosidase activity was measured spectrophotometrically in crude
extracts using o-nitrophenol-ß-galactoside as described
(9)
.
Western blot analysis and glucose phosphorylating activity
Freeze-dried muscles were cut very finely. Samples of 10 mg were
homogenized in 50 volumes of ice-cold buffer 50 mM Tris-HCl buffer (pH
7.4) with 1 mM EDTA, 100 mM KCl, 300 mM sucrose, 10 mM
ß-mercaptoethanol, 1 mM leupeptin, and 1 mM benzamidin and
centrifuged at 10,000 x g for 15 min. The resulting
supernatants were used for the determination of enzyme activities and
Western blot analysis. Protein concentration was measured with Bio-Rad
protein assay reagent. For Western blotting, samples of 20 µg of
protein were prepared by standard procedures and immunoblotting was
performed using a polyclonal antibody as described (6
, 10)
. Glucose phosphorylating activity was determined at 100 mM
glucose as described (11)
in a Cobas Fara autoanalyzer.
Crude membranes for GLUT4 analysis were prepared and immunoblot
analysis was performed as described previously (12)
.
Polyclonal anti-GLUT4 antibody was kindly provided by Dr. A. Zorzano.
Measurement of circulating glucose, insulin, lactate, and glucose
tolerance test
To test glucose tolerance, 18 h fasted or ad
libitum-fed animals were anesthetized with pentobarbital, and a
glucose bolus (2 g/kg of body weight) was administered by
intraperitoneal (i.p.) injection. Animals were subsequently bled via
the tail vein. Blood glucose concentration was monitored with the aid
of a Reflotron photometer (Boehringer Mannheim) and serum L-lactate
levels were measured enzymatically. Serum insulin was determined by an
ELISA test (Crystal Chem).
In vivo 2-deoxyglucose uptake
To measure in vivo 2-deoxyglucose uptake,
2-deoxy[3H]glucose (0.45 µCi/kg body weight)
was injected 10 or 90 min after glucose bolus via the tail vein.
Gastrocnemius and soleus muscles were excised and freeze-dried 10 or 30
min after 2-deoxyglucose injection. Aliquots of the muscle tissue were
used to determine
2-deoxy[3H]glucose-6-phosphate accumulation
according to the Somogyi procedure (13)
.
Statistical analysis
Data are expressed as means ± SE. The
statistical significance was determined by an unpaired Student’s
t test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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); the central part was less affected and the soleus muscle was seldom
reached. In the gastrocnemius, areas of higher intensity could be
detected that presumably corresponded to the multiple sites of
injection (three). The percentage of fibers expressing
ß-galactosidase in positive animals was estimated to range from 8%
to 68% of fibers and the median value (n=4) was around
40%. Potential dissemination of the transgene to other tissues,
including liver, which is the preferential adenoviral target tissue
(7)
, was analyzed in the animals injected with AdCMV-lacZ.
No ß-galactosidase activity was detected by spectrophotometric
enzymatic analysis in tissues other than the infected leg (data not
shown).
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In animals injected with AdCMV-GKL, GK expression in gastrocnemius
muscle was analyzed 1 month after gene transfer. A majority of the
AdCMV-GKL-treated animals expressed GK protein in muscle, as measured
by immunoblot analysis. The amount of GK in muscle of
AdCMV-GKL-injected animals was quantified by densitometric analysis and
normalized to the endogenous GK content in liver of 1-month-old
untreated rats (Fig. 2
). Among 10 animals positive for GK expression in muscle, levels varied
over a range from 0.5- to 4-fold the GK content in the control livers,
likely due to variations in the gene delivery efficiency. To cope with
these differences in expression, positive GK-treated animals were
divided into two groups (Fig. 3A
). The high expressing group was composed of animals with GK
protein content in gastrocnemius muscle twofold higher than control
livers, while the low expressing group included animals with GK levels
below this threshold. Total glucose phosphorylating activity was also
analyzed in muscle extracts (Fig. 3B
). The glucose
phosphorylating activity, estimated at 100 mM glucose in the
supernatant fractions of gastrocnemius muscle, was higher in
GK-expressing muscles relative to the counterpart uninfected muscle by
30% and 100% for the low and high expressing groups, respectively.
Liver GK activity was not affected by AdCMV-GKL injection (11.86±1.75
mU/mg dry liver weight) when compared to control animals (9.41±0.7 dry
liver weight), confirming that viruses were not escaping to other
tissues.
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Glucose disposal
We next evaluated the effect of GK expression in muscle on glucose
disposal. Prior to administration of the glucose bolus, no differences
in blood glucose were observed among positive AdCMV-GKL-treated
compared with AdCMV-lacZ-treated rats in either fasting or fed
conditions (Fig. 4
). However, clearance of the glucose bolus was clearly enhanced in
AdCMV-GKL-treated fed animals (Fig. 4A
). Maximal differences
between the AdCMV-GKL treated and control animals were noted 10 to 20
min after glucose administration. Moreover, more efficient glucose
clearance was observed in the high GK-expressing group compared to the
low GK-expressing group, suggesting that GK expression level was the
determining factor for the improvement in glucose clearance. In
contrast, in fasted animals that exhibited a slower rate of glucose
extraction (Fig. 4B
), no difference in the glucose clearance
profile during an IPGTT (i.p. glucose tolerance test) was found among
AdCMV-GKL-treated and control rats.
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Serum lactate concentrations during the IPGTT in fed animals were
determined (Fig. 5
). No differences in basal lactate levels were found between
AdCMV-GKL-treated and untreated fed rats, whereas a 48% and 55%
increase in circulating lactate was detected in the low and high
GK-expressing animals relative to controls 20 min after injection of
glucose, suggesting an enhanced rate of glucose metabolism in muscle of
GK-expressing rats. Insulin levels in fasted animals were not different
in AdCMV-GKL-treated (0.46±0.13 ng/ml) as compared to untreated
(0.46±0.04 ng/ml) rats. Fed animals had higher basal insulin levels
than fasted and there were no differences in insulin levels between
AdCMV-GKL-treated (GK-low 1.85±0.17 ng/ml and GK-high 1.03±0.49
ng/ml) animals and control (1.17±0.46 ng/ml) fed animals in the basal
state or 20 min after the glucose bolus (9.65±0.92 ng/ml, 11.15±0.60
ng/ml and 9.29±1.45 ng/ml for untreated, GK-low and GK-high,
respectively).
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The nonmetabolizable glucose analog 2-deoxyglucose (2-DOG) was used to
assess whether the enhanced glucose disposal in positive
AdCMV-GKL-treated animals was linked to an increase in glucose uptake.
3-[3H]-2-DOG was injected intravenously 10 or
90 min after i.p. administration of the glucose load; 10 and 30 min
later, respectively, gastrocnemius muscles were excised to assay the
accumulation of 3-[3H]2-DOG-6-P. Since the GK
cDNA was delivered to one of the two hind legs, the counterpart
gastrocnemius provided an internal control for
3-[3H]-2-DOG uptake. Therefore, the
accumulation of 3-[3H]2-DOG-6-P was expressed
in every animal as a ratio between the content of
3-[3H]-2-DOG-6-P in the adenovirus-infected
(right) vs. uninfected (left) gastrocnemius muscle bed (Fig. 6
). In control animals, this ratio was 0.94. The low and high
GK-expressing groups exhibited 25 and 38.9% increases in ratio values,
respectively, when 3-[3H]-2-DOG uptake was
measured 20 min after glucose injection. No differences in
3-[3H]2-DOG-6-P ratio values were found between
controls (0.98±0.15) and low or high (0.91±0.04 and 1.04±0.09)
GK-treated animals when 3-[3H]-2-DOG was
administered after glucose returned to basal levels (90 min after
glucose challenge).
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GLUT4 expression
We analyzed whether expression of GK in gastrocnemius muscle
affects GLUT4 expression (Fig. 7
). No significant differences in GLUT4 content, as assessed by
immunoblot analysis of total crude membranes, were observed between
AdCMV-GKL-treated and untreated samples of whole gastrocnemius muscle.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 15)
. To limit the immunological response to adenovirus
(16)
, recombinant viruses were administered to newborn (2-
to 3-day-old) rats by i.m. injection to the hind limb. With this
approach, GK expression in muscle was maintained for 1 month in
multiple animals. This time frame of expression allowed us to study
animals at 1 month of age, which is relevant because at this stage rats
have an adult nutritional status, including expression of liver
glucokinase gene at adult levels (17)
. Considerable
variation in the expression levels of GK in the muscles injected with
AdCMV-GKL were noted in terms of GK protein content and increments in
total hexokinase activity of muscle extracts. In GK-positive muscles,
GK protein content varied in a range from 50% to 400% of the levels
present in the liver of 1-month-old rats. These differences are
attributable to the variability in the efficiency of gene delivery by
i.m. targeting of viruses.
We show that expression of GK in muscle results in improved
glucose disposal during an IPGTT and that this appeared to be related
to the increased glucose uptake capacity of GK-expressing muscle. A
clear impact of muscle GK expression on glucose homeostasis was found
only in the period immediately after glucose administration, whereas no
differences in basal glucose levels were found in fasted or fed
animals. Consistent with this, in vivo 2-deoxyglucose uptake
in GK-expressing muscles was higher than in control muscles only when
3-[3H]-2-DOG was administered during the
glucose clearance period. Therefore, our data show that by altering the
muscle glucose phosphorylating capacity, glucose tolerance rather than
basal glycemic control is altered. Moreover, we found that GK
expression in muscle only affected glucose clearance in fed rats. The
rate of whole body glucose utilization, which has been shown to
correspond to that of muscle glucose utilization, is a function of
circulating insulin and glucose concentrations (2)
.
Glucose transport and insulin stimulation of this process are
considered the rate-limiting steps of muscle glucose utilization,
although it has been proposed that a more distal step becomes
rate-limiting under hyperinsulinemic hyperglycemic conditions (1
, 2)
. These latter findings are consistent with those of the
current study, in which glucokinase expression in muscle was only able
to increase the rate of glucose extraction in fed animals. Furthermore,
transgenic mice overexpressing hexokinase II in skeletal muscle
exhibited no alterations in glucose tolerance or in fasting or fed
blood glucose levels relative to normal controls (4)
,
whereas isolated muscles from these transgenic animals exhibited a
marked increase in glucose uptake after maximal stimulation by insulin
(4)
. This line of reasoning may also be used to explain
our earlier findings, in which treatment of in vitro
cultures of human myocytes with AdCMV-GKL resulted in a marked
increment in glucose disposal that did not require insulin stimulation
(5)
. Muscle cells in vitro, unlike muscle
in vivo, express high levels of GLUT1, which resides in the
cell membrane, leading to a high glucose transport rate independent of
insulin stimulation. Therefore, these results all seem to indicate that
the metabolic impact of glucose phosphorylating enzymes in muscle is
limited to conditions in which glucose transport is highly stimulated
and exceeds glucose phosphorylation capacity.
Strikingly, our data show that delivery of GK in muscle of one hind
limb is sufficient to exert, under certain metabolic conditions, a
clear effect on whole body glucose disposal. When evaluating the
metabolic impact of this manipulation, we should consider that
1) the levels of expression of GK achieved in muscle are,
per mg of total protein, on the order of 0.5- to 4-fold the endogenous
expression in liver, and 2) in 1-month-old rats the total
dry weight of gastrocnemius from one hind limb is ~0.18 ± 0.005
g compared to the liver dry weight of ~1.5 g. Thus, in relative terms
the total GK levels achieved in gastrocnemius muscle represent 10 to
50% of the total GK content in liver. With respect to the distribution
of transgene, we found that i.m. injection of AdCMV-lacZ resulted in
detectable ß-galactosidase expression in an average of 40% of the
gastrocnemius muscle fibers. Since the major part of a glucose load is
known to be disposed of by muscle under conditions of hyperglycemia and
hyperinsulinemia (18)
, it is conceivable that expression
of GK via gene therapy in a significant fraction of fibers within a
single leg muscle could affect glucose tolerance. The precedent for
this idea comes from research on exercise training, which is a
physiological mechanism for increasing metabolic capacity of those
muscle fibers involved in the exercise (19
, 20)
. For
instance, training by running in a rodent treadmill predominantly
up-regulates glucose oxidation capacity in the muscles involved during
this exercise, namely, red gastrocnemius and soleus, while also
improving whole body insulin-stimulated glucose disposal
(19)
.
In summary, our results demonstrate that expression of GK in muscle leads to an enhanced capacity for muscle glucose disposal during IPGTT. A remarkable finding of this study is that engineering of just a fraction of the whole body mass for GK expression is sufficient for improving whole body glucose homeostasis. The relatively simple technique of adenovirus-mediated gene transfer into muscle can now be applied to the study of various animal models of non-insulin-dependent diabetes mellitus to determine whether GK expression enhances glucose clearance in the face of insulin resistance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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