| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 274, Issue 45, 31833-31838, November 5, 1999
§,¶,
,, and**
From the Department of Biochemistry and Molecular
Biology, University of Barcelona, E-08028 Barcelona, Spain and the
Gifford Laboratories for Diabetes Research, Departments of
Biochemistry and Internal Medicine, University of Texas
Southwestern Medical Center, Dallas, Texas 75235
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
Zucker diabetic fatty rats develop type 2 diabetes concomitantly with peripheral insulin resistance. Hepatocytes from these rats and their control lean counterparts have been cultured, and a number of key parameters of glucose metabolism have been determined. Glucokinase activity was 4.5-fold lower in hepatocytes from diabetic rats than in hepatocytes from healthy ones. In contrast, hexokinase activity was about 2-fold higher in hepatocytes from diabetic animals than in healthy ones. Glucose-6-phosphatase activity was not significantly different. Despite the altered ratios of glucokinase to hexokinase activity, intracellular glucose 6-phosphate concentrations were similar in the two types of cells when they where incubated with 1-25 mM glucose. However, glycogen levels and glycogen synthase activity ratio were lower in hepatocytes from diabetic animals. Total pyruvate kinase activity and its activity ratio as well as fructose 2,6-bisphosphate concentration and lactate production were also lower in cells from diabetic animals. All of these data indicate that glucose metabolism is clearly impaired in hepatocytes from Zucker diabetic fatty rats.
Glucokinase overexpression using adenovirus restored glucose metabolism
in diabetic hepatocytes. In glucokinase-overexpressing cells, glucose
6-phosphate levels increased. Moreover, glycogen deposition was greatly
enhanced due to the activation of glycogen synthase. Pyruvate kinase
was also activated, and fructose-2,6-bisphosphate concentration and
lactate production were increased in glucokinase-overexpressing diabetic hepatocytes. Overexpression of hexokinase I did not increase glycogen deposition. In conclusion, hepatocytes from Zucker diabetic fatty rats showed depressed glycogen and glycolytic metabolism, but
glucokinase overexpression improved their glucose utilization and storage.
Non-insulin-dependent diabetes mellitus
(NIDDM)1 is associated with
insulin secretory defects that occur together with insulin resistance
(1-3). The insensitivity to insulin and relative insulin deficiency in
NIDDM lead to a decrease in glucose utilization by liver, muscle, and
adipose tissue and to an increase in the hepatic glucose production
(4). Neither the role of hepatic metabolism in the development of type
2 diabetes nor its alterations are well established.
The development of diabetes in male Zucker diabetic fatty rats (ZDF
rats) has many features in common with human NIDDM (5). Male ZDF rats
develop progressive insulin resistance and glucose intolerance between
3 and 8 weeks of age and usually become completely diabetic between 8 and 10 weeks, whereas their lean counterparts (ZLC rats) develop
neither insulin resistance nor diabetes. Therefore, they are considered
a genetic model of type 2 diabetes. ZDF rats are hyperinsulinemic
throughout development.
In mammalian cells, glucose is phosphorylated to glucose 6-phosphate
(Glu-6-P) by members of the hexokinase family. Hepatocytes from healthy
rats contain mainly hexokinase IV, usually called glucokinase (GK), and
small amounts of hexokinases I, II, and III. GK differs from the rest
of hexokinases in that it is approximately half as large, it is not
allosterically inhibited by Glu-6-P, and it has a much higher
S0.5 for glucose (8 mM versus
50-100 µM). In recent studies, we have shown that
overexpression of GK, but not hexokinase I, in healthy Wistar rat
hepatocytes by adenovirus-mediated gene transfer leads to marked
enhancement of glycolysis and glycogen synthesis (6, 7). Other studies
in transgenic mice showed the same effect of GK overexpression in
vivo and concluded that its overexpression can correct
hyperglycemia in streptozotocin-treated mice, a model of IDDM (8,
9).
We studied various parameters of glucose metabolism in cultured
hepatocytes from ZDF rats and compared them with those determined from
hepatocytes isolated from ZLC rats. We show that hepatocytes from
Zucker diabetic fatty rats have a depressed glycogen and glucose
metabolism, but glucokinase overexpression improves their glucose
utilization and storage.
Preparation of Recombinant Adenoviruses--
Recombinant
adenoviruses containing the cDNA encoding rat liver GK (AdCMV-GKL)
and HK I (AdCMV-HKI) were prepared as described previously (10,
11).
Hepatocyte Isolation, Culture, and Transfection with Recombinant
Adenovirus--
Male ZDF (ZDF/Gmi-fa/fa) and ZLC
(ZLC/+/fa and +/+) rats
were purchased from Genetic Models (Indianapolis, IN). The animals were
used when they were 12-13 weeks old. At this age, the blood glucose
levels (9:00-10:00) were 24 ± 4 mM in ZDF and
5.2 ± 0.5 mM in ZLC rats. Hepatocytes were isolated
from 24-h fasted animals by collagenase perfusion as described (12).
Cells were suspended in Dulbecco's modified Eagle's medium
(Whittaker) supplemented with 10 mM glucose, 10% fetal
bovine serum (Whittaker), 100 nM insulin (Roche Molecular
Biochemicals), and 100 nM dexamethasone (Sigma) and seeded
onto plastic plates of 60-mm diameter treated with 0.1% gelatin
(Sigma) at a final density of 6 × 104
cell/cm2. After cell attachment (5 h at 37 °C),
hepatocytes were treated with stocks of AdCMV-GKL or AdCMV-HKI at a
multiplicity of infection of 10 for 2 h at 37 °C.
After treatment with the virus, the medium was replaced with
Dulbecco's modified Eagle's medium supplemented with 1 mM
glucose, 1 nM dexamethasone, and 1 nM insulin.
Cells were incubated for 16 h, and then experimental manipulations
were performed as detailed in the text and figure legends. At the end
of each experimental manipulation, cell monolayers were frozen in
liquid N2 until analysis.
Enzyme Activity Assay--
To measure enzyme activities, 100 µl of homogenization buffer consisting of 10 mM Tris-HCl
(pH 7.0), 150 mM KF, 15 mM EDTA, 600 mM sucrose, 15 mM 2-mercaptoethanol, 10 µg/ml
leupeptin, 1 mM benzamidine, and 1 mM
phenylmethylsulfonyl fluoride was added to frozen plates containing the
cell monolayers, and cells were collected with a plastic scraper. Cells
were lysed by thawing and checked under light microscopy for
completeness. Homogenates were collected in Eppendorf tubes and
centrifuged at 10,000 × g for 15 min at 4 °C, and
supernatants and pellets were used for determinations. Protein
concentration was measured as described by Bradford (13) using the
Bio-Rad assay reagent. Glucose-phosphorylating activity was measured
spectrophotometrically in 10,000 × g supernatant fractions of hepatocyte extracts at 37 °C in the presence of 100 mM and 0.5 mM glucose as described (14) The
phosphorylating capacity obtained at 0.5 mM glucose is
considered the hexokinase activity, while the subtraction of the
activity measured at 0.5 mM glucose from the activity
measured at 100 mM glucose is considered the glucokinase
activity of the extract. Glycogen synthase activity was measured in the
presence or absence of 6.6 mM Glu-6-P in both supernatant
and pellet fractions as described (15). The activity measured in the
absence of Glu-6-P represents the active form of the enzyme (I or a
form), whereas the activity tested in the presence of 6.6 mM Glu-6-P is a measure of total activity. The ratio of
these two activities is known as the glycogen synthase activity ratio
and is an estimate of the extent of the activation of the enzyme.
glucose-6-phosphatase activity was measured in both supernatant and
pellet fractions as described (16).
Pyruvate kinase (PK) activity was measured spectrophotometrically.
Frozen plates were scraped with 100 µl of a homogenization buffer at
pH 7.4 with 50 mM glycylglycine, 15 mM EDTA,
100 mM KF, and 5 mM potassium phosphate. The
homogenates were centrifuged at 10,000 × g for 15 min
at 4 °C, and total PK activity and activity ratio
(V0.15/V, measured at 0.15 and 5 mM phosphoenolpyruvate, respectively) were determined in
the supernatants as described (17).
Metabolite Determinations--
Glycogen content was measured by
scraping cells into 100 µl of 30% KOH, and boiling the extract for
15 min. Glycogen was measured as described (18). Fructose
2,6-bisphosphate (Fru-2,6-P2) levels were measured by
scraping cells into 300 µl of ice-cold 50 mM NaOH, and
incubating at 80 °C for 10 min. The homogenates were centrifuged at
10,000 × g for 15 min at 4 °C, and the content was
determined as described (19). The intracellular concentrations of
Glu-6-P were measured by spectrophotometric assays (20). L-Lactate levels in the incubation medium were measured as
described (21).
Statistical Methods--
Results are given as the mean ± S.E. for the indicated number of experiments. Comparisons among the
different experimental groups were carried out by unpaired Student's
t test. Differences were considered statistically
significant at p < 0.05.
Glucose-phosphorylating Capacity, Glucose-6-phosphatase Activity,
and Glu-6-P Levels in Hepatocytes from ZLC and ZDF Rats
Glucose-phosphorylating capacity and glucose-6-phosphatase
activity were measured in homogenates of hepatocytes from ZDF and ZLC
rats (Table I). GK activity, measured as
the difference between the glucose-phosphorylating capacity of the
extract assayed at 100 and 0.5 mM glucose, was about
4.5-fold lower in hepatocytes from ZDF rats than in hepatocytes from
ZLC rats (3.5 ± 0.4 milliunits/106 cells compared
with 16.2 ± 1.7 milliunits/106 cells). In contrast,
hexokinase activity, measured as the glucose-phosphorylating capacity
of the extract assayed at 0.5 mM glucose, was higher in ZDF
rats than in ZLC rats (6.5 ± 0.9 milliunits/106 cells
compared with 3.8 ± 0.6 milliunits/106 cells).
Glucose-6-phosphatase activity was not significantly different (Table
I). ZDF and ZLC hepatocytes preincubated for 16 h in 1 mM glucose and then transferred to media containing 1, 5, 10, and 25 mM glucose for 2 h showed a glucose
concentration-dependent increase in Glu-6-P levels (Fig.
1). Glu-6-P rose from 0.27 ± 0.04 nmol/106 cells and 0.38 ± 0.08 nmol/106
cells to 0.89 ± 0.09 nmol/106 cells and 0.81 ± 0.08 nmol/106 cells in ZLC and ZDF hepatocytes,
respectively, when glucose was increased from 1 to 25 mM.
Despite altered ratios of GK to hexokinase activity, no significant
differences in Glu-6-P concentration were observed between hepatocytes
from ZLC and ZDF rats. This probably reflects the higher efficiency of
hexokinase due to its lower Km for glucose.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Enzyme activities in ZLC and ZDF hepatocytes
View larger version (14K):
[in a new window]
Fig. 1.
Glu-6-P levels and glycogen content in ZLC
( 
) and ZDF (
) hepatocytes. ZDF and ZLC hepatocytes were
incubated for 16 h in the presence of 1 mM glucose.
Cells were then exposed to the concentrations of glucose indicated for
2 h and collected for measurement of intracellular Glu-6-P levels
(A) and glycogen content (B). Data represent the
mean ± S.E. for five independent experiments. *,
p < 0.01; **, p < 0.001 compared with
ZLC cells. a, p < 0.01 compared with cells
incubated with 1 mM glucose.
Glycolysis in Hepatocytes from ZDF and ZLC Rats
To determine the glycolytic flux of the hepatocytes from ZLC and
ZDF rats, lactate production, PK activity, and Fru-2,6-P2 concentration were analyzed. Lactate accumulation was measured using an
experimental approach identical to that described earlier for the
determination of Glu-6-P. We found a sharp
glucose-dependent increase in lactate production and in
Fru-2,6-P2 concentration in hepatocytes from ZLC rats but
not in hepatocytes from ZDF rats (Fig.
2). Lactate production in hepatocytes
from ZLC rats was higher than in hepatocytes from ZDF rats at all
glucose concentrations tested. Hepatocytes from ZDF rats hardly
modified their lactate production in response to an increase in glucose
concentration, and only a slight increase in Fru2,6P2
concentration was observed.
|
Total PK activity was 5.4-fold lower in ZDF hepatocytes than in ZLC
hepatocytes (Table I). The PK activity ratio was also lower in ZDF than
in ZLC hepatocytes (0.20 ± 0.03 versus 0.31 ± 0.04). This activity ratio was not altered when cells were incubated in
different glucose concentrations (Fig.
3). These results could explain the
difference in lactate accumulation observed in ZDF hepatocytes in
comparison with ZLC hepatocytes. PK is one of the rate-limiting steps
in glycolysis; therefore, a decrease in its activity is clearly
reflected in the overall flux of glycolysis.
|
Glycogen Synthesis in Hepatocytes from ZDF and ZLC Rats
To evaluate glycogen synthesis in ZDF and ZLC hepatocytes, a
number of assays were performed. We determined the glycogen content using an experimental approach identical to that described earlier for
the determination of Glu-6-P levels. A glucose
concentration-dependent increase in glycogen content in ZLC
hepatocytes was found. In contrast, ZDF hepatocytes deposited only
negligible amounts of glycogen in response to glucose (Fig. 1). In
light of this dramatic difference, we determined the activity of the
rate-limiting enzyme for glycogenesis, glycogen synthase. Total
glycogen synthase activity, measured in the presence of 6.6 mM Glu-6-P, was not significantly different in ZDF and ZLC
hepatocytes (2.0 ± 0.2 milliunits/106 cells
versus 2.1 ± 0.2 milliunits/106 cells).
Moreover, the amount of glycogen synthase protein measured by
immunoblotting was also the same in the two groups (data not shown). In
addition, glycogen phosphorylase activity was not significantly different in ZDF and ZLC hepatocytes (data not shown). The glycogen synthase activation state, calculated as the ratio of the enzyme activity measured in the absence of Glu-6-P to that in the presence of
Glu-6-P, was determined in the 10,000 × g supernatant
and pellet fractions of extracts prepared from hepatocytes treated as
described earlier for the Glu-6-P measurements. The glycogen synthase
(
Glu-6-P/+Glu-6-P) activity ratio increased in a glucose
concentration-dependent fashion in ZLC hepatocytes in both
pellet and supernatant fractions (Fig. 4,
A and B). In contrast, the ratio was
significantly lower in ZDF hepatocytes and did not change in response
to glucose either in the supernatant or in the pellet. These findings
explain their low capacity to synthesize glycogen.
|
Metabolic Effects of GK Overexpression in Hepatocytes from ZDF Rats
We have previously shown that GK overexpression greatly enhances glucose utilization and glycogen deposition in hepatocytes from healthy rats (6, 7). It was then interesting to study the impact of GK overexpression in hepatocytes of this genetic model of NIDDM and evaluate the possibility that GK overexpression could correct the metabolic alterations observed in ZDF rat hepatocytes.
GK Overexpression in ZDF Hepatocytes--
The efficiency of the
recombinant adenovirus for overexpression of GK in rat hepatocytes is
well documented (6, 7). AdCMV-GKL-treated ZDF hepatocytes showed a GK
activity of 168 ± 19 milliunits/106 cells. This value
was 48- and 10-fold higher than in untreated ZDF and ZLC hepatocytes,
respectively. Untreated and AdCMV-GKL-treated ZDF hepatocytes
preincubated for 16 h in 1 mM glucose and then transferred to media containing 1, 5, 10, and 25 mM glucose
for 2 h showed a glucose concentration-dependent
increase in Glu-6-P levels (Fig. 5).
AdCMV-GKL-treated cells had higher levels than untreated hepatocytes in
all conditions studied, but the effect was more pronounced at higher
glucose concentration. The maximal difference was observed at 25 mM glucose, where Glu-6-P intracellular concentration was
15-fold higher than in ZDF-untreated cells.
|
Effect of GK Overexpression in ZDF Hepatocytes on
Glycolysis--
In order to evaluate the effect of GK overexpression
in ZDF hepatocytes on the glycolytic flux, lactate production, PK
activity, and Fru-2,6-P2 concentration were determined.
AdCMV-GKL-treated cells produced about 3-fold more lactate and had
6-7-fold more Fru-2,6-P2 than untreated ZDF in all
conditions studied (Fig. 6). PK activity
ratio was higher in AdCMV-GKL-treated (0.33 ± 0.04) than in
untreated ZDF hepatocytes (0.20 ± 0.03) (Fig.
7B). Moreover, PK total
activity was also increased at high glucose concentrations in
GK-overexpressing cells (80 ± 6.8 milliunits/106 cells)
compared with untreated ones (57.5 ± 5.1 milliunits/106
cells) (Fig. 7B). These results may explain the enhanced
glycolytic flux observed in AdCMV-GKL-treated cells.
|
|
Effect of GK Overexpression in ZDF Hepatocytes on Glycogen
Synthesis--
Glycogen levels were 9-13-fold higher in
GK-overexpressing hepatocytes than in untreated cells in all conditions
studied (Fig. 5). Total glycogen synthase activity was not
significantly different in GK-overexpressing and untreated ZDF
hepatocytes (2.1 ± 0.1 milliunits/106 cells
versus 2.2 ± 0.2 milliunits/106 cells),
and the amount of glycogen synthase protein measured by immunoblotting
was also the same in the two groups (data not shown). However, in
GK-overexpressing ZDF hepatocytes, the capacity of glycogen synthase to
be activated in a glucose-dependent manner in both
supernatant and pellet fractions was restored. The glycogen synthase
(Glu-6-P/+Glu-6-P) activity ratio, determined in the 10,000 × g supernatant and pellet fractions, was much higher in AdCMV-GKL-treated cells than in untreated ZDF cells (Fig.
8, A and B). The
maximal difference in the activity ratio was observed at 25 mM glucose in supernatants (0.46 ± 0.02 versus 0.17 ± 0.02) and in pellets (0.48 ± 0.03 versus 0.16 ± 0.02). The higher activation state of
glycogen synthase in AdCMV-GKL-treated ZDF hepatocytes explains their
enhanced capacity to synthesize glycogen in comparison with untreated
ZDF hepatocytes.
|
Metabolic Effects of HK I Overexpression in Hepatocytes from ZDF Rats
In our previous work (6, 7), we observed that HK I overexpression did not affect glycogen deposition in hepatocytes from healthy rats. We thus attempted to determine whether HK overexpression affects glycogen in hepatocytes from ZDF rats.
The efficiency of AdCMV-HKI for overexpression of HK I in rat hepatocytes is well known (6, 7). AdCMV-HKI-treated ZDF hepatocytes showed a 4-fold increase in HK activity. Glycogen levels were not significantly different in HK I-overexpressing hepatocytes and untreated cells (Table II) at any of the concentrations of glucose tested.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
ZDF rats develop type 2 diabetes concomitantly with peripheral
insulin resistance. Although a good deal of information about the
-cell function of this model is available, much less is known about
the carbohydrate metabolism in the liver of these animals. In this
study, we have analyzed the response to glucose of cultured hepatocytes
from ZDF rats in comparison with ZLC hepatocytes, their nondiabetic
counterparts. ZDF hepatocytes clearly showed a decrease in their
capacity to store glucose as glycogen and a fall in glycolytic flux.
These result in a low rate of glucose utilization, which is a
characteristic pattern of a diabetic phenotype. These defects are
probably a consequence of the low activity of GK and PK in these cells.
Decreased GK and PK activities have also been observed in the liver of
ZDF rats,2 and these
alterations are preserved when hepatic cells are isolated and cultured.
This indicates that ZDF cultured hepatocytes are a good model to study
the alterations of carbohydrate metabolism in these animals.
In the liver, glucose is stored as glycogen. The synthesis of this polysaccharide is a process that contributes to control of glycemia in the postprandial state. NIDDM is associated with an impairment of glycogen synthesis, which results in a decrease of glucose utilization by the liver, contributing to hyperglycemia. ZDF rat hepatocytes show a clear decrease in the ability to synthesize glycogen, although they have no alteration in total glycogen synthase activity. As described (22-24), glucose induces the activation of liver glycogen synthase through an increase in Glu-6-P, which causes the covalent activation of glycogen synthase, thus leading to an increase in glycogen accumulation. Although ZDF and ZLC hepatocytes show similar increases in the intracellular levels of Glu-6-P when incubated with glucose, glycogen synthase is normally activated in ZLC, while it remains essentially inactive in ZDF rat hepatocytes. A critical observation is that GK activity is very low in ZDF cells, while conversely HK activity is higher. Therefore, in ZLC hepatocytes Glu-6-P is mainly produced by GK, whereas in ZDF cells glucose phosphorylation is catalyzed mainly by other hexokinases. These data provide further support for our recent proposal of a differential regulatory impact of Glu-6-P produced by hexokinase versus GK in liver cells (7). Since Glu-6-P is mainly produced by hexokinase in ZDF hepatocytes, this metabolite is not able to trigger activation of glycogen synthase and for this reason glucose does not induce glycogen synthesis. All of these results suggest that GK plays a key role in the activation of glycogen synthase and that the lack of GK activity is most probably responsible for the deficiency in glycogen synthesis of ZDF hepatocytes.
Further support for this hypothesis was obtained by GK overexpression in ZDF hepatocytes. The increase in the intracellular levels of Glu-6-P produced by GK in the diabetic cells led to the activation of glycogen synthase and to an increase in glycogen accumulation, as we have observed in healthy hepatocytes (7). Therefore, GK overexpression is able to rescue the ability of ZDF cells to synthesize glycogen in response to glucose. In clear agreement with our previous results (6, 7) overexpression of HK I was without effect on glycogen deposition.
On the other hand, glycolytic flux is also decreased in ZDF hepatocytes. Lactate production is much lower in ZDF hepatocytes than in ZLC hepatocytes. This result can be explained by the observed deficit in total and active PK activity and the decrease in the levels of Fru-2,6-P2. This metabolite controls a key step of glycolysis regulating the activity of phosphofructose kinase-1 and phosphofructose phosphatase-1. PK is also one of the limiting steps of glycolysis. PK activity is 5-6-fold lower in ZDF hepatocytes than in ZLC hepatocytes, and, moreover, its activation state is also lower.
GK-overexpressing cells showed an increased glycolytic flux. Lactate production was 3-fold higher in ZDF hepatocytes treated with AdCMV-GKL. This result can be explained by the sum of several factors. First, the increase in GK activity produced a great increase in Glu-6-P levels that exerted a push effect and also resulted in an increase in the levels of Fru-2,6-P2, which raised the flux through phosphofructose kinase-1. Second, in GK-overexpressing cells, PK total activity as well as its activation state were increased. This increase in PK total activity can be explained by an enhancement in PK gene transcription by Glu-6-P as described in Refs. 25 and 26. The net result of all of these factors would be an increase of the rate of lactate production.
In summary, GK overexpression induced an increase in glucose utilization both by triggering glucose storage into glycogen and by increasing glycolysis. This finding indicates that GK overexpression can overcome the deficiency in glucose utilization characteristic of the hepatocytes isolated from ZDF rats and suggests gene therapy strategies that may be relevant to type 2 diabetes. In normal fasted animals, the liver has a net glucose synthesis and output, and after feeding it switches to net glucose uptake and storage. In type 2 diabetes, the liver fails to respond to postprandial increases in glucose. Studies in a variety of animal models of diabetes have led to the suggestion that lowering of the GK/glucose-6-phosphatase enzyme activity ratio, whether by a decrease in GK, an increase in glucose-6-phosphatase, or a combination of the two, may impair hepatic glucose metabolism and contribute to the etiology of diabetes (27-29). In our model of type 2 diabetes, ZDF rats, we also observed a decrease in the GK/glucose-6-phosphatase ratio caused by the significant decrease in GK activity. Our results suggest that GK supplementation in ZDF rats may enhance hepatic glucose clearance, possibly to an extent that will result in a decrease in circulating glucose concentration.
However, a cautionary note in this regard comes from a recent study in
normal rats in which adenovirus-mediated overexpression of glucokinase
in liver resulted in lowering of blood glucose, but with accompanying
increases in free fatty acid and triglyceride levels (30). It remains
to be determined whether similar complications will arise when
glucokinase is overexpressed in liver of NIDDM models such as the ZDF rat.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Anna Adrover for skilled technical assistance, Dr. Joan C. Ferrer and Dr. Luisa María Lois for helpful suggestions, and R. Rycroft for assistance in preparing the English manuscript.
| |
FOOTNOTES |
|---|
* This study was supported by Spanish government Grant DGICYT PB96-0992 (to J. J. G.) and National Institutes of Health Grant P50H2598801 (to C. B. N.).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.
§ Recipient of an Formación de Personal Investigador fellowship from the Spanish government.
¶ Recipient of an Formació d'Investigadors fellowship from the Generalitat de Catalunya.
** To whom correspondence should be addressed: Dept. de Bioquímica i Biologia Molecular, Facultat de Química, Universitat de Barcelona, Martí i Franquès, 1, E-08028 Barcelona, Spain. Tel.: 34-93-4021206; Fax: 34-93-4021219; E-mail: guino@sun.bq.ub.es.
2 A. Barberà and J. J. Guinovart, unpublished data.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: NIDDM, non-insulin-dependent diabetes mellitus; ZDF, Zucker diabetic fatty; ZLC, Zucker lean control; HK, hexokinase, GK, glucokinase; PK, pyruvate kinase; AdCMV-GKL, recombinant adenovirus containing the cDNA of rat liver glucokinase; AdCMV-HKI, , recombinant adenovirus containing the cDNA of hexokinase I.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Porte, D., Jr. (1991) Diabetes 40, 166-180[Abstract] |
| 2. | Defronzo, R. A., Bonadonna, R. C., and Ferrannini, E. (1992) Diabetes Care 15, 318-368[Abstract] |
| 3. | O'Meara, N. M., and Polonsky, K. S. (1994) in Joslin's Diabetes Mellitus (Kahn, C. R. , and Weir, G. C., eds) , pp. 81-96, Lea and Febiger, Philadelphia |
| 4. |
McGarry, J. D.
(1992)
Science
258,
766-770 |
| 5. | Peterson, R. G., Shaw, W. N., Neel, M.-A., Little, L. A., and Eichberg, J. (1990) ILAR News 32, 16-19 |
| 6. |
O'Doherty, R. M.,
Lehman, D. L.,
Seoane, J.,
Gómez-Foix, A. M.,
Guinovart, J. J.,
and Newgard, C. B.
(1996)
J. Biol. Chem.
271,
20524-20530 |
| 7. |
Seoane, J.,
Gómez-Foix, A. M.,
O'Doherty, R. M.,
Gómez-Ara, A. M.,
Newgard, C. B.,
and Guinovart, J. J.
(1996)
J. Biol. Chem.
271,
23756-23760 |
| 8. |
Ferré, T.,
Pujol, A.,
Riu, E.,
Bosch, F.,
and Valera, A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7225-7230 |
| 9. | Hariharan, N., Farrelly, D., Hagan, D., Hillyer, D., Arbeeny, C., Sabrah, T., Treloar, A., Brown, K., Kalinowski, S., and Mookhtiar, K. (1997) Diabetes 46, 11-16[Abstract] |
| 10. |
Becker, T. C.,
BeltrandelRio, H.,
Noel, R. J.,
Johnson, J. H.,
and Newgard, C. B.
(1994)
J. Biol. Chem.
269,
21234-21238 |
| 11. |
Becker, T. C.,
Noel, R. J.,
Johnson, J. H.,
Lynch, R. M.,
Hirose, H.,
Tokuyama, Y.,
Bell, G. I.,
and Newgard, C. B.
(1996)
J. Biol. Chem.
271,
390-394 |
| 12. | Massague, J., and Guinovart, J. J. (1977) FEBS Lett. 82, 317-320[CrossRef][Medline] [Order article via Infotrieve] |
| 13. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
| 14. |
Kuwajima, M.,
Newgard, C. B.,
Foster, D. W.,
and McGarry, J. D.
(1986)
J. Biol. Chem.
261,
8849-8853 |
| 15. | Thomas, J. A., Schlender, K. K., and Larner, J. (1968) Anal. Biochem. 25, 486-499[CrossRef][Medline] [Order article via Infotrieve] |
| 16. | Alegre, M., Ciudad, C. J., Fillat, C., and Guinovart, J. J. (1988) Anal. Biochem. 173, 185-189[CrossRef][Medline] [Order article via Infotrieve] |
| 17. | Feliu, J. E., Hue, L., and Hers, H. G. (1977) Eur. J. Biochem. 81, 609-617[Medline] [Order article via Infotrieve] |
| 18. | Chan, T. M., and Exton, J. H. (1976) Anal. Biochem. 71, 96-105[CrossRef][Medline] [Order article via Infotrieve] |
| 19. | Van Shaftingen, E., Lederer, B., Bartrons, R., and Hers, H. G. (1982) Eur. J. Biochem. 129, 191-195[Medline] [Order article via Infotrieve] |
| 20. | Lang, G., and Michal, G. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed), Vol. 3 , pp. 1238-1242, Academic Press, Inc., New York |
| 21. | Gutmann, I., and Wahlefel, A. W. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed), Vol. 3 , pp. 1464-1468, Academic Press, Inc., New York |
| 22. | Ciudad, C. J., Carabaza, A., and Guinovart, J. J. (1986) Biochem. Biophys. Res. Commun. 141, 1195-1200[CrossRef][Medline] [Order article via Infotrieve] |
| 23. | Carabaza, A., Ciudad, C. J., Baque, S., and Guinovart, J. J. (1992) FEBS Lett. 296, 211-214[CrossRef][Medline] [Order article via Infotrieve] |
| 24. |
Massillon, D.,
Bollen, M.,
DeWulf, H.,
Overloop, K.,
Vanstapel, F.,
Van Hecke, P.,
and Stalmans, W.
(1995)
J. Biol. Chem.
270,
19351-19356 |
| 25. | Vaulont, S., and Kahn, A. (1994) FASEB J. 8, 28-35[Abstract] |
| 26. | Girard, J., Perdereau, D., Foufelle, F., Prip-Buus, C., and Ferré, P. (1994) FASEB J. 8, 36-42[Abstract] |
| 27. | Burchell, A., and Cain, D. I. (1985) Diabetologia 28, 852-856[CrossRef][Medline] [Order article via Infotrieve] |
| 28. |
Barzilai, N.,
and Rossetti, L.
(1993)
J. Biol. Chem.
268,
25019-25025 |
| 29. |
Massillon, D.,
Barzilai, N.,
Chen, W.,
Hu, M.,
and Rossetti, L.
(1996)
J. Biol. Chem.
271,
9871-9874 |
| 30. | O'Doherty, R. M., Lehman, D. L., Telemaque-Potts, S., and Newgard, C. B. (1999) Diabetes 48, 2022-2027[Abstract] |
This article has been cited by other articles:
|
|
 |
|
  D. Cifuentes, C. Martinez-Pons, M. Garcia-Rocha, A. Galina, L. R. de Pouplana, and J. J. Guinovart Hepatic Glycogen Synthesis in the Absence of Glucokinase: THE CASE OF EMBRYONIC LIVER J. Biol. Chem., February 29, 2008; 283(9): 5642 - 5649. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Fujimoto, E. P. Donahue, and M. Shiota Defect in glucokinase translocation in Zucker diabetic fatty rats Am J Physiol Endocrinol Metab, September 1, 2004; 287(3): E414 - E423. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Shao, L. Qiao, and J. E. Friedman Prolactin, progesterone, and dexamethasone coordinately and adversely regulate glucokinase and cAMP/PDE cascades in MIN6 {beta}-cells Am J Physiol Endocrinol Metab, February 1, 2004; 286(2): E304 - E310. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Gao, K. Walder, T. Sunderland, L. Kantham, H. C. Feng, M. Quick, N. Bishara, A. de Silva, G. Augert, J. Tenne-Brown, et al. Elevation in Tanis Expression Alters Glucose Metabolism and Insulin Sensitivity in H4IIE Cells Diabetes, April 1, 2003; 52(4): 929 - 934. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. R. Gomis, E. Cid, M. Garcia-Rocha, J. C. Ferrer, and J. J. Guinovart Liver Glycogen Synthase but Not the Muscle Isoform Differentiates between Glucose 6-Phosphate Produced by Glucokinase or Hexokinase J. Biol. Chem., June 21, 2002; 277(26): 23246 - 23252. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hawkins, I. Gabriely, R. Wozniak, C. Vilcu, H. Shamoon, and L. Rossetti Fructose Improves the Ability of Hyperglycemia Per Se to Regulate Glucose Production in Type 2 Diabetes Diabetes, March 1, 2002; 51(3): 606 - 614. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. J. Desai, E. D. Slosberg, B. R. Boettcher, S. L. Caplan, B. Fanelli, Z. Stephan, V. J. Gunther, M. Kaleko, and S. Connelly Phenotypic Correction of Diabetic Mice by Adenovirus-Mediated Glucokinase Expression Diabetes, October 1, 2001; 50(10): 2287 - 2295. [Abstract] [Full Text]
|
|
|
|
|
|
A. Basu, R. Basu, P. Shah, A. Vella, C. M. Johnson, M. Jensen, K. S. Nair, W. F. Schwenk, and R. A. Rizza Type 2 Diabetes Impairs Splanchnic Uptake of Glucose but Does Not Alter Intestinal Glucose Absorption During Enteral Glucose Feeding: Additional Evidence for a Defect in Hepatic Glucokinase Activity Diabetes, June 1, 2001; 50(6): 1351 - 1362. [Abstract] [Full Text]
|
|
|
|
|
|
M. C. Muñoz, A. Barberà, J. Domínguez, J. Fernàndez-Alvarez, R. Gomis, and J. J. Guinovart Effects of Tungstate, a New Potential Oral Antidiabetic Agent, in Zucker Diabetic Fatty Rats Diabetes, January 1, 2001; 50(1): 131 - 138. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
) or
left untreated (
