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Diabetes Research and Training Center, Departments of Medicine and Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Glucokinase (GK) catalyzes the phosphorylation
of glucose in 
-cells and hepatocytes, and mutations in the GK gene
have been implicated in a form of human diabetes. To investigate the
relative role of partial deficiencies in the hepatic vs. pancreatic GK activity, we examined insulin secretion, glucose disposal, and hepatic
glucose production (HGP) in response to hyperglycemia in transgenic
mice 1) with one disrupted GK
allele, which manifest decreased GK activity in both liver and
-cells (GK+/
), and 2) with
decreased GK activity selectively in -cells (RIP-GKRZ). Liver GK
activity was decreased by 35-50% in the GK+/ but not in
the RIP-GKRZ compared with wild type (WT) mice. Hyperglycemic clamp
studies were performed in conscious mice with or without concomitant
pancreatic clamp. In all studies
[3-3H]glucose was
infused to measure the rate of appearance of glucose and HGP during 80 min of euglycemia (Glc ~5 mM) followed by 90 min of hyperglycemia
(Glc ~17 mM). During hyperglycemic clamp studies, steady-state plasma
insulin concentration, rate of glucose infusion, and rate of glucose
disappearance (Rd) were
decreased in both GK+/ and RIP-GKRZ compared with WT mice.
However, whereas the basal HGP (at euglycemia) averaged ~22
mg · kg
1 · min1
in all groups, during hyperglycemia HGP was suppressed by only 48% in
GK+/ compared with ~70 and 65% in the WT and RIP-GKRZ mice,
respectively. During the pancreatic clamp studies, the ability of
hyperglycemia per se to increase
Rd was similar in all groups. However, hyperglycemia inhibited HGP by only 12% in GK+/, vs. 42 and 45%, respectively, in the WT and RIP-GKRZ mice. We conclude that, although impaired glucose-induced insulin secretion is common to
both models of decreased pancreatic GK activity, the marked impairment
in the ability of hyperglycemia to inhibit HGP is due to the specific
decrease in hepatic GK activity.
transgenic mice; glucose cycling; gluconeogenesis; glycogen; maturity-onset diabetes of the young
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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GLUCOKINASE (GK) is a low-affinity hexokinase that is
largely responsible for glucose phosphorylation in pancreatic -cells and hepatocytes (24). In -cells, GK catalyzes a rate-limiting step
in glucose metabolism and is considered a "glucose sensor" for
regulation of insulin secretion by extracellular glucose. Partial
deficiencies in the hepatic glucose phosphorylation capacity occur in
humans with diabetes mellitus and may contribute to its pathophysiology
(6). Mutations in the GK gene are responsible for a form of
maturity-onset diabetes of the young (MODY 2) (13). Although in the
case of MODY 2 this enzymatic defect is inherited (13), it may also be
acquired secondarily to associated hormonal and metabolic alterations
in some patients with insulin-dependent diabetes mellitus (8, 19) and
in others with non-insulin-dependent diabetes mellitus (6). The rate of
hepatic glucose phosphorylation is largely dependent on the mass effect
of glucose, i.e., its portal concentration, and on the in vivo activity
of GK (26, 27, 32). Acutely, hyperglycemia per se promotes hepatic
glucose uptake, decreases net liver glycogenolysis, and inhibits
hepatic glucose production (HGP) (26-28). We have suggested that
the increase in the rates of hepatic glucose phosphorylation, which is
induced by hyperglycemia, is pivotal to its ability to inhibit HGP
(26).
In a recent communication (1) we reported that the disruption of one
allele of the GK gene (GK+/) in mice resulted in a modest
decrease (~30%) in the level of gene expression and/or activity of hepatic GK and in a decreased response of HGP and direct
pathway of glycogen synthesis to acute changes in the plasma glucose
concentrations (1). However, the presence of a concomitant partial
deficit in the -cell GK and the lower plasma insulin concentrations
during the hyperglycemic clamp studies could have accounted for some or
all of the hepatic metabolic alterations in this mouse model (1, 10).
In fact, chronic changes in the circulating insulin levels may have
been sufficient to alter the hepatic gene expression and activity of GK
and other key hepatic enzymes. Furthermore, the differences in the
circulating plasma insulin concentrations observed during the in vivo
studies complicate the interpretation of the glucose flux data. In the
present study we aimed to investigate the specific regulatory
components of whole body glucose homeostasis that are affected by
long-term partial deficits in the activities of GK in the -cells of
the pancreas and in the parenchymal cells of the liver. In particular, we wished to discern whether the metabolic changes in hepatic glucose
fluxes and in their responses to changes in extracellular glucose
levels, which are present in mice and humans with whole body GK
deficiency, are due to a specific hepatic defect or are secondary to
chronic and/or short-term alterations in circulating insulin
levels. To this end, the responses of insulin secretion and of hepatic
and peripheral glucose fluxes to standardized hyperglycemic challenges
were compared in two transgenic models with a moderate deficit in
glucose phosphorylation, selectively in the pancreatic -cells or in
both liver and pancreas. Thus mice with a disrupted GK allele
(GK+/) (1) were compared with a mouse model with a selective
disruption of GK gene expression and activity in -cells that was
generated with a GK ribozyme (10). Additionally, we made use of a
pancreatic clamp technique that allowed us to investigate the role of a
sustained decrease in liver GK in the regulation of hepatic glucose
fluxes in the presence of similar and fixed pancreatic hormone levels
(26).
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Animals
Three groups of male mice were studied with two experimental protocols. Group 1 included 11 control (C57BL/6J) mice (Jackson Breeding Laboratories, Bar Harbor, ME); group 2 included 16 GK+/ mice
(1); and group 3 included 14 RIP-GKRZ
mice (10). Figure 1 provides a schematic
representation of the two experimental protocols.
Protocol 1 consisted of a
hyperglycemic clamp study (n = 6 or
more for each group). Protocol 2 combined pancreatic and hyperglycemic clamp studies
(n = 5 or more for each group).
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At 4-6 mo of age, all mice (28-35 g) were anesthetized with chloral hydrate (400 mg/kg of body wt ip), and an indwelling catheter was inserted into the right internal jugular vein, as previously described (23, 25). The venous catheter was used for the multiple infusions; blood samples were obtained from the tail vein. Mice were studied 4-6 days postsurgery.
Euglycemic and Hyperglycemic Clamp Studies
Studies were performed in awake, unrestrained, chronically catheterized mice by use of the pancreatic and hyperglycemic clamp techniques (1, 23, 25, 26) in combination with high-performance liquid chromatography (HPLC)-purified [3-3H]glucose and [U-14C]lactate infusions, as previously described (15, 26). Food was removed for 8 h before the in vivo study infusions. All studies lasted 170 min and included an 80-min euglycemic period for assessment of basal turnover rates and a 90-min hyperglycemic clamp period. Eighty minutes before the start of glucose, insulin, and/or somatostatin infusions, a prime-continuous infusion of HPLC-purified [3-3H]glucose (New England Nuclear, Boston, MA; 10 µCi bolus and 0.1 µCi/min) was initiated and maintained throughout the remainder of the study. [U-14C]lactate (5 µCi bolus and 0.25 µCi/min) was infused during the last 10 min of the study.Protocol 1. Briefly, a variable infusion of a 25% glucose solution was started at time 0 and periodically adjusted to sequentially clamp the plasma glucose concentration at ~5 mM for 80 min (euglycemic period) and at ~17 mM for 90 min (hyperglycemic period).
Protocol 2.
Briefly, a primed-continuous infusion of somatostatin (3 µg · kg1 · min1),
insulin (~0.3
mU · kg1 · min1),
and glucagon (5 ng · kg1 · min1)
was administered, and a variable infusion of a 25% glucose solution was started at time 0 and periodically
adjusted to sequentially clamp the plasma glucose concentration at ~5
mM for 80 min (euglycemic period) and at ~17 mM for 90 min
(hyperglycemic period).
30, 0, 20, 40, 60, 75, and 90 min during the study. Additional plasma samples for the determination of plasma glucose concentration (~20 µl) were obtained at 40 and
20 min and at 10-min intervals thereafter. The total volume of
blood withdrawn was ~0.9 ml/study; to prevent volume depletion and
anemia, a solution (1:1, vol/vol) of ~1.2 ml of fresh blood (~0.6
ml obtained by heart puncture from littermates of the test animals) and
heparinized saline (10 U/ml) was infused at a rate of 7 µl/min.
Furthermore, after larger samples at time
0 and 40, 60, and 90 min, red blood cells were
resuspended in saline and immediately returned through the infusion
catheter. All determinations were also performed on portal vein blood
obtained at the end of the experiment. To minimize stress during the
sampling procedures, all mice were accustomed to handling and tail
sampling, were freely moving within a large cage, and were allowed
sufficient time for postsurgical recovery (4 days or more). Blood
samples for assessment of glucose concentration were obtained every 10 min from a cut at the tip of the tail (1, 23, 25).
At the end of the in vivo studies, mice were anesthetized
(pentobarbital sodium, 60 mg/kg body wt iv), the abdomen was quickly opened, portal vein blood was obtained, and the liver was
freeze-clamped in situ with aluminum tongs precooled in liquid
nitrogen. The time from the injection of the anesthetic until
freeze-clamping of the liver was <45 s. All tissue samples were
stored at 80°C for subsequent analysis.
The study protocol was reviewed and approved by the Institutional
Animal Care and Use Committee of the Albert Einstein College of
Medicine.
Analytic Procedures
Plasma glucose was measured by the glucose oxidase method (Glucose Analyzer II, Beckman Instruments, Palo Alto, CA). Plasma insulin was measured by radioimmunoassay with use of rat and porcine insulin standards. Plasma [3H]glucose radioactivity was measured in duplicates in the supernatants of Ba(OH)2 and ZnSO4 precipitates (Somogyi procedure) of plasma samples (25 µl) after evaporation to dryness to eliminate tritiated water. Uridine 5'-diphosphate-glucose (UDP-Glc), uridine 5'-diphosphate-galactose (UDP-Gal), and phosphoenolpyruvate (PEP) concentrations and specific activities in the liver were obtained through two sequential chromatographic separations, as previously reported (14, 15, 23, 26).GK Activity
Hepatic GK activity was measured by a continuous spectrophotometric method (3, 9, 26). Liver homogenates (~200 mg) were prepared in (mM) 50 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 100 KCl, 1 EDTA, 5 MgCl2, and 2.5 dithioerythritol. Homogenates were centrifuged at 100,000 g for 45 min to sediment the microsomal fraction. The postmicrosomal fraction was assayed at 37°C in a medium containing 50 mM HEPES (pH 7.4), 100 mM KCl, 7.5 mM MgCl2, 5 mM ATP, 2.5 mM dithioerythritol, 10 mg/ml albumin, and glucose at 0.5 mM (for hexokinases activity), 18 mM (for GK activity at in vivo glucose levels), or 100 mM (maximal glucose phosphorylation capacity), 0.5 mM NAD+, 4 U glucose-6-phosphate 1-dehydrogenase (L. mesenteroides), and the equivalent of ~1 mg of wet liver. The reaction was initiated by the addition of ATP, and the rate of NAD+ reduction was measured at 340 nm. Glucose phosphorylation was determined as the absorbency change in the complete medium minus the absorbency change in the absence of ATP, under conditions in which the absorbency is increasing linearly with time (usually from 20 to 40 min). Kinetic analysis of GK was also performed at glucose concentrations of 0.5, 8, 10, 15, 18, 25, and 100 mM from livers obtained at the end of the in vivo studies.Calculations
Under steady-state conditions for plasma glucose concentrations, the rate of glucose disappearance (Rd) equals the rate of glucose appearance (Ra). The latter was calculated as the ratio of the rate of infusion of [3-3H]glucose [disintegra-tions · min1
(dpm) · min1]
and the steady-state plasma
[3H]glucose specific
activity (dpm/mg). When exogenous glucose was given, the rate of
endogenous glucose production was calculated as the difference between
Ra and the infusion rate of
glucose (GIR). The percentage of the hepatic glucose 6-phosphate pool directly derived from plasma glucose was calculated as the ratio of
[3H]UDP-Glc and plasma
[3H]glucose specific
activities. The percentage of the hepatic glucose 6-phosphate pool
derived from PEP gluconeogenesis was calculated as the ratio of the
specific activities of
[14C]UDP-Glc and
2× [14C]PEP
after in vivo labeling with
[U-14C]lactate (15,
26).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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General Characteristics of the Experimental Animals
Three groups of conscious male mice were studied: 11 wild type (WT) controls, 16 mice with one GK allele disrupted by homologous recombination in mouse embryonic stem cells (GK+/), and 14 transgenic mice whose GK activity was attenuated specifically in the
-cells of the pancreas by a GK ribozyme approach (RIP-GKRZ). Some of the data obtained in 5 of the 16 GK+/ mice were included in a previous publication (1) and are reported here solely to facilitate comparison with the RIP-GKRZ mice. There were no differences in the
mean body weights among the three groups of mice (Table
1). After a ~6-h fast
(postabsorptive state), the plasma glucose and insulin concentrations
and the basal rate of HGP were also similar in the three experimental
groups (Table 1).
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Impact of Genotype on Hepatic GK Activity
We examined the kinetic parameters of hepatic GK in extracts prepared from liver samples obtained at the completion of the in vivo studies. Because the insulin promoter targets the ribozyme to the pancreatic-cells, GK gene expression and activity should not be affected in
the liver of RIP-GKRZ mice, whereas the disruption of one allele of the
GK gene is expected to result in a reduction in the hepatic enzymatic
activity. Indeed, as shown in Table 2, hepatic GK maximum velocity
(Vmax) was
significantly decreased in the GK+/ mice in liver samples
obtained at the completion of both protocol
1 (by 35% vs. WT) and protocol
2 (by 50% vs. WT). Conversely, GK activity was
unchanged in the RIP-GKRZ group compared with WT. GK
Km was similar
(~12 mM) in all groups (Table 2). Thus these two transgenic models
differ considerably in their glucose phosphorylation capacity in the
liver, and their comparison should allow one to gain insight into the
specific impact of a decrease in hepatic GK per se on whole body and
hepatic glucose fluxes and their regulation by hyperglycemia.
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Effect of Hyperglycemia on Insulin Secretion and Glucose Fluxes
Protocol 1 was designed to examine the effect of a similar increase in the circulating glucose concentrations on insulin secretion and on peripheral and hepatic glucose fluxes. The steady-state plasma glucose concentration averaged ~5.5 mM during the euglycemic period, whereas it was raised by ~12 mM during the hyperglycemic period. Steady-state conditions for plasma glucose concentration and specific activity were achieved within 40 min during the basal and clamp periods of the studies. However, the steady-state plasma insulin concentration was significantly decreased in the two groups of mice with decreased GK activity in the pancreatic-cells
(Table 3). Thus, in keeping with previous
observations in conscious mice (1) and the perfused pancreas (10), the
ability of an increase in glucose concentration to elicit insulin
secretion was partially impaired in both GK+/ and RIP-GKRZ mice.
Interestingly, the decline in the plasma insulin concentration compared
with WT was more evident and reproducible in the RIP-GKRZ mice than in
the GK+/ mice. The latter may reflect our previous finding of a
~70% decrease in islet GK activity in the RIP-GKRZ model (10)
compared with a ~40% decrease in islet GK activity in the GK+/ model (1). Our experimental approach does not allow one to
identify the glycemic threshold at which the defect in GK activity results in decreased insulin secretion.
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The whole body Rd and the GIR
during the hyperglycemic clamp study are depicted in Fig.
2. GIR and
Rd were decreased by ~30% in
both GK+/ and RIP-GKRZ mice compared with WT
(P < 0.01 for all). Figure
3 depicts the rate of glucose production
during the basal period and its suppression by hyperglycemia and
endogenous hyperinsulinemia. Basal HGP was similar and averaged ~22
mg · kg1 · min1
in the three experimental groups. In contrast, HGP during the hyperglycemic clamp studies was significantly (63%) higher in the
GK+/ mice compared with the WT. Similarly, when the inhibition of HGP during the hyperglycemic clamp was expressed as a percent decrease from basal levels, it was significantly impaired in
GK+/ mice (48% inhibition) compared with WT (70% inhibition;
P < 0.01). Conversely, the absolute
rate of HGP and the percent inhibition from basal were unchanged in the
RIP-GKRZ mice (Fig. 3).
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Effect of Hyperglycemia Per Se on Glucose Fluxes
The steady-state plasma glucose concentration was kept at the basal level (~5.5 mM) during the euglycemic period, whereas it was raised by ~12 mM during the hyperglycemic period (protocol 2 in Table 3). Steady-state conditions for plasma glucose concentration and specific activity were achieved within 40 min during the basal and clamp periods of the studies. In this protocol, however, the plasma insulin concentration was kept at similar levels (~9 ng/ml) in all groups during the hyperglycemic clamp studies, allowing us to discern the metabolic responses to hyperglycemia per se. Under these experimental conditions the whole body Rd was similar in the three experimental groups (Fig. 4A). However, the average GIR required to maintain the target hyperglycemic level during the last 50 min of the clamp study was significantly less in the GK+/ mice than in either WT or RIP-GKRZ mice (Fig.
4B). HGP was significantly and
similarly inhibited by ~45% in response to hyperglycemia in WT and
RIP-GKRZ (Fig. 5). In contrast,
hyperglycemia caused only a 12% decline in HGP in GK+/ mice,
and the rate of HGP was 45% higher in the latter group compared with
WT (Fig. 5; P < 0.01). These data
support the hypothesis that hepatic glucose phosphorylation fails to
properly adapt to increased circulating glucose levels, leading to the
blunting of the suppression of HGP by hyperglycemia in the GK+/
mice. Estimates of the relative contributions of the direct
phosphorylation of glucose and of gluconeogenesis to the hepatic
glucose 6-phosphate pool can be derived by assessment of the specific
activities of hepatic substrates after the infusion of labeled lactate
and glucose (15, 23, 26).
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Effect of Reduced GK Activity on Stimulation of the "Direct Pathway" of Liver UDP-Glc Formation by Hyperglycemia
An impairment in hepatic glucose phosphorylation may affect the relative contribution of plasma glucose to the hepatic glucose 6-phosphate pool. Table 4 displays the [3H]UDP-Glc, [3H]UDP-Gal, and [3H]Glc specific activities that are used to calculate the contribution of plasma glucose ("Direct Contribution" in Table 4) to the hepatic glucose 6-phosphate pool. The UDP-Gal specific activities confirmed the values obtained with UDP-Glc, suggesting rapid and complete isotopic equilibration between the two intracellular pools. The ratio of the specific activities of 3H-labeled hepatic UDP-Glc and UDP-Gal and portal vein plasma glucose provided an estimate of the contribution of the direct pathway. As shown in Table 4, the contributions of the direct pathway to the hepatic UDP-hexose pool measured at the end of both protocols 1 and 2 were significantly diminished in the GK+/ mice compared with both WT
and RIP-GKRZ mice. These changes in the composition of the intrahepatic
UDP-hexose pools provide evidence for decreased flux through GK in vivo
in GK+/ mice, which likely reflects the decrease in GK activity
measured in vitro.
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Effect of Reduced GK Activity on the "Indirect Pathway" of Liver UDP-Glc Formation
Table 5 displays the [14C]UDP-Glc, [14C]UDP-Gal, and [14C]PEP specific activities that are used to calculate the contribution of gluconeogenesis ("Indirect Contribution" in Table 5) to the hepatic glucose 6-phosphate pool. The indirect pathway accounted for 30-35% of the hepatic UDP-Glc pool in WT and RIP-GKRZ mice. This contribution was increased to 45-55% in the GK+/ mice.
These data indicate that the reduced liver GK activity in the
GK+/ mice leads to a significantly higher proportion of hepatic
glucose 6-phosphate being derived from gluconeogenesis vs. plasma
glucose compared with WT and RIP-GKRZ mice.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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A fundamental premise of our experimental design was that the genetic
manipulations implemented in our animal models resulted in discordant
effects on the hepatic activity of GK. The extent of deficit in the
-cell and liver glucose phosphorylating capacity induced by the
disruption of one allele of the GK gene has been debated regarding both
human MODY and animal models (1, 4, 17, 30, 31). In humans, in whom the
enzymatic activity of the GK protein encoded by the mutant allele is
generally negligible (16), GK activity should be ~50% of the normal
levels. However, a recent study has suggested that compensatory
mechanisms may be activated in -cells of individuals with MODY 2 and
may account for an insulin secretory function that is higher than
expected (5, 29). In mouse models in which one allele of the GK gene had been disrupted, the decrease in GK activity in -cells varied between 37 and 50% (1, 17, 30). Similarly, the decrease in the hepatic
GK activity in the same mouse models varied between 28 and 44% (1,
17). The hepatic isoform of GK is generated by a different promoter
than the pancreatic isoform (20-22). Because hepatic GK is
regulated by insulin, it has been postulated that changes in
circulating levels of the hormone are likely to upregulate the
transcription of the normal GK allele in the liver and perhaps diminish
the impact of this genetic manipulation at this site (1, 3, 22). Our
findings show that the activity of GK in vitro was decreased in the
GK+/ mice by 35 and 50% at the end of
protocols 1 and
2, respectively. Interestingly, the
decrease in hepatic GK activity was more severe and reproducible at the end of the pancreatic clamp studies, in which the plasma insulin concentration was kept at near basal levels, than at the completion of
the hyperglycemic clamp studies performed at high circulating insulin
levels. It can be postulated that the intact GK allele is quite
sensitive to insulin regulation and that the degree of compensation may
vary on the basis of the nutritional and hormonal status of the animal
at the time of sampling. Overall, the present finding of a partial
decrease in the hepatic GK activity in GK+/, but not in
RIP-GKRZ, mice is consistent with previous reports (1, 10) and allowed
us to use these two experimental models to further dissect the
metabolic impact of a partial and chronic inhibition of hepatic GK.
During the hyperglycemic clamp studies, the ability of hyperglycemia to
promote insulin secretion was significantly diminished, particularly in
the RIP-GKRZ mice. This observation reproduces in the intact animal the
decreased glucose-induced insulin secretion we had previously observed
in this model with use of the perfused pancreas technique (10). This is
also consistent with elegant secretory studies performed in humans
carrying a mutant GK allele (5). Peripheral glucose uptake did not
appear to be specifically affected by the alterations in GK activity.
In fact, although moderate decreases in the rates of whole body
Rd were observed in both
GK-deficient models during the hyperglycemic clamp studies, this
observation appeared to be due to the lower plasma insulin concentrations. However, our findings in protocol
1 may also indicate lower insulin sensitivity in the
GK+/ than in the RIP-GKRZ mice. In fact, the
Rd was similar in these two groups
in the presence of higher insulin levels in the GK+/ mice. It
should be pointed out that the differences in circulating plasma
insulin concentrations between these two groups did not achieve
statistical significance because of the large variability in the levels
measured in the GK+/ mice. Finally, the decreased
Rd was not reproduced when the
circulating insulin levels were kept at the same levels in all groups
(protocol 2). Overall, our data do
not allow one to suggest or to exclude a modest effect of either mild
glucose intolerance or of decreased GK activity in other tissues
(brain) on peripheral insulin action in the GK+/ mice.
Conversely, reproducible and specific features of liver glucose
metabolism were demonstrated in the GK+/ mice. In fact, our
findings indicate that the diminished glucose phosphorylation capacity
in the liver of GK+/ mice causes an impairment in the ability of
hyperglycemia to inhibit HGP. This defect is not observed in mice in
which GK activity is reduced solely in -cells. Furthermore, these
observations cannot be ascribed to differences in pancreatic hormone
levels among the groups, because they were reproduced during
hyperglycemic-pancreatic clamp studies. The ability of hyperglycemia to
inhibit HGP and the contribution of the direct pathway to the hepatic
glucose 6-phosphate pool were markedly decreased in the GK+/
group compared with both WT and RIP-GKRZ groups. Several recent
findings support the independent role of a decrease (even modest) in
hepatic GK activity in the pathophysiology of carbohydrate intolerance
in MODY and in animal models. Hepatic insulin resistance appears to
represent an early finding in patients with MODY 2 (7), and impaired
hepatic glycogen synthesis and decreased contribution of the direct
pathway have recently been reported in an elegant study using
13C nuclear magnetic resonance
spectroscopy in a group of MODY 2 patients (31). We have also shown
that transient and short-term inhibition of hepatic GK activity with
use of glucosamine can reproduce some of the defects in hepatic glucose
fluxes (2). Finally, two recent studies have demonstrated improved
glucose tolerance in transgenic mice with liver-specific overexpression of GK (11, 18).
An important finding in the present study is that the decreased flux
through GK was paralleled by a marked increase in the contribution of
the gluconeogenic pathway to the hepatic glucose 6-phosphate pool. The
increased HGP in the GK+/ mice was associated with a marked
increase in the relative contribution of gluconeogenesis to HGP.
Interestingly, a transient and acute inhibition of hepatic GK activity
in rats resulted in similar effects on HGP and the direct pathway of
hepatic glycogen repletion, but not on gluconeogenesis (2). In fact,
the increased HGP in the latter rat study was due to an increased rate
of glycogenolysis. Conversely, a recent study by Velho et al. (31) in
MODY 2 patients demonstrated that the decreased contribution of direct
hepatic phosphorylation of glucose to postmeal glycogen synthesis was
compensated in part by a parallel increase in the contribution of the
indirect or gluconeogenic pathway. This apparent discrepancy regarding
the metabolic consequences of short-term vs. chronic decreases in the
hepatic GK activity may be due to the noteworthy role of hepatic glucose phosphorylation in regulating gene expression of key hepatic enzymes in the gluconeogenic and glycolytic pathways, such as phosphoenolpyruvate carboxykinase and
L-type pyruvate kinase. Indeed, Ferre and co-workers (11, 12) reported
that the hepatic overexpression of GK leads to a marked increase in
pyruvate kinase mRNA and activity. Thus a prolonged moderate decrease
in the rate of hepatic glucose phosphorylation is likely to alter the
intrahepatic distribution of glucose fluxes, probably through
regulation of gene expression of key enzymes. In conclusion, our data
indicate that decreased activity of GK in the liver can cause increased HGP in the face of hyperglycemia.
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ACKNOWLEDGEMENTS |
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The authors thank Rong Liu, Robin Squeglia, and Anton Svetlanov for their excellent technical assistance.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grants R01-DK-45024, R01-DK-48321, and the Albert Einstein Diabetes Research and Training Center Grant DK-20541; the Juvenile Diabetes Foundation; and the American Diabetes Association. S. Efrat is a recipient of the NIDDK J. A. Shannon Award. N. Barzilai is supported by a grant from the National Institute on Aging (KO8-AG-00639). M. Hawkins is the recipient of a postdoctoral fellowship from the Juvenile Diabetes Foundation International. L. Rossetti and S. Efrat are the recipients of Career Scientist Awards from the Irma T. Hirschl Trust.
Address for reprint requests: L. Rossetti, Div. of Endocrinology, Dept. of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461.
Received 11 April 1997; accepted in final form 3 July 1997.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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