Vol. 91, Issue 5, 2041-2046, November 2001
Sucrose diets increase glucose-6-phosphatase and
glucose release and decrease glucokinase in hepatocytes
Michael E.
Bizeau1,
Jeffrey S.
Thresher2, and
Michael J.
Pagliassotti1,2
1 University of Colorado Health Sciences Center, Department
of Medicine, Division of Endocrinology, Metabolism, and Diabetes,
Denver, Colorado 80262; and 2 Exercise and Sport Research
Institute, Arizona State University, Tempe, Arizona 85287
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ABSTRACT |
A high-sucrose diet (SU)
decreases insulin action in the liver (Pagliassotti MJ, Shahrokhi KA,
and Moscarello M. Am J Physiol Regulatory Integrative Comp
Physiol 266: R1637-R1644, 1994). The present study was
conducted to characterize the effect of SU on glucagon action in
isolated periportal (PP) and perivenous (PV) hepatocytes by measuring
glucagon-stimulated glycogenolysis and glucose release. Male rats were
fed a SU (68% sucrose) or starch diet (ST, 68% starch) for 1 wk, and
hepatocytes were isolated from PP or PV regions (n = 4/diet/cell population). Hepatocytes were incubated for 1 h in the
presence of varying concentrations of glucagon (0-100 nM). In PP
and PV cells, glucagon stimulation of glucose release and
glycogenolysis (sum of glucose release and lactate accumulation) was
not significantly different between SU and ST cells. However, in the SU
PP cells, glucose release was increased compared with ST PP cells, both
in the absence of glucagon (76.1 ± 4 vs. 54.8 ± 3 nmol · h
1 · mg cell wet
wt1) and at all glucagon concentrations. In SU-fed PV
cells, glucose release was increased compared with ST PV cells in the
absence of glucagon (79.3 ± 5 vs. 56.4 ± 5 nmol · h1 · mg cell wet
wt1) and at low glucagon concentrations. Maximal
glucose-6-phosphatase activity (in
nmol · min1 · mg protein1)
was elevated in SU compared with ST cells (61.4 ± 3 vs. 37.5 ± 4 in PP and 37.5 ± 4 vs. 29.5 ± 3 in PV cells). In
contrast, maximal glucokinase activity (in
nmol · min1 · mg protein1)
was elevated in ST compared with SU cells (15.9 ± 2 vs. 12.1 ± 1 in PP and 19.4 ± 2 vs. 14.2 ± 1 in PV cells). These
data demonstrate that SU increases the capacity for glucose release in
both PP and PV hepatocytes, in part because of reciprocal changes in
glucose-6-phosphatase and glucokinase.
liver; diet composition; glucose production
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INTRODUCTION |
DIETARY NUTRIENTS CAN RAPIDLY
AND DIRECTLY influence hepatic metabolism. For example, in rats,
1 wk of high- sucrose diet (SU) feeding decreases the ability of
insulin to suppress hepatic gluconeogenesis in vitro (19)
and glucose production in vivo (19, 21) and increases the
capacity for gluconeogenesis in both isolated hepatocytes
(22) and perfused livers (20). After 1 wk of
SU feeding, insulin levels and basal glucose production are not
increased (19, 21). Thus, despite insulin resistance and
an increased capacity for hepatic gluconeogenesis, the liver maintains
appropriate rates of basal glucose production. Hepatic adaptations in
this dietary model can be used to further the understanding of
environmental factors that contribute to the development of impaired
glucose tolerance and Type 2 diabetes as well as to the autoregulation
of hepatic glucose production.
Glucagon is an important regulator of hepatic glucose production,
stimulating both glycogenolysis and gluconeogenesis (9). Although SU feeding does not significantly change circulating levels of
glucagon, it is presently unknown whether glucagon action to stimulate
glucose release or glycogenolysis in the liver is altered by this diet.
Isolated hepatocytes are an ideal system to study glucagon action in
the liver because in vivo experiments using glucagon infusions induce
hyperglycemia that becomes a variable in itself that must be controlled
for. The hepatic acinus, the functional unit of the liver, can be
divided into periportal (PP) and perivenous (PV) regions with respect
to blood flow (23). Within the acinus, glucagon receptors
as well as the enzymes of carbohydrate metabolism are distributed in a
heterogeneous pattern (12). The PP region of the acinus
contains greater amounts of the glucagon receptor and glucagon receptor
mRNA as well as gluconeogenic enzymes (14). Therefore,
glucagon may exert a greater action in the PP region of the acinus,
perhaps leading to greater glucose release in PP cells with glucagon
stimulation. In addition, it has been demonstrated that PP and PV
hepatocytes do not adapt homogeneously to dietary perturbations
(4, 24), thus raising the following question: If a SU diet
alters glucagon action, are both the PP and PV cell population
similarly affected? Therefore, isolation and study of these two
hepatocyte populations can provide valuable information as to how the
liver adapts to dietary nutrients.
The glycogenolytic and gluconeogenic pathways both provide
glucose-6-phosphate (G-6-P) that can be dephosphorylated by
the enzyme glucose-6-phosphatase (G6Pase) to glucose. Glucose can be
released from the hepatocyte or rephosphorylated by the enzyme glucokinase (GK) and retained within the hepatocyte. Thus glucose release from the hepatocyte is a function of the activities of GK and
G-6-P. The present study was undertaken to examine the effects of SU on glucagon action and the enzymes involved in the terminal step of glucose release from the hepatocyte.
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METHODS |
Experimental animals and feeding protocol.
Male Sprague-Dawley rats weighing ~180 g were obtained from an
institutional breeding stock. All animals were housed individually in a
temperature-controlled room with a 12:12-h light-dark cycle and free
access to food and water. All procedures for animal use were approved
by the Institutional Animal Care and Use Committee at Arizona State
University. On initiation of the study, all animals were provided free
access to a semipurified high-starch diet (ST; % of total
calories = 68 cornstarch, 20 protein, 12 fat) for a 2-wk baseline
period. Food intake was measured daily, and body weight was recorded
weekly. After the 2-wk baseline period, rats were switched to either SU
(% of total calories = 68 sucrose, 20 protein, 12 fat) or
remained on ST for 1 wk. During this week, rats were fed 95% of the
average food intake recorded during the second week of baseline
feeding. Feeding 95% of baseline calories during the experimental
feeding period results in rats with similar rates of weight gain and
body composition (21). Complete diet composition is
presented in Table 1.
Hepatocyte isolation.
PP and PV hepatocytes were isolated from rats fed normally the previous
evening according to the basic procedures developed by Lindros and
Penttila (15) as modified by Jones and Titheradge (11). For preparation of PV-enriched hepatocytes, rats
were anesthetized with an intramuscular injection of ketamine (50 mg/kg), xylazine (10 mg/kg), and acepromazine (5 mg/kg). The abdominal cavity was opened, and the portal vein and superior vena cava were
cannulated. The liver was perfused in the anterograde direction with
calcium-free Krebs-Ringer bicarbonate buffer equilibrated with 95%
O2-5% CO2 at 37°C and pH 7.4. Once the liver
was cleared of blood, ~50 ml of the initial perfusate was allowed to
drain to waste. Digitonin (2 ml of 10 mg/ml in Krebs-Ringer, 20 mM
HEPES, pH 7.4) was then injected into the portal vein until the
reticular pattern described by Lindros and Penttila (15)
was observed. Flow was rapidly switched to the retrograde direction to
remove digitonin and continued at ~45 ml/min until 50 ml of perfusate was collected. For PP hepatocytes, digitonin (2.5 ml) was injected via
the superior vena cava until the characteristic dot pattern of PV
destruction was observed (15). Digitonin was removed by perfusing in the anterograde direction at ~45 ml/min until 50 ml of
perfusate was collected. After digitonin washout, hepatocytes were
prepared by using standard collagenase perfusion methods (2). The initial quality of the cell preparation was
assessed by trypan blue exclusion (0.2% final concentration). Only
preparations with >90% dye exclusion were used in cell incubations.
Hepatocyte incubations.
Before use in incubations, hepatocytes were suspended at a final
concentration of 30 mg wet weight/ml in Krebs-Ringer bicarbonate buffer
containing 1% gelatin and equilibrated for 25 min with 95%
O2-5% CO2 at 37°C. Cell suspensions (2.0 ml)
were incubated for 1 h with or without glucagon (in nM: 0, 0.01, 0.1, 1, 10, and 100). For determination of glucose and lactate, 250 µl of the cell suspensions were removed before (0 min) and after (60 min) incubations and added to an equal amount of 0.6 M perchloric acid.
Neutralization of the perchloric acid supernatants was performed by
using 1 M KHCO3. For glycogen determinations, a 250-µl
aliquot of cell suspension was removed before (0 min) and after (60 min) the incubations and rapidly centrifuged to pellet the cells. The supernatant was discarded, and the cell pellet was frozen in liquid nitrogen and stored at 
80°C until analysis.
Enzyme analysis.
G6Pase activity was measured, according to the methods of Burchell et
al. (5), in freeze-thawed cell suspensions containing ~30 mg cells/ml at G-6-P concentrations of (in mM) 0.5, 1, 2.5, 5, and 10. Nonspecific phosphatase activity was estimated by using paranitrophenylphosphate as substrate. Alanine aminotransferase (Sigma
kit) and glutamate dehydrogenase (17) were measured
spectrophotometrically in freeze-thawed cell suspensions pretreated
with 0.1% Triton X-100. GK activity was determined on aliquots of cell
suspensions (~60 mg cells/ml) that were frozen in a medium containing
150 mM KCl, 50 mM HEPES, and 1.5 mg/ml dithiothreitol at pH 7.5. The aliquots were thawed and centrifuged at 20,300 g for 30 min.
The supernatant was then assayed for GK activity as described by
Davidson and Arion (8). All enzyme activities were
determined on aliquots of cell suspensions taken before the start of incubations.
Metabolite assays.
Glucose and lactate release into the incubation medium were measured
enzymatically (Sigma kits). Glycogen was measured by using the method
of Chan and Exton (6). Protein was determined by the
method of Lowry et al. (16).
Statistical procedures.
Data from each experiment were represented as the average value of each
triplicate incubation. Data were analyzed using either a one-way or a
two-way ANOVA where appropriate. If the overall F value
obtained from the ANOVA was significant, comparisons between mean values were made by using a Student-Newman-Keuls test.
Significance was set at P < 0.05 for all comparisons.
All data are presented as means ± SE.
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RESULTS |
General animal and cell characteristics.
After 1 wk on the respective diets, there were no differences in body
weight between groups (SU rats = 356 ± 5.9 g, ST
rats = 363 ± 8.1 g). As has been previously
demonstrated by using the digitonin perfusion method (7,
12), alanine aminotransferase activity was greater in PP
hepatocytes, whereas glutamate dehydrogenase activity was greater in PV
hepatocytes (Table 2). There were no diet
effects on either alanine aminotransferase or glutamate dehydrogenase
in PP and PV cell populations. Total cell yield per liver was greater
from PP compared with PV hepatocyte isolations (Table 2).
Glucose release in isolated hepatocytes.
In both PP and PV hepatocytes, glucagon action to increase glucose
release was not different between diet groups. In PP hepatocytes, glucose release was significantly elevated in SU compared with ST at
all glucagon concentrations (Fig.
1A). In PV cells, glucose release was significantly elevated in SU vs. ST at 0 and
1011 M glucagon only (Fig. 1B). Maximal
glucose release occurred at 108 M glucagon in all groups
and was lower in ST PP cells compared with all other groups.
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Fig. 1.
Dose-dependence curves for glucose release in isolated
periportal (PP; A) and perivenous (PV; B)
hepatocytes in response to glucagon. Data are expressed as nmol
glucose · h1 · mg cell wet
wt1. ST, starch diet; SU, high sucrose diet. Values are
means ± SE (n = 4 animals per group). *SU
significantly different from ST for a given cell population
(P < 0.05).
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Glycogen degradation.
Initial glycogen levels were higher in PV compared with PP cells (Fig.
2). There was no effect of diet on
initial glycogen concentration or glycogenolysis (sum of glucose
release and lactate accumulation) in either PP or PV hepatocytes (Fig.
3). Estimated net glycogenolysis, based
on pre- to postincubation glycogen concentration, produced similar
results.
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Fig. 2.
Initial liver glycogen content in PP and PV hepatocytes
isolated from normally fed ST and SU animals. Values are means ± SE (n = 4 animals per group). *PV significantly
different from PP (P < .05).
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Fig. 3.
Net glycogenolysis in PP (A) and PV
(B) hepatocytes isolated from ST and SU animals. Data are
expressed as nmol glucose units · h1 · mg
cell wet weight1. Data were calculated from summing
glucose release and lactate accumulation. Values are means ± SE
(n = 4 animals per cell type per diet group).
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Lactate accumulation.
In PP cells, lactate accumulation decreased as a function of increasing
glucagon concentration such that net removal of lactate was observed at
109 M glucagon (Fig.
4A). PV cells displayed a
different pattern of lactate accumulation at low-glucagon
concentrations but still exhibited net removal of lactate at
~109 M glucagon (Fig. 4B). Under all
conditions, the lactate concentration in the incubation medium was <8
µM and endogenous in origin.
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Fig. 4.
Dose-dependence curves for the rate of net lactate
accumulation in isolated PP (A) and PV (B)
hepatocytes in response to glucagon. Data are presented as nmol
lactate · h1 · mg cell wet
weight1. Values are means ± SE (n = 4 animals per group).
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Enzyme activities.
GK activity was significantly increased in ST compared with SU in both
PP and PV cells (Fig. 5). In contrast to
GK activity, G6Pase activity was increased in SU vs. ST in both PP and
PV cells (Fig. 6). There were no
differences, either between cell populations or diets, in nonspecific
phosphatase activity determined by using paranitrophenylphosphate as a
substrate (data not shown).
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Fig. 5.
Glucokinase kinetics in PP (A) and PV
(B) hepatocytes from ST and SU animals. Freeze-thawed cell
suspensions (30 mg/ml) were assayed for glucokinase activity as
described in METHODS. Data are presented as
nmol · min1 · mg protein1.
Values are means ± SE (n = 4 animals per cell
type per diet group). *SU significantly different from ST for a given
cell population (P < 0.05).
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Fig. 6.
Glucose-6-phosphatase (G6Pase) kinetics in PP
(A) and PV (B) hepatocytes from ST and SU
animals. Freeze-thawed cell suspensions (30 mg/ml) were assayed for
G6Pase activity as described in METHODS. Data are presented
as nmol · min1 · mg
protein1. Values are means ± SE (n = 4 animals per cell type per diet group). *SU significantly different
from ST for a given cell population (P < 0.05).
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DISCUSSION |
The goal of the present study was to examine the effect of SU on
glucagon-stimulated glucose release and glycogenolysis in isolated PP
and PV hepatocytes. Therefore, PP and PV hepatocytes were isolated
after a normal night's feeding period. Initial glycogen concentrations
in the hepatocytes obtained in the present study were 10- to 20-fold
higher compared with hepatocytes obtained after a 24-h period of
starvation using similar isolation procedures (4).
Hepatocytes were isolated by using a modification of the digitonin
perfusion method. Cells obtained by this method demonstrated a pattern
of marker enzyme activity typically associated with PP and PV cell
preparations isolated by this procedure (7, 12). This
pattern was characterized by increased alanine aminotransferase activity in cells isolated from the PP region and increased glutamate dehydrogenase activity in cells isolated from the PV region. Thus cell
preparations designated as PP were enriched with PP cells and those
designated PV were enriched with PV cells.
Data from the present study indicate that, in the absence of glucagon,
glucose release was significantly increased in both PP and PV cells by
SU. The increased glucose release was maintained across all glucagon
concentrations in the PP cell, but only at lower glucagon
concentrations in the PV cells. The only other study to examine glucose
release in response to glucagon in PP and PV hepatocytes isolated from
fed animals demonstrated greater glucose release in PV compared with PP
cells (10). This result is consistent with the data from
the ST group obtained in the present study, where the PP-to-PV ratio
for glucose release was ~0.7. In contrast, this ratio was ~0.96 in
the SU group. Thus SU produced a liver that was characterized by a more
homogenous pattern of glucose release across the acinus but did not
significantly alter the dose-response relationship between glucagon and
glucose release. The increased homogeneity of glucose release in the SU group appears to be the result of adaptations in the PP population.
Increased glucose release in the absence of glucagon could result from
increased glycogenolysis, increased partitioning of G-6-P
toward glucose, or increased gluconeogenesis. Glycogenolysis was not
significantly increased in either PP or PV cells after SU feeding. In
addition, the rate of glycogenolysis exceeded glucose release both in
the absence of and in the presence of glucagon. Although
gluconeogenesis was not estimated in the present study, the
contribution of gluconeogenesis to net glucose release was probably
minimal. The minimal contribution of gluconeogenesis in this study was
likely because of the absence of added gluconeogenic precursors in the
incubation medium. Therefore, SU appears to increase the partitioning
of glycogenolysis-derived G-6-P toward glucose release.
Net glucose release from the liver is a function of the relative
activities of the enzymes responsible for glucose phosphorylation (GK)
and G-6-P dephosphorylation (G6Pase). In the current study, SU increased G6Pase activity in both PP and PV cell populations compared with ST. Additionally, in both the SU and ST animals, G6Pase
activity was greater in the PP than in the PV population. Greater
G6Pase activity in the PP compared with the PV population has been
observed previously in hepatocytes isolated from 24-h-fasted rats fed
SU (4). Thus a primary adaptation induced by SU is to
increase G6Pase activity in the liver. It should be noted that changes
in G6Pase activity were not because of effects on nonspecific phosphatases because diet effects were not observed when
paranitrophenylphosphate was used as a substrate. In addition to the
increased activity of G6Pase in the livers of SU animals, GK activity
in both the PP and PV cell populations was lower compared with the ST
group. Lower GK activity would act to augment the effect of the
increased G6Pase on glucose release from the cell.
In summary, 1 wk of SU does not alter glucagon action on glycogenolysis
or stimulation of glucose release in either PP or PV hepatocytes.
Increased G6Pase and lower GK activity were found in both PP and PV
cell populations from SU animals. Thus the greater G-6-P-to-GK activity ratio observed in both PP and PV cells
from the SU animals could in part explain the greater rates of glucose release observed in the SU animals in the absence and presence of glucagon.
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ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-55386 and DK-47416.
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FOOTNOTES |
Address for reprint requests and other correspondence:
M. E. Bizeau, Univ. of Colorado Health Sciences Center, Dept. of
Medicine, Division of Endocrinology, Metabolism and Diabetes, Campus
Box B151, 4200 E. Ninth Ave., Denver, CO 80262 (E-mail:
michael.bizeau{at}uchsc.edu).
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.
Received 31 January 2001; accepted in final form 11 July 2001.
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ABSTRACT
INTRODUCTION
