-Cell adaptation in 60% pancreatectomy rats that preserves
normoinsulinemia and normoglycemia
Ye Qi
Liu,
Peter W.
Nevin, and
Jack L.
Leahy
Division of Endocrinology, Diabetes and Metabolism, University
of Vermont, Burlington, Vermont 05405
 |
ABSTRACT |
Islet
-cells are the regulatory element of the glucose homeostasis system.
When functioning normally, they precisely counterbalance changes in
insulin sensitivity or
-cell mass to preserve normoglycemia. This
understanding seems counter to the dogma that
-cells are regulated
by glycemia. We studied 60% pancreatectomy rats (Px) 4 wk postsurgery
to elucidate the
-cell adaptive mechanisms. Nonfasting glycemia and
insulinemia were identical in Px and sham-operated controls. There was
partial regeneration of the excised
-cells in the Px rats, but it
was limited in scope, with the pancreas
-cell mass reaching 55% of
the shams (40% increase from the time of surgery). More consequential
was a heightened glucose responsiveness of Px islets so that glucose
utilization and insulin secretion per milligram of islet protein were
both 80% augmented at normal levels of glycemia. Investigation of the
biochemical basis showed a doubled glucokinase maximal
velocity in Px islets, with no change in the glucokinase
protein concentration after adjustment for the different
-cell mass
in Px and sham islets. Hexokinase activity measured in islet extracts
was also minimally increased, but the glucose 6-phosphate concentration
and basal glucose usage of Px islets were not different from those in
islets from sham-operated rats. The dominant
-cell adaptive response
in the 60% Px rats was an increased catalytic activity of glucokinase.
The remaining
-cells thus sense, and respond to, perceived
hyperglycemia despite glycemia actually being normal.
-Cell mass and
insulin secretion are both augmented so that whole pancreas insulin
output, and consequently glycemia, are maintained at normal levels.
glucokinase; glucose metabolism; glycolysis; glucose 6-phosphate; islets of Langerhans; insulin secretion
 |
INTRODUCTION |
THE ISLET
-CELL through its secretion of
insulin regulates the storage and metabolism of cellular fuels. Not
surprisingly, the regulatory system is complex, with multiple factors
affecting insulin secretion, proinsulin synthesis, and the mass of
pancreatic
-cells. The best studied factor is glucose; all of these
functional aspects are glucose responsive, and it is well accepted that
glycemia is the major regulator of
-cell function. However, the
primary effect of
-cell activity is to maintain a normal metabolic
milieu. When
-cells function normally, insulin secretion precisely
meets tissue insulin needs so that normoglycemia is maintained (16). Moreover, whole body insulin sensitivity varies throughout life (puberty, pregnancy, and aging are insulin-resistant states), yet most
humans do not develop diabetes. As such, an unanswered question in
regard to the glucose homeostasis system is how
-cell adaptation
occurs in the absence of ongoing changes in glycemia.
Insight has come from studies of rodents with genetic-based
insulin resistance or pregnancy; their
-cells are supersensitive to
glucose so that insulin secretion is augmented at normoglycemia (4,
6-8, 11, 30). Kahn et al. (15) reported the same finding in
healthy humans made insulin resistant by a 14-day nicotinic acid
infusion. Thus a disassociation of the normal coupling between glucose
concentration and insulin secretion characterizes the
-cell
adaptation to insulin resistance. The mechanism of this effect is
unknown. We studied nondiabetic insulin-resistant spontaneously hypertensive rats (SHR) and found that catalytic activity of the
-cell glucose sensor enzyme, glucokinase, was enhanced (6). However,
alternate mechanisms have been reported in other models (3, 34, 40). In
particular, a recent suggestion is that elevated
-cell fatty acid
(FA) metabolism mediates the heightened glucose sensing for insulin
secretion, because hypertriglyceridemia commonly is found with insulin
resistance (32), and culturing islets with FA shifts to the left the
glucose concentration-insulin secretion curve (12, 28, 42). It remains
unclear how to reconcile these different findings.
Little is known about how glucose homeostasis is preserved when the
-cell mass is lowered. Biobreeding rats before onset of autoimmune
diabetes have a left-shifted glucose concentration-insulin secretion
curve (36). We made the same observation after a 60% pancreatectomy in
rats (20). Thus a variable
-cell sensitivity to glucose also
operates when the
-cell mass is lowered. Whether the biochemical
details are the same as for insulin-resistant states is not known.
The current study investigated rats after a 60% pancreatectomy (Px);
these rats maintain normal plasma levels of insulin and glucose despite
the considerable loss of
-cells (14, 19, 29), making them an
excellent model for the investigation of
-cell adaptive mechanisms.
Rats were studied 4 wk after the Px surgery to minimize any possibility
that the observed changes were in transition and not reflective of the
completed
-cell adaptation process.
 |
METHODS |
60% Px model and islet isolation.
One hundred-gram Sprague-Dawley rats underwent 60% Px by use of our
previously described method (19). Briefly, the portion of the pancreas
bordered by the spleen and stomach extending to the small flap of
pancreas attached to the pylorus was removed by use of gentle abrasion
with cotton applicators. The removed portion was 57 ± 3% of the
pancreas weight (19). Control (sham) rats underwent laparotomy and
mobilization of the pancreas with gentle rubbing between the fingers.
Postoperatively, all rats received standard chow and tap water ad
libitum until being studied 4 wk after the surgery. Islets were
isolated using an adaptation of the Gotoh method (10): pancreas duct
infiltration with collagenase (Serva, Heidelberg, Germany), Histopaque
gradient separation (Sigma, St. Louis, MO), and hand picking. Islet DNA
content was measured by the Labarca method (17), protein by a
commercial kit (Bio-Rad, Hercules, CA) with bovine albumin as standard,
and insulin content after acid ethanol extraction with an insulin RIA
(1). Freshly isolated islets were used in all experiments.
Oral glucose tolerance test and meal challenge.
Both tests were preceded by an overnight fast. Px and sham rats were
administered 1 g/kg of glucose (0.5 g/ml) by gavage tube. Blood for
plasma glucose determination was obtained by tail snipping at 0, 30, 60, and 120 min. The meal challenge was performed 3 days later. Px and
sham rats were given free access to chow at 9:00 AM (time 0),
and plasma glucose values were measured at 0, 30, 60, and 120 min.
Islet insulin secretion and glucose utilization.
Islets underwent 30 min of preincubation in warmed and oxygenated
Krebs-Ringer buffer (KRB) with 2.8 mM glucose and 0.5% BSA. Insulin
secretion was assessed using triplicate batches of 10 islets in glass
vials containing 1 ml of KRB with 0.5% BSA and 2.8, 5.5, 8.3, or 16.7 mM glucose for 60 min in a 37°C shaking water bath. Medium was
separated by gentle centrifugation and stored at
20°C
pending insulin measurement by RIA (1). Islet glucose usage was
measured as previously described (6) under the same experimental
conditions with a method based on quantifying conversion of
D-[5-3H]glucose (NEN, Boston, MA)
to [3H]H2O (2).
Islet glucokinase/hexokinase kinetics.
Glucose phosphorylation was measured in islet extracts as previously
described (6) with a method based on quantifying conversion of
NAD+ to NADH by exogenous glucose-6-phosphate dehydrogenase
(22). Islet homogenates were centrifuged at 12,000 g for 10 min, and the supernatants were incubated at 10 glucose concentrations
(0.03-100 mM) to measure glucose phosphorylation. Maximal velocity
(Vmax) and Michaelis-Menten constant
(Km) values for glucokinase and hexokinase were
calculated by linear regression from an Eadie-Scatchard plot
(volume/substrate concn) and 10 cycles of the method of
Spears et al. (35) to identify each enzyme's activity.
Glucokinase immunoblots.
Glucokinase immunoblots were performed as described (6) using sheep
antiserum raised against an Escherichia coli-derived B1 isoform
of rat glucokinase (gift from Dr. Mark Magnuson, Vanderbilt University). Bound antibody was detected by chemiluminescence and
quantified by densitometry. Islets used under denaturing conditions were homogenized in 80 mM Tris (pH 6.8), 0.5% NP-40, 0.5% Triton X, 5 mM EDTA, 0.2 mM N-ethylmaleimide, and 1 mM phenylmethylsulfonyl fluoride, followed by heating at 95°C for 5 min in Laemmli sample buffer that contained 2% SDS and by running on a 10% acrylamide gel
with stacking and separating buffers that contained 0.4% SDS. Nondenaturing gels were performed using islets that were homogenized in
20 mM K2HPO4, 1 mM EDTA, 110 mM KCl, and 2 mM
dithiothreitol, placed in Laemmli sample buffer without SDS, and run on
a 15% acrylamide gel with buffers without SDS.
Islet glucose 6-phosphate concentration.
Px and sham islets (20 per tube) were incubated in prewarmed and
oxygenated KRB with 0.5% BSA and 2.8, 8.3, or 16.7 mM glucose for 60 min in a 37°C shaking water bath, followed by rapid lysis in 10 µl of 40 mM NaOH. Then they were placed on ice for 10 min, and 3 µl
of 0.15 M HCl were added with incubation at 75°C for 20 min to
destroy cellular enzymes to ensure stability of glucose 6-phosphate
(G-6-P) content. G-6-P was measured using an adaptation of the Lowry and Passonneau method (24) as previously described (23).
Analytical methods.
Plasma glucose was measured with a Beckman Glucose Analyzer II
(Beckman, Fullerton, CA). The insulin RIA used charcoal separation (1)
and rat insulin standards (Lilly, Indianapolis, IN). Plasma triglycerides and free fatty acids were measured by commercial kits
(Sigma and Wako Chemicals, Richmond, VA, respectively).
Data analysis and statistical methods.
Data are expressed as means ± SE. The listed n values for the
isolated islet data are the numbers of experiments that were performed
using islets from separate isolation days. Islet data are expressed per
protein or DNA content to adjust for the difference in cell mass of
isolated islets from Px and sham rats. Western blots were performed
using islets from single Px and sham rats, so the listed n
value is the number of animals studied. The densitometry results from
Western blots were expressed in relative terms by comparing the Px band
on each gel to the sham band (assigned a value of 100%). Statistical
significance was determined with the unpaired Student's
t-test, except for the Western blot results, for which the
one-tailed t-test was used.
 |
RESULTS |
General characteristics.
Body weight and nonfasting glycemia, insulinemia, triglyceridemia, and
plasma free fatty acids were equal in the 60% Px and sham-operated
rats (Table 1). An oral glucose tolerance
test and a meal challenge were performed to confirm the normoglycemia of the 60% Px rats; glycemic responses were identical in the two groups (Fig. 1).

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Fig. 1.
Rats 4 wk after 60% ( , n = 8) or sham ( , n = 4)
pancreatectomy underwent an oral glucose tolerance test (OGTT,
left) followed 3 days later by a meal challenge
(right). Both tests were preceded by an overnight fast.
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|
One possibility for these results was regeneration of the excised
-cells. We had previously addressed that issue 7 wk after 60% Px:
-cell mass was 55% of the sham rats, which is a 40% increase from
the postsurgery period, and islet non-
-cell mass was unchanged at
45% of control, which suggests that the islet regeneration was
-cell specific (19). We now report identical findings in isolated
islets from 60% Px rats 4 wk after the surgery. Islet DNA, protein,
and insulin content were 40% increased compared with the sham-operated
animals (Table 2). Consequently, the 60% Px induced some
-cell regeneration, but it was incomplete.
Islet glucose sensing/responsiveness.
We assessed insulin secretion and glucose utilization in isolated
islets from the 60% Px and sham-operated rats (Fig.
2); results are expressed per milligram of
protein to compensate for the different islet cell mass. Both
parameters showed an upregulated response in the Px islets, with no
change in the dose giving a half-maximal response (ED50),
and a 70-80% increase at 8.4 mM glucose, which approximates the
usual nonfasting plasma glucose level of the Px and sham rats (Table
1).

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Fig. 2.
Insulin secretion (A, n = 3 experiments) and glucose
utilization (B, n = 4 experiments) in isolated islets
from rats 4 wk after 60% ( ) or sham ( ) pancreatectomy.
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|
The pattern of the increase suggested augmented glucokinase activity in
the Px islets, which was confirmed by measuring glucose phosphorylation
in islet extracts over a range of 0.03-100 mM glucose (Fig.
3; derived enzyme kinetics shown in Table
3). Glucose phosphorylation over the range
of glucose concentrations that approximated normoglycemia was 60%
increased in the Px islets, in close agreement with the results in Fig.
2. The reason was a twofold increase in glucokinase
Vmax (P < 0.002) with no change in
ED50. Hexokinase Vmax also was somewhat
increased. However, the absence of increased basal glucose usage in the
Px islets (Fig. 2) suggested that its activity within the intact cell
was not substantially changed. Neither was there a difference in
G-6-P concentration between the Px and sham islets at 2.8, 8.3, and 16.7 mM glucose (Fig. 4), which is a
potent allosteric inhibitor of hexokinase (9). The reason for the
raised basal insulin secretion in the Px islets was thus not clear from
these results.

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Fig. 3.
Glucose phosphorylation (n = 5 experiments) measured after
90-min incubations at the shown glucose concentrations in islet
extracts from rats 4 wk after 60% ( ) or sham ( ) pancreatectomy.
The reaction was measured at 30°C as the appearance of NADH from
NAD+ by exogenous glucose-6-phosphate dehydrogenase.
Left: incubations carried out at 0.03-0.5 mM glucose,
primarily representing hexokinase activity; right: incubations
carried out at 6-100 mM glucose, primarily representing
glucokinase activity.
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Fig. 4.
Glucose 6-phosphate (G-6-P) concentration (n = 3 experiments) of isolated islets from rats 4 wk after 60% ( ) or sham
( ) pancreatectomy. Freshly isolated islets were incubated in KRB and
2.8, 8.3, or 16.7 mM glucose for 60 min, followed by measurement of
G-6-P content, as described in the text.
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|
Glucokinase immunoblot.
Glucokinase immunoblots were performed in Px and sham islets; Fig.
5A shows a standard denaturing gel
with samples from 2 Px and 2 sham rats. A small increase in glucokinase
was noted in the Px islets, which averaged 142 ± 12% of control
(P < 0.02 based on 6 Px and 6 sham rats). However, the gels
were performed using equivalent amounts of islet protein from Px and
sham islets and thus failed to adjust for the 40%
-cell enrichment
of the Px islets (Table 1); the close agreement between these figures suggested that the increased band intensity of the Px islets on immunoblot simply reflected differences in how much
-cell protein was loaded. Additional support for this conclusion is shown in Fig.
5B, which is a nondenaturing gel with comparable Px and sham islet extracts. Nondenaturing conditions leave relatively intact protein-protein interactions or allosteric influences that modulate a
protein's function. Striking differences were seen compared with the
denaturing gel; the Px band intensity was now less than that of the
sham, confirming the presence of a glucokinase posttranslational change
in Px islets. Thus the doubled glucokinase Vmax per
kilogram DNA in the Px islets stemmed from an enhanced catalytic
activity of this enzyme, as opposed to a changed
-cell expression.

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Fig. 5.
Glucokinase Western blots of isolated islets from rats 4 wk after 60%
(Px) or sham (C) pancreatectomy. A: 30-µg protein aliquots
from 2 separate sham-operated and 60% Px rats run under denaturing
conditions, as described in text. The top band eluted at 52 kDa and is
glucokinase. B: 40-µg protein aliquots from a sham and a 60%
Px rat run under nondenaturing conditions, as described in text.
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|
 |
DISCUSSION |
This study has identified the
-cell adaptive responses in 60% Px
rats that kept insulinemia, and thus glycemia and triglyceridemia, indistinguishable from sham-operated rats. One element was a limited regeneration of the excised
-cells, which brought the pancreas
-cell mass from 40% of normal at the time of surgery to slightly more than one-half of normal several weeks later (19). The more consequential effect was a changed relationship between the glucose concentration and islet glucose utilization so that normal levels of
glycemia elicited a raised flux of islet glucose metabolism, and
consequently greater than normal insulin secretion. The latter reflects
-cell glycolytic flux being a key regulator of glucose-induced insulin secretion through well characterized effects on
-cell ion
channels and membrane potential (27, 31). The heightened glycolytic
flux stemmed from an enhanced activity of glucokinase, which is the
main regulator of
-cell glucose usage and is termed the
-cell
glucose sensor (26). The mechanism was an increased catalytic activity
of this enzyme, as opposed to a change of its
-cell concentration,
which was confirmed by the novel observation of divergent denaturing
and nondenaturing glucokinase immunoblots in the Px islets. Crucial to
the workings of this regulatory system was the observation that the
degree of change in glucokinase activity was small; glucose
phosphorylation and, consequently, glucose metabolism were 60-80%
increased in Px islets at the physiologically relevant 8.4 mM glucose,
compared with the multifold changes in gene expression that make up
many cellular regulatory systems, such as the fivefold increase in
proinsulin gene expression reported after short-term exposure of
-cells to a high glucose concentration (21). The reason is that
glucokinase is the rate-limiting
-cell glycolytic enzyme and is
without allosteric or end-product influences (26, 27). Its activity
thus directly modulates
-cell glucose utilization and insulin
secretion (38). This effect was evident in the current study by the
close agreement between the degree of changes in glucokinase activity,
glucose utilization, and insulin secretion in the Px islets. As such,
insulin release at 8.4 mM glucose also increased 80%, which is very
significant physiologically. By this reasoning, it is of interest that
the rise in
-cell mass to 55% of normal (19), together with the
92% increased glucokinase activity per cell mass shown in Table 3,
results in whole pancreas glucokinase activity in the Px rats being
identical to that in the sham rats. Our results are consistent with the
recent study of Martín et al. (25), which concluded that an
upregulation of islet glucose metabolism was the basis for the
-cell
glucose hypersensitivity in normoglycemic 60% Px mice, although the
study provided no insight as to the mechanism (25). Note, glucose utilization was assessed in this study only as conversion of
[5-3H]glucose to
[3H]H2O, and a measure of affected
pathways and metabolites is needed to fully understand how the change
in glucokinase activity affects islet-cell glucose metabolism,
analogous to the studies of others (38, 39).
Our finding of increased glucokinase catalytic activity in the Px
islets agrees with our studies of normoglycemic insulin-resistant SHR
rats (6) and rats that were hyperinsulinemic and normoglycemic secondary to glucose infusions (5). Making the same observation in
three diverse rat models of successful
-cell adaptation suggests that variable glucokinase activity is a core
-cell adaptive response of the glucose homeostasis system that is initiated by changing metabolic demands for insulin secretion of multiple types. The basis
for this effect is not known, although evidence for a protein interaction affecting
-cell glucokinase activity has recently appeared (37). However, this is not the only mechanism that alters
-cell glucose sensing in a compensatory fashion, because others have
been identified in specialized states. Best studied is pregnancy, a
state of insulin resistance, in which upregulation of
-cell
glucokinase and hexokinase cellular levels occurs, presumably on the
basis of the distinctive mediators prolactin and placental hormones
(30, 34). A second mode regards the compensatory hyperinsulinemia in
models of insulin resistance with marked hypertriglyceridemia, which
has been linked to high levels of islet FA metabolites (12, 28,
42). We showed that the mechanism is increased activity of
phosphofructokinase that lowers the G-6-P level through
accelerated G-6-P metabolism (23). G-6-P is a
potent inhibitor of hexokinase (9), and our results thus focused on
deinhibition of hexokinase as the basis for the enhanced basal
-cell
glucose sensing and insulin secretion with excess FA. Viewed in
toto, there is a repertoire of
-cell adaptive responses depending on
the stimulus, which have in common an ability to alter insulin
secretion through changes in the
-cell sensitivity to
glucose and thus do not require a change in glycemia. The importance of
this effect reflects the myriad of cellular dysfunctions
attributed to chronic hyperglycemia (so-called glucose toxicity) (20,
33, 41).
The above discussion has focused on normoglycemic states, i.e., when
the
-cell adaptive mechanism is successful. Studies of hyperglycemic
states have suggested that there is the occurrence of an additional
element that affects
-cell glucose sensing. We studied 90% Px rats
that develop mild hyperglycemia, and we observed that increased
hexokinase Vmax was the dominant change affecting
islet glucose phosphorylation (13), in tandem with a substantial rise
in basal insulin secretion (20). We also studied 48-h glucose-infused
rats and showed that islet hexokinase Vmax and
basal insulin secretion were increased in hyperglycemic rats but not in
those that were normoglycemic/hyperinsulinemic (5). Cockburn et al. (7)
studied diabetes-prone Zucker diabetic fatty rats before and after the
onset of diabetes; the prediabetes time point was characterized by
increased islet glucokinase activity (analogous to our results in 60%
Px rats), as opposed to hexokinase activity and basal insulin secretion
being markedly increased when hyperglycemia was established (7). These
results suggest that hyperglycemia or an associated metabolic defect
upregulates
-cell hexokinase activity, and consequently basal
insulin secretion, through an undefined mechanism. Moreover, we have
argued that a hyperstimulated insulin secretion, leading to the
-cell insulin content falling below a required level for normal
secretory responses, is a mechanism of
-cell dysfunction with
chronic hyperglycemia (18). It may be that this increased hexokinase
activity is an important causative factor of this sequence.
In summary, our results provide an explanation for the conundrum
regarding the glucose homeostasis system whereby
-cells are known to
be glucose responsive at multiple functional levels, so that glycemia
is considered their main regulatory influence, coupled with the
understanding that glycemia varies little throughout life because of
offsetting compensations of insulin secretion. The regulatory system
observed herein is based on a changeable rate of
-cell glucose
metabolism so that
-cells are "tricked" to perceive altered
glycemia in the face of normoglycemia, allowing glucose-regulated
-cell adaptive responses to occur when glycemia is unaltered.
 |
ACKNOWLEDGEMENTS |
This work was supported by a grant from the American Diabetes
Association (to J. L. Leahy).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: J. L. Leahy, Univ. of Vermont College of Medicine, Given C331, Burlington, VT
05405 (E-mail: jleahy{at}zoo.uvm.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. §1734 solely to indicate this fact.
Received 9 August 1999; accepted in final form 25 January 2000.
 |
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