1 Departments of Biochemistry and 3 Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214; and 2 Center for Metabolism and Nutrition, Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio 44109
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ABSTRACT |
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Artificial rearing of 4-day-old rat pups on a high-carbohydrate (HC) milk formula results in the immediate onset of hyperinsulinemia. To evaluate these early changes, studies on pancreatic function were carried out on 12-day-old HC rats and compared with age-matched mother-fed (MF) pups. The plasma insulin and glucagon contents were increased sixfold and twofold, respectively, in HC rats compared with MF rats. There was a distinct leftward shift in the glucose-stimulated insulin secretory pattern for HC islets. HC islets secreted insulin in the absence of any added glucose and in the presence of Ca2+ channel inhibitors. The activities of glucokinase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate dehydrogenase complex were significantly increased in HC islets compared with MF islets. The protein contents of GLUT-2 and hexokinase were significantly increased in HC islets. These findings indicate that a nutritional intervention in the form of a HC formula only during the suckling period has a profound influence on pancreatic function, causing the onset of hyperinsulinemia.
newborn rats; nutritional modification; insulin secretion; -cell
metabolism
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INTRODUCTION |
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THE ENDOCRINE PANCREAS undergoes significant structural
modifications during the late intrauterine and suckling periods in the
rat, involving differentiation, neogenesis, and apoptosis (13). These
changes modulate the insulin secretory capacity of the pancreas.
Nutrition during the early phases of life is of major importance for
proper tissue development and functional maturation (9). Earlier
studies from this laboratory have demonstrated the consequences of a
dietary modification in the form of a high-carbohydrate (HC) milk
formula given only during the suckling period (12, 29). The "pup in
a cup" model, adapted in this laboratory, involves the artificial
rearing of 4-day-old pups on a HC milk formula (56% of the total
calories being derived from carbohydrates compared with 8% in rat
milk) until postnatal day
24 when they are weaned onto lab chow.
This nutritional intervention, only during the suckling period, results
in the immediate onset of hyperinsulinemia, which persists into
adulthood without any further nutritional stimulus (12, 29). The growth
rate of HC rats increases around postnatal
day
55, and these animals are distinctly
obese by day 100 (12, 29). In 100-day-old HC rats,
hypertrophy of the -cells and an altered glucose-stimulated insulin
secretory pattern have been reported (12, 16). HC females spontaneously
transmit these characteristics to their progeny (30). In addition,
alterations in the insulin-signaling pathway for the activation of
glycogen synthase in liver, muscle, and adipose tissue have also been
reported in 100-day-old HC rats (25, 26).
Because the timing of the dietary modification in this rat model coincides with the neonatal endocrine pancreatic development, it appears that this HC milk formula potentiates certain changes in the pancreas (both structural and functional), causing the onset of hyperinsulinemia in these pups. It also appears that these changes are "imprinted" into adulthood. It follows that hyperinsulinemia is an early response in this model, leading to the onset of obesity and insulin resistance in adult life. Hence, understanding the mechanism(s) that causes the initial onset of hyperinsulinemia in these rats is fundamentally pertinent.
Insulin secretion from -cells in the endocrine pancreas is regulated
by nutrient and nonnutrient secretagogue (19). The insulinotropic
action of glucose and other nutrient secretagogues requires their
metabolism in the
-cells (the fuel hypothesis), resulting in an
increase in the ATP-to-ADP ratio in the
-cell, which closes the
KATP channels; this leads to
membrane depolarization, opening of voltage-dependent
Ca2+ channels, influx of
Ca2+, and a rise in the cytosolic
free Ca2+ concentration; the
elevation of intracellular Ca2+
concentration directly triggers insulin exocytosis (21). To understand
the effect of the HC milk formula on the insulin secretory pattern,
insulin secretion in response to various stimuli (a range of glucose
concentrations and modulators of
Ca2+ and
K+ channels) was studied in islets
isolated from 12-day-old HC rats and compared with the pattern obtained
from the islets isolated from age-matched MF rats.
Nutrient transport and metabolism are important components of the insulin secretory process in the islets. Because the onset of hyperinsulinemia is an early response to the HC milk formula in the HC rat, we focused our attention in this study on the insulin secretory pattern, as well as glucose transport and metabolism, in islets from 12-day-old rats. The hypothesis is that changes in the insulin secretory pattern and in glucose transport and/or metabolism are responsible for the onset and persistence of hyperinsulinemia in this rat model.
Our results show significant changes in the insulin secretory pattern with concomitant changes in glucose transport and metabolism in HC islets. These observations indicate that a dietary modification during the suckling period has a profound influence on pancreatic function in these pups and that these changes have implications for the onset of pathological conditions in adulthood.
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MATERIALS AND METHODS |
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Materials. Collagenase type IV was from Worthington Biochemicals (Freehold, NJ). GLUT-2 antibody was from Biogenesis (Sandown, NH). The antibody to the low Michaelis-Menten constant (Km) hexokinase was a kind gift from Dr. J. E. Wilson (Michigan State University). The insulin radioimmunoassay kit was from Linco Research (St. Louis, MO). 2-Deoxy-D-glucose, mannoheptulose, glibenclamide and iodoacetate, and kits for assay of glucose and triglycerides and all other reagent grade chemicals were from Sigma (St. Louis, MO). The kit for the assay of free fatty acids (FFA) was from Boehringer-Mannheim (Indianapolis, IN). Nimodipine was from Calbiochem (San Diego, CA), and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid BAPTA was from Molecular Probes (Eugene, OR). The protein assay kit was from Bio-Rad (Hercules, CA). [1-14C]pyruvate and the reagents for chemiluminiscence were from NEN-Du Pont (Boston, MA).
Animal protocols. All animal protocols
were approved by the Institutional Animal Care and Use Committee. Timed
pregnant Sprague-Dawley rats were obtained from Zivic Miller
Laboratories (Zellenople, PA). The newborn pups were pooled and
assigned to each nursing mother (11 pups/dam) and were left with the
mothers until postnatal day
4. On postnatal
day
4, pups were assigned randomly to
control and experimental groups. In the mother-fed (MF) control group, pups were reared by their nursing mothers, whereas pups in the experimental group were reared artificially on a high-carbohydrate (HC)
formula, wherein 56% of the calories are derived from carbohydrate, 24% from protein, and 20% from fat (Table
1). The artificial-rearing technique
employed in this study has been described in detail previously (29).
Intragastric cannulas were placed while the animals were under light
anesthesia, and the pups were raised artificially on the HC milk
formula in isolation from their dams. Pups were individually housed in
styrofoam cups floating in a temperature-regulated water bath at
37°C. Milk was delivered for 20- to 25-min periods every 2 h at a
rate of 0.45 kcal · g body wt1 · day
1.
The volume of HC formula fed was calculated such that the growth rate
of the HC rats mimicked that of age-matched MF rats. Twice daily the
pups were stroked in the anal-genital region to promote urination and
defecation. The daily routine included cleaning, weighing, and
adjusting the rate of formula delivery. On
day
12, the rats were killed by
decapitation, trunk blood was collected, and the pancreas was processed
for the isolation of islets.
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Earlier reports from this laboratory established that the artificial rearing of 4-day-old pups on a high-fat milk formula similar in macronutrient composition (protein 24%, carbohydrate 8%, and lipid 68%) to rat milk, per se had no influence on the onset of hyperinsulinemia and adult onset obesity (12, 29).
Isolation of islets. Pancreatic islets were isolated from 12-day-old pups by a modification of the method described previously (33). Briefly, pancreases from two 12-day-old pups were pooled and digested with 3 mg of collagenase (type IV, Worthington) in Hank's buffer, pH 7.4, at 37°C in a shaking water bath. The digestion was stopped after 10-12 min by the addition of ice-cold Krebs-Ringer bicarbonate (KRB) buffer containing 0.2% bovine serum albumin (BSA), pH 7.4. After being washed two times with the same buffer, islets were picked manually under a stereomicroscope.
Insulin secretion. Four batches of islets were isolated, and islets from the same batch were used to compare insulin secretion at the different glucose concentrations. Equal numbers (30) of freshly isolated islets from 12-day-old MF and HC pups were preincubated at 37°C in KRB buffer containing 16 mM HEPES, 1 mM glucose, 0.01% BSA, pH 7.4, for 30 min under an atmosphere of 95% O2-5% CO2 in a shaking water bath. The islets were then resuspended in fresh KRB buffer (0.5 ml), and an aliquot of the buffer was removed for determination of zero-time insulin levels. Islets were further incubated with glucose at final concentrations of 1, 2.8, 5.5, or 16.7 mM, and aliquots of buffer were withdrawn at 10 and 60 min for determination of insulin. Zero-time insulin levels were subtracted from insulin levels determined at later times for determination of insulin release.
Plasma insulin, glucagon, glucose, triglyceride, and
FFA levels. Pups (12-day-old) were killed by
decapitation, and trunk blood was collected in heparinized tubes.
Plasma was separated by centrifugation at 8,000 rpm for 10 min and
stored at 70°C. Plasma insulin and glucagon levels were
measured by radioimmunoassay. Plasma glucose, triglycerides, and FFA
were measured with kits according to the protocols described by suppliers.
Pancreatic insulin content. A piece of
the pancreas was accurately weighed and homogenized in 200 µl of acid
alcohol solution (75 ml ethanol, 1.5 ml 12 N HCl, and 23.5 ml of
distilled water). The pancreatic extracts were centrifuged at 10,000 rpm for 5 min at 4°C, and the supernatants were stored at
20°C until assayed for insulin as described earlier.
Glyceraldehyde-3-phosphate dehydrogenase assay. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was carried out as described previously (5). Briefly, islets (~100) were washed with homogenization buffer containing 50 mM glycylglycine, pH 7.0, 10 mM EDTA, 100 mM sodium fluoride, and 0.5 mM dithiothreitol and were homogenized by sonication for 10 s in 100 µl of the same buffer. The homogenate was centrifuged at 10,000 rpm for 30 s, and the supernatant was used for enzyme assay. GAPDH activity was measured in buffer containing 50 mM triethanolamine pH 7.6, 50 mM arsenate, 100 mM glyceraldehyde 3-phosphate, 2.4 mM reduced glutathione, and homogenate (1-3 µg protein). The tubes were preincubated in a water bath for 5 min at 37°C after which a baseline fluorometric reading was recorded at 347 nm excitation and 448 nm emission. NAD+ (250 µM final concentration) was added to start the reaction, and the linear change in fluorescence was recorded for 5 min.
Assay for glucose-phosphorylating
activities. Glucose-phosphorylating activity
measurements were carried out by a modification of the method described
previously (27). Briefly, islets (~300) were sonicated in ice-cold
buffer containing 20 mM
K2HPO4,
1 mM EDTA, 110 mM KCl, and 5 mM dithiothreitol (pH 7.4). The sonicated material was then centrifuged at 12,000 g at 4°C for 20 min. The pellet
was resuspended in homogenization buffer. Ten microliters of
supernatant or the resuspended pellet was added to 200 µl of assay
buffer containing 50 mM HEPES, pH 7.4, 100 mM KCl, 7.4 mM MgCl2, 15 mM -mercaptoethanol,
0.5 mM NAD+, 0.05% BSA, 3 µg/ml
glucose 6-phosphate dehydrogenase, 5 mM ATP, and either 0.5 or 100 mM
glucose. The low and high
Km glucose activities were measured at 0.5 and 100 mM glucose, respectively. After
being incubated for 90 min at 30°C, the reaction was stopped by the
addition of 2 ml of 500 mM NaHCO3
buffer (pH 9.4), and fluorescence was measured at 347 nm emission and
448 nm excitation. In each assay, blanks were obtained by incubating
0.5 or 100 mM glucose in the absence of ATP. For glucokinase activity,
correction for the hexokinase activity was applied by subtracting the
activity measured at 0.5 mM glucose from the activity measured at 100 mM glucose.
Pyruvate dehydrogenase complex assay. Pyruvate dehydrogenase complex (PDC) activity was measured essentially as described previously (14). Briefly, ~250 islets were homogenized in 150 µl of 50 mM HEPES (pH 7.5), 0.2 mM KCl, 3 mM EDTA, 5 mM dithiothretiol, 0.5 µg/µl leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, and 0.25% (vol/vol) Triton-X 100. The homogenate was centrifuged for 10 min at 500 g, and the supernatant was twice frozen and thawed. The active form of PDC was measured in the supernatant as such, and the total form was measured after activation with lambda protein phosphatase (New England BioLabs). Enzyme activity was assayed by measuring the rate of decarboxylation of [1-14C]pyruvate. The reactions were stopped by addition of trichloroacetic acid. The liberated 14CO2 was absorbed and quantified with a scintillation counter.
Western blot of glucose transporter-2 protein (GLUT-2) and hexokinase protein. Islets were lysed in 80 mM Tris, pH 6.8, containing 5% SDS, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, and 0.2 mM N-ethylmaleimide. Equal amounts of protein were separated by electrophoresis on a 10% SDS-PAGE gel, transferred to nitrocellulose membrane, and probed with the respective antibodies. Protein bands were visualized by chemiluminescence. The immunoblots were scanned with a densitometric scanner.
Protein assay. Protein assays were carried with kits from Bio-Rad according to the instructions of the manufacturer.
Statistical analysis. The results are means ± SE. The significance of differences between MF and HC groups was analyzed by Student's t-test or by ANOVA with Tukey's honestly significant difference test.
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RESULTS |
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Effects of HC diet on physiological parameters and
insulin secretion. Table 2
shows the physiological characteristics of the 12-day-old MF and HC
rats. The characteristic feature is the marked increase (>6-fold) in the plasma insulin levels of HC rats compared with age-matched MF rats. An increase (>2-fold) in plasma glucagon level is also observed. The plasma insulin-to-glucagon molar
ratio is increased from 0.4 in MF rats to 1.1 in HC rats. Pancreatic
insulin levels are also higher in HC rats. Despite the hyperinsulinemia
in the HC rats, plasma glucose levels are comparable between the two
groups of rats. Plasma FFA and triglycerides are significantly
decreased in the HC rats compared with age-matched MF rats. There is no
significant change in the body weight of the HC rats compared with MF
rats.
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Figure 1 depicts the glucose-stimulated
insulin secretory pattern of islets from 12-day-old MF and HC rats at
10 and 60 min. MF islets did not secrete any measurable amount of
insulin at 1 mM glucose (A: 10 min;
B: 60 min) or at 2.8 mM glucose at 10 min and secreted a very small amount of insulin (0.23 fmol/30 islets)
at 60 min. In contrast, islets isolated from 12-day-old HC rats
secreted insulin at 1 and 2.8 mM glucose at both 10 and 60 min (Fig.
1). At 5.5 mM glucose, HC islets secreted ~15- and 9-fold more
insulin compared with MF islets at 10 and 60 min, respectively. At 16.7 mM glucose, the response by HC islets was markedly higher compared with
MF islets at both 10 and 60 min. Intragroup comparison showed that MF
islets have a higher (16.7 to 5.5 mM glucose) ratio for
insulin secretion at both 10 and 60 min (6- and 10.5-fold) compared
with HC islets (1.4- and 3.46-fold). But for each glucose concentration
studied, HC islets secreted significantly greater amounts of insulin
compared with MF islets. These results clearly indicate an altered
insulin secretory pattern for islets isolated from 12-day-old HC rats,
with a marked leftward shift (1 mM glucose) for
glucose-stimulated insulin secretion.
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Effects of inhibitors and nonnutrient stimuli on
insulin secretion. In the presence of
Ca2+ channel inhibitors
(nimodipine and BAPTA) HC islets secreted ~40% of the amount of
insulin they secrete at 5.5 mM glucose (Table 3). Similar results were obtained in the
presence of EGTA (500 µM) or
Ca2+ free buffer. MF islets did
not secrete any measurable amount of insulin under these conditions.
The amount of insulin secreted by HC islets under these conditions is
almost 3.5-fold higher than the amount of insulin secreted by MF islets
in the presence of Ca2+ at 5.5 mM
glucose. Under conditions where K+
channels are closed, as in the presence of 100 µM glibenclamide or 25 mM potassium chloride, and the membrane is in a depolarized state, MF
islets secreted significantly more insulin compared with their basal
levels, whereas this treatment had no effect on HC islets. In the
presence of the glucokinase inhibitor, mannoheptulose, there was no
effect on insulin secretion at 5.5 mM glucose, whereas at 16.7 mM
glucose the insulin secreted was almost reduced to basal levels
(5.5 mM glucose) in both MF and HC islets. It was also
observed that HC islets could secrete insulin in the absence of added
glucose, in the presence of the nonmetabolizable sugar 2-deoxyglucose,
and also in the presence of iodoacetate (an inhibitor of glycolysis) in
amounts similar to what they secrete in the presence of
Ca2+ channel inhibitors (~1.60
fmol · 30 islets1 · 60 min
1; Table 3). On the
basis of these findings, it is clear that basal insulin release is
fundamentally altered in the HC islets.
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Glucose metabolism in pancreatic
islets. Glucose is transported into the pancreatic
-cell where it is metabolized via both the glycolytic pathway and
the tricarboxylic acid cycle, resulting in ATP production and
initiation of insulin secretion. Because glucose transport into the
islet cells is the first step in the pathway leading to insulin
secretion, GLUT-2 protein content was measured in islets from MF and HC
pups. Western blot analysis of islet homogenates from 12-day-old MF and
HC pups indicated a significant increase (~70%) in GLUT-2 protein
content in HC islets compared with MF islets (Fig.
2).
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Glucokinase (high
Km for glucose)
and hexokinase (low
Km for glucose)
activities were measured in the islet homogenates (supernatant and
pellet fractions). Preliminary experiments indicated that glucokinase
was present predominantly in the supernatant fraction and hexokinase
was present in both the pellet and supernatant fractions. Glucokinase
activities in the supernatant fractions of MF and HC islet homogenates
showed an increase of ~40% in activity for the HC rats (Fig.
3). Hexokinase activities in the
supernatant and pellet fractions of the islet homogenates were observed
in nearly equal amounts for both the groups but were increased
significantly in both the supernatant and pellet fractions from HC
islets compared with MF islets (an increase of ~100% in the
supernatant fraction and 60% for the pellet fraction).
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Because a significant difference was observed in the low
Km hexokinase
activity in MF and HC islets, hexokinase protein was also quantified.
Hexokinase protein was significantly increased (60%) in islets from HC
rats (Fig. 4). No change was observed in
the glucokinase protein content in islets from MF and HC rats (data not
shown).
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The glucose 6-phosphate formed as a result of glucokinase-hexokinase
activities is further metabolized in the glycolytic pathway, and GAPDH
is a key enzyme in this pathway. Because of the importance of GAPDH in
the coupling of glucose metabolism to insulin secretion, its activity
was measured in islet supernatants from 12-day-old MF and HC pups. A
significant increase in GAPDH (~30%) was observed in islet extracts
from HC pups compared with MF pups (Fig.
5).
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Acetyl-CoA is formed from pyruvate by oxidative decarboxylation of
pyruvate catalyzed by the PDC, and the activity of the active form of
PDC is crucial for glucose oxidation. Hence, the active and total forms
of PDC were measured in islet homogenates from 12-day-old MF and HC
rats. The active form of PDC is increased by ~43% in islet
homogenates from HC islets compared with MF islets (Fig.
6). The total form of the enzyme, obtained
after conversion of the inactive form to the active form by treatment
with a partially purified phosphatase, was also significantly increased
by almost twofold in islet extracts from HC pups (Fig. 6). The above
observations indicate significant changes in glucose transport and
metabolism in HC islets.
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DISCUSSION |
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Hyperinsulinemia is a very early event in the HC rats. Hence, this study was designed primarily to characterize the effects of the nutritional intervention, in the form of a HC milk formula during the suckling period, on pancreatic islet function. The influence of this HC formula on islet function becomes all the more significant because the suckling period is a critical window in the development of the endocrine pancreas. This report clearly indicates that an increase in carbohydrate-derived calories during the suckling period has a profound influence on pancreatic function, resulting in hyperinsulinemia during this period and forming the basis for adult-onset obesity as described previously (12).
A comparison of the plasma profiles of the 12-day-old MF and HC rats indicates a significant increase in insulin content in the plasma of HC rats (Table 2). The immediate onset of hyperinsulinemia in these rats may represent an early adaptive response to the HC formula and indicates that the pancreas may be an initial target in the HC rats. An earlier study from this laboratory demonstrated that even during the first 24 h after the rats are on the HC formula, there is a steep increase in circulating insulin levels (10). The significant increase in plasma glucagon in HC rats may be a regulatory adaptation to maintain glucose homeostasis in the face of rising insulin levels. The hyperinsulinemic HC rats maintain normoglycemia, indicating that the counterregulatory effects of glucagon balance the effects of hyperinsulinemia and offset the onset of hypoglycemia in the HC pups. Interestingly, although the plasma glucagon levels in the HC rats are comparable with the plasma glucagon levels in age-matched MF rats on days 54 and 100, these rats remain normoglycemic but hyperinsulinemia persists throughout adult life (12). Even 1-yr-old HC rats remain euglycemic although they are hyperinsulinemic (unpublished observations). It is interesting to note that in the 12-day-old HC rats, the glycogen content of liver and muscle is significantly increased compared with age-matched MF rats (data not shown), indicating hypersensitivity to insulin action (higher insulin-to-glucagon molar ratio) in the peripheral tissues. Elevated FFA levels have been implicated to regulate pancreatic function and to modulate insulin levels in rodent models of obesity (20, 28). FFA levels were significantly lower in HC rats, indicating that they may not have a regulatory role to play in the augmented insulin secretion during this period. The nature of the milk consumed by these rats during the suckling period (high fat for MF rats and HC for HC rats) may account for the low levels of FFA and triglycerides in the HC rats compared with MF rats. There was no significant difference in the body weight between the two groups of rats on day 12, because the volume of HC formula fed was adjusted to give the same growth rate for these pups as their age-matched MF pups.
The primary short-term regulation of insulin secretion is achieved by elevated glucose levels. However, this stimulus is absent in the HC rats, because their glucose levels were not significantly different compared with age-matched MF rats (Table 2). Hence, some other functional and/or structural changes must contribute to maintain this hyperinsulinemia. Earlier we had shown that the number of insulin-positive cells and islet size were increased by ~1.6-fold and 1.3-fold, respectively, in islets from 12-day HC rats (12). But these reported increases do not appear to account for an approximately sixfold increase seen in circulating plasma insulin levels.
A leftward shift in the glucose dose response for insulin secretion was observed in islets from 12-day-old HC rats (Fig. 1). The lowering of the threshold for glucose-stimulated insulin secretion (1 mM glucose for HC islets) may be responsible to a large extent for maintaining the >6-fold increase in circulating plasma insulin levels. This leftward shift in insulin secretion makes it possible for a large increase in insulin secretion at basal and subbasal glucose levels. Similar lowering in the threshold for glucose-stimulated insulin release has been reported in obese Zucker rats and in pregnancy (conditions in which hyperinsulinemia is present) (20, 31).
Ca2+ is an important mediator of
glucose-stimulated insulin secretion (32). From our data, it is evident
that HC islets secrete a modest amount of insulin in the absence of
extracellular Ca2+ or when the
intracellular Ca2+ stores are
depleted or the voltage-gated Ca2+
channels are blocked (Table 3). This indicates two possibilities: 1) a
Ca2+-independent insulin secretion
pathway is also operative in HC islets and/or
2) structural alterations in
-cells of HC rats facilitate insulin secretion in the absence of
Ca2+. Komatsu et al. (15) have
shown that a Ca2+-dependent and a
Ca2+-independent GTP-dependent
insulin secretion pathways, both of which require glucose metabolism,
exist in rat pancreatic islets. The fact that the HC islets secrete as
much insulin in the presence of 2-deoxyglucose or in the absence of
glucose or in the presence of iodoacetate, an inhibitor of glycolysis,
as they do in the absence of Ca2+
suggests some structural alterations in HC islets. These results also
suggest that a common mechanism may be responsible for insulin secretion under such conditions by HC islets. Depolarization of the
membrane by glibenclamide or high
K+ does not appear to alter
insulin secretion by HC islets, indicating that
Ca2+ stores are already elevated
in the HC islets and may contribute partly to the elevated basal
insulin release. Under similar conditions, insulin secretion by MF
islets is significantly increased above basal levels. It has been
suggested that neurotransmitters and incretins simultaneously activate
protein kinase C and protein kinase A activities, causing an increase
in GTP levels and insulin secretion (15). It is possible that a similar
mechanism may be operative in the HC islets and account for the
increased basal insulin secretion in the HC rats.
Due to the central importance of glucose metabolism in insulin
secretion by the -cells, it was of interest to evaluate the adaptive
changes occurring in glucose metabolism in the islets of the HC rats
(Fig. 7). It is plausible that initial
changes in glucose metabolism are imprinted into adulthood and form the basis for the onset of pathological conditions. The first step in the
pathway for glucose-stimulated insulin secretion by the
-cells is
the facilitated diffusion of glucose by GLUT-2. Our studies indicate an
approximate increase of 70% in GLUT-2 protein content in islets from
12-day HC rats (Fig 2). Weinhaus et al. (31) have reported a similar
increase in GLUT-2 protein in islets of normoglycemic and
hyperinsulinemic pregnant rats. In contrast, in the hyperinsulinemic
fa/fa
Zucker rats GLUT-2 expression and function were normal (20). In rodent
models of overt hyperglycemia, the loss of glucose-induced insulin
secretion has been attributed to a reduction in GLUT-2 protein content
in the
-cells (18). However, islets cultured in a medium containing
a high glucose concentration demonstrate an increase in GLUT-2 protein
levels (34). It appears that the expression and function of GLUT-2 may
be specifically modified according to the physiological and metabolic
environment of the model being studied.
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Glucose is metabolized in islets to serve as stimulus for insulin secretion. Both the low and high Km glucose-phosphorylating activities were significantly increased in HC islets compared with MF islets (Fig. 3). Several reports suggest that under some conditions, increased activity of the low Km glucose-phosphorylating enzyme could contribute to basal hyperinsulinemia (1, 2, 4, 8, 20). Overexpression of the hexokinase I gene in transfected rat islets caused a significant increase in insulin secretion at low glucose levels (1). Similar results were reported when yeast hexokinase B gene was overexpressed in islets of transgenic mice (8). Male JCR:LA/N-cp rats exhibited a fourfold increase in hexokinase activity and a leftward shift in glucose-induced insulin secretion (2). In the hyperinsulinemic Zucker diabetic and Zucker fatty rats, hexokinase activity was significantly increased in islets (20). In the preobese and prediabetic stage of the Zucker diabetic and Zucker fatty rats, increased hexokinase activity was associated with a leftward shift in insulin secretion (4). It appears that the marked increase in the low Km hexokinase activity in both the supernatant and pellet fractions of islet homogenates from HC rats coupled with the significant increase in hexokinase protein content plays a significant role in sustaining basal hyperinsulinemia in the HC rats (Figs. 3 and 4). The modest increase in glucokinase activity may also have a role to play in the altered insulin secretory pattern in the 12-day-old HC rats (Fig. 3). Glucokinase activity is increased, without changes in hexokinase activity, in rat islets during pregnancy (31) and in the islets of spontaneously hypertensive rats (3), both of which are normoglycemic but hyperinsulinemic. Because both hexokinase and glucokinase activities are significantly increased in HC islets, it appears that the overall increase in the glucose-phosphorylating activities contributes significantly to the hyperinsulinemic state in the 12-day-old HC rats (Fig. 7).
Both GAPDH and PDC (active and total) activities were significantly
increased in islets from 12-day HC pups (Figs. 5 and 6). GAPDH activity
was reported to be increased in islets from 100-day-old HC male rats
(16). Moreover, -cells cultured in a high glucose medium showed
increased GAPDH expression (24). An increase in GAPDH is associated
with increased ATP production, which mediates insulin secretion (6).
PDC catalyzes the oxidation of pyruvate to acetyl-CoA for ATP
production in the tricarboxylic acid cycle and has been shown to be
decisive for glucose oxidation in several tissues (23). In the diabetic
db/db
mouse model, diminished glucose-induced insulin secretion has been
attributed to a decrease in PDC activity in islets (35). Our results
indicate that increased PDC and GAPDH activities support increased
glucose metabolism and increased insulin secretion by islets from
12-day HC rats (Fig. 7).
Recently, Leahy et al. (17) reported that in the normoglycemic,
hyperinsulinemic, and spontaneously hypertensive rats, pancreatic -cell insulin content was increased, whereas no changes occurred in
proinsulin mRNA levels, biosynthesis, or degradation. These observations suggest that some other regulatory mechanism(s) modulating insulin secretion may be operative in these rats. In the HC rat model,
it appears that the onset of hyperinsulinemia is the primary event and
is not a compensatory response to either insulin resistance or overt
hyperglycemia in these 12-day-old HC rats. A recent study (21) showed
that qualitative and quantitative differences in fatty acid content of
the diet influence glucose-induced insulin secretion by islets. In the
HC formula, fatty acids are qualitatively similar to rat milk (11), but
the fat-derived calories are reduced compared with rat milk and are
compensated for by an increase in carbohydrate-derived calories.
Circulating FFA levels are reduced in the HC rat, and considering that
the fat-derived calories are reduced in the HC formula, it appears that
the increase in carbohydrate-derived calories primarily contributes to
the basal hyperinsulinemia in this model. The lowering of the glucose
threshold for the insulin secretory response sustained by a marked
increase in hexokinase protein content and activity observed in these
rats may, in part, contribute to the development of the
hyperinsulinemic state. It is not clear what mechanism is responsible
for the modest insulin release observed in the absence of any stimulus
or in the presence of Ca2+ channel
inhibitors by islets from HC rats (Table 3). However, insulin release
observed under such conditions could contribute substantially to the
basal hyperinsulinemia of the HC rats. Other regulatory factors, such
as storage and trafficking of insulin, the influence of a variety of
peptide factors produced by the
-cells, and the incretins produced
by the gut, may have a role to play in the regulation of secretion of
insulin by the HC islets. The diet-induced rat model for obesity
reported from this laboratory differs from other reported animal models
for obesity in that the increased consumption of carbohydrate-derived
calories only during the suckling period causes the immediate onset of
hyperinsulinemia, which is the primary event, and subsequently leads to
adult onset obesity. Our results clearly emphasize the importance of
the role played by early nutrition on the development of pancreatic
function and the implications of changes in its function for the onset of obesity and insulin resistance in adulthood. It appears that altering the nature of the fuel source during the suckling period has
profound implications for functional alterations of the pancreas. The
HC milk given during the suckling period induces modifications in the
-cells resulting in the onset of hyperinsulinemia. This model
therefore provides a unique opportunity to evaluate the importance of
the early onset of hyperinsulinemia to the development of conditions
like obesity, insulin resistance, and possibly non-insulin-dependent diabetes mellitus in adulthood.
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ACKNOWLEDGEMENTS |
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The authors are grateful to G. Goldberg for technical assistance with animal care. The authors are also grateful to Dr. J. Aletta (Department of Pharmacology and Toxicology, State University of New York at Buffalo) for critical reading of the manuscript.
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FOOTNOTES |
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This work was supported in part by National Institute of Child Health and Human Development Grant HD-11089 and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-51601.
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.
Address for reprint requests and other correspondence: M. S. Patel, Dept. of Biochemistry, School of Medicine and Biomedical Sciences, State Univ. of New York at Buffalo, 140 Farber Hall, 3435 Main St., Buffalo, NY 14214 (E-mail: mspatel{at}buffalo.edu).
Received 30 April 1999; accepted in final form 28 July 1999.
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