Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214
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ABSTRACT |
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To investigate the influence of a high carbohydrate (HC) intake during the suckling period on pancreatic function in adult life, neonatal rats were artificially reared on a HC milk formula during the preweaning period and then weaned onto lab chow. In the adult HC rat, hyperinsulinemia is sustained by a variety of biochemical and molecular adaptations induced in the HC islets during the suckling period. The adult HC islets showed a distinct left shift in the glucose-stimulated insulin-secretory pattern. HC islets were also able to secrete moderate levels of insulin in the absence of glucose and in the presence of Ca2+ channel inhibitors. In addition, the mRNA levels of preproinsulin, somatostatin transcription factor-1, upstream stimulatory factor-1, stress-activated protein kinase-2, phosphatidylinositol kinase, and GLUT-2 genes were significantly increased in HC islets. These results show that consumption of a HC formula during the suckling period programs pancreatic islet function in adult rats, resulting in the maintenance of hyperinsulinemia in the postweaning period and eventually leading to the development of obesity in adult life.
neonatal nutrition; metabolic adaptations; insulin secretion; obesity
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INTRODUCTION |
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IT IS NOW INCREASINGLY CLEAR that dietary influences exerted early in life have long-term consequences leading to the development of pathological conditions in adulthood (2). Several studies on animal models and human epidemiological data support the notion that fetal and immediate postnatal nutritional adaptations have permanent effects on cellular structure, physiology, and metabolism in different organs (7). The late fetal and early postnatal periods in the rat have been recognized as being critical for pancreatic islet ontogeny (10). Previous studies from this laboratory have illustrated both the immediate and long-term consequences of a dietary modification in the form of a high-carbohydrate (HC) diet fed to neonatal rats in the preweaning period (8, 32). Artificial rearing of neonatal rat pups on a HC milk formula (56% of total calories from carbohydrate vs. 8% in rat milk) results in the immediate onset of hyperinsulinemia and alterations in lipid metabolism in these rats (8). These changes persist into adulthood with the development of adult-onset obesity (8).
The ability of -cells to secrete insulin is regulated by nutrient,
hormonal, and neuronal stimuli. Glucose metabolism has been widely
accepted to play a principal role in insulin secretion by its effects
on the ATP-sensitive K+ (KATP) and
Ca2+ channels (26). Glucose metabolism coupled
to the activation of protein kinase A (PKA) and protein kinase C (PKC)
can augment insulin secretion by the Ca2+-independent
pathway (12). Long-term regulation of insulin secretion is
regulated at the level of transcription of the preproinsulin gene and
translation of its mRNA. In response to the HC dietary intervention
during the suckling period, several adaptations at the molecular,
cellular, and biochemical levels take place in islets isolated from
12-day-old HC rats. Molecular adaptations include upregulation of the
specific transcription factor genes, thereby facilitating increased
preproinsulin gene transcription (29a). In
addition, the transcription of several islet-specific transcription
factors that modulate cellular development of the pancreas is also
increased (29a). Considering the fact that this dietary
treatment overlaps the critical window of pancreatic development in
rat, the increased gene transcription of these transcription factors
may be critical for the altered ontogeny of the HC pancreas during this period. Cellular alterations in 12-day-old HC islets include changes in the number and size of the islets and rate of
proliferation and apoptosis (22). Biochemical
adaptations in 12-day-old HC islets include extensive changes in both
proximal and distal sites in the insulin-secretory pathway (1,
29). Significant among these changes are the ability of
the HC islets to secrete insulin in a glucose- and
Ca2+-independent manner, a marked left shift in the
response to a glucose stimulus, and upregulation of a glucagon-like
peptide-1 (GLP-1)-mediated process in islets (29).
Our hypothesis is that early adaptations in islets from HC neonatal rats (29) are programmed into adulthood and contribute to the hyperinsulinemic state of 100-day-old HC rats (32). Hence, we have analyzed biochemical and molecular changes in islets from 100-day-old male HC rats and have observed that the early events in 12-day-old HC islets are sustained into adulthood, indicating the importance of the consequences of a nutritional stimulus during the critical period of organ development.
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MATERIALS AND METHODS |
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Materials. 2-Deoxyglucose, mannoheptulose, glibenclamide, iodoacetate, collagenase type I, diprotonin, GLP-1, acetylcholine (ACh), kits for assay of triglycerides and glucose, and all other reagent-grade chemicals were from Sigma Chemical (St. Louis, MO). The insulin RIA kit was from Linco Research (St. Louis, MO). The assay kit for the estimation 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). TRIzol reagent, murine leukemia virus reverse transcriptase, and all the primers were from GIBCO-BRL (Grand Island, NY). pGEM-3Z vector was from Promega (Madison, WI).
Animal protocol.
The Institutional Animal Care and Use Committee approved all animal
protocols used in this study. Timed-pregnant Sprague-Dawley rats were
obtained from Zivic Miller Laboratories (Zelienople, PA) and were
housed in a temperature- and light-controlled room with free access to
laboratory chow and water. Newborns of several mothers were pooled and
randomly distributed to nursing mothers (12 pups/dam). On postnatal
day 4, pups were randomly assigned to either control or
experimental group. In the mother-fed (MF) control group, pups were
allowed to be nursed by their dams. The pups in the experimental group
were reared artificially on a HC formula wherein 56% of the total
daily calories were derived from carbohydrates compared with 8% in rat
milk. The artificial rearing technique employed in this study has been
described in detail previously (8). In brief, the pups
were anesthetized, and intragastric cannulas were introduced. They were
housed in Styrofoam cups floating in a 37°C waterbath and were fed HC
formula at the rate of 0.45 kcal · g body
wt1 · day
1. The pups were stroked
in the urogenital region to promote urination and defecation every day.
On day 18, the cannulas of the pups were cut close to the
skin, and the pups were housed in plastic cages and continued on the HC
formula provided in feeding tubes until day 24. On postnatal
day 24, control and experimental pups were weaned onto
laboratory stock diet. Food and water were provided ad libitum, and
both groups were killed on day 100. Blood and pancreases
were collected at the time of killing and processed as described below.
Plasma levels of insulin, GLP-1, glucose, triglyceride, and free
fatty acids.
One hundred-day-old HC and MF rats were killed by decapitation, and
trunk blood was collected in heparinized tubes (containing 50 µM
diprotinin for GLP-1). Plasma was collected by centrifugation at 5,000 rpm for 10 min and stored at 70°C. Plasma insulin was measured by
RIA with rat insulin as standard. Plasma glucose, triglycerides, and
free fatty acids (FFA) were quantitated using kits according to
manufacturers' instructions. Plasma GLP-1 (active form) was measured
by the Assay Services of Linco Research.
Islet isolation and insulin secretion.
Pancreatic islets were isolated from 100-day-old HC and MF rats by
collagenase digestion (34). The islets were hand picked under a stereomicroscope and were used fresh for studies related to
insulin secretion or were stored frozen at 80°C until further use.
For insulin secretion experiments, islets (10/tube) were preincubated
in Krebs-Ringer bicarbonate (KRB) buffer containing 16 mM HEPES, 5.5 mM
glucose, and 0.01% BSA, pH 7.4, for 30 min under an atmosphere of 95%
02-5% C02 in a shaking waterbath at 37°C. At
the end of the preincubation period, the entire solution was aspirated,
fresh KRB buffer containing the appropriate glucose concentration and
the required agonist/antagonist was introduced, and an aliquot of the
buffer was removed for determination of insulin levels at time
0, followed by subsequent removal of aliquots at 10 and 60 min for
the determination of insulin release. For insulin secretion studies in
the absence of glucose, preincubation was also done in the absence of
glucose. Similarly, for a stringent Ca2+-deprived
condition, both preincubation and incubation were carried out under a
stringent Ca2+-depleted condition. Insulin levels in the
samples were determined by RIA with rat insulin as standard. Duplicate
islet incubations were performed for each animal. The results are
expressed as femtomoles of insulin released per 10 islets per time
indicated, because there were no significant differences in the protein
or DNA content of islets between MF and HC rats (results not shown).
Measurement of glucose-phosphorylating activity. Glucose-phosphorylating activity was assayed by a modification of the method described previously (31). Briefly, islets (~200) were sonicated in ice-cold buffer containing (in mM) 20 potassium phosphate (pH 7.4), 1 EDTA, 110 KCl, and 5 dithiothreitol (DTT) (pH 7.4). The homogenate was then centrifuged at 12,000 g at 4°C for 20 min. The pellet was resuspended in the same buffer. Glucose-phosphorylating activity in islet extracts (pellet and supernatant fractions) was measured at 0.5 or 100 mM glucose, as described previously (1). 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.
RNA isolation and cDNA synthesis.
Total RNA was isolated from islets obtained from 100-day-old HC and
age-matched MF control rats by use of the TRIzol
reagent-phenol-chloroform procedure (GIBCO-BRL). cDNA was prepared
using 6 µg of total islet RNA and 20 pmol random hexamers in a
30-µl solution containing (in mM) 50 Tris · HCl, pH 8.4, 75 KCl, 4 MgCl2, 10 DTT, and 0.125 of each dNTP and 200 units
of Moloney murine leukemia virus reverse transcriptase. After
incubation for 1 h at 42°C, the reaction mixture was heated to
70°C for 15 min to inactivate the reverse transcriptase. The cDNA was
stored at 20°C.
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Protein assay. Protein assays were carried out using a kit from Bio-Rad according to the instructions of the manufacturer.
Statistical analysis. The results are means ± SE. The significance of difference between MF and HC groups was analyzed by use of Student's t-test. Differences were considered significant at P < 0.05.
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RESULTS |
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Effect of feeding HC diet to neonates on physiological parameters
and insulin secretion by islets of adult rats.
Earlier (32), we reported that HC rats reared artificially
on a HC formula during the suckling period maintained hyperinsulinemia with normoglycemia in the postweaning period and gained body weight at
a higher rate starting around day 55 compared with MF
controls. The present study (Table 2)
indicates that the body weight of 100-day-old HC rats is significantly
higher than age-matched MF rats and that they continue to be
hyperinsulinemic (~3-fold increase) but are normoglycemic. Because
obesity is also associated with the changes in plasma FFA and
triglyceride levels in other obesity-related models, we determined
their levels in the present study. The results presented in Table 2
show that there are no significant differences in the plasma levels of
glucose, FFA, and triglycerides between MF and HC groups. Thus these
factors may not be contributing to the maintenance of hyperinsulinemic
state in adult HC rats.
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Effect of channel inhibitors and modulators on insulin secretion by
HC and MF islets.
According to the paradigm proposed for glucose-stimulated insulin
secretion, there are at least three target sites important for the
regulation of insulin release: 1) glucose metabolism via the
glycolytic and mitochondrial pathways for ATP production, 2)
closing of the KATP channel, and 3) opening of
the voltage-sensitive Ca2+ channel (16). In
accordance with the above paradigm, we examined the alterations at all
of the three target sites by using inhibitors and modulators of various
pathways on insulin secretion. In the presence of iodoacetate (an
inhibitor of glycolysis), HC islets secreted ~15% (~6
fmol · 10 islets1 · 60 min
1) of the amount they secreted in the presence of
basal glucose concentration (Table 3) and
similar to MF islets at basal glucose concentration. Notably, the
entire amount (~6 fmol · 10 islets
1 · 60 min
1) was secreted
during the late phase of insulin secretion (Table 3). Furthermore, the
HC islets secreted a similar amount of insulin in the presence of
2-deoxyglucose (a nonmetabolizable analog of glucose). Under conditions
where the K+ channels are closed and the membrane is
depolarized in the presence of 100 µM glibenclamide, MF islets
secreted significantly more insulin compared with their basal values
both at 10 and 60 min; however, this treatment had no effect on HC
islets (Table 3). The exposure to diazoxide (pharmacological activator
of KATP channel) abolished the basal insulin secretion by
MF islets, but HC islets secreted ~15% of the amount they had
secreted under basal condition at 60 min (Table 3). When the islets
were treated with the L-type Ca2+ channel blocker
nimodipine (1 µM) or BAPTA (1 µM), the same pattern of insulin
secretion (~6 fmol · 10 islets
1 · 60 min
1) by the HC islets was observed as with the treatment
of diazoxide and as in the complete absence of glucose (Table 3). These
results indicate that the altered basal insulin secretion in the HC
islets is affected through modifications at all of the three target
sites discussed earlier.
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Insulin secretion via the
Ca2+-independent augmentation pathway in
adult HC rats.
It is known that Ca2+ is required for both the
KATP channel-dependent and -independent pathways for
insulin secretion. Additionally, there exists a
Ca2+-independent augmentation pathway that is glucose
dependent and requires the simultaneous activation of PKA and PKC
(11). The possible alteration in this pathway and the
contribution of this pathway to the increased insulin secretion was
examined in the HC and MF islets under a stringent
Ca2+-deprived condition at three glucose concentrations (0, 5.5, and 16.7 mM). Because the response under this condition was seen
only in the late phase, the results of this experiment are reported for
the late phase only. As indicated in the previous section, the HC
islets secreted a significant amount of insulin (6 fmol · 10 islets1 · 60 min
1) under
glucose-deprived and Ca2+-free conditions, and this
secretion was significantly increased in the presence of GLP-1 plus ACh
(Fig. 2A). There was no effect of these agents for MF islets (Fig. 2A). At 5.5 mM glucose,
the activators of PKA and PKC caused insulin secretion (~2 fmol) in the MF islets, whereas HC islets secreted 17 fmol of insulin (Fig. 2B). At a higher glucose concentration (16.7 mM), the
combined presence of PKA and PKC activators increased HC islet insulin secretion twofold compared with the amount secreted by the MF islets
under identical conditions (Fig. 2C).
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Glucose metabolism in adult HC and MF islets.
In view of the known relationship between glucose metabolism and
insulin secretion (20) and also the fact that we have
observed increased oxidation and utilization of glucose in adult HC
islets (unpublished observation), experiments were carried out
to determine whether increased insulin secretion would reflect
increases in the activity of glucose-metabolizing enzymes. Glucose is
transported via the action of GLUT-2 into the pancreatic -cell,
where it is metabolized via both the glycolytic pathway and the
tricarboxylic acid pathway, resulting in ATP production. Glucose is
phosphorylated by the glucose-phosphorylating enzymes glucokinase
[high Michaelis-Menten constant (Km)] and
hexokinase (low Km), and this step is considered to be the rate-limiting step in glycolytic flux; hence, these activities were measured in the islet homogenates (supernatant and
pellet fractions). Glucokinase activity in the supernatant fraction of
HC homogenate showed an increase of 12% compared with that seen in the
MF islets (Fig. 3). Hexokinase activities
in the supernatant and pellet fractions of the islet homogenate were observed in nearly equal levels for both groups but were increased significantly in both fractions from HC islets compared with MF islets.
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Upregulation of gene expression in HC islets.
In the short term, insulin secretion demands are met by the translation
of preexisting mRNA. However, to meet the chronic demand for insulin
secretion, appropriate alterations have to take place at the level of
gene expression. In keeping with this hypothesis of expression of the
preproinsulin gene, several transcriptional factors and related
proteins were investigated by measuring mRNA levels using a
semiquantitative RT-PCR procedure (27). As depicted in
Fig. 4A, the preproinsulin
mRNA level was increased about threefold in the HC islets compared with
the MF islets. The mRNA level of GLUT-2 was also increased ~3.5-fold
in the HC islets compared with the MF islets (Fig. 4B).
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DISCUSSION |
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Earlier studies on the HC rat model have shown that the mere switch from high-fat-derived calories in rat milk to high-carbohydrate-derived calories in the HC formula causes extensive adaptations at the molecular, cellular, and biochemical levels in islets isolated from 12-day-old HC rats (Refs. 22, 29, 29a). The late fetal and immediate postnatal periods are critical periods in pancreatic ontogeny in the rat. Nutritional experiences early in life as an etiological factor in the development of adulthood diseases have been reported. For example, dietary restriction during pregnancy in rats, particularly of proteins, produces reduction in birth weight and causes the onset of hypertension and hyperglycemia in adult offspring, even in the absence of nutritional insult (13, 14, 23). Our results presented here clearly indicate that several of the adaptations induced in pancreatic islets during the suckling period due to rearing of neonatal pups on the HC milk formula are programmed into adulthood and form the basis for the adult-onset obesity seen in 100-day-old HC rats. Hyperinsulinemia and the associated changes in islets from HC rats are unique to neonatal exposure of islets to the HC diet, because when neonates are fed a formula high in fat, they do not show hyperinsulinemia (32).
Despite the withdrawal of the HC formula at the time of weaning, circulating insulin levels continued to be significantly increased in 100-day-old HC rats compared with age-matched MF rats, indicating persistence of the altered insulin-secretory capacity of the HC islets. In 12-day-old HC rats, the plasma GLP-1 level was markedly higher compared with age-matched MF controls, and the GLP-1-mediated signal for augmented insulin release was shown to be one of the mechanisms supporting the hyperinsulinemic state in these rats (29). However, GLP-1-mediated events appear not to be programmed into adulthood in the adult HC rats, as is evident from the similar circulating GLP-1 level between MF and HC rats (Table 2). Milburn et al. (18) have suggested that the increased plasma levels of FFA frequently associated with obesity may increase insulin secretion in obese individuals with normoglycemia. Such an association does not appear functional in 100-day-old HC rats, because their plasma FFA levels were normal.
The primary short-term regulation of insulin secretion is achieved by elevated glucose levels. Considering that both plasma glucose and FFA levels were normal in 100-day-old HC rats, it is tempting to speculate that a non-nutrient-mediated process (at the structural, biochemical, or molecular levels) as a consequence of the early adaptive response experienced by the islets appears more likely as a contributory factor for the hyperinsulinemic state of the HC rats in adult life. We have recently documented extensive cellular adaptations in 12-day-old HC islets (22). Earlier, we reported that there was an increase in insulin positive mass in adult HC islets (32), suggesting that persistence of cellular adaptations induced during the suckling period may account, in part, for the hyperinsulinemic state of the HC adult rats.
The left shift in glucose dependency observed in the adult HC rats points to intrinsic differences in low-Km glucose metabolism. Activities of both hexokinase (low Km) and glucokinase (high Km) were significantly increased in HC islets compared with MF islets (Fig. 3). A wealth of data supports the notion that, under some conditions, increased activity of a low-Km glucose-phosphorylating enzyme could contribute to basal hyperinsulinemia. Overexpression of low-Km hexokinase in isolated islets or islet cell lines supported increased basal insulin secretion (3). In obese Zucker rats, low-Km hexokinase activity was increased in islets (18). Overexpression of glucokinase activity in normal islets resulted in insulin secretion without any change in glucose usage or lactate production (20). Mannoheptulose is reported to competitively block the hexose binding site of glucokinase to produce inhibition of insulin secretion (17). In the present study, neither the MF or the HC islets showed any susceptibility to inhibition by mannoheptulose (Table 3) due to the basal concentration of glucose employed in the secretion studies. Because the activities of these two crucial enzymes are significantly increased in adult HC islets, it emerges that the overall increase in the glucose-phosphorylating activities contributes significantly to the hyperinsulinemic state in the 100-day-old HC rats. It appears that adaptations with respect to glucose metabolism observed in islets from 12-day-old HC rats (1) are programmed and continued to be expressed in islets from 100-day-old HC rats.
Ca2+ is an important element required for metabolically regulated insulin secretion. Although the majority of insulin secretion (~85%) is inhibited in the absence of extracellular Ca2+, a small but significant amount of insulin (~15% of the amount secreted in the basal state by adult HC islets) was secreted in the complete absence of extracellular Ca2+ or when the Ca2+ channels were blocked (Table 3). Secretion of insulin from Ca2+-depleted islets is indicative of either structural changes in islets or the possibility of the existence of a Ca2+ channel-independent pathway (11). Depolarization of the membrane by glibenclamide did not appear to alter insulin secretion by HC islets, indicating that the Ca2+ stores were already elevated in the HC islets and partly contributed to the elevated secretion of basal insulin. Again, these altered insulin-secretory capacity of 100-day-old islets reflects the persistence of the early adaptive responses observed in 12-day-old islets.
Recently, it has been shown that, in islets, a Ca2+-independent augmentation pathway operates that stimulates insulin secretion under stringent calcium-depleted conditions but requires glucose metabolism and simultaneous activation of PKA and PKC (11). In the present study, 100-day-old HC islets released moderate amounts of insulin under stringent glucose- and calcium-deprived conditions in the presence of physiological activators of PKA and PKC (Fig. 2). In the presence of glucose, still higher amounts of insulin were secreted, indicating that the Ca2+-independent augmentation pathway is upregulated in HC islets compared with MF islets and may play an important role in the maintenance of hyperinsulinemia in adult rats.
The ability of the -cell to hypersecrete insulin presumably results
from an altered pattern of preproinsulin gene expression. The chronic
hyperinsulinemic state of the HC rats warrants continuous replenishment
of insulin stores on a minute-to-minute basis by compensation at the
molecular level by increases in the rates of translation of the
preproinsulin mRNA and transcription of the preproinsulin gene. In
100-day-old HC islets, the level of preproinsulin mRNA was markedly
increased compared with the MF islets. That preproinsulin gene
expression is modulated according to demand is illustrated by decreases
in mRNA levels associated with a decreased insulin content in Zucker
diabetic rats (30) and increased mRNA levels in
corticosterone-induced insulin resistance (6, 21).
Transcription of the preproinsulin gene is dependent on the specific
cis-acting sequences located within the proximal promoter (up to 350 bp from the transcription start site) (4).
Specifically, the E boxes located at
104 and
232 and the A boxes
located at
76,
130,
210 and
312 are important (5).
The E1 box binds insulin enhancer factor-1, and the A boxes bind a
number of homeodomian transcription factors, of which STF-1 is the most
abundant in the
-cells (25). In the present study, the
level of STF-1 mRNA is increased about threefold in the HC rat islets
compared with MF islets, and this increase may contribute to the
increased transcription of the preproinsulin gene. In addition to its
effect on preproinsulin gene transcription, STF-1 transactivates
several other islet genes, including the GLUT-2 gene, which were also
significantly increased in HC islets. The altered expression of other
genes may also explain the coordinated upregulation of insulin gene
transcription. For example, the enhanced level of USF-1 mRNA may
account for the increased expression of STF-1, because USF-1
specifically binds to the STF-1 promoter and can thereby function as an
upstream regulator of STF-1 gene expression in HC islets
(24).
Glucose can modulate the STF-1 DNA binding activity via a
phosphorylation cascade involving SAPK-2 (15). In
pancreatic -cells, high glucose is shown to activate SAPK-2 via PI
3-kinase (15). Activation of SAPK-2 leads to
phosphorylation in the cytoplasm of an inactive 31-kDa form of STF-1
that is modified to a 46-kDa active form that is translocated to the
nucleus and causes increased preproinsulin gene transcription
(33). In the present study, the mRNA levels of both PI
3-kinase and SAPK-2 were increased significantly in the HC islets
compared with MF islets. Considering the fact that these two play an
important role in the phosphorylation cascade leading to the activation
of STF-1 and its binding to its cognate DNA binding site, it is
reasonable to conclude that their increased levels do contribute to the
overall increase in preproinsulin gene transcription observed in this study.
In conclusion, the results from this study on islets from 100-day-old
HC rats clearly indicate that the adaptive responses induced in
12-day-old HC islets are sustained in 100-day-old islets. For example,
the altered insulin-secretory pattern with leftward shift in response
to a glucose stimulus, the increased glucose-phosphorylating activities, the ability to secrete insulin in a calcium and
glucose-independent manner, the upregulation of the calcium-independent
augmentation pathway, and the increased preproinsulin gene expression
supported by upregulation of immediate upstream events are all
programmed into adulthood. The question is raised how the augmented
expression of each of the mRNAs studied might relate to the observed
phenotypic change. It is possible that a common molecular mechanism may
be responsible for these alterations such as the increased expression of a key transcription factor(s) that is required for normal expression of -cell genes. The expression of this factor is probably regulated by glucose-derived metabolite(s) in neonatal rats as the phenotypic change observed in adult HC rats is induced by a mere switch in the
type of calories consumed (from fat to carbohydrates) during the
suckling period.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. S. G. Laychock (Dept. of Pharmacology and Toxicology) and Dr. G. Willsky (Dept. of Biochemistry) of the State University of New York at Buffalo Medical school 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.
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.
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 18 December 2000; accepted in final form 30 April 2001.
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