Departments of 1 Biochemistry and 2 Pharmacology and Toxicology, 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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Artificial rearing of neonatal rats on a high-carbohydrate (HC) milk formula resulted in the immediate onset of hyperinsulinemia. This study examines, in islets of 12-day-old HC rats, adaptive changes that support the hyperinsulinemic state. Increases in plasma glucagon-like peptide-1 (GLP-1) levels and islet GLP-1 receptor mRNA supported increased insulin secretion by HC islets. Isolated HC islets, but not mother-fed (MF) islets, secreted moderate amounts of insulin in a glucose- and Ca2+-independent manner. Under stringent Ca2+-free conditions and in the presence of glucose, GLP-1 plus acetylcholine augmented insulin release to a larger extent in HC islets. Levels of adenylyl cyclase type VI mRNA and activities of protein kinase A, protein kinase C, and calcium calmodulin kinase II were increased in HC islets. A tenfold increase in norepinephrine concentration was required to inhibit insulin secretion in HC islets compared with MF islets, indicating reduced sensitivity to adrenergic signals. This study shows that significant alterations at proximal and distal sites of the insulin secretory pathway in HC islets may support the hyperinsulinemic state of these rats.
hyperinsulinemia; nutritional intervention; protein kinase C; protein kinase A; glucagon-like peptide-1; adenylyl cyclase
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INTRODUCTION |
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STUDIES IN ANIMAL
MODELS and human epidemiological and clinical studies indicate
that both the quantity and quality of nutrition during critical periods
of early development (fetal and neonatal) have lifetime consequences,
supporting the concept of the metabolic programming of early adaptive
responses into adulthood (30). These adaptive responses
occur at the cellular, biochemical, and molecular levels
(46). The late fetal and early postnatal periods of rat
pancreatic islet ontogeny constitute a period of vulnerability to a
nutritional stimulus/insult (6). During these ontogenic periods, the endocrine pancreas undergoes structural and functional adaptations to a nutritional intervention that are memorized and that
prime the organism for adult-onset conditions. For example, gestational
diabetes and protein malnutrition during pregnancy alter the
insulin-secretory capacity of -cells of adult progeny (2,
8).
The consequences of a nutritional intervention in the form of a high-carbohydrate (HC) milk formula only during the suckling period have been studied extensively in this laboratory. With the use of the "pup-in-the-cup" model, 4-day-old pups are artificially reared on a HC milk formula (56% of calories being derived from carbohydrate vs. 8% in rat milk) until postnatal day 24, when they are weaned onto laboratory chow (18, 19). This nutritional intervention results in the immediate onset of hyperinsulinemia, which persists into adulthood (15, 19). The HC animals remain normoglycemic during the suckling period as well as in adulthood (44); they are obese by postnatal day 100 (19). Hypertrophy of the islets and altered insulin secretory patterns are seen in both 12- and 100-day-old rats (27, 44).
Insulin secretion by the -cells is subject to a wide variety of
stimulatory and inhibitory influences by nutrients, hormones, and
neuronal factors. The major pathway for the stimulation of insulin
secretion is the KATP channel-dependent pathway
(25). In contrast, the KATP
channel-independent pathway of insulin secretion augments glucose
effects after the increase in intracellular Ca2+ (12,
37). The activation of protein kinase A (PKA) and protein kinase
C (PKC) potentiates glucose-stimulated insulin secretion (5) even under stringent Ca2+-free conditions
(Ca2+-independent augmentation pathway) (25).
On the other hand, partial or complete inhibition of insulin secretion
is brought about by several agents, including norepinephrine (NE)
(38). Insulin secretion at any particular time is thus a
balance between stimulation and inhibition.
In a recent study, a leftward shift in the insulin secretory response to a glucose stimulus with a concomitant increase in the low Km hexokinase activity was seen in islets isolated from 12-day-old HC rats (1). However, the leftward shift in the insulin secretory pattern does not totally account for the sixfold increase observed in circulating insulin levels in 12-day-old HC rats compared with age-matched MF rats. Hence, efforts have continued to be made to understand other primary adaptations that could support the hyperinsulinemic state. The hypothesis tested is that signal transduction mechanisms including adenylyl cyclase, PKA, PKC, and calcium calmodulin kinase II (CaM kinase II) regulated by G protein-coupled receptors mediate insulin secretion in islets from 12-day-old HC islets and contribute to the early onset of hyperinsulinemia in these rats.
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MATERIALS AND METHODS |
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Materials.
Collagenase (type IV) was from Worthington Biochemicals (Freehold, NJ).
The insulin radioimmunoassay kit was from Linco Research (St. Louis,
MO). Iodoacetate, diazoxide, norepinephrine (NE), glucagon-like
peptide-1 (GLP-1), acetylcholine (ACh), and all other reagent-grade
chemicals were from Sigma (St. Louis, MO). Nimodipine,
bis-indolylmaleimide 1, PKA inhibitor 14-22 amide and
autocamtide-2-related inhibitory peptide were from Calbiochem (San
Diego, CA).
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
methyl ester (BAPTA-AM) was from Molecular Probes (Eugene, OR).
The assay kits for PKA, PKC, and CaM kinase II and TRIzol reagent,
murine leukemia virus reverse transcriptase, Superscript preamplification kit, Superscript II ribonuclease H reverse
transcriptase kit, and the primers used for the amplification of GLP-1
receptor were all from GIBCO-Life Technologies (Grand Island, NY). The TOPO TA cloning kit was from Invitrogen (Carlsbad, CA); PGEM-3Z vector
was from Promega (Madison, WI); [-32P]ATP was from Du
Pont-NEN (Boston, MA); and the protein assay reagent was from Bio-Rad
(Hercules, CA).
Animal protocol.
The Institutional Animal Care and Use Committee approved all animal
protocols. Timed pregnant Sprague-Dawley rats (Zivic Miller Laboratories, Zellenople, PA) were given access to stock laboratory diet and water ad libitum. Newborns of all mothers were pooled and
distributed to nursing mothers (12 pups/dam). On postnatal day
4, pups were removed from their dams and assigned randomly to
either control or experimental diet groups. In the mother-fed (MF)
group, pups were nursed by their dams. The pups in the experimental group (HC group) were artificially reared on a HC milk formula [caloric content: carbohydrate 56%, protein 24%, and fat 20%; caloric content of rat milk: carbohydrate 8%, protein 24%, and fat
68% (18)]. The artificial rearing technique has been
described in detail elsewhere (18). Briefly, intragastric
cannulas were introduced under mild anesthesia, and the pups were
housed in Styrofoam cups floating in a 37°C water bath. Milk was
given for 20- to 25-min periods every 2 h at the rate of 0.45 kcal · g body wt1 · day
1 to
match the body weight of age-matched MF rats. Pups from both groups
were killed on postnatal day 12, and blood and pancreas were
collected and processed as described below.
Isolation of islets. Pancreatic islets were isolated from the 12-day-old rat pups by collagenase digestion as described previously (47). Briefly, pancreas from two rats were pooled and digested with collagenase, and the islets were hand picked under a dissecting microscope.
Insulin secretion.
Insulin secretion was measured by static incubation. Agonist/antagonist
was included during preincubation and secretion periods, as indicated
in the legends to the figures. In a typical experiment, 30 freshly
isolated islets were incubated in 500 µl of Krebs-Ringer bicarbonate
(KRB) buffer containing 16 mM HEPES, 1 mM glucose, and 0.01% BSA, pH
7.4, for 30 min at 37°C (preincubation) under an atmosphere of 95%
O2 and 5% CO2. The preincubation medium was then removed by aspiration; 500 µl of fresh KRB buffer containing agonist/antagonist and desired glucose concentration were introduced, and an aliquot of the sample was withdrawn for determination of zero-time insulin. The incubation was further continued, and aliquots of the samples were withdrawn at 10 and 60 min for determination of
insulin levels. All samples were kept frozen at 20°C until assayed
for insulin by radioimmunoassay with rat insulin as standard. When a
stringent Ca2+-depleted condition was needed, KRB buffer
devoid of calcium and with EGTA (500 µM), nimodipine (1 µM), and
BAPTA (1 µM) was used during the preincubation and incubation
periods. When NE was used, it was present during the incubation period only.
Plasma GLP-1 content.
Twelve-day-old pups were killed by decapitation, and trunk blood was
collected in heparinized tubes (containing 50 µM diprotin A). Plasma
was separated by centrifugation and stored at 70°C. The assay for
the plasma level of the active form of GLP-1 (7-37) was carried out by the Assay Services Facility of Linco Research.
GLP-1 receptor mRNA content of islets. Total RNA was isolated from pancreatic islets obtained from 12-day-old HC and MF rat pups using the TRIzol-chloroform procedure. GLP-1-receptor cDNA was prepared by use of 6 µg of total islet RNA using the SuperScript preamplification kit according to manufacturer's instructions. The relative level of GLP-1 receptor mRNA was determined by PCR in the presence of a synthetic competitor DNA (21). The competitor DNA for measuring mRNA of GLP-1 receptor was prepared by introduction of a small internal deletion into the cloned GLP-1 receptor cDNA using the PCR-based mutagenesis procedure (40). The primers that were used for making competitor DNA were sense 5'-ACTTCCTTGTCTTCATCC-3' and antisense 5'-GTTTCATGCTGCTGTCCCTCCATCTGGACCTCATTG-3'. The competitor DNA was deleted by 50 bp and cloned into PGEM-3Z vector. A semiquantitative RT-PCR assay (39), in which the same amount of competitor template added to each reaction, was used to compare the levels of GLP-1-receptor mRNAs in islets from HC and MF rats. The primers that were used to amplify the GLP-1 receptor cDNA were sense 5'-ACTTCCTTGTCTTCATCC-3' and antisense 5'-GTTTCATGCTGCTGTCCCT-3'. PCR reaction was carried out as described previously (39). The PCR products were separated by electrophoresis in 2% agarose gel, analyzed with Bio-Rad Gel Doc 1000 and Molecular Analyst software for quantitative analysis, and normalized using competitor control.
Semiquantitative analysis of adenylyl cyclase isoform mRNAs in isolated islets. Total RNA was extracted from rat pancreatic islets using TRIzol. First strand cDNA was obtained by RT from 0.5 µg of total RNA by use of a random hexamer and SuperScript II ribonuclease H reverse transcriptase kit as described previously (28). Islet cDNA was amplified with primers from a highly conserved sequence among adenylyl cyclase isoforms (types I-VIII), as reported previously (13): sense 5'-GCTCTAGACCATCGGTAGCACCTACATGGC-3' and antisense 5'-GCGACATTCACTGCATTICCCCAGATGTCATA-3'. PCR analysis of islet cDNA produced three bands of ~300, 270, and 250 bp, which were purified and cloned using a TOPO TA cloning kit with pCR2.1-TOPO vector, and the plasmids purified from positive clones were sequenced using M13 reverse primer.
To quantitate adenylyl cyclase isoform transcript levels, PCR was carried out as described previously (28). The sequences of primer pairs for adenylyl cyclase genes andAssay of protein kinases. Activities of PKA, PKC, and CaM kinase II were determined in islets isolated from 12-day-old MF and HC rats by means of assay kits from GIBCO-Life Technologies. Details of the assay conditions are as per manufacturer's instructions.
Protein assay. Protein assays were carried out by the Bradford method with the use of the protein assay reagent from Bio-Rad.
Statistical analysis. The results are means ± SE, and statistical significance between the two 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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Glucose- and Ca2+-independent insulin secretion by HC
islets.
In an earlier report (1), we observed that islets from
12-day-old HC rats secreted significant amounts of insulin in the absence of any nutrient stimuli (~1.92 fmol · 30 islets1 · 60 min
1). Under identical
conditions, MF islets did not secrete any measurable amount of insulin.
To understand the mechanisms supporting insulin secretion by HC islets
under these conditions, insulin secretion was studied in the presence
of iodoacetate (an inhibitor of glycolysis), diazoxide (pharmacological
activator of KATP channels), and under a stringent
Ca2+-deprived condition (Fig.
1). When insulin secretion was measured in the absence of glucose but in the presence of iodoacetate, HC islets
secreted similar amounts of insulin as they did when both glucose and
iodoacetate were absent (Fig. 1). When diazoxide was used in the
absence of glucose to activate KATP channels and exclude
Ca2+ influx through voltage-dependent Ca2+
channels, HC islets secreted similar amounts of insulin as under only-glucose-deprived conditions (Fig. 1). To rule out the possibility of increased influx and availability of Ca2+ supporting
insulin secretion in the absence of glucose, insulin secretion was
assayed in the absence of glucose and under a stringent Ca2+-deprived condition (Ca2+-free buffer and
in the combined presence of EGTA, BAPTA, and nimodipine). HC islets
secreted a similar amount of insulin under the
Ca2+-deprived and control conditions, indicating the
ability of these islets to secrete insulin in a glucose- and
Ca2+-independent manner (Fig. 1).
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GLP-1: plasma level and receptor mRNA
level in islets.
The greater insulin secretory response to ingested glucose compared
with intravenous glucose administration is principally the result of
incretin hormones secreted from the gastrointestinal tract in response
to the glucose absorbed (23). Plasma levels of GLP-1, one
of the incretin hormones, were measured in 12-day-old MF and HC rats. A
significant increase (approximately threefold) was observed in plasma
GLP-1 levels in HC rats compared with MF rats (Fig.
2). GLP-1 binds to its cognate receptor
in the -cell and transmits the signal to activate insulin secretion
(23). The relative mRNA levels of the GLP-1 receptor in
islets from 12-day-old MF and HC rats were quantitated using a
semiquantitative PCR method. A significant increase (approximately
threefold) in the mRNA level of GLP-1 receptor in HC islets was also
seen (Fig. 3).
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PKA, PKC, and CaM kinase II activation in HC islets. GLP-1 and ACh, via activation of PKA and PKC respectively, stimulate insulin secretion (25). The effect of GLP-1 and ACh on insulin secretion by islets isolated from 12-day-old MF and HC rats in the presence of either 0 or 5.5 mM glucose was studied. GLP-1 or ACh did not augment insulin secretion by MF or HC islets at 10 min (data not shown). In the absence of glucose, insulin secretion from MF islets was not detectable, whereas HC islets secreted a moderate amount of insulin (Table 2). When no glucose was available, either GLP-1 or ACh stimulated insulin secretion to a significantly higher level in HC islets compared with MF islets (Table 2). In the presence of 5.5 mM glucose, insulin secretion by HC islets was at least twofold higher compared with MF islets in the presence of glucose alone or under either GLP-1- or ACh-stimulated conditions (Table 2).
The activities of PKA, PKC, and CaM kinase II, which modulate insulin secretion by the
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Adenylyl cyclase isoform expression in MF and
HC islets.
There are at least 10 adenylyl cyclase isoforms reported to date
(42), and five of these isoforms (III, IV, V, VI, and VII) have been reported to be present in rat pancreatic islets
(29). In Fig. 5A,
expression of adenylyl cyclase isoforms in rat islets confirms the
presence of types III, V, and VI and shows, in addition, that adenylyl
cyclase type II mRNA is present (Fig. 5A). Adenylyl cyclase type II mRNA was also identified by RT-PCR to be present in
RINm5F insulinoma cells (data not shown). The effects of the HC milk
formula on adenylyl cyclase mRNA expression levels were determined in
islets from 12-day-old MF and HC rats. Figure 5B shows
representative RT-PCR results for adenylyl cyclase isoforms and
-actin in 12-day-old MF and HC rat islets. After correction for
dilution differences between the isoforms, analysis of the relative
levels of adenylyl cylase isoform mRNA showed that type VI mRNA was the
most highly expressed isoform among adenylyl cyclases II, III, V, and
VI in both MF and HC islets (Fig. 5C). The HC rat islets
showed a marked increase in the relative level of adenylyl cyclase type
VI mRNA (137% of MF; Fig. 5C). The levels of mRNA for the
other adenylyl cyclase isoforms quantitated in this study (II, III, and
V) did not show any significant changes in response to the dietary
intervention (Fig. 5C).
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Ca2+ channel-independent augmentation pathway in
insulin secretion by HC islets.
In addition to the KATP channel-dependent and
KATP channel-independent augmentation pathways for insulin
secretion that require Ca2+ for their action, there exists
a Ca2+-independent augmentation pathway that is glucose
dependent and requires the simultaneous activation of PKA and PKC
(24). Insulin secretion by MF and HC islets was studied
under stringent Ca2+-deprived conditions
(Ca2+-free buffer plus BAPTA, nimodipine, and EGTA) at 0, 5.5, and 16.7 mM glucose in the simultaneous presence of the
physiological activators of PKA (GLP-1) and PKC (ACh). The potentiated
glucose response was seen only at 60 min; hence, results for only the late phase of insulin secretion are reported. As indicated earlier (Fig. 1), in the absence of glucose, HC islets secreted significant amounts of insulin under a stringent Ca2+-deprived
condition. This secretion was significantly increased in the presence
of GLP-1 plus ACh (153% of the insulin secreted in the absence of
glucose; Fig. 6). There was no effect on
MF islets under this condition. In the presence of the activators of
PKA and PKC at 5.5 mM glucose, ~1 fmol of insulin was secreted by MF
islets, whereas HC islets secreted 2.8 fmol of insulin
(P < 0.001 compared with MF; Fig. 6). At 16.7 mM
glucose in the presence of GLP-1 and ACh, HC islets secreted
approximately twofold more insulin compared with the amount of insulin
secreted by MF islets under identical conditions (Fig. 6). With
increasing concentrations of glucose, a concentration-dependent
response to activation by GLP-1 and ACh with respect to insulin
secretion was observed for both MF and HC islets, with a greater
response being evident in the case of HC islets (Fig. 6).
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Effect of NE on insulin secretion by HC islets. NE is a physiological antagonist of insulin and has been reported to inhibit insulin secretion by isolated islets and cultured islet insulinoma cell lines (34). Because HC islets secrete considerable amounts of insulin in the absence of glucose and under stringent Ca2+-deprived conditions, it was of interest to see whether NE could inhibit insulin secretion under these conditions. NE (10 µM) totally inhibited insulin secretion by both MF and HC islets at 0, 5.5, and 16.7 mM glucose in the absence (results not shown) and in the presence of GLP-1 plus ACh (Fig. 6).
Because NE completely inhibited insulin secretion by HC islets, the concentration response to this antagonist was determined to assess whether the pattern of inhibition on insulin secretion by HC islets differed from that observed for MF islets. Figure 7 depicts the concentration-response curve for the effect of NE on insulin secretion at 16.7 mM glucose at 10 and 60 min for both MF and HC islets. For MF islets, the IC50 for NE inhibition of insulin secretion at 10 min was 0.007 µM (Fig. 7). In contrast, for HC islets, the IC50 for NE-induced inhibition of insulin secretion at 10 min was 0.065 µM (Fig. 7). Thus an ~10-fold increase in NE concentration was required to achieve the same degree of inhibition in HC islets compared with MF islets at 10 min. At 60 min, the IC50 for inhibition of insulin secretion by NE for MF and HC islets was ~0.007 µM and 0.03 µM, respectively. These results indicate that the sensitivity of the response to NE is altered in HC islets.
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DISCUSSION |
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Our results indicate that a nutritional switch from the natural program of fat-derived calories in rat milk to a tailor-made formula rich in carbohydrate-derived calories during the suckling period in rats has several profound consequences for their islet function. Because the HC formula is given to these rats during the critical window of their pancreatic development, it is anticipated that several adaptive changes must take place in the islets of these rats. Because the immediate onset of hyperinsulinemia persists into adulthood, even after the withdrawal of the nutritional stimulus, it appears that these early changes in the HC pancreas are most likely programmed into adulthood. Hence, these early adaptations in HC islets have a profound impact in this model.
Recently, we reported a marked leftward shift in glucose-stimulated insulin secretion accompanied by a significant increase in the low Km hexokinase activity supporting basal hyperinsulinemia in 12-day-old HC rats (1). It is apparent that other adaptive changes must occur to account for the more than sixfold increase seen in circulating insulin levels in 12-day-old HC rats (1). The results from the present study indicate that several changes at proximal and distal sites in the insulin secretory pathway of the HC islets may contribute to the immediate onset and persistence of hyperinsulinemia in HC rats.
A surprising observation was the ability of islets from 12-day-old HC
rats to secrete substantial amounts of insulin in the absence of any
stimulus at 60 min (HC islets secrete ~40% of the insulin they
secrete at 5.5 mM glucose and ~3.5-fold the insulin secreted by MF
islets at 5.5 mM glucose under these conditions) (1). The
availability of endogenous glucose from glycogen has been excluded by
use of iodoacetate (Fig. 1). Diazoxide in the absence of glucose
elicited an identical response to no glucose (control) alone, excluding
the influence of increased influx of calcium to support insulin
secretion when no stimulus is present. HC islets secreted similar
amounts of insulin in a medium containing no glucose and under a
simultaneous stringent Ca2+-deprived condition, indicating
that the availability of calcium is not limiting for insulin secretion
by HC islets when no glucose is available. When the glucose-stimulated
insulin secretion experiment was performed by carrying out the
preincubation and stimulation of islets on ice, HC islets secreted
moderate and identical amounts of insulin at 0, 5.5, and 16.7 mM
glucose (our personal observations). Collectively, these results
indicate that HC islets secrete a moderate amount of insulin through a
glucose- and calcium-independent mechanism that does not involve the
proximal steps requiring the metabolism of glucose and increased
concentrations of intracellular Ca2+. This mode of insulin
secretion has so far not been reported in any other system and
represents the unique ability of neonatal HC islets to secrete insulin
under these conditions to adapt to the demands of the HC formula.
Vargas et al. (45) have shown that the net -cell
response may result from a unique integration of cells with
individually specific stimulus-coupling characteristics. For example,
even at very low glucose levels, some
-cells appear to be tonically
"on" (45). Basal hyperinsulinemia observed in fa/fa Zucker rats has been attributed to the
presence of an increased number of
-cells that have a heightened
response to basal glucose (7). On the basis of these
observations, it is suggested that the dietary intervention in this
model results in a subset of
-cells in HC islets that can secrete
insulin independently of glucose metabolism and the presence of calcium.
The islet cells of the pancreas are the core of the endocrine mechanism
controlling fuel metabolism in the body; hence, they receive several
afferent signals that influence secretion of the various pancreatic
hormones (9). The incretin effect, which is the augmented
insulin release in response to ingested glucose, plays a significant
role, enabling the coupling by the gastrointestinal tract hormones of
the glucose absorbed and the insulin secreted (11). GLP-1 is one of the principal incretin
hormones (33), and its circulating plasma levels are
significantly increased (about threefold) in the HC rats (Fig. 2).
GLP-1 transduces its signal by binding to its cognate receptor in the
plasma membrane of target cells (23) (Fig.
8). The presence of receptors for GLP-1
has been demonstrated for -cells as well as for the brain (23). The mRNA level of the receptor for GLP-1 in the HC
islets is increased approximately threefold compared with MF islets
(Fig. 3), suggesting that ingestion of a diet high in
carbohydrate-derived calories regulates GLP-1 receptor expression. The
binding of GLP-1 to its receptor increases the activity of adenylyl
cyclase via specific G protein activation with resultant increases in
cAMP content and PKA activity (23). In addition to
activating PKA, cAMP binds to other target proteins directly and
modulates intracellular Ca2+ levels (20). cAMP
can also exert a direct action on insulin secretion at a point distal
to the rise in free intracellular Ca2+ concentration
(3). GLP-1 could directly augment insulin secretion by
increasing
-cell mass by its effects on neogenesis and replication in the
-cells (48). Previously, it was reported that HC
islets have increased mass and insulin content compared with MF islets (44). The results of this study suggest that heightened
basal insulin release in HC islets may be attributed to increased
plasma levels of GLP-1 and augmented GLP-1 receptor/PKA response in the HC
-cells, leading to stimulation of distal steps in exocytosis (Fig. 8). It is possible that, in addition to the effects of GLP-1, there may be yet unidentified additional factors that also contribute to the hyperinsulinemic state of the HC rats.
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The potent incretin activity of GLP-1 and the increased circulating
levels of GLP-1 in HC rats suggest that adenylyl cyclase may be an
important pathway for modulating insulin secretion in this model.
Although at least five isoforms of adenylyl cyclase were previously
identified in rat islets (29), this study is the first to
characterize the presence of adenylyl cyclase type II in rat islets and
to report the relative abundance of the isoforms. This study is also
the first demonstration that adenylyl cyclase mRNA levels can be
selectively regulated in a physiological manner in pancreatic islets.
Artificially raising suckling rats on the HC milk formula also resulted
in an increase in adenylyl cyclase type VI mRNA without any effect on
the other isoforms. Adenylyl cyclase type VI is responsive to
GS protein and PKC stimulation and to Gi,
Ca2+, and PKA inhibition (29, 42, 43).
Therefore, changes in the level of adenylyl cyclase type VI have the
potential to have an impact on the regulated production of cAMP in
-cells and the secretion of insulin.
The role of PKA, PKC, and CaM kinase II in the regulation of insulin
secretion by the -cells is well documented (22). CaM kinase II, PKA, and PKC are activated by increases in the levels of
intracellular Ca2+, cAMP and diacylglycerol, respectively
(22). The activities of CaM kinase II, PKA, and PKC were
significantly increased in islets from 12-day-old HC rats compared with
islets from age-matched MF rats. Insulin secretion responses in the
presence of specific inhibitors of these kinases indicate that these
enzymes act in synergy. Individually, only the specific inhibitor of
CaM kinase II partially inhibited insulin secretion, and this
inhibition was evident only at 16.7 mM glucose. It has been reported
that inhibition of PKC alone did not inhibit the secretory response to
glucose (41), consistent with our observation that the
specific inhibitors of PKA and PKC individually did not inhibit insulin secretion by HC or MF islets. The use of these inhibitors in
combination caused increased inhibition of insulin secretion by HC
islets at both 5.5 and 16.7 mM glucose, indicating a role for these
kinases in sustaining hyperinsulinemia in HC rats. In addition, HC
islets secreted increased amounts of insulin in a
Ca2+-independent manner in the presence of physiological
activators of PKA and PKC and increasing concentrations of glucose
compared with MF islets. Even when no glucose was present, under a
stringent calcium-deprived condition, the simultaneous activation of
PKA and PKC increased insulin secretion by HC islets. These results indicate that the Ca2+-independent augmentation pathway for
insulin secretion (measurable only at the late phase) is upregulated in
HC islets compared with MF islets and that the glucose- and
Ca2+-independent pathway that is evident in HC islets (not
seen in MF islets under these conditions) is also activated by PKA and PKC. The previously described data showing an increase in adenylyl cyclase type VI mRNA in HC islets also support the argument that cAMP
levels are increased in vivo and contribute to activation of PKA and
elevated insulin secretion. The large increase in insulin secretion
observed in HC islets in the presence of either GLP-1 or ACh complement
the results obtained for PKA and PKC activities in HC islets.
Collectively, these results suggest that increased activities of PKA
and PKC contribute to sustain the altered insulin secretory pattern
observed in the HC islets and that these phenomena occur in vivo, even
in the absence of overt hyperglycemia.
Our findings are consistent with several other reports on the role of
the protein kinases in the regulation of insulin secretion by the
-cells. It has been suggested that the possible localization of CaM
kinase II to the insulin secretory granules and its regulatory properties endow it with the ability to drive the secretory demands on
the
-cells (10). CaM kinase II has been implicated in
the modulation of insulin secretion by ACh (14). An
increase in the activities of PKA and PKC has been reported to result
in a large exocytotic response for insulin by phosphorylation of
proteins that directly modulate exocytosis (4). ACh and
cholecystokinin activate PKC and can potentiate insulin secretion via
desensitization of
2-adrenergic receptor sensitivity
(16, 17).
Islets are extensively innervated, and activation of the parasympathetic nervous system via vagal cholinergic nerves during feeding is important for nutrient-induced insulin secretion (36). Cholinergic mechanisms coupled with enhanced carbohydrate availability and the alteration in GLP-1 reported in this study could contribute significantly to the altered insulin secretory pattern of the HC islets. Collectively, these results suggest adaptive changes at the enteropancreatic axis as well as the neuropancreatic axis in the HC rats (Fig. 8).
In contrast to the incretins, NE (10 µM) completely inhibited the
insulin secretion seen in the absence of glucose and under essentially
calcium-free conditions, suggesting that this pathway is sensitive to
2-adrenergic receptor signals. Although it is widely
recognized that
2-adrenergic receptor-mediated signals inhibit adenylyl cyclase activity, NE has also been reported to inhibit
insulin secretion at a point distal to cAMP generation (26). The dose-response curve to the inhibitory
effects of NE on insulin secretion by HC islets indicates reduced
sensitivity of the
2-adrenergic signaling pathway
(receptor G protein coupling mediated) in HC islets. It is possible
that signals arising from incretin (GLP-1) and neuronal inputs (ACh)
counteract/inhibit the NE signals and support hyperinsulinemia in the
HC rats (Fig. 8). Alternatively, mechanisms currently unknown could
contribute to downregulation or desensitization of the
2-adrenergic receptor in HC islets.
The immediate onset of hyperinsulinemia and its persistence into
adulthood, despite withdrawal of the nutritional stimulus, are
indicative of a complex array of adaptations in the pancreas of HC
rats. Our earlier and present results establish some of these
adaptations at both proximal and distal sites in the process of insulin
secretion by HC islets. Distal elements in signal transduction in
islets have been proposed to contribute to the hyperglycemic state in
Goto Kakizaki rats (32) and may include the soluble N-ethylmaleimide-sensitive fusion protein
receptor proteins involved in the vesicle docking/fusion step
of insulin exocytosis (35). Our results summarized in Fig.
8 provide evidence for a key role for protein kinase-induced signal
transduction pathways in modulating adaptive responses in the -cells
of HC islets. Figure 8 also indicates the possible sites that could
contribute to the reduced sensitivity to NE signaling. Taken together,
it is apparent that multiple adaptations in the islets of young HC rats
contribute to the immediate onset of hyperinsulinemia and its
programming into adulthood.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institute of Child Health and Human Development Grant HD-11089 (M. S. Patel) and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-51601 (M. S. Patel) and DK-25705 (S. G. Laychock).
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FOOTNOTES |
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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).
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 3 May 2000; accepted in final form 31 July 2000.
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REFERENCES |
---|
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---|
1.
Aalinkeel, R,
Srinivasan M,
Kalhan SC,
Laychock SG,
and
Patel MS.
A dietary intervention (high carbohydrate) during the neonatal period causes islet dysfunction in rats.
Am J Physiol Endocrinol Metab
277:
E1061-E1069,
1999
2.
Aerts, L,
Holemans K,
and
Van Assche FA.
Maternal diabetes during pregnancy: consequences for the offspring.
Diabetes Metab Rev
6:
147-167,
1990[ISI][Medline].
3.
Ammala, C,
Ashcroft FM,
and
Rorsman P.
Calcium-independent potentiation of insulin release by cyclic AMP in single beta-cells.
Nature
363:
356-358,
1993[ISI][Medline].
4.
Ammala, C,
Eliasson L,
Bokvist K,
Berggren P,
Honkanen RE,
Sjoholm A,
and
Rorsman P.
Activation of protein kinases and inhibition of protein phosphatases play a central role in the regulation of exocytosis in mouse pancreatic cells.
Proc Natl Acad Sci USA
91:
4343-4347,
1994[Abstract].
5.
Ashcroft, SJ.
Protein phosphorylation and -cell function.
Diabetologia
37:
21-29,
1994.
6.
Bramblett, DE,
Huang HP,
and
Tsai MJ.
Pancreatic islet development.
Adv Pharmacol
47:
255-315,
2000[Medline].
7.
Chan, CB,
MacPhail RM,
Sheu L,
Wheeler MB,
and
Gaisano HY.
Cell hypertrophy in fa/fa rats is associated with basal glucose hypersensitivity and reduced SNARE protein expression.
Diabetes
48:
997-1005,
1999[Abstract].
8.
Dahri, S,
Reusens B,
Remacle C,
and
Hoet JJ.
Nutritional influences on pancreatic development and potential links with non-insulin-dependent diabetes.
Proc Nutr Soc
54:
345-356,
1995[ISI][Medline].
9.
D'Alessio, D.
Peptide hormone regulation of islet cells.
Horm Metab Res
29:
297-300,
1997[ISI][Medline].
10.
Easom, RA.
CaM kinase II: a protein kinase with extraordinary talents germane to insulin exocytosis.
Diabetes
48:
675-684,
1999[Abstract].
11.
Fehmann, HC,
Goke R,
and
Goke B.
Cell and molecular biology of the incretin hormones glucagon-like peptide-I and glucose-dependent insulin releasing polypeptide.
Endocr Rev
16:
390-410,
1995[ISI][Medline].
12.
Gembal, M,
Gilon P,
and
Henquin JC.
Evidence that glucose can control insulin release independently from its action on ATP-sensitive K+ channels in mouse cells.
J Clin Invest
89:
1288-1295,
1992[ISI][Medline].
13.
Granneman, JG.
Expression of adenylyl cyclase subtypes in brown adipose tissue: neural regulation of type III.
Endocrinology
136:
2007-2012,
1995[Abstract].
14.
Gromada, J,
Hoy M,
Renstrom E,
Bokvist K,
Eliasson L,
Gopel S,
and
Rorsman P.
CaM kinase II-dependent mobilization of secretory granules underlies acetylcholine-induced stimulation of exocytosis in mouse pancreatic -cells.
J Physiol (Lond)
518:
745-759,
1999
15.
Haney, PM,
Raefsky-Estrin C,
Caliendo A,
and
Patel MS.
Precocious induction of hepatic glucokinase and malic enzyme in artificially reared rat pups fed a high-carbohydrate diet.
Arch Biochem Biophys
244:
787-794,
1986[ISI][Medline].
16.
Harris, TE,
Persaud SJ,
and
Jones PM.
Atypical isoforms of PKC and insulin secretion from pancreatic beta-cells: evidence using Go 6976 and Ro 31-8220 as PKC inhibitors.
Biochem Biophys Res Commun
227:
672-676,
1996[ISI][Medline].
17.
Harris, TE,
Persaud SJ,
Saermark T,
and
Jones PM.
A myristoylated pseudosubstrate peptide inhibitor of protein kinase C: effects on glucose- and carbachol-induced insulin secretion.
Mol Cell Endocrinol.
121:
133-141,
1996[ISI][Medline].
18.
Hiremagalur, BK,
Vadlamudi S,
Johanning GL,
and
Patel MS.
Alterations in hepatic lipogenic capacity in rat pups artificially reared on a milk-substitute formula high in carbohydrate or medium-chain triglycerides.
J Nutr Biochem
3:
474-480,
1992[ISI].
19.
Hiremagular, BK,
Vadlamudi S,
Johanning GL,
and
Patel MS.
Long-term effects of feeding of high carbohydrate diet in preweaning by gastrosomy: a new rat model for obesity.
Int J Obes
17:
495-502,
1993[ISI].
20.
Holz, GG,
Leech CA,
and
Habener JF.
Activation of a cAMP-regulated Ca(2+)-signaling pathway in pancreatic beta-cells by the insulinotropic hormone glucagon-like peptide-1.
J Biol Chem
270:
17749-17757,
1995
21.
Iwashima, Y,
Kondoh-Abiko A,
Seino S,
Takeda J,
Eto M,
Polonsky KS,
and
Makino I.
Reduced levels of messenger ribonucleic acid for calcium channel, glucose transporter-2, and glucokinase are associated with alterations in insulin secretion in fasted rats.
Endocrinology
135:
1010-1017,
1994[Abstract].
22.
Jones, PM,
and
Persaud SJ.
Protein kinases, protein phosphorylation, and the regulation of insulin secretion from pancreatic -cells.
Endocr Rev
19:
429-461,
1998
23.
Kieffer, TJ,
and
Habener JF.
The glucagon-like peptides.
Endocr Rev
20:
876-913,
1999
24.
Komatsu, M,
Schermerhorn T,
Aizawa T,
and
Sharp GW.
Glucose stimulation of insulin release in the absence of extracellular Ca2+ and in the absence of any increase in intracellular Ca2+ in rat pancreatic islets.
Proc Natl Acad Sci USA
92:
10728-10732,
1995[Abstract].
25.
Komatsu, M,
Schermerhorn T,
Noda M,
Straub SG,
Aizawa T,
and
Sharp GWG
Augmentation of insulin release by glucose in the absence of extracellular Ca2+.
Diabetes
46:
1928-1938,
1997[Abstract].
26.
Laychock, SG.
Alpha 2-adrenoceptor stimulation affects total glucose utilization in isolated islets of Langerhans.
Mol Pharmacol
32:
241-248,
1987[Abstract].
27.
Laychock, SG,
Vadlamudi S,
and
Patel MS.
Neonatal rat dietary carbohydrate affects pancreatic islet insulin secretion in adults and progeny.
Am J Physiol Endocrinol Metab
269:
E739-E744,
1995
28.
Lee, B,
Bradford PG,
and
Laychock SG.
Characterization of inositol 1,4,5-trisphosphate receptor isoform mRNA expression and regulation in rat pancreatic islets, RINm5F cells and betaHC9 cells.
J Mol Endocrinol
21:
31-39,
1998
29.
Leech, CA,
Castonguay MA,
and
Habener JF.
Expression of adenylyl cyclase subtypes in pancreatic beta-cells.
Biochem Biophys Res Commun
254:
703-706,
1999[ISI][Medline].
30.
Lucas, A.
Programming by early nutrition: an experimental approach.
J Nutr
128:
401S-406S,
1998[ISI][Medline].
31.
Manolopoulos, VG,
Liu J,
Unsworth BR,
and
Lelkes PI.
Adenylyl cyclase isoforms are differentially expressed in primary cultures of endothelial cells and whole tissue homogenates from various rat tissues.
Biochem Biophys Res Commun
208:
323-331,
1995[ISI][Medline].
32.
Metz, SA,
Meredith M,
Vadakekalam J,
Rabaglia ME,
and
Kowluru A.
A defect late in stimulus-secretion coupling impairs insulin secretion in Goto-Kakizaki diabetic rats.
Diabetes
48:
1754-1762,
1999[Abstract].
33.
Mojsov, S,
Weir GC,
and
Habener JF.
Insulinotropin: glucagon-like peptide I (7-37) co-encoded in the glucagon gene is a potent stimulator of insulin release in the perfused rat pancreas.
J Clin Invest
79:
616-619,
1987[ISI][Medline].
34.
Morgan, NG,
and
Montague W.
Studies on the mechanism of inhibition of glucose-stimulated insulin secretion by noradrenaline in rat islets of Langerhans.
Biochem J
226:
571-576,
1985[ISI][Medline].
35.
Nagamatsu, S,
Nakamichi Y,
Yamamura C,
Matsushima S,
Watanabe T,
Ozawa S,
Furukawa H,
and
Ishida H.
Decreased expression of t-SNARE, Syntaxin 1 and SNAP-25 in pancreatic cells is involved in impaired insulin secretion from diabetic GK rat islets.
Diabetes
48:
2367-2373,
1999[Abstract].
36.
Rasmussen, H,
Zawalich KC,
Ganesan S,
Calle R,
and
Zawalich WS.
Physiology and pathophysiology of insulin secretion.
Diabetes Care
13:
655-666,
1990[Abstract].
37.
Sato, Y,
Aizawa T,
Komatasu M,
Okada N,
and
Yamda T.
Dual functional role of membrane depolarization/Ca2+ influx in rat pancratic cell.
Diabetes
41:
438-443,
1992[Abstract].
38.
Sharp, GWG
Mechanisms of inhibition of insulin release.
Am J Physiol Cell Physiol
271:
C1781-C1799,
1996
39.
Siebert, PD,
and
Larrick JW.
Competitive PCR.
Nature
359:
557-558,
1992[ISI][Medline].
40.
Siebert, PD,
and
Larrick JW.
PCR MIMICS: competitive DNA fragments for use as internal standards in quantitative PCR.
Biotechniques
14:
244-249,
1993[ISI][Medline].
41.
Sjoholm, A.
Glucose stimulates islet -cell mitogenesis through GTP-binding proteins and by protein kinase C-dependent mechanisms.
Diabetes
46:
1141-1147,
1997[Abstract].
42.
Sunahara, RK,
Dessauer CW,
and
Gilman AG.
Complexity and diversity of mammalian adenylyl cyclases.
Annu Rev Pharmacol Toxicol
36:
461-480,
1996[ISI][Medline].
43.
Tang, WJ,
Yan S,
and
Drum CL.
Class III adenylyl cyclases: regulation and underlying mechanisms.
Adv Second Messenger Phosphoprotein Res
32:
137-151,
1998[Medline].
44.
Vadlamudi, S,
Hiremagalur BK,
Tao S,
Kalhan SC,
Kalaria RN,
Kaung HC,
and
Patel MS.
Long-term effects on pancreatic function of feeding a HC formula to rats during the preweaning period.
Am J Physiol Endocrinol Metab
265:
E565-E571,
1993
45.
Vargas, de, LM.,
Sobolewski J,
Siegel R,
and
Moss LG.
Individual cells within the intact islet differentially respond to glucose.
J Biol Chem
272:
26573-26577,
1997
46.
Waterland, RA,
and
Garza C.
Potential mechanisms of metabolic imprinting that lead to chronic diseases.
Am J Clin Nutr
69:
179-197,
1999
47.
Xia, M,
and
Laychock SG.
Insulin secretion, myo-inositol transport, and Na(+)-K(+)-ATPase in glucose-desensitized rat islets.
Diabetes
42:
1392-1400,
1993[Abstract].
48.
Xu, G,
Stoffers DA,
Habener JF,
and
Bonner-Weir S.
Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats.
Diabetes
48:
2270-2276,
1999[Abstract].