Adaptive changes in insulin secretion by islets from neonatal rats raised on a high-carbohydrate formula

Malathi Srinivasan1, Ravikumar Aalinkeel1, Fei Song1, Bumsup Lee2, Suzanne G. Laychock2, and Mulchand S. Patel1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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); [gamma -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 wt-1 · 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 and beta -actin were as previously reported (28, 31). PCR was performed for 30 cycles under the following conditions: denaturation for 1 min at 94°C, annealing for 1 min at 60°C, and extension for 2 min at 72°C, with the final extension lasting 7 min. Preliminary studies established the linearity of amplification rates under the conditions used for these experiments. Polymerization reactions were carried out using 10 µl 1:2.5 dilution cDNAs (for adenylyl cyclase types II and V), 1:5 dilution cDNAs (for adenylyl cyclase type III), 1:20 dilution cDNAs (for adenylyl cyclase type VI), or 1:200 dilution cDNAs (for beta -actin) as templates in a 25-µl reaction volume for which amplification was in the exponential phase. The amplimers were separated by electrophoresis in a 1.5% agarose gel and analyzed as described above. The image densities of PCR products for adenylyl cyclase transcripts were compared with the density of coamplified beta -actin to determine the ratio of expression. Values are expressed as relative levels of adenylyl cyclase isoforms/beta -actin mRNA after correcting for the dilution factors.

Assay 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.


    RESULTS
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ABSTRACT
INTRODUCTION
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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 islets-1 · 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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Fig. 1.   Insulin secretion by islets isolated from 12-day-old mother-fed (MF) and high-carbohydrate-fed (HC) rats. Equal numbers of islets (n = 30) from each group were preincubated in Krebs-Ringer bicarbonate (KRB) buffer containing no glucose (control) for 30 min. For experiments carried out under stringent Ca2+-deprived conditions, preincubation was carried out in KRB buffer devoid of calcium and glucose but containing EGTA (500 µM), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 1 µM), and nimodipine (1 µM). Islets were then washed and further incubated in buffer containing no glucose and either diazoxide (250 µM) or iodoacetate (1 mM) or under stringent Ca2+-deprived conditions for another 60 min. At 60 min, an aliquot was withdrawn and frozen for analysis of insulin. Results are means ± SE of 4 independent experiments.

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 beta -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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Fig. 2.   Plasma levels of the active form of glucagon-like peptide (GLP-1) in 12-day-old MF and HC rats. Results are means ± SE of 5 different samples. a P < 0.001 compared with MF.



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Fig. 3.   A: RT-PCR analysis of GLP-1 receptor mRNA from 12-day-old MF and HC islets. Each reaction contained equal amounts of competitor template, which was designed to be amplified by the same primer oligonucleotides as the target sequence. B: densitometric scan of agarose gels. The intensity of the bands of the target sequences was quantitated in relation to the competitor. Results (means ± SE) are presented as relative expression (MF value as 1) calculated from 4 samples in each group. a P < 0.05 compared with MF.

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 beta -cells, were measured in islet extracts from 12-day-old MF and HC rats. The activity of the active form of PKA was increased approximately twofold in HC islets compared with MF islets (Fig. 4). There was no change (P > 0.05) in the total activity of PKA stimulated by cyclic AMP between the two groups (3,252 ± 310 vs. 3,446 ± 412 pmol phosphate incorporated · min-1 · mg protein-1 for MF and HC islets, respectively). The activity of PKC was also significantly increased in the HC islets compared with MF islets (Fig. 4). The activity of CaM kinase II was increased by ~33% in HC islets compared with MF islets (Fig. 4). To investigate whether these changes had any effect on insulin secretion, the effect of specific inhibitors of PKA (PKA inhibitor 14-22 amide), PKC (bisindolylmaleimide 1), and CaM kinase II (autocamtide-2-related inhibitory peptide) on insulin secretion by MF and HC islets at 1, 5.5, and 16.7 mM glucose at both 10 and 60 min was studied. When used singly, the specific inhibitors of PKA and PKC did not inhibit insulin secretion by either MF or HC islets at either 10 or 60 min at 1, 5.5, or 16.7 mM glucose (data not shown). The specific inhibitor of CaM kinase II significantly inhibited insulin secretion only at 16.7 mM glucose at both 10 and 60 min by both MF and HC islets. The extent of inhibition was almost similar for both groups of islets (~60% for the 10-min phase and 83 and 71% for the 60-min phase for MF and HC islets, respectively; data not shown).


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Fig. 4.   Activities of protein kinase A (PKA, A), PKC (B), and calcium calmodulin (CaM) kinase II (C) in islets from 12-day-old MF and HC rats. MF and HC islets were sonicated in the respective extraction buffers, and the homogenates were used for enzyme assays. Results are means ± SE of 4 independent experiments. Compared with MF: a P < 0.002 in A, b P < 0.01 in B, and c P < 0.003 in C.

Because the specific inhibitors of PKA and PKC singly did not cause any change in insulin secretion, the effect of these inhibitors in combination was evaluated on insulin secretion by MF and HC islets at 0, 5.5, and 16.7 mM glucose. These inhibitors did not affect the 60-min phase of insulin release by HC islets at 0 glucose (Table 1). Although neither the 10- nor the 60-min phases of insulin release at 5.5 mM glucose by MF islets was affected by the combination of these inhibitors, the 60-min phase for HC islets at 5.5 mM glucose was reduced by ~30% (Table 1). At 16.7 mM glucose, an ~40% decrease was observed for the early phase of insulin secretion by the HC islets in the presence of a combination of the specific inhibitors, and the late-phase insulin secretion was inhibited to a larger extent (64%; Table 1). In contrast, the inhibitors did not affect the early insulin release response of MF islets, but they did inhibit the 60-min phase by ~56% at 16.7 mM glucose (Table 1).

                              
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Table 1.   Insulin secretion by islets from 12-day-old MF and HC rats in the simultaneous presence of the specific inhibitors of PKA and PKC


                              
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Table 2.   Insulin secretion by islets from 12-day-old MF and HC rats in the presence of 0 and 5.5 mM glucose and either GLP-1 or ACh

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 beta -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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Fig. 5.   Relative levels of adenylyl cyclase (AC) isoform mRNA in rat islets. Total RNA was extracted from rat islets and reverse transcribed. A: characterization of PCR products of adult rat islet AC types II, III, V, and VI and beta -actin (beta ) by densitometric analysis of an ethidium bromide-stained agarose gel. M, molecular size marker. Data are representative experimental results. B: PCR products for AC isoforms and beta  in 12-day-old MF and HC rat islets. Data are representative experimental results. C: relative levels of AC isoforms' mRNA in 12-day-old MF and HC rat islets. Values are means ± SE of 6 experiments. a P < 0.02 compared with MF.

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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Fig. 6.   Calcium-independent insulin secretion by islets from 12-day-old MF and HC rats. Equal numbers of islets (n = 30) were preincubated in KRB buffer containing no glucose and under stringent Ca2+-deprived conditions for 30 min. The islets were washed, and the incubation was continued under stringent Ca2+-deprived conditions in the presence of 0, 5.5, or 16.7 mM glucose and in the simultaneous presence of GLP-1 (100 nM) and ACh (1 µM). At the end of 60 min, an aliquot was withdrawn and frozen for assay of insulin. The same experiment was repeated with the additional inclusion of norepinephrine (NE, 10 µM) only during the incubation period. Results are means ± SE of 4 independent experiments. a P < 0.001 compared with insulin secretion in the absence of GLP-1 plus ACh for each group; * no detectable insulin secretion under the experimental condition.

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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Fig. 7.   Concentration-dependent inhibition of insulin secretion by NE in 12-day-old MF and HC islets. Equal numbers (n = 30) of islets from MF and HC rats were preincubated in KRB buffer containing 5.5 mM glucose for 30 min. At the end of the preincubation period, they were incubated in KRB buffer containing 16.7 mM glucose and various concentrations of NE (0.001-10 µM). Aliquots were withdrawn at 10 and 60 min for assay of insulin. Results are means ± SE of 4 independent experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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 beta -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 beta -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 beta -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 beta -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 beta -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 beta -cell mass by its effects on neogenesis and replication in the beta -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 beta -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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Fig. 8.   Summary of the signaling pathways induced by hormonal and neuronal inputs in HC islets in response to the high-carbohydrate intervention. R, receptor; ER, endoplasmic reticulum; IP3, inositol 1,4,5-triphosphate; DAG, diacylglycerol; (up-arrow ), increase in activity or mRNA content; (odash ), reduced sensitivity to NE-sensitive pathways. The positive influence of GLP-1- and ACh-mediated signals, as well as reduced sensitivity to NE-induced signals, are indicated. (This figure is adapted from Ref. 25).

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 beta -cells and the secretion of insulin.

The role of PKA, PKC, and CaM kinase II in the regulation of insulin secretion by the beta -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 beta -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 beta -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 alpha 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 alpha 2-adrenergic receptor signals. Although it is widely recognized that alpha 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 alpha 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 alpha 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 beta -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.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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