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Am J Physiol Endocrinol Metab 292: E1364-E1377, 2007. First published January 16, 2007; doi:10.1152/ajpendo.00672.2006
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Autonomic involvement in the permanent metabolic programming of hyperinsulinemia in the high-carbohydrate rat model

Paul Mitrani, Malathi Srinivasan, Catherine Dodds, and Mulchand S. Patel

Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York

Submitted 18 December 2006 ; accepted in final form 11 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Exposure to a high-carbohydrate (HC) milk formula during the suckling period results in permanent metabolic programming of hyperinsulinemia in HC rats. Previous studies have shown that hyperinsulinemia in HC rats involves a programmed hyperresponsiveness to glucose. However, the immediate onset and persistence of enhanced insulin secretion throughout life suggests a role for numerous factors that control insulin secretion. Present in vivo and in vitro studies have shown a role for altered autonomic activity, including increased parasympathetic and decreased sympathetic activities, in the maintenance of hyperinsulinemia in 100-day-old HC rats. HC rats were shown to be more sensitive to cholinergic-induced potentiation of glucose-stimulated insulin secretion (GSIS) in response to acetylcholine and showed increased sensitivity to blockade of cholinergic-induced insulin secretion by the muscarinic-type 3 receptor-specific antagonist 4-diphenylacetoxy-N-methylpiperidine. In addition, HC rats were less sensitive to adrenergic-induced inhibition of insulin secretion by oxymetazoline, whereas treatment with yohimbine resulted in increased GSIS. Furthermore, HC rats showed greater reductions in plasma insulin levels after vagotomy, as well as an attenuation of yohimbine-induced potentiation of GSIS, suggesting that yohimbine-mediated changes are mediated by parasympathetic activity. Changes in autonomic regulation of GSIS are supported by increased mRNA levels of the parasympathetic signaling molecules muscarinic-type 3 receptor, phospholipase Cbeta1, and protein kinase C-{alpha} and decreased levels of {alpha}2a-adrenergic receptors in islets from adult HC rats. In conclusion, metabolic programming of hyperinsulinemia throughout adulthood of HC rats involves changes in autonomic activity in response to the HC dietary intervention in the suckling period.

high-carbohydrate milk formula; parasympathetic nervous system; and sympathetic nervous system

STUDIES OF THE RISING PREVALENCE of obesity, type 2 diabetes mellitus (48), and metabolic syndrome (19) suggest that the origins of these growing epidemics involve an acquired susceptibility to altered nutritional environments during critical periods of early development, including both the fetal and neonatal periods (9, 47). This vulnerability manifests as a metabolic programming phenomenon in which a stimulus or insult during critical periods of organogenesis in early life induces permanent alterations in the development and function of affected organs at the cellular, biochemical, and molecular levels to increase the chance of survival in altered nutritional environments (52). These adaptations result in an increased susceptibility to metabolic diseases in adulthood, including obesity, type 2 diabetes mellitus, and metabolic syndrome (9, 47). In the rat, development of the endocrine pancreas is vulnerable to nutritional insults during the late fetal period and the immediate postnatal period, during which permanent structural and functional alterations can occur (12). It has been found that gestational diabetes and maternal protein restriction during pregnancy (2, 16), as well as over- and undernutrition during the early postnatal period (6, 47), result in offspring with impaired insulin secretory responses to glucose throughout life due to altered programming of pancreatic function.

Studies of an early nutritional intervention in the form of a high-carbohydrate (HC) milk formula during the immediate postnatal period (days 4–24) (22, 23) have found that the immediate onset of hyperinsulinemia involves a metabolic programming phenomenon that results in permanent hyperinsulinemia throughout life despite the removal of the HC nutritional stimulus at weaning. In addition to the persistent hyperinsulinemia, adult HC rats show impaired glucose tolerance compared with controls, although they are euglycemic, suggesting a state of insulin resistance (47, 51). Although insulin secretion is primarily a response to circulating glucose levels (27), the level of insulin secretion at any one time is a dynamic balance between stimulatory and inhibitory signals that act to maintain glucose homeostasis. For example, other secretagogues, including the neurotransmitters acetylcholine (ACh) and norepinephrine, are known to modify glucose-stimulated insulin secretion (GSIS) to maintain euglycemia (13, 44). Although chronic hyperinsulinemia in adult HC rats has been shown to involve enhanced insulin secretory response to glucose stimulation through numerous cellular, molecular, and biochemical changes that regulate glucose metabolism (23, 46, 51), the exact mechanisms responsible for the persistence of hyperinsulinemia throughout adulthood are still incompletely understood. Therefore, the metabolic programming of hyperinsulinemia in adult HC rats may involve compensatory responses in multiple systems that regulate insulin secretion (47).

Recent studies have shown that impaired nutritional status during development (29), as well as prolonged changes in blood glucose levels during adulthood (8, 28, 33), can result in alterations in autonomic regulation of GSIS. Normally, blood glucose levels are tightly regulated within a narrow range through a complex system of central and peripheral mechanisms (39). Peripheral control of insulin secretion involves directly regulation by circulating glucose levels, whereas central control involves the indirect mechanisms mediated by the central nervous system (CNS) and the autonomic nervous system (ANS) (10, 26). ANS regulation of insulin secretion involves two opposing pathways, the parasympathetic nervous system (PNS) and the sympathetic nervous system (SNS) (14), which are known to extensively innervate the pancreas (40, 46). Parasympathetic stimulation induces release of ACh, which activates muscarinic type 3 receptors (M3Rs), resulting in enhanced GSIS (5, 20, 30, 34). Sympathetic stimulation induces release of norepinephrine, which binds to the {alpha}2A-adrenergic receptor ({alpha}2A-AR) (25, 35), resulting in G protein-mediated inhibition of adenylate cyclase and subsequent inhibition of insulin secretion (15). Thus, the PNS and SNS act in opposition to regulate insulin secretion in response to circulating glucose and maintain euglycemia. Furthermore, a complex network of neural interconnections (11) between the PNS and SNS ensures that an appropriate balance between the two systems results in optimal levels of blood glucose and plasma insulin in relation to metabolic demand.

Altered activity of the ANS due to prolonged hyperglycemia contributes to enhanced insulin secretory responses to glucose through increases in parasympathetic activity and decreases in sympathetic activity (8, 28, 33). Conversely, decreased parasympathetic and increased sympathetic activity have been associated with impaired insulin secretion in offspring of rats fed a low-protein diet during pregnancy (29). Due to the fact that the HC dietary intervention overlaps with a critical period of postnatal ANS development (54), alterations in autonomic control of insulin secretion in the HC rat in the immediate postnatal period (unpublished data) may contribute to permanent programming of hyperinsulinemia throughout life. The present studies evaluated the possibility that permanent changes in ANS activity in adult HC rats, such as increased vagal tone or decreased sympathetic tone, contribute to the metabolic programming of insulin hypersecretion throughout life. Evaluation of changes in autonomic activity in response to an early nutritional intervention will further our understanding of the mechanisms involved in permanent programming of hyperinsulinemia and provide insight into the causes of the growing obesity, type 2 diabetes mellitus, and metabolic syndrome epidemics.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal Protocols

All animal protocols were approved by the Institutional Animal Care and Use Committee of the University at Buffalo, State University of New York. Timed pregnant Sprague-Dawley rats were obtained from Zivic Miller Laboratories (Zellenople, PA) and had access to a standard rodent laboratory diet (Harlan-Teklad, Madison, WI) and water ad libitum. The newborn pups were pooled and randomly assigned to each nursing mother (10 pups/dam) and then left with the mothers until postnatal day 4. On postnatal day 4, pups were assigned randomly to control and experimental groups. In the mother-fed (MF) control group, pups were reared by their nursing mothers (caloric distribution in rat milk: 8% carbohydrate, 24% protein, and 68% fat), whereas pups in the HC experimental group were artificially reared on a HC formula (caloric distribution in HC milk: 56% carbohydrate, 24% protein, and 20% fat) (23). The high-fat (HF) control group was artificially reared on a HF milk formula with a caloric distribution similar to that of rat milk (caloric distribution in HF milk: 8% carbohydrate, 24% protein, and 68% fat). The artificial rearing technique employed in this study has been described in detail previously (51). Animals were weaned on postnatal day 24 and given ad libitum access to standard rodent laboratory diet and water. Each rat was housed individually and kept at a constant temperature (23°C) on a fixed (12:12-h) artificial light cycle until time of adult experiments described below.

Glucose Tolerance Tests in 100-Day-Old Rats

Intravenous glucose tolerance tests (IVGTTs) were performed by a modification of previously reported techniques (8, 49). Briefly, MF, HC, and HF rats were anesthetized, the external jugular vein was exposed, and a 15-cm cannula (MRE 040 cannula; Braintree Scientific, Braintree, MA) was introduced and secured with silk suture thread (absorbable 4-0 Vicryl sutures; Ethicon, Piscataway, NJ). Animals were allowed to recover for 1 wk. After the recovery period, animals were fasted for 14 h prior to IVGTT. First, rats were given an intraperitoneal injection of the designated treatment (0.9% saline, agonist, or antagonist in saline) at time –10 min. At designated time 0, rats were given an intravenous injection of glucose (0.5 g/kg body wt) via the jugular vein catheter. Blood glucose levels were measured with a glucose meter (Ascensia Elite meter; Ascensia, Minnedosa, MB, Canada) immediately before (time 0) and at 5, 10, 15, 20, 30, 45, and 60 min after glucose administration. For insulin measurements, 100-µl blood samples were collected from the tail vein in heparinized micropipettes immediately before (time 0) and at 10, 20, 30, and 60 min after glucose administration. Plasma was separated by centrifugation and stored at –20°C. Plasma insulin levels were measured by radioimmunoassay according to the manufacturer's instructions (Linco Research, St. Charles, MO). The total changes in blood glucose ({Delta}G) and plasma insulin levels ({Delta}I) over the entire 60-min time period for each study were determined and used to calculate the insulinogenic index ({Delta}I/{Delta}G), which is a measure of the insulin secretory response to glucose, and stimulation by the designated agonists or antagonists.

Dose response experiments for ACh chloride (cholinergic agonist) and oxymetazoline (OM) hydrochloride ({alpha}2A-AR specific agonist) were carried out as above with the following concentrations: ACh at 0.55, 2.75, and 13.75 µmol/kg body wt and OM at 33.7, 169, and 674 nmol/kg body wt. Agonist and antagonist treatments for other experiments were as follows: ACh (2.75 µmol/kg body wt), 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; 0.21 µmol/kg body wt; M3R antagonist), yohimbine (Yoh) hydrochloride (10 µmol/kg body wt; {alpha}2-adrenergic antagonist), or OM (33.7 nmol/kg body wt). All drugs were from Sigma-Aldrich (St. Louis, MO).

Vagotomy

Subdiaphragmatic vagotomy was performed using a modified technique previously described by Dixon et al. (17). Briefly, rats were fasted for 14 h before vagotomy. Rats were anesthetized, and a midline incision was made to expose the peritoneal cavity. The dorsal and ventral branches of the vagus nerve, which travel through the diaphragm with the esophagus, were visualized and dissected clean. Subsequently, the nerve trunks were severed and removed, followed by removal of any remaining nerve fascicles on the serosal surface of the esophagus. The abdominal incision was sutured closed, and animals were allowed to recover for 30 min before IVGTT was performed as before. In the sham operations, the experimental surgery was replicated with all branches of the vagus left intact and touched only with the tip of a moistened cotton swab.

Isolation of Islets From 100-Day-Old Rats

Pancreatic islets were isolated from the 100-day-old rats by a modification of a collagenase digestion protocol described previously (21, 53). Briefly, adult rats were killed and the pancreas was inflated with a 15 mg/kg body wt collagenase solution (Crescent Chemical, Islandia, NY) via the pancreatic duct. The pancreas was digested in a 37°C water bath for 20–25 min, and islets were separated by shear force. Islets were hand-picked under a dissecting microscope.

Insulin Secretion From Islets Isolated From 100-day-old Rats

Insulin secretion was measured by static incubation as described previously (53). For each preparation, five freshly isolated islets were collected per tube and were preincubated in 500 µl of Krebs-Ringer bicarbonate buffer containing 16 mM HEPES, 5.5 mM glucose, and 0.01% bovine serum albumin, pH 7.4, for 30 min at 37°C under an atmosphere of 95% O2 and 5% CO2. Islets were then incubated with 500 µl of fresh buffer containing agonist/antagonist (as noted in figure legends) and desired glucose concentration (5.5 or 16.7 mM). Sample aliquots for determination of insulin secretion were taken at 10- and 60-min time points and stored at –20°C.

Real-Time RT-PCR

RNA was isolated from islets of 100-day-old MF, HC, and HF rats using the TRIzol reagent phenol-chloroform procedure (Gibco, Rockville, MD). Total RNA was quantified, and mRNA samples were reverse-transcribed into cDNAs by using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Levels of {alpha}2A-AR, M3R, phospholipase Cbeta1 (PLCbeta1), and protein kinase C{alpha} (PKC{alpha}) mRNA in pancreatic islets were measured via real-time RT-PCR using the iCycler system (Bio-Rad). Primer sequences, which were designed to span at least one exon-exon junction of the target mRNA to prevent amplification of contaminating genomic DNA, were synthesized by Invitrogen (Carlsbad, CA) and are presented in Table 1. The mRNA levels detected by SYBR Green (Bio-Rad) analysis were normalized to 18S mRNA levels (QuantumRNA Classic II 18S Internal Standard, 324 bp; Ambion, Austin, TX). PCR efficiency was examined by serially diluting the template cDNA, and melting curve data were collected to assess PCR specificity. Each cDNA sample was run in triplicate, and a corresponding mRNA sample that had not been subjected to reverse transcription was included as a negative control in each run. Relative mRNA levels were calculated according to the comparative {Delta}{Delta}CT method.


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  		Table 1. Primers for real-time PCR

 
Western Blot Analysis

Protein was extracted from islets isolated from 100-day-old HC, HF, and MF rats. Briefly, 300 fresh islets were resuspended in 75 µl of solubilization buffer (51) and then homogenized by sonication. Islet homogenates were centrifuged, and total cell protein in the supernatant was measured by the BCA method (Bio-Rad). Equal amounts of protein (75 µg) for each preparation were added to an equal volume of 2x Laemmli sample buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (51). Protein was transferred to a nitrocellulose and blocked with 5% skimmed milk in 0.1% Tween-Tris-buffered saline (TTBS) for 2 h at 4°C, followed by incubation with 3% dry milk TTBS with rabbit anti-{alpha}2A-AR (1:1,000; Affinity BioReagents, Golden, CO), rabbit anti-M3R (1:1,000; Novus Biological, Littleton, CO), mouse monoclonal anti-PLCbeta1 (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse monoclonal anti-PKC{alpha} (1:1,000; Santa Cruz Biotechnology) antibodies overnight at 4°C. Membranes were then washed and incubated with goat-anti-rabbit or goat anti-mouse IgG-HRP (1:1,000; Bio-Rad) in TTBS with 3% dry milk for 2 h at room temperature. Protein bands were visualized using chemiluminescence (PerkinElmer, Wellesley, MA), and densitometry analysis was performed using the Quantity One program.

Statistical Analysis

Results are expressed as means ± SE of 6–8 independent experiments. Statistical analysis of experimental group vs. control group was done using Student's t-test for MF and HC groups and the analysis of variance followed by a Tukey's test for MF, HC, and HF groups. P values <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In Vivo Glucose-Stimulated Insulin Secretion in 100-Day-Old Rats

Saline treatment. Glucose tolerance tests were performed in 100-day-old MF, HC, and HF rats to assess in vivo GSIS responses to an intraperitoneal injection of saline 10 min before an intravenous glucose load, and sequential blood samples were taken to measure changes in blood glucose and plasma insulin levels. HC rats showed significant reductions in glucose tolerance compared with MF and HF rats despite similar fasting blood glucose levels, as seen by the elevations in blood glucose at every time point after glucose administration (Fig. 1A). In addition, HC rats showed significant elevations in GSIS compared with MF and HF rats at every time point (Fig. 1B), including zero time, which suggests that HC rats also exhibit a state of insulin resistance that possibly contributes to reduced glucose tolerance.


Figure 1
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  		Fig. 1. Intravenous glucose tolerance tests (IVGTTs) in 100-day-old mother-fed (MF; {circ}); high-carbohydrate-fed (HC; {triangleup}), and high-fat-fed (HF; {square}) rats treated with saline. Changes in blood glucose levels (A) and changes in plasma insulin levels (B) in response to glucose load (0.5 g/kg iv). Values are given as means ± SE. Each group consisted of ≥6 rats. ANOVA, followed by post hoc analysis with a Tukey test, was performed between MF, HC, and HF rats. *P < 0.01 vs. MF; #P < 0.05 vs. MF.

 
ACh treatment. Dose-response experiments using ACh were carried out to evaluate sensitivity to cholinergic-induced potentiation of GSIS in 100-day-old MF and HC rats. Although no significant changes were seen in blood glucose levels for HC and MF rats at any concentration of ACh (Fig. 2, A and C), ACh treatment resulted in dose-dependent increases in plasma insulin levels in both MF (Fig. 2B) and HC rats (Fig. 2D). Each concentration of ACh resulted in significant increases in {Delta}I/{Delta}G for HC rats, whereas only the two highest doses of ACh resulted in significant increases in {Delta}I/{Delta}G for MF rats compared with saline-treated rats (Table 2). In addition, ACh-induced increases in {Delta}I/{Delta}G compared with saline treatment were significantly higher in HC rats compared with MF rats (Table 2), suggesting that HC rats exhibited increased in vivo sensitivity to ACh-induced potentiation of GSIS.


Figure 2
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  		Fig. 2. IVGTTs in 100-day-old MF and HC rats treated with saline ({circ}) and different concentrations of acetylcholine [ACh; ACh1 = 0.55 (bullet), ACh2 = 2.75 ({triangleup}), and ACh3 = 13.75 µmol ACh/kg body wt ({blacktriangleup})]. ACh-induced changes in blood glucose levels (A) and plasma insulin levels (B) in MF rats in response to glucose load (0.5 g/kg body wt). Changes in blood glucose levels (C) and plasma insulin levels (D) in HC rats in response to glucose load. Values are given as means ± SE. Each group consisted of ≥6 rats. Student's t-test was performed between treated and untreated rats. *P < 0.01 vs. saline treatment.

 

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  		Table 2. Values for {Delta}G, {Delta}I, and {Delta}I/{Delta}G during the entire 60 min of IVGTTs in 100-day-old MF and HC rats in response to increasing concentrations of ACh and OM

 
OM treatment. Dose-response experiments using OM were carried out to evaluate sensitivity to adrenergic-induced inhibition of GSIS in 100-day-old MF and HC rats. OM dose-dependently increased blood glucose levels for MF (Fig. 3A) and HC rats (Fig. 3C). Whereas MF rats showed significant OM-induced reductions in plasma insulin levels (Fig. 3B) and {Delta}I/{Delta}G at all concentrations of OM (Table 2), significant reductions in plasma insulin levels (Fig. 3D) and {Delta}I/{Delta}G (Table 2) were seen only at higher doses of OM in HC rats compared with saline-treated animals. In addition, OM-induced reductions in {Delta}I/{Delta}G in relation to saline treatment were significantly less in HC rats compared with MF rats (Table 2), suggesting that HC rats exhibited decreased in vivo sensitivity to OM-induced inhibition of insulin secretion.


Figure 3
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  		Fig. 3. IVGTTs in 100-day-old MF and HC rats treated with saline ({circ}) and different concentrations of oxymetazoline [OM; OM1 = 33.7 (bullet), OM2 = 169 ({triangleup}), OM3 = 674 nmol/kg body wt ({blacktriangleup})]. OM-induced changes in blood glucose levels (A) and plasma insulin levels (B) in MF rats in response to glucose load (0.5 g/kg body wt). Changes in blood glucose levels (C) and plasma insulin levels (D) in HC rats in response to glucose load. Values are given as means ± SE. Each group consisted of ≥6 rats. Student's t-test was performed between treated and untreated rats. *P < 0.01 vs. saline treatment.

 
4-DAMP treatment. Experiments using 4-DAMP were performed to assess the effects of in vivo blockade of the M3R on GSIS. Treatment with 4-DAMP resulted in significantly reduced GSIS, with significant increases in blood glucose levels in 100-day-old MF, HC, and HF rats compared with saline-treated animals (Fig. 4C; HF results not shown). In addition, 4-DAMP treatment resulted in significant reductions in plasma insulin levels that were more pronounced in HC rats (Fig. 4D). However, the combination of increased blood glucose and decreased plasma insulin levels resulted in significant reductions in {Delta}I/{Delta}G for all groups compared with saline treatments (Table 3). Therefore, in vivo blockade of M3R may mediate significant decreases in {Delta}I/{Delta}G by inhibiting cholinergic-induced potentiation of GSIS.


Figure 4
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  		Fig. 4. Parasympathetic-induced changes in IVGTTs in 100-day-old MF [A and B: {circ}, saline; bullet, 2.75 µmol ACh/kg body wt. C and D: bullet, 0.21 µmol 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP)/kg body wt] and HC rats (A and B: {triangleup}, saline; {blacktriangleup}, 2.75 µmol ACh/kg body wt. C and D: {blacktriangleup}, 0.21 µmol 4-DAMP/kg body wt). ACh-induced changes in blood glucose (A) and plasma insulin levels (B) in response to glucose load. 4-DAMP-induced changes in blood glucose (C) and plasma insulin levels (D) in response to glucose load. HF data are not included in the graphs due to similarity to MF data. Values are given as means ± SE. Each group consisted of ≥6 rats. Student's t-test was performed between similarly-treated MF rats. *P < 0.01 vs. saline treatment; #P < 0.05 vs. saline treatment.

 

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  		Table 3. Values for {Delta}G, {Delta}I, and {Delta}I/{Delta}G during the entire 60 min of IVGTTs in 100-day-old MF, HC, and HF rats in response to ACh, 4-DAMP, Yoh and OM

 
Yoh treatment. Experiments using Yoh were performed to assess the effects of in vivo blockade of the {alpha}2A-AR on adrenergic-induced inhibition of GSIS. Although treatment with Yoh resulted in no significant changes in blood glucose levels for any group (Fig. 5C; HF results not shown), significant increases in plasma insulin levels were seen in MF, HC, and HF rats (Fig. 5D; HF results not shown). Due to the increases in plasma insulin levels, {Delta}I/{Delta}G values were significantly increased in MF, HC, and HF rats (Table 3). Therefore, in vivo blockade of {alpha}2AR may result in increases in GSIS by inhibiting adrenergic-induced inhibition of insulin secretion. It is of note that, although {Delta}I/{Delta}G values were increased in all groups in response to Yoh, there were no differences in the fold increases of {Delta}I/{Delta}G between MF, HC, and HF rats (Table 3).


Figure 5
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  		Fig. 5. Sympathetic-induced changes in IVGTTs in 100-day-old MF [A and B: {circ}, saline; bullet, 33.7 nmol OM/kg body wt. C and D: bullet, 10 µmol yohimbine (Yoh)/kg body wt] and HC rats (A and B: {triangleup}, saline; {blacktriangleup}, 33.7 nmol OM/kg body wt; C and D: {blacktriangleup}, 10 µmol Yoh/kg body wt). OM-induced changes in blood glucose (A) and plasma insulin levels (B) in response to glucose load. Yoh-induced changes in blood glucose (C) and plasma insulin levels (D) in response to glucose. HF data are not included in the graphs due to similarity to MF data. Values are given as means ± SE. Each group consisted of ≥6 rats. Student's t-test was performed between similarly-treated MF and HC rats and between treated vs. untreated rats. *P < 0.01 vs. saline treatment; #P < 0.05 vs. saline treatment.

 
In Vivo Glucose-Stimulated Insulin Secretion in Vagotomized 100-Day-Old Rats

Vagatomy treatment. IVGTT was performed in vagotomized (VgX) 100-day-old MF and HC rats to assess the effects of parasympathetic denervation of the pancreas on in vivo insulin secretion in response to glucose and autonomic agonism and antagonism. Fasting blood glucose levels were 5.3 ± 0.1 mM for MFVgX rats and 5.7 ± 0.2 mM for HCVgX rats (Fig. 6A). Fasting plasma insulin levels were 38 ± 5 pM for MFVgX rats and 54 ± 3 pM for HCVgX rats (Fig. 6B), which were significantly reduced in HC rats compared with HCIntact rats (Fig. 6B). During the IVGTT, peak plasma insulin levels were reached at 10 min for MFVgX (78 ± 2 pM) and HCVgX rats (114 ± 5 pM) and were significantly lower than in intact animals (Fig. 6B). The insulinogenic index ({Delta}I/{Delta}G) was significantly lower for both MFVgX and HCVgX rats compared with intact rats (Table 4). Consequently, vagatomy resulted in significant attenuation of the differences in {Delta}I/{Delta}G values between HC and MF rats (HCIntact/MFIntact = 163 ± 11% vs. HCVgX/MFVgX = 136 ± 7%), suggesting that parasympathetic input to the pancreas is important for HC hyperinsulinemia.


Figure 6
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  		Fig. 6. Changes in IVGTTs in 100-day-old MF ({circ} and bullet) and HC rats ({triangleup} and {blacktriangleup}) in intact ({circ} and {triangleup}) and vagotomized rats (bullet and {blacktriangleup}). Changes in blood glucose levels (A) and plasma insulin levels (B) in response to glucose (0.5 g/kg body wt). Values are given as means ± SE. Each group consisted of ≥6 rats. Student's t-test was performed between intact and vagotomized rats. *P < 0.01 vs. intact rats; #P < 0.05 vs. intact rats.

 

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  		Table 4. Values for {Delta}G, {Delta}I, and {Delta}I/{Delta}G during the entire 60 min of IVGTTs in intact and vagotomized 100-day-old MF and HC rats in response to ACh, 4-DAMP, Yoh, and OM

 
Treatment of VgX rats with ACh and 4-DAMP. Treatment with ACh resulted in partial normalization of the effects of vagatomy on blood glucose levels in both MFVgX and HCVgX rats (Fig. 7A) but only significantly increased plasma insulin levels in HCVgX rats compared with saline-treated VgX HC rats (Fig. 7B). However, ACh treatment increased {Delta}I/{Delta}G values for both MFVgX and HCVgX rats compared with saline-treated VgX animals (Table 4). Furthermore, treatment with 4-DAMP had no effect on blood glucose or plasma insulin levels (Fig. 7, C and D) or {Delta}I/{Delta}G values (Table 4) in either MFVgX or HCVgX rats.


Figure 7
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  		Fig. 7. Parasympathetic-induced changes in IVGTTs in vagotomized 100-day-old (A and B: {circ}, saline; bullet, 2.75 µmol ACh/kg body wt. C and D: bullet, 0.21 µmol 4-DAMP/kg body wt) and HC rats (A and B: {triangleup}, saline; {blacktriangleup}, 2.75 µmol ACh/kg body wt; C and D: {blacktriangleup}, 0.21 µmol 4-DAMP/kg body wt). ACh-induced changes in blood glucose (A) and plasma insulin levels (B) in response to glucose. 4-DAMP-induced changes in blood glucose (C) and plasma insulin levels (D) in response to glucose load. Values are given as means ± SE. Each group consisted of ≥6 rats. Student's t-test was performed between treated vs. untreated vagotomized rats from the same group. *P < 0.01 vs. saline-treated vagotomized rats; #P < 0.05 vs. saline-treated vagotomized rats.

 
Treatment of VgX rats with OM and Yoh. Treatment with OM significantly reduced GSIS in both MFVgX and HCVgX rats, as seen by significant increases in blood glucose (Fig. 8A) and decreases in plasma insulin levels in MFVgX + OM and HCVgX + OM rats (Fig. 8B) compared with saline-treated vagotomized rats. In addition, {Delta}I/{Delta}G values were significantly decreased in MFVgX + OM and HCVgX + OM rats compared with saline-treated vagotomized rats (Table 4). Furthermore, the magnitude of OM-induced inhibition of {Delta}I/{Delta}G in HCVgX rats was less pronounced compared with HCIntact rats (HCIntact = 28% vs. HCVgX = 14%, P < 0.05), suggesting that the ability of OM to reduce in vivo insulin secretion may also be related to regulation of parasympathetic activity. Yoh treatment did not significantly alter blood glucose levels in either MFVgX or HCVgX rats (Fig. 8C) but did increase plasma insulin levels in HCVgX rats compared with saline-treated vagotomized rats (Fig. 8D). Yoh treatment in MFVgX rats did not result in significant increases in plasma insulin levels (Fig. 8D) compared with saline-treated vagotomized rats. Yoh treatment also significantly increased {Delta}I/{Delta}G values for HCVgX rats, but not MFVgX rats, compared with saline-treated vagotomized animals (Table 4). In addition, vagatomy resulted in a significant attenuation of Yoh-induced potentiation of insulin secretion in HC rats, suggesting that the ability of Yoh to increase GSIS may be through regulation of parasympathetic activity (fold increase in {Delta}I/{Delta}G between saline and Yoh treatment: HCIntact = 1.8-fold vs. HCVgX = 1.2-fold).


Figure 8
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  		Fig. 8. Sympathetic-induced changes in IVGTTs in vagotomized 100-day-old MF (A and B: {circ}, saline; bullet, 33.7 nmol OM/kg body wt; C and D: {circ}, saline; bullet, 10 µmol Yoh/kg body wt) and HC rats (A and B: {triangleup}, saline; {blacktriangleup}, 33.7 nmol OM/kg body wt. C and D: {triangleup}, saline; {blacktriangleup}, 10 µmol Yoh/kg body wt). OM-induced changes in blood glucose (A) and plasma insulin levels (B) in response to glucose load. Yoh-induced changes in blood glucose (C) and plasma insulin levels (D) in response to glucose. Values are given as means ± SE. Each group consisted of ≥6 rats. Student's t-test was performed between treated vs. untreated rats from the same group. *P < 0.01 vs. saline-treated vagotomized rats; #P < 0.05 vs. saline-treated vagotomized rats.

 
In Vitro Insulin Secretion From Islets From 100-Day-Old Rats

The effects of autonomic agonists and antagonists on in vitro GSIS was studied using islets isolated from 100-day-old MF, HC, and HF rats and treatment with two glucose, basal (5.5 mM), and stimulatory (16.7 mM) concentrations. Adult HC islets secreted significantly higher levels of insulin in response to glucose alone compared with MF and HF islets at both glucose concentrations for 10 and 60 min (no treatment; Fig. 9).


Figure 9
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  		Fig. 9. Insulin secretion by islets isolated from 100-day-old (open bars), HC (black bars), and HF rats (gray bars) in the presence of 5.5 and 16.7 mM glucose and 100 µM ACh, 100 µM 4-DAMP (4-DP), or 100 µM ACh and 100 µM 4-DP. A: insulin secretion at 5.5 mM glucose after 10 min. B: insulin secretion at 5.5 mM glucose after 60 min. C: insulin secretion at 16.7 mM glucose after 10 min. D: insulin secretion at 16.7 mM glucose after 60 min. Values are given as means ± SE (n = 6–8 for each experiment). ANOVA was performed for insulin secretion by MF, HC, and HF islets between glucose treatments alone and compared with similarly-treated MF rats. *P < 0.01, insulin secretion vs. MF insulin secretion; #P < 0.01, insulin secretion vs. glucose alone (no treatment); <P < 0.01, fold change vs. MF insulin secretion; >P < 0.01, %decrease vs. ACh treatment.

 
ACh treatment (100 µM) resulted in significant potentiation of insulin secretion by MF, HC, and HF islets compared with glucose treatment alone (Fig. 9). In addition, ACh-induced potentiation of GSIS above treatment with 5.5 mM glucose alone was significantly higher in HC islets compared with MF and HF islets (Fig. 9, A and B, and Table 5). ACh treatment at 16.7 mM glucose also resulted in a greater potentiation of insulin secretion above glucose treatment alone in HC islets compared with MF and HF islets (Fig. 9, C and D, and Table 5).


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  		Table 5. %Change in insulin secretion by MF, HC, and HF islets vs. %saline treatment

 
Whereas treatment with 4-DAMP alone had no effect on GSIS by MF, HC, and HF islets at either glucose concentration (Fig. 9), 4-DAMP (100 µM) in the presence of ACh at 5.5 mM glucose resulted in significant reductions in cholinergic-induced potentiation of insulin secretion by MF, HC, and HF islets at both the 10- and 60-min time points (Fig. 9, A and B, and Table 5). Treatment with 4-DAMP at 16.7 mM resulted in similar reductions in ACh-induced potentiation of insulin secretion by MF, HC, and HF islets (Fig. 9, C and D, and Table 5). This suggests that ACh-induced potentiation of GSIS is mediated through activation of the M3R and may be important to HC insulin hypersecretion. No differences were seen in insulin secretion between MF and HF islets treated with 4-DAMP or ACh in the presence of 4-DAMP (Fig. 9).

OM treatment (10 µM) resulted in significant reductions in insulin secretion by MF, HC, and HF islets compared with treatment with glucose alone (Fig. 10 and Table 5). Whereas treatment with Yoh alone (10 µM) had no effect on GSIS by MF, HC, and HF islets at either glucose concentration (Fig. 10), Yoh in the presence of OM at 5.5 mM glucose reduced adrenergic-induced inhibition of insulin secretion and resulted in significant normalization of insulin secretion by MF, HC, or HF islets compared with treatment with OM alone at both 10- and 60-min time points (Fig. 10, A and B, and Table 5). Yoh treatment at 16.7 mM glucose resulted in similar normalization of insulin secretion by MF, HC, and HF islets (Fig. 10, C and D, and Table 5). This suggests that OM-induced inhibition of GSIS is mediated through activation of the {alpha}2A-AR and may be important to HC insulin hypersecretion.


Figure 10
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  		Fig. 10. Insulin secretion by islets isolated from 100-day-old MF (open bars), HC (black bars), and HF rats (gray bars) in the presence of 5.5 and 16.7 mM glucose and 10 µM OM, 10 µM Yoh, or 10 µM OM and 10 µM Yoh. A: insulin secretion at 5.5 mM glucose after 10 min. B: insulin secretion at 5.5 mM glucose after 60 min. C: insulin secretion at 16.7 mM glucose after 10 min. D: insulin secretion at 16.7 mM glucose after 60 min. Values are given as means ± SE (n = 6–8 for each experiment). ANOVA was performed for insulin secretion by MF, HC, and HF islets between glucose treatments alone and compared with similarly-treated MF rats. *P < 0.01 insulin secretion vs. MF insulin secretion; #P < 0.01 insulin secretion vs. glucose alone (no treatment); >P < 0.01 increased insulin secretion vs. OM treatment.

 
mRNA Levels in Islets From 100-Day-Old Rats

The long-term effects of the HC dietary intervention on cholinergic and adrenergic signaling pathways in pancreatic islets isolated from 100-day-old MF, HC, and HF rats were evaluated using quantitative real-time PCR. Specifically, changes in adrenergic signaling pathways were assessed by measuring islet levels of {alpha}2A-AR mRNA, whereas changes in cholinergic signaling pathways were assessed by measuring islet expression of M3R, PLCbeta1, and PKC{alpha} mRNAs. The level of {alpha}2A-AR was reduced in HC islets (53%) compared with MF (100%) and HF (106%) islets (Fig. 11). In addition, levels of cholinergic signaling molecules were significantly increased in HC islets (M3R = 254%, PLCbeta1 = 271%, and PKC{alpha}-288%) compared with MF islets (each at 100%) and HF islets (M3R = 103%, PLCbeta1 = 110%, and PKC{alpha} = 107%) (Fig. 11). No significant differences in islet mRNA levels were seen between MF and HF rats (Fig. 11).


Figure 11
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  		Fig. 11. Measurements of {alpha}2a-adrenergic receptor ({alpha}2A-AR), muscarinic type 3 receptor (M3R), phospholipase Cbeta1 (PLCbeta1), and PKC{alpha} mRNA levels in islets isolated from 100-day-old rats. A: quantitative real-time PCR analysis of {alpha}2A-AR, M3R, PLCbeta1, and PKC{alpha} mRNAs in pancreatic islets isolated from 100-day-old MF (open bars), HC (black bars), and HF rats (gray bars). Values were normalized using 18S as an internal standard, and fold difference was calculated using the critical threshold cycle (CT) method ({Delta}{Delta}CT). Individual samples were run in triplicate, and values are given as means ± SE of 6 experiments. Student's t-test was performed between islet mRNA levels from MF and HC islets or HF islets. *P < 0.01 vs. MF mRNA levels.

 
Protein Content in Islets From 100-Day-Old Rats

Western blot analysis of islet protein content was performed (Fig. 12A) to confirm the changes in cholinergic and adrenergic signaling pathways that were seen in real-time PCR experiments. HC islets had lower levels of {alpha}2A-AR (35% reduction) compared with MF islets (100%) (Fig. 12B). In addition, HC islets showed increased PLCbeta1 (137%) and PKC{alpha} (147%) content compared with MF (Fig. 12B). M3R content of HC islets (107%) was similar to MF islets (100%) (Fig. 12B).


Figure 12
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  		Fig. 12. Measurements of {alpha}2A-AR, M3R, PLCbeta1, and PKC{alpha} protein content in islets isolated from 100-day-old rats. A: Western blot. B: Western blot analysis of {alpha}2A-AR, M3R, PLCbeta1, and PKC{alpha} content in islets isolated from 100-day-old MF (open bars) and HC rats (black bars). Values are given as means ± SE of 6 experiments. Student's t-test was performed between islet protein content of MF and HC rats. #P < 0.05 vs. MF protein content.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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This study confirms our previous reports about the presence of chronic hyperinsulinemia in adult HC rats (1, 47) and extends the findings showing the involvement of the ANS in the maintenance of a chronic hyperinsulinemia in HC rats. Present in vivo and in vitro findings of insulin secretion in response to ACh, 4-DAMP, Yoh, and OM in intact and vagotomized HC rats provide evidence that altered autonomic activity is involved in the chronic hyperinsulinemia in adult HC rats through increased sensitivity to cholinergic-induced potentiation of GSIS, as well as reduced islet sensitivity to adrenergic-induced inhibition of insulin secretion. In addition, molecular changes within pancreatic islets, including increased cholinergic and decreased adrenergic signaling, may contribute to enhanced insulin secretion in response to glucose.

Parasympathetic-Stimulated Insulin Secretion in 100-Day-Old Rats

Whereas glucose is the primary stimulus for insulin secretion by beta-cells, substantial evidence exists that other secretagogues, including ACh and norepinephrine, are involved in the regulation of GSIS (13, 47). Previous in vitro studies of insulin secretion have found that islets isolated from 12-day-old HC rats treated with ACh and GLP-1 resulted in greater increases in GSIS compared with MF islets, suggesting increased beta-cell responsiveness to cholinergic and incretin stimulation during the neonatal period in HC rats (46, 47). Furthermore, evidence in 12-day-old HC rats showed that altered autonomic activity is involved in the immediate onset of hyperinsulinemia in response to the HC dietary intervention (31).

ACh treatment resulted in significant increases in GSIS for HC islets compared with MF and HF islets at basal (5.5 mM) and stimulatory (16.7 mM) glucose concentrations (Fig. 9), suggesting that HC islets were more responsive to cholinergic-induced potentiation of GSIS. This finding is consistent with other studies showing that parasympathetic-induced potentiation of GSIS from islets is related to cholinergic primed beta-cells that secrete more insulin in response to glucose (33, 56). For example, ACh-primed beta-cells show a greater enhancement of GSIS compared with unprimed beta-cells when treated with a subsequent glucose challenge (24, 56). Also, islets isolated from hyperinsulinemic monosodium L-glutamate-treated obese rats showed greater carbachol-induced potentiation of GSIS compared with controls, suggesting a role for increased parasympathetic potentiation of islet insulin secretion that resulted in hyperinsulinemia in these obese rats (7). Although treatment with 4-DAMP alone had no effect on GSIS by HC or control islets, 4-DAMP in the presence of ACh resulted in significant reductions in cholinergic-induced insulin secretion by both HC and control islets (Fig. 9), indicating that ACh-induced potentiation of GSIS is mediated through activation of M3R.

In vivo insulin secretion in MF and HC rats during IVGTT in response to parasympathetic agonists and antagonists also showed that enhanced responsiveness to cholinergic activity contributed to HC hyperinsulinemia. Specifically, 100-day-old HC rats showed significantly greater potentiation of GSIS in response to ACh compared with MF and HF rats as well as increased sensitivity to blockade of the M3R (Figs. 4 and 7). Parasympathetic involvement in HC hyperinsulinemia was further supported by a greater reduction in GSIS by vagatomy in HC rats compared with MF rats. It is important to note that in vivo increases in insulin secretory responses to glucose in HC and control rats after ACh treatments were due to the ACh itself and not to alterations in the glycemic profile after glucose loading. This is supported by the fact that {Delta}G values were not different between untreated intact rats and intact rats receiving ACh, whereas {Delta}G in vagotomized rats actually decreased after treatment with ACh (data not shown).

Parasympathetic activity is involved in maintenance of normal glucose tolerance and insulin secretion, as evidenced by the role of cholinergic activity in the cephalic phase of insulin secretion (3, 4). Acute changes in blood glucose levels have been associated with altered parasympathetic nerve activity in rats, with vagal firing rates being correlated with blood glucose over a wide range of concentrations (36). Evidence for parasympathetic activity in the cephalic phase of insulin secretion and the coupling of parasympathetic firing rates to blood glucose levels (36) suggested that early exposure to a HC milk formula during the immediate postnatal period may result in an adaptive increase in parasympathetic activity in HC rats that facilitates increased insulin secretion in response to increased glucose stimulation. Furthermore, this increased parasympathetic activity may result in increased ACh stimulation of HC islets, which sensitizes pancreatic beta-cells to glucose stimulation. This would be consistent with evidence that ACh mediates beta-cell sensitization to glucose stimulation. ACh stimulates phosphoinositide turnover (55), resulting in increased generation of diacylglycerol and activation of the Ca2+-dependent protein kinase (37), which enhances insulin secretion in response to glucose (34, 50, 56). Therefore, enhanced ACh stimulation of HC islets may result in permanent programming of enhanced sensitivity to glucose.

In the present study, adult HC islets showed increased levels (2.5- to 3.0-fold) of M3R, PLCbeta1, and PKC{alpha} mRNAs compared with MF and HF islets (Fig. 11). These changes in the cholinergic signaling pathway are similar to changes seen in 12-day-old HC rats, suggesting that altered responses to autonomic stimulation are permanently programmed into the insulin secretory pathways of HC rats. Western blot analysis of islet protein content in adult HC islets confirmed the PCR findings showing increased PLCbeta1 and PKC{alpha} content compared with MF islets (Fig. 12). The fact that increases in M3R protein content of HC islets did not reach statistical significance suggests that increased expression of M3R mRNA may not be realized at the protein level. Alternatively, the use of the collagenase digestion protocol for the isolation of pancreatic islets may have modified membrane proteins such as M3R. Therefore, early exposure to a carbohydrate-rich diet may permanently alter gene expression patterns in islets and result in an adaptive upregulation of cholinergic signaling pathways that result in chronic hyperinsulinemia in response to glucose.

Sympathetic Inhibition of Insulin Secretion in 100-Day-Old Rats

Sympathetic activity results in release of norepinephrine (5), which inhibits insulin secretion through activation of {alpha}2A-ARs located on beta-cells. Previous in vitro studies of insulin secretion have found that islets isolated from HC rats were less sensitive to norepinephrine-induced inhibition of insulin secretion (46). Treatment of adult islets with OM resulted in a significantly lesser inhibition in GSIS for HC islets compared with MF and HF islets (Fig. 10). The involvement of the sympathetic nervous system in beta-cell hyperresponsiveness to glucose in HC rats is evident from the results of in vivo glucose tolerance tests in intact and vagotomized rats using OM and Yoh (Figs. 5 and 8). Although Yoh treatment resulted in significantly increased plasma insulin levels in intact HC rats compared with saline-treated HC rats, the ability of vagatomy to abolish these effects suggests that Yoh-induced increases in insulin secretion may be due to indirect stimulation of parasympathetic activity.

Regulation of autonomic activity involves a complex system of peripheral and central interactions in which PNS and SNS activities are balanced by each other. For example, the dorsal motor nucleus of the vagus nerve, which regulates PNS activity, expresses adrenergic receptors that inhibit parasympathetic activity and further enhance sympathetic inhibition of insulin secretion (45). Studies of adrenergic denervation of the dorsal motor nuclei with 6-hydroxydopamine resulted in increased GSIS and hyperinsulinemia that was reversed by bilateral vagotomy (45), suggesting that sympathetic-induced inhibition of insulin secretion involves central inhibition of parasympathetic output as well as direct effects on pancreatic islets (45). Yoh data in vagotomized HC rats are consistent with evidence showing that parasympathetic activity is regulated by adrenergic inputs to the dorsal motor nucleus of the vagus nerve and suggest that Yoh may be acting centrally to reduce adrenergic blockade of vagal activity, resulting in enhanced parasympathetic-induced potentiation of insulin secretion. The role of parasympathetic activity in Yoh-induced potentiation of GSIS is further supported by the finding that vagotomy resulted in an attenuation of insulin secretion in response to Yoh.

Although altered responses to Yoh seemed to involve central regulation of vagal outflow to the pancreas, the reduced inhibitory effect of the {alpha}2A-AR-specific agonist OM in intact HC rats and the fact that vagotomy attenuated the inhibitory effect of OM in HC rats suggested that reductions in OM-induced inhibition of insulin secretion may involve reduced sensitivity at the level of pancreatic islets as well as reduced adrenergic inhibition of parasympathetic activity. This would be consistent with our previous studies (46, 47) showing that islets isolated from HC rats exhibited a 10-fold reduction in sensitivity to inhibition of insulin secretion by norepinephrine. Furthermore, a study of selective surgical denervation of pancreatic sympathetic innervation resulted in increased GSIS in association with impaired adrenergic stimulation of beta-cells (38). Reduced sensitivity to adrenergic agonism in HC rats may be the result of decreased adrenergic activity at the level of the islets due to reductions in sympathetic outflow to the pancreas. Therefore, reduced sympathetic activity may play an important role in the development and maintenance of chronic hyperinsulinemia in HC rats. This is supported by decreased levels of {alpha}2A-AR mRNA (53%) and {alpha}2A-AR protein content (35%) in islets from 100-day-old HC rats compared with MF islets. Several studies provide a framework for downregulation of {alpha}2A-AR in relation to reduced HC islet sensitivity to adrenergic-induced inhibition of insulin secretion. For example, increased cAMP levels are known to induce reductions in {alpha}2A-AR expression (41). Previous findings have shown that HC islets exhibited increased levels of adenylate cyclase type VI mRNA (46), which may result in enhanced cAMP generation in these islets. Increased levels of cAMP may contribute to the observed reduced expression of {alpha}2A-AR in HC islets. In this context it is interesting to note that Dolz et al. (18) demonstrated that, in the Goto-Kakizaki rat beta-cell, ACh-stimulated cAMP generation was instrumental in the ACh-triggered insulin release at low glucose concentrations.

Another factor contributing to reduced sympathetic activity in HC islets could be leptin resistance in these islets. It is of interest to note that plasma leptin levels are markedly increased in the adult HC male rats (unpublished data). One of the important peripheral actions of leptin involves the inhibition of insulin biosynthesis and secretion by pancreatic beta-cells (43). Mizuno et al. (32) suggested that regulation of plasma insulin levels by leptin could be due to its stimulatory effects on the sympathetic nerve endings around the pancreatic islets. Despite the increased circulating levels of leptin, hyperinsulinemia is observed in the HC rats, which suggests a state of leptin resistance in the islets resulting in reduced sympathetic activity. This is in keeping with a similar observation in rodent models of obesity wherein hypothalamic leptin resistance is observed despite increased circulating levels of leptin (42).

In conclusion, chronic hyperinsulinemia in adult HC rats is associated with altered autonomic activity through increased parasympathetic and decreased sympathetic activities. These changes in autonomic activity may function to preserve the enhanced insulin secretory capacity of beta-cells in HC rats, which helps to maintain insulin hypersecretion throughout life. Since adult HC rats appear to exhibit a state of insulin resistance due to hyperinsulinemia in the presence of euglycemia (1), it is possible that programmed changes in autonomic activity are an adaptive mechanism that ensures compensatory increases in insulin secretion and prevents hyperglycemia due to insulin resistance. It also suggests that early exposure to glucose during the HC dietary intervention may result in permanent alterations in the threshold for activation of autonomic activity, resulting in compensatory increases in PNS and decreases in SNS activities throughout life to meet the increased insulin demand caused by insulin resistance. However, additional studies are required to determine the full extent of autonomic involvement in HC hyperinsulinemia, including recordings of parasympathetic and sympathetic firing rates and the role of altered control of autonomic activity. These studies will further our understanding of metabolic programming in response to early nutritional environments and will help address the growing epidemics of obesity and type 2 diabetes.


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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-61518.


   ACKNOWLEDGMENTS
 
We thank Dr. Suzanne Laychock of the School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, for fruitful discussions and help in setting up the real-time PCR technique used in this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. S. Patel, Dept. of Biochemistry, School of Medicine and Biomedical Sciences, Univ. at Buffalo, State University of New York, 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.


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