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Am J Physiol Gastrointest Liver Physiol 293: G112-G120, 2007. First published May 17, 2007; doi:10.1152/ajpgi.00442.2006
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HORMONES AND SIGNALING

Pioglitazone reverses insulin resistance and impaired CCK-stimulated pancreatic secretion in eNOS(–/–) mice: therapy for exocrine pancreatic disorders?

Raju C. Reddy,1 Yibai Hao,2 Sae-Hong Lee,2 Srinivasa R. Gangireddy,1 Chung Owyang,2 and Matthew J. DiMagno2

1Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, and 2Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of Michigan School of Medicine, Ann Arbor, Michigan

Submitted 26 September 2006 ; accepted in final form 7 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In mice, eNOS (endothelial nitric oxide synthase) maintains in vivo pancreatic secretory responses to carbachol or cholecystokinin octapeptide (CCK-8), maintains insulin sensitivity, and modulates pancreatic microvascular blood flow (PMBF). eNOS(–/–) mice are insulin resistant, and their exocrine pancreatic secretion is impaired. We hypothesized that the reduced exocrine pancreatic secretion in eNOS(–/–) mice is due to insulin resistance or impaired PMBF. To test this hypothesis, we gave eNOS(–/–) and wild-type (WT) mice pioglitazone (20 or 50 mg·kg–1·day–1), an insulin-sensitizing peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) activator, and measured pancreatic protein secretion evoked by CCK-8 (160 pmol·kg–1·h–1, a maximal stimulus). We also measured insulin resistance, serum glucose, C-peptide, insulin, pancreatic RNA digestive enzyme expression, and PMBF (microsphere technique). In WT mice, pioglitazone did not increase CCK-8-stimulated protein output over baseline. In eNOS(–/–) mice, however, pioglitazone substantially increased the low CCK-8-stimulated protein output that is characteristic of these mutant mice (P < 0.005). Pioglitazone abolished the CCK-8-evoked hyperinsulinemia (P < 0.005) and increased insulin sensitivity of eNOS(–/–) mice (P < 0.05), the latter based on hyperinsulinemic-euglycemic clamp studies. Pioglitazone had no effect on PMBF or pancreas mRNA expression of insulin or digestive enzymes. We conclude that in hyperinsulinemic eNOS(–/–) mice, a nonobese model of insulin resistance relevant to diabetes mellitus and possibly chronic pancreatitis, reduced pancreatic secretion is caused, at least in part, by insulin resistance. Insulin-sensitizing PPAR-{gamma} agonists such as pioglitazone may thus simultaneously correct endocrine and exocrine pancreatic disorders.

nitric oxide; insulin; diabetes; cholecystokinin

IN A PREVIOUS IN VIVO STUDY in mice that was designed to clarify a controversy regarding the effect of nitric oxide (NO) on secretagogue-stimulated pancreatic exocrine secretion (15), we found that nonselective nitric oxide synthase (NOS) inhibition or endothelial NOS (eNOS) gene deletion reduced cholecystokinin octapeptide (CCK-8)- and carbachol-stimulated pancreatic secretion by ~50%. In contrast, neuronal NOS (nNOS) gene deletion augmented CCK-8- but not carbachol-stimulated pancreatic secretion. This indicates that eNOS has a dominant stimulatory role on pancreatic protein secretion and that nNOS has a minor inhibitory effect. Because protein expression of eNOS was weak and nNOS absent in pancreatic acini compared with whole tissue preparations (17) and secretory responses were intact in isolated acini from eNOS(–/–) mice or wild-type (WT) mice treated with a nonselective NOS inhibitor (15), we surmised that NO modulates extra-acinar events. Furthermore, because immunohistochemical studies indicate that nNOS localizes to pancreatic nerves and eNOS localizes to the vasculature, we proposed that nNOS tonically inhibits acetylcholine release from pancreatic neurons and that eNOS may act on the pancreatic microvasculature to increase pancreatic microvascular blood flow (PMBF) (15). In addition, decreased pancreatic exocrine secretion in eNOS gene-deleted mice, a nonobese model of insulin resistance (12, 18, 58, 59), may also occur through effects on insulin signaling because pancreatic exocrine secretion decreases with insulin resistance (5, 55, 72–74) and insulinopenia (3, 11, 13, 20, 27, 33, 36, 44, 50). Finally, eNOS may also have complex biological effects on neural and other nonacinar cell types to maintain pancreatic exocrine secretion (15).

To help clarify how eNOS maintains pancreatic exocrine secretion, we tested the hypothesis that the reduced exocrine pancreatic secretion in eNOS(–/–) mice is due to insulin resistance or impaired PMBF. To this end, we determined in eNOS(–/–) mice whether pioglitazone improves secretagogue-stimulated in vivo pancreatic exocrine secretion (15). We chose pioglitazone because it can increase sensitivity to insulin (i.e., reverse insulin resistance) (45, 57) and thereby improve insulin and glucose homeostasis. Also, pioglitazone is an agonist for peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) (45, 57), and such agents are currently being used in the treatment of patients with type 2 diabetes mellitus. This is consistent with findings that PPAR-{gamma} is a ligand-activated transcription factor that regulates metabolic pathways involved in insulin resistance and hyperlipidemia (45, 57). In the pancreas, PPAR-{gamma} is localized to pancreatic acini (4, 23, 39, 61) and beta-cells (78). PPAR-{gamma} agonists consist of the thiazolidinedione class of drugs, such as rosiglitazone and pioglitazone, which are potent activators of PPAR-{gamma} both in vitro and in vivo.

To obtain a more comprehensive picture of glucose/insulin homeostasis and pancreatic secretion in eNOS(–/–) mice, we acquired data on several related variables. For example, to assess pancreatic secretion, we measured biliary-pancreatic juice protein output, previously validated as an indicator of pancreatic enzyme secretion (15), referred to as pancreatic exocrine secretion. Measures of insulin homeostasis included serum glucose, serum insulin, serum C-peptide, and pancreatic insulin transcription. We assessed insulin sensitivity by using a hyperinsulinemic-euglycemic clamp (12, 58, 59). Additional surrogate markers of insulin resistance were also assessed by measuring serum insulin and hepatic mRNA expression of two key insulin-signaling intermediates, insulin receptor substrate isoforms IRS-1 and IRS-2. IRS-2 is primarily responsible for hepatic insulin signaling and is a potential therapeutic target because of its major role in maintaining insulin sensitivity (28, 32, 52, 76). We also investigated whether pioglitazone augments PMBF [assessed by a microsphere method (17)] in eNOS(–/–) mice during in vivo secretagogue-stimulated pancreatic exocrine secretion and whether increases in pancreatic secretion and PMBF are parallel or unassociated events in WT mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. CCK-8 was obtained from Research Plus (Bayonne, NJ), SYBR Green 1 dye was from Sigma Chemical (St. Louis, MO), and TaqMan reverse transcription reagents were from Applied Biosystems (Foster City, CA). All other reagents were from Sigma.

Animal care, selection, and feeding. All mice were obtained from the Jackson Laboratory. Animals were 6–8 wk old, weighed 18–24 g, were maintained in a climate-controlled room kept at 22°C, were exposed to a 12-h on and 12-h off light-dark cycle, and were fed standard laboratory chow and given water ad libitum. In vivo pancreatic secretion was studied in mice with a targeted gene deletion of eNOS (60), bred on a C57BL/6J genetic background. Control mice were age and sex matched and came from an identical genetic background. Pioglitazone or vehicle (0.25% carboxymethylcellulose) was administered to mice by oral gavage once daily for five consecutive days. All experiments were approved by the University of Michigan Committee on Use and Care of Animals.

In vivo pancreatic secretion. As previously described (15, 16), mice were anesthetized with intraperitoneal injections of 80 mg/kg ketamine plus 5 mg/kg xylazine (Rompun) to produce 30–45 min of anesthesia, redosing when necessary, with the original ketamine dose (without xylazine). With the use of a surgical, x4-magnifying, Leica MZ6 stereo microscope, a PE-10 cannula was inserted into the right internal jugular vein for secretagogue infusion. A superficial, 1.5- to 2.0-cm midline abdominal incision was made, and a PE-10 catheter was inserted into the extraduodenal, intrapancreatic common bile-pancreatic duct. The abdominal wound was covered with sterile gauze, and the mouse was placed on a protective pad, overlying a heating pad, to maintain body temperature at 37°C.

Experiments began following a 30-min stabilization period. From the collected bile-pancreatic juice, volume and protein concentration were measured. The latter was determined spectrophotometrically using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). Juice was collected every 15 min, and basal protein output was obtained from the second of two 15-min baseline, mixed-juice samples, based on our prior studies showing that basal bile-pancreatic protein output in anesthetized mice stabilizes 15 min after bile-pancreatic duct cannulation and remains constant for 90 min (15). CCK-8 was infused intravenously using a Harvard PHD 2000 Push/Pull Syringe Pump (Harvard Apparatus, Holliston, MA) at a rate of 160 pmol·kg–1·h–1, which we have previously shown to maximally stimulate bile-pancreatic protein output in C57BL/6J mice (15). Sodium chloride (0.9%) infusion was used as a vehicle control. Protein output was expressed as µg/min, and statistical analyses were performed on the pooled peak CCK-8 response, determined by subtracting basal from pooled peak secretagogue responses between 45 and 75 min (16).

Hyperinsulinemic-euglycemic clamp studies. These studies were performed in mice 5 days after insertion of jugular catheters, which allowed animals to recover from surgery, as indicated by behavior and weight recovery. Studies were performed on 12-h fasted, awake, unrestrained mice. During experiments, the insulin infusion rate was clamped at 20 mU·kg–1·min–1 as previously described (12, 18, 59). Plasma glucose levels were measured every 10 min during the clamp. Differences in insulin sensitivity were determined from the final glucose infusion rates (GIRs) at 90 min.

Total RNA isolation, reverse transcription, and quantitative real-time PCR. As previously described (16), total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) and further purified with an RNeasy kit from QIAGEN (Valencia, CA) using the RNase-Free DNase Step (no. 79254) according to the manufacturer's instructions. RNA purity and concentration were examined by optical density (OD)260/OD280 ratios and by 1% agarose gel electrophoresis and ethidium bromide staining. Total RNA (200 ng) was reverse transcribed using TaqMan reverse transcription reagents with random hexamers as primers, and cDNA was quantified in a Genesys 5 spectrophotometer. Quantitative PCR was carried out, and data were analyzed using an I-Cycler IQ Real-Time PCR detection system (Bio-Rad Laboratories). Each reaction contained 2 µl of cDNA (0.5 µg/µl), 10.3 µl of HPLC water, 2 µl of 10x PCR buffer, 2.2 µl of MgCl2 (50 mM), 1 µl of fluorescein, 0.4 µl of dNTP (10 mM), and 2 µl of primer mixtures [consisting of 0.1 nmol/µl concentration of each forward and reverse primer (Table 1) and 1 µl of 100x SYBR Green 1 dye per 100 µl volume]. Quantitative PCR was performed at 95°C for 2 min, followed by 60°C for 1 min (repeated 40 times) and 55°C for 1 min. The specificity of the products (in contrast to the primer-dimers) was determined by monitoring the melt curve, obtained in increments of 0.5°C every 10-s interval from 55°C to 96°C. As a negative control, real-time PCR runs included the primers alone (and PCR cocktail) without cDNA. Finally, primer pairs were also validated with a standard dilution curve. The PCR product abundance was indicated by the fluorescence resulting from the incorporation of SYBR Green 1 dye into the double-stranded DNA produced during the PCR reaction. The relative abundance of individual gene transcripts was expressed as a ratio of values in eNOS(–/–) mice compared with those in WT mice. This ratio was calculated as 2n, where n is the difference between the mean CT (threshold cycle) value for a specific gene transcript in WT mice minus the value for an eNOS(–/–) mouse sample. As an internal control, data were normalized to the CT value of the appropriate "housekeeping" gene (Rb 23-kDa), measured from the same RNA sample and PCR run, and expressed as a fold change compared with WT mice, which were assigned the value 1.


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  		Table 1. Primers used for quantitative real-time PCR of mouse pancreas and liver RNA

 
Gene sequences were obtained from the GenBank NCBI Sequence Viewer (http://www.ncbi.nlm.nih.gov), and primers for quantitative RT-PCR (Table 1) were designed using Primer Express Primer software (Applied Biosystems) and synthesized by Invitrogen.

Measurement of serum glucose and islet hormones. Mice were euthanized by carbon dioxide asphyxiation, mixed arteriovenous blood was collected from the decapitated body and centrifuged at 4°C, and serum was stored at –70°C. Serum glucose and insulin were measured in the Chemistry Core of the Michigan Diabetes Research and Training Center. Glucose was measured enzymatically and serum insulin by RIA using a commercially available rat insulin assay (Linco Research, St. Louis, MO) and rat insulin standards. Serum somatostatin and C-peptide were measured by RIA using commercially available kits from Phoenix Pharmaceuticals (Belmont, CA) and rat somatostatin and C-peptide standards, respectively.

PMBF studies. Mice were anesthetized and prepared for surgery as described above (see In vivo pancreatic secretion). With the use of a surgical, x4-magnifying, Leica MZ6 stereo-microscope, a 30-cm PE-10 cannula was flushed with heparinized (100 U/ml), sterile SanSaline (BioPal, Wellesley Hills, MA), inserted into the right internal carotid artery, advanced 1 cm toward the aortic arch, secured and used for microsphere injection. A second PE-10 cannula was similarly prepared and inserted into the right iliac artery for blood sample collection.

PMBF was measured as previously described (16). Microspheres (15-µm diameter, BioPal) were infused (80,000/100 µl) over 10 s into the aorta via the internal carotid artery cannula, and a 100-µl blood sample was simultaneously removed over 1 min from the external iliac artery using a Harvard PHD 2000 Push/Pull Syringe Pump. The weight of blood samples confirmed the measured volume removed. The total blood removed for a single PMBF measurement was 100 µl, equal to 6–8% of the circulating blood volume. Following euthanasia, pancreas and kidneys were removed, washed in SanSaline, blotted, weighed, dried overnight (75°C), and commercially analyzed by neutron activation for blood flow determination (BioPal). Adequate mixing of microspheres in the arterial circulation was considered to be accomplished when blood flow measurements for the two kidneys differed by < 10%. Blood flow measurements were expressed as ml·min–1·g–1 tissue.

Statistical analyses. The data reported represent the means ± SE from multiple determinations obtained from three or more experiments. Statistical comparisons were performed using the Student's t-test when comparing only two groups. When comparing three or more groups, ANOVA was used followed by post hoc testing with Fisher's protected least significant differences test using StatView software (SAS Institute, Cary, NC). Statistical significance was assumed for P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Gross appearance of the pancreas and cannulation of the biliary-pancreatic duct. The gross appearance of the pancreas and the ease of cannulating the bile-pancreatic duct were similar in eNOS(–/–) and WT mice. Although we successfully cannulated all mice, anesthesia-related deaths occurred in a small number of knockout and WT mice (n = 2–4/group) before completion of experiments. The incomplete data from these experiments were excluded from further analysis.

Pioglitazone restores pancreatic exocrine secretion and abolishes hyperinsulinemia in CCK-8-stimulated eNOS(–/–) mice. Pioglitazone had no effect on basal bile-pancreatic protein output in WT mice (Fig. 1A) or eNOS(–/–) mice (Fig. 1B). Also, pioglitazone had no effect on net CCK-8-stimulated secretion in WT mice, the latter determined by subtracting basal from pooled peak CCK-8 protein output between 45 and 75 min (Fig. 1A). In contrast, in eNOS(–/–) mice, 20 mg/kg pioglitazone increased net CCK-8-stimulated output (P = not significant), and 50 mg/kg pioglitazone completely normalized net CCK-8-stimulated output (P < 0.005) (Fig. 1B). eNOS(–/–) and WT mice had similar baseline blood glucose (Fig. 2A) and serum insulin levels (Fig. 2B), both expressed as % saline-treated WT mice (that were assigned a value of 100%). Blood glucose values were not affected by eNOS genotype, CCK-8, or pioglitazone (Fig. 2A). Blood insulin increased 93% in the CCK-8- vs. saline-treated eNOS(–/–) group (P < 0.005), but this increase was prevented in pioglitazone-pretreated, CCK-8-treated eNOS(–/–) mice (Fig. 2B). Blood insulin was not affected by CCK-8 or pioglitazone in the WT groups. These data indicate that correction of hyperinsulinemia, a marker of insulin resistance, is associated with correction of reduced pancreatic exocrine secretion in eNOS(–/–) mice.


Figure 1
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  		Fig. 1. Dose response of pioglitazone (Pio) on in vivo cholecystokinin octapeptide (CCK-8)-stimulated pancreatic secretion in wild-type (WT; A) and eNOS(–/–) (B) mice. Pioglitazone (20 or 50 mg/kg) or vehicle was administered by daily gavage for 5 days. Bile-pancreatic protein output was collected every 15 min. Basal output was determined from 15–30 min, and intravenous CCK-8 (160 pmol·kg–1·h–1) was infused starting at 30 min and ending at 90 min. CCK-8-stimulated bile-pancreatic protein output is expressed as µg/min. Data points are shown as flat lines to reflect sample collection over 15-min intervals. Statistical analysis was performed on the CCK-8-dependent pancreatic protein secretion (CCK stimulated response, top, right), determined by subtracting basal from pooled peak secretagogue response between 45 and 75 min. Data shown are means ± SE (n = 10–14 mice/group). *P < 0.05 vs. vehicle-pretreated eNOS(–/–) mice. KO, eNOS(–/–).

 

Figure 2
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  		Fig. 2. Serum glucose and insulin homeostasis in eNOS(–/–) and WT mice during pancreatic secretion, evoked in vivo by CCK-8 (160 pmol·kg–1·h–1). Mice were pretreated with vehicle or pioglitazone (Pio; 50 mg/kg) for 5 days by oral gavage. A: glucose was measured enzymatically, and values were normalized to the saline-treated eNOS(+/+) group (assigned a value of 100%). B: serum insulin was measured by RIA, and values were normalized to the saline-treated eNOS(+/+) group (assigned a value of 100%). Columns are means ± SE (n = 3–4 mice/group). *P < 0.005 vs. the eNOS(+/+) control group.

 
mRNA for pancreatic digestive enzymes in eNOS(–/–) and WT pancreas. Pancreatic amylase 2, lipase 1, chymotrypsin B, proelastase 1, and trypsinogen gene expression were unaffected by eNOS genotype or the experimental conditions (Table 2), indicating that increased enzyme secretion in eNOS(–/–) mice in response to pioglitazone and then CCK-8 treatment was not due to increased enzyme synthesis.


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  		Table 2. Constitutive gene expression of pancreatic digestive enzymes

 
The inhibitory islet peptide somatostatin, which is suppressed by insulin, is not increased in CCK-8-treated eNOS(–/–) vs. WT groups. In contrast to observations in type 1 diabetes mellitus (36, 46, 49), circulating somatostatin levels (Table 3) and pancreatic tissue somatostatin mRNA expression (Table 3) were not increased in the insulin-resistant eNOS(–/–) mice (12, 18, 59) compared with WT mice, indicating that somatostatin is not responsible for reducing CCK-8-evoked in vivo pancreatic exocrine secretion in eNOS(–/–) mice.


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  		Table 3. Insulin and somatostatin tissue gene expression or serum protein levels

 
Hyperinsulinemia is unrelated to insulin transcription and secretion in eNOS(–/–) vs. WT mice in response to CCK-8. Insulin mRNA expression in pancreatic tissue was similar in eNOS(–/–) and WT mice under all experimental conditions during pancreatic secretion experiments (Table 3). CCK-8-treated eNOS(–/–) and WT mice (Table 4) had similar or lower (P = not significant) serum C-peptide values, expressed as % CCK-treated WT mice (assigned 100%). These findings indicate that the hyperinsulinemia of CCK-8-treated eNOS(–/–) mice (see Fig. 2B) is not associated with increased insulin transcription or secretion.


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  		Table 4. Serum C-peptide levels

 
Pioglitazone augments hepatic gene expression of a key insulin signaling intermediate in CCK-treated eNOS(–/–) vs. WT mice. Expression of mRNA for IRS-1 in hepatic tissue was similar in eNOS(–/–) and WT mice under all experimental conditions during pancreatic secretion experiments (Fig. 3A). In contrast, expression of mRNA for IRS-2 in hepatic tissue increased 125% in eNOS(–/–) mice pretreated with pioglitazone (vs. vehicle) and subsequently treated with CCK-8 (P < 0.001). It was 80% greater in eNOS(–/–) (vs. WT) mice receiving pioglitazone and then CCK-8 (P < 0.005; Fig. 3B). These observations are consistent with recent findings that pioglitazone preferentially increases IRS-2 but not IRS-1 mRNA expression in cultured human adipose tissue (63) and that IRS-2 plays a major role in maintaining insulin sensitivity (28, 32, 52, 76).


Figure 3
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  		Fig. 3. Hepatic gene expression of key insulin signaling intermediates. Insulin receptor substrate-1 (IRS-1; A) and IRS-2 (B) in eNOS(–/–) and WT mice during pancreatic secretion, evoked in vivo by CCK-8 (160 pmol·kg–1·h–1). Mice were pretreated with vehicle or pioglitazone (50 mg/kg) for 5 days by oral gavage. Whole liver mRNA gene expression for IRS-1 and IRS-2 was quantitated by real-time PCR. Data are expressed as fold change compared with WT, which was assigned a value of 1. Values were normalized to the Rb 23-kDa "housekeeping" gene. Columns are means ± SE (n = 6–7 mice/group). *P < 0.005 vs. the pioglitazone-pretreated, CCK-8-treated eNOS (+/+) group. **P < 0.001 vs. the CCK-8-treated eNOS(–/–) group.

 
Pioglitazone augments insulin sensitivity in eNOS(–/–) mice. Steady-state GIRs were highest in the vehicle-pretreated WT mice (95.0 ± 6.4 mg·kg–1·min–1) and lowest in the vehicle-pretreated eNOS mice (65.7 ± 7.0 mg·kg–1·min–1) (P < 0.05; Fig. 4). Pioglitazone increased the GIR in eNOS(–/–) mice (87.5 ± 4.7 mg·kg–1·min–1) to levels similar to that of WT mice (P = not significant).


Figure 4
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  		Fig. 4. Hyperinsulinemic-euglycemic clamp study. Mice were pretreated with vehicle or pioglitazone (50 mg/kg) for 5 days by oral gavage. Steady-state glucose levels were achieved by 50 min, and glucose infusion rates were then determined. Data shown are means ± SE (n = 4 mice/group). *P < 0.05 vs. other groups. Veh, vehicle-treated mice.

 
Maximal physiological secretory doses of CCK-8 do not increase PMBF in eNOS(–/–) or WT mice. Doses of CCK that are physiological and that produce a maximal secretory effect had no effect on PMBF in eNOS(–/–) or WT mice (Fig. 5, A and B), and pioglitazone did not affect PMBF in response to stimulation by physiological doses of CCK-8 (Fig. 5A). However, PMBF increased in WT mice by 1.6- and 2.4-fold in response to 400 and 1,600 pmol·kg–1·h–1 CCK-8, respectively (P < 0.05; Fig. 5B). Thus pancreatic secretory and circulatory events are not associated in mice, and pioglitazone's augmentation of pancreatic exocrine secretion in eNOS(–/–) mice is not explained by changes in PMBF.


Figure 5
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  		Fig. 5. Pancreatic microvascular blood flow (PMBF) increases during pancreatic secretion, evoked in vivo in mice by CCK-8. PMBF was measured using neutron-activated microspheres (see MATERIALS AND METHODS) and was expressed as ml·min–1·g–1 tissue. A: eNOS (–/–) mice were pretreated with vehicle or pioglitazone (50 mg/kg) for 5 days by oral gavage. PMBF was measured after a 45-min intravenous infusion of saline or CCK-8, respectively. Data represent means ± SE (n = 3 mice/group). B: in WT mice, PMBF was measured at baseline and in response to increasing CCK-8 doses (40, 160, 400, or 1,600 pmol·kg–1·h–1) infused intravenously for 45 min. Data represent means ± SE (n = 3 mice/group). *P < 0.05 vs. baseline.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
NO, a gaseous neurotransmitter, vasodilator, and paracrine regulator, regulates diverse but potentially related physiological processes in the pancreas, including pancreatic exocrine secretion (22, 29, 30, 42, 48, 75), insulin secretion (29, 53, 54, 58), and PMBF (30, 48). Our data support the hypothesis that exocrine pancreatic function couples at least partially with endocrine function and that eNOS and insulin sensitivity are critical to this relationship.

Insulin signaling maintains pancreatic exocrine function by potentiating postprandial, CCK- and secretin-stimulated pancreatic secretion by approximately fivefold in rats (3, 27, 36) and humans (11, 13, 20, 33, 50). Insulin likely acts on intrapancreatic targets rather than on vagal cholinergic pathways regulated by CCK (47) because insulinopenia reduces secretagogue-stimulated exocrine secretion in the isolated perfused rat pancreas and in the intact rat (27, 44), and bethanechol coadministration with secretagogues does not correct pancreatic secretion in diabetic patients (19). Why decreasing insulin signaling reduces pancreatic exocrine secretion remains unclear. Possibilities include 1) hyperglycemia, which induces insulin resistance (51) and reduces pancreatic exocrine secretion (38), 2) the loss of trophic effects on the exocrine pancreas (31), and 3) upregulation of the expression of inhibitory peptides, particularly somatostatin (35, 36, 46, 49).

However, none of these considerations adequately explains reduced pancreatic exocrine secretion in eNOS(–/–) mice. First, serum glucose was unaffected by eNOS genotype, CCK-8, or pioglitazone. Second, our data do not support the possibility that eNOS(–/–) mice have impaired digestive enzyme synthesis. eNOS(–/–) mice had similar levels of pancreatic mRNA for amylase and other digestive enzymes, corroborating our previous report that dispersed pancreatic acini from eNOS(–/–) mice had an intact, CCK-8-evoked, amylase secretory response (15). Finally, tissue and circulating levels of the inhibitory peptide somatostatin were similar in eNOS(–/–) vs. WT mice. Hence, these mechanisms associated with insulinopenia do not explain decreased pancreatic enzyme secretion in eNOS(–/–) mice.

Similar to insulinopenic conditions, nondiabetic models of insulin resistance also have reduced pancreatic exocrine function, observed in obese (5, 55, 72–74) and nonobese (12, 18, 43, 58, 59, 77) models. Adult obese rats (Zucker) (55) and mice (ob/ob) (73) have hyperinsulinemia (55, 73), reduced pancreatic amylase activity and mRNA expression (55, 73), and reduced pancreatic exocrine secretion evoked in vivo by CCK (5). Hyperinsulinemia develops from insulin hypersecretion to maintain euglycemia and/or from impaired hepatic clearance. Mirroring our observations, Shankar et al. (59) showed in eNOS(–/–) vs. WT mice that steady-state fasting blood insulin and glucose were not increased. Indeed, fasting insulin levels were somewhat lower in eNOS(–/–) vs. WT mice, but this was attributed to data variability. In response to CCK-8-evoked exocrine pancreatic secretion, we observed hyperinsulinemia in eNOS(–/–) mice, but again, insulin hypersecretion is likely not involved. CCK-8-treated eNOS(–/–) vs. WT mice have a similar or a lower serum C-peptide level, indicating that insulin secretion is not increased but may be decreased (P = not significant). This observation substantiates the known differential effect of NO on insulin secretion; only islet (nNOS) but not extraislet (eNOS) sources of NO stimulate insulin release (53, 54).

To explain hyperinsulinemia, we argue but do not directly prove that impaired insulin clearance is responsible for CCK-evoked hyperinsulinemia in eNOS(–/–) mice. First, eNOS(–/–) mice have hepatic (and peripheral) insulin resistance, based on hyperinsulinemic-euglycemic studies by Shankar et al. (59). Second, CCK increases circulating insulin but not C-peptide levels in eNOS(–/–) vs. WT mice, a discordance that occurs when hepatic clearance of insulin is impaired or renal clearance of C-peptide is accelerated. Finally, because IRS-2 is a central transducer of hepatic insulin signaling that maintains insulin sensitivity (28, 32, 52, 76), it is noteworthy that pioglitazone concurrently increases hepatic mRNA expression of IRS-2 greater than other groups (P < 0.005) and abolishes hyperinsulinemia in eNOS(–/–) mice, possibly by increasing hepatic clearance of insulin.

Of therapeutic importance relevant to all models of insulin resistance, PPAR-{gamma} agonists improve insulin resistance irrespective of cause (45, 57) even when beta-cell function is partially reduced (71). In obese rats, the PPAR-{gamma} agonist ciglitazone corrected hyperinsulinemia and restored glucose homeostasis, but also normalized pancreatic amylase mRNA expression (72, 74). Similarly, we report that pioglitazone treatment of nonobese eNOS(–/–) mice improves insulin sensitivity (clamp data) and abolishes CCK-evoked hyperinsulinemia, but also completely restores pancreatic exocrine secretion. Thus although the mechanisms remain obscure, these data indicate that insulin resistance and reduced pancreatic exocrine function are at least parallel phenomena, and they support our hypothesis that normal insulin sensitivity is necessary for normal pancreatic exocrine function.

An alternative explanation for reduced pancreatic exocrine secretion in eNOS(–/–) mice is decreased PMBF or islet blood flow (IBF). The hypothesis that decreasing PMBF reduces pancreatic exocrine secretion is at least 50 years old (70). eNOS activity preserves endothelial function and modulates vasodilation and blood perfusion of organs (40). We recently reported that eNOS gene deletion in mice prevents augmentation of PMBF during experimental acute pancreatitis but has no effect on baseline PMBF (17). Others showed that NOS inhibitors reduced PMBF and pancreatic protein secretion evoked by in vivo stimulation (30, 48), but the association between secretory and circulatory events was secretagogue dependent (48). In our study, we found that circulatory and exocrine secretory changes are dissociated events. First, PMBF in the eNOS(–/–) group did not increase over basal in response to CCK-8 and pioglitazone. Second, experiments using WT mice showed that PMBF increased over basal only with supramaximal (and not maximal secretory) CCK-8 doses, similar to a dose that inhibits pancreatic exocrine secretion (15), increases PMBF, and causes acute pancreatitis (17). These observations further clarify the relationship between exocrine pancreatic circulatory and secretory changes (see review in Ref. 15) and indicate a dissociation of these events, but do not exclude the possibility that increased O2 extraction occurs with stimulation of secretion (56), a consideration beyond the scope of the present study.

Conceivably, changes in IBF can influence coupling of pancreatic endocrine and exocrine function due to modulation of islet-acinar arterial cascades. Because fractional IBF is ~10% of total murine PMBF (25), measurements of PMBF are insensitive to IBF changes. Individual conditions may increase IBF preferentially (10, 68) or together with PMBF (2, 6, 7, 26, 64, 66, 69), because islet capillaries can feed or bypass the exocrine pancreas (25) and because hormonal, neural, and local vasoactive factors regulate IBF independently of exocrine PMBF (25). Increased IBF is associated with conditions of increased islet beta-cell stress (25) but is more closely associated with hyperglycemia than with insulin release (6, 8, 24, 41, 67) because short-term pharmacological normalization of hyperglycemia decreases islet capillary pressure in adult Goto-Kakizaki (GK) rats (9). These data indicate to us that CCK-8-treated, insulin-resistant eNOS(–/–) and WT mice have similar IBF (reflecting similar blood glucose), but we do not have direct evidence for this deduction, and examination of IBF is beyond the scope of the present study.

In summary, we show that pioglitazone reduces insulin resistance and restores pancreatic exocrine secretion in eNOS(–/–) mice but does not increase PMBF. These findings support the hypothesis that insulin resistance and impaired insulin signaling reduce CCK-8-stimulated pancreatic exocrine secretion in eNOS(–/–) mice. Our findings in eNOS(–/–) mice are clinically relevant for understanding normal pancreatic exocrine physiology, the pathogenesis of (pre-) diabetes (43, 77) and exocrine and endocrine pancreatic function in chronic pancreatitis. Alcohol and smoking are major risk factors for chronic pancreatitis and both uncouple eNOS from NO production and cause insulin resistance (1, 21, 37, 62, 65). Insulin-sensitizing PPAR-{gamma} agonists such as pioglitazone improve insulin resistance or diabetes [common in chronic pancreatitis (14)], avoid hypoglycemia associated with insulin therapy (34), and are potential novel treatments for reduced pancreatic exocrine secretion. Conceivably, earlier treatment may halt progression of chronic pancreatitis because insulin-sensitizing PPAR-{gamma} agonists ameliorate inflammation and fibrosis and even reverse established chronic pancreatitis in rat models (14). Hence, the relationships among eNOS and endothelial, endocrine, and exocrine function have potential importance for understanding pancreatic physiology and treating pancreatic disease.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants HL-070068 (R. C. Reddy), DK-48419 (C. Owyang), and DK-073298 (M. J. DiMagno), and the Michigan Gastrointestinal Peptide Center Grant P30-DK-34933. This work also utilized the Chemistry Core of the Michigan Diabetes Research and Training Center P60-DK-20572.


   ACKNOWLEDGMENTS
 
We thank David Oldfield for assistance with the somatostatin RIA, Becky Cai for assistance with the hyperinsulinemic clamp studies, and the Virchow Research Institute (Hyderabad, India) for pioglitazone.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. J. DiMagno, Dept. of Internal Medicine, Division of Gastroenterology and Hepatology, Univ. of Michigan School of Medicine, 1150 W. Medical Center Drive, 6520 MSRB 1, Ann Arbor, MI 48109 SPC 5682 (e-mail: mdimagno{at}umich.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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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