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Endocrinology, doi:10.1210/en.2005-0900
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Endocrinology Vol. 147, No. 3 1488-1497
Copyright © 2006 by The Endocrine Society

Attenuated Insulin Release and Storage in Fetal Sheep Pancreatic Islets with Intrauterine Growth Restriction

Sean W. Limesand, Paul J. Rozance, Gary O. Zerbe, John C. Hutton and William W. Hay, Jr.

Perinatal Research Center (S.W.L., P.J.R., W.W.H.), Department of Pediatrics, University of Colorado Health Sciences Center, Aurora, Colorado 80010; and Department of Preventative Medicine and Biometrics (G.O.Z.) and Barbara Davis Center for Childhood Diabetes (J.C.H.), University of Colorado Health Sciences Center, Denver, Colorado 80262

Address all correspondence and requests for reprints to: Sean W. Limesand, Department of Animal Sciences, University of Arizona, P.O. Box 210035, Tucson, Arizona 85721-0035. E-mail: Limesand{at}ag.arizona.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We determined in vivo and in vitro pancreatic islet insulin secretion and glucose metabolism in fetuses with intrauterine growth restriction (IUGR) caused by chronic placental insufficiency to identify functional deficits in the fetal pancreas that might be caused by nutrient restriction. Plasma insulin concentrations in the IUGR fetuses were 69% lower at baseline and 76% lower after glucose-stimulated insulin secretion (GSIS). Similar deficits were observed with arginine-stimulated insulin secretion. Fetal islets, immunopositive for insulin and glucagon, secreted insulin in response to increasing glucose and KCl concentrations. Insulin release as a fraction of total insulin content was greater in glucose-stimulated IUGR islets, but the mass of insulin released per IUGR islet was lower because of their 82% lower insulin content. A deficiency in islet glucose metabolism was found in the rate of islet glucose oxidation at maximal stimulatory glucose concentrations (11 mmol/liter). Thus, pancreatic islets from nutritionally deprived IUGR fetuses caused by chronic placental insufficiency have impaired insulin secretion caused by reduced glucose-stimulated glucose oxidation rates, insulin biosynthesis, and insulin content. This impaired GSIS occurs despite an increased fractional rate of insulin release that results from a greater proportion of releasable insulin as a result of lower insulin stores. Because this animal model recapitulates the human pathology of chronic placental insufficiency and IUGR, the ß-cell GSIS dysfunction in this model might indicate mechanisms that are developmentally adaptive for fetal survival but in later life might predispose offspring to adult-onset diabetes that has been previously associated with IUGR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NUMEROUS HUMAN EPIDEMIOLOGICAL studies have shown that prenatal nutritional insufficiency is associated with later life metabolic disorders, such as obesity, insulin resistance, overt type 2 diabetes mellitus, and hypertension (1, 2, 3). Mechanisms that might be causally responsible for this association have not been clearly defined, but glucose ß-cell deficiency is a component of type 2 diabetes (4). A specific example of inadequate ß-cell function leading to diabetes is the autosomal dominant genetic mutation of transcription factors that regulate ß-cell function identified as maturity-onset diabetes of the young (reviewed in Ref.5). Therefore, one possible mechanism for adult type 2 diabetes is that certain organs, for example the pancreas, are differentially more inhibited in their development and function by nutrient deficiency in the fetus. Such nutrient deficiency also causes generalized fetal growth restriction [intrauterine growth restriction (IUGR)] (6). Pancreatic ß-cells secrete the anabolic hormone, insulin, in response to nutrient changes during the second half of gestation (7), likely coordinating fetal growth rate with fetal nutrient supply (8, 9) and making the ß-cell a potential target for nutritional adaptation in utero (2).

An ovine model of IUGR induced by placental insufficiency replicates many of the complications found in human pregnancies with marked fetal growth restriction (10, 11, 12, 13, 14, 15, 16, 17, 18), including clinical abnormalities in umbilical artery Doppler velocimetry (19), which is associated with fetal hypoglycemia and hypoxia (20, 21). In sheep fetuses from this model of IUGR, we previously showed a 76% reduction in ß-cell mass in fetuses near term (0.9 of gestation) as a result of decreased rates of ß-cell proliferation and neoformation (18). Similarly, less than normal amounts of pancreatic endocrine tissue were found in a cohort of human fetuses with severe IUGR (<1.5 kg at term gestation) (22). Examples of altered pancreatic endocrine mass also have been found in rodent models of IUGR (23, 24, 25), and nutrient-restricted pregnant rats produce offspring with deficits in ß-cell mass (26, 27, 28, 29). These similar observations in human, rat, and sheep fetuses with IUGR indicate that reduced ß-cell mass in the fetus is a common mammalian response to nutrient restriction in fetuses with placental insufficiency and IUGR.

In this study, we examined two critical functions of fetal islets, insulin secretion and glucose metabolism, to determine how placental insufficiency-induced nutrient deprivation impairs fetal ß-cell development and function. Insulin secretion in fetal sheep with placental insufficiency and IUGR has not been determined, limiting the capacity to understand the impact of nutrient deficiencies on fetal ß-cell function. Deficiencies in ß-cell function occur in human IUGR fetuses with hypoglycemia and hypoxia, both general outcomes of placental insufficiency and IUGR in all species (30). After birth, ß-cell dysfunction has been associated with type 2 diabetes mellitus in adult humans who had low birth weights (31, 32, 33, 34). Other mammalian models of IUGR, such as the glucose-deprived fetal sheep (35) or the pregnant rat fed a low-protein diet (23), have reduced fetal insulin secretion. For the glucose-deprived sheep fetus, glucose deprivation during the final 20% of gestation negatively influences fetal insulin secretion in utero, which persists after the insult is corrected by returning the fetus to euglycemia for 5 d (35). Similarly, offspring from rat dams fed a low-protein diet during pregnancy and lactation were unable to respond to an oral glucose challenge at 70 d of age. Also, low-birth-weight mice from undernourished dams exhibit dysregulation of insulin secretion later in life (23, 25, 36). Taken together, these previous data from humans, rodents, and sheep indicate that fetal and early neonatal nutritional deficits negatively impact ß-cell function in utero and continue into adulthood, at least for humans and rodent models, but these particular animal models of IUGR only partially replicate complications observed in human IUGR pregnancies. Therefore, we conducted the present studies with the model of placental insufficiency-induced IUGR in fetal sheep, analyzed ß-cell function in the fetus in vivo and in isolated fetal islets of IUGR fetuses, and compared the results with normal fetuses. We identified impaired islet glucose oxidation in response to high glucose concentrations, decreased islet insulin synthesis and content, and total insulin secretion in the IUGR fetuses and their isolated pancreatic islets, despite increased fractional rates of islet insulin secretion (i.e. the islets were more efficient at secreting stored insulin, but this did not compensate for the reduced islet insulin content, thus resulting in impaired overall islet and pancreatic insulin secretion). These results indicate specific metabolic defects in pancreatic islets from nutrient-restricted, IUGR fetuses that limit overall insulin production and thereby insulin secretion independently of their reduced ß-cell replication and the capacity of each islet to secrete stored insulin in response to glucose stimulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Ovine model of IUGR
Cheviot ewes carrying singleton pregnancies were purchased from Ovis (Canton, SD). All animal procedures were conducted at the Perinatal Research laboratories at the University of Colorado Health Sciences Center (UCHSC) with approval of the Institutional Animal Care and Use Committee. The Perinatal Research laboratories and UCHSC are accredited for animal research by the U.S. Department of Agriculture, the National Institutes of Health, and the American Association for Accreditation of Laboratory Animal Care. IUGR fetuses were generated by exposing pregnant ewes to elevated ambient temperatures with a diurnal cycle of 40 C for 12 h and 35 C for 12 h, from 39 ± 2 d gestation age (dGA) (mean ± SD) until 96 ± 5 dGA as previously described (11, 18, 37). Control fetuses were from pair-fed, gestational age-matched, pregnant ewes maintained at 25 C. Twenty-six pregnant ewes were divided into the two treatment groups. All of the control fetuses (n = 13) survived treatment, surgical procedures, and in vivo studies. Ewes exposed to hyperthermia exhibited an increased abortion rate (30% loss), and two animals did not survive fetal surgery, resulting in seven IUGR fetuses with completed in vivo experiments.

Surgical preparation
At approximately 125 dGA, indwelling polyvinyl catheters were surgically placed in the fetus for blood sampling and glucose infusion as previously described (35). Fetal catheters for blood sampling were placed in the abdominal aorta via hind limb pedal arteries. Fetal catheters for infusion were placed in the femoral veins via the saphenous veins. Maternal catheters were placed in the femoral artery for sampling and femoral vein for infusion. All catheters were tunneled sc to the ewe’s flank, exteriorized through a skin incision, and kept in a plastic pouch sutured to the ewe’s skin. Ewes were allowed to recover for 5–7 d before physiological studies were conducted to determine fetal insulin secretion responsiveness to iv infusions of glucose and arginine.

Glucose- and arginine-simulated insulin secretion (GSIS and ASIS)
A square-wave hyperglycemic clamp was used to determine insulin secretion in response to glucose as previously reported (35). Briefly, a continuous transfusion of maternal blood into the fetus (12 ml/h) was started 45 min before baseline sampling and maintained for the duration of the study to compensate for blood collection. All sample times are presented relative to the start of the fetal glucose bolus and continuous infusion. Whole blood collected in syringes lined with EDTA (Sigma-Aldrich, Inc., St. Louis, MO) was centrifuged (14,000 x g) for 3 min at 4 C. Plasma was aspirated from the pelleted red blood cells and stored at –70 C for hormone and amino acid measurements. Baseline plasma glucose and insulin concentrations were determined at –35, –25, –15, and –5 min. Blood gas and oxygen saturations were measured in blood collected in syringes lined with heparin (Elkins-Sinn, Inc., Cherry Hill, NJ). The hyperglycemic clamp was initiated with a dextrose in water bolus (230 ± 22 mg/kg) into the fetus followed by a constant infusion of 33% dextrose in water to increase and maintain fetal arterial plasma concentration at 2.4 mmol/liter, which in preliminary experiments using various glucose concentrations (2–11 mmol/liter) was found to produce maximal insulin concentrations in fetal sheep. Fetal arterial plasma samples were collected at 5, 10, 15, 20, 30, 45, 60, 75, 90, and 105 min. During basal (–45 to 0 min) and steady-state hyperglycemic clamp (60–105 min) periods, fetal blood was collected in a final concentration of 0.5 mM EDTA and 0.33 mM reduced glutathione from nine controls and seven IUGR fetuses for measurement of fetal plasma catecholamine concentration. After the 105-min hyperglycemic sample, a bolus of arginine (0.5 mmol/kg estimated fetal weight) in 5 ml of 0.4 M sodium acetate and 0.9% sodium chloride was injected (4 min) into the fetal circulation, and plasma samples were collected at 5, 10, 20, and 30 min for subsequent measurement of insulin concentrations.

Biochemical analysis
Blood oxygen saturation and hemoglobin concentrations were measured with an OSM III hemoximeter (Radiometer, Copenhagen, Denmark). Oxygen content was determined as the product of oxygen saturation and oxygen capacity. The pH, pO2, and hematocrit were determined for 39.1 C using an ABL 520 blood gas analyzer (Radiometer). Plasma glucose and lactate concentrations were measured immediately using a YSI model 2700 select biochemistry analyzer (Yellow Springs Instruments, Yellow Springs, OH). Arterial amino acid concentrations were measured using a Dionex 300 model 4500 amino acid analyzer (Dionex, Sunnyvale, CA) after deproteinization with sulfosalicyclic acid.

Plasma insulin concentrations were measured by an ovine insulin ELISA (Alpco Diagnostics, Windham, NH). Other hormones measured included glucagon with a glucagon RIA (Linco Research Inc., St. Charles, MO), cortisol with a salivary cortisol ELISA (Alpco Diagnostics), and catecholamines by HPLC using Dionex methodology.

Fetal pancreatic islet isolation
The ewes and fetuses were anesthetized with ketamine (4.4 mg/kg) and diazepam (0.11 mg/kg) given iv. After a hysterotomy, the fetus was removed, blotted dry, and weighed. The ewe was then killed with iv concentrated sodium pentobarbital (10 ml, Sleepaway; Fort Dodge Animal Health, Fort Dodge, IA). Islets were obtained from the fetal pancreas with a retrograde perfusion of digestive solution into the pancreatic ducts. The digestion solution was Liberase BlendZyme III (0.175 mg/ml; Roche, Indianapolis, IN) in Krebs Ringer buffer (KRB; 118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, KH2PO4, 25 mM NaHCO3, pH 7.3) supplemented with 0.2% DNase I (Roche). The pancreatic duct at the duodenum was clamped, and a 20- or 22-gauge polyvinyl catheter was inserted into the common bile duct at the junction of the hepatic branch. Digestion solution was infused until the pancreas was fully distended. The pancreas was dissected free, placed into an additional 20 ml of digestion buffer, and incubated at 37 C for 35–45 min until digested. The digested tissue was diluted with 3–5 vol of KRB containing 0.25% BSA by allowing the cell clusters to settle for 10 min. Islets were purified over a discontinuous gradient of 10 ml of a 2:1 solution of Histopaque (1.119 g/ml; Sigma) and KRB/BSA and centrifuged at 800 x g for 20 min. Cell clusters were removed from the interface and rinsed once in KRB/BSA media with a 1-min centrifugation (800 x g). Islets were hand picked, and purity was confirmed by fluorescent immunostaining for insulin and glucagon in initial preparations as previously reported (18). Islets were cultured overnight at 37 C in 95% O2/5% CO2 in RPMI 1640 media containing 2.8 mmol/liter glucose (Sigma) supplemented with 1% fetal bovine serum and 1x penicillin-streptomycin-neomycin (50 U, 50 µg, and100 µg, respectively) solution (Sigma).

Static islet incubation
Before static incubations to determine insulin secretion, fetal islets were washed twice in KRB/BSA media and once in KRB/BSA media supplemented with 10 µmol/liter Forskolin (Sigma-Aldrich, Inc.) equilibrated to 37 C and 95% O2/5% CO2. Forskolin was added to the incubations to activate adenylate cyclase and augment insulin release. Ten fetal islets from nine control and seven IUGR fetuses were hand picked and placed into a 1.5-ml tube (n ≥ 3 per incubation condition), and static incubations were performed for 1 h in KRB/BSA/Forskolin media containing 0, 1.1, or 11.1 mmol/liter glucose concentration or 30 mmol/liter KCl without glucose. Test islet incubations on ice or in the presence of 10 µmol/liter epinephrine at 37 C were performed in the presence of 11 mmol/liter glucose to distinguish cellular breakdown and intact islet responsiveness for each islet harvest. After 1 h at 37 C, islets were pelleted by a 3-min centrifugation at 800 x g, and media were aspirated and frozen for insulin determination. Insulin was acid-ethanol extracted (1 mol/liter HCl/70% ethanol) at –20 C for 24 h from the fetal islets and cellular debris removed by centrifugation (20 min at 15,000 x g). Islet DNA content was determined by resuspending cellular debris in 10 mmol/liter Tris-HCl, 1 mmol/liter EDTA (pH 7.5) containing 0.1% SDS using the PicoGreen dsDNA Quantitation Kit (Molecular Probes Inc., Eugene, OR). Insulin concentrations were determined with ovine insulin ELISA on dilutions of the incubation media and acid-ethanol extract to determine insulin released (media) and total islet insulin content (media plus islet extract). Insulin secretion was normalized by islet number (nanograms per islet per hour) or as the fraction released of the total islet insulin content.

Islets from seven control and seven IUGR fetuses were used to measure islet glucose utilization rate. Islet glucose utilization rate was determined for 25 islets per fetus in a Nunc CryoTube (1 ml; Nalge Nunc International, Naperville, IL) attached with epoxy inside a 20-ml plastic scintillation vial (Beckman Instruments Inc, Brea, CA) that contained 0.5 ml H2O. Islets were incubated for 1 h in 95% O2/5% CO2 at 37 C in 150 µl KRB/BSA supplemented with either 1.1 or 11.1 mmol/liter glucose and 6 or 10 µCi/ml [5-3H]D-glucose (New England Nucleotides, Perkin-Elmer Life Sciences, Inc., Boston, MA). Four or more replicates for each incubation condition per fetus were used to determine 3H2O formation from [5-3H]D-glucose, and an identical number of incubations with no islets was performed to obtain background levels. After the incubation, the reaction was stopped with 100 µl of 1 mol/liter HCl, and the 3H2O produced was allowed to equilibrate with the H2O in the scintillation vial for 18 h at 37 C. The recovery efficiency of 3H2O was determined for each experiment by determining the proportion of 3H2O recovered for a known amount of 3H2O added. The inner tube containing the fetal islets was removed, BioSafe II (RPI, Mount Prospect, IL) scintillation fluid was added, and the amount of 3H2O was determined by liquid scintillation counting. Specific activity of glucose (dpm/mole) for the KRB/BSA incubation media was determined using the YSI select biochemistry analyzer II and the Packard Tri-Carb 2300TR liquid scintillation analyzer (PerkinElmer, Boston, MA). The rate of islet glucose utilization (moles per islet per hour) was calculated by subtracting the blank dpm from the sample dpm, correcting for the recovery efficiency, dividing by the glucose-specific activity, and dividing by the hours of incubation.

Islet glucose oxidation rate was determined for 25 islets from each fetus (n = 7 fetuses per group) placed in a 1-ml Nunc CryoTube affixed with epoxy inside a 20-ml glass scintillation vial (n ≥ 4 per incubation condition) and sealed with a Suba-Seal Rubber Septa (Sigma-Aldrich). Islets were incubated in KRB/BSA containing D-glucose (1.1 or 11 mmol/liter) and [U-14C]D-glucose (8 or 16 µCi/ml, respectively) preequilibrated in 95% O2/5% CO2 atmosphere at 37 C for 2 h. Blank reactions were included to determine background levels. Reaction vials were quickly cooled and the reaction stopped with 100 µl of 1 mol/liter HCl. Solvable (0.5 ml; PerkinElmer) was added to the base of the scintillation vial to trap the CO2. Vials were incubated at 37 C with gentle agitation for 90 min. The rubber stopper and reaction tube were removed, Ultima Gold scintillation fluid (PerkinElmer) was added, and captured 14CO2 dpm was determined with a Packard Tri-Carb 2300TR liquid scintillation analyzer. The specific activity of glucose in the KRB/BSA was determined as described for the utilization. The rate of glucose oxidized (moles per islet per hour) was calculated by subtracting the blank dpm from the islet incubated dpm, dividing by the specific activity for glucose, and dividing by the time of the incubation.

Statistical analysis
Insulin concentrations during the GSIS basal and hyperglycemic steady states were subjected to a repeated-measures ANOVA with both fixed effects for treatment group and draw time and random effects for sheep (SAS Proc MIXED) (38). Statistical analysis for ASIS was an ANOVA that accounted for the fetal sheep as a random effect (SAS Proc MIXED) (38). Biochemical, hematological, and hormone measurements were analyzed by ANOVA and individual means were compared by Student t test (least significant difference) (38). Similar analysis was used to distinguish differences between basal and hyperglycemic periods of the GSIS study. The hierarchical nonlinear stochastic parameter regression model, Sij = {delta} + {alpha}Gij/(ß + Gij) was fit to the fraction of insulin secretion (Sij) vs. glucose concentrations (Gij) for fetus i in glucose concentration j. Insulin release from isolated fetal sheep islets was analyzed using the Mixed Procedure (38) with insulin secretion as the dependent variable and fetal sheep as a random effect.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experimental parameters during treatment
Before entering the environmental chamber, the average core body temperatures for the control and IUGR animals were 38.8 ± 0.2 and 39.0 ± 0.2 C, respectively. Ten days after entering the environmental chamber, the ewes’ average core body temperature increased significantly (P < 0.01) to 39.5 ± 0.1 C, and for the duration in the environmental chamber their mean core body temperature was 39.7 ± 0.1 C, significantly greater than the control ewes’ average, 38.8 ± 0.1 C. After removing the ewes from the environmental chamber to the control environment at 96 ± 5 dGA for surgery and physiological studies, their average body temperature decreased to 38.9 ± 0.1 C, not different from the control ewes at 38.8 ± 0.1 C.

Control ewes were pair-fed in accord with ewes in the environmental chamber; however, control ewes were not discarded if their pair-fed companion aborted, in which case these control ewes were fed according to a previous history of feed intake for ewes of the same gestational age in the environmental chamber. In the environmental chamber, the average feed intake was 1.2 ± 0.1 kg/d, and for the same period the control ewes received 1.0 ± 0.1 kg/d (P = not significant). After removing ewes from the environmental chamber, pair feeding continued with an average food intake of 1.4 ± 0.1 kg/d for the IUGR and control animals. After surgery, feed consumption was not different between control ewes (1.2 ± 0.4 kg/d) and IUGR ewes (1.0 ± 0.2 kg/d).

Maternal and fetal hematological value
Hematological variables for the mother and fetus during the basal period of the GSIS study are shown in Tables 1Go and 2Go as mean ± SEM. In addition to fetal plasma glucose concentrations, pO2 and O2 content were lower in the IUGR fetuses, but fetal pH and hematocrit were not different. Maternal hematological values (Table 1Go) were not different between the two groups. Increased maternal-fetal arterial gradients of blood O2 content, pO2, and plasma glucose concentration were found for IUGR fetuses (Table 1Go), which are indicative of reduced uteroplacental and fetal uptakes of glucose and oxygen from the maternal circulation (10, 11).


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  		TABLE 1. Maternal and fetal arterial hematological parameters for control and IUGR treatment groups

 

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  		TABLE 2. Fetal plasma glucose and insulin concentrations during the GSIS clamp periods

 
Arterial plasma concentrations for two amino acids, arginine (control 107.5 ± 7.6 µmol/liter vs. IUGR 52.0 ± 7.1 µmol/liter) and serine (control 501.1 ± 41.2 µmol/liter vs. IUGR 286.3 ± 63.7 µmol/liter), were decreased (P < 0.05) in IUGR fetuses. In contrast, arterial plasma concentrations were greater (P < 0.05) in IUGR fetuses for taurine (control 19.9 ± 4.8 µmol/liter vs. IUGR 118.1 ± 28.2 µmol/liter), proline (control 115.5 ± 6.2 µmol/liter vs. IUGR 193.7 ± 20.1 µmol/liter), alanine (control 275.8 ± 16.7 µmol/liter vs. IUGR 420.3 ± 46.5 µmol/liter), and cystine (control 7.4 ± 0.9 µmol/liter vs. IUGR 10.8 ± 1.2 µmol/liter).

Fetal insulin secretion response to glucose
The mean plasma glucose concentration for IUGR fetuses at baseline was 48% lower (P < 0.001) than controls (Table 2Go and Fig. 1AGo). Similarly, the mean plasma insulin concentration for IUGR fetuses during the baseline measurements was 69% lower (P < 0.05) than the control mean (Table 2Go and Fig. 1BGo). The dextrose bolus administered to increase glucose concentrations to approximately 2.4 mmol/liter was 230 ± 22 mg/kg in control fetuses and 445 ± 39 mg/kg in IUGR fetuses (P < 0.001). The continuous dextrose infusion to maintain hyperglycemia at 2.4 mmol/liter was not different between the control group (8.8 ± 0.5 mg·min–1·kg–1) and the IUGR group (10.1 ± 0.8 mg·min–1·kg–1) Although plasma glucose concentrations were not different during the hyperglycemic clamp (Fig. 1AGo and Table 2Go), the mean plasma insulin concentration for the IUGR fetuses was 76% lower (P < 0.01) than the control fetuses during the hyperglycemic clamp (Table 2Go and Fig. 1BGo).


Figure 1
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  		FIG. 1. Fetal GSIS. Fetal plasma glucose (A) and insulin (B) concentrations determined for the in vivo GSIS study are presented as the mean ± SEM for control (n = 13) and IUGR (n = 7) fetuses. Administration of the glucose bolus and infusion were started at 0 min. Statistical analyses are presented in Results.

 
Fetal plasma insulin accumulation was attenuated in IUGR fetuses, showing a decreased capacity of pancreatic ß-cells to increase circulating plasma insulin concentration in response to glucose. The change in mean plasma insulin concentration for the control fetuses was 0.33 ± 0.02 ng/ml, a significant increase (P < 0.01) over basal concentrations, whereas the plasma insulin concentration in the IUGR fetuses increased only marginally by 0.06 ± 0.03 ng/ml (P < 0.05), significantly less (P < 0.01) than controls. The GSIS shows that the amount of insulin released in response to glucose was significantly diminished in vivo for the IUGR fetuses compared with control fetuses, resulting in significantly lower plasma insulin concentrations.

The mean plasma insulin-to-glucose concentration ratio during the basal period was not different between IUGR and control fetuses (P = 0.19; Table 2Go). During the hyperglycemic period of the GSIS study, however, this ratio was significantly lower (P < 0.05) in IUGR fetuses (Table 2Go). Because the rates of glucose infusion were not different between control and IUGR fetuses, these data indicate that either insulin sensitivity or glucose clearance, or both, was enhanced in the IUGR fetuses.

ASIS
Insulin concentrations after the administration of arginine reached maximum values after 5 min in all fetuses in both treatment groups (Fig. 2Go). Mean maximal insulin concentration in response to arginine was significantly (P < 0.01) less for IUGR fetuses (0.8 ± 0.1 ng/ml) compared with the control mean plasma insulin concentration (2.9 ± 0.5 ng/ml). The change in insulin concentrations in response to arginine was lower (P < 0.001) in the IUGR fetuses compared with control fetuses, 0.6 vs. 2.3 ng/ml, respectively.


Figure 2
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  		FIG. 2. ASIS. Mean fetal plasma insulin concentrations (ng/ml, y-axis) after the arginine bolus (minutes, x-axis) are presented for control (n = 13) and IUGR (n = 7) fetuses.

 
Glucagon, cortisol, and catecholamine concentrations
Glucagon concentrations in the fetal plasma were significantly (P < 0.05) greater in IUGR fetuses (79.0 ± 18.3 pg/ml) compared with control fetuses (39.8 ± 7.3 pg/ml) at the baseline sampling period. A similar increase in plasma glucagon concentrations was observed during the hyperglycemic period of the GSIS with IUGR fetuses at 84.2 ± 23.6 pg/ml and control fetuses at 37.5 ± 6.9 pg/ml. Stimulation of the {alpha}-cells with the administration of arginine elicited glucagon release into the fetal circulation, but the average augmentation was not different between the control and IUGR fetuses, 2.2 ± 0.5-fold and 2.3 ± 0.5-fold, respectively, demonstrating no deleterious effects of IUGR on fetal {alpha}-cell responsiveness to arginine.

Plasma cortisol concentrations for the control and IUGR fetuses were not different (P = 0.4), 4.4 ± 0.5 vs. 8.6 ± 4.2 ng/ml, respectively. The high variation for the IUGR fetuses was attributed to a single fetus with a plasma cortisol concentration of 37.6 ng/ml. The gestational age of this fetus was not different, 135 dGA, but this fetus had the lowest oxygen content (0.7 mmol/liter) and the highest plasma lactate concentration (8.15 mmol/liter) of all of the fetuses in either group. Exclusion of this fetus’s value from the analysis showed a mean plasma cortisol concentration in IUGR fetuses of 4.4 ± 1.0 ng/ml, not different from control fetuses.

The mean plasma norepinephrine concentration in the IUGR fetuses was elevated 4-fold (P < 0.001), 2564 ± 344 pg/ml compared with 636 ± 115 pg/ml in control fetuses. The plasma norepinephrine concentrations for all fetuses measured were negatively associated with the total arterial blood oxygen content (R2 = 0.84; Fig. 3Go).


Figure 3
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  		FIG. 3. Norepinephrine vs. oxygen content in fetal sheep. Comparison of fetal plasma norepinephrine (pg/ml, y-axis) and fetal blood oxygen content (mmol/liter, x-axis) are presented for control ({square}, n = 9) and IUGR (bullet, n = 7) fetuses. The regression line (y = –1007.3x + 4117.3; R2 = 0.84) for all fetuses is depicted on the graph.

 
Fetal and organ weights at autopsy
After the in vivo evaluation of ß-cell function, the fetuses were returned to basal conditions and then euthanized and autopsied (Table 3Go) between 129 and 137 dGA. The ratio of males to females was not different between treatment groups, 62% males in the control group and 71% males in the IUGR group. Significant reductions (P < 0.001) in fetal weight (56%), placental weight (59%), and crown-rump length (23%) were found in IUGR fetuses (Table 3Go). The liver, heart, lung, and kidney weights were significantly less in IUGR fetuses, but their fractions of total fetal mass were unchanged between treatments. Average brain weights were significantly less (P < 0.001) for IUGR fetuses compared with control fetuses, 32.0 ± 2.2 vs. 42.3 ± 1.1 g. As a fraction of total body weight, however, the brain made up a greater portion of the IUGR fetal weight (Table 3Go), and the brain to liver ratio was 1.6-fold higher for IUGR fetuses, 0.70 ± 0.06, compared with controls, 0.42 ± 0.02. The spleen and carcass weights were affected to a greater extent by placental insufficiency than the other organs; their fractions of fetal weight were significantly lower in IUGR fetuses (Table 3Go).


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  		TABLE 3. Postmortem data and proportion of organ weight to fetal weight

 
Islet responsiveness in vitro
Endocrine cell clusters isolated from the fetal pancreases were immunostained for insulin and glucagon to confirm purification of islets of Langerhans (supplemental Fig. 1A, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Hand-picked clusters contained cells that expressed insulin or glucagon. The fraction of insulin released in the absence of glucose in the incubation medium was 0.06 and reached a mean maximum level of 0.14 at medium glucose concentrations greater than 7.5 mmol/liter (supplemental Fig. 1B). The half-maximal insulin release occurred at a glucose concentration of 2.7 mmol/liter. In addition, insulin release with 11 mmol/liter glucose was inhibited by low temperatures (ice) or 10 µmol/liter epinephrine (data not shown). These data show that purified fetal sheep islets secrete insulin in response to glucose and function in vitro as they do in vivo.

In vitro islet insulin release was determined for control and IUGR fetuses. The fraction of total insulin released in 11 mmol/liter glucose was significantly (P < 0.05) greater than basal for control and IUGR islets (Fig. 4AGo) and was inhibited with low temperature and 10 µmol/liter epinephrine. Furthermore, incubating islets in 30 mmol/liter potassium chloride (KCl) to depolarize the endocrine cells significantly augmented (P < 0.001) the fraction of insulin release compared with basal conditions in both treatment groups. The IUGR islets secreted a greater (P < 0.01) portion of their total insulin in the presence of 11 mmol/liter glucose or 30 mmol/liter KCl (Fig. 4AGo). However, the islet insulin content of the IUGR fetuses was 82% less than the control fetal islet insulin content (22.3 ± 2.7 vs. 3.9 ± 1.1 ng insulin per islet in IUGR fetuses; P < 0.001; Fig. 4BGo). An 83% reduction for insulin content in IUGR islets also was found when islet insulin content was normalized by DNA content, 30.4 ± 4.5 vs. 5.2 ± 2.1 ng insulin/ng DNA, indicating that a lower islet cell number was not the cause for less insulin content. Therefore, the amount of insulin released per islet from the IUGR fetal islets was significantly less than from control fetal islets incubated in 11 mmol/liter glucose or 30 mmol/liter KCl (Fig. 4CGo). These data indicate that the response of IUGR islets to secrete insulin was not impaired; rather, the major limitation for insulin release was because of less insulin stored in the IUGR islet.


Figure 4
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  		FIG. 4. Comparison of control and IUGR islets. Fetal sheep islets isolated from control (white bars, n = 9) and IUGR (black bars, n = 7) treatment groups were evaluated for the fraction of insulin released (A), insulin content (B), and nanograms of insulin secreted per islet (C). *, P < 0.05, differences between treatment groups for the incubation condition (A and C) and difference between control and IUGR fetal islets insulin content (B).

 
In addition to insulin secretion, glucose metabolism was determined to evaluate islet function. Glucose utilization rates of control fetal islets increased with increased incubation medium glucose concentration (11 vs. 1.1 mmol/liter; P < 0.01; Fig. 5AGo). Islet glucose utilization rates were not different between control and IUGR fetal islets in either 1.1 mmol/liter (equal to normal physiological values) or 11 mmol/liter (~10-fold greater than normal physiological values in fetal sheep) glucose incubations (Fig. 5AGo). Glucose oxidation rates in the islets were similar for IUGR and control islets at 1.1 mmol/liter glucose concentrations but were only significantly increased for control with 11 mmol/liter glucose (P < 0.05; Fig. 5BGo).


Figure 5
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  		FIG. 5. Fetal sheep islet glucose utilization and oxidation. The rate of glucose utilization (A) and rate of glucose oxidation (B) for control (white bars, n = 7) and IUGR (black bars, n = 7) fetal islets were determined for four or more replicates at 1.1 mmol/liter glucose (normal ovine fetal glucose concentration) or 11.1 mmol/liter glucose (stimulated concentrations). A significant reduction (*, P < 0.05) in the rate of glucose oxidation was found for IUGR islets at 11.1 mmol/liter glucose, but no difference was identified at normal glucose concentrations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we compared insulin secretion in the fetus and isolated fetal pancreatic islets in normal (control) fetuses and IUGR fetuses produced by a model of placental insufficiency. We showed that fetal plasma insulin concentrations were significantly lower in IUGR fetuses during basal and hyperglycemic conditions and had smaller increases in insulin concentrations in response to secretagogues. The primary cause of the reduced insulin release in response to an increase in glucose concentration in the IUGR islets was their decreased insulin content, which was not compensated for by their greater fractional insulin release. Furthermore, the rate of islet glucose utilization was not different between treatment groups, indicating that glucose entered glycolysis at the same rate in both control and IUGR islets; however, the rate of glucose oxidation was decreased in the IUGR islets at very high glucose concentrations, which indicates impairment of islet oxidative glucose metabolism. These observations show that decreased islet insulin content, in addition to reduced ß-cell mass, was responsible for lower insulin secretion in IUGR fetuses. The pancreatic islet/ß-cell dysfunction demonstrated in the IUGR fetuses indicates discordance between islet responsiveness, which was increased, and ß-cell insulin biosynthesis, which was diminished.

Fetuses with placental insufficiency-induced IUGR exhibit greater disparity in ß-cell dysfunction compared with fetuses made chronically hypoglycemic during the final 20% of gestation. Fetuses with placental insufficiency have a larger reduction in fetal plasma insulin accumulation. The change in insulin in response to glucose is 82% lower for placental insufficiency-induced IUGR fetuses compared with control fetuses (Fig. 1Go), almost twice as great a reduction in GSIS as in the chronically hypoglycemic fetuses (45% lower) (35, 39). The fetal plasma glucose concentrations during the basal period of the GSIS were equivalent between placental insufficiency and chronically hypoglycemic fetuses (Table 2Go) (35), indicating additional factors, such as gestational timing of the nutritional deficiency, duration of the nutrient deprivation, or type of nutrient restriction, impair different processes regulating insulin secretion in the fetal ß-cell.

The discrepancy between insulin secretion in control and placental insufficiency-induced IUGR fetuses can be explained partially by a significant reduction in ß-cell mass of 76% (18). We previously determined that pancreatic ß-cell area was 42% lower and pancreas weight was 59% less for this model of IUGR at the same gestation age (133 dGA) because of lower rates of ß-cell mitosis and neoformation (18).

It also is possible, however, that the significantly lower fetal blood oxygen content contributed to the lower rate of insulin secretion in the IUGR fetuses in vivo. This hypoxemic effect could be caused directly by the reduced amount of oxygen or indirectly by the increased norepinephrine concentrations, because catecholamines are known to impair insulin secretion through {alpha}2-adrenergic receptors in fetal sheep ß-cells (40, 41, 42). We observed a negative association between oxygen content and norepinephrine concentrations in the present study in the IUGR fetuses (Fig. 3Go), likely suppressing insulin release with respect to fetal hypoxia. The fact that insulin release was attenuated in the IUGR fetal islets in vitro in the absence of catecholamines in the incubation medium indicates that the attenuated in vivo insulin secretion in the IUGR fetuses was in part independent of catecholamine inhibition of insulin secretion. This persistence of attenuated insulin secretion by the IUGR islets in vitro, therefore, must represent different pathways of regulation of insulin secretion.

Arginine has been shown to depolarize the ß-cell membrane to elicit insulin release rather than being metabolized, like glucose, to produce ATP (43, 44, 45, 46). ASIS was significantly less in the IUGR fetuses, by 74% of control fetuses (Fig. 2Go). This decrease in ASIS was greater than the 34% decrease found for chronically hypoglycemic fetuses (35), providing additional evidence for a role of reduced ß-cell mass/insulin content (18) and/or inhibitory hormones in the placental insufficiency-induced IUGR islets.

In contrast to the significant dysfunction observed for ß-cells, fetal {alpha}-cell responsiveness in the IUGR fetuses, measured by glucagon secretion after administration of arginine, was not different from control fetuses. Thus, the IUGR fetuses primarily had a pancreatic ß-cell defect, confirming previous pancreatic morphometric analysis that identified reductions in ß-cell mass and insulin content but found no change in glucagon-positive ({alpha}-cell) area and glucagon content (18).

During the GSIS studies, the fetal weight-specific glucose infusion rates to maintain the hyperglycemic steady-state condition were similar for control and IUGR fetuses, indicating comparable rates of glucose clearance between control and IUGR fetuses. In such hyperglycemic conditions, it is unlikely that there would be persistent fetal glucose production. Thus, the steady-state plasma disposal rate of glucose in the IUGR fetuses during the hyperglycemic conditions was occurring with significantly lower insulin concentrations than in the control fetuses (Table 2Go). Such increased glucose clearance could result from increased concentrations of glucose transporters or augmentation of insulin action to promote glucose transporter 4 translocation from inactive intracellular storage pools to active sites in the cell membrane. Preliminary data for glucose transporter levels in muscle and liver show no differences between control and IUGR fetuses (Smith, D. L., S. W. Limesand, and W. W. Hay, unpublished data). Enhancement of several proximal steps in the insulin/IGF-I signaling cascade that regulate glucose metabolism has been described for this fetal sheep model of IUGR (47). Therefore, we postulate that the increased glucose clearance in these IUGR fetuses might represent increased insulin sensitivity in response to the decreased nutrient supplies from the placenta and the low insulin concentrations. If persistent, such increased glucose clearance and possible insulin sensitivity might represent mechanisms that would account for a thrifty phenotype that has been characterized for other IUGR models (3).

It is noteworthy that isolated fetal sheep islets in our experiments secreted insulin in response to glucose, which has not been observed previously with pancreatic explants or islet-like cell clusters from fetal sheep (48, 49) or from isolated islets/pancreatic explants from human (50, 51, 52), porcine (51), or rat (23, 53) fetuses. The in vitro islet incubations in the present studies also showed that fetal sheep islets were left-shifted (higher insulin secretion rates at lower glucose concentrations) in response to glucose compared with adult rodent islets (54, 55), which might be a species trait, because plasma glucose concentrations in adult sheep average only 2.5 ± 0.5 mmol/liter naturally (56) compared with 5.1 ± 0.7 mmol/liter in rats (24). Furthermore, our islet incubations were performed in the presence of forskolin, an activator of adenylate cyclase, which has been shown to enhance insulin secretion in other species (57, 58, 59, 60) and may have contributed to the robust glucose-stimulated insulin release from our fetal sheep islets.

Comparison of control and IUGR fetal sheep islets identified a defect in insulin biosynthesis and/or storage rather than ß-cell responsiveness. In fact, the fraction of insulin released per insulin content was greater for IUGR fetal islets compared with control fetal islets under maximal stimulated conditions with 11 mmol/liter glucose or 30 mmol/liter KCl (Fig. 4AGo), even though insulin release per IUGR islet was significantly less than control islets (Fig. 4CGo). In contrast, fetal rats from pregnant dams fed a low-protein diet have a reduced fraction of insulin release when stimulated with amino acids but no deficit in islet insulin content (23, 53). Results similar to the low-protein rat model are found in chronically hypoglycemic fetal sheep that have limited islet insulin secretion capacity but no deficiencies in islet insulin content (Rozance, P. J., S. W. Limesand, J. C. Hutton, and W. W. Hay, unpublished data). It also is possible that our IUGR islets might have a secretion defect, which was overcome by including forskolin in the islet incubation. This has been recognized in GK rat islets, for example (61).

Histological analysis of the IUGR fetal pancreas showed lower islet area with reduced proportional contribution of ß-cells (identified as insulin-positive cells), resulting in a 52% reduction in islet ß-cell area but no changes in {alpha}-cell or {delta}-cell area (18). In the present study, islet DNA content was not different between treatments, and an identical reduction in insulin content was found, normalized to either islet number or DNA content. Isolated fetal sheep islets have a similar size resulting from islet size selection by the isolation procedure. However, the 22% lower proportion of ß-cells within the IUGR islets might still occur but does not account for the much greater 82% reduction in insulin content per islet (Fig. 4BGo). Therefore, insulin content is reduced to a greater extent than islet ß-cell mass in the IUGR islets, indicating that insulin biosynthesis and/or storage is significantly lower in the IUGR islets.

Glucose metabolism in the ß-cell plays an important role in insulin release and insulin biosynthesis (62, 63). In control fetal sheep islets, an expected increase of approximately 4.5-fold was found for both glucose utilization and oxidation rates when glucose concentration in the incubation medium was increased from the physiologically normal value of 1.1 mmol/liter to 11.1 mmol/liter. Glucose utilization and oxidation rates were not different between IUGR and control islets at 1.1 mmol/liter incubation medium glucose concentrations (Fig. 5Go). The ratio of glucose oxidation to utilization was 0.28 at 1.1 mmol/liter glucose and 0.37 at 11.1 mmol/liter, similar to the ratios reported in normal rat islets (36). The rate of glucose oxidation for IUGR islets incubated in 11.1 mmol/liter glucose, however, was less than control islets, resulting in a decreased oxidation/utilization ratio distinguishing a defect in oxidative glucose metabolism in the IUGR islets.

The lower rate of glucose oxidation in the IUGR islets cannot be explained by a lower fraction of ß-cells as was the case for the insulin content, indicating potential changes in glucose metabolism within the IUGR ß-cell itself. Glucose utilization data show similar rates of glucose entry into glycolysis, but complete oxidative metabolism in IUGR islets was impaired during glucose stimulation (Fig. 5Go). Alternative pathways for glucose molecules might include anaerobic metabolism, redistribution of glucose flux to the pentose pathway, or glycerol phosphate and malate/aspartate shuttles, but none of these possibilities is conclusive for the ß-cell (62). Because signals derived from the oxidative metabolism of glucose are implicated in the regulation of ß-granule exocytosis, proinsulin biosynthesis, and insulin gene transcription, a defect in the islet’s ability to increase oxidative metabolism in response to an increase in extracellular glucose concentration has major implications in terms of ß-cell function. As in the case of type 2 diabetes models in adult animals, a failure of stimulus-secretion and stimulus-biosynthesis coupling would explain the defect in both secretion rates and insulin content seen in these IUGR animals and warrants further investigation.

In conclusion, the present study demonstrates impaired insulin secretion in response to glucose and depolarizing agents in fetal sheep islets with IUGR caused by fetal nutrient deficits. This insulin secretion deficit was a result of a major reduction in the insulin content within the IUGR islets resulting from deficient insulin storage and/or biosynthesis, compounding the effects of decreased ß-cell mass previously observed in this IUGR model. In contrast, fractional islet insulin secretion was increased, indicating that there was no deficit in mechanisms regulating insulin exocytosis. Although reduced pancreatic development and insulin secretion in undernourished fetuses might appropriately limit the metabolic cost of growth when nutrients are scarce, the impaired development of fetal islets (decreased replication and number of ß-cells) and insulin biosynthesis and/or storage might create a somatic cell inheritance that could predispose to neonatal complications, particularly hyperglycemia, and type 2 diabetes. Hyperglycemia is commonly observed in low-birth-weight, IUGR newborn infants receiving parenteral glucose infusions (64, 65, 66). Such infants also frequently have stress-induced increases of cortisol, catecholamines, and glucagon concentrations. If the reduced pancreatic islet insulin content and decreased overall insulin secretion rate in response to hyperglycemia observed in this study occur in such infants, they might augment the hyperglycemia by reducing the normal effects of insulin to inhibit hepatic glucose production and promote glucose utilization in peripheral tissues. The latter also might negatively impact neonatal growth. If these conditions producing decreased insulin secretion persist into adulthood, they also might limit insulin secretion in response to glucose stimulation and insulin resistance, thereby predisposing IUGR offspring to type 2 diabetes.


   Footnotes
 
This work was supported by National Institutes of Health (NIH) Grants RO1 HD42815 (to W.W.H.), NRSA DK60300 (to S.W.L.), and KO1 DK067393 (to S.W.L.). P.J.R. was supported by NIH Institutional Training Grant HD07186 (to W.W.H.) and the NIH-Colorado Clinical Nutrition Research Unit (P30 DK048520-11, J.Hill, principal investigator). Histological core services were provided by the UCHSC Barbara Davis Center for Childhood Diabetes, which is supported by NIH Diabetes and Endocrinology Research Center Grant P30-DK57516 (J. C. Hutton, principal investigator).

First Published Online December 8, 2005

Abbreviations: ASIS, Arginine-simulated insulin secretion; dGA, days gestation age; GSIS, glucose-simulated insulin secretion; IUGR, intrauterine growth restriction; KRB, Krebs Ringer buffer.

Received July 19, 2005.

Accepted for publication December 1, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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S. W. Limesand, P. J. Rozance, L. D. Brown, and W. W. Hay Jr.
Effects of chronic hypoglycemia and euglycemic correction on lysine metabolism in fetal sheep
Am J Physiol Endocrinol Metab, April 1, 2009; 296(4): E879 - E887.
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K. L. Gatford, S. N. B. Mohammad, M. L. Harland, M. J. De Blasio, A. L. Fowden, J. S. Robinson, and J. A. Owens
Impaired {beta}-Cell Function and Inadequate Compensatory Increases in {beta}-Cell Mass after Intrauterine Growth Restriction in Sheep
Endocrinology, October 1, 2008; 149(10): 5118 - 5127.
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P. J. Rozance, S. W. Limesand, J. S. Barry, L. D. Brown, S. R. Thorn, D. LoTurco, T. R. H. Regnault, J. E. Friedman, and W. W. Hay Jr.
Chronic late-gestation hypoglycemia upregulates hepatic PEPCK associated with increased PGC1{alpha} mRNA and phosphorylated CREB in fetal sheep
Am J Physiol Endocrinol Metab, February 1, 2008; 294(2): E365 - E370.
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S. W. Limesand, P. J. Rozance, D. Smith, and W. W. Hay Jr.
Increased insulin sensitivity and maintenance of glucose utilization rates in fetal sheep with placental insufficiency and intrauterine growth restriction
Am J Physiol Endocrinol Metab, December 1, 2007; 293(6): E1716 - E1725.
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J. A. Owens, K. L. Gatford, M. J. De Blasio, L. J. Edwards, I. C. McMillen, and A. L. Fowden
Restriction of placental growth in sheep impairs insulin secretion but not sensitivity before birth
J. Physiol., November 1, 2007; 584(3): 935 - 949.
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S. M. Husted, M. O. Nielsen, M. P. Tygesen, A. Kiani, D. Blache, and K. L. Ingvartsen
Programming of intermediate metabolism in young lambs affected by late gestational maternal undernourishment
Am J Physiol Endocrinol Metab, August 1, 2007; 293(2): E548 - E557.
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P. J. Rozance, S. W. Limesand, G. O. Zerbe, and W. W. Hay Jr.
Chronic fetal hypoglycemia inhibits the later steps of stimulus-secretion coupling in pancreatic beta-cells
Am J Physiol Endocrinol Metab, May 1, 2007; 292(5): E1256 - E1264.
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 Am. J. Physiol. Endocrinol. Metab.Home page
P. J. Rozance, S. W. Limesand, and W. W. Hay Jr.
Decreased nutrient-stimulated insulin secretion in chronically hypoglycemic late-gestation fetal sheep is due to an intrinsic islet defect
Am J Physiol Endocrinol Metab, August 1, 2006; 291(2): E404 - E411.
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