INTRODUCTION

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STUDIES IN LATE-GESTATION fetal sheep have shown that fetal plasma glucose and insulin concentrations independently regulate fetal whole body glucose utilization rates (15-17). A central question from these studies is whether the effects of fetal plasma glucose and insulin concentrations to increase fetal glucose utilization rate are dependent on the process of glucose transport into fetal cells. Glucose transport is mediated by glucose transporter proteins, which consist of a family of facilitative transmembrane glycoproteins that move glucose into cells down a concentration gradient (4, 10, 24, 27-29). Of the different major isoforms, Glut 1 is found in all tissue types in the fetus, whereas Glut 4 is present primarily in insulin-responsive tissues, such as skeletal muscle, but in lower concentrations relative to the adult (31, 32, 34). Under normoglycemic and normoinsulinemic conditions, Glut 1 and Glut 4 reside in the cell membrane and in separate endosomal storage pools within the cytoplasm, respectively (12). Glut 1 is largely constituent in cell membranes, providing for basal glucose uptake. In contrast, Glut 4 is found primarily in the endosomal stores and much less at the cell membrane (1, 26). In adult human, animal, and insulin-responsive cell models, increased amount and translocation of Glut 1 and Glut 4 from intracellular storage pools to the cell membrane mediate increased rates of glucose uptake by cells in response to higher plasma concentrations of glucose and insulin, respectively (3, 7, 13, 36, 37). In contrast to these adult studies, there is minimal information in the fetus to correlate in vivo fetal glucose utilization rates with the amount of these transporters.

The purpose of the present study was to determine whether there are changes in Glut 1 and Glut 4 glucose transporter protein concentrations in skeletal muscle in fetal sheep under conditions of selective fetal hyperglycemia or hyperinsulinemia when glucose utilization rate is increased. We hypothesized that increased glucose utilization by the fetus and, in particular, by fetal skeletal muscle would be associated with increases in fetal skeletal muscle cell Glut 1 and Glut 4 transporter protein concentrations in response to selective hyperglycemia or hyperinsulinemia. We anticipated that the relative expression of Glut 1 would be increased by selective hyperglycemia and that the relative expression of Glut 4 would be increased by selective hyperinsulinemia. Skeletal muscle was chosen for study specifically because it contains both Glut 1 and Glut 4 (10, 28), shows increased rates of glucose uptake and utilization in response to increased plasma concentrations of glucose and insulin (2, 8, 41), and accounts for the majority of glucose consumption in the late-gestation fetus (15, 23, 30, 35).

	METHODS

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Animal preparation. We studied 33 pregnant Columbia-Rambouillet ewes obtained from a commercial breeder (Nebeker Ranch, Santa Monica, CA) at 90% of a 147-day gestation. Surgery was performed at 120 days gestation to place indwelling polyvinyl catheters in the ewe and fetus. The ewes were fasted for 2 days before surgery. The ewes were sedated with a bolus injection (14 ml) of pentobarbital sodium solution (50 mg/ml) via an external jugular vein catheter. Anesthesia was provided by a single spinal injection of 2 ml tetracaine hydrochloride 1%. Sedation was maintained during surgery by intermittent 1- to 2-ml intravenous injections of pentobarbital sodium. With the use of a standard hysterotomy approach, fetal catheters for infusion were placed in the inferior vena cava via hindlimb pedal veins, and sampling catheters were placed in the umbilical vein directly and in the fetal aorta via hindlimb pedal arteries. A maternal femoral arterial sampling catheter and femoral venous infusion catheters were placed via a single groin incision. All catheters were filled with 0.9% (wt/vol) sodium chloride in water containing 100 U/ml of sodium heparin. The catheters were tunneled subcutaneously to exit through a flank incision and were kept in a plastic pouch secured to the ewe's flank. The catheters were flushed every other day with the heparinized sodium chloride solution. For infection prophylaxis, the ewe was given intramuscular injections of gentamicin (80 mg; American Pharmaceutical Partners, Los Angeles, CA) and procaine penicillin G (600,000 U; Vedco, St. Joseph, MO) just before surgery, and the fetus was given intra-amniotic ampicillin (500 mg; Apothecon, Bristol-Meyers Squibb, New York, NY) at the end of surgery. A minimum 72-h recovery period followed surgery. During this time, the ewes had free access to food, minerals, and water. Sheep were kept in standard plastic carts, at least two in a room. The room was kept at 60 ± 2°F. Eight hours of darkness and 16 h of light were provided each day. This study was approved by the University of Colorado Health Sciences Center Institutional Animal Care and Use Committee. All procedures and studies were performed at the University of Colorado Health Sciences Center Perinatal Research Facility, which is accredited by the United State Drug Administration, National Institutes of Health, and the American Association for Accreditation of Laboratory Animal Care.

Study design. Animals were divided into two study groups. One group of fetuses was made hyperglycemic while maintaining normal plasma insulin concentrations, and the other was made hyperinsulinemic while maintaining normal plasma glucose concentrations (19). The two study groups were subdivided into three experimental time periods: 1, 2.5, and 24 h.

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Hyperglycemia studies. Hyperglycemia with normal fetal plasma insulin concentration was produced in study group 1 by infusing 50% dextrose in water (D₅₀W) into the mother and somatostatin into the fetus to prevent increased fetal insulin secretion in response to the experimental hyperglycemia. Somatostatin (6 mg) was mixed in 0.9% (wt/vol) sodium chloride in water (20 ml) to a concentration of 300 µg/ml and was given to the fetus as a priming bolus (100 µg/kg) followed by a constant infusion (4 µg · min¹ · kg¹). The dextrose infusion into the ewe was initiated 60 min after starting the somatostatin infusion into the fetus. The ewe received a priming bolus of D₅₀W (~300 mg/kg) followed by an initial infusion rate of ~2.3 mg · kg¹ · h¹. Glucose clamp technique (18) was used to keep fetal arterial plasma glucose concentration at ~40 mg/dl, twice the mean control period value. To establish a constant fetal glucose concentration, fetal arterial plasma glucose concentration was measured every 10 min, and the maternal dextrose infusion rate was adjusted until the fetal arterial plasma glucose concentration was stable at the target concentration. Once the clamp concentration was established, only infrequent measurements of fetal glucose concentration were necessary to verify continued stability.

Hyperinsulinemia studies. Hyperinsulinemia with euglycemia was produced in study group 2 by infusing insulin into the fetus and adjusting a D₅₀W infusion into the ewe to keep fetal arterial plasma glucose concentration at the mean control period value. Regular insulin (100 U/ml) was mixed with normal saline to a concentration of 60 mU/ml. The fetus received a 30-mU/kg priming bolus of insulin followed by a constant infusion at 1 mU · min¹ · kg¹. Fetal arterial plasma glucose concentration was measured every 10 min, and the maternal dextrose infusion rate was adjusted to keep the fetal arterial plasma glucose concentrations at the mean control period value.

Biochemical analyses. Plasma glucose and lactate concentrations were measured in duplicate using a YSI model 2700 analyzer (Yellow Springs Instrument, Yellow Springs, OH). To measure plasma insulin concentrations, sampled blood was immediately centrifuged at 4°C for 3 min, and the plasma was stored at 70°C until analysis with a Linco rat insulin RIA kit (St. Charles, MO) using ovine insulin standards (Eli Lily, Indianapolis, IN). Blood oxygen saturation, oxygen content, and hemoglobin concentration were measured using a Radiometer OSM3 hemoximeter (Copenhagen, Denmark).

Calculations. Umbilical blood flow was calculated by the transplacental steady-state diffusion technique using tritiated water (³H₂O) as the tracer (40). Net umbilical (fetal) glucose uptake rate (UGU_f) was calculated by application of the Fick principle as UGU_f (mg · min¹ · kg¹) = umbilical blood flow (ml/min) × Gv-Ga (mg/dl), where Gv and Ga are the umbilical venous and the fetal arterial plasma glucose concentrations, respectively. UGU_f was considered equal to fetal glucose utilization rate, because there is no net fetal glucose production when the fetus receives normal or above-normal rates of glucose supply from the placenta and has normal to high plasma glucose and/or insulin concentrations (14, 21).

Glucose transporter protein measurement. Thoroughly washed ovine fetal skeletal muscle samples were homogenized using a Tekmar Tissuemizer (Cincinnati, OH). The samples were then sonicated (60 sonic, Dismembrator, Fisher Scientific, Pittsburgh, PA) using 2-s cycles of 5-7 W to ensure adequate homogenization of the tissue. Protein content was assessed by the Bio-Rad dye-binding assay (Bio-Rad, Richmond, CA). Fifty micrograms of protein in the tissue homogenates were subjected to discontinuous 10% SDS-polyacrylamide gel electrophoresis followed by electroblot transfer to nitrocellulose (Nytran: Schleicher & Schuell, Keene, NH). Equality of loading and efficiency of transfer were evaluated by Coomassie blue staining of the gel and transfer of prestained standards, respectively. The nitrocellulose membranes were incubated for 1-2 h in 5% nonfat dry milk in PBS, 0.1% Tween 20 to decrease nonspecific binding of the antibody. This was followed by incubation for 1 h at room temperature with an affinity-purified rabbit anti-rat antibody that was generated against the hemocyanin-limpet linked rat Glut 1 (1:2,000 dilution) or rat Glut 4 (1:500 dilution) COOH terminal 16 amino acids. These antibodies have been previously characterized, and the isoform specificity has been confirmed (9). Ovine Glut 1 and Glut 4 proteins were readily detected with the anti-rat glucose transporter antibodies as previously described by Das et al. (9). After the membranes were washed in PBS-0.1% Tween 20, those that were treated with the Glut 1 or Glut 4 antibodies were incubated with a peroxidase-linked goat anti-rabbit IgG (1:2,500 dilution) for 1 h at room temperature and subsequently exposed to a chemiluminescence reagent (Amersham Pharmacia Biotech, Little Chalfont, UK). The chemiluminescence was captured by autoradiography over an optimal period of time (1-5 min). Glucose transporter protein concentrations were assessed by densitometry once the presence of linearity between the time of autoradiographic exposure and the optical density was established. The results were expressed as percentages of the means of the corresponding control for each experimental condition.

Subcellular localization studies. Cryostat (8 µm) sections of skeletal muscle were obtained at a cross-sectional level and mounted on Superfrost/Plus slides. A few sections were obtained in the longitudinal orientation. Comparable sections at the four different times (0, 1, 2.5, and 24 h) from the two experimental groups were all mounted on a single slide and subjected to immunohistochemical analysis to overcome interassay variability. The tissue sections were fixed in acetone for 10 min at 4°C and then washed with PBS (pH 7.4) for 10 min. The fixed tissue sections were incubated overnight with an optimal concentration of the rabbit anti-rat Glut 1 (1:500 dilution) or Glut 4 (1:200 dilution) IgGs in PBS at 4°C in a humidified chamber. Glut 1 or Glut 4 preabsorbed respective antibodies served as the appropriate negative controls. The sections were subsequently incubated with FITC linked with goat anti-rabbit secondary antibody (1:100; Sigma Chemical, St. Louis, MO) for 1.5 h at 23°C. After extensive washes in PBS, the sections were mounted in a commercially available mounting solution (Biomeda, Foster City, CA), placed on a coverslip, and visualized using an Olympus microscope with an epifluorescence attachment using the appropriate filter. Cellular localization of Glut 1 and Glut 4 immunoreactivity was undertaken using the Simple 32 image analyzer software program (Compix, Imaging Systems, Cranberry Township, PA).

Data analysis. All results are expressed as means ± SE. When two groups were compared, the Student's t-test was used. Differences were determined by the Kruskal-Wallis test followed by a post hoc t-test when comparing more than two time points, given the small sample size limitation in sheep investigations.

	RESULTS

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There were no differences among groups in gestational age at surgery or study or fetal weight at study (Table 1). Fetal arterial blood oxygen saturation values, fetal arterial oxygen content, and fetal hematocrit were unchanged by experimental conditions (Table 2). The arterial oxygen saturation values and fetal arterial oxygen content were within the normal range for late-gestation fetal sheep (5, 16).

Immunolocalization of the fetal glucose transporter proteins did show Glut 1 transporter at the skeletal muscle cell sarcolemma under control conditions and at all experimental time points (Fig. 1). Glut 4 transporter was seen primarily in the intracellular space at all time points (Fig. 2). Changes in the basal distribution patterns of Glut 1 and Glut 4 under different experimental conditions were not significant.

View larger version (48K): [in this window] [in a new window]   Fig. 1.   Representative photomicrograph showing labeled Glut 1 transporter at the fetal ovine skeletal muscle cell sarcolemma (arrow).

View larger version (69K): [in this window] [in a new window]   Fig. 2.   Representative photomicrograph showing labeled Glut 4 transporter protein (punctate staining) in the intracellular space of fetal ovine skeletal muscle cells (arrow).

Figure 3 shows evidence of selective hyperglycemia with normal fetal arterial plasma insulin concentrations in study group 1. The mean control period arterial plasma glucose concentration was 19.9 ± 0.7 mg/dl. The mean study period glucose concentrations were 43.2 ± 2.7 mg/dl at 1 h (n = 6), 37.8 ± 2.8 mg/dl at 2.5 h (n = 6), and 41.2 ± 2.0 mg/dl at 24 h (n = 5), all twice the mean control period glucose concentration (P < 0.05 for all). Mean fetal arterial plasma insulin concentration did not change over the study periods (Fig. 3), averaging 14.5 ± 1.5 µU/ml in the control period, 11.8 ± 2.2 µU/ml at 1 h, 12.9 ± 2.9 µU/ml at 2.5 h, and 18.0 ± 4.8 µU/ml at 24 h. Table 3 shows that the rate of fetal glucose uptake in this study was maximally increased at 1 h, remained significantly increased at 2.5 h, and had returned to the control rate by 24 h of glucose stimulation. Under these experimental conditions of fetal hyperglycemia and euinsulinemia, Glut 1 protein amount in the skeletal muscle (Fig. 4) increased fourfold at 1 h (P < 0.05) but was not significantly different than the control concentration at other study times, although a strong trend toward increase was evident at 24 h. Glut 4 protein amount was significantly increased twofold at 1 h (P < 0.05 vs. control). Although the change in Glut 4 protein concentration did not show continued statistically significant elevation at 2.5 and 24 h, it remained above control at those time points.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Clamp data showing selective fetal hyperglycemia to approximately twice the mean control period glucose concentration with no change in fetal insulin concentration (hyperglycemic-euinsulinemic experimental condition). Values are means ± SE. Control (Con), n = 5; 1 h, n = 6; 2.5 h, n = 6; 24 h, n = 5.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Data from the hyperglycemic-euinsulinemic experiments showing Glut 1 and Glut 4 transporter protein increased above control amount at 1, 2.5, and 24 h, reaching statistical significance at 1 h. Changes in Glut 1 and Glut 4 protein amount during selective hyperglycemia are shown in a representative Western blot and graphically. Chart shows Glut 1 protein (open bars) and Glut 4 protein (filled bars) in arbitrary units expressed as a %control period value. Values are means ± SE. Con, n = 5; 1 h, n = 6; 2.5 h, n = 6; 24 h, n = 5.

Figure 5 shows evidence of selective hyperinsulinemia with maintenance of normal fetal arterial plasma glucose concentrations in study group 2. Mean basal fetal arterial plasma insulin concentration in the hyperinsulinemic-euglycemic group was 18.4 ± 2.0 µU/ml, not different from the mean basal insulin concentration measured in the hyperglycemic-euinsulinemic group. Insulin concentrations were increased two- to threefold at all study times, to 80.6 ± 8.5 µU/ml at 1 h (n = 5), 59.3 ± 7.6 µU/ml at 2.5 h (n = 6), and 53.1 ± 4.8 µU/ml at 24 h (n = 5; P < 0.05 for all). Fetal arterial plasma glucose concentration did not change significantly from the control period value of 21.9 ± 1.0 mg/dl, averaging 17.8 ± 3.9 mg/dl at 1 h, 23.0 ± 2.0 mg/dl at 2.5 h, and 19.5 ± 0.6 mg/dl at 24 h. The mean net fetal glucose uptake/utilization rate in the control period in this group was 6.4 ± 0.6 mg · min¹ · kg¹, which was not different from the control fetal glucose uptake/utilization rate observed in the hyperglycemia study (Table 3). As shown in Table 3, glucose uptake/utilization rate was twice the control rate at all study times. Glut 1 protein amount in the skeletal muscle did not change with fetal hyperinsulinemia (Fig. 6). Glut 4 protein amount increased 1.5- to 2-fold at 1 and 2.5 h, achieving statistical significance only at 2.5 h of hyperinsulinemic stimulation (P < 0.05 vs. control; Fig. 6).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Clamp data showing selective fetal hyperinsulinemia to ~3 times the mean control period insulin concentration with no change in fetal glucose concentration (hyperinsulinemic-euglycemic experimental condition). Values are means ± SE. Con, n = 5; 1 h, n = 5; 2.5 h, n = 6; 24 h, n = 5.

View larger version (40K): [in this window] [in a new window]   Fig. 6.   Data from the hyperinsulinemic-euglycemic experiments. No change in Glut 1 protein amount was measured in response to insulin stimulation at any time point. An acute increase in Glut 4 protein was measured after 1 h of insulin stimulation. The increase reached statistical significance at 2.5 h but returned to control amount by 24 h. Changes in glucose transporter protein amounts are shown in a representative Western blot and graphically. Glut 1 (open bars) and Glut 4 (filled bars) protein amounts are in arbitrary units expressed as %control period amount. Values are means ± SE. Con, n = 5; 1 h, n = 5; 2.5 h, n = 6; 24 h, n = 5.

	DISCUSSION

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These results show that selective increases in fetal plasma glucose or insulin concentrations in late-gestation fetal sheep have independent in vivo effects on fetal glucose uptake/utilization rate and on Glut 1 and Glut 4 glucose transporter protein concentration within skeletal muscle cells. The effects of glucose and insulin are independent, time dependent, and glucose transporter-isoform specific.

Significant changes in variables other than glucose and insulin concentrations, such as fetal arterial oxygen saturation or oxygen content, which could affect fetal glucose transporter concentration or intracellular location, were not seen in these studies. Doses for insulin infusion in this study were well below the pharmacological doses used in some previous studies from this laboratory (20, 41) and resulted in fetal hyperinsulinemia only two- to threefold above control concentrations. Also, the fetal sheep were transfused with maternal blood after the control blood draws to avoid the hypoxemia and acidosis that have been reported previously with anemia and hemodilution by infusates (16). As shown in Table 2, there was no experimental effect on fetal oxygen saturation, fetal arterial oxygen content, or hematocrit with either hyperglycemia or hyperinsulinemia at any time point. In a similar hyperglycemic clamp experiment using somatostatin (11), no significant changes in fetal blood flow, fetal umbilical oxygen uptake, or fetal arterial oxygen content were measured as a result of somatostatin infusion alone.

Under basal conditions of euglycemia and euinsulinemia, immunofluorescence assays showed an expected association of Glut 1 primarily with the cell membrane/sarcolemma and Glut 4 primarily within the cells. The lack of significant and/or consistent change in these distribution patterns, particularly when correlated with the protein concentration changes, was surprising. This may represent a limitation of this type of assay, especially in light of the relatively low concentrations of Glut 4 in fetal tissue at any time or under any condition. Subcellular fractionation procedures may yield a more quantitative assessment of the translocation process.

There was a clear temporal relationship between the initial increase in the rate of fetal glucose utilization and the total amount of Glut 1 and Glut 4 protein during experimental hyperglycemia. This early increase in net glucose uptake in response to hyperglycemia probably represents the acute response to increased substrate availability, because transport of glucose is energy independent and follows its concentration gradient from the plasma into tissues and cells. The acute and immediate increase in fetal skeletal muscle Glut 1 and Glut 4 protein concentrations in response to a perturbation in circulating glucose concentrations has been reported here for the first time. This increase reflects a net balance between glucose transporter protein synthesis and degradation, processes that will require investigation in the future.

The relative decrease in skeletal muscle sarcolemma-associated Glut 1 protein concentration at 24 h of the hyperglycemic condition compared with the 1-h concentration correlated with the return of net fetal glucose utilization to control. Downregulation of the ovine fetal skeletal muscle Glut 1 transporter protein with associated decreases in glucose transport has been previously observed with more sustained hyperglycemia (9), an observation that correlates with decreases in fetal glucose utilization (6). Under acute hyperglycemic conditions, such a downregulation from control values was not evident, but rather an absence of a change or increase was noted. In contrast, Glut 4 concentrations are either unchanged or increased from baseline at 24 h. This increase in intracellular Glut 4 protein does not correlate with fetal glucose utilization. Thus it appears that while acute fetal hyperglycemia correlates with an increase in fetal glucose utilization and increased Glut 1 and Glut 4 protein, Glut 1 may be active in transporting glucose via its sarcolemmal association, whereas intracellular Glut 4, in the absence of hyperinsulinemia, may be inactive.

In contrast to the hyperglycemic condition, only Glut 4 protein concentration in the skeletal muscle was increased with hyperinsulinemia. The increase in Glut 4 protein peaked at 2.5 h and was transient, despite ongoing hyperinsulinemia and a sustained increase in glucose uptake and utilization above control. This observation suggests that sustained hyperinsulinemia fails to alter fetal skeletal muscle Glut 1 or Glut 4 concentrations. However, no long-term investigations characterizing the chronic effects of fetal hyperinsulinemia on fetal skeletal muscle glucose transporter proteins exist.

Temporal changes in glucose uptake in organs other than skeletal muscle could contribute to the overall decline in fetal glucose uptake with hyperglycemia and the sustained increase in glucose uptake with hyperinsulinemia. Thus glucose transporter expression in the skeletal muscle alone would not necessarily translate into whole fetus glucose flux. Support for this in the fetus comes from previous observations of an acute increase in the amount of Glut 1 transporter protein in fetal myocardial muscle, brain, and liver at 2-48 h of experimental hyperglycemia followed by decreased amounts of transporter protein after chronic periods of hyperglycemia (9). Additionally, experiments in adult rats have shown time dependency of insulin action on skeletal muscle and adipose tissues (25). Hence, whereas whole fetal glucose utilization rates provide a surrogate for fetal skeletal muscle glucose utilization, it does not replace the in vivo skeletal muscle glucose transport and uptake assessments, which are necessary in future investigations.

The results of these studies indicate that substrate- and hormone-induced changes in the expression of Glut 1 and Glut 4 in ovine fetal skeletal muscle cells play a significant role in regulating the effects of hyperglycemia and hyperinsulinemia on fetal glucose uptake. It is interesting that whereas Glut 1, as anticipated, responded only to glucose stimulation, Glut 4 transporter protein responded to both hyperglycemia and hyperinsulinemia. Both transporters are found in skeletal muscle tissues but, in the adult, are selectively responsive to either glucose or insulin. This pattern of response by late-gestation fetal glucose transporters is quite different from that seen in the adult. It also has been shown that insulin stimulation of glucose transport is developmentally regulated (22, 38) and that Glut 1 is the predominant isoform in most fetal rat tissues (39). Thus the function of Glut 4 in fetal tissues, as opposed to its insulin responsiveness and rate-limiting role in adult tissue glucose uptake (28), remains to be determined. Further studies in the fetus are indicated to more specifically determine the temporal and kinetic relationships among changes in plasma glucose and insulin concentrations, Glut 1 and Glut 4 subcellular localization, and skeletal muscle glucose uptake.

Perspectives