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Am J Physiol Regul Integr Comp Physiol 284: R1138-R1146, 2003. First published January 16, 2003; doi:10.1152/ajpregu.00560.2002
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Vol. 284, Issue 4, R1138-R1146, April 2003

Insulin-induced translocation of facilitative glucose transporters in fetal/neonatal rat skeletal muscle

Jing He1,*, M. Thamotharan2,*, and Sherin U. Devaskar2

1 University of Pittsburgh School of Medicine, Magee-Womens Research Institute, Pittsburgh, Pennsylvania 15213-3180; and 2 Divisions of Neonatology and Developmental Biology, Department of Pediatrics, David Geffen School of Medicine at University of California-Los Angeles, Los Angeles, California 90095-1752


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the effect of insulin on fetal/neonatal rat skeletal muscle GLUT-1 and GLUT-4 concentrations and subcellular distribution by employing immunohistochemical analysis and subcellular fractionation followed by Western blot analysis. We observed that insulin did not alter total GLUT-1 or GLUT-4 concentrations or the GLUT-1 subcellular distribution in fetal/neonatal or adult skeletal muscle in 60 min. The basal and insulin-induced changes in subcellular distribution of GLUT-4 were different between the fetal/neonatal and adult skeletal muscle. Under basal conditions, sarcolemma-associated GLUT-4 was higher in the newborn compared with the adult, translating into a higher glucose transport. In contrast, insulin-induced translocation of GLUT-4 to the sarcolemma- and insulin-induced glucose transport was lower in the newborn compared with the adult. This age-related change results in enhanced basal glucose transport to fuel myocytic proliferation and differentiation while relatively curbing the insulin-dependent glucose transport in the newborn.

development; glucose transport; subcellular localization


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GLUCOSE, AN ESSENTIAL SUBSTRATE for cellular oxidative metabolism, is transported across the bilipid cellular membranes by a process of facilitative diffusion (2, 11). This process is mediated by a family of structurally related membrane-spanning glycoproteins termed the glucose transporters (2, 6, 11, 12, 19, 20, 23, 24). Of the various facilitative glucose transporter isoforms (GLUT-1 to GLUT-12) (2, 6, 11, 12, 19, 20, 23, 24), GLUT-1 (basal isoform) and GLUT-4 (insulin-responsive isoform) appear to be the predominant isoforms that are expressed in insulin-sensitive tissues (8, 27). The skeletal muscle glucose uptake represents a majority of the body's insulin-mediated glucose disposal (10). In the adult, insulin has both an acute and chronic effect on GLUT-4 subcellular distribution and synthesis, respectively (5, 21). Studies in adult skeletal muscle have revealed that GLUT-1 is expressed in the sarcolemma and GLUT-4 is sequestered predominantly in intracellular compartments under basal conditions (13, 29). Insulin stimulation causes translocation of GLUT-4 to the sarcolemma and transverse tubules (34), thereby mediating insulin-induced glucose transport.

Fetal/neonatal skeletal muscle responds to insulin by increasing glucose use (1, 30), an increase that is lower than that observed in the adult (4, 18). However, similar to the adult, in the fetal rat, we previously demonstrated GLUT-1 to reside primarily in the sarcolemma, whereas GLUT-4 occupied intracellular compartments (27). Although investigations describing insulin's effect on fetal rat skeletal muscle GLUT-1 exist (30), there is limited information regarding insulin's effect on fetal/newborn rat GLUT-4. We hypothesized that the fetal/neonatal skeletal muscle GLUT-4 alone would demonstrate a different pattern of insulin-responsive subcellular distribution compared with the adult. To test this hypothesis, we examined the effect of high circulating insulin on fetal/neonatal rat skeletal muscle GLUT-1 and GLUT-4 concentrations, subcellular distribution, and glucose transport.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Sprague-Dawley pregnant (19 day gestation) and nonpregnant female rats (300-350 g) (Taconic Farm, Germantown, NY) were housed in individual cages, exposed to 12:12-h light/dark cycles at 21-23°C and allowed access to standard rat chow (Purina, St. Louis, MO) ad libitum. As approved by the Magee-Womens Research Institute's Animal Care and Use Committee at the University of Pittsburgh and the Animal Research Committee of the University of California, Los Angeles, the guidelines of the National Institutes of Health were followed.

Insulin Study

Fetal studies. On day 20 of gestation (term 22-23 days), the maternal rats (n = 15) were anesthetized intraperitoneally with xylazine (8 mg/kg) and ketamine (40 mg/kg), and both the uterine horns were exposed via laparotomy. The fetuses within both uterine horns received either 0.05 U of human insulin (Eli Lilly, Indianapolis, IN) (n = 10) or an equal volume of vehicle (0.9% NaCl) (n = 5) intraperitoneally via a transmural access across the intact uterine wall. After intrauterine insulin therapy, the uterine horns were returned to the abdominal cavity, and the abdominal wall was approximated.

Postnatal studies. Some of the pregnant animals were allowed to deliver spontaneously, the litter size randomly reduced to eight at birth, and the progeny at 1-2 days of life received either 0.05 U/pup (immunohistochemical studies; n = 6) or 8 U/kg (subfractionation studies; n = 10) of pup body weight (equal to 0.048 U of insulin/pup) of human insulin (Eli Lilly, Indianapolis, IN) or an equal volume of vehicle (0.9% NaCl) (n = 16) intraperitoneally and the animals were returned to their mothers.

Adult studies. Nonpregnant female adult rats received either 8 U/kg of insulin (n = 20) or an equal volume of vehicle (0.9% NaCl) (n = 20) intraperitoneally, and the animals were returned to their cages with ad libitum access to food and water.

Blood and Tissue Collection

At timed intervals of 10, 20, 40, and 60 min postinsulin or vehicle treatment, the fetal, neonatal, and adult rats were euthanized either by decapitation (fetal) or with 100 mg/kg of pentobarbital sodium intraperitoneally. Pooled fetal and neonatal and single adult jugular venous blood was obtained, and the plasma was separated and stored in aliquots at -70°C until further analysis. Fetal and neonatal hindlimb mixed skeletal muscle [gastrocnemius, soleus, and extensor digitorum longus (EDL) were all pooled], and adult soleus (oxidative fibers; type I) and EDL (glycolytic fibers; type II) muscles were rapidly dissected free of bone, fascia, and skin. The skeletal muscle was quickly frozen in isopentane and cooled in liquid nitrogen, and the frozen tissue was embedded in OCT freezing compound (Miles, Elkart, IN); tissue blocks were stored at -70°C for immunohistochemical analysis. In addition, mixed skeletal muscle was obtained from the newborn and adult hindlimb, cooled in liquid nitrogen, and stored at -70°C until the subfractionation experiments were undertaken. Freshly dissected mixed skeletal muscle was employed for the sarcolemmal vesicular 2-deoxy-glucose transport experiments.

Plasma Assays

Plasma glucose concentrations were measured by the glucose oxidase method (Sigma Chemical). Plasma insulin concentrations were assessed by the double antibody radioimmunoassay using species-specific antibodies and standards (Linco Research, St. Louis, MO). The sensitivity of the insulin assay is 0.1 ng/ml. The interassay variability was 9%, and intra-assay variability was 2%.

Subcellular Localization Studies

Cryostat (8 µm) sections of the skeletal muscle were obtained at a cross-sectional level and mounted on Superfrost/Plus slides. Comparable sections at four different times (10, 20, 40, and 60 min) from the insulin- and vehicle-treated groups at a given age were all mounted on a single slide and subjected to immunohistochemical analysis to overcome interassay variability. The tissue sections were fixed in 100% 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 container. GLUT-1 or GLUT-4 peptide preabsorbed respective antibodies served as the appropriate negative control. The sections were subsequently incubated with FITC-linked goat anti-rabbit secondary antibody (1:100 dilution) (Sigma Chemical) for 1.5 h at 23°C (30). After extensive washes in PBS, the sections were mounted in a commercially available mounting solution (Biomeda, Foster City, CA), placed under a cover slip, and visualized using an Olympus microscope with an epifluorescence attachment using the appropriate filter (1).

Semi-quantification of GLUT-1 and GLUT-4 immunoreactivity was undertaken in a blinded fashion using the Simple 32 image analyzer software program (Compix, Imaging Systems, Cranberry Township, PA). The number of cells expressing sarcolemma-associated GLUT-1 or GLUT-4 immunoreactivity was expressed as a percent of the total number of myotubes/myocytes per high power field (×40 magnification). In addition, the sarcolemmal-associated GLUT-1 or GLUT-4 immunoreactivity was assessed as a percent of the total cellular immunoreactivity. This was accomplished by determining the intensity of the immunoreactivity × the total area of a cell that represented the total cell immunoreactivity. Intracellular immunoreactivity was assessed by semi-quantifying the intensity of the immunoreactivity × the area of the cell with the perimeter just internal to the sarcolemmal region. The sarcolemmal-associated immunoreactivity was calculated as the difference between total cellular immunoreactivity and intracellular immunoreactivity. In all cases, the net immunoreactive intensity was derived based on a gray scale (black = 0 and white = 255) after subtracting the background. Ten cells per high power field in a section were arbitrarily assessed in this manner. The sarcolemmal-associated and the intracellular-associated immunoreactivity were compared between the insulin and vehicle treatment groups at a given age and at the different time points after insulin or vehicle treatments.

Skeletal Muscle Subfractionation Studies

Fetal and neonatal skeletal muscle (hindlimb) and adult EDL and soleus muscles were initially examined. After noting that fetal skeletal muscle consisted primarily of myotubes and the neonatal skeletal muscle of myocytes, further membrane subfractionation experiments were undertaken in mixed skeletal muscle obtained only from the 1- to 2-day-old newborn and adult. In addition, the gastrocnemius muscle (oxidative and glycolytic fibers) from the adult was also examined. Both ages were treated with 8 U/kg of insulin or an equal volume of saline 20 min before isolation. This time point was chosen as the optimal time point after initial time course experiments.

Subcellular fractions were prepared at 4°C. One-half to one gram of previously trimmed skeletal muscle that was snap-frozen and crushed was transferred into ice-cold 30 ml of sucrose buffer (250 mM sucrose, 20 mM HEPES-Tris, 1 mM EDTA, and 100 µM PMSF, pH 7.4). The muscle was then homogenized on ice with a Polytron homogenizer (30-mm probe at setting 3) for one 15-s burst. This homogenate was filtered through two layers of cheese cloth to remove residual connective tissue. Some of the homogenate was saved for Western blot analysis and enzyme assays, and the rest was centrifuged at 3,000 g for 10 min. The supernatant was saved for preparing the low-density microsome (LDM)-enriched fraction. The pellet was resuspended in 10 mM Tris · HCl, pH 8.0, using a loose-fitting Teflon homogenizer (Thomas C600, Fisher Scientific) with three nonshearing strokes. The suspension was then centrifuged, and the supernatant was discarded. The pellet was washed twice, and the final pellet was suspended in 10 mM Tris · HCl, pH 8.0, in a ratio of 15-30 ml:1-2 g of the original skeletal muscle (wt/vol) and homogenized using three strokes of the Teflon pestle, as before, and the suspension was transferred to a 40-ml glass beaker. A 9 × 25 mm diameter Teflon-coated magnet was added to each beaker, covered with parafilm, and stored at 4°C for 16 h. Subsequently, 50 mM lithium bromide (200 µl:10 ml of the suspension) was added to the beakers and magnetically stirred at a setting of three for 2.5 h on a multiport magnetic stirring base (Labline multimagnestir, LabLine Instruments, Melrose, IL) to extract all contractile elements. The LiBr-treated suspension was then transferred to a 50 ml centrifuge tube and diluted with the addition of 20 ml of 10 mM Tris · HCl, pH 8.0, and centrifuged at 10,000 g for 10 min. The resultant pellet was resuspended in 10 ml of 10 mM Tris · HCl, pH 8.0, using three strokes of the Teflon pestle. The resuspended pellet was centrifuged at 6,000 g for 10 min, and the pellet obtained was treated with 25% potassium bromide (KBr) [15 ml per 1-2 g of trimmed muscle (wt/vol)]; the resultant pellet was resuspended again using three strokes of the Teflon pestle followed by centrifugation at 10,000 g for 30 min. The KBr-treated pellet was washed once with 250 mM sucrose buffer. This was done to remove residual KBr using 20 strokes of the Teflon pestle, and the suspension was recentrifuged at 17,000 g at 4°C for 10 min. The final sarcolemmal pellet was suspended in 200-300 µl of sucrose buffer and frozen at -70°C until the Western blot analysis, enzyme assays, or glucose uptake measurements were performed. The supernatant saved earlier in the procedure for preparing the LDM subfraction was centrifuged at 48,000 g for 30 min. The supernatant from this centrifugation was further subjected to a second centrifugation at 250,000 g over 1 h in an ultracentrifuge to yield the final LDM subfraction, which was also stored at -70°C until Western blot analysis and enzyme assays were undertaken (28).

Enzyme Assays

Spectrophotometric assays of marker enzymes were undertaken to establish the relative purity of the subfractions isolated. K+-sensitive p-nitrophenol phosphatase was used as a marker for the plasma membrane and EGTA-sensitive Ca2+-ATPase as a marker for assessing contamination by the sarcoplasmic reticulum (3).

Western Blot Analysis

The skeletal muscle homogenates and fractionated sarcolemma/plasma membrane (PM) and LDM samples were sonicated (60 sonic, Dismembrator, Fisher Scientific, Pittsburgh, PA) using two 50-s cycles of 5-7 W. The resulting suspension was centrifuged at 10,000 g at 4°C for 10 min, and the supernatant was saved for Western blot analysis. Predetermined optimal protein concentrations of the homogenates (25 µg), PM (15 µg), or LDM (5 µg) subfractions obtained from skeletal muscle were subjected to discontinuous 10% SDS-polyacrylamide gel electrophoresis followed by electroblot transfer to nitrocellulose (Nytran, Schleicher & Schuell, Keene, NH). The nitrocellulose filters were rinsed once in PBS-T and blocked for 1 h in 5% nonfat dry milk at 22°C. The filters were washed three times (1 × 15 min and 2 × 10 min) in PBS-T followed by incubation for 1-2 h at 23°C with an affinity-purified rabbit anti-rat antibody (0.5 µg/ml) that was generated against the hemocyanin-limpet linked rat GLUT-1 (1:1,000 dilution) and rat GLUT-4 (1:2,500 dilution) COOH terminal 16 amino acids (1), which were synthesized as oligopeptides. After washing the filters three times with PBS-T, they were treated with the peroxidase-linked goat anti-rabbit IgG and subsequently exposed to a chemiluminescence reagent (Amersham Life Science, Little Chalfont, Buckinghamshire, UK). The chemiluminescence was captured on X-ray film over a range of exposure times (1 to 5 min) (1) to determine the optimal exposure time. GLUT-1 and GLUT-4 protein concentrations were assessed by quantification of the protein bands by densitometry. The presence of linearity between the time of X-ray film exposure and the optical density of the GLUT-1 or GLUT-4 bands was initially ensured.

2-Deoxy-Glucose Uptake in Isolated Sarcolemmal Vesicles

Plasma membrane fractions prepared from either the neonatal or adult rat skeletal muscle were homogenized in 250 mM sucrose, 20 mM HEPES-Tris, pH 7.2, using a Potter-Elvehjem homogenizer and centrifuged at 48,000 g for 30 min. 2-Deoxy-glucose transport studies using sarcolemmal vesicles were conducted at 37°C using the Millipore filtration technique. 2-[14C]deoxyglucose uptake experiments were initiated by mixing 5 µl membrane suspension with 45 µl of radiolabeled incubation medium (same as vesicle loading buffer) having either 1 mM 2-[14C]deoxyglucose or both 1 mM 2-[14C]deoxyglucose and 0.1 mM cytochalasin B. Uptake of 2-[14C]deoxyglucose was terminated by injecting 20 µl of the reaction mixture into 2 ml of ice-cold stop solution (same composition as the incubation medium without the radiolabeled tracer). This was then filtered through a Millipore filter (0.2 mm) and was washed with another 8 ml of ice-cold stop solution. Filters containing the washed vesicles were placed in scintillation cocktail, and the radioactivity was assessed in a 1219 Beta scintillation spectrometer (LKB Wallace) (14). All isotope transport values were corrected for a "vesicle blank" obtained by adding the incubation medium and the vesicles directly into the stop solution. Glucose uptake in the presence of cytochalasin B was subtracted from the total glucose uptake to calculate the carrier-mediated glucose transport by these skeletal muscle sarcolemmal vesicles (14). Carrier-mediated glucose transport is the same as cytochalasin B-inhibitable glucose uptake.

Data Analysis

Data are expressed as means ± SE. Comparisons between the age-matched and time-matched insulin- and vehicle-treated groups were undertaken by Student's t-test, with P < 0.05 considered as significant. When more than three groups were compared simultaneously, such as the inter-time comparisons per treatment group, the one-way analysis of variance was employed followed by a post hoc Newman-Keuls test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Table 1 demonstrates the plasma glucose and insulin concentrations in the fetus, newborn, and adult in both the vehicle- and insulin-treated groups employed in the immunohistochemical investigations. As can be seen, at all three ages, intraperitoneal administration of insulin led to a substantial increase in circulating insulin concentrations spanning from 20 to 60 min. In the fetus and newborn, the increase in plasma insulin concentrations ranged from 6- to 20-fold, and in the adult the increase was 20-fold. Concomitantly, a threefold decline in circulating glucose levels was observed from 20 through 60 min in the fetus and adult, but only a 1.5-fold decline in glucose concentrations was noted in the newborn, although the absolute plasma glucose values after insulin treatment were the same at all three ages. The 10 min insulin and glucose concentrations were not assessed. The fetal vehicle group reflected a glucose concentration closer to the adult level, perhaps reflective of the stress associated with the surgical procedure necessary for delivering insulin directly into the fetus.

                              
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  		Table 1.   Plasma insulin and glucose concentrations

In response to the in vivo insulin treatment that led to hyperinsulinemia and hypoglycemia, we observed no substantial change in either GLUT-1 or GLUT-4 total amounts in the fetal and neonatal skeletal muscle homogenates from 10 to 60 min after the treatment (Table 2) compared with the time- and age-matched vehicle treatment group. In the adult, although GLUT-1 was undetectable on Western blot analysis, GLUT-4 quantification in both the EDL and soleus revealed no change due to insulin treatment (reflects the insulin-responsive state) between 10 and 60 min compared with the vehicle-treated group (reflects the basal state).

                              
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  		Table 2.   GLUT-4 and GLUT-1 concentrations in skeletal muscle

Figure 1 depicts the GLUT-1 immunolocalization results. GLUT-1 immunoreactivity was observed in the sarcolemmal membrane of the fetal skeletal myotubes (Fig. 1A), the neonatal myocytes (Fig. 1B), and minimally in adult myocytes (Fig. 1C) (EDL and soleus) in the vehicle-treated sections (Fig. 1, A-C). However, the most prominent GLUT-1 localization in the adult was not to the myocytes but to the perineural sheaths. In contrast, as seen in Fig. 2, GLUT-4 immunoreactivity was noted in low amounts in the fetal myotubes (Fig. 2A) and neonatal myocytes (Fig. 2B), but significant amounts were noted in adult myocytes (Fig. 2C) of the vehicle-treated sections (Fig. 2, A-C). GLUT-4 immunoreactivity was not associated with the sarcolemma, but mainly was in intracellular compartments, causing a punctate appearance (Fig. 2, A-C). Insulin treatment led to no change in GLUT-1 immunolocalization in the fetus (Fig. 2D), newborn (Fig. 2E), or adult (Fig. 2F) skeletal muscle at 10, 20, 40, or 60 min compared with the time- and age-matched vehicle treated group (Fig. 1, D-F). Similarly, insulin did not perceptibly affect the fetal or neonatal skeletal muscle diffuse subcellular GLUT-4 localization pattern (Fig. 2, D and E). However, in the adult, a more prominent sarcolemma-associated GLUT-4 with a relative diminution in the diffuse subcellular punctuate GLUT-4 immunoreactivity was observed in the EDL muscle (Fig. 2F). Semi-quantification of the number of myotubes/myocytes that demonstrated sarcolemmal association of GLUT-4 increased in response to insulin only in the adult muscle (EDL shown) (Fig. 3C), but not in the fetal or neonatal skeletal muscle (Fig. 3, A and B). Quantification of the immunoreactivity that was associated with the sarcolemmal region revealed no effect of insulin on the fetal and neonatal GLUT-1 (fetus: vehicle = 94 ± 2.3 vs. insulin = 94 ± 2.2; newborn: vehicle = 98.25 ± 0.6 vs. insulin = 98.75 ± 0.58%) and GLUT-4 immunoreactivity (Fig. 3, A and B). In contrast, in the adult EDL, a 1.5-fold increase in the sarcolemma GLUT-4 was noted as a percent of total myocytic GLUT-4 immunoreactivity in response to insulin (Fig. 3D). A similar phenomenon was observed in the soleus muscle as well (data not shown).


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  		Fig. 1.   Skeletal muscle GLUT-1 immunolocalization studies. Representative GLUT-1 immunoreaction (shown by arrows) in vehicle (A-C)- and insulin (D-F)-treated fetus (A, D), newborn (B, E), and adult (C, F) skeletal myotubes (T), myocytes (M), and perineural sheaths (PN) is seen after 20 min of intervention (insulin or vehicle). Adult extensor digitorum longus (EDL) muscle is shown here. Scale bar (1 cm) = 0.01 mm. Magnification ×40. d, Day.



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  		Fig. 2.   Skeletal muscle GLUT-4 immunolocalization studies. Representative GLUT-4 immunoreaction (shown by arrows) in vehicle (A-C)- and insulin (D-F)-treated fetus (A, D), newborn (B, E), and adult (C, F) skeletal myotubes and myocytes is seen after 20 min of intervention (insulin or vehicle). Adult EDL muscle is shown here. Scale bar (1 cm) = 0.01 mm. Magnification ×40.



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  		Fig. 3.   Semi-quantification of skeletal muscle GLUT-4 immunolocalization. Percent myotubes/myocytes with sarcolemmal GLUT-4 among the total number of myotubes/myocytes in fetal (A), newborn (B), and adult (C) skeletal muscle (EDL) sections is depicted. Sarcolemmal GLUT-4 as a percent of the total myocytic cellular GLUT-4 immunoreactivity is also shown only in the adult skeletal muscle where insulin-induced translocation was observed (D). n is shown by each bar.

Plasma insulin concentrations in animals employed for the subfractionation studies revealed a 20-fold increase in the newborn (control = 2.47 ± 0.83 vs. insulin treated = 43 ± 6.42 ng/ml; P < 0.001) and a similar increase in the adult (control = 1.32 ± 0.34 vs. insulin treated = 36.84 ± 7.18 ng/ml; P < 0.001). Subfractionation experiments revealed minimal GLUT-4 in the adult sarcolemmal membrane fraction in the vehicle treatment group. Insulin treatment led to a fourfold increase in sarcolemmal-associated GLUT-4 at 20 min. Concomitantly, although a large amount of GLUT-4 was present in the LDM fraction in the vehicle-treated group, insulin treatment led to an insignificant decline in the LDM GLUT-4 concentrations compared with the vehicle-treated group (Fig. 4B).


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  		Fig. 4.   Western blot analysis-membrane subfractionation studies. Western blot analysis demonstrating GLUT-4 in the newborn (A) and adult (B) sarcolemma/plasma membrane (PM) and low-density microsomes (LDM) prepared from the hindlimb mixed skeletal muscle obtained from the vehicle (Veh)- and insulin (Ins)-treated groups. Western blot analysis demonstrating GLUT-1 in the newborn (C) and GLUT-4 in the adult gastrocnemius muscle (D) in the vehicle- and insulin-treated groups. Representative Western blots are shown at top and quantification of the GLUT-1 and GLUT-4 bands at bottom. n is shown by each bar.

In the newborn skeletal muscle, compared with the adult, a higher amount of GLUT-4 was associated with the sarcolemma under vehicle-treated basal conditions. However, after insulin treatment, a twofold increase in sarcolemmal GLUT-4 is observed with a simultaneous decline in LDM GLUT-4 (Fig. 4A) compared with the vehicle treatment group. In contrast, insulin led to no change from that of the vehicle-treated group in the neonatal skeletal muscle sarcolemma-associated GLUT-1 concentrations (Fig. 4C). Similarly no change in the LDM GLUT-1 concentrations was observed secondary to insulin treatment. Analysis of the adult gastrocnemius muscle that is 60% enriched in the oxidative fiber type revealed a sixfold increase in sarcolemma-associated GLUT-4 concentrations due to insulin treatment (Fig. 4D). This insulin-induced increase in sarcolemmal GLUT-4 concentrations is higher than the fourfold increase observed in the adult mixed muscle fiber type (Fig. 4B).

The sarcolemma fraction isolated from the adult and newborn skeletal muscle and used in this study revealed an eightfold enrichment in the K+-sensitive p-nitrophenol phosphatase compared with the homogenate and the EGTA-sensitive Ca2+-ATPase activity was not detectable. The LDM fraction did not reveal any K+-sensitive p-nitrophenol phosphatase or EGTA-sensitive Ca2+-ATPase activity.

2-Deoxy-glucose uptake studies in isolated vesicles revealed a threefold higher amount of basal cytochalasin B inhibitable 2-deoxy-glucose uptake, which translates into glucose transport in the newborn vehicle-treated group compared with the adult counterpart. Whereas the ultimate cytochalasin B inhibitable 2-deoxy-glucose uptake achieved after insulin stimulation (basal + insulin responsive) was no different between the adult and newborn, the insulin-induced glucose uptake was twofold lower in the newborn compared with the adult (Fig. 5).


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  		Fig. 5.   2-Deoxy-glucose uptake studies. Cytochalasin B-inhibitable 2-deoxy-glucose uptake by sarcolemmal vesicles obtained from mixed skeletal muscle from the vehicle- and insulin-treated groups in the newborn and adult age groups. n is shown by each bar. *P < 0.05 vs. vehicle; #P < 0.05 vs. newborn.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We demonstrated differential subcellular localization of GLUT-4 in the fetus/newborn and adult skeletal muscle. In the adult, while sarcolemma GLUT-1 constitutively present in small amounts mediates some basal glucose transport (16, 27), GLUT-4 is present in sarcolemma vesicles, transverse tubular membranes, and terminal cisternae of the sarcoplasmic reticulum in the triadic junctions and mediates insulin-responsive glucose transport (34). In the present study, the age-related difference in insulin responsiveness of GLUT-4 may be due to several factors, one of them being that the basal concentration of GLUT-4 in the sarcolemma fraction is greater in the newborn skeletal muscle compared with the adult. This may reflect the higher circulating fetal insulin concentrations that the newborn was exposed to in utero compared with the vehicle-treated adult. The difference in circulating endogenous insulin concentrations between the fetus and adult is fourfold. Thus chronic exposure to endogenous concentrations of fetal insulin may lead to a cascade of events that culminate in greater sarcolemma GLUT-4 concentrations. The intracellular insulin signaling pathway may trigger either enhanced translocation of GLUT-4 in the basal state or decrease endocytosis (7, 22, 31). Negating this theory are the relatively similar circulating insulin concentrations in the vehicle-treated newborn and adult groups. Hence the basal GLUT-4 changes noted in the newborn may not be reflective of the innate insulin-induced alterations in the neonatal intracellular signaling mechanisms. However, if the higher fetal insulin concentrations initiate changes in the subcellular distribution of GLUT-4, changes that persist in the newborn and do not revert back on removal from the high insulin fetal environment, the insulin-induced cascade of events culminating in sarcolemma association of GLUT-4 in the newborn is plausible.

Exogenous insulin administration led to a lower increase in plasma insulin concentrations in the newborn compared with the fetus and adult; nevertheless, the increase was 5- to 10-fold higher than the basal values. Insulin-induced translocation that led to a twofold increase in the sarcolemma-associated GLUT-4 concentrations in the fetus/newborn although detected by subfractionation experiments was not detected by immunolocalization. Although the newborn skeletal muscle demonstrated insulin-induced translocation of GLUT-4, this induction was lower than that seen in the adult. Whereas the final plasma insulin concentrations achieved by insulin treatment were similar in the newborn and adult (i.e., 43 ± 6.4 vs. 36.84 ± 7 ng/ml), due to a relatively higher basal insulin concentration in the newborn (2.47 ± 0.83 ng/ml) compared with the adult (1.32 ± 0.34 ng/ml), a 17-fold increase from the basal value (vehicle treated) was observed in the former and a 27-fold increase in the latter. Hence, 40 U/kg dose of insulin was also tried in the newborn and yielded a similar level of GLUT-4 translocation to that observed with the 8 U/kg dose (unpublished observations).

Another contributory factor may be the existent higher basal concentrations of sarcolemmal GLUT-4. Prior exposure to higher endogenous fetal insulin concentrations may activate a cascade of events involved in forming the soluble N-ethyl-maleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex (22, 31), which is essential for GLUT-4 translocation, the effects of which persist in the newborn. These newborn specific changes in sarcolemma GLUT-4 are paralleled by alterations in glucose transport. Thus 2-deoxy-glucose uptake in the newborn probably reflects the function of both sarcolemma GLUT-4 and GLUT-1 unlike in the adult where GLUT-4 predominantly contributes toward the net glucose uptake. An alternate explanation for this age-dependent difference in insulin's effect on GLUT-4 subcellular distribution may relate to lower total amounts of GLUT-4 present in the fetus and newborn (35, 36). This may lead to a proportional decrease in the cellular GLUT-4 pool that is amenable to insulin-induced translocation. Perhaps a certain threshold of GLUT-4 is essential for activation of/interaction between the vesicular proteins (SNARE complex) (22, 31) before translocation. Alternatively, perhaps the translocation machinery is immature, or the process of unmasking the GLUT 4 COOH-terminus region involving a conformational change, or disruption of a protein-protein interaction (34), is not fully developed in the fetus/newborn. Contrasting this speculation are the increased amounts of neonatal sarcolemma GLUT-4 in the basal state compared with the adult. Insulin-stimulated inhibition of endocytosis mediated by interactions of dynamin II with amphiphysin to form the clathrin-coated vesicles being underdeveloped or dysregulated may form a more likely basis for this observation (33).

Regardless of the mechanism responsible for the present observation, fetal hyperglycemia decreases total concentrations of skeletal muscle GLUT-4 (8), whereas insulin-induced fetal hypoglycemia (8, 27) or uteroplacental insufficiency causing intrauterine growth restriction and fetal hypoglycemia (25) produces no change. However, despite the fetal hyperglycemia-induced changes in total GLUT-4 concentrations, an inefficient insulin-inducible translocation system may deter the functionality of GLUT-4 during the perinatal period. This makes the fetal/neonatal skeletal muscle glucose transport heavily weighted toward basal transport, thereby supporting cellular proliferation and growth while being relatively insulin resistant, especially when insulin action is defined in "classical" metabolic terms involving GLUT-4 alone.

Insulin does increase fetal/neonatal glucose uptake and use, although the effect is not comparable to that of the adult (4, 18). In vitro studies in fetal rat skeletal muscle explants demonstrated an enhanced insulin-induced glucose uptake by 24 h. This effect, however, was not mediated by GLUT-4, but rather by a pretranslational increase in GLUT-1 concentrations (30). In vivo studies in fetal sheep demonstrated a similar effect of insulin on skeletal muscle GLUT-1 concentrations by 60 min (1). Thus it is feasible that in the fetus/newborn compared with the adult, the effect of insulin may be mediated via increased sarcolemma GLUT-1 concentrations due to greater synthesis or decreased degradation rather than by insulin-induced posttranslational events. This fact is also supported by the presence of greater GLUT-1 amounts in the fetus/newborn (25, 26). Alternatively, but concomitantly, the consumption of a high-fat milk diet during the suckling phase may contribute to the low levels of GLUT-4 present in the newborn. In the adult, a high-fat diet is known to cause insulin resistance by altering insulin-induced GLUT-4 translocation compared with control rats maintained on a regular rat chow (17, 35). Thus the aberrant insulin-induced GLUT-4 translocation in the newborn mimicked that previously reported in adult rats maintained on a high-fat diet (17, 35).

Fetal insulin infusions cause an increase in fetal ovine skeletal muscle total GLUT-4 concentrations and glucose uptake by 60 min (1). The fetal sheep skeletal muscle is more mature than the rat, because fetal sheep skeletal muscle consists of myocytes similar to the newborn rat (1), whereas the fetal rat skeletal muscle consists of myotubes. Furthermore, skeletal muscle in the rat is undifferentiated until 1-3 wk of postnatal age (15, 32); hence it is not feasible to examine different muscle fiber types at 1-2 days of age. To make the newborn to adult comparison valid, we concentrated mainly on mixed muscle fiber type in the adult. It is quite possible that the insulin-induced translocation in the adult EDL and soleus may prove to be different from what we observed in mixed muscle. This is supported by our observation in the adult gastrocnemius muscle (oxidative:glycolytic ratio = 60:40), which demonstrated greater sensitivity to insulin than the adult mixed muscle fiber type. In fact, specific adult muscle fiber types, e.g., the oxidative type, may demonstrate better insulin responsiveness than the mixed muscle, thereby making the limitation of insulin-induced GLUT-4 translocation in the fetus/newborn compared with the adult more prominent.

Although we concentrated only on insulin-induced subcellular localization of GLUT-4, insulin is also known to enhance capillary blood flow (9) and induce conformational changes in skeletal muscle, leading to a 50% increase in the diameter of transverse tubules (34). Thus, in addition to differences in the GLUT-4 subcellular localization, both or one of these processes may also be immature in the fetus/newborn.

We conclude that in vivo high circulating concentrations of insulin do not acutely alter total GLUT-1 or GLUT-4 concentrations in skeletal muscle of the rat fetus, newborn, or adult. In contrast, whereas insulin enhances translocation of GLUT-4 to the adult sarcolemma/transverse tubules, a lesser effect is visible in the fetus/newborn. At all three ages, no effect of insulin is observed on the subcellular distribution of GLUT-1 that remains highly associated with the sarcolemma. Paralleling these observations, insulin increases glucose transport in adult skeletal muscle by fourfold, whereas it only increases it by twofold in the neonatal skeletal muscle. We speculate that while insulin is capable of translocating GLUT-4 in the newborn skeletal muscle, the concomitant effects of prior exposure to higher fetal insulin concentrations and/or an immature GLUT-4 translocation/endocytosis machinery is further compromised by the abundant GLUT-1 that exists during this developmental stage. Thus while GLUT-4 may to some extent mediate both basal and insulin responsive glucose transport in the fetus/newborn, the time-dependent increase in fetal/neonatal GLUT-1 due to insulin treatment may play a predominant role in mediating insulin's effect on glucose transport. The effect of GLUT-4-mediated insulin responsiveness of glucose transport, while present in the fetus/newborn, may be overshadowed by GLUT-1 and be fully realized only when the adult potential is achieved.


    ACKNOWLEDGEMENTS

This work was supported by grants from the National Institutes of Health (HD-42130, HD-25024, and HD-33997).


    FOOTNOTES

* J. He and M. Thamotharan contributed equally to this work.

Address for reprint requests and other correspondence: S. U. Devaskar, 10833 Le Conte Ave., MDCC B2-375, Los Angeles, CA 90095-1752.

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.

First published January 16, 2003;10.1152/ajpregu.00560.2002

Received 11 September 2002; accepted in final form 11 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Regul Integr Comp Physiol 284(4):R1138-R1146
0363-6119/03 $5.00 Copyright © 2003 the American Physiological Society


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