1 Department of Pediatrics,
Women & Infants Hospital, Brown University School of Medicine,
Providence, Rhode Island 02905-2401;
2 Robert Schwartz MD Center for
Metabolism and Nutrition, Three- to six-day-old lambs infused with 100 mU · kg
neonatal glucose homeostasis
THE EUGLYCEMIC HYPERINSULINEMIC CLAMP TECHNIQUE, as
described by DeFronzo et al. (9), is a useful approach to assess the isolated effects of hyperinsulinemia on glucose metabolism without the
associated insulin counterregulatory hormonal effects from hypoglycemia. By combining the clamp with infusion of a stable isotope,
endogenous glucose production can be quantified, and the contribution
of changes in glucose production vs. altered peripheral sensitivity to
insulin can be evaluated.
Farrag et al. (12) applied these techniques to the human preterm
neonate and reported that, in contrast to the adult, the neonate has
persistent glucose production and greater peripheral sensitivity to
insulin. In a subsequent investigation to evaluate the developmental
response to insulin, Farrag et al. (11) evaluated a group of preterm
infants early in the immediate neonatal period and again at the
conclusion of the neonatal period. Greater peripheral sensitivity to
insulin was noted in the preterm neonate early in the neonatal period,
but not later, compared with the term neonate. The investigators
concluded that an adult-like response to insulin requires maturation
past the neonatal period in the human neonate.
In this investigation, we combined our experience with the lamb as an
animal model for evaluation of glucose homeostasis in the newborn
period (6-8) and the euglycemic hyperinsulinemic clamp technique
(11, 12) to test the hypothesis that increased insulin sensitivity of
the neonate is due in part to an alteration in the expression of
glucose transport proteins. We have focused our attention on the effect
of euglycemic hyperinsulinemia on 1)
endogenous glucose production and GLUT-2 protein expression in the
liver and 2) glucose utilization and
GLUT-4 protein expression in muscle.
Animals and Research Study Design
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 · min
1
insulin required greater amounts of glucose to maintain euglycemia during a euglycemic hyperinsulinemic clamp compared with 31- to 35-day-old insulin-infused lambs (15.87 ± 3.47 vs. 4.30 ± 1.11 mg · kg
1 · min
1,
P < 0.05, respectively). Endogenous
glucose production persisted in both groups; however, the percent
decrease compared with age-matched lambs receiving no insulin was
greater in the younger group compared with the older group (53%,
P < 0.001, vs. 34%,
P < 0.01). The younger animals
showed greater glucose utilization compared with the older animals (215 vs. 96%, respectively, P < 0.01).
No effect of insulin was noted on GLUT-4 protein expression in either
group. GLUT-2 expression was increased in older vs. younger lambs.
Older insulin-infused lambs showed lower GLUT-2 expression than older 0 insulin-infused lambs [0.94 ± 0.07 vs. 1.64 ± 0.10 (OD) units, P < 0.005].
Increased sensitivity to insulin in the younger animals was not related
to acute changes in GLUT-4 expression. Increased GLUT-2 expression with
age, as well as decreased expression with hyperinsulinemia, is
consistent with the development of an insulin-resistant state in the adult.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5 days before either delivery or study with ad
libitum food and water. To ensure that the lambs were in a
postabsorptive state for the study, early groups (3- to 6-day-old
newborns) were fasted overnight for 12 h. The late groups (31- to
35-day-old lambs) were fasted for 72 h, with water being withheld for
the final 12 h. The mean weight of the animals at the time of study was
4.43 ± 0.22 g for the early groups and 11.59 ± 0.76 g for the
late groups. Lambs were secured on a surgery table, their necks were
shaved, and lidocaine was administered as the anesthetic. Cotton
blindfolds were placed on the animals to help calm them. An incision,
~1.5 in. long, was made over the jugular vein. A 5-Fr polyurethane
catheter was inserted ~8 cm into the vessel to facilitate the
administration of study solutions. A similar procedure was used for the
carotid artery for blood sampling. Animals were placed in a mesh sling for the remainder of the study. No additional sedation was utilized. Animals were kept blindfolded and primarily slept throughout the study.
Euglycemic Hyperinsulinemic Clamp Procedure
Each study lasted 300 min and began after a 1-h recovery from surgery. A blood sample was obtained immediately before the study to measure blood glucose concentration and background isotopic enrichment of glucose. The first 180 min constituted the basal period of the study. This period started with a prime plus constant infusion of deuterated glucose (D-[6,6-2H2]glucose) at a rate of 0.04 mg · kgStable isotope tracer. Deuterated glucose (D-[6,6-2H2]glucose) was obtained from Cambridge Isotope Laboratories (Woburn, MA) and used as the stable isotopic tracer. The lyophilized material was prepared as a separate stock solution in 0.45% saline solution and tested for sterility and pyrogens by Ethide Sterilizing (Coventry, RI) according to Food and Drug Administration standards. The solution was stored in sterile standard pharmacy containers at 4°C.
Preparation of infusates. All reagents were prepared on the morning of each study. Regular human insulin (Humulin R, recombinant DNA origin, Eli Lilly, Indianapolis, IN) was prepared in 0.45% saline solution containing 1% albumin to a final concentration of 20 mU/ml. Ten percent dextrose water was used for exogenous glucose delivered by a Medfusion pump model 2010 (Medfusion, Duluth, GA)
Preparation of blood samples. Blood was centrifuged to obtain plasma. Plasma 2H2 enrichment glucose turnover was determined as follows. Plasma proteins were precipitated with 70% acetone, and the resultant supernate was passed over an anion (Dowex AG-1-X8), cation (Dowex AG-50-W-X8) exchange column and rinsed with H2O. The pentacetate derivative was prepared, and the enrichment was determined by gas chromatography-mass spectrometry (GC-MS) on a Hewlett-Packard (Palo Alto, CA) 5988B GC-MS by electron impact ionization at mass-to-charge ratios (m/z) 200 and m/z 202. Sample enrichment was calculated relative to a standard curve run simultaneously.
The following parameters were measured during the basal and clamp periods. Insulin concentration was measured by radioimmunoassay (Diagnostic Product, Los Angeles, CA). Glucagon concentration was measured by radioimmunoassay (ICN Pharmaceuticals, Costa Mesa, CA). Cortisol was measured by radioimmunoassay (INCSTAR, Stillwater, MN). Blood gases were measured on a Ciba-Corning 238 pH/blood gas analyzer (Ciba-Corning, Norwood, MA). Heart rate and systolic and diastolic pressures were recorded on a Corometrics 556 Monitor (Corometrics Medical Systems, Wallingford, CT).Calculations. The glucose turnover was calculated according to the equations of Steele as they apply to isotopic non-steady-state conditions (21), as we have utilized previously (12).
The insulin sensitivity index (ISI) at euglycemia, as reported by Bergman et al. (1), was calculated by the following
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Western Blot Analysis
At the conclusion of the clamp study, the animals were immediately euthanized, and tissue samples were rapidly dissected. Tissue collection was completed as quickly as possible (i.e., within 3-5 min). The tissues were then snap-frozen in liquid nitrogen and stored atTissue samples were homogenized on ice in homogenization buffer (0.25 M
sucrose, 0.5 mM EDTA, 50 mM HEPES, pH 7.4, containing aprotinin,
leupeptin, and 4-(2-amino ethyl)-benzenesulfonyl
fluoride). Samples were then centrifuged at 1,200 g for 10 min. The resulting supernatant was collected and saved on ice. The pellet was
rehomogenized and centrifuged at 1,200 g for 10 min. The supernatants were
pooled and centrifuged at 9,000 g for
10 min. The resulting supernatant was centrifuged at 100,000 g for 2 h (10, 15, 16, 19). The pellet
was then resuspended in buffer, and protein was determined by the
bicinchoninic acid method (Pierce, Rockford, IL). Samples were
solubilized for 30 min at room temperature in sample buffer (0.5 M
Tris · HCl, pH 6.8, 10% SDS, glycerol,
-mercaptoethanol, 2H2O,
and 0.5% bromophenol blue) and loaded onto 10% gels, 50 or 100 µg
protein/lane. Prestained molecular mass markers, positive controls, negative controls, and a protein pool sample, to assess the
efficiency of transfer, were run on each gel. Proteins were transferred
to polyvinylidene fluoride membrane using a semi-dry transfer apparatus
(Fisher Scientific, Pittsburgh, PA). Equality of loading and transfer
efficiency were also assessed by Coomassie Blue staining of the gel.
Membranes were then blocked at 37°C for 1 h in 5% (wt/vol) nonfat
dry milk in Tris-buffered saline with Tween 20 (TBST). This was
followed by incubation with either rabbit anti-rat GLUT-2 or rabbit
anti-rat GLUT-4 antibody (1:500 dilution with 5% normal donkey serum)
for 1 h at room temperature. After extensive washing in TBST, the
membranes were incubated with donkey anti-rabbit horseradish peroxidase
immunoglobulin G (Amersham, Arlington Heights, IL) at 1:5,000 with 5%
nonfat dry milk for 1 h at room temperature. Membranes were again
extensively washed in TBST, and immunoreactivity was detected using ECL
on Hyper film. Autoradiograms were quantified by scanning densitometry, and the results are reported in arbitrary optical density (OD) units.
Statistics
Statistical analysis was by SAS Proc Mixed (SAS Institute, Cary, NC). The data obtained during the baseline and clamp periods were averaged to determine the mean value for each concentration (i.e., blood glucose) or measurement (i.e., heart rate) for each group. The groups were subsequently compared using between- and within-animal comparisons over time. Statistical significance was at a level of P ![]() |
RESULTS |
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Figure
1A
depicts the blood glucose concentration over time for the basal and
euglycemic hyperinsulinemic clamp periods. Euglycemia was maintained
during all of the steady-state insulin infusions. Blood glucose
concentrations during the clamp period are listed in Table
1. There were no significant differences
between the basal and clamp periods within any group. The early groups
did have significantly greater glucose concentrations compared with the
late groups in the basal time periods: 88 ± 7 mg/dl (early basal 0 insulin) vs. 58 ± 7 mg/dl (late basal 0 insulin)
(P < 0.01); 91 ± 6 mg/dl (early
basal 100 mU · kg1 · min
1
insulin) vs. 61 ± 3 mg/dl (late basal 100 mU · kg
1 · min
1
insulin) (P < 0.01). The
early groups also had significantly greater glucose concentrations
compared with the late groups in the clamp periods: 88 ± 7 mg/dl
(early clamp 0 insulin infused) vs. 58 ± 1 mg/dl (late clamp 0 insulin infused); 92 ± 6 mg/dl (early clamp 100 mU · kg
1 · min
1
insulin infused) vs. 61 ± 2 mg/dl (late clamp 100 mU · kg
1 · min
1
insulin infused), P < 0.01.
|
|
Figure 1B depicts the total glucose
appearance rates, which are listed in Table 1 for the clamp period. The
total glucose appearance rates were significantly higher in the early
groups vs. the late groups during the basal period: 5.86 ± 0.03 mg · kg1 · min
1
(early basal 0 insulin infused) vs. 2.91 ± 0.33 mg · kg
1 · min
1
(late basal 0 insulin infused), P < 0.05; 7.33 ± 0.53 mg · kg
1 · min
1
(early basal 100 mU · kg
1 · min
1
insulin infused) vs. 2.93 ± 0.04 mg · kg
1 · min
1
(late basal 100 mU · kg
1 · min
1
insulin infused), P < 0.01. There
were no significant differences in the clamp periods when 0 insulin was
infused compared with the corresponding basal periods: 5.49 ± 0.03 mg · kg
1 · min
1
(early clamp 0 insulin infused) vs. 5.86 ± 0.03 mg · kg
1 · min
1
(early basal 0 insulin infused); 2.82 ± 0.30 mg · kg
1 · min
1
(late clamp 0 insulin infused) vs. 2.93 ± 0.04 mg · kg
1 · min
1
(late basal 0 insulin infused). During the hyperinsulinemic clamp period, there were significant increases in glucose appearance rate
from basal values in the 100 mU · kg
1 · min
1
insulin groups: 22.56 ± 2.24 mg · kg
1 · min
1
(early clamp 100 mU · kg
1 · min
1
insulin infused) vs. 7.33 ± 0.53 mg · kg
1 · min
1
(early basal 100 mU · kg
1 · min
1
insulin infused), P < 0.01; 5.86 ± 1.00 mg · kg
1 · min
1
(late clamp 100 mU · kg
1 · min
1
insulin infused) vs. 2.93 ± 0.04 mg · kg
1 · min
1
(late basal 100 mU · kg
1 · min
1
insulin infused), P < 0.001.
Figure 1C depicts endogenous glucose
production (EGP) rates, which are also listed in Table 1. EGP rates
were significantly higher in the early groups vs. the late
groups during the basal period: 5.83 ± 0.33 mg · kg1 · min
1
(early basal 0 insulin infused) vs. 2.86 ± 0.33 mg · kg
1 · min
1
(late basal 0 insulin infused); 7.29 ± 0.53 mg · kg
1 · min
1
(early basal 100 mU · kg
1 · min
1
insulin infused) vs. 2.89 ± 0.04 mg · kg
1 · min
1
(late basal 100 mU · kg
1 · min
1
insulin infused), P < 0.01. There
were no significant differences in the clamp periods when 0 insulin was
infused compared with the corresponding basal periods: 5.46 ± 0.33 mg · kg
1 · min
1
(early clamp 0 insulin infused) vs. 5.83 ± 0.33 mg · kg
1 · min
1
(early basal 0 insulin infused); 2.74 ± 0.30 mg · kg
1 · min
1
(late clamp 0 insulin infused) vs. 2.86 ± 0.33 mg · kg
1 · min
1
(late basal 0 insulin infused). During the hyperinsulinemic clamp periods there were significant decreases in glucose production rates
from basal values in the insulin-infused groups: 3.61 ± 1.18 mg · kg
1 · min
1
(early clamp 100 mU · kg
1 · min
1
insulin infused) vs. 7.29 ± 0.53 mg · kg
1 · min
1
(early basal 100 mU · kg
1 · min
1
insulin infused), P < 0.01; 1.55 ± 0.33 mg · kg
1 · min
1
(late clamp 100 mU · kg
1 · min
1
insulin infused) vs. 2.89 ± 0.04 mg · kg
1 · min
1
(late basal 100 mU · kg
1 · min
1
insulin infused), P < 0.05.
Plasma insulin concentrations were not significantly different between
the groups during the basal period: 8 ± 2 µU/ml (early basal 0 insulin infused), 17 ± 1 µU/ml (late basal 0 insulin
infused), 10 ± 2 µU/ml (early basal 100 mU · kg1 · min
1
insulin infused), 13 ± 2 µU/ml (late basal 100 mU · kg
1 · min
1
insulin infused). During the clamp period, plasma insulin
concentrations were significantly higher in the insulin-infused groups,
as listed in Table 1.
Plasma cortisol concentrations were not significantly different between
the groups in the basal period: 8.9 ± 2.1 µg/dl (early basal 0 insulin infused), 14.2 ± 1.3 µg/dl (late basal 0 insulin infused), 9.7 ± 2.7 µg/dl (early basal 100 mU · kg1 · min
1
insulin infused), 6.1 ± 1.9 µg/dl (late basal 100 mU · kg
1 · min
1
insulin infused). Plasma cortisol concentrations during the clamp period were also not significantly different between the groups, as
listed in Table 1.
Plasma glucagon concentrations were not significantly
different between the groups during the basal period: 500 ± 192 pg/ml (early basal 0 insulin infused), 650 ± 93 pg/ml (late basal 0 insulin infused), 526 ± 103 pg/ml (early basal 100 mU · kg1 · min
1
insulin infused), 376 ± 46 pg/ml (late basal 100 mU · kg
1 · min
1
insulin infused). There were no differences in the glucagon
concentrations during the clamp period, as listed in Table 1.
The late groups of animals had a lower heart rate than the early groups (P < 0.01), but no group showed a change in rate between basal and clamp periods. Systolic and diastolic pressures were measured during both the basal and clamp periods, and there were no significant differences between the groups. No significant differences were detected in pH, PO2, or PCO2 between the groups during either the basal or clamp periods.
Figure 2 depicts the percent decrease in
EGP and the percent increase in glucose utilization. EGP was
significantly reduced at both ages in the insulin-infused groups
compared with the age-matched groups receiving no insulin (53%
reduction in the early group, P < 0.001, and 34% reduction in the late group,
P < 0.01). There was a significant
difference in percent reduction between the two insulin-infused groups
(P < 0.05).
|
The increase in glucose utilization was significant in the infused groups compared with the noninfused groups (P < 0.001, respectively, for early and late infused groups vs. age-matched noninfused groups). The early animals showed a significantly greater increase in utilization compared with the late groups of animals (215 vs. 96%, respectively, P < 0.01).
Figure 3 shows the ISI at euglycemia, as
well as the MCR of insulin calculated for the early and late groups
during infusion of 100 mU · kg1 · min
1
insulin. The early group had an ISI of 0.115 ± 0.048, which was significantly greater than the ISI for the late group (0.033 ± 0.019), P < 0.005. The MCR was not
different between the early and late insulin-infused groups: 6.81 ± 0.61 (early group) vs. 6.65 ± 0.50 (late group).
|
Figure 4A
shows representative Western blots of GLUT-4 protein in muscle and
GLUT-2 protein in liver. Fifty micrograms of crude muscle or 100 µg
of crude liver membrane protein were loaded per lane. Five-day-old 0 insulin, 5-day-old 100 mU · kg1 · min
1
insulin, 30-day-old 0 insulin, and 30-day-old 100 mU · kg
1 · min
1
insulin groups are shown. Positive and negative rat membrane protein
controls were run simultaneously on each gel. For muscle gels, the
positive controls were rat muscle membrane proteins, and the negative
controls were rat liver membrane proteins. For the liver gels, the
positive controls were rat liver membrane proteins, and the negative
controls were rat muscle membrane proteins.
|
Figure 4B demonstrates results quantified by scanning densitometry of autoradiograms. Data are means ± SE reported in OD units. GLUT-4 protein concentration was not significantly altered with insulin infusion in any group: 5-day-old 0 insulin, 5.67 ± 0.12; 5-day-old 100 insulin, 5.99 ± 0.01; 30-day-old 0 insulin, 5.67 ± 0.01; 30-day-old 100 insulin, 5.70 ± 0.01. GLUT-2 protein concentration increased with age: 5-day-old 0 insulin, 0.77 ± 0.01 vs. 30-day-old 0 insulin, 1.52 ± 0.12, P < 0.005. Euglycemic hyperinsulinemia decreased GLUT-2 expression in the older animals: 30-day-old 0 insulin, 1.52 ± 0.12 vs. 0.94 ± 0.07, P < 0.005.
Table 1 shows the metabolic data during the clamp period of the studies. The amount of glucose infused to maintain euglycemia in the late group was significantly lower than that in the early group (P < 0.05).
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DISCUSSION |
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In this investigation we examined glucose homeostasis throughout the neonatal period, utilizing our published lamb model (6-8). To evaluate glucose homeostasis during this developmental period at the cellular level, we also examined changes in the glucose transport system. We studied lambs at 3-6 days of age, early in the neonatal period, and at 31-35 days, late in the neonatal period. Muscle and liver tissue samples were harvested at the conclusion of euglycemic hyperinsulinemic clamp studies for glucose transporter evaluation.
Kliegman et al. (14) first used the euglycemic hyperinsulinemic clamp
in newborn beagle puppies. At comparable plasma insulin concentrations,
glucose production in the adult group was completely suppressed,
whereas in the newborns complete suppression was not achieved. The
investigators attributed the persistent glucose production to hepatic
insulin resistance. Farrag et al. (12) recently reported the use of the
hyperinsulinemic euglycemic clamp for the first time in the human
neonate. Persistent glucose production was apparent across a wide range
of insulin infusion rates (0.2-4.0 mU · kg1 · min
1).
EGP was sensitive to low insulin concentrations, plateaued quickly, and
became nonresponsive to higher insulin concentrations. Beginning at the
insulin infusion rate of 0.5 mU · kg
1 · min
1
in the human preterm neonate, there was a significant reduction in EGP
ranging from 41-58% in the groups studied. If suppression of
glucose production is the maximal effect of insulin on the liver, then
this effect was not achieved in the human preterm neonate. Peripheral
glucose utilization increased over basal rates only at insulin infusion
rates of 2 and 4 mU · kg
1 · min
1.
The investigators were not able to determine the maximal effect on
glucose utilization because a plateau was not reached at the insulin
infusion rates employed. However, the neonatal glucose utilization
response to insulin far exceeded the maximal response reported in the adult.
In the present study, we have successfully employed the euglycemic
hyperinsulinemic clamp in the neonatal lamb. The choice of 100 mU · kg1 · min
1
insulin infusion rate was based on a series of preliminary studies that
used a range of rates from 2.0 to 500 mU · kg
1 · min
1.
At lower infusion rates no effects of insulin were detected. Not until
rates of 25-50
mU · kg
1 · min
1
insulin were used did effects on glucose production and utilization begin to appear. Responses at 100 and 500 mU · kg
1 · min
1
insulin were not significantly different, and therefore we concluded that a maximal insulin response could be obtained at the 100 mU · kg
1 · min
1
insulin infusion rate. This rate is comparable to that used by Kleigman
et al. (14) in the newborn beagle puppy model but
considerably higher than the rate used by Farrag et al. (12) in the
human neonate.
Similar to data in the human neonate, EGP was not completely suppressed in the lamb despite very high plasma insulin concentrations. Interestingly, the early neonatal group appeared to be more responsive to insulin, resulting in a significantly greater percent decrease in EGP than in the late group (i.e., 53 vs. 38%, respectively, P < 0.05). Relative to peripheral glucose utilization, at comparable insulin infusion rates, the early group responded with a 215% rise over basal. This was significantly higher than the 96% increase in the late group. The late group also required significantly lower glucose infusion rates to maintain euglycemia during insulin infusion compared with the early group, as noted in Table 1. These data are consistent with data from the human preterm neonate compared with the adult, because the neonate showed persistent glucose production as well as greater peripheral sensitivity to insulin.
The late groups had significantly lower blood glucose concentrations
compared with the early groups. We attribute this to the fact that, for
the older animals, food was withheld for 72 h, as advised by the Food
and Drug Administration and with the approval of the animal care
committees, because by 30 days these animals can be weaned and
considered true ruminants. Because of this difference in basal and,
therefore, clamp glucose concentrations between the groups, we utilized
the ISI to compare the peripheral sensitivity to insulin among the
groups (1). As was noted with the early preterm human neonate vs. the
late preterm neonate (11), the early group of lambs had significantly
greater peripheral sensitivity to insulin compared with the late group.
The MCR for insulin was not different between the groups. It may be
that at an infusion rate of 100 mU · kg1 · min
1
insulin there is a plateau in the clearance rate.
The appropriate distribution of whole body glucose is, at least in
part, regulated by the tissue-specific expression and regulation of
several glucose transporter isoforms with distinct kinetic properties.
GLUT-2 is the major glucose transporter isoform expressed in
hepatocytes, -cells, and kidney. The distinguishing feature of this
isoform is that it is a low-affinity high-turnover transport system.
Coupled with the kinetically similar hexokinase, glucokinase in
hepatocytes, and
-cells, GLUT-2 forms part of a glucose-sensing apparatus that responds to subtle changes in blood glucose with alterations in the rate of glucose uptake into the cell. GLUT-4 glucose
transporter is expressed in adipocytes and muscle cells. These are the
"insulin-sensitive" cell types, so called because they respond to
insulin with a rapid and reversible increase in glucose transport.
Glucose transport in insulin-sensitive tissues has received attention
because of the importance of this process in the maintenance of whole
body glucose homeostasis (15, 19).
In this study there appeared to be a developmental increase in GLUT-2 in the late groups vs. the early groups (P < 0.05). This increase may signal the onset of an insulin-resistant state in the ruminant (3, 4, 13, 20). Similar patterns of increased expression have been noted in studies of rats made diabetic by streptozotocin administration. After an initial decrease, GLUT-2 expression increased with time (5, 17). The reduction in expression of GLUT-2 with euglycemic hyperinsulinemia is in agreement with clamp studies in the diabetic rat. Forty eight to 72 h after streptozotocin injection, GLUT-2 protein levels increased in the liver of the diabetic rat. Physiological insulin infusion decreased GLUT-2 protein to levels below control levels (5). It should also be noted that, in ruminant liver, a high level of GLUT-5 mRNA has also been detected, implying that GLUT-5 may also be involved in the uptake and release of glucose (13). We have yet to explore this possibility in our model.
Acute euglycemic hyperinsulinemia caused no change in the expression of GLUT-4. This is consistent with previous studies showing that acute hyperinsulinemia is not a regulator of GLUT-4 expression (18). We speculate that changes in GLUT-4 expression are not directly responsible for the changes in insulin sensitivity.
In summary, we found persistent glucose production during the infusion
of 100 mU · kg1 · min
1
of insulin in both groups of animals. We noted that the late group of
insulin-infused animals were not sensitive to the effects of insulin in
that they 1) required very little
glucose infused to maintain euglycemia,
2) showed a lesser percent decrease
in EGP, and 3) had a lower percent
increase in glucose utilization compared with the early group. At the
level of the glucose transporter, GLUT-4 protein expression was not
significantly different between the two ages and was not affected by
acute euglycemic hyperinsulinemia. The expression of GLUT-2 appeared to
change with the age of the animals, increasing significantly as they
matured. Insulin infusion resulted in no change in the early animals
but a significant decrease in the late group.
In conclusion, in the newborn lamb as in the human neonate: 1) persistent glucose production during insulin infusion was noted in all lambs studied, 2) an adult-like response to insulin requires maturation beyond the neonatal period. Although conclusions for other species should be made with caution because of differences in the regulation of glucose transporters between ruminants and nonruminants, insulin-sensitive GLUT-4 does not appear to be a pivotal protein in the control of glucose uptake and metabolism during acute euglycemic hyperinsulinemia in the neonatal period in the lamb. GLUT-2 expression increases with age and decreases with acute insulin infusion in the older animals. This appears to be consistent with the development of insulin resistance in the adult sheep.
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FOOTNOTES |
---|
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. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: R. M. Cowett, Dept. of Neonatology, The Children's Hospital M-62, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195-0001 (E-mail: cowettr{at}ccf.org).
Received 18 December 1998; accepted in final form 17 August 1999.
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