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. 2010 Mar 16;151(5):1990–1997. doi: 10.1210/en.2009-0777

The Ontogeny of Insulin Signaling in the Preterm Baboon Model

Cynthia L Blanco 1, Hanyu Liang 1, Joaquin Joya-Galeana 1, Ralph A DeFronzo 1, Donald McCurnin 1, Nicolas Musi 1
PMCID: PMC2869258  PMID: 20233798

Abstract

Hyperglycemia, a prevalent condition in premature infants, is thought to be a consequence of incomplete suppression of endogenous glucose production and reduced insulin-stimulated glucose disposal in peripheral tissues. However, the molecular basis for these conditions remains unclear. To test the hypothesis that the insulin transduction pathway is underdeveloped with prematurity, fetal baboons were delivered, anesthetized, and euthanized at 125 d gestational age (GA), 140 d GA, or near term at 175 d GA. Vastus lateralis muscle and liver tissues were obtained, and protein content of insulin signaling molecules [insulin receptor (IR)-β, IR substate-1, p85 subunit of phosphatidylinositol 3-kinase, Akt, and AS160] and glucose transporters (GLUT)-1 and GLUT4 was measured by Western blotting. Muscle from 125 d GA baboons had markedly reduced GLUT1 protein content (16% of 140 d GA and 9% of 175 d GA fetuses). GLUT4 and AS160 also were severely reduced in 125 d GA fetal muscle (43% of 175 d GA and 35% of 175 d GA, respectively). In contrast, the protein content of IR-β, IR substate-1, and Akt was elevated by 1.7-, 5.2-, and 1.9-fold, respectively, in muscle from 125 d GA baboons when compared with 175 d GA fetuses. No differences were found in the content of insulin signaling proteins in liver. In conclusion, significant gestational differences exist in the protein content of several insulin signaling proteins in the muscle of fetal baboons. Reduced muscle content of key glucose transport-regulating proteins (GLUT1, GLUT4, AS160) could play a role in the pathogenesis of neonatal hyperglycemia and reduced insulin-stimulated glucose disposal.

Changes in insulin signaling proteins in muscle during normal fetal development are implicated in the development of transient hyperglycemia of premature infants.

The neonate lacks a precise control of glucose homeostasis when compared with the adult (1). In particular, premature infants weighing less than 1000 g have 18 times the risk of developing hyperglycemia when compared with those weighing more than 2000 g (2). Transient neonatal hyperglycemia has been reported in up to 80% of extremely premature infants and has been associated with intraventricular hemorrhage, retinopathy of prematurity, and increased risk of death (2,3,4,5). Our understanding of glucose metabolism in the neonate remains fragmentary. There is evidence that the hyperglycemia of the extremely low birth weight infant (birth weight <1000 g) may be due to inadequate suppression of endogenous glucose production (1,6). In addition, extremely low birth weight infants have elevated plasma insulin levels and an increased proinsulin to insulin ratio (7), indicating that these infants are insulin resistant. Nonetheless, the molecular basis for the reduced insulin-stimulated glucose disposal of prematurity remains unclear. In addition, there is a limited understanding regarding the developmental changes that occur within the insulin signaling pathway.

Skeletal muscle is the main site responsible for insulin-stimulated glucose disposal (8). For insulin to exert its effects on cellular glucose metabolism, this hormone must first bind to its receptor (IR) and initiate a signaling cascade that includes tyrosine phosphorylation of the IR and insulin receptor substrate (IRS)-1, followed by activation of phosphatidylinositol (PI) 3-kinase (9). Insulin-stimulated activation of PI3-kinase leads to translocation of the glucose transporter (GLUT)-4, resulting in an increase in glucose transport rate across the plasma membrane (10). The precise mechanism by which activation of PI 3-kinase causes GLUT4 translocation is still unknown, although phosphorylation/activation of phosphoinositide-dependent kinase 1 (11), Akt (12), protein kinase C (13), and AS160 (14) have been implicated. Several abnormalities distinguish insulin-resistant muscle from normal muscle, including decreased insulin-stimulated GLUT4 translocation, decreased IR and IRS-1 tyrosine phosphorylation, and decreased PI 3-kinase activity (15).

There is evidence suggesting developmental differences in the insulin signaling pathway (16,17). Studies performed in rats have found that insulin-stimulated GLUT4 translocation in muscle is lower in term rats when compared with adult animals (17,18). Others reported that aging is associated with changes in several insulin signaling proteins in the rat, including decreased phosphorylation of IRS-1, Akt, and AS160 (19).

Baboons have close (97%) phylogenetic proximity with humans (20). In addition, baboons can develop insulin resistance and diabetes spontaneously, a feature that distinguishes this animals from rodents, a frequently used model in diabetes research that very rarely develops insulin resistance and diabetes spontaneously (20). Previously we have shown that the adult baboon is a pertinent nonhuman primate model to examine the underlying cellular/molecular mechanisms responsible for insulin resistance (20). In this study we describe the incidence of hyperglycemia in a preterm baboon model. We also examined developmental differences of key insulin signaling proteins in skeletal muscle and liver in fetal baboons delivered prematurely at 67, 75, and 94% gestation. We hypothesize that the insulin signal transduction pathway is not fully developed in the liver and muscle of preterm baboons.

Materials and Methods

Incidence of hyperglycemia in preterm baboons

Thirty-two baboons were delivered prematurely at 125 d gestational age (GA) via cesarean section under general anesthesia from healthy, nondiabetic mothers at the Southwest National Primate Research Center at the Southwest Foundation for Biomedical Research (SFBR; San Antonio, TX). All studies were approved by the Institutional Animal Care Committee at the SFBR. Animal experiments were conducted in accord with accepted standards of humane animal care. Animals were intubated immediately after birth and chronically ventilated to maintain alive for up to 14 d. Central lines were placed for fluid management and parenteral nutrition. Minimal enteral feeds were initiated after 5 d of age if bowel gas pattern was considered normal. Sepsis was defined by at least one positive blood, urine, or cerebrospinal fluid culture. Antibiotics were provided accordingly. Plasma glucose concentrations were measured from central arterial lines soon after birth and then at a minimum of every 6 h. Hyperglycemia was defined as a plasma glucose concentration of 150 mg/dl or greater (8.3 mmol/liter) on at least two different occasions during the first 10 d of life; this level has been previously identified as being clinically relevant (5). Intravenous insulin was given at a dose of 0.5 IU/kg to treat plasma glucose greater than 200 mg/dl.

Measurement of insulin signaling and glucose transporter proteins

We studied 18 fetal baboons (10 males, eight females) that were delivered at SFBR. Animals were delivered prematurely via cesarean section under general anesthesia from healthy, nondiabetic mothers at 125 d GA, 140 d GA, or near term at 175 d GA (full term = 185 d GA) and euthanized immediately after birth. Immediately after euthanasia, vastus lateralis muscle and liver tissue samples were obtained, snap frozen in liquid nitrogen, and stored at −80 C.

Materials

Primary antibodies against IR-β, phosphotyrosine, and Akt-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), IRS-1, p85 subunit of PI 3-kinase, Akt-2, GLUT1, and GLUT4 were from Millipore (Chicago, IL), and glyceraldehyde-3-phosphate dehydrogenase, AS160, phospho-IR-β Tyr1361, phospho-Akt Ser473, and total Akt were from Cell Signaling Technology (Danvers, MA). Electrophoresis reagents were purchased from Bio-Rad (Hercules, CA), and enhanced chemiluminescence Western blot reagents and film were from Amersham (Arlington Heights, IL). Serum insulin levels were measured with 1-2-3 UltraSensitive human insulin ELISA (ALPCO Diagnostics, Windham, NH); plasma glucose was measured using the Analox GM9 analyzer (Analox Instruments, London, UK) and the AU 640 immunoanalyzer (Olympus Inc., Center Valley, PA).

Western blot analysis and immunoprecipitation

Muscle samples were homogenized in ice-cold lysis buffer [containing 20 mmol/liter Tris (pH 7.5), 10 mmol/liter sodium pyrophosphate, 100 mmol/liter sodium fluoride, 2 mmol/liter sodium orthovanadate, 5 mmol/liter EDTA (pH 8.0), 1% Nonidet P-40, 1 mmol/liter phenylmethylsulfonyl fluoride, 3 mmol/liter benzamidine, 10 μg/ml leupeptin, and 10 μg/ml aprotinin]. Homogenates were rotated for 1 h at 4 C and then centrifuged at 14,000 × g for 10 min at 4 C. The supernatants were collected and protein concentrations measured by the Bradford assay. Lysate proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. After blocking in Tris-buffered saline with 5% nonfat dry milk, the membranes were incubated overnight at 4 C with the primary antibody indicated above. Bound antibodies were detected with antirabbit immunoglobulin-horseradish peroxidase-linked whole antibody and by using ECL reagents. The intensities of the bands were quantified by densitometry using the NIH imaging program (National Institutes of Health, Bethesda, MD) with the results reported in arbitrary OD units. For measurement of IRS-1, tyrosine phosphorylation proteins were immunoprecipitated with anti-IRS-1 antibody followed by incubation with protein A agarose beads. The beads were washed extensively, and proteins eluted with Laemmli buffer were separated by SDS-PAGE and transferred to nitrocellulose membrane, followed by incubation with an antiphosphotyrosine antibody.

Statistical analysis

Statistical calculations and demographic distributions were performed with SPSS for Windows (version 11.5, SPSS, Inc., Chicago, IL). Differences between groups were determined using one-way ANOVA, followed by the Tukey test. P < 0.05 was considered to be statistically significant.

Results

Hyperglycemia in preterm baboons

Baboons studied had a mean birth weight of 370 ± 43 g and the mean survival was 12.3 ± 3.8 d. Fifty percent of the baboons were males. Twenty-eight of the 32 animals included in this study (87%) developed hyperglycemia. The mean plasma glucose levels per day are depicted in Fig. 1; a total of 1644 plasma glucose measurements were included in the final analysis. Insulin was used for the treatment of hyperglycemia in 60% of all animals (mean 1.7 ± 1.8 d of life). The mean glucose infusion rate during the peak glucose level found on d 1 was 5.0 ± 0.4 mg/kg · min. Three baboons developed sepsis during the study period.

Figure 1.

Figure 1

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Daily plasma glucose from 125 d GA baboons who survived longer than 7 d. Graphical data are means ± se.

Developmental differences of key insulin signaling proteins in skeletal muscle and liver

Animal characteristics

Eighteen animals were delivered at three different gestations (125 d GA, 140 d GA, and 175 d GA). Animal characteristics are shown in Table 1. Plasma insulin and glucose concentrations at birth were similar between groups (data not shown).

Table 1.

Animal characteristics of fetal baboons used to investigate developmental differences of key insulin signaling proteins in skeletal muscle and liver

Group Gestation (% of term) n Male/female Birth weight (g)
125 d GA 67 6 4/2 412 ± 33
140 d GA 75 6 1/5 552 ± 60
175 d GA 94 6 5/1 1046 ± 104
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sd is shown. 

Content of IR-β, IRS-1, and p85 subunit of PI3-kinase

Figure 2A shows that the protein content of IR-β in muscle was elevated by 1.9-fold in the most immature animals (P < 0.01). Consistent with this finding, IR-β Tyr1361 phosphorylation also was increased in the muscle of immature animals (P < 0.05) (Fig. 2B). The protein content of IRS-1 was also higher in the muscle of the preterm animals compared with the term animals (P < 0.01) (Fig. 2C). Similarly, IRS-1 tyrosine phosphorylation was elevated in the most immature animals (P < 0.05) (Fig. 2D). The muscle protein content of the p85 subunit of PI3-kinase was similar across all gestational ages (Fig. 2E).

Figure 2.

Figure 2

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Insulin signaling protein content and phosphorylation in muscle from fetal baboons born at different gestational ages. IR-β (A), phospho-IR-β Tyr 1361 (B), IRS-1 (C), IRS-1 tyrosine phosphorylation (D), and p85 subunit of PI3-kinase (E) were measured by Western blotting and immunoprecipitation (n = 6/group). Representative blots from three animals per group also are shown. Graphical data are means ± se. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *, P < 0.05; **, P < 0.01.

Muscle Akt content

Total Akt content was elevated by 1.7-fold in the muscle from 125 d GA baboons (P < 0.001) and 1.4-fold higher in baboons of 140 d GA (P < 0.05) when compared with 175 d GA fetuses (Fig. 3A). In line with Akt protein content, Ser 473 phosphorylation tended to be higher in the muscle of the most immature animals, although this difference did not reach statistical significance (Fig. 3B). Figure 3, C and D, shows that increases in the content of both Akt-1 and Akt-2 account for the elevation in total Akt protein observed in immature baboons.

Figure 3.

Figure 3

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Akt protein content and phosphorylation in muscle from fetal baboons. Total Akt (A), phospho-Akt-Ser 473 (B), Akt-1 (C), and Akt-2 (D) were measured by Western blotting (n = 6/group). Representative blots from three animals per group also are shown. Graphical data are means ± se. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. *, P < 0.05; **, P < 0.01.

Glucose transporter and AS160 content in muscle

In contrast to IR-β, IRS-1, and Akt, the muscle of 125 d GA fetal baboons had markedly lower GLUT1 protein content in the basal state (16% of 140 d GA and 9% of 175 d GA fetuses, P < 0.05) (Fig. 4A). GLUT4 protein also was significantly reduced in the fetal muscle at 125 d GA (35% of 140 d GA and 43% of 175 d GA, P < 0.05) (Fig. 4B). These reductions in GLUT1 and GLUT4 in 125 d GA fetuses were accompanied by lower content of the Rab GTPase activating protein AS160 (47% of 140 d GA and 35% of 175 d GA, P < 0.01) (Fig. 4C).

Figure 4.

Figure 4

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Glucose transporter and AS160 protein content in muscle from fetal baboons: GLUT1 (A), GLUT4 (B), and AS160 (C) protein content (n = 6/group). Representative blots from three animals per group also are shown. Graphical data are means ± se. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. *, P < 0.05; **, P < 0.01.

Insulin signaling molecules in liver

Opposite to our findings in muscle, the liver from fetal baboons had similar content of insulin signaling and glucose transporter proteins across gestational ages (Fig. 5, A–F).

Figure 5.

Figure 5

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Protein content of insulin signaling proteins and glucose transporters in liver from fetal baboons born at different gestational ages. GLUT1 (A), GLUT4 (B), IR-β (C), IRS-1 (D), p85 subunit of PI 3-kinase (E), and total Akt protein content (F) was measured by Western analysis (n = 6/group). Representative blots from three animals per group also are shown. Graphical data are means ± se. *, P < 0.05; **, P < 0.01.

Discussion

Hyperglycemia is a common condition that leads to significant morbidity in premature infants (2,3,4). However, the molecular basis for the hyperglycemia of prematurity is poorly understood. The purpose of this study was to examine whether developmental differences exist in the content of key insulin signaling proteins in muscle and liver during the fetal period. We used a baboon model because these animals share 97% homology with humans at the DNA level and spontaneously develop many diseases similar to those in man, including obesity and diabetes (20). In addition, we recently sequenced genes and proteins of several insulin signaling molecules in the baboon, which were found to be about 98% identical with those of man (20).

We found gestational differences in the protein content of several insulin signaling and glucose transporter proteins in the muscle of fetal baboons. The most striking differences observed were reductions in the content of GLUT1 and GLUT4 in immature baboons. GLUT4 is the predominant insulin-sensitive transporter in muscle and its translocation from an intracellular compartment to the plasma membrane is required for the ability of insulin to promote glucose transport (21). The physiological importance of GLUT4 in muscle is highlighted by studies in GLUT4 knockout mice, which showed that these animals are glucose intolerant and have severely depressed insulin-stimulated glucose transport in muscle (22). Because GLUT4 is ultimately responsible for transporting glucose into the cell in response to insulin, the reduction in GLUT4 content observed in immature animals likely plays an important role in the alterations of glucose homeostasis that occur in prematurity.

In line with a reduction in GLUT4 in more preterm baboons, these animals also had severely reduced content in GLUT1. Whereas GLUT4 is the main glucose transporter involved in postprandial glucose transport, when insulin concentrations in the circulation are the highest, GLUT1 is responsible for the constitutive, insulin-independent glucose transport that takes place in all cells, including muscle (23). Studying the role that GLUT1 plays on maintaining glucose homeostasis using knockout models has been limited because deletion of this gene is embryonically lethal (24). However, GLUT1 overexpression results in a 3- to 4-fold increase in basal glucose transport in muscle ex vivo, in fasting hypoglycemia and improved glucose tolerance (25), suggesting that GLUT1 also plays an important role in maintaining whole-body glucose homeostasis.

Along with reductions in GLUT1 and GLUT4, the most immature baboons had reduced content of AS160 in muscle. AS160 is a Rab-GAP (GTPase-activating protein), and phosphorylation of AS160 on phospho-Akt substrate motifs on insulin stimulation is thought to remove its inhibitory GAP activity and increases GLUT4 trafficking to the cell membrane (26,27). Therefore, our finding of reduced AS160 content in muscle from immature baboons is consistent with the observed reductions in the content of distal proteins involved in glucose transport.

In contrast to the reductions observed in distal signaling molecules, we found that some proximal signaling proteins such as IR-β, IRS-1, and Akt are increased in muscle from the more preterm animals. Akt, the IR, and IRS-1 play other roles besides insulin signaling. During embryonic development, IGFs function to promote growth, as evidenced by mutants lacking IGF-I or IGF-II, which develop severe intrauterine growth restriction (28). This growth-promoting function is mediated by two receptors, IR and the IGF-I receptor, with the role of the former being predominantly related to IGF-II signaling and growth control during most of the embryonic period (29). The role of IR in growth is further evidenced in infants with leprechaunism, a disease that results from mutations in the IR gene and leads to growth restriction and death (30). Regarding IRS-1, mice with targeted deletion of this protein are born alive but retarded in embryonal and postnatal growth (31). In human fetuses, variations in insulin concentrations contribute to alterations in fetal growth. For example, elevated insulin concentrations, as seen during uncontrolled maternal diabetes, is known to promote fetal overgrowth, whereas isolated fetal insulin deficiency under a stable maternal environment can cause fetal growth restriction (32,33). In addition, insulin promotes skeletal muscle protein synthesis and growth, which decreases progressively with maturity (34). Therefore, the increase in IR-β and IRS-1 content seen in immature animals could be related to the accelerated growth that occurs during early gestation.

Against our hypothesis, we found that total Akt protein content decreased progressively with age. Insulin-responsive tissues are enriched in Akt (35), and this protein is thought to be a key mediator of both cell growth and metabolism (12,36). Akt has three isoforms; in muscle, the most abundant are Akt-1 and Akt-2 (37,38). It has been proposed that Akt-1 and Akt-2 transmit distinct biological signals (39). In particular, Akt-1 is implicated in cell growth, whereas Akt-2 is thought to play a more important role in regulating glucose metabolism (39). Upon our observation of increased total Akt content in immature baboons, we expected that the increase in total Akt content would be accounted mainly by elevated Akt-1 due to the rapid rate of growth that occurs during early gestation. Nonetheless, we found significant increases in the content of both Akt isoforms in early gestation. Even though Akt-2 is thought to play a role in regulating glucose metabolism, there is scarce evidence of its specific physiological implications in animal models and humans. In addition, because insulin action seems to change with maturity, developmental differences in Akt isoforms may coexist (20). We speculate that the up-regulation in IR-β and Akt protein content in the muscle of the most immature baboons may be related to preferential insulin signaling toward growth and/or compensation to overcome the reductions in the content of distal insulin signaling proteins/glucose transporters.

In this study, we demonstrate that hyperglycemia is a common condition in preterm baboons, affecting up to 87% of the animal population studied. The incidence of hyperglycemia is similar of that reported in extremely premature infants (2,3,4,5,6). Moreover, fetal baboons were delivered at comparable gestational ages as seen in humans. In clinical practice, infants of youngest gestation are commonly delivered between 24–27 wk and have a birth weight less than 1000 g (60–67% gestation, compared with 125 d baboons delivered at 67% gestation); premature infants delivered at 30 wk weigh between 1000 and 1500 g (75% gestation, equivalent to 140 d baboons); and infants delivered at term are at least 37 wk of gestation (92% gestation, compared with 94% gestation of 175 d baboons). Due to the high incidence of hyperglycemia and reduced insulin-stimulated glucose disposal reported in premature infants of lowest gestation (24–27 wk, or <1000 g) (2,3,4,6), finding a significant down-regulation of GLUT1, GLUT4, and AS160 proteins in the immature baboons when compared with older gestations is very relevant to the hyperglycemia that occurs in premature infants.

It is possible that developmental changes in insulin signaling protein content in the muscle of preterm baboons could be due to concurrent variations in fetal insulin and/or glucose concentrations. However, plasma glucose and insulin levels measured at birth in the preterm animals evaluated for insulin signaling protein content were the same as the levels measured at birth in term animals.

In contrast to our findings in muscle, there were no differences in the content of insulin signaling proteins and glucose transporters in the liver across the different gestational ages. The lack of differences is consistent with the findings that hepatic dysfunction is a late event in the development and progression of diabetes (40).

In summary, this study provides evidence that hyperglycemia is a common condition in preterm baboons and that variations in insulin signaling proteins exist in muscle during normal fetal development. No differences were present in the liver from preterm animals. Down-regulation of AS160, GLUT1, and GLUT4 muscle protein content may be implicated in the development of transient hyperglycemia of premature infants. Future studies will be needed to evaluate whether preterm baboons have defects in the insulin transduction pathways under insulin-stimulated conditions.

Acknowledgments

We thank the personnel at the University of Texas Health Science Center at San Antonio, the Texas Diabetes Institute, and the Southwest Foundation for Biomedical Research for their dedication and support for this project.

Footnotes

This work was supported by grants from the American Diabetes Association (to N.M. and R.A.D.), Grant HL52636 from the National Institutes of Health (to Bronchopulmonary Dysplasia Resource Center), Grants AG030979 and DK080157 (to N.M.), Grant DK24092 (to R.A.D), Grant P51RR13986 for facility support at the Southwest Foundation for Biomedical Research, the San Antonio Nathan Shock Center (to N.M), the University of Texas Health Science Center at San Antonio Executive Research Committee (to N.M.), the South Texas Health Research Center (to N.M.), and the U.S. Department of Veterans Affairs (to R.A.D.).

Disclosure Summary: The authors have nothing to disclose.

First Published Online March 16, 2010

Abbreviations: GA, Gestational age; GLUT, glucose transporter; IR, insulin receptor; IRS, IR substrate; PI, phosphatidylinositol; SFBR, Southwest Foundation for Biomedical Research.

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