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Endocrinology Vol. 140, No. 3 1442-1448
Copyright © 1999 by The Endocrine Society

ARTICLES

Regulation of Hepatic Glycogen in the Insulin-Like Growth Factor II-Deficient Mouse1

Mary Frances Lopez, Pieter Dikkes, David Zurakowski, Lydia Villa-Komaroff and Joseph A. Majzoub

Departments of Medicine/Endocrinology (M.F.L., J.A.M.), Neurology (P.D.), and Research Computing and Biostatistics (D.Z.), Children’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; and the Department of Neurology, Northwestern University (L.V.K.), Evanston, Illinois 60208

Address all correspondence and requests for reprints to: Mary Frances Lopez, Ph.D., Division of Endocrinology, Enders 416, Children’s Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115. E-mail: lopez_m{at}a1.tch.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-like growth factor II (IGF-II), a polypeptide hormone with structural homologies to insulin-like growth factor I (IGF-I) and insulin, regulates the metabolism and growth of many tissues. In this study, we examined the role of IGF-II in hepatic glycogen metabolism in normal and growth-retarded IGF-II-deficient (knockout) mice. Liver glycogen content was significantly lower in the IGF-II knockout than in control livers during embryonic day 18 and postnatal day 0. Biochemical results were verified histologically using a glycogen-specific stain. The enzymatic activity of glycogen synthase, the rate-limiting enzyme for glycogen synthesis, was significantly lower in livers of knockout mice than in livers from wild-type controls on embryonic day 18 and postnatal day 0. The levels of glycogen synthase messenger RNA were not different between the two groups at any age studied, indicating that IGF-II acts posttranscriptionally. Hepatic glycogen content, measured in newborns after food withdrawal, was significantly lower in knockout mice compared with that in wild-type mice after 0, 3, and 6 h of fasting. Blood glucose was significantly lower in knockouts vs. wild-type newborn mice before fasting and was similar in both genotypes after 6 h of fasting. Consistent with this, only 23% of IGF-II knockout newborn mice survived fasting for 12 h, whereas 93% of wild-type mice survived this treatment. These results indicate that IGF-II is required for the regulation of glycogen metabolism of the mouse in the perinatal period, possibly via stimulation of glycogen synthase activity. IGF-II, via perinatal regulation of glycogen synthesis, may regulate fetal growth as well as play an important role in the transition from fetal to postnatal life by protecting the neonate against hypoglycemia during periods of fasting.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AT BIRTH the newborn has to make several adjustments to adapt to extrauterine life, including the maintenance of normoglycemia (1). Before birth, fetal glucose levels are maintained by transplacental passage of glucose from the mother. However, there is a critical period between birth and the establishment of suckling when the newborn depends on its hepatic glycogen stores to maintain blood glucose. Thus, the presence of appropriate hepatic glycogen stores at birth would enhance survival during this critical transitional period. In the rat, it has been shown that intrauterine growth-retarded fetuses have smaller hepatic glycogen stores than normal mice (2, 3). In the human similar observations have been made (4). Hepatic glycogen synthesis and storage increase at the end of gestation in most mammals, including man and rodents. The accumulation of glycogen stores parallels the increase in activity of the rate-limiting enzyme for glycogen synthesis, glycogen synthase (2, 5). In the adult, insulin regulates glycogen synthesis and the activity of glycogen synthase (6). In the fetus, however, it is not clear whether insulin is the main regulator of glycogen synthesis or whether other hormones, such as insulin-like growth factors, play a role in this regard (7).

Insulin-like growth factor II (IGF-II), a polypeptide hormone that shares 48% amino acid identity to proinsulin and is expressed in high amounts by almost every fetal tissue (8), may be involved in fetal carbohydrate metabolism. IGF-II has been shown to stimulate glycogen synthesis in fetal hepatocytes, hepatoma cell lines, and fetal limb buds (9, 10, 11, 12). In addition, when IGF-II is injected into hypophysectomized rats, blood glucose is decreased, and tissue glycogen and lipid synthesis is stimulated (13). Evidence for an effect of IGF-II in carbohydrate metabolism in humans comes from patients with nonislet cell tumor-associated hypoglycemia (14). These tumors produce and release high levels of IGF-II, which lead to hypoglycemia by stimulating hepatic and tissue glucose uptake (14).

IGF-II is also important for normal fetal and placental growth. Mice with targeted disruption of the IGF-II gene exhibit both fetal and placental growth retardation (15, 16, 17). We have recently shown that placentas from IGF-II-deficient mice have lower glycogen content than those from wild-type (WT) mice (17). As IGF-II is present at much higher concentrations than insulin in the fetus (18) and is capable of anabolic functions in the fetus, we asked whether IGF-II could be an important regulator of carbohydrate metabolism during fetal development. If so, the decreased growth rate seen in the IGF-II knockout (KO) fetus and placenta might be due not only to interference with a mitogenic function of IGF-II (16), but possibly also to inadequate production of carbohydrate fuel sources needed for proper growth.

In the present study, we used the IGF-II KO mouse to determine whether the absence of IGF-II affects perinatal hepatic carbohydrate metabolism. If we found decreased hepatic glycogen stores in IGF-II-deficient mice, we wanted to determine whether this would have important functional consequences, such as decreased survival after a period of fasting.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The isotopes [14C]uridine diphosphoglucose (UDPG), [32P]deoxy-CTP, and GeneScreen hybridization transfer membranes were purchased from DuPont-New England Nuclear (Boston, MA). UDPG-glucose, glucose 6-phosphate, rabbit liver glycogen (type II), mes-(2-[N-morpholino]ethanesulfonic acid), and Trizma [Tris(hydroxymethyl)-aminomethane] were purchased from Sigma Chemical Co. (St. Louis, MO).

Animals and tissue collection
Mice carrying the inactivated IGF-II gene (IGF-II KO mice) were originally provided by Dr. Argiris Efstratiadis through the courtesy of Dr. Virginia Papaioanou (15). The colony of mice was generated initially from a heterozygous x heterozygous mating, and the two genotypes were subsequently maintained and bred separately. The mice were maintained on a 14-h light, 10-h dark schedule (lights on at 0700 h) and allowed free access to food and water. Six- to 8-week-old females were housed with adult male mice and examined daily for vaginal plugs. The presence of a vaginal plug was designated day 0 of pregnancy. Newborn mice were placed in a temperature-controlled environment for the duration of the food withdrawal experiments. Livers and serum samples from IGF-II KO and WT mice were collected between 0800–1000 h and snap-frozen in liquid nitrogen on embryonic day 15 (e15), e18, postnatal day 0 (p0), p7, p14, p21, and p28. Samples were stored at -80 C until used. A minimum of three IGF-II KO and three WT litters were used for each experiment. All experiments were approved by the Children’s Hospital animal care and use committee.

Histological analysis
Livers were fixed by immersion in Rossman’s fixative for 24 h. All tissues used in this study were embedded in paraffin using an automated tissue processor. Serial 5-µm sections were cut using a Reichert-Jung Biocut rotatory microtome (Leica Instruments,Nussloch, Germany). Sections were floated on a 45 C water bath and mounted onto subbed slides (0.5% gelatin and 0.05% chromium potassium sulfate). The slides were allowed to dry overnight at 40 C. Before staining, paraffin was removed from the sections with three changes of xylene, followed by rehydration in descending concentrations of ethanol (100%, 90%, 70%, and 50%) and water. Sections were stained with Best’s carmine for glycogen content (19). Diastase treatment was used before or after Best’s carmine staining to show that staining was specific to glycogen.

Glycogen determination
The liver glycogen concentration was measured as described previously (20). Briefly, tissue was digested with 30% KOH saturated with Na2SO4 at 95 C for 30 min. To precipitate the glycogen out of the digested tissue, 95% ethanol was added. Glycogen precipitates were dissolved in water and analyzed by the phenol sulfuric acid calorimetric method (20).

RNA isolation and Northern analysis
Total RNA was isolated from livers using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH). Twenty micrograms of total RNA from both KO and WT livers were electrophoresed on a formaldehyde/agarose gel and transferred onto a nylon membrane by capillary transfer (21). Prehybridization, hybridization, and membrane washing were performed as previously described (21). Briefly, blots were prehybridized overnight at 42 C in 50% formamide, 5 x SSC (1 x SSC = 0.15 M NaCl and 15 mM sodium citrate, pH 7), 10 x Denhardt’s (0.2% Ficoll, 0.02% polyvinylpyrolidone, and 0.2% BSA), 0.1% SDS, 100 µg/ml denatured salmon sperm DNA, and water. Hybridization was performed using the same solution and temperature as those described above plus 10% dextran sulfate and radioactive probe. After hybridization, membranes were washed in 0.1 x SSC and 0.4% SDS and exposed to Kodak X-Omat AR film (Eastman Kodak Co., Rochester, NY) for 48 h. Glycogen synthase complementary DNA, a gift from Dr. Ernest Y. C. Lee (22), was labeled by random priming using the [32P]dCTP and Klenow enzyme labeling kit (Boehringer Mannheim, Indianapolis, IN). One million counts per min/lane were added during the hybridization procedure. The intensity of the glycogen synthase bands was normalized to that of the 28S ribosomal band to account for differences in the amount of RNA loaded. Results were quantified by scanning autoradiograms with an LKB Ultrascan X laser densitometer (Rockville, MD).

Glycogen synthase assay
The glycogen synthase assays were performed as described previously (23, 24). Briefly, livers were homogenized in cold buffer containing 100 mM NaF, 10 mM EDTA, 50 mM glycylglycine, and 0.5 dithiothreitol at a final pH of 7. Homogenates were used to determine both active and total glycogen synthase activities and for protein determination. For active glycogen synthase, 20 µl homogenate were added to 50 µl reaction mixture [50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 1% glycogen, 15 mM Na2SO4, 1.5 mM UDPG, and [14C]UDPG; 10,000–15,000 dpm/tube]. For total glycogen synthase, 10 mM UDPG and 10 mM glucose-6-phosphate were added to the incubation mixture, and Na2SO4 was omitted. Reactions were incubated for 30 min and were terminated by adding 50 µl reaction mixture to anion exchange resin columns (Dowex I-X8; 100–200 cm pore size mesh; chloride form; bed volume, 1.5 ml) and were washed four times with 0.5 ml water. Eluates were collected and mixed with 10 ml Ecolite liquid scintillation cocktail (ICN Biomedicals, Inc., Costa Mesa, CA), and radioactivity was counted with an LS 6000 liquid scintillation counter system (Beckman Coulter, Inc., Fullerton, CA). One unit of glycogen synthase activity was defined as the amount of enzyme that incorporated 1 µmol substrate (UDPG) into product per min at 30 C. The protein concentration of liver homogenates was determined using a bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL).

Serum glucose determination
Serum glucose was measured by the glucose oxidase method using an APEC automated glucose analyzer (APEC, Inc., West Peabody, MA)

Insulin RIA
Serum insulin was measured using a rat insulin RIA kit (Linco Research, Inc., St. Charles, MO). The rat insulin antibody in this kit cross-reacts 100% with mouse insulin antibody.

Statistics
Data are expressed as the mean and SEM. Two-way factorial ANOVA was used to compare means between WT and IGF-II KO animals at different measurements across time. An interaction F test was used to test whether group differences were time dependent. Simple main effects tests with a Bonferroni correction to maintain an overall {alpha} level of 0.05 were used to compare groups at each time point (25). One-way ANOVAs were performed to examine differences in glycogen levels across time within each group. Levene’s test was used to assess the homogeneity of variance (26). Given the small sample sizes and as there was some indication of unequal variances, two-tailed P < 0.01 was considered statistically significant for all tests using ANOVA. No significant departures from a Gaussian distribution were detected for insulin levels; therefore, mean values were compared between IGF-II KO and WT animals at each time point using Student’s two-sample t test with a Bonferroni correction. The proportions of surviving newborns that were food-deprived for 12 h were compared between groups using Fisher’s two-tailed exact test. Two-sample (unpaired) t tests were calculated to ascertain mean differences in blood glucose levels between KO and wild-type animals in the food deprivation studies, with P < 0.05 taken as a significant difference. Statistical analysis was performed using the SAS software package (version 6.12, SAS Institute, Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Glycogen levels in the livers of IGF-II KO and WT mice
In wild-type mice, glycogen stores in the fetal liver increased significantly during the second half of gestation, from an average of 4.4 ± 3 mg/g tissue on embryonic day 15 to 69.4 ± 4 mg/g tissue on embryonic day 18 (P < 0.0001; Fig. 1Go). On the day of birth, glycogen levels decreased to 51.4 ± 8 mg/g tissue and remained low until they increased on p28 (42.4 ± 2 mg/g tissue). The hepatic glycogen content of the IGF-II KO mice differed from normal during the neonatal period. A less dramatic, although significant, elevation in glycogen stores was seen before birth, increasing from 1.2 ± 0.4 mg/g tissue on e15 to 15.4 ± 0.8 on e18 (P < 0.0001). Glycogen concentrations at birth were not very different from those on e18 (14.6 ± 1.6 mg/g tissue). When comparisons were made between IGF-II KO and WT mice, we found that WT mice had significantly higher glycogen stores on e18 and p0 (P < 0.0001). No significant differences in glycogen levels between KO and WT mice during e15 or at any other postnatal age studied were observed. Glycogen concentrations were obtained using three animals for each age group. IGF-II KO and WT mice for each age group were obtained from different litters.



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  		Figure 1. Hepatic glycogen concentrations in IGF-II KO (squares) and WT (circles) mice at different ages. Mean values were significantly different between IGF-II KO and WT mice on e18 and p0 (P < 0.0001), as denoted by the asterisks.

 
Histological studies using a glycogen-specific stain were performed at different ages. Results obtained from these experiments are in agreement with the above biochemical data. The amount of glycogen stain in liver sections from WT mice was higher than that in KO mice on e18 and p0, but not on p28 (Fig. 2Go).



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  		Figure 2. Glycogen in liver sections from WT (A, C, and E) and IGF-II KO (B, D, and F) mice on e18 (A and B), p0 (C and D), and p28 (E and F). Sections were stained with Best’s carmine. Note the differences in the amount of glycogen staining between the genotypes on e18 and p0, but not on p28. A–F are the same magnification. Bar, 100 µm.

 
Glycogen synthase activity and messenger RNA (mRNA) levels in the livers of IGF-II KO and WT mice
Because glycogen levels are lower in livers of IGF-II KO than in WT mice before and at birth, we determined whether this reduction was due to lower activity of glycogen synthase, the rate-limiting enzyme for glycogen synthesis. Glycogen synthase activity was examined at different ages to determine whether there was a correlation between the amount of hepatic glycogen present and glycogen synthase activity (n = 3 for each age, except on e18, where n = 6). We found that the activity of glycogen synthase I, the independent form of glycogen synthase that is fully active in the absence of any effector, was significantly lower in the livers of KO than in WT mice on day 18 of gestation (P < 0.0001) and at birth (P < 0.001; Fig. 3Go). Total glycogen synthase activity, which includes the glycogen synthase D form and requires glucose 6-phosphate for maximal activity, was also significantly lower in KO than in WT mice on e18 (P < 0.0001) and p0 (P < 0.001), but not at other ages studied (Fig. 3Go).



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  		Figure 3. Glycogen synthase I (A) and glycogen synthase D (B) in livers from IGF-II KO (hatched bars) and WT (dark bars) mice. Mean values were significantly different between the genotypes on e18 (P < 0.0001) and p0 (P < 0.001), as denoted by the asterisks.

 
To determine whether this difference was due to different levels of glycogen synthase mRNA in the two genotypes, Northern blot analysis was performed. No differences in liver glycogen synthase mRNA levels were detected between KO and WT mice on e18, p0, or p28 (Fig. 4Go). Levels of glycogen synthase mRNA appeared to be constant at different ages, with no significant differences observed in the levels of glycogen synthase mRNA among prenatal, postnatal, and young adult animals (n = 3 for each group on each day).



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  		Figure 4. Liver glycogen synthase mRNA levels in IGF-II KO and WT mice. Tissue was collected on e18, p0, and p28. Glycogen synthase (GS) densitometric values were normalized to the corresponding 18S RNA densitometric values. There were no differences between IGF-II KO mice (hatched bars) and WT (dark bars) in the amounts of hepatic glycogen synthase mRNA on any day.

 
Glucose levels in blood of IGF-II KO and wild-type mice under free feeding conditions
Glucose levels were analyzed in serum samples obtained from IGF-II KO and WT mice at different ages during free access to food (Table 1Go). The glucose profiles of KO and wild-type mice are very similar, with levels falling at birth, followed by a steady increase until adulthood. Basal glucose levels were consistently higher for WT animals than for KO mice at each time point, and no interaction was found between group and time (P = 0.52). Using a conservative criterion of P < 0.01, wild-type mice had significantly higher glucose levels on p21 (P < 0.01) and marginally higher levels on p7 (P = 0.018) and p28 (P = 0.017). The differences observed between the two groups on e18, p0, and p14 were not significant.


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  		Table 1. Glucose levels in IGF-II KO and wild-type mice at different ages

 
Insulin levels in IGF-II KO and WT mice under free feeding conditions
Serum insulin was measured in IGF-II KO and WT mice on e18, p0, p7, and p21 during free access to food (Table 2Go). Insulin levels decreased at birth and then increased during the first week of life for both genotypes. No statistical differences were found between IGF-II KO and WT mice at any of the ages studied (P > 0.01 in each case).


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  		Table 2. Insulin levels on embryonic day 18 and postnatal days 0, 7, and 21

 
Glycogen and glucose levels after food deprivation in newborn IGF-II KO and WT mice
To determine the relationship between liver glycogen content and plasma glucose, we fasted both KO and WT newborn pups immediately after birth. Their livers were collected 0, 3, 6, 9, and 12 h after birth. Figure 5Go shows hepatic glycogen concentrations of newborn IGF-II KO and WT fasted pups. As seen previously (Fig. 1Go), glycogen content at birth was much greater in wild-type compared with IGF-II KO pups. Glycogen concentrations in the livers of WT mice were reduced by approximately 50% after 3 h of food withdrawal (from 62 ± 5 mg/g tissue after birth to 28 ± 9 after 3 h; P < 0.001). This sharp decline in glycogen levels continued until 9 h, after which the levels reached a plateau. A significant difference in the amount of hepatic glycogen was found between KO and WT pups at birth (P < 0.0001) and after 3 h (P < 0.001) and 6 h (P < 0.01) of food withdrawal. No differences were found between the groups after 9 h of food withdrawal (P > 0.80). Fisher’s exact test indicated that the proportion of animals surviving 12 h of food withdrawal was significantly higher among wild-type than IGF-II KO mice (P < 0.001). One of 15 wild-type mice died before completion of a 12-h food withdrawal period (93% survival), whereas 10 of 13 KO pups died before the 12-h food withdrawal was completed (23% survival). Glucose concentrations were also measured in mice immediately after birth and after 6 h of food deprivation. Table 3Go shows that glucose levels were significantly lower in the IGF-II KO mice within 1 h after birth (P < 0.001) but were not different after 6 h of food deprivation (P = 0.2).



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  		Figure 5. Glycogen concentrations in livers of IGF-II KO (hatched bars) and WT (solid bars) newborn mice 3, 6, 9, and 12 h after food withdrawal. Mean values were significantly different between IGF-II KO and WT mice before food withdrawal (P < 0.0001) as well as 3 h (P < 0.001) and 6 h (P < 0.01) after withdrawal, as denoted by the asterisks.

 

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  		Table 3. Glucose levels in newborn IGF-II KO and wild-type mice before suckling (0 h) and 6 h after food deprivation

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we report that the absence of IGF-II gene expression during the perinatal period is associated with a marked reduction in hepatic glycogen content, lower blood glucose after birth, and higher neonatal mortality after fasting. Fetal hepatic glycogen content, which is maximal just before birth, is 5-fold greater in wild-type mice compared with that in IGF-II KO mice. The lower levels of glycogen in the IGF-II KO mice during the perinatal period are probably due to a decrease in glycogen synthesis, because these mice also have lower glycogen synthase activity compared with wild-type mice. Our results indicate that IGF-II may regulate glycogen synthase, the rate-limiting enzyme for glycogen synthesis. Differences in glycogen synthase activity between IGF-II KO and WT mice are likely to occur at the posttranscriptional level, as the steady state mRNA levels for this enzyme are similar in both genotypes. As the original mouse line was on a mixed strain background (129/57B6), and there would have been a random assortment of chromosomes from the two mouse strains in the original littermates and subsequent generations, this might contribute in part to the difference observed. However, the data are strong enough that the effects seen are probably not due to strain differences.

During late gestation, most mammals synthesize large quantities of fetal glycogen and store it in tissues such as liver and lung (27). Although glycogen in the lung is used as a substrate for surfactant synthesis immediately before birth, glycogen in the liver is used during the perinatal period to maintain levels of glycemia as the newborn makes the adjustment to extrauterine life (27). Therefore, the liver glycogen accumulated during the later stages of gestation is rapidly hydrolyzed to glucose immediately after birth. Earlier studies have suggested that deposition of glycogen in the fetal liver is due to an increase in glycogen synthase activity. Glycogen in the fetus accumulates in parallel with the increase in glycogen synthase, and postnatally, levels of both glycogen and glycogen synthase also decline in parallel (5). It has been shown that the glycogen reserves at birth last only for a limited time if food is not supplied. It is known that in the rat, glycogen alone is sufficient to provide energy for 2–6 h after birth, whereas in humans, glycogen can provide energy for a maximum of 24 h (28).

One of the classical metabolic actions of insulin is to stimulate glycogen synthesis. However, the effect of insulin in the fetus appears to be distinct from that in the adult. Insulin has been shown to stimulate glycogen synthesis in fetal hepatocytes (9, 29, 30). However, many investigators have demonstrated that in the absence of cortisol, insulin is incapable of stimulating glycogen synthesis in isolated fetal hepatocytes, whereas adult hepatocytes respond appropriately in the absence of cortisol (29, 31). In addition, insulin has been shown to significantly augment the amount of glucose incorporated into glycogen by adult cells, but not in fetal cells (7, 32). A direct infusion of insulin also failed to exert a significant effect on glycogen synthesis by the isolated near-term fetal monkey liver (33). Moreover, in fetal rats, the hepatic glycogen content is normal despite fetal hypoglycemia and hypoinsulinemia induced by maternal hyperinsulinemia (2). However, pathologically elevated levels of insulin and glucose in the fetus, as occur in diabetic pregnancies, can stimulate fetal growth (34) and liver glycogen accumulation (35). In our studies, we have found that serum insulin levels were not significantly different between KO and WT mice, suggesting that the deficiency seen in glycogen stores was probably not due to the lack of insulin. However, insulin may be responsible for stimulating the glycogen stores present in the IGF-II KO embryos. Results from our study suggest that IGF-II may be a perinatal insulin-like hormone capable of regulating hepatic glycogen synthesis. Consistent with this possibility, the structure of IGF-II is homologous to that of insulin (8), and when made by tumors in excessive amounts can cause hypoglycemia, presumably by the insulin-like actions of promotion of glucose utilization, glycogen synthesis, and blockade of hepatic glycogenolysis.

IGF-II-deficient mice not only have lower hepatic glycogen stores during the perinatal period, but also have lower glycogen synthase activity, indicating that IGF-II may regulate glycogen synthesis by affecting the activity of glycogen synthase. We found evidence for posttranscriptional regulation of glycogen synthase activity by IGF-II. Glycogen synthase exists in a dephosphorylated form, glycogen synthase I, and a phosphorylated form, glycogen synthase D. An intermediate form of this enzyme has also been reported (36). The dephosphorylated enzyme is more active, whereas the phosphorylated enzyme requires glucose 6-phosphate for maximal activity (37). Thus, IGF-II may regulate the amount of enzyme that is being synthesized in the hepatic liver or/and the level of phosphorylation. IGF-II may be affecting phosphorylation of glycogen synthase either by acting at the level of protein phosphatases, which are capable of dephosphorylating and activating glycogen synthase, or by decreasing the activity of another enzyme(s), such as glycogen synthase kinase 3, which increases the phosphorylation of glycogen synthase and therefore reduces its activity (38, 39).

It is not known through which receptor IGF-II might exert its cellular effects in the fetal liver. Both the type 1 and type 2 IGF receptors as well as insulin receptors are known to be expressed in the fetal liver (40). It is possible that IGF-II mediates its effects via the type 1 IGF receptor, as most of the effects of the IGFs are known to be mediated through this receptor (8). We cannot rule out the possibility, however, that IGF-II may be interacting with the insulin receptor. A recent study has shown that IGF-II can bind to the insulin receptor with very high affinity to cause mitogenic effects (41). Louvi et al. (42) have also elegantly shown that IGF-II is an important ligand for both the insulin and IGF type 1 receptors in the fetus. They have suggested that the insulin receptor in the mouse embryo is related to IGF-II signaling rather than to mediation of the functions of insulin (42). In any event, both the type 1 IGF and insulin receptors have tyrosine kinase activity that is known to regulate enzymes involved in glycogen synthesis (43).

As the newborn enters the extrauterine environment, many stresses are encountered, including a variable period of fasting before the initiation of suckling. Normal liver glycogen levels help to maintain proper levels of glycemia during this time. If hepatic glycogen stores are low at birth, as seen in some cases of intrauterine growth retardation, the newborn is in danger of hypoglycemia if hepatic glycogen is depleted (4). In the case of the IGF-II KO mouse, in which fetal growth retardation occurs, we have shown that not only are hepatic glycogen stores low before and at birth, but the newborn mice are hypoglycemic and are not able to survive long periods of food deprivation. This result suggests that IGF-II may play an important role in the successful survival of the neonate.

If IGF-II has a nutritional impact on the fetus or newborn, it could explain in part the poor intrauterine growth seen in IGF-II KO mice (15). Efstratiadis et al. have postulated this to be due to IGF-II causing a decrease in cellular mitotic activity (15). However, a nutritional deficiency in utero, secondary to either maternal food deprivation (2) or placental insufficiency, is well known to cause fetal growth retardation associated with hepatic glycogen depletion. We suggest that in addition to decreased cellular mitotic activity, IGF-II deficiency and the associated low hepatic and placental glycogen stores might contribute to nutritional deprivation and growth failure.


   Acknowledgments
 
We thank Drs. Argiris Efstratiadis and Virginia Papaioanou for the kind gift of the IGF-II knockout mice, and Ms. Allison Carrigan for helping with the mouse colony.


   Footnotes
 
1 This work was supported by NIH Grants 1-R03-DK-52276 (to M.F.L.) and 3-R01-DK-50511 (to J.A.M.), and Mental Retardation Center Grant HD-18655. Back

Received June 29, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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