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Perturbed Skeletal Muscle Insulin Signaling in the Adult Female Intra-Uterine

Growth Restricted Rat

Shilpa A. Oak, Cang Tran, Gerald Pan, Mannikkavasagar Thamotharan, and

Sherin U. Devaskar*

Division of Neonatology & Developmental Biology, Department of Pediatrics, David

Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1752.

Running Title: Skeletal Muscle Insulin Signaling in IUGR Offspring

*Address Correspondence to:

10833, Le Conte Avenue, MDCC-B2-375

Los Angeles, CA 90095-1752

Email address: sdevaskar@mednet.ucla.edu

Articles in PresS. Am J Physiol Endocrinol Metab (January 31, 2006). doi:10.1152/ajpendo.00437.2005

Copyright © 2006 by the American Physiological Society.

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Abstract

To determine the molecular mechanism(s) linking fetal adaptations in intra-

uterine growth restriction (IUGR) to adult mal adaptations of type 2 diabetes mellitus, we

investigated the effect of prenatal semi-nutrient restriction, modified by early postnatal

ad lib access to nutrients (CM/SP) or semi-nutrient restriction (SM/SP), versus early

postnatal semi-nutrient restriction alone (SM/CP) or control nutrition (CM/CP), on

skeletal muscle post-receptor insulin signaling pathway in the adult offspring. The

altered in-utero hormonal/metabolic milieu was associated with no change in basal total

IRS1, p85 and p110β subunits of PI-3-kinase, PKCθ and PKCζ concentrations, but an

increase in basal IRS2 (p<0.05) only in the CM/SP group, and an increase in basal p-

PDK-1 (p<0.05), p-Akt (p<0.05) and p-PKCζ (p<0.05) concentrations in the CM/SP and

SM/SP groups. Insulin stimulated increase in p-PDK-1 (p<0.05) and p-Akt (p<0.0007)

with no increase in p-PKCζ, was seen both in CM/SP and SM/SP groups. SHP2

(p<0.03) and PTP1B (p<0.03) increased only in SM/SP with no change in PTEN in

CM/SP and SM/SP groups. Aberrations in kinase and phosphatase moieties in the adult

IUGR offspring were initiated in-utero but further sculpted by the early postnatal

nutritional state. While the CM/SP group demonstrated enhanced kinase activation, the

SM/SP group revealed an added increase in phosphatase concentrations, with the net

result of heightened basal insulin sensitivity in both groups. The inability to further

respond to exogenous insulin was due to the key molecular distal road block consisting

of a resistance to phosphorylate and activate PKCζ necessary for GLUT4 translocation.

This protective adaptation may become maladaptive and serve as a forerunner for

gestational and type 2 diabetes mellitus.

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Key Words: IUGR, insulin signaling, skeletal muscle, PKCζ, metabolic programming.

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Introduction

Epidemiological studies have linked prenatal nutrient restriction presenting with

low birth weight to the metabolic syndrome consisting of insulin resistance/type 2

diabetes mellitus, obesity, hypertension, and coronary artery disease during adult life

(7,29,46,51,57,60). Mimicking these human epidemiological observations, we

developed an animal model consisting of prenatal nutrient restriction that caused IUGR

in the rat (53). Using this animal model, we previously demonstrated aberrant skeletal

muscle expression and insulin induced translocation of the insulin-responsive glucose

transporter isoform (GLUT4) that persisted in the adult offspring as the mechanism

responsible for the subsequent development of glucose intolerance and insulin

resistance (53). In addition, various early postnatal nutrient intake regimens causing

differing adult phenotypes were observed not to affect the skeletal muscle GLUT4

imprint acquired in-utero. However, the mechanism(s) behind the post-translational

perturbations of GLUT4 translocation remain unknown.

Previous investigations in the adult type 2 diabetic have revealed aberrations in

the insulin induced translocation of GLUT4 related to changes in the post-receptor

insulin signaling pathway involving either the kinase (IRS, P-I-3-kinase, PDK, Akt,

atypical PKC) (34,44) and/or phosphatase (SHP2, PTP-1B, PTEN) arms (15,32,49).

Using the isocaloric selective nutrient (protein) restricted maternal rat model, changes in

the activating arm of the skeletal muscle insulin signaling pathway indicating increased

insulin sensitivity have been observed (55), while studies in white adipose tissue

demonstrated alterations supporting insulin resistance (45). We therefore hypothesized

that intra-uterine nutrient restriction alters the skeletal muscle insulin signaling pathway

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with adaptive perturbations in the kinase- and/or phosphatase arms that are further

modified by early postnatal nutrient intake. To test this hypothesis, we employed the mid

to late gestation maternal semi-nutrient restricted pregnant rat model with early

postnatal nutrient restriction or ad lib access to nutrients, and examined the effect on

skeletal muscle insulin signaling pathway in basal and insulin stimulated states. This

was accomplished by cross-fostering of animals that generated four experimental

groups. Thus, the control mother with ad lib access to nutrients fed the intra-uterine

semi-nutrient restricted progeny (CM/SP) and represented intra-uterine nutrient

restriction with early postnatal ad lib nutrition, while the semi-nutrient restricted mother

fed the intra-uterine growth restricted progeny (SM/SP) and represented intra-uterine

and early postnatal nutrient restriction. These groups were compared to the control

mother fed control progeny (CM/CP), that served as the "gold standard", and the semi-

nutrient restricted mother fed control progeny (SM/CP), that represented early postnatal

nutrient restriction in the absence of IUGR (figure 1).

Experimental Procedures

Animals: Sprague-Dawley rats (60 day old; 200-250g) (Charles River Laboratories,

Hollister, CA) were housed in individual cages, exposed to 12h light/dark cycles at 21-

230C, and allowed ad lib access to standard rat chow. The National Institutes of Health

guidelines were followed as approved by the Animal Research Committee of the

University of California, Los Angeles.

Maternal Semi-Nutrient Restriction Model: Pregnant rats received 50% of their daily

food intake (11g/day) beginning from d11 through d21 of gestation (SM), which

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constitutes mid- to late gestation, as compared to their control counterparts (CM) that

received ad lib access to rat chow (~22g/day). Both groups had ad lib access to drinking

water (53).

Postnatal Animal Maintenance: At birth, the litter size was culled to six. In addition, the

newborn rats born to the semi-nutrient restricted mothers (SP) were cross-fostered to

be reared by either a mother who continued to be semi-nutrient restricted by receiving

20 g/day food intake through lactation (27) (SM/SP) or a control mother with ad lib

access to rat chow (~40g/day) (CM/SP). Similarly, newborn pups born to control

mothers (CP) were cross-fostered to be reared by either a mother who continued to be

semi-nutrient restricted through lactation (27) (SM/CP) or a control mother with ad lib

access to rat chow (CM/CP). This food restriction scheme ensured that the semi-

nutrient restricted maternal rats received about 50% of the ad libitum food intake

through mid to late pregnancy and lactation (1,27,28). Thus four groups were created,

with control mothers rearing control (CM/CP) or prenatal semi-nutrient restricted pups

(CM/SP), and pre- and postnatal semi-nutrient restricted mothers rearing prenatal semi-

nutrient restricted pups (SM/SP) or control pups (SM/CP) (figure 1). At d21, the pups

were weaned from the mother and maintained in individual cages on a similar diet of

standard rat chow (53).

In-vivo insulin administration: At d60 female animals from all four experimental groups

received either vehicle or insulin (8U/kg) intra-peritoneally (figure 1). After 20 min, the

predetermined optimal time point (23,53), the animals were deeply anesthetized with

inhalational isoflurane to maintain organ blood flow, and skeletal muscle from the hind

limb was harvested.

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Skeletal muscle preparation: Skeletal muscle was rapidly separated from surrounding

tissues, quickly snap frozen in liquid nitrogen, and stored at -700C. Homogenates were

prepared as previously described (53). Briefly, skeletal muscle was powdered under

liquid nitrogen and suspended in three volumes of cell lysis buffer (Cell Signaling

Technology, Beverly, MA). The samples were homogenized with a handheld

homogenizer for 1-2 min at half speed followed by 30 min incubation on ice. The

samples were then subjected to further homogenization with 20 up and down strokes

using a tight fitting Potter-Elvenjhem homogenizer. These homogenates were then

centrifuged at 10,000 rpm at 40C for 10 min and stored at -700C until western blot

analysis was undertaken.

Antibodies: Rabbit polyclonal anti-Akt, anti-p-Akt (Ser-473), anti-PDK1, anti-p-PDK1

(Ser-241), anti-SHP2, and anti-p-PKCζ/λ (Thr-410/403) were from Cell Signaling

Technology (Beverly, MA). Anti-PI-3-kinase p85 (p85PAN), anti-p85, anti IRS1 and anti

IRS2 antibodies were purchased from Upstate Biotechnology (Lake placid, NY). Rabbit

polyclonal anti-PTEN, anti-p110β, and mouse monoclonal PKCθ antibodies were from

Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PTP1B antibody was purchased from

BD transduction laboratories (Lexington, KY). Anti-vinculin antibody was from Sigma

Chemical Co. (St. Louis, MO). Rabbit polyclonal anti-AKT2 antibody was a generous gift

from Dr. M. J. Birnbaum (University of Pennsylvania School of Medicine, PA).

Western blot analysis: The homogenates were sonicated (60 sonic, Dismembrator,

Fisher Scientific, Pittsburgh, PA) using two 50-sec cycles of 5-7 watts. The resulting

suspension was centrifuged at 10,000g at 40C for 10 min and the supernatant subjected

to Western blot analysis as previously described (53). Briefly, 50 µg of skeletal muscle

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homogenates were separated on SDS-PAGE and electroblotted onto nitrocellulose

membranes. Membranes were then blocked in 5% nonfat dry milk in phosphate

buffered saline containing 0.1% Tween-20 (PBST) for 1h, incubated with the specific

primary antibody (1:1000 dilution for AKT, p-AKT, PDK1, p-PDK1, p85, p110β and

SHP2, 1:100 for PKCθ and PKCζ, 1:200 for PTEN, 1:2500 for PTP1B, 1:500 for IRS2

and p-PKCζ [Thr410], 1:2000 for IRS1 and AKT2, and 1:4000 for vinculin) overnight at

40C with gentle agitation. The membranes were then washed with PBST three times for

15 min each. The membranes were then incubated with the appropriate secondary

horseradish peroxidase conjugated antibody for 1h at room temperature. After washing

the membranes three times for 15 min each, protein bands were visualized by using the

enhanced chemiluminescence method (Amersham Biosciences, Piscataway, NJ). The

quantification of protein bands was performed by densitometry using the Scion Image

software. The presence of linearity between the time of X-ray film exposure and the

optical density of the various protein bands was initially ensured (53).

PKCζ activity assay: PKCζ activity assay was performed as previously described

(37,41) with slight modifications. One mg of skeletal muscle homogenate was pre-

cleared by incubation with 50 µl of 50% slurry of Protein A/G–agarose (Santa Cruz

Biotechnology, Santa Cruz, CA) for 2 hr at 40C. Five µg of anti-PKCζ rabbit polyclonal

antiserum raised against a peptide in the T-loop of PKCζ was incubated with 50 µl of

50% slurry of Protein A/G–agarose for 2 hr at 40C on the rotor. The antibody bound

beads were washed three times with the cell lysis buffer and incubated with the pre-

cleared homogenate overnight at 40C on the rotor. The precipitated agarose beads

were then washed four times with the cell lysis buffer followed by two washes with the

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kinase buffer and resuspended in kinase buffer without myelin basic protein (MBP,

substrate) and ATP. In vitro kinase assay was carried out for 30 min at 25°C in 50 µl

buffer containing 35 mM Tris, pH 7.4, 10 mM MgCl2, 1 mM EGTA, 2 mM Na3VO4, 20

µg/ml leupeptin, 0.5 mM ATP, 4 µg MBP and 0.4 µCi [ϒ-32P] ATP. After incubation, 32P-

labeled substrate was trapped on P-81 filter papers. P81 filter papers were then washed

four times with 0.75% phosphoric acid and once with acetone and counted in a liquid

scintillation counter. Samples that contain no antibody or no substrate were used as

controls to account for background and endogenous phosphorylation respectively.

Data Analysis: Data are expressed as mean ± standard deviation. The analysis of

variance models were used to compare the various treatment groups and the F-values

obtained. Inter-group differences were determined by the Fisher's PLSD test when

ANOVA revealed significance. Significance was assigned when the p value was ≤ 0.05.

Results

The insulin signaling pathway as it pertains to GLUT4 translocation is

schematically represented in figure 2.

Activating Arm

Upregulation of skeletal muscle total IRS2 but not IRS1, p85 and p110β subunits

of P-I-3 kinase.

Figure 3A shows the expression levels of IRS1 and IRS2 in the three

experimental groups compared to the control group. IRS1 protein expression levels are

unchanged in the CM/SP, SM/SP, and SM/CP groups compared to the CM/CP group.

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However, IRS2 protein levels are significantly higher in CM/SP (p<0.05 Vs CM/CP,

p<0.05 Vs SM/SP) and SM/CP (p<0.05 Vs CM/CP, p<0.05 Vs SM/SP).

Figure 3B shows the total protein expression of p85, the regulatory subunit and

p110β the catalytic subunit of P-I-3-kinase. As can be seen both proteins remain

unaltered in the CM/SP, SM/SP and SM/CP groups compared to the CM/CP group. The

total protein expression of p50α and p55α was also analyzed and found to be no

different from control (data not shown).

Increased phosphorylation of skeletal muscle PDK1 in the basal state.

Figure 4A demonstrates the phosphorylation status of PDK1 in basal and insulin

stimulated states. CM/SP (p<0.05), SM/SP (p=0.004) and SM/CP (p=0.015) groups

show higher phosphorylation of PDK1 in the basal state compared to the control,

CM/CP. All the four groups namely CM/CP (p=0.02), CM/SP (p=0.0002), SM/SP

(p=0.005), and SM/CP (p=0.0003) respond to insulin stimulation resulting in a further

increase of PDK1 phosphorylation. CM/SP group shows the highest extent of PDK1

phosphorylation in response to insulin stimulation. SM/SP and SM/CP groups exhibit a

similar extent of insulin induced PDK1 phosphorylation compared to the CM/CP group.

Figure 4B demonstrates total PDK1 protein concentrations in the CM/SP, SM/SP and

SM/CP groups to be no different from the control, CM/CP.

Increased phosphorylation of skeletal muscle AKT (Ser 473) at the basal state.

Figure 5A shows the phosphorylation status of AKT at basal state and in

response to insulin stimulation. CM/SP (p<0.05), and SM/SP (p=0.004) groups show

higher phosphorylation of AKT in the basal state compared to the “control”, CM/CP.

SM/CP group shows an overall tendency towards increased phosphorylation of AKT in

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the basal state although not significantly higher than CM/CP. All the four groups namely

CM/CP (p=0.0006), CM/SP (p<0.0001), SM/SP (p=0.0003) and SM/CP (p=0.002)

respond to insulin stimulation with a further increase in phosphorylation of AKT. CM/SP

group shows the highest extent of phosphorylation of AKT in response to insulin

stimulation. SM/SP and SM/CP groups exhibit a similar extent of insulin stimulated

phosphorylation of AKT compared to the CM/CP group. Figure 5B demonstrates higher

AKT2 expression in SM/SP (p=0.011) and SM/CP (p=0.027) groups compared to

CM/CP group. CM/SP group shows a trend towards increased AKT2 expression that is

not significantly different from the CM/CP group. The combined expression of AKT1, 2,

3 in the three groups namely CM/SP, SM/SP, and SM/CP is no different from control,

the CM/CP group.

Increased phosphorylation and kinase activity of skeletal muscle PKCζ in basal

state with no further response to insulin.

Figure 6A shows that protein expression of PKCζ and PKCθ are unaltered in the

CM/SP, SM/SP and SM/CP groups regardless of prenatal or early postnatal nutrient

restriction compared to the CM/CP group. Figure 6B shows the phosphorylation of

PKCζ in the basal and insulin stimulated states in the four groups. CM/CP group shows

insignificant phosphorylation in the basal state that significantly increases after insulin

stimulation (p=0.002). In contrast, CM/SP group (p=0.04) and SM/SP group (p=0.015)

show significantly higher basal phosphorylation of PKCζ compared to CM/CP group.

However, both groups fail to show further phosphorylation of PKCζ in response to

insulin stimulation unlike the control, CM/CP group. Interestingly, SM/CP group does not

show significantly higher phosphorylation of PKCζ in the basal state but still fails to

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show a significant insulin induced increase in the phosphorylation of PKCζ. Figure 6C

shows the kinase activity of PKCζ in the basal and insulin stimulated states of the four

groups. CM/CP group shows minimal kinase activity in the basal state that significantly

increases after insulin stimulation (p<0.0001). In contrast, CM/SP and SM/SP groups

show a tendency towards higher basal kinase activity of PKCζ compared to the CM/CP

group although not statistically significant. However, both groups fail to show a further

increase in the kinase activity of PKCζ in response to insulin stimulation unlike the

control, CM/CP group. In agreement with the phosphorylation of PKCζ in Figure 6B,

SM/CP group does not show significantly higher activity of PKCζ at the basal state but

still fails to show an insulin induced increase in the phosphorylation of PKCζ.

De-activating Arm

Increased skeletal muscle protein tyrosine phosphatase1 B and SHP2.

Figure 7 demonstrates total protein concentrations of phosphatases namely

PTP1B, PTEN, and SHP2, in the four groups. Only the SM/SP group shows augmented

protein expression of PTP1B (p<0.05 Vs CM/CP, p<0.05 Vs SM/CP) and SHP2 (p<0.05

Vs CM/CP, p<0.05 Vs SM/CP) when compared to the CM/CP and SM/CP groups.

Increased PTP1B protein concentrations in the CM/SP group were not consistently

observed thereby not achieving statistical significance. The SHP2 protein expression in

CM/SP and SM/CP groups is no different from that of control, the CM/CP group. PTEN

levels remain unaltered in the four groups.

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All changes in the insulin signaling pathway detected in all three experimental

groups (CM/SP, SM/SP, and SM/CP) versus the CM/CP group have been summarized

in table 1 within the context of the phenotype and skeletal muscle GLUT4 changes (53).

Discussion

Our present study focused on the female offspring unlike other investigations that

have predominantly described the male progeny and reported changes consistent with

insulin resistance in adipose tissue (45) and skeletal muscle (47,48). Unlike a single

previous study that examined aging females (18), we examined the adult stage (~6-8

wk) when pregnancy is most likely to occur to allow assessment of the pre-conceptional

metabolic imprint in these IUGR adult animals. Other groups have previously

demonstrated that the IUGR adult female offspring is prone to gestational diabetes

mellitus (8) hence information regarding the pre-conceptional state may provide hints

regarding the predisposition towards gestational metabolic disturbances.

Previously using the four experimental groups described in this report (table 1)

we delineated the phenotype of the adult offspring where the CM/SP group expressed a

near normal pancreatic insulin response to a glucose load followed subsequently by

abnormally enhanced growth. In contrast the SM/SP and the SM/CP group had a

decreased insulin response to the same glucose load with the former demonstrating

persistent growth restriction while the latter subsequently attained normal growth (53).

In skeletal muscle, the rate-limiting step in insulin-induced glucose uptake is the insulin

responsive glucose transport. Despite inter-group differences in the adult phenotype we

previously determined that both the IUGR groups exhibited altered sub-cellular GLUT4

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localization in skeletal muscle. While there was heightened basal accumulation of

GLUT4 in the plasma membrane in both the CM/SP and SM/SP groups, there was no

further increase in plasma membrane associated GLUT4 concentrations in response to

exogenous insulin. Unlike the two IUGR groups, the early postnatal nutrient restricted

group of SM/CP demonstrated an intermediate response with retention of some insulin

responsiveness of the GLUT4 sub-cellular localization (53).

Since the post-receptor insulin signaling pathway is the predominant mechanism

by which GLUT4 translocation is regulated, we investigated this pathway in order to

determine the point at which this pathway is altered in the IUGR offspring. While total

IRS-1 was unaltered, IRS-2 the isoform that mediates glucose metabolism was

increased in CM/SP and SM/CP groups. Although IRS-2 is activated by

phosphorylation, increased total amounts support enhanced availability of substrate for

activation (19). The absence of a similar increase in the SM/SP group may reflect the

inhibitory check imposed by the counter-regulatory increase in PTP1B concentrations.

The total protein expression of the components of P-I-3 kinase, p85 and p110β were no

different than that of the control group, unlike previous observations of a decline in a

protein restricted IUGR rat model (47). Recently, McCurdy et al reported that the

regulatory isoforms p50α and p55α levels were approximately 40% lower in skeletal

muscle of nutrient restricted versus the ad lib fed adult rats that were unmanipulated

(43). However, in our model of the IUGR adult offspring we did not see any difference in

the experimental groups compared to the control.

Phosphoinositide-dependent protein kinase-1 (PDK-1) is a serine/threonine

kinase, activated by a lipid product of P-I-3 kinase, phosphatidylinositol 3,4,5-

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triphosphate [PI(3,4,5)P3] (13,38,54,58). PDK-1 directly phosphorylates and activates

downstream effectors of P-I-3 kinase, such as AKT and atypical PKCs that mediate

physiological actions of insulin (2,13,14,19,38). AKT is activated in response to insulin in

a variety of cell types (35). Previously, AKT2 was shown to be the critical isoform in

insulin dependent glucose uptake and maintenance of glucose homeostasis (42). In our

present study, while total AKT1, 2, 3 changes were imperceptible, increased AKT2

expression in SM/SP and SM/CP groups and a trend towards an increase in the CM/SP

group, may contribute towards heightened basal insulin sensitivity. We also observed a

basal increase in p-PDK-1 and p-AKT in the CM/SP and SM/SP groups when compared

to the CM/CP group. These changes are consistent with programming towards

increased endogenous insulin signaling resulting in heightened insulin sensitivity with

increased basal GLUT4 concentrations in the plasma membrane. In the presence of

exogenous insulin that mimics a post-prandial insulin surge, however a further

enhancement of p-PDK-1 and p-AKT was observed contrary to the insulin resistance of

GLUT4 translocation previously described (53). This suggests that the perturbation

responsible for this insulin resistant event must be either downstream of PDK-1/AKT or

belong to an alternate pathway.

Studies demonstrate that the atypical protein kinase C (PKC) isoforms λ and ζ

are downstream mediators of P-I-3 kinase (52) and PDK-1 (3) (figure 2). Over

expression of constitutively active PKCλ in adipocytes (36) or wild-type PKCζ in muscle

in vivo (17) enhances both basal and insulin-stimulated glucose transport. Early growth

restriction by maternal selective nutrient (protein) restriction led to a down regulation of

total PKCζ that was associated with insulin resistance of skeletal muscle glucose

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uptake (48). Increased expression of PKCθ in skeletal muscle of diabetic patients has

also been reported previously (31). These studies cumulatively suggest that atypical

PKCs play a vital role in mediating insulin’s effect on GLUT4 translocation thereby

constituting an alternate mediator to AKT. However in contrast to previous reports, we

observed no difference in total PKCζ and PKCθ protein expression in CM/SP, SM/SP

and SM/CP groups compared to that of control, the CM/CP group. However, in the

basal state, there was increased phosphorylation of PKCζ in CM/SP and SM/SP groups

when compared to the CM/CP group. These changes are also in keeping with the

activated kinase arm in the basal state and may contribute along with increased IRS2,

AKT2, pPDK1, and pAKT to the basal heightened insulin sensitivity evident as the

increase in plasma membrane associated GLUT4 concentrations (53).

In addition to the components of the kinase arm, protein tyrosine phosphatases

(PTPs) form a large family of enzymes that serve as key regulators in signal

transduction pathways. Since the kinase (activating) arm of the insulin signaling

pathway is tempered by the phosphatase (de-activating) arm, our studies demonstrated

that while PTEN changes were imperceptible, SHP2 and PTP1B basal levels were

increased in the SM/SP group alone. In agreement with our results, muscle specific

deletion of PTEN that regulates insulin signaling at the P-I-3-kinase level did not result

in increased insulin sensitivity when the mice were fed a normal chow diet (61).

Recently, however both PTEN mRNA and protein concentrations increased in soleus

muscle of obese Zucker rats (Fa/Fa) versus the age-matched lean group and fructose-

fed lean Zucker rats (fa/fa) versus the regular chow fed group, supporting a role for

PTEN gene expression in insulin resistance (39). SHP2 and PTP1B have been reported

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to regulate insulin signaling at the IRS level (16,40). Over expression of the dominant

negative SHP2 lacking the protein tyrosine phosphatase domain (∆PTP) in transgenic

mice led to end-organ insulin resistance despite physiological insulin concentrations

(40). Thus, SHP2, a protein tyrosine phosphatase contrary to conventional expectations

of a phosphatase may act as a positive regulator of insulin signaling at the IRS level.

In contrast, PTP1B knock out mice are not only insulin sensitive but also maintain

euglycemia in the fed state, with one-half the level of insulin observed in wild-type

littermates. On the other hand, over expression of PTP1B inhibits insulin signaling in

cultured cells (12,16,33,50,59). The expression and/or activity of skeletal muscle PTP1B

are increased in insulin-resistant rodents and humans. In addition, transgenic over

expression of PTP1B in muscle causes insulin resistance (62). Over expression of

PTP1B in adipocytes inhibits insulin-stimulated phosphoinositide-3-kinase activity

without altering glucose transport or AKT/Protein kinase B activation (59). Recently an

increased association of PTP1B with insulin receptor in spite of enhanced insulin

stimulated glucose uptake and insulin signaling was reported in 10 day old rat pups

born to food restricted mothers (20). This suggests that the increase in PTP1B in the

IUGR offspring may reflect an adaptive response to exaggerated insulin sensitivity. In

keeping with this concept, our observed increase in PTP1B expression in IUGR pups

that continue to be exposed to early postnatal nutrient restriction (SM/SP) may

represent an adaptation towards providing a regulatory check to the heightened insulin

activating pathway acquired in-utero. The need for this PTP1B check is not consistently

seen in the CM/SP group that is exposed to a shorter period of in-utero nutrient

restriction alone. Our previous report supports this concept as well demonstrating higher

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whole body insulin sensitivity based on a lower insulin response in maintaining glucose

tolerance in the SM/SP versus the CM/SP group (53). Adaptations in the basal insulin

signaling pathway while individualized in each IUGR group, led to a common imprint of

a higher basal accumulation of GLUT4 in plasma membrane in both CM/SP and SM/SP

groups.

In addition to heightened basal insulin activation, PTP1B-overexpressing mice

exhibit impaired insulin stimulated skeletal muscle activation of PKCζ/λ (62). Further,

over expression of a dominant-negative mutant of PKCζ or PKCλ abrogates insulin-

induced GLUT4 translocation and glucose transport in adipose (6,36) and muscle cells

(5). Defective insulin-induced GLUT4 translocation is associated with increased basal

activity and insulin unresponsiveness of protein kinase C ζ/λ in skeletal muscle of high-

fat fed rats compared to controls (56). Insulin stimulated protein kinase C ζ/λ activity is

also impaired in skeletal muscle of humans with obesity and type 2 diabetes (34). Thus

along with pAKT activation of PKCζ/λ is required for GLUT4 translocation mediated

maximal insulin stimulated glucose uptake (4,36,52). The mechanism underlying this

process involves PKCζ mediated serine phosphorylation of VAMP2 (vesicle-associated

membrane protein 2) in GLUT4 containing intracellular vesicles thereby increasing

glucose transport in skeletal muscle (9). In addition, insulin induces PKCζ interaction

with Munc18c thereby regulating GLUT4 translocation to the plasma membrane, a

process that can be abrogated by a P-I-3-kinase inhibitor (LY294002) (25). More

recently, PKCζ has been observed to interact with 80K-H (26) that is expressed

abundantly in major insulin sensitive tissues including fat and muscle cells (21), and is

related to vacuolar system associated protein-60 (VASAP-60) that is involved in

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vesicular trafficking (11). Additionally, 80K-H serves as a substrate for atypical PKC (24)

and interacts with both PKCζ and munc18c in an insulin-dependent manner. Based on

this accumulated information, it is suggested that insulin triggers the formation of a

PKCζ-80K-H-munc18c complex that then separates munc18c from syntaxin-4 (t-

SNARE protein component) (25,26) freeing the latter to allow easy interaction with

VAMP-2 (v-SNARE protein component) via the coil-coiled sequences called SNARE

motifs towards formation of the SNARE (N-ethylmaleimide sensitive factor attachment

protein receptors) complex. The SNARE complex is responsible for insulin induced

vesicular trafficking by intracellular membrane fusion to the plasma membrane (22), a

process that underlies GLUT4 translocation (30).

In our present investigation, insulin responsive p-PKCζ concentrations and

activity increased only in the CM/CP group, but failed to show a similar response in the

CM/SP, SM/SP, and SM/CP groups. This impaired insulin induced PKCζ

phosphorylation and activation may underlie the aberrant insulin induced GLUT4

translocation observed previously (53). Thus, while the basal insulin signaling

mechanisms involving the activating arm globally set the stage for enhanced basal

GLUT4 concentrations in the plasma membrane (53), the insulin resistance of GLUT4

translocation can only be explained by the absence of increased phosphorylation and

activation of PKCζ. Thus this step forms a critical distal road block in insulin signaling

that may in turn be responsible for compensatory increases in insulin stimulated

proximal steps consisting of phosphorylation of PDK1 and AKT that respectively

constitute the immediate upstream or alternate steps to PKCζ. The cellular adaptive

mechanisms targeted at survival consisting of heightened basal skeletal muscle insulin

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sensitivity may raise the set point for glucose delivery and therefore become protective

against further insulin action in response to either exogenous insulin or a post-prandial

insulin surge. Thus in-utero changes in p-PKCζ may be a key adaptive mechanism that

sets the stage for the subsequent development of insulin resistance of GLUT4

translocation in the adult IUGR offspring.

Insulin induced GLUT4 translocation to the skeletal muscle plasma membrane

translates directly into insulin responsive glucose transport (10). In a protein restricted

IUGR model alterations in skeletal muscle GLUT4 distribution have directly altered

skeletal muscle glucose transport (47). Based on this, we expect an increased basal

glucose transport into skeletal muscle with a resistance to further increase in response

to exogenous insulin in both the CM/SP and SM/SP groups. These changes would

support a dysregulation of skeletal muscle glucose transport. However these skeletal

muscle specific changes need to be placed in the context of the whole body

glucose/insulin kinetics to make physiological sense of tissue-specific aberrations. We

have previously demonstrated no difference in whole body peripheral insulin sensitivity

based on insulin tolerance testing at d60 (53) in the female CM/SP and SM/SP offspring

when compared to CM/CP. In addition, others have demonstrated no change in total

body glucose utilization in response to a hyperinsulinemic glucose clamp in d100

females in groups comparable to CM/SP and SM/SP (28). Thus skeletal muscle specific

changes may serve as necessary adaptations targeted at normalizing the whole body

peripheral glucose/insulin homeostasis and thereby the physiology, i.e. skeletal muscle

mechanisms adapt towards preserving insulin sensitivity and glucose delivery to other

organs (e.g. adipose tissue, liver, myocardium and brain) as a protective response to in-

21

utero nutrient restriction. Thus, while the IUGR adult offspring may present with near

normal whole body insulin sensitivity during the pre-conceptional stage (53) due to fine

tuning achieved by various cellular adaptive/protective mechanisms, certain

circumstances such as pregnancy and cryptic adiposity can tip this precariously

balanced metabolic homeostasis, causing maladaptive predisposition towards the

development of insulin resistance, a forerunner of gestational and type 2 diabetes

mellitus.

In summary, we have described phenotype specific adaptive alterations in

skeletal muscle of the adult female IUGR offspring that enhances basal but resists

insulin responsive insulin signaling targeted at GLUT4 translocation to the plasma

membrane. These changes consist of an increase in the activating kinase arm and in

certain cases the phosphatase arm in the basal state. In response to insulin stimulation

while a further increase in kinase activation was observed proximal to PKCζ, the key

step mediating insulin resistance of GLUT4 translocation was the phosphorylation and

activation of PKCζ. Hence therapeutic targets of p-PKCζ may help reverse the

subsequent development of insulin resistance under maladaptive circumstances in the

IUGR female adult offspring, thereby aborting the development of gestational diabetes

and propagation of the epidemic of type 2 diabetes mellitus.

Acknowledgements: This work was supported by grants from the National Institutes of

Health HD 41230, HD 25024, and HD 46979. Marielle Nguyen participated initially while

conditions for the AKT experiments were being established.

22

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Table 1: Summary of changes in the skeletal muscle insulin signaling pathway in

the CM/SP, SM/SP and SM/CP groups compared to CM/CP, the control group.

Compared to CM/CP (control -ad lib access to nutrients pre-and postnatally)

CM/SP (prenatal semi-nutrientrestriction)

SM/SP (pre- and early post natal semi-nutrientrestriction)

SM/CP (early post-natal semi-nutrientrestriction)

Phenotype: Body wt. gain & GTT

Higher growth rateGlucose tolerant

Lower growth rateGlucose tolerant

Catch up to CM/CPGlucose tolerant

Insulin response to a glucose load& ITTGLUT4 – basalGLUT – Insulinstimulated

↔ (no change)↔ (no change)

↑↔ (no change) from basal

↓↔ (no change)

↑↔ (no change)from basal

↓↔ (no change)↔ (no change)slight ↑ from basalcondition

Kinase arm ↑ IRS2 protein expression ↔ (no change)

↑ IRS2 protein expression

Basal condition ↑p-AKT↑p-PDK1↑p-PKCζ & trend towards higher PKCζ activity

↑p-AKT↑p-PDK1↑p-PKCζ & trend towards higher PKCζ activity

↔ (no change) in p-AKT, p-PDK1, p-PKCζ and activity.

Insulin stimulated condition(compared to the corresponding basal condition and CM/CP).

↑ p-AKT (same as CM/CP)↑p-PDK1 (same as CM/CP)↔ p-PKCζ &activity from basal(unlike CM/CP)

↑ p-AKT (same as CM/CP)↑ p-PDK1 (same as CM/CP)↔ p-PKCζ & activity from basal (unlike CM/CP)

↑ p-AKT (same as CM/CP)↑ p-PDK1 (same as CM/CP)↔ p-PKCζ & activityfrom basal (unlike CM/CP)

Total AKT, PDK1, PKCζ, pPKCζ, IRS1, P110β, and P85 protein expression

↔ (no change) ↔ (no change) ↔ (no change)

Phosphatase arm ↔ (no change) in PTP1B, SHP2, PTEN

↑PTP1B↑SHP2↔ PTEN

↔ (no change) in PTP1B, SHP2, PTEN

33

Figure Legends

Figure 1: Study design demonstrating the four experimental groups obtained by

cross-fostering postnatal rat pups: CM/CP = control mothers rearing control pups,

CM/SP = control mothers rearing in-utero semi-nutrient restricted pups, SM/SP =

semi-nutrient restricted mothers rearing in-utero semi-nutrient restricted pups,

and SM/CP = semi-nutrient restricted mothers rearing control pups. Semi-nutrient

restricted mothers received 50% of daily nutrient intake from mid to late

pregnancy (embryonic [e]11 to e21) through lactation (postnatal [pn] d1 to d21).

At d60 the female offspring in each group received vehicle (basal, INS -) or

insulin (insulin stimulated, INS +).

Figure 2: Schematic representation of the post-receptor insulin signaling

pathway beginning from insulin binding to its receptor to GLUT4 translocation via

vesicular trafficking. Ins = insulin and IR = insulin receptor, “P” =

phosphorylation.

Figure 3: Skeletal muscle IRS1, IRS2, p85 and p110β subunits of P-I-3 kinase

concentrations under basal conditions: Top panels show representative western

blots demonstrating A) IRS1, IRS2, and vinculin (Vin; internal control). B) p85,

p110β, and vinculin (Vin; internal control). Bottom panels in A and B show

corresponding densitometric analyses of the protein bands of interest/vinculin

34

shown as % of CM/CP and presented as mean ± S.D (n=6 per group). *p <0.05

Vs CM/CP, $p< 0.05 Vs SM/SP.

Figure 4: Skeletal muscle phosphorylated PDK1 (pPDK1) and total PDK1 under

basal and insulin stimulated conditions. Top panels show representative western

blots demonstrating A) pPDK1 and vinculin (Vin; internal control), B) PDK1 and

vinculin (Vin; internal control). Bottom panels in A) and B) show corresponding

densitometric analyses of the protein bands of interest/vinculin shown as % of

CM/CP and presented as mean ± S.D (n=6 per group and per treatment).

$p<0.05 Vs CM/CP basal, *p<0.05 insulin stimulated (INS+) Vs the basal states

(INS-) of the same group, $p < 0.05 Vs the CM/CP basal state (INS-), $$p< 0.05

Vs the insulin stimulated CM/CP (INS+).

Figure 5: Skeletal muscle phosphorylated AKT and total AKT (1,2&3) & AKT2

under basal and insulin stimulated conditions. Top panels show representative

western blots demonstrating A) p-AKT and Vinculin (Vin; internal control), B)

AKT1, 2, 3, AKT2, and Vinculin (Vin; internal control). Bottom panels in A) and B)

show corresponding densitometric analyses of the protein bands of

interest/vinculin shown as % of CM/CP and presented as mean ± S.D. (n=6 per

group and per treatment). *p<0.05 insulin stimulated (INS+) Vs basal states (INS-

) of the same group, $p<0.05 Vs CM/CP basal (INS-), ##p< 0.05 Vs insulin

stimulated CM/CP, %p<0.05 Vs insulin stimulated SM/CP.

35

Figure 6: Skeletal muscle total protein kinase C ζ and protein kinase C θ

concentrations under basal and phosphorylation & activity of PKCζ under basal

and insulin stimulated conditions. Top panels show representative western blots

demonstrating A) PKCζ, PKCθ and vinculin (Vin; internal control), B) p-PKCζ,

and vinculin (Vin; internal control). Bottom panels show densitometric analysis of

the protein bands of interest/vinculin shown as % of CM/CP and presented as

mean ± S.D. (n=6 per group [panels A&B] and per treatment [panel B]). C) PKCζ

activity is shown as cpm/min (mean±S.D.; n=6 per group and per treatment).

*p<0.05 insulin stimulated (INS+) Vs basal states (INS-) of the same group,

$p<0.05 Vs CM/CP basal (INS-).

Figure 7: Skeletal muscle protein tyrosine phosphatase 1B (PTP1B), SHP2 and

PTEN under basal conditions. Top panel shows representative western blots

demonstrating PTP1B, SHP2, PTEN, and vinculin (Vin; internal control). Bottom

panel shows densitometric analyses of the protein bands of interest/vinculin

shown as % of CM/CP and presented as mean ± S.D. (n=6 per group). *p <0.05

Vs CM/CP, $$ p< 0.05 Vs SM/CP.

36

37

38

39

40

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