Alterations in the IU and perinatal environment have a significant impact on fetal development and susceptibility to MetS in adult life. Given the genetic contribution associated with T2DM and obesity we sought to determine the effect of maternal HF feeding on a mouse model genetically predisposed to develop features of MetS, the G4+/− mouse 
. As previously published, HF feeding for two weeks prior to mating and during pregnancy did not result in maternal obesity 
. This model therefore allows us to investigate the interaction between HF exposure IU/L and genotype in the absence of any confounding effect of maternal obesity.
WT and G4+/− mice exposed to HF IU were smaller at e18.5 (decreased fetal weight and CRL) and developed features of MetS compared to mice exposed to C IU/L including decreased glucose and insulin tolerance and increased adiposity. Interestingly, MetS developed despite animals being weaned onto a low fat diet, suggesting that “permanent adaptations" occur in the IU environment that may determine future physiological features, regardless of good dietary and exercise habits. Future studies will address the impact these adaptations may have on susceptibility to MetS when mice are weaned onto a high fat diet.
Reduced fetal/birth weight in response to HF IU has been reported in some studies 
but not others 
. Some reasons for these discrepancies include: duration of HF exposure; presence/absence of maternal obesity; diet composition; and species studied. Low birth weight or normal birth weight accompanied by rapid weight gain during the first year of life both increase the risk of obesity 
. In addition, small size at birth correlates with increased fat mass 
. Increased postnatal adiposity plays a role in the pathogenesis of the MetS 
. In this study HF exposure during pregnancy and lactation increased offspring adiposity independent of genotype. Because HF exposure occurred during both pregnancy and lactation we cannot conclude whether there is a critical period of exposure to HF that determines the trajectory of postnatal growth.
Although both G4+/− and WT HF IU/L offspring developed features of MetS, genotype dependent differences were observed. In WT offspring, HF IU/L increased fed serum glucose and PAI-1 levels and decreased adiponectin levels consistent with insulin resistance, inflammation and obesity 
. G4+/− C IU/L and G4+/− HF IU/L mice had a similar serum profile to WT HF IU/L offspring. Increased glucose levels in G4+/− C IU/L mice are, most probably, a result of decreased glucose transport into GLUT4 expressing tissues such as skeletal muscle and adipose tissue 
. This data demonstrates that, as early as 6 wks of age, WT HF IU/L offspring exhibit a metabolic profile similar to G4+/− C IU/L mice, a genetic model of MetS.
HF IU/L did not exacerbate the serum profile in G4+/− mice, but G4+/− HF IU/L offspring did exhibit increased SBP compared to WT littermates. This data indicates that hypertension, a feature of MetS, is exacerbated by exposure to HF IU/L in mice with a hemizygous lesion in GLUT4 (GLUT4+/−).
To begin to address the etiology of MetS in response to HF IU/L the metabolic and molecular effects of HF IU on fetal liver were investigated. Liver was selected as it plays a critical role in regulating metabolic processes in response to nutrient availability and has been demonstrated to be highly susceptible to programming IU 
. Evidence suggests that alterations in the IU environment programs epigenetic modifications in liver that may impact metabolism. By assessing gene expression, our aim was to identify pathways that maybe targets of epigenetic modifications in utero. Gene expression data suggests that HF IU produces a phenotype in fetal liver similar to that observed with fasting.
Fetal serum ketones, glycerol and TG levels, and hepatic glycogen levels were not altered in response to HF IU suggesting that alterations in gene expression were a compensatory adaptation. Decreased fetal serum glucose levels in G4+/− HF fetus compared to WT HF may be due to the 12-fold increase in gene expression of Slc2a2, the glucose transporter GLUT2 (p<0.01) in G4+/− HF IU liver. Increased Slc2a2 expression may be a compensatory adaptation by the G4+/− “at risk” liver and may explain why G4+/− HF IU offspring maintained similar glucose levels to G4+/− C IU offspring. Alternatively, GLUT4 expression, which has been detected in mouse placenta as early as e12 
, may be decreased in G4+/− placenta resulting in decreased fetal-placenta glucose transport.
Expression of genes involved in glycolysis (PFK) and gluconeogenesis (PCK1, G6Pase) were increased in HF fetal liver. Increased PCK1 gene expression increases basal hepatic glucose production (HGP), triggering impaired glucose tolerance 
. Increased expression of genes involved in gluconeogenesis and glycolysis accompany hepatic insulin resistance in rodent models of IU programming 
Although changes in HGP may not be directly inferred from gene expression data 
changes in fetal hepatic gene expression are consistent with increased HGP and may, in part, explain the increase in fed glucose levels observed in 6 wk old WT HF IU offspring. In a NHP model, chronic maternal consumption of HF increased expression of gluconeogenic genes. This was accompanied with fetal hepatic lipid deposition 
In contrast to that NHP model 
, increased lipid (TG and cholesterol) accumulation in HF fetal liver was not observed. Despite no change in hepatic TG levels, expression of CIDEC mRNA was increased in WT, but not G4+/− HF fetuses. CIDEC is associated with the formation of lipid droplets. Proinflammatory genes such as IFN-γ, which was upregulated in G4+/− HF fetal liver, repress CIDEC expression in adipocytes 
. Expression of the transcription factor SREBF2, which regulates expression of genes involved in cholesterol synthesis 
, which is associated with insulin resistance 
, was decreased in G4+/− HF IU fetal liver compared with G4+/− C IU. Similar to CIDEC, expression of SREBP is also regulated by inflammation and HF 
suggesting that altered gene expression between WT and G4+/− HF fetuses could be the result of a different inflammatory response to HF IU.
Fetal lipid accumulation is thought to be primarily maternally derived since rates of de novo
lipogenesis in fetal liver are low 
. In a C57Bl/6 mouse model of acute maternal HF feeding, more lipids were transported to the fetus when more lipids were consumed by the mother 
. The amount of lipid consumed directly correlated with fetal growth 
. In our model HF IU fetuses were smaller than C IU, this combined with the absence of hepatic TG accumulation, suggests that the phenotype observed in our model may be a result of decreased maternal lipid transfer. Consistent with the NHP model, 2 wks HF in increased maternal serum glycerol and NEFA levels suggesting increased lipolysis, however maternal serum TG levels were decreased 
. Therefore, it is possible that in our CD1 model, maternal HF results in altered availability of lipids that affect the growth of the fetus.
HF IU increased hepatic expression of genes associated with insulin resistance (TNFα, SOCS3, PAI-1) 
and cellular stress (TXM, MAFF and DUSP) 
. We speculate that this gene expression profile could predispose HF IU/L offspring to develop hepatic insulin resistance and steatosis in adult life 
Inflammatory cytokines are a proposed link between obesity, insulin resistance and metabolic disease 
. Markers of oxidative stress and inflammation are increased in livers of NHP fetuses 
and livers of 15wk old mice exposed to HF IU 
. Fetal inflammation is associated with several neonatal diseases, such as brain damage and chronic lung disease 
. However, the role of fetal inflammation in DOHaD remains unresolved. Catalano et al., reported that maternal inflammation does not translate into inflammation of the fetal compartment 
. In contrast, altered cytokines and inflammatory markers in cord blood have been associated with altered fetal growth associated with placental insufficiency 
. G4+/− HF IU had increased levels of IFNγ, MCP-1, RANTES and M-CSF compared to G4+/− C IU. In contrast, WT HF IU serum levels were not significantly different than WT C IU. Elevated chemokine levels, such as MCP-1 which contributes to insulin resistance and hepatic steatosis 
, or RANTES which has been associated with obesity 
may have long term implications for susceptibility to MetS in later life 
Inflammatory and redox responses to HF IU was genotype dependent. WT HF fetuses upregulated expression of HMOX1 mRNA, which inhibits leukocyte migration 
. G4+/− HF fetuses upregulated mRNA expression of ICAM 1, a molecule associated with the recruitment of inflammatory cells 
. Expression of the retinol dehydrogenase RDH12, a NADP+
-dependent oxidoreductase 
, was downregulated in G4+/−, but not WT HF IU liver. Both medium-chain aldehydes and retinoids exert biological activities that can lead to cytotoxic effects 
. These results demonstrate that fetal genotype and IU environment interact to regulate components of the innate immune system.
One possible explanation for the metabolic differences between genotypes could be the developmental regulation of glucose transporters. GLUT4 mRNA and protein are expressed during fetal life in brown adipose tissue, heart and skeletal muscle 
and is sensitive to alterations in maternal nutrient intake 
. Differences in the phenotype between genotypes may be related to the specific expression of glucose transporters that play an important role in the regulation of glucose uptake and metabolism under diverse nutritional environments.