Changes in the maternal environment leading to an altered intrauterine milieu can result in subtle insults to the fetus, promoting increased lifetime disease risk and/or disease acceleration in childhood and later in life. Particularly worrisome is that the prevalence of NAFLD is rapidly increasing among children and adults, and is being diagnosed at increasingly younger ages, pointing towards an early-life origin. A wealth of evidence, in humans and non-human primates, suggests that maternal nutrition affects the placenta and fetal tissues, leading to persistent changes in hepatic metabolism, mitochondrial function, the intestinal microbiota, liver macrophage activation and susceptibility to NASH postnatally. Deleterious exposures in utero include fetal hypoxia, increased nutrient supply, inflammation and altered gut microbiota that might produce metabolic clues, including fatty acids, metabolites, endotoxins, bile acids and cytokines, which prime the infant liver for NAFLD in a persistent manner and increase susceptibility to NASH. Mechanistic links to early disease pathways might involve shifts in lipid metabolism, mitochondrial dysfunction, pioneering gut microorganisms, macrophage programming and epigenetic changes that alter the liver microenvironment, favouring liver injury. In this Review, we discuss how maternal, fetal, neonatal and infant exposures provide developmental clues and mechanisms to help explain NAFLD acceleration and increased disease prevalence. Mechanisms identified in clinical and preclinical models suggest important opportunities for prevention and intervention that could slow down the growing epidemic of NAFLD in the next generation.NAFLD is a general term used to describe a broad spectrum of liver abnormalities, ranging from simple, uncomplicated hepatic steatosis to NASH, accompanied by different degrees of Correspondence to J.E.F. jed.friedman@ucdenver.edu. Author contributionsAll authors researched data for the article, provided a substantial contribution to discussion of content, wrote the article, and reviewed and edited the manuscript before submission. Competing interests statementThe authors declare no competing interests. HHS Public AccessAuthor manuscript Nat Rev Gastroenterol Hepatol. Author manuscript; available in PMC 2017 December 12. Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript inflammation and fibrosis. NAFLD is increasing in prevalence, especially in adolescents 1 , despite the levellingoff of the obesity epidemic from 2003-2004 to 2013-2014 in children and adolescents aged 2-19 years (REF.2). The most common chronic liver disease worldwide, NAFLD increases the risk of cardiovascular events eightfold and type 2 diabetes mellitus (T2DM) threefold 3 , and is a strong risk factor for hepatocellular carcinoma (HCC) 4 . At the time of paediatric NAFLD diagnosis, 25-50% of children have NASH and 10-25% have advanced fibrosis 5 . For many decades, it was thought that NAFLD was predominantly driven by obesity in the setting of genetic suscep...

Key pointsr Adults who were affected by intrauterine growth restriction (IUGR) suffer from reductions in muscle mass, which may contribute to insulin resistance and the development of diabetes.r We demonstrate slower hindlimb linear growth and muscle protein synthesis rates that match the reduced hindlimb blood flow and oxygen consumption rates in IUGR fetal sheep. These adaptations resulted in hindlimb blood flow rates in IUGR that were similar to control fetuses on a weight-specific basis.r Net hindlimb glucose uptake and lactate output rates were similar between groups, whereas amino acid uptake was significantly lower in IUGR fetal sheep.r Among all fetuses, blood O 2 saturation and plasma glucose, insulin and insulin-like growth factor-1 were positively associated and norepinephrine was negatively associated with hindlimb weight.r These results further our understanding of the metabolic and hormonal adaptations to reduced oxygen and nutrient supply with placental insufficiency that develop to slow hindlimb growth and muscle protein accretion.Abstract Reduced skeletal muscle mass in the fetus with intrauterine growth restriction (IUGR) persists into adulthood and may contribute to increased metabolic disease risk. To determine how placental insufficiency with reduced oxygen and nutrient supply to the fetus affects hindlimb blood flow, substrate uptake and protein accretion rates in skeletal muscle, late gestation control (CON) (n = 8) and IUGR (n = 13) fetal sheep were catheterized with aortic and femoral catheters and a flow transducer around the external iliac artery. Muscle protein kinetic rates were measured using isotopic tracers. Hindlimb weight, linear growth rate, muscle protein accretion rate and fractional synthetic rate were lower in IUGR compared to CON (P < 0.05). Absolute hindlimb blood flow was reduced in IUGR (IUGR: 32.9 ± 5.6 ml min −1 ; CON: 60.9 ± 6.5 ml min −1 ; P < 0.005), although flow normalized to hindlimb weight was similar between groups. Hindlimb oxygen consumption rate was lower in IUGR (IUGR: 10.4 ± 1.4 μmol min −1 100 g −1 ; CON: 14.7 ± 1.3 μmol min −1 100 g −1 ; P < 0.05). Hindlimb glucose uptake and lactate output rates were similar between groups, whereas amino acid uptake was lower in IUGR (IUGR: 1.3 ± 0.5 μmol min −1 100 g −1 ; CON: 2.9 ± 0.2 μmol min −1 100 g −1 ; P < 0.05). Blood O 2 saturation (r 2 = 0.80, P < 0.0001) and plasma glucose (r 2 = 0.68, P < 0.0001), insulin (r 2 = 0.40, P < 0.005) and insulin-like growth factor (IGF)-1 (r 2 = 0.80, P < 0.0001) were positively associated and norepinephrine (r 2 = 0.59, P < 0.0001) was negatively associated with hindlimb weight. Slower hindlimb linear growth and muscle protein synthesis rates match reduced hindlimb blood flow and oxygen consumption rates in the IUGR fetus. Metabolic adaptations to slow hindlimb growth are probably hormonally-mediated by mechanisms that include increased fetal norepinephrine and reduced IGF-1 and insulin.

Intrauterine growth-restricted (IUGR) fetal sheep, produced by placental insufficiency, have lower oxygen concentrations, higher lactate concentrations, and increased hepatic glucose production that is resistant to suppression by insulin. We hypothesized that increased lactate production in the IUGR fetus results from reduced glucose oxidation, during basal and maximal insulin-stimulated conditions, and is used to support glucose production. To test this, studies were performed in late-gestation control (CON) and IUGR fetal sheep under basal and hyperinsulinemic-clamp conditions. The basal glucose oxidation rate was similar and increased by 30-40% during insulin clamp in CON and IUGR fetuses (P < 0.005). However, the fraction of glucose oxidized was 15% lower in IUGR fetuses during basal and insulin-clamp periods (P = 0.05). IUGR fetuses also had four-fold higher lactate concentrations (P < 0.001) and lower lactate uptake rates (P < 0.05). In IUGR fetal muscle and liver, mRNA expression of pyruvate dehydrogenase kinase (PDK4), an inhibitor of glucose oxidation, was increased over fourfold. In IUGR fetal liver, but not skeletal muscle, mRNA expression of lactate dehydrogenase A (LDHA) was increased nearly fivefold. Hepatic expression of the gluconeogenic genes, phosphoenolpyruvate carboxykinase (PCK)1, and PCK2, was correlated with expression of PDK4 and LDHA. Collectively, these in vivo and tissue data support limited capacity for glucose oxidation in the IUGR fetus via increased PDK4 in skeletal muscle and liver. We speculate that lactate production also is increased, which may supply carbon for glucose production in the IUGR fetal liver.

ObjectiveNon-alcoholic fatty liver disease (NAFLD) risk begins in utero in offspring of obese mothers. A critical unmet need in this field is to understand the pathways and biomarkers underlying fetal hepatic lipotoxicity and whether maternal dietary intervention during pregnancy is an effective countermeasure.MethodsWe utilized a well-established non-human primate model of chronic, maternal, Western-style diet induced obesity (OB-WSD) compared with mothers on a healthy control diet (CON) or a subset of OB-WSD mothers switched to the CON diet (diet reversal; OB-DR) prior to and for the duration of the next pregnancy. Fetuses were studied in the early 3rd trimester.ResultsFetuses from OB-WSD mothers had higher circulating triglycerides (TGs) and lower arterial oxygenation suggesting hypoxemia, compared with fetuses from CON and OB-DR mothers. Hepatic TG content, oxidative stress (TBARs), and de novo lipogenic genes were increased in fetuses from OB-WSD compared with CON mothers. Fetuses from OB-DR mothers had lower lipogenic gene expression and TBARs yet persistently higher TGs. Metabolomic profiling of fetal liver and serum (umbilical artery) revealed distinct separation of CON and OB-WSD groups, and an intermediate phenotype in fetuses from OB-DR mothers. Pathway analysis identified decreased tricarboxylic acid cycle intermediates, increased amino acid (AA) metabolism and byproducts, and increased gluconeogenesis, suggesting an increased reliance on AA metabolism to meet energy needs in the liver of fetuses from OB-WSD mothers. Components in collagen synthesis, including serum protein 5-hydroxylysine and hepatic lysine and proline, were positively correlated with hepatic TGs and TBARs, suggesting early signs of fibrosis in livers from the OB-WSD group. Importantly, hepatic gluconeogenic and arginine related intermediates and serum levels of lactate, pyruvate, several AAs, and nucleotide intermediates were normalized in the OB-DR group. However, hepatic levels of CDP-choline and total ceramide levels remained high in fetuses from OB-DR mothers.ConclusionsOur data provide new metabolic evidence that, in addition to fetal hepatic steatosis, maternal WSD creates fetal hypoxemia and increases utilization of AAs for energy production and early activation of gluconeogenic pathways in the fetal liver. When combined with hyperlipidemia and limited antioxidant activity, the fetus suffers from hepatic oxidative stress and altered intracellular metabolism which can be improved with maternal diet intervention. Our data reinforce the concept that multiple “first hits” occur in the fetus prior to development of obesity and demonstrate new biomarkers with potential clinical implications for monitoring NAFLD risk in offspring.

Adults who were affected by intrauterine growth restriction (IUGR) suffer from reductions in muscle mass and insulin resistance, suggesting muscle growth may be restricted by molecular events that occur during fetal development. To explore the basis of restricted fetal muscle growth, we used a sheep model of progressive placental insufficiency-induced IUGR to assess myoblast proliferation within intact skeletal muscle in vivo and isolated myoblasts stimulated with insulin in vitro. Gastrocnemius and soleus muscle weights were reduced by 25% in IUGR fetuses compared to controls (CON). The ratio of Pax7+ nuclei (a marker of myoblasts) to total nuclei was maintained in IUGR muscle compared to CON, but the fraction of Pax7+ myoblasts that also expressed Ki-67 (a marker of cellular proliferation) was reduced by 23%. Despite reduced proliferation in vivo, fetal myoblasts isolated from IUGR biceps femoris and cultured in enriched media in vitro responded robustly to insulin in a dose- and time-dependent manner to increase proliferation. Similarly, insulin stimulation of IUGR myoblasts up-regulated key cell cycle genes and DNA replication. There were no differences in the expression of myogenic regulatory transcription factors that drive commitment to muscle differentiation between CON and IUGR groups. These results demonstrate that the molecular machinery necessary for transcriptional control of proliferation remains intact in IUGR fetal myoblasts, indicating that in vivo factors such as reduced insulin and IGF1, hypoxia, and/or elevated counter-regulatory hormones may be inhibiting muscle growth in IUGR fetuses.

In a sheep model of intrauterine growth restriction (IUGR) produced from placental insufficiency, late gestation fetuses had smaller skeletal muscle mass, myofiber area, and slower muscle protein accretion rates compared with normally growing fetuses. We hypothesized that IUGR fetal muscle develops adaptations that divert amino acids (AAs) from protein accretion and activate pathways that conserve substrates for other organs. We placed hindlimb arterial and venous catheters into late gestation IUGR ( n = 10) and control (CON, n = 8) fetal sheep and included an external iliac artery flow probe to measure hindlimb AA uptake rates. Arterial and venous plasma samples and biceps femoris muscle were analyzed by mass spectrometry-based metabolomics. IUGR fetuses had greater abundance of metabolites enriched within the alanine, aspartate, and glutamate metabolism pathway compared with CON. Net uptake rates of branched-chain AA (BCAA) were lower by 42%–73%, and muscle ammoniagenic AAs (alanine, glycine, and glutamine) were lower by 107%–158% in IUGR hindlimbs versus CON. AA uptake rates correlated with hindlimb weight; the smallest hindlimbs showed net release of ammoniagenic AAs. Gene expression levels indicated a decrease in BCAA catabolism in IUGR muscle. Plasma purines were lower and plasma uric acid was higher in IUGR versus CON, possibly a reflection of ATP conservation. We conclude that IUGR skeletal muscle has lower BCAA uptake and develops adaptations that divert AAs away from protein accretion into alternative pathways that sustain global energy production and nitrogen disposal in the form of ammoniagenic AAs for metabolism in other organs.

Placental insufficiency is associated with reduced supply of amino acids to the fetus and leads to intrauterine growth restriction (IUGR). IUGR fetuses are characterized by lower glucose-stimulated insulin secretion, smaller pancreatic islets with less β-cells, and impaired pancreatic vascularity. To test whether supplemental amino acids infused into the IUGR fetus could improve these complications of IUGR we used acute (hours) and chronic (11 d) direct fetal amino acid infusions into a sheep model of placental insufficiency and IUGR near the end of gestation. IUGR fetuses had attenuated acute amino acid-stimulated insulin secretion compared with control fetuses. These results were confirmed in isolated IUGR pancreatic islets. After the chronic fetal amino acid infusion, fetal glucose-stimulated insulin secretion and islet size were restored to control values. These changes were associated with normalization of fetal pancreatic vascularity and higher fetal pancreatic vascular endothelial growth factor A protein concentrations. These results demonstrate that decreased fetal amino acid supply contributes to the pathogenesis of pancreatic islet defects in IUGR. Moreover, the results show that pancreatic islets in IUGR fetuses retain their ability to respond to increased amino acids near the end of gestation after chronic fetal growth restriction.

Fetal hypoxemia is associated with pregnancy conditions that cause an early activation of fetal glucose production. However, the independent role of hypoxemia to activate this pathway is not well understood. We hypothesized that fetal hypoxemia would activate fetal glucose production by decreasing umbilical glucose uptake and increasing counter-regulatory hormone concentrations. We induced hypoxemia for 9 days with maternal tracheal N2 gas insufflation to reduce maternal and fetal arterial Po2 by ~20% (HOX) compared with fetuses from ewes receiving intratracheal compressed air (CON). At 0.9 of gestation, fetal metabolic studies were performed ( n = 7 CON, 11 HOX). Umbilical blood flow rates, net fetal oxygen and glucose uptake rates, and fetal arterial plasma glucose concentrations were not different between the two groups. Fetal glucose utilization rates were lower in HOX versus CON fetuses but not different from umbilical glucose uptake rates, demonstrating the absence of endogenous glucose production. In liver tissue, mRNA expression of gluconeogenic genes G6PC (P < 0.01) and PCK1 ( P = 0.06) were six- and threefold greater in HOX fetuses versus CON fetuses. Increased fetal norepinephrine and cortisol concentrations and hepatic G6PC and PCK1 expression were inversely related to fetal arterial Po2. These findings support a role for fetal hypoxemia to act with counter-regulatory hormones to potentiate fetal hepatic gluconeogenic gene expression. However, in the absence of decreased net fetal glucose uptake rates and plasma glucose concentrations, hypoxemia-induced gluconeogenic gene activation is not sufficient to activate fetal glucose production.