Isolating mononuclear cells from fetal bone and liver for metabolic, functional, and immunophenotypic analyses in nonhuman primates.

Studying fetal hematopoiesis is challenging as hematopoiesis transitions from the liver to bone marrow. Obtaining human samples is not possible, and small animal models may not provide sufficient biological material. Here, we present a protocol for isolating hematopoietic cells from the nonhuman primate fetal liver and bone. We describe steps for using cells from the same fetus for fluorescence lifetime imaging microscopy to measure metabolism, assessing cellular function, and flow cytometry for immunophenotyping at the single-cell level. For complete details on the use and execution of this protocol, please refer to Nash et al. (2023).1.

Endotoxin biomarkers, hepatic fat fraction, liver volume and liver stiffness among adolescents at high-risk for non-alcoholic fatty liver disease: The HEROES study.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is on the rise among youth. Identifying biomarkers of NAFLD progression/risk can aid in prevention efforts. AIMS: This pilot study investigated associations of two endotoxin biomarkers-lipopolysaccharide-binding protein (LBP) and anti-endotoxin core immunoglobulin G (EndoCab)-with markers of NAFLD among 99 Latino/Latina adolescents (11-19 years) with obesity. MATERIALS & METHODS: We used linear regression to examine associations of each endotoxin biomarker (per 1-SD) with hepatic fat fraction (HFF), liver volume, and liver stiffness. RESULTS: We found positive associations of LBP with HFF and liver volume. Each 1-SD increment in LBP corresponded with 2.35% (95% CI: 0.46%, 4.23%) higher HFF and 0.14 (0.06, 0.23) L greater liver volume after adjusting for age, sex, and maternal education. Accounting for abdominal adiposity and Tanner stage did not change results. Excluding 72 participants with NAFLD attenuated associations of LBP with HFF but associations with liver volume persisted (0.11 [0.01, 0.21] L). EndoCab was not associated with any liver outcomes. Neither endotoxin biomarker predicted liver stiffness. DISCUSSION/CONCLUSION: While additional research is warranted, our results support LBP as a biomarker of NAFLD risk/progression in high-risk youth.

Maternal diet alters long-term innate immune cell memory in fetal and juvenile hematopoietic stem and progenitor cells in nonhuman primate offspring.

Maternal overnutrition increases inflammatory and metabolic disease risk in postnatal offspring. This constitutes a major public health concern due to increasing prevalence of these diseases, yet mechanisms remain unclear. Here, using nonhuman primate models, we show that maternal Western-style diet (mWSD) exposure is associated with persistent pro-inflammatory phenotypes at the transcriptional, metabolic, and functional levels in bone marrow-derived macrophages (BMDMs) from 3-year-old juvenile offspring and in hematopoietic stem and progenitor cells (HSPCs) from fetal and juvenile bone marrow and fetal liver. mWSD exposure is also associated with increased oleic acid in fetal and juvenile bone marrow and fetal liver. Assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling of HSPCs and BMDMs from mWSD-exposed juveniles supports a model in which HSPCs transmit pro-inflammatory memory to myeloid cells beginning in utero. These findings show that maternal diet alters long-term immune cell developmental programming in HSPCs with proposed consequences for chronic diseases featuring altered immune/inflammatory activation across the lifespan.

Maternal Western diet is associated with distinct preclinical pediatric NAFLD phenotypes in juvenile nonhuman primate offspring.

Pediatric NAFLD has distinct and variable pathology, yet causation remains unclear. We have shown that maternal Western-style diet (mWSD) compared with maternal chow diet (CD) consumption in nonhuman primates produces hepatic injury and steatosis in fetal offspring. Here, we define the role of mWSD and postweaning Western-style diet (pwWSD) exposures on molecular mechanisms linked to NAFLD development in a cohort of 3-year-old juvenile nonhuman primates offspring exposed to maternal CD or mWSD followed by CD or Western-style diet after weaning. We used histologic, transcriptomic, and metabolomic analyses to identify hepatic pathways regulating NAFLD. Offspring exposed to mWSD showed increased hepatic periportal collagen deposition but unchanged hepatic triglyceride levels and body weight. mWSD was associated with a downregulation of gene expression pathways underlying HNF4α activity and protein, and downregulation of antioxidant signaling, mitochondrial biogenesis, and PPAR signaling pathways. In offspring exposed to both mWSD and pwWSD, liver RNA profiles showed upregulation of pathways promoting fibrosis and endoplasmic reticulum stress and increased BiP protein expression with pwWSD. pwWSD increased acylcarnitines and decreased anti-inflammatory fatty acids, which was more pronounced when coupled with mWSD exposure. Further, mWSD shifted liver metabolites towards decreased purine catabolism in favor of synthesis, suggesting a mitochondrial DNA repair response. Our findings demonstrate that 3-year-old offspring exposed to mWSD but weaned to a CD have periportal collagen deposition, with transcriptional and metabolic pathways underlying hepatic oxidative stress, compromised mitochondrial lipid sensing, and decreased antioxidant response. Exposure to pwWSD worsens these phenotypes, triggers endoplasmic reticulum stress, and increases fibrosis. Overall, mWSD exposure is associated with altered expression of candidate genes and metabolites related to NAFLD that persist in juvenile offspring preceding clinical presentation of NAFLD.

Maternal Pyrroloquinoline Quinone Supplementation Improves Offspring Liver Bioactive Lipid Profiles throughout the Lifespan and Protects against the Development of Adult NAFLD.

Maternal obesity and consumption of a high-fat diet significantly elevate risk for pediatric nonalcoholic fatty liver disease (NAFLD), affecting 10% of children in the US. Almost half of these children are diagnosed with nonalcoholic steatohepatitis (NASH), a leading etiology for liver transplant. Animal models show that signs of liver injury and perturbed lipid metabolism associated with NAFLD begin in utero; however, safe dietary therapeutics to blunt developmental programming of NAFLD are unavailable. Using a mouse model of maternal Western-style diet (WD), we previously showed that pyrroloquinoline quinone (PQQ), a potent dietary antioxidant, protected offspring of WD-fed dams from development of NAFLD and NASH. Here, we used untargeted mass spectrometry-based lipidomics to delineate lipotoxic effects of WD on offspring liver and identify lipid targets of PQQ. PQQ exposure during pregnancy altered hepatic lipid profiles of WD-exposed offspring, upregulating peroxisome proliferator-activated receptor (PPAR) α signaling and mitochondrial fatty acid oxidation to markedly attenuate triglyceride accumulation beginning in utero. Surprisingly, the abundance of very long-chain ceramides, important in promoting gut barrier and hepatic function, was significantly elevated in PQQ-treated offspring. PQQ exposure reduced the hepatic phosphatidylcholine/phosphatidylethanolamine (PC/PE) ratio in WD-fed offspring and improved glucose tolerance. Notably, levels of protective n - 3 polyunsaturated fatty acids (PUFAs) were elevated in offspring exposed to PQQ, beginning in utero, and the increase in n - 3 PUFAs persisted into adulthood. Our findings suggest that PQQ supplementation during gestation and lactation augments pathways involved in the biosynthesis of long-chain fatty acids and plays a unique role in modifying specific bioactive lipid species critical for protection against NAFLD risk in later life.

Endotoxin Biomarkers Are Associated With Adiposity and Cardiometabolic Risk Across 6 Years of Follow-up in Youth.

CONTEXT: Metabolic endotoxemia may be a shared mechanism underlying childhood obesity and early-onset metabolic diseases (eg, type 2 diabetes, nonalcoholic fatty liver disease). OBJECTIVE: Examine prospective associations of serum endotoxin biomarkers lipopolysaccharide (LPS) and its binding protein, LPS binding protein (LBP), and anti-endotoxin core immunoglobulin G (EndoCab IgG) with adiposity and cardiometabolic risk in youth. DESIGN/SETTING: This prospective study included 393 youth in the Exploring Perinatal Outcomes Among Children cohort in Colorado. Participants were recruited from 2006 to 2009 at age 10 years (baseline) and followed for 6 years (follow-up). We examined associations of endotoxin biomarkers at baseline with adiposity [body mass index (BMI) z-score, visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), skinfolds, waist circumference] and cardiometabolic risk (insulin, glucose, adipokines, lipid profile, blood pressure) across both visits using mixed-effects regression, and with hepatic fat fraction (HFF) at follow-up using linear regression. RESULTS: Higher LPS and LBP predicted greater adiposity across follow-up. Each 1-unit log-transformed LPS corresponded with 0.23 (95% CI 0.03, 0.43) units BMI z-score, 5.66 (95% CI 1.99, 9.33) mm3 VAT, 30.7 (95% CI 8.0, 53.3) mm3 SAT, and 8.26 (95% CI 4.13, 12.40) mm skinfold sum. EndoCab IgG was associated with VAT only [3.03 (95% CI 0.34, 5.71) mm3]. LPS was associated with higher insulin [1.93 (95% CI 0.08, 3.70) µU/mL] and leptin [2.28 (95% CI 0.66, 3.90) ng/mL] and an adverse lipid profile. No association was observed with HFF. Accounting for pubertal status and lifestyle behaviors did not change findings. However, adjustment for prepregnancy BMI and gestational diabetes attenuated most associations. CONCLUSIONS: Serum endotoxin may be a marker of pathophysiological processes underlying development of childhood obesity and cardiometabolic conditions associated with exposure to fetal overnutrition.

Western diet-induced shifts in the maternal microbiome are associated with altered microRNA expression in baboon placenta and fetal liver.

Maternal consumption of a high-fat, Western-style diet (WD) disrupts the maternal/infant microbiome and contributes to developmental programming of the immune system and nonalcoholic fatty liver disease (NAFLD) in the offspring. Epigenetic changes, including non-coding miRNAs in the fetus and/or placenta may also underlie this risk. We previously showed that obese nonhuman primates fed a WD during pregnancy results in the loss of beneficial maternal gut microbes and dysregulation of cellular metabolism and mitochondrial dysfunction in the fetal liver, leading to a perturbed postnatal immune response with accelerated NAFLD in juvenile offspring. Here, we investigated associations between WD-induced maternal metabolic and microbiome changes, in the absence of obesity, and miRNA and gene expression changes in the placenta and fetal liver. After ~8-11 months of WD feeding, dams were similar in body weight but exhibited mild, systemic inflammation (elevated CRP and neutrophil count) and dyslipidemia (increased triglycerides and cholesterol) compared with dams fed a control diet. The maternal gut microbiome was mainly comprised of Lactobacillales and Clostridiales, with significantly decreased alpha diversity (P = 0.0163) in WD-fed dams but no community-wide differences (P = 0.26). At 0.9 gestation, mRNA expression of IL6 and TNF in maternal WD (mWD) exposed placentas trended higher, while increased triglycerides, expression of pro-inflammatory CCR2, and histological evidence for fibrosis were found in mWD-exposed fetal livers. In the mWD-exposed fetus, hepatic expression levels of miR-204-5p and miR-145-3p were significantly downregulated, whereas in mWD-exposed placentas, miR-182-5p and miR-183-5p were significantly decreased. Notably, miR-1285-3p expression in the liver and miR-183-5p in the placenta were significantly associated with inflammation and lipid synthesis pathway genes, respectively. Blautia and Ruminococcus were significantly associated with miR-122-5p in liver, while Coriobacteriaceae and Prevotellaceae were strongly associated with miR-1285-3p in the placenta; both miRNAs are implicated in pathways mediating postnatal growth and obesity. Our findings demonstrate that mWD shifts the maternal microbiome, lipid metabolism, and inflammation prior to obesity and are associated with epigenetic changes in the placenta and fetal liver. These changes may underlie inflammation, oxidative stress, and fibrosis patterns that drive NAFLD and metabolic disease risk in the next generation.

Maternal Western diet exposure increases periportal fibrosis beginning in utero in nonhuman primate offspring.

Maternal obesity affects nearly one-third of pregnancies and is a major risk factor for nonalcoholic fatty liver disease (NAFLD) in adolescent offspring, yet the mechanisms behind NAFLD remain poorly understood. Here, we demonstrate that nonhuman primate fetuses exposed to maternal Western-style diet (WSD) displayed increased fibrillar collagen deposition in the liver periportal region, with increased ACTA2 and TIMP1 staining, indicating localized hepatic stellate cell (HSC) and myofibroblast activation. This collagen deposition pattern persisted in 1-year-old offspring, despite weaning to a control diet (CD). Maternal WSD exposure increased the frequency of DCs and reduced memory CD4+ T cells in fetal liver without affecting systemic or hepatic inflammatory cytokines. Switching obese dams from WSD to CD before conception or supplementation of the WSD with resveratrol decreased fetal hepatic collagen deposition and reduced markers of portal triad fibrosis, oxidative stress, and fetal hypoxemia. These results demonstrate that HSCs and myofibroblasts are sensitive to maternal WSD-associated oxidative stress in the fetal liver, which is accompanied by increased periportal collagen deposition, indicative of early fibrogenesis beginning in utero. Alleviating maternal WSD-driven oxidative stress in the fetal liver holds promise for halting steatosis and fibrosis and preventing developmental programming of NAFLD.

Gestational Diabetes Is Uniquely Associated With Altered Early Seeding of the Infant Gut Microbiota.

Gestational diabetes mellitus (GDM) is a worldwide public health problem affecting up to 27% of pregnancies with high predictive values for childhood obesity and inflammatory diseases. Compromised seeding of the infant gut microbiota is a risk factor for immunologic and metabolic diseases in the offspring; however, how GDM along with maternal obesity interact to alter colonization remains unknown. We hypothesized that GDM individually and in combination with maternal overweight/obesity would alter gut microbial composition, diversity, and short-chain fatty acid (SCFA) levels in neonates. We investigated 46 full-term neonates born to normal-weight or overweight/obese mothers with and without GDM, accounting for confounders including cesarean delivery, lack of breastfeeding, and exposure to antibiotics. Gut microbiota in 2-week-old neonates born to mothers with GDM exhibited differences in abundance of 26 microbial taxa; 14 of which showed persistent differential abundance after adjusting for pre-pregnancy BMI. Key pioneering gut taxa, including potentially important taxa for establishing neonatal immunity, were reduced. Lactobacillus, Flavonifractor, Erysipelotrichaceae, and unspecified families in Gammaproteobacteria were significantly reduced in neonates from mothers with GDM. GDM was associated with an increase in microbes involved in suppressing early immune cell function (Phascolarctobacterium). No differences in infant stool SCFA levels by maternal phenotype were noted; however, significant correlations were found between microbial abundances and SCFA levels in neonates. Our results suggest that GDM alone and together with maternal overweight/obesity uniquely influences seeding of specific infant microbiota in patterns that set the stage for future risk of inflammatory and metabolic disease.

Pediatric Non-Alcoholic Fatty Liver Disease: Nutritional Origins and Potential Molecular Mechanisms.

Non-alcoholic fatty liver disease (NAFLD) is the number one chronic liver disease worldwide and is estimated to affect nearly 40% of obese youth and up to 10% of the general pediatric population without any obvious signs or symptoms. Although the early stages of NAFLD are reversible with diet and lifestyle modifications, detecting such stages is hindered by a lack of non-invasive methods of risk assessment and diagnosis. This absence of non-invasive means of diagnosis is directly related to the scarcity of long-term prospective studies of pediatric NAFLD in children and adolescents. In the majority of pediatric NAFLD cases, the mechanisms driving the origin and rapid progression of NAFLD remain unknown. The progression from NAFLD to non-alcoholic steatohepatitis (NASH) in youth is associated with unique histological features and possible immune processes and metabolic pathways that may reflect different mechanisms compared with adults. Recent data suggest that circulating microRNAs (miRNAs) are important new biomarkers underlying pathways of liver injury. Several factors may contribute to pediatric NAFLD development, including high-sugar diets, in utero exposures via epigenetic alterations, changes in the neonatal microbiome, and altered immune system development and mitochondrial function. This review focuses on the unique aspects of pediatric NAFLD and how nutritional exposures impact the immune system, mitochondria, and liver/gastrointestinal metabolic health. These factors highlight the need for answers to how NAFLD develops in children and for early stage-specific interventions.

Maternal Fat-1 Transgene Protects Offspring from Excess Weight Gain, Oxidative Stress, and Reduced Fatty Acid Oxidation in Response to High-Fat Diet.

Overweight and obesity accompanies up to 70% of pregnancies and is a strong risk factor for offspring metabolic disease. Maternal obesity-associated inflammation and lipid profile are hypothesized as important contributors to excess offspring liver and skeletal muscle lipid deposition and oxidative stress. Here, we tested whether dams expressing the fat-1 transgene, which endogenously converts omega-6 (n-6) to omega-3 (n-3) polyunsaturated fatty acid, could protect wild-type (WT) offspring against high-fat diet induced weight gain, oxidative stress, and disrupted mitochondrial fatty acid oxidation. Despite similar body mass at weaning, offspring from fat-1 high-fat-fed dams gained less weight compared with offspring from WT high-fat-fed dams. In particular, WT males from fat-1 high-fat-fed dams were protected from post-weaning high-fat diet induced weight gain, reduced fatty acid oxidation, or excess oxidative stress compared with offspring of WT high-fat-fed dams. Adult offspring of WT high-fat-fed dams exhibited greater skeletal muscle triglycerides and reduced skeletal muscle antioxidant defense and redox balance compared with offspring of WT dams on control diet. Fat-1 offspring were protected from the reduced fatty acid oxidation and excess oxidative stress observed in offspring of WT high-fat-fed dams. These results indicate that a maternal fat-1 transgene has protective effects against offspring liver and skeletal muscle lipotoxicity resulting from a maternal high-fat diet, particularly in males. Altering maternal fatty acid composition, without changing maternal dietary composition or weight gain with high-fat feeding, may highlight important strategies for n-3-based prevention of developmental programming of obesity and its complications.

Author Correction: The gut microbiota in infants of obese mothers increases inflammation and susceptibility to NAFLD.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Microplate Assays for Spectrophotometric Measurement of Mitochondrial Enzyme Activity.

Spectrophotometric analysis of metabolic enzyme activity from homogenized tissues is a valuable method for investigating mitochondrial content and capacity. Enzyme activity is normally measured in single cuvette spectrophotometers, requiring a large sample volume and low throughput. Here, we describe microplate assays for high-throughput analysis of mitochondrial enzymes citrate synthase, ß-hydroxyacyl CoA dehydrogenase, aconitase, and mitochondrial electron transport system (ETS) complexes I, II, III, and IV.

Switching obese mothers to a healthy diet improves fetal hypoxemia, hepatic metabolites, and lipotoxicity in non-human primates.

OBJECTIVE: Non-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. METHODS: We 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. RESULTS: Fetuses 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. CONCLUSIONS: Our 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.

The gut microbiota in infants of obese mothers increases inflammation and susceptibility to NAFLD.

Maternal obesity is associated with increased risk for offspring obesity and non-alcoholic fatty liver disease (NAFLD), but the causal drivers of this association are unclear. Early colonization of the infant gut by microbes plays a critical role in establishing immunity and metabolic function. Here, we compare germ-free mice colonized with stool microbes (MB) from 2-week-old infants born to obese (Inf-ObMB) or normal-weight (Inf-NWMB) mothers. Inf-ObMB-colonized mice demonstrate increased hepatic gene expression for endoplasmic reticulum stress and innate immunity together with histological signs of periportal inflammation, a histological pattern more commonly reported in pediatric cases of NAFLD. Inf-ObMB mice show increased intestinal permeability, reduced macrophage phagocytosis, and dampened cytokine production suggestive of impaired macrophage function. Furthermore, exposure to a Western-style diet in Inf-ObMB mice promotes excess weight gain and accelerates NAFLD. Overall, these results provide functional evidence supporting a causative role of maternal obesity-associated infant dysbiosis in childhood obesity and NAFLD.

Placental lipoprotein lipase activity is positively associated with newborn adiposity.

INTRODUCTION: Recent data suggest that in addition to glucose, fetal growth is related to maternal triglycerides (TG). To reach the fetus, TG must be hydrolyzed to free fatty acids (FFA) and transported across the placenta, but regulation is uncertain. Placental lipoprotein lipase (pLPL) hydrolyzes TG, both dietary chylomicron TG (CM-TG) and very-low density lipoprotein TG (VLDL-TG), to FFA. This may promote fetal fat accretion by increasing the available FFA pool for placental uptake. We tested the novel hypothesis that pLPL activity, but not maternal adipose tissue LPL activity, is associated with newborn adiposity and higher maternal TG. METHODS: Twenty mothers (n = 13 normal-weight; n = 7 obese) were prospectively recruited. Maternal glucose, insulin, TG (total, CM-TG, VLDL-TG), and FFA were measured at 14-16, 26-28, and 36-37 weeks, and adipose tissue LPL was measured at 26-28 weeks. At term delivery, placental villous biopsies were immediately analyzed for pLPL enzymatic activity. Newborn percent body fat (newborn %fat) was assessed by skinfolds. RESULTS: Placental LPL activity was positively correlated with birthweight (r = 0.48;P = 0.03) and newborn %fat (r = 0.59;P = 0.006), further strengthened by correcting for gestational age at delivery (r = 0.75;P = 0.0001), but adipose tissue LPL was not. Maternal TG and BMI were not correlated with pLPL activity. Additionally, pLPL gene expression, while modestly correlated with enzymatic activity (r = 0.53;P < 0.05), was not correlated with newborn adiposity. DISCUSSION: This is the first study to show a positive correlation between pLPL activity and newborn %fat. Placental lipase regulation and the role of pLPL in pregnancies characterized by nutrient excess and fetal overgrowth warrant further investigation.

Pyrroloquinoline quinone prevents developmental programming of microbial dysbiosis and macrophage polarization to attenuate liver fibrosis in offspring of obese mice.

Increasingly, evidence suggests that exposure to maternal obesity creates an inflammatory environment in utero, exerting long-lasting postnatal signatures on the juvenile innate immune system and microbiome that may predispose offspring to development of fatty liver disease. We found that exposure to a maternal Western-style diet (WD) accelerated fibrogenesis in the liver of offspring and was associated with early recruitment of proinflammatory macrophages at 8-12 weeks and microbial dysbiosis as early as 3 weeks of age. We further demonstrated that bone marrow-derived macrophages (BMDMs) were polarized toward an inflammatory state at 8 weeks of age and that a potent antioxidant, pyrroloquinoline quinone (PQQ), reversed BMDM metabolic reprogramming from glycolytic toward oxidative metabolism by restoring trichloroacetic acid cycle function at isocitrate dehydrogenase. This resulted in reduced inflammation and inhibited collagen fibril formation in the liver at 20 weeks of age, even when PQQ was withdrawn at 3 weeks of age. Beginning at 3 weeks of age, WD-fed mice developed a decreased abundance of Parabacteroides and Lactobacillus, together with increased Ruminococcus and decreased tight junction gene expression by 20 weeks, whereas microbiota of mice exposed to PQQ retained compositional stability with age, which was associated with improved liver health. Conclusion: Exposure to a maternal WD induces early gut dysbiosis and disrupts intestinal tight junctions, resulting in BMDM polarization and induction of proinflammatory and profibrotic programs in the offspring that persist into adulthood. Disrupted macrophage and microbiota function can be attenuated by short-term maternal treatment with PQQ prior to weaning, suggesting that reshaping the early gut microbiota in combination with reprogramming macrophages during early weaning may alleviate the sustained proinflammatory environment, preventing the rapid progression of nonalcoholic fatty liver disease to nonalcoholic steatohepatitis in offspring of obese mothers. (Hepatology Communications 2018;2:313-328).

Altered gene expression and metabolism in fetal umbilical cord mesenchymal stem cells correspond with differences in 5-month-old infant adiposity gain.

The intrauterine period is a critical time wherein developmental exposure can influence risk for chronic disease including childhood obesity. Using umbilical cord-derived mesenchymal stem cells (uMSC) from offspring born to normal-weight and obese mothers, we tested the hypothesis that changes in infant body composition over the first 5 months of life correspond with differences in cellular metabolism and transcriptomic profiles at birth. Higher long-chain acylcarnitine concentrations, lipid transport gene expression, and indicators of oxidative stress in uMSC-adipocytes were related to higher adiposity at 5 months of age. In uMSC-myocytes, lower amino acid concentrations and global differential gene expression for myocyte growth, amino acid biosynthesis, and oxidative stress were related to lower infant percent fat-free mass at 5 months of age, particularly in offspring of obese mothers. This is the first evidence of human infant adipocyte- or myocyte-related alterations in cellular metabolic pathways that correspond with increased adiposity and lower fat-free mass in early infancy. These pathways might reflect the effects of an adverse maternal metabolic environment on the fetal metabolome and genome. Our findings suggest that programmed differences in infant stem cell metabolism correspond with differences in body composition in early life, a known contributor to obesity risk.

Maternal obesity alters fatty acid oxidation, AMPK activity, and associated DNA methylation in mesenchymal stem cells from human infants.

OBJECTIVE: Infants born to mothers with obesity have greater adiposity, ectopic fat storage, and are at increased risk for childhood obesity and metabolic disease compared with infants of normal weight mothers, though the cellular mechanisms mediating these effects are unclear. METHODS: We tested the hypothesis that human, umbilical cord-derived mesenchymal stem cells (MSCs) from infants born to obese (Ob-MSC) versus normal weight (NW-MSC) mothers demonstrate altered fatty acid metabolism consistent with adult obesity. In infant MSCs undergoing myogenesis in vitro, we measured cellular lipid metabolism and AMPK activity, AMPK activation in response to cellular nutrient stress, and MSC DNA methylation and mRNA content of genes related to oxidative metabolism. RESULTS: We found that Ob-MSCs exhibit greater lipid accumulation, lower fatty acid oxidation (FAO), and dysregulation of AMPK activity when undergoing myogenesis in vitro. Further experiments revealed a clear phenotype distinction within the Ob-MSC group where more severe MSC metabolic perturbation corresponded to greater neonatal adiposity and umbilical cord blood insulin levels. Targeted analysis of DNA methylation array revealed Ob-MSC hypermethylation in genes regulating FAO (PRKAG2, ACC2, CPT1A, SDHC) and corresponding lower mRNA content of these genes. Moreover, MSC methylation was positively correlated with infant adiposity. CONCLUSIONS: These data suggest that greater infant adiposity is associated with suppressed AMPK activity and reduced lipid oxidation in MSCs from infants born to mothers with obesity and may be an important, early marker of underlying obesity risk.

Metabolic Inflexibility with Obesity and the Effects of Fenofibrate on Skeletal Muscle Fatty Acid Oxidation.

This study was designed to investigate mechanisms of lipid metabolic inflexibility in human obesity and the ability of fenofibrate (FENO) to increase skeletal muscle fatty acid oxidation (FAO) in primary human skeletal muscle cell cultures (HSkMC) exhibiting metabolic inflexibility. HSkMC from 10 lean and 10 obese, insulin resistant subjects were treated with excess fatty acid for 24 h (24hFA) to gauge lipid-related metabolic flexibility. Metabolically inflexible HSkMC from obese individuals were then treated with 24hFA in combination with FENO to determine effectiveness for increasing FAO. Mitochondrial enzyme activity and FAO were measured in skeletal muscle from subjects with prediabetes (n=11) before and after 10 weeks of fenofibrate in vivo. 24hFA increased FAO to a greater extent in HSkMC from lean versus obese subjects (+49% vs. +9%, for lean vs. obese, respectively; p<0.05) indicating metabolic inflexibility with obesity. Metabolic inflexibility was not observed for measures of cellular respiration in permeabilized cells using carbohydrate substrate. Fenofibrate co-incubation with 24hFA, increased FAO in a subset of HSkMC from metabolically inflexible, obese subjects (p<0.05), which was eliminated by PPARα antagonist. In vivo, fenofibrate treatment increased skeletal muscle FAO in a subset of subjects with prediabetes but did not affect gene transcription or mitochondrial enzyme activity. Lipid metabolic inflexibility observed in HSkMC from obese subjects is not due to differences in electron transport flux, but rather upstream decrements in lipid metabolism. Fenofibrate increases the capacity for FAO in human skeletal muscle cells, though its role in skeletal muscle metabolism in vivo remains unclear.