Angiopoietin-like protein 4 induces growth hormone variant secretion and aggravates insulin resistance during pregnancy, linking obesity to gestational diabetes mellitus.

Angiopoietin-like protein 4 (ANGPTL4) is a secretory glycoprotein involved in regulating glucose homeostasis in non-pregnant subjects. However, its role in glucose metabolism during pregnancy and the pathophysiology of gestational diabetes mellitus (GDM) remains elusive. Thus, this study aimed to clarify the relationship between ANGPTL4 and GDM and investigate the pathophysiology of placental ANGPTL4 in glucose metabolism. We investigated this issue using blood and placenta samples in 957 pregnant women, the human 3A-sub-E trophoblast cell line, and the L6 skeletal muscle cell line. We found that ANGPTL4 expression in the placenta was higher in obese pregnant women than in lean controls. Palmitic acid significantly induced ANGPTL4 expression in trophoblast cells in a dose-response manner. ANGPTL4 overexpression in trophoblast cells resulted in endoplasmic reticulum (ER) stress, which stimulated the expression and secretion of growth hormone-variant (GH2) but not human placental lactogen. In L6 skeletal muscle cells, soluble ANGPTL4 suppressed insulin-mediated glucose uptake through the epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinases 1/2 (ERK 1/2) pathways. In pregnant women, plasma ANGPTL4 concentrations in the first trimester predicted the incidence of GDM and were positively associated with BMI, plasma triglyceride, and plasma GH2 in the first trimester. However, they were negatively associated with insulin sensitivity index ISI0,120 in the second trimester. Overall, placental ANGPTL4 is induced by obesity and is involved in the pathophysiology of GDM via the induction of ER stress and GH2 secretion. Soluble ANGPTL4 can lead to insulin resistance in skeletal muscle cells and is an early biomarker for predicting GDM.

The association between plasma angiopoietin-like protein 4, glucose and lipid metabolism during pregnancy, placental function, and risk of delivering large-for-gestational-age neonates.

BACKGROUND: Large-for-gestational-age (LGA) neonates have increased risk of adverse pregnancy outcomes and adult metabolic diseases. We aimed to investigate the relationship between plasma angiopoietin-like protein 4 (ANGPTL4), a protein involved in lipid and glucose metabolism during pregnancy, placental function, growth factors, and the risk of LGA. METHODS: We conducted a prospective cohort study and recruited women with singleton pregnancies at the National Taiwan University Hospital between 2013 and 2018. First trimester maternal plasma ANGPTL4 concentrations were measured. RESULTS: Among 353 pregnant women recruited, the LGA group had higher first trimester plasma ANGPTL4 concentrations than the appropriate-for-gestational-age group. Plasma ANGPTL4 was associated with hemoglobin A1c, post-load plasma glucose, plasma triglyceride, plasma free fatty acid concentrations, plasma growth hormone variant (GH-V), and birth weight, but was not associated with cord blood growth factors. After adjusting for age, body mass index, hemoglobin A1c, and plasma triglyceride concentrations, plasma ANGPTL4 concentrations were significantly associated with LGA risk, and its predictive performance, as measured by the area under the receiver operating characteristic curve, outperformed traditional risk factors for LGA. CONCLUSIONS: Plasma ANGPTL4 is associated with glucose and lipid metabolism during pregnancy, plasma GH-V, and birth weight, and is an early biomarker for predicting the risk of LGA.

Lunasin ameliorates glucose utilization in C2C12 myotubes and metabolites profile in diet-induced obese mice benefiting metabolic disorders.

AIMS: Obesity is the main cause of low-grade inflammation and oxidation, resulting in insulin resistance. This study aimed to investigate the effects of a seed peptide lunasin on glucose utilization in C2C12 myotubes and the metabolite profiles in obese mice. MAIN METHODS: C2C12 myotubes were challenged by palmitic acid (PA) to mimic the obese microenvironment and inflammation, cell vitality, and glucose utilization were determined. C57BL6/j mice were divided into low-fat diet (LF), high-fat diet (HF), and HF with intraperitoneally injected lunasin (HFL) groups. Glucose intolerance and metabolite profiles of the tissues were analyzed. KEY FINDINGS: In vitro, C2C12 myotubes treated with lunasin showed decreased proinflammatory cytokines and increased cell vitality under palmitic acid conditions. Lunasin improved glucose uptake and glucose transporter 4 expression by activating insulin receptor substrate-1 and AKT phosphorylation. Next-generation sequencing revealed that lunasin regulates genes expression by promoting insulin secretion and decreasing oxidative stress. In vivo, HF mice showed increased tricarboxylic acid cycle and uric acid metabolites but decreased bile acids metabolites and specific amino acids. Lunasin intervention improved glucose intolerance and modulated metabolites associated with increased insulin sensitivity and decreased metabolic disorders. SIGNIFICANCE: This study is the first to reveal that lunasin is a promising regulator of anti-inflammation, anti-oxidation, and glucose utilization in myotubes and ameliorating glucose uptake and metabolite profiles in obese mice, contributing to glucose homeostasis and benefiting metabolic disorders.

Lipidomics of children and adolescents exposed to multiple industrial pollutants.

BACKGROUND: There are limited studies on the lipidomics of children and adolescents exposed to multiple industrial pollutants. OBJECTIVES: In this study, we aimed to investigate lipid profile perturbations in 99 children and adolescents (aged 9-15) who lived in a polluted area surrounding the largest petrochemical complex in Taiwan. Previous studies have reported increased risks of acute and chronic diseases including liver dysfunctions and chronic kidney disease (CKD) in residents living in this area. METHODS: We measured urinary concentrations of 11 metals and metalloids and polycyclic aromatic hydrocarbons (PAHs) metabolite 1-hydroxypyrene (1-OHP) as exposure biomarkers, and urinary oxidative stress biomarkers and serum acylcarnitines as early health effect biomarkers. The association between individual exposure biomarkers and early health effect biomarkers were analyzed using linear regression, while association of combined exposure biomarkers with four oxidative stress biomarkers and acylcarnitines were analyzed using weighted quantile sum (WQS) regression. Lipid profiles were analyzed using an untargeted liquid chromatography mass spectrometry-based technique. "Meet-in-the-middle" approach was applied to identify potential lipid features that linked multiple industrial pollutants exposure with early health effects. RESULTS: We identified 15 potential lipid features that linked elevated multiple industrial pollutants exposure with three increased oxidative stress biomarkers and eight deregulated serum acylcarnitines, including one lysophosphatidylcholines (LPCs), four phosphatidylcholines (PCs), and two sphingomyelins (SMs) that were up-regulated in high exposure group compared to low exposure group, and two LPCs, four PCs, and two phosphatidylinositols (PIs) down-regulated in high exposure group compared to low exposure group. CONCLUSION: Our findings could provide information for understanding the health effects, including early indicators and biological mechanism identification, of children and adolescents exposed to multiple industrial pollutants during critical stages of development.

Maternal Plasma Lipids During Pregnancy, Insulin-like Growth Factor-1, and Excess Fetal Growth.

CONTEXT: Maternal lipids during pregnancy and placental growth factors are associated with excess fetal growth. However, how these factors interact to increase the risk of delivering large-for-gestational-age (LGA) neonates remains unclear. In this study, we investigated the relationship between maternal plasma triglycerides (TGs) and free fatty acids (FFAs) during pregnancy, cord blood insulin-like growth factors (IGF), and LGA. OBJECTIVE: In a cell model, we studied the effect of different FAs on placental IGF-1 secretion. METHODS: This cohort study included pregnant women with term pregnancy and without diabetes or hypertensive disorders in pregnancy. Maternal fasting plasma TGs and FFAs were measured in the second trimester. Cord blood IGF-1, IGF-2, and IGF binding protein-1 and protein-3 were measured at the time of delivery. A human trophoblast cell line, 3A-sub-E, was used to evaluate the effect of different FFAs on placental IGF-1 secretion. RESULTS: We recruited 598 pregnant women-neonate pairs. Maternal plasma TG (180 mg/dL [152.5-185.5 mg/dL] vs 166 mg/dL [133-206 mg/dL], P = .04) and cord blood IGF-1 concentrations (72.7 ±â€…23.0 vs 54.1 ±â€…22.8 ng/mL, P < .001) were higher in the LGA group and were significantly associated with birth weight z score. Maternal plasma free palmitic acid (PA) and stearic acid (SA), but not oleic acid (OA) or linoleic acid (LA), were significantly associated with cord blood IGF-1 concentrations. In 3A-sub-E cells, treatment with PA, SA, and LA, but not OA, induced IGF-1 expression and secretion. CONCLUSION: Certain FFAs can induce placental IGF-1 secretion, which suggests a potential pathophysiology linking maternal plasma lipids and LGA.

Dihydroceramide desaturase regulates the compartmentalization of Rac1 for neuronal oxidative stress.

Disruption of sphingolipid homeostasis is known to cause neurological disorders, but the mechanisms by which specific sphingolipid species modulate pathogenesis remain unclear. The last step of de novo sphingolipid synthesis is the conversion of dihydroceramide to ceramide by dihydroceramide desaturase (human DEGS1; Drosophila Ifc). Loss of ifc leads to dihydroceramide accumulation, oxidative stress, and photoreceptor degeneration, whereas human DEGS1 variants are associated with leukodystrophy and neuropathy. In this work, we demonstrate that DEGS1/ifc regulates Rac1 compartmentalization in neuronal cells and that dihydroceramide alters the association of active Rac1 with organelle-mimicking membranes. We further identify the Rac1-NADPH oxidase (NOX) complex as the major cause of reactive oxygen species (ROS) accumulation in ifc-knockout (ifc-KO) photoreceptors and in SH-SY5Y cells with the leukodystrophy-associated DEGS1H132R variant. Suppression of Rac1-NOX activity rescues degeneration of ifc-KO photoreceptors and ameliorates oxidative stress in DEGS1H132R-carrying cells. Therefore, we conclude that DEGS1/ifc deficiency causes dihydroceramide accumulation, resulting in Rac1 mislocalization and NOX-dependent neurodegeneration.

Cell metabolomics analyses revealed a role of altered fatty acid oxidation in neurotoxicity pattern difference between nab-paclitaxel and solvent-based paclitaxel.

Peripheral neuropathy (PN) is a dose-limiting, painful adverse reaction associated with the use of paclitaxel. This common side effect was often partially attributed to the solvent used for solubilization of the highly hydrophobic drug substance. Therefore, the development of alternative formulations thrived, which included that of Abraxane® containing nanoparticle albumin-bound paclitaxel (nab-paclitaxel). However, studies demonstrated inconsistent conclusions regarding the mitigation of PN in comparison with the traditional formulation. The mass spectrometry-based cell metabolomics approach was used in the present study to explore the potentially associated mechanisms. Although no significant difference in the effects on cell viability was observed, fold changes in carnitine, several acylcarnitines and long-chain fatty acid(s) were significantly different between treatment groups in differentiated and undifferentiated SH-SY5Y cells. The most prominent difference observed was the significant increase of octanoylcarnitine in cells treated with solvent-based paclitaxel, which was found to be associated with significant decrease of medium-chain acyl-CoA dehydrogenase (MCAD). The findings suggested the potential role of altered fatty acid oxidation in the different neurotoxicity patterns observed, which may be a possible target for therapeutic interventions worth further investigation.

Differences in Fatty Acid Oxidation between Nab-Paclitaxel- and Solvent-Based Paclitaxel-Treated A549 Cells Based on Metabolomics.

The pharmacokinetics, safety, and anticancer efficacy profiles of nanoparticle albumin-bound (nab)-paclitaxel formulations are superior to those of solvent-based paclitaxel formulations. The aims of the present study were to study the effects of nab-paclitaxel and solvent-based paclitaxel formulations on the metabolic profiles of the model cell line (A549) and attempt to elucidate the associated metabolic pathways. A mass spectrometry-based cell metabolomics approach and viability evaluation were used to explore the potential difference. Western blotting was utilized to measure the levels of relevant proteins, and carnitine palmitoyltransferase 1 (CPT1) activities were quantified. Fold changes normalized to controls in levels of carnitine and several acylcarnitines were significantly different (p < 0.05) between A549 cells treated with nab-paclitaxel and those treated with solvent-based paclitaxel. Relative to the controls, there were also significant fold change differences in palmitic and linoleic acid levels in the cell lysates, mitochondrial CPT1 activities, and mitochondrial medium-chain acyl-CoA dehydrogenase (MCAD) protein levels in the A549 cells subjected to the nab-paclitaxel and solvent-based paclitaxel formulations. Results suggested that the two formulations differentially modulated fatty acid oxidation in the A549 cells. While cell viability results did not reveal significant differences, the findings implied that a mass spectrometry-based cell metabolomics approach could be a sensitive tool to explore the differences caused by formulation changes without using animals. Since uncertainties of products containing nanomaterials warrant holistic screening to address safety concerns, the aforementioned approach may be of regulatory importance and is worth further investigation.

Using matrix-induced ion suppression combined with LC-MS/MS for quantification of trimethylamine-N-oxide, choline, carnitine and acetylcarnitine in dried blood spot samples.

Recently, there has been significant interest in the influences of the human gut microbiota on many diseases, such as cardiovascular disease (CVD) and metabolic disorders. Trimethylamine N-oxide (TMAO) is one of the most frequently discussed gut-derived metabolites. Dried blood spot (DBS) sampling has been regarded as an attractive alternative sampling strategy for clinical studies and offers many advantages. For DBS sample processing, whole-spot analysis could minimize hematocrit-related bias, but it requires blood volume calibration. This study developed a method combining matrix-induced ion suppression (MIIS) with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to estimate blood volume and quantify TMAO and its precursors and derivatives, including choline, carnitine and acetylcarnitine, in DBSs. The MIIS method used an ion suppression indicator (ISI) to measure the extent of ion suppression caused by the blood matrix, which was related to the blood volume. The results showed that the volume estimation accuracy of the MIIS method was within 91.7-109.7%. The combined MIIS and LC-MS/MS method for quantifying TMAO, choline, carnitine and acetylcarnitine was validated in terms of linearity, precision and accuracy. The quantification accuracy was within 91.2-113.2% (with LLOQ <119%), and the imprecision was below 8.0% for all analytes. A stability study showed that the analytes in DBSs were stable at all evaluated temperatures for at least 30 days. The validated method was applied to quantify DBS samples (n = 56). Successful application of the new method demonstrated the potential of this method for real-world DBS samples and to facilitate our understanding of the gut microbiota in human health.

Characterization of TMAO productivity from carnitine challenge facilitates personalized nutrition and microbiome signatures discovery.

The capability of gut microbiota in degrading foods and drugs administered orally can result in diversified efficacies and toxicity interpersonally and cause significant impact on human health. Production of atherogenic trimethylamine N-oxide (TMAO) from carnitine is a gut microbiota-directed pathway and varies widely among individuals. Here, we demonstrated a personalized TMAO formation and carnitine bioavailability from carnitine supplements by differentiating individual TMAO productivities with a recently developed oral carnitine challenge test (OCCT). By exploring gut microbiome in subjects characterized by TMAO producer phenotypes, we identified 39 operational taxonomy units that were highly correlated to TMAO productivity, including Emergencia timonensis, which has been recently discovered to convert γ-butyrobetaine to TMA in vitro. A microbiome-based random forest classifier was therefore constructed to predict the TMAO producer phenotype (AUROC = 0.81) which was then validated with an external cohort (AUROC = 0.80). A novel bacterium called Ihubacter massiliensis was also discovered to be a key microbe for TMA/TMAO production by using an OCCT-based humanized gnotobiotic mice model. Simply combining the presence of E. timonensis and I. massiliensis could account for 43% of high TMAO producers with 97% specificity. Collectively, this human gut microbiota phenotype-directed approach offers potential for developing precision medicine and provides insights into translational research. Video Abstract.

Investigating the Association of the Biogenic Amine Profile in Urine with Therapeutic Response to Neoadjuvant Chemotherapy in Breast Cancer Patients.

Neoadjuvant treatment (NAT) can downstage breast cancer and can be utilized for different clinical applications. However, the response to NAT varies among individuals. Having effective biomarkers is important to optimize the treatment of breast cancer. Concentrations of biogenic amines have been found to show an association with cancer cell proliferation, but their clinical utility remains unclear. This study developed a postcolumn-infused internal standard (PCI-IS)-assisted liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) method for profiling biogenic amines in human urine. Putrescine-d8 was selected as the PCI-IS to calibrate the errors caused by matrix effects in the urine sample. The optimized method was applied to investigate the association between changes in 14 amines and the therapeutic response to NAT in breast cancer patients. Urine samples were collected before initiation of chemotherapy (n = 60). Our results indicated that the levels of N1-acetylspermine, spermidine, norepinephrine, and dopamine were significantly higher in the responder group than the nonresponder group. These metabolites were incorporated with clinical factors to identify NAT responders, and the prediction model showed an area under the curve value of 0.949. These observations provide remarkable insights for future studies in elucidating the roles of biogenic amines in breast cancer. Additionally, the PCI-IS-assisted amine profiling method can facilitate these studies.

Specific diacylglycerols generated by hepatic lipogenesis stimulate the oncogenic androgen receptor activity in male hepatocytes.

BACKGROUND: Obesity-induced hepatocellular carcinoma (HCC) is more prevalent in males than in females, but the underlying mechanism remains unclear. The influence of hepatic androgen receptor (AR) pathway on the gender difference of HCC has been well documented. Here we investigated the role of hepatic lipogenesis, which is elevated in the livers of obese and nonalcoholic fatty liver disease (NAFLD) patients, in stimulating the AR pathway for the male preference of obesity induced HCC. METHODS: Male C57BL/6J mice were fed a fructose-rich high carbohydrate diet (HCD) to induce hepatic lipogenesis. The effect of hepatic lipogenesis on AR was examined by the expression of hydrodynamically injected AR reporter and the endogenous AR target gene; the mechanism was delineated in hepatoma cell lines and validated in male mice. RESULTS: The hepatic lipogenesis induced by a fructose-rich HCD enhanced the transcriptional activity of hepatic AR in male mice, which did not happen when fed a high fat diet. This AR activation was blocked by sh-RNAs or inhibitors targeting key enzymes in lipogenesis, either acetyl-CoA carboxylase subunit alpha (ACCα), or fatty acid synthase (FASN), in vivo and in vitro. Further mechanistic study identified that specific unsaturated fatty acid, the oleic acid (C18:1 n-9), incorporated DAGs produced by hepatic lipogenesis are the key molecules to enhance the AR activity, through activation of Akt kinase, and this novel mechanism is targeted by metformin. CONCLUSIONS: Our study elucidates a novel mechanism underlying the higher risk of HCC in obese/NAFLD males, through specific DAGs enriched by hepatic lipogenesis to increase the transcriptional activity of hepatic AR, a confirmed risk factor for male HCC.

Improved Dried Blood Spot-Based Metabolomics Analysis by a Postcolumn Infused-Internal Standard Assisted Liquid Chromatography-Electrospray Ionization Mass Spectrometry Method.

Dried blood spots (DBSs) have gained increasing attention recently with their growing importance in precision medicine. DBS-based metabolomics analysis provides a powerful tool for investigating new biomarkers. Until now, very few studies have discussed measures for improving analytical accuracy with the consideration of the special characteristics of DBSs. The present study proposed a postcolumn infused-internal standard (PCI-IS) assisted strategy to improve data quality for DBS-based metabolomics studies. An efficient sample preparation protocol with 80% acetonitrile as the extraction solvent was first established to improve the metabolite recovery. The PCI-IS assisted liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was used to simultaneously estimate the blood volume and correct the signal change caused by ion source contamination and the matrix effect to evaluate the spot volume effect and hematocrit (Hct) variation effect on target metabolites. Phenylalanine-d8 was selected as the single PCI-IS to correct the matrix effect. For calibration of errors caused by the blood volume difference, 75% of the test metabolites showed good correlation (R2 ≥ 0.9) between the spot volume and the signal intensity after PCI-IS correction compared to less than 50% metabolites with good correlation before calibration. The spot volume was further calibrated by the same PCI-IS. Investigation of the Hct variation effect on target metabolites revealed that it affected the concentrations of metabolites in the DBS samples depending on their abundance in the red blood cell (RBC) or plasma; it is essential to preinvestigate the distribution of metabolites in blood to minimize the comparison bias in metabolomics studies. Finally, the PCI-IS assisted method was applied to study acetaminophen-induced liver toxicity. The results indicated that the proposed PCI-IS strategy could effectively remove analytical errors and improve the data quality, which would make the DBS-based metabolomics more feasible in real-world applications.

Metabolomics of Children and Adolescents Exposed to Industrial Carcinogenic Pollutants.

Studies on metabolomes of carcinogenic pollutants among children and adolescents are limited. We aim to identify metabolic perturbations in 107 children and adolescents (aged 9-15) exposed to multiple carcinogens in a polluted area surrounding the largest petrochemical complex in Taiwan. We measured urinary concentrations of eight carcinogen exposure biomarkers (heavy metals and polycyclic aromatic hydrocarbons (PAHs) represented by 1-hydroxypyrene), and urinary oxidative stress biomarkers and serum acylcarnitines as biomarkers of early health effects. Serum metabolomics was analyzed using a liquid chromatography mass spectrometry-based method. Pathway analysis and "meet-in-the-middle" approach were applied to identify potential metabolites and biological mechanisms linking carcinogens exposure with early health effects. We found 10 potential metabolites possibly linking increased exposure to IARC group 1 carcinogens (As, Cd, Cr, Ni) and group 2 carcinogens (V, Hg, PAHs) with elevated oxidative stress and deregulated serum acylcarnitines, including inosine monophosphate and adenosine monophosphate (purine metabolism), malic acid and oxoglutaric acid (citrate cycle), carnitine (fatty acid metabolism), and pyroglutamic acid (glutathione metabolism). Purine metabolism was identified as the possible mechanism affected by children and adolescents' exposure to carcinogens. These findings contribute to understanding the health effects of childhood and adolescence exposure to multiple industrial carcinogens during critical periods of development.

Identification of TMAO-producer phenotype and host-diet-gut dysbiosis by carnitine challenge test in human and germ-free mice.

OBJECTIVE: The gut microbiota-derived metabolite, trimethylamine N-oxide (TMAO) plays an important role in cardiovascular disease (CVD). The fasting plasma TMAO was shown as a prognostic indicator of CVD incident in patients and raised the interest of intervention targeting gut microbiota. Here we develop a clinically applicable method called oral carnitine challenge test (OCCT) for TMAO-related therapeutic drug efforts assessment and personalising dietary guidance. DESIGN: A pharmacokinetic study was performed to verify the design of OCCT protocol. The OCCT was conducted in 23 vegetarians and 34 omnivores to validate gut microbiota TMAO production capacity. The OCCT survey was integrated with gut microbiome, host genotypes, dietary records and serum biochemistry. A humanised gnotobiotic mice study was performed for translational validation. RESULTS: The OCCT showed better efficacy than fasting plasma TMAO to identify TMAO producer phenotype. The omnivores exhibited a 10-fold higher OR to be high TMAO producer than vegetarians. The TMAO-associated taxa found by OCCT in this study were consistent with previous animal studies. The TMAO producer phenotypes were also reproduced in humanised gnotobiotic mice model. Besides, we found the faecal CntA gene was not associated with TMAO production; therefore, other key relevant microbial genes might be involved. Finally, we demonstrated the urine TMAO exhibited a strong positive correlation with plasma TMAO (r=0.92, p<0.0001) and improved the feasibility of OCCT. CONCLUSION: The OCCT can be used to identify TMAO-producer phenotype of gut microbiota and may serve as a personal guidance in CVD prevention and treatment. TRIAL REGISTRATION NUMBER: NCT02838732; Results.