Cholesterol, Amyloid Beta, Fructose, and LPS Influence ROS and ATP Concentrations and the Phagocytic Capacity of HMC3 Human Microglia Cell Line.

Research has found that genes specific to microglia are among the strongest risk factors for Alzheimer's disease (AD) and that microglia are critically involved in the etiology of AD. Thus, microglia are an important therapeutic target for novel approaches to the treatment of AD. High-throughput in vitro models to screen molecules for their effectiveness in reversing the pathogenic, pro-inflammatory microglia phenotype are needed. In this study, we used a multi-stimulant approach to test the usefulness of the human microglia cell 3 (HMC3) cell line, immortalized from a human fetal brain-derived primary microglia culture, in duplicating critical aspects of the dysfunctional microglia phenotype. HMC3 microglia were treated with cholesterol (Chol), amyloid beta oligomers (AßO), lipopolysaccharide (LPS), and fructose individually and in combination. HMC3 microglia demonstrated changes in morphology consistent with activation when treated with the combination of Chol + AßO + fructose + LPS. Multiple treatments increased the cellular content of Chol and cholesteryl esters (CE), but only the combination treatment of Chol + AßO + fructose + LPS increased mitochondrial Chol content. Microglia treated with combinations containing Chol + AßO had lower apolipoprotein E (ApoE) secretion, with the combination of Chol + AßO + fructose + LPS having the strongest effect. Combination treatment with Chol + AßO + fructose + LPS also induced APOE and TNF-α expression, reduced ATP production, increased reactive oxygen species (ROS) concentration, and reduced phagocytosis events. These findings suggest that HMC3 microglia treated with the combination of Chol + AßO + fructose + LPS may be a useful high-throughput screening model amenable to testing on 96-well plates to test potential therapeutics to improve microglial function in the context of AD.

Transcriptomic and glycomic analyses highlight pathway-specific glycosylation alterations unique to Alzheimer's disease.

Glycosylation has been found to be altered in the brains of individuals with Alzheimer's disease (AD). However, it is unknown which specific glycosylation-related pathways are altered in AD dementia. Using publicly available RNA-seq datasets covering seven brain regions and including 1724 samples, we identified glycosylation-related genes ubiquitously changed in individuals with AD. Several differentially expressed glycosyltransferases found by RNA-seq were confirmed by qPCR in a different set of human medial temporal cortex (MTC) samples (n = 20 AD vs. 20 controls). N-glycan-related changes predicted by expression changes in these glycosyltransferases were confirmed by mass spectrometry (MS)-based N-glycan analysis in the MTC (n = 9 AD vs. 6 controls). About 80% of glycosylation-related genes were differentially expressed in at least one brain region of AD participants (adjusted p-values < 0.05). Upregulation of MGAT1 and B4GALT1 involved in complex N-linked glycan formation and galactosylation, respectively, were reflected by increased concentrations of corresponding N-glycans. Isozyme-specific changes were observed in expression of the polypeptide N-acetylgalactosaminyltransferase (GALNT) family and the alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase (ST6GALNAC) family of enzymes. Several glycolipid-specific genes (UGT8, PIGM) were upregulated. The critical transcription factors regulating the expression of N-glycosylation and elongation genes were predicted and found to include STAT1 and HSF5. The miRNA predicted to be involved in regulating N-glycosylation and elongation glycosyltransferases were has-miR-1-3p and has-miR-16-5p, respectively. Our findings provide an overview of glycosylation pathways affected by AD and potential regulators of glycosyltransferase expression that deserve further validation and suggest that glycosylation changes occurring in the brains of AD dementia individuals are highly pathway-specific and unique to AD.

Human fasting modulates macrophage function and upregulates multiple bioactive metabolites that extend lifespan in Caenorhabditis elegans: a pilot clinical study.

BACKGROUND: Periodic prolonged fasting (PF) extends lifespan in model organisms and ameliorates multiple disease states both clinically and experimentally owing, in part, to its ability to modulate the immune system. However, the relationship between metabolic factors, immunity, and longevity during PF remains poorly characterized especially in humans. OBJECTIVE: This study aimed to observe the effects of PF in human subjects on the clinical and experimental markers of metabolic and immune health and uncover underlying plasma-borne factors that may be responsible for these effects. METHODS: In this rigorously controlled pilot study (ClinicalTrial.gov identifier, NCT03487679), 20 young males and females participated in a 3-d study protocol including assessments of 4 distinct metabolic states: 1) overnight fasted baseline state, 2) 2-h postprandial fed state, 3) 36-h fasted state, and 4) final 2-h postprandial re-fed state 12 h after the 36-h fasting period. Clinical and experimental markers of immune and metabolic health were assessed for each state along with comprehensive metabolomic profiling of participant plasma. Bioactive metabolites identified to be upregulated in circulation after 36 h of fasting were then assessed for their ability to mimic the effects of fasting in isolated human macrophage as well as the ability to extend lifespan in Caenorhabditis elegans. RESULTS: We showed that PF robustly altered the plasma metabolome and conferred beneficial immunomodulatory effects on human macrophages. We also identified 4 bioactive metabolites that were upregulated during PF (spermidine, 1-methylnicotinamide, palmitoylethanolamide, and oleoylethanolamide) that could replicate these immunomodulatory effects. Furthermore, we found that these metabolites and their combination significantly extended the median lifespan of C. elegans by as much as 96%. CONCLUSIONS: The results of this study reveal multiple functionalities and immunological pathways affected by PF in humans, identify candidates for the development of fasting mimetic compounds, and uncover targets for investigation in longevity research.

The role of FUT8-catalyzed core fucosylation in Alzheimer's amyloid-ß oligomer-induced activation of human microglia.

Fucosylation, especially core fucosylation of N-glycans catalyzed by α1-6 fucosyltransferase (fucosyltransferase 8 or FUT8), plays an important role in regulating the peripheral immune system and inflammation. However, its role in microglial activation is poorly understood. Here we used human induced pluripotent stem cells-derived microglia (hiMG) as a model to study the role of FUT8-catalyzed core fucosylation in amyloid-ß oligomer (AßO)-induced microglial activation, in view of its significant relevance to the pathogenesis of Alzheimer's disease (AD). HiMG responded to AßO and lipopolysaccharides (LPS) with a pattern of pro-inflammatory activation as well as enhanced core fucosylation and FUT8 expression within 24 h. Furthermore, we found increased FUT8 expression in both human AD brains and microglia isolated from 5xFAD mice, a model of AD-like cerebral amyloidosis. Inhibition of fucosylation in AßO-stimulated hiMG reduced the induction of pro-inflammatory cytokines, suppressed the activation of p38MAPK, and rectified phagocytic deficits. Specific inhibition of FUT8 by siRNA-mediated knockdown also reduced AßO-induced pro-inflammatory cytokines. We further showed that p53 binds to the two consensus binding sites in the Fut8 promoter, and that p53 knockdown abolished FUT8 overexpression in AßO-activated hiMG. Taken together, our evidence supports that FUT8-catalyzed core fucosylation is a signaling pathway required for AßO-induced microglia activation and that FUT8 is a component of the p53 signaling cascade regulating microglial behavior. Because microglia are a key driver of AD pathogenesis, our results suggest that microglial FUT8 could be an anti-inflammatory therapeutic target for AD.

Whole egg consumption increases plasma choline and betaine without affecting TMAO levels or gut microbiome in overweight postmenopausal women.

As a crucial part of the symbiotic system, the gut microbiome is metabolically connected to many diseases and conditions, including cardiovascular diseases (CVD). Trimethylamine (TMA) is produced by gut bacteria from dietary choline, betaine, or L-carnitine, and is then converted in the liver to Trimethylamine N-oxide (TMAO), which in turn affects hepatic and intestinal lipid metabolism. Circulating TMAO is positively associated with CVD risk. Because eggs are rich in choline, it has been speculated that their consumption may increase plasma TMAO. In this study, we hypothesized that 2 eggs per day increases plasma TMAO level by altering gut microbiome composition in mildly hypercholesterolemic postmenopausal women. In this randomized, cross-over study, 20 overweight, postmenopausal women were given 2 whole eggs and the equivalent amount of yolk-free substitute as breakfast for 4 weeks, in randomized order, with a 4-week washout in between. Fasting blood draws and stool were collected at the beginning and end of each treatment period. Plasma TMAO, choline, betaine and other metabolites were analyzed using LC/MS, while gut microbiome composition was analyzed using 16S amplicon sequencing. Plasma choline and betaine were significantly increased after whole egg but not yolk-free substitute, however TMAO level was not significantly affected by treatments. Gut microbiome composition showed large inter-individual variability at baseline and in response to the treatments. The consumption of 2 eggs per day in overweight, postmenopausal mildly hypercholesterolemic women significantly increased plasma choline and betaine, but did not increase plasma TMAO or alter gut microbiome composition.

HDL Glycoprotein Composition and Site-Specific Glycosylation Differentiates Between Clinical Groups and Affects IL-6 Secretion in Lipopolysaccharide-Stimulated Monocytes.

The goal of this pilot study was to determine whether HDL glycoprotein composition affects HDL's immunomodulatory function. HDL were purified from healthy controls (n = 13), subjects with metabolic syndrome (MetS) (n = 13), and diabetic hemodialysis (HD) patients (n = 24). Concentrations of HDL-bound serum amyloid A (SAA), lipopolysaccharide binding protein (LBP), apolipoprotein A-I (ApoA-I), apolipoprotein C-III (ApoC-III), α-1-antitrypsin (A1AT), and α-2-HS-glycoprotein (A2HSG); and the site-specific glycovariations of ApoC-III, A1AT, and A2HSG were measured. Secretion of interleukin 6 (IL-6) in lipopolysaccharide-stimulated monocytes was used as a prototypical assay of HDL's immunomodulatory capacity. HDL from HD patients were enriched in SAA, LBP, ApoC-III, di-sialylated ApoC-III (ApoC-III2) and desialylated A2HSG. HDL that increased IL-6 secretion were enriched in ApoC-III, di-sialylated glycans at multiple A1AT glycosylation sites and desialylated A2HSG, and depleted in mono-sialylated ApoC-III (ApoC-III1). Subgroup analysis on HD patients who experienced an infectious hospitalization event within 60 days (HD+) (n = 12), vs. those with no event (HD-) (n = 12) showed that HDL from HD+ patients were enriched in SAA but had lower levels of sialylation across glycoproteins. Our results demonstrate that HDL glycoprotein composition, including the site-specific glycosylation, differentiate between clinical groups, correlate with HDL's immunomodulatory capacity, and may be predictive of HDL's ability to protect from infection.

Comprehensive profiles of human milk oligosaccharides yield highly sensitive and specific markers for determining secretor status in lactating mothers.

Human milk oligosaccharides (HMOs), as an abundant and bioactive component of breast milk, work in many ways to promote the health of breast fed infants. The expression of HMOs has been shown to vary in accordance with Lewis blood type and secretor status, as women of different blood types differ in the expression of α1,2 fucosyltransferase (FUT2) and α1,3/4 fucosyltransferase (FUT3). In this study, HMOs were extracted from the milk of 60 women from The Gambia, Africa with various Lewis and secretor blood types. The HMOs were profiled using high resolution HPLC-Chip/TOF mass spectrometry. Notably, the amounts of fucosylation varied significantly between Le(a+b-) nonsecretors, Le(a-b+) and Le(a-b-) secretors, and Le(a-b-) nonsecretors. With higher frequency of expression of the recessive Lewis negative and nonsecretor phenotypes in West African populations, the HMO profiles of several milks from women of these phenotypes were examined, demonstrating decreased amounts of total oligosaccharide abundance and lower relative amounts of fucosylation. Also in this study, four specific fucosylated structures (2'FL, LNFP I, LDFT, and LNDFH I) were determined to be specific and sensitive glycan markers for rapidly determining secretor status without the need for serological testing.

Quantitative lipid metabolomic changes in alcoholic micropigs with fatty liver disease.

BACKGROUND: Chronic ethanol consumption coupled with folate deficiency leads to rapid liver fat accumulation and progression to alcoholic steatohepatitis (ASH). However, the specific effects of alcohol on key liver lipid metabolic pathways involved in fat accumulation are unknown. It is unclear whether lipid synthesis, lipid export, or a combination of both is contributing to hepatic steatosis in ASH. METHODS: In this study we estimated the flux of fatty acids (FA) through the stearoyl-CoA desaturase (SCD), phosphatidylethanolamine-N-methyltransferase (PEMT), and FA elongation pathways in relation to liver triacylglycerol (TG) content in Yucatan micropigs fed a 40% ethanol folate-deficient diet with or without supplementation with S-adenosyl methionine (SAM) compared with controls. Flux through the SCD and PEMT pathways was used to assess the contribution of lipid synthesis and lipid export respectively on the accumulation of fat in the liver. Liver FA composition within TG, cholesterol ester (CE), phosphatidylethanolamine, and phosphatidylcholine classes was quantified by gas chromatography. RESULTS: Alcoholic pigs had increased liver TG content relative to controls, accompanied by increased flux through the SCD pathway as indicated by increases in the ratios of 16:1n7 to 16:0 and 18:1n9 to 18:0. Conversely, flux through the elongation and PEMT pathways was suppressed by alcohol, as indicated by multiple metabolite ratios. SAM supplementation attenuated the TG accumulation associated with alcohol. CONCLUSIONS: These data provide an in vivo examination of liver lipid metabolic pathways confirming that both increased de novo lipogenesis (e.g., lipid synthesis) and altered phospholipid metabolism (e.g., lipid export) contribute to the excessive accumulation of lipids in liver affected by ASH.